Final Report - Medizinische Fakultät Mannheim

Transcrição

International Research Training Group
GRK 880 “Vascular Medicine”
Final Report
International Research Training Group
GRK 880
Table of Contents
1. General Information
3
1.1 Title in German and English
3
1.2 Funding period
3
1.3 Spokespersons
3
1.3.1 Mannheim / Heidelberg
3
1.3.2 Groningen
3
1.4 Participating researchers (affiliation and research area)
3
1.4.1 Mannheim / Heidelberg
3
1.4.2 Groningen
7
2. Research Results
11
3. Qualification, Supervision, Cooperations
14
Appendix 1: List of the Graduates, Postdocs and Research Students
n.a.
1a) Graduates with stipend (GRK 880/1)
n.a.
Graduates with stipend (GRK 880/2)
n.a.
Graduates with stipend (GRK 880/2-3)
n.a.
Graduates with stipend (GRK 880/3)
n.a.
1b) Graduates without stipend (GRK 880/1-4)
n.a.
1d) Postdoctoral researcher (Funding by the DFG)
n.a.
1h) Research students (Funding by the DFG)
n.a.
Appendix 2: Individual Reports of the Graduates and Postdoctoral Researcher
17
2a) Graduates (GRK 880/2)
17
Graduates (GRK 880/2-3)
51
Graduates (GRK 880/3)
64
2b) Postdoctoral researcher (GRK 880/3)
111
Appendix 3: Publication list (Graduates, Research Students, Postdoctoral
Researcher)
n.a.
2
1. General Information
1.1 Title in German and English
Vaskuläre Medizin / Vascular Medicine
Vaskuläre Schäden und Reparatur / Vascular Damage and Repair
Vaskuläre Schäden / Vascular Damage
1.2 Funding Period
GRK 880/1 (01.01.2004-31.12.2006)
GRK 880/2 (01.01.2007-30.06.2008)
GRK 880/3 (01.07.2008-31.12.2012)
GRK 880/4 (01.01.2013-31.12.2013) (Completion funding)
1.3. Spokespersons
University of Heidelberg
Prof. Dr. Fokko van der Woude† (GRK 880/1)
Prof. Dr. Hans-Peter Hammes (GRK 880/2- GRK 880/4)
University of Groningen
Prof. Dr. Lou de Leij (GRK 880/1)
Prof. Dr. Han Moshage (GRK 880/2- GRK 880/4)
1.4 Participating Researchers
Name
1.4.1 Mannheim/ Heidelberg
Time Period Research Area
Prof. Dr.
Hellmut G.
Augustin
01.01.200830.09.2013
Vascular
biology,
tumor biology
Prof. Dr.
Grietje
Beck
01.01.200431.12.2006
Anaesthesiology
critical care,
sepsis
microcirculation
Dr.
Urs
Benck
01.01.200431.12.2006
Nephrology
Prof. Dr.
Angelika
Bierhaus†
01.01.200415.04.2012
Experimental
Diabetology
Prof. Dr.
Martin
Borggrefe
01.07.200831.12.2012
Cardiology,
arrhythmia,
cardiomyopathies
Affiliation
Vascular Biology & Tumor Angiogenesis; Medical
Faculty Mannheim, University of Heidelberg;LudolfKrehl-Str. 13-17, 68167 Mannheim
+49-621-383-9962 :+49-621-383-9961
[email protected]
http://www.angiolab.de
HSK, Dr. Horst Schmidt Kliniken GmbH
Ludwig-Erhard-Str. 100, 65199 Wiesbaden
+49-611 - 43 2176
+49-611 - 43 2521
[email protected]
http://www.hsk wiesbaden.de/abteilungen/anae.html
V. Medical Dept., Medical Faculty Mannheim;
University of Heidelberg, Theodor-Kutzer-Ufer 1-3,
68167 Mannheim
+49-621-383-2495 +49-621-383- 733525
[email protected]
http://www.ma.uniheidelberg.de/inst/med5/
Medizinische Universitätsklinik Heidelberg
Innere Medizin I
Im Neuenheimer Feld 410
D-69120 Heidelberg
I. Medical Dept., Medical Faculty Mannheim;
68167 Mannheim
+49-621-383-2204 +49-621-383-2012
[email protected]
http://www.umm.uni-heidelberg.de/inst/med1/
3
Name
Time Period
Research Area
Affiliation
Prof. Dr.
Michael
Deuschle
01.01.200431.12.2006
Central Institute of Mental Health, Dept. of Psychiatry
and Psychotherapy; J5, 68159 Mannheim
+49-621-1703-2321 +49-621-1703-2325
[email protected]
http://www.zi-mannheim.de/
Prof. Dr.
Matthias
Goebeler
01.01.200730.06.2013
Prof. Dr.
Christian
Fink
01.07.200831.12.2012
Depression,
sleep medicine,
stress
physiology,
sequelae of
stress-related
disorders
Vascular
signalling,
endothelial
inflammation
and migration,
innate immunity,
dermatology
Cardiothoracic
Imaging
PD Dr.
Andreas
Fischer
01.07.200831.12.2013
Cardiovascular
biology and
pathophysiolog,
clinical
chemistry
Prof. Dr.
Sergij
Goerdt
01.01.200431.12.2013
Prof. Dr.
HermannJosef
Gröne
01.01.200731.12.2012
Dermatooncology,
vascular
biology,
dermatoimmunology
Cellular and
molecular
pathology
Prof. Dr.
Hans-Peter
Hammes
01.01.200431.12.2013
Endocrinology,
diabetology,
diabetic
retinopathy
Prof. Dr.
Markus
Hecker
01.01.200730.09.2013
Cardiovascular
physiology and
pathophysiology
Clinic for Dermatology, Venerology and Allergology,
University Hospital Würzburg, Josef-Schneider-Str. 2
D-97080 Würzburg
+49- 0931/201-26351
[email protected]
http://www.hautklinik.uk-wuerzburg.de/ueberuns.html
Allgemeines Krankenhaus Celle
Siemensplatz 4
D-29223 Celle
+49-5141 72-1701
+49-5141 72-1709
[email protected]
http://www.akh-celle.de
Deutsches Krebsforschungszentrum
Im Neuenheimer Feld 280, 69120 Heidelberg
06221 42 4150
06221 42 4159
[email protected]
http://www.dkfz.de/de/vaskulaeresignaltransduktion/index.php
Dept. of Dermatology, Venerology and Allergology,
Medical Faculty Mannheim, University of Heidelberg;
Theodor-Kutzer Ufer 1-3, 68167 Mannheim
+49-621-383-2280 +49-621-383-3815
[email protected]
http://www.umm.de/4088.0.html#c20230
Department of Cellular and Molecular Pathology
German Cancer Research Center (DKFZ) Im
Neuenheimer Feld 280, 69120 Heidelberg
+49-621-6221-4350 +49-621-6221-4352
[email protected]
http://www.dkfz-heidelberg.de/index.html
68167 Mannheim
+49-621-383-2663 +49-621-383-2663
[email protected]
http://www.ma.uniheidelberg.de/inst/med5/
Institute of Physiology and Pathophysiology; Division
of Cardiovascular Physiology, University of
Heidelberg;
Im Neuenheimer Feld 326, 69120 Heidelberg,
+49-6221-54-4035 +49-6221-54-4038
[email protected];
http://www.physiologie.uni-hd.de
4
Name
Time period
Dr.
01.01.2004Ralf
31.12.2006
Jesenofsky
Prof. Dr.
Thomas
Korff
01.01.200730.09.2013
Prof. Dr.
Jens
Kroll
01.07.200830.09.2013
Prof. Dr.
Julia
Kzhyshkowska
01.01.200428.02.2013
Prof. Dr.
Florian
Lederbogen
01.01.200431.12.2006
01.07.200831.12.2013
Prof. Dr.
Matthias
Löhr
01.01.200431.12.2006
Prof. Dr.
Susanne
Lutz
01.07.200831.01.2010
Prof. Dr.
Peter Paul
Nawroth
01.01.200431.12.2013
Research Area
Affiliation
Experimental
pancreatology
and GI oncology
II. Medical Dept., Medical Faculty Mannheim,
68167 Mannheim
+49-621-383- 5982 +49-621-383-3805
[email protected]
http://www.gastric.de/typo3_mannheim/index.php
Vascular biology Institute of Physiology and Pathophysiology; Division
of Cardiovascular Physiology, University of
Heidelberg;
Im Neuenheimer Feld 326, 69120 Heidelberg,
+49-6221-54-4131 +49-6221-54-4038
[email protected];
http://www.physiologie.uni-hd.de
Vascular
Vascular Biology & Tumor Angiogenesis; Medical
biology,
Faculty Mannheim, University of Heidelberg ; Ludolfangiogenesis,
Krehl-Str. 13-17, 68167 Mannheim
developmental
+49-621-383-9965 :+49-621-383-9961
biology,
[email protected]
zebrafish
http://www.angiolab.de
Immunology,
cellular and
molecular
biology
+49-621-383-2440 +49-621-383-3815
[email protected]
http://www.umm.de/4088.0.html#c20230
Stress research, Central Institute of Mental Health, Dept. of Psychiatry
platelet
activation
+49-621-1703-2402 +49-621-1703-2325
[email protected]
Tumour biology, Institutionen för Klinisk Vetenskap, Intervention och
pancreas
Teknik (CLINTEC),Karolinska Universitetssjukhuset,
carcinoma,
Huddinge, K54, 141 86 Stockholm
animal models
+46-8-5858-9591 +46-8-5858-2340
[email protected]
http://ki.se/ki/jsp/polopoly.jsp?d=9292&l=sv
Cardiovascular
Center for Pharmacology and Toxicology, University
G protein
of Göttingen, Robert-Koch-Str. 40, 37075 Göttingen
signaling
+49- 551-39-10665 +49- 551-39-5699
[email protected]
http://www.med.unigoettingen.de/de/content/ueberuns/217_262.html
Clinical and
University Hospital Heidelberg; Dept. of Medicine I
experimental
and Clinical Chemistry; Im Neuenheimer Feld 410,
diabetology,
69120 Heidelberg
diabetic
+49-6221-56-8600 +49-6221-56-5226
complications
[email protected]
http://www.klinikum.uniheidelberg.de/UEberblick.879.0.html
5
Name
Time period
Research Area
Affiliation
Prof. Dr.
Liliana
Schaefer
01.01.200731.12.2012
Matrix biology,
immunology,
nephrology
Prof. Dr. Dr.
Lothar
Schilling
01.01.200431.05.2013
Cerebrovascular
function –
physiology and
pathophysiology
Dr.
Kai Schledzewski
01.01.200431.12.2013
Vascular
biology,
endothelial
differentiation,
tumor biology
Prof. Dr.
Patrick
Schloss
01.07.200831.12.2013
Prof. Dr.
Marc
Schmidt
01.01.200730.06.2013
Prof. Dr.
Stefan
Schönberg
01.07.200831.12.2012
Neuronal
differentiation of
stem cells,
monoaminergic
neurotransmitter
systems
Vascular
signalling,
endothelial
inflammation
and migration,
innate immunity
Radiology,
cardiovascular
imaging,
oncologic
imaging
Institute for Pharmacology und Toxicology
Division Nephropharmacologie
Klinikum der Johann Wolfgang Goethe Universität
Theodor-Stern Kai 7, Bau 75 - 1.126
60590 Frankfurt a.M., Germany
+49-69-6301-7899 +49-69-6301-7942
[email protected]
http://www.kgu.de/zpharm/allg/
Dept. of Neurosurgery, Medical Faculty Mannheim,
University of Heidelberg, Theodor-Kutzer Ufer 1-3,
68167 Mannheim
+49-621-383-1623 +49-621-383-1937
[email protected]
http://www.umm.uni-heidelberg.de/inst/nch/
+49-621-383-3103 +49-621-383-3815
[email protected]
http://w3.umm.de/1424.0.html.0.html
Central Institute of Mental Health, Dept. of Psychiatry
+49-621-1703-2901 +49-621-1703-2325
[email protected]
Prof. Dr.
Tim
Süselbeck
01.07.200831.12.2012
Cardiology,
Coronary artery
disease
Prof. Dr.
Peter
Vajkoczy
01.01.200431.12.2006
Neurosurgery
Prof. Dr.
Thomas
Wieland
01.07.200831.12.2013
Molecular
pharmacology,
vascular signal
transduction,
vascular biology
Clinic for Dermatology, Venerology and Allergology,
University Hospital Würzburg, Josef-Schneider-Str. 2
D-97080 Würzburg
+49- 0931/201-26396
[email protected]
http://www.hautklinik.ukw.de/forschung.html
Institute for Clinical Radiology, Medical Faculty
Mannheim, University of Heidelberg, Theodor-KutzerUfer 1-3, 68167 Mannheim
+49-621-383-2276 +49-621-383-3817
[email protected]
http://www.umm.uni-heidelberg.de/inst/ikr/
Kardiologische Praxisklinik Ludwigshafen,LudwigGuttmann-Str. 11, 67071 Ludwigshafen
+49- 621 - 53 82 50 +49-621 - 53 82 525
[email protected]
http://www.kardiologieludwigshafen.de/Kardiologie-Start.html
Department of Neurosurgery, Charité, Augustenburger
Platz , 13353 Berlin
+49-30-450-560-002
+49-30-450-560-900
http://neurochirurgie.charite.de/
Institute of Experimental and Clinical Pharmacology
and Toxicology, Mannheim Medical Faculty, University
of Heidelberg,Maybachstraße 14, 68169 Mannheim
+49-621-383-9610 +49-621-383-9611
[email protected]
http://www.ma.uniheidelberg.de/inst/phar/epm/index.html
6
Name
Time period
Research Area
Affiliation
Prof. Dr.
Fokko
van der
Woude†
Prof. Dr.
Benito
Yard
01.01.200404.12.2006
Vasculitis, organ
transplantation,
diabetic
nephropathy
Experimental
diabetic
nephropathy
68167 Mannheim
01.01.200431.12.2013
V. Medical Dept.; Medical Faculty Mannheim,
University of Heidelberg; Theodor-Kutzer-Ufer 1-3,
68167 Mannheim
+49-621-383-3212 +49-621-383-2610
[email protected]
http://www.ma.uni-heidelberg.de/inst/med5/
1.4.2. Groningen
Name
Time Period
Research Area
Affiliation
Dr.
Stephan
Bakker
01.01.200431.12.2012
Diabetic
nephropathy,
cardiovascular
disease, renal
transplantation
Prof. Dr.
J. A. den
Boer
01.01.200431.12.2006
Mental stress
Dr.
Jacob
van den
Born
01.01.200731.12.2012
Experimental
nephrology
Prof. Dr.
Reinold
Gans
01.01.200431.12.2009
Clinical
diabetology
Dr.
Martin
Harmsen
01.01.200731.12.2012
Cardio-vascular
regenerative
medicine
Dr.
Wijnand
Helfrich
01.01.200431.12.2006
Tumor
Immunology
Dept. of Internal Medicine; UMCG, University of
Groningen, Hanzeplein 1, 9713 GZ Groningen
+31-50-361-3677 +31-50-361-9310
[email protected]
http://www.rug.nl/umcg/faculteit/disciplinegroepe/
internegeneeskunde/algemeneinterne/index
Dept. of Psychiatry, UMCG, University of Groningen,
Hanzeplein 1, 9713 GZ Groningen
+31 50 361 2056 +31 50 361 9132
[email protected]
http://www.rug.nl/staff/disciplines?discipline=Psychia
try
Laboratory of Experimental Nephrology; UMCG;
University of Groningen, Hanzeplein 1, De Brug 4.042;
9713 GZ Groningen
+31-50-361-0475 +31-50-361-9310
[email protected]
http://www.umcg.nl/NL/UMCG/overhetumcg/
organisatie/Specialismen/nefrologie/Pages/default.asp
x
Dept. of Internal Medicine; UMCG, University of
Groningen, Hanzeplein 1, 9713 GZ Groningen,
+31-50-361-2587 +31-50-361-3677
[email protected]
http://www.umcg.nl
Dept. of Pathology & Laboratory Medicine;
Cardiovascular Regenerative Medicine Research
Group (CAVAREM); UMCG, University of Groningen;
Hanzeplein 1, 9700 RB Groningen,
+ 31-50-361-4776 + 31-50-361-9911
[email protected]
http://cavarem.eu
Department of Surgery - Surgery Research
Laboratory, UMCG, University of Groningen
Hanzeplein 1,9713 GZ Groningen
+31 50 361 3733
[email protected]
http://www.rug.nl/staff/w.helfrich/index
7
Name
Time Period
Research Area
Prof. Dr.
Robert
Henning
01.01.200731.12.2012
Vascular
pharmacology,
renocardiovascular
disease,
hibernation
Dr.
Peter
Heeringa
01.01.200731.12.2012
Prof. Dr.
01.01.2007Jan-Luuk
31.12.2012
Hillebrands
Dr.
Jan
Kamps
01.01.200731.12.2012
Prof. Dr.
Cees
Kallenberg
01.01.200431.12.2012
Prof. Dr.
Lou de Leij
01.01.200431.12.2009
Dr.
Henri
Leuvenink
01.07.200831.12.2012
Prof. Dr.
Jan Jakob
Mooij
01.01.200431.12.2006
Affiliation
Dept. Clinical Pharmacology; Faculty of Medicine,
UMCG, University of Groningen; Ant. Deusinglaan 1,
FB20, 9713 AV Groningen
+31-50-363-2810 +31-50-363-2812
[email protected]
http://www.rug.nl/umcg/faculteit/disciplinegroepen
/klinischefarmacologie/index
Immunology
Dept of Pathology and Medical Biology, Medical
Biology section UMCG, University of Groningen,
+31 50-361-0789/8043 +31 50-361-9911
[email protected]
http://www.rug.nl/umcg/faculteit/disciplinegroepen/pl
g/medbiol/research/ec/index
Diabetes and
Dept. of Pathology & Medical Biology – Pathology;
transplantation- UMCG,University of Groningen; PO box 30.001 EA10,
associated
9700 RB Groningen
vascular
+31-50-361-2875 +31-50-361-9107
disease
[email protected]
http://www.umcg.nl/NL/UMCG/Afdelingen/Pathologie
/Research/
Lipid based drug Dept. of Phathology & Laboratory Medicine, section
delivery systems Medical Biology, UMCG, University of Groningen
and vascular
drug targeting
+31-50-361-1293 +31-50-361-9911
[email protected]
http://www.rug.nl/umcg/faculteit/disciplinegroepen/pl
g/medbiol/research/ec/index
Clinical
Dept. of Rheumatology and Clinical Immunology,
immunology,
T3.242, UMCG, University of Groningen
systemic
P.O. Box 30.001, 9700 RB Groningen
autoimmune
+31 50 3612945 [email protected]
diseases and
http://www.umcg.nl/EN/Research/Faculty/Pages/Kall
vasculitis
enberg.aspx
Endothelial cell
Currently: Dean of research
biology
UMCG; University of Groningen; Hanzeplein 1; 9713
GZ Groningen; +31-50-3639167; +31-503632883; [email protected]
http://www.rug.nl/staff/l.f.m.h.de.leij/index
Organ
Dept of Surgery, Surgical Research Laboratory,
preservation
UMCG, University of Groningen,
transplantation
Hanzeplein 1, 9700 RB Groningen
+31 50-361-9801 +31 50-363-2796
[email protected]
http://www.rug.nl/staff/h.g.d.leuvenink/index
Brain tumours
Dept. Neurosurgery,UMCG, University of Groningen
+31 50 361 2837 [email protected]
http://www.rug.nl/staff/j.j.a.mooij/index
8
Name
Time Period
Research Area
Affiliation
Prof. Dr.
Grietje
Molema
01.01.200431.12.2012
Microvascular
endothelial
pharmacology
and vascular
drug targeting
Prof. Dr.
Han
Moshage
01.01.200731.12.2012
Experimental
hepatology and
gastroenterology, graduate
education
Prof. Dr.
Gerjan
Navis
01.01.200731.12.2012
Experimental
nephrology
Prof. Dr.
Matthijs
Oudkerk
01.07.200831.12.2012
Cardiovascular
imaging
Prof. Dr.
Rutger
Ploeg
01.01.200431.12.2012
Transplantation
medicine
Dr.
Gieta
Lammersvan der
Pompe
Prof. Dr.
Martina
Schmidt
01.01.200431.12.2006
Biological
psychiatry
Dept. Pathology and Medical Biology, Medical Biology
section, Laboratory for Endothelial Biomedicine &
Vascular Drug Targeting research; UMCG, University
of Groningen; University of Groningen; Hanzeplein
1,IPC EA11, 9713 GZ Groningen,
+31-50-361-8043/0115 +31-50-361-9911
[email protected]
http://www.rug.nl/umcg/faculteit/
disciplinegroepen/plg/medbiol/index
Dept. of Gastroenterology and Hepatology; UMCG,
University of Groningen, Hanzeplein 1, 9713 GZ
Groningen
+ 31-50-361-2364 + 31-50-361-2612
[email protected]
http://www.rug.nl/umcg/faculteit/
disciplinegroepen/maagdarmenlever/apoptosis
Dept. of Medicine, Division of Nephrology; UMCG,
University of Groningen, Hanzeplein 1, 9700 RB
Groningen
+31-50-361-2955 +31-50-361-9310
[email protected]
http://www.rug.nl/staff/g.j.navis/index
Dept. of Radiology, UMCG, University of Groningen,
+31-50-3614260 +31-50-3619168
[email protected]
http://www.rug.nl/staff/m.oudkerk/index
Dept. of Surgery,UMCG, University of Groningen,
Hanzeplein 1, 9713 GZ Groningen,
+31-50 361-2283 +31-50 361-4873
[email protected]
http://www.rug.nl/umcg/faculteit/disciplinegroepen/ch
irurgie/index
Past address: Dept. of Biological Psychiatry, University
of Groningen, Hanzeplein 1, 9713 GZ Groningen.
050-361 2056/06 – 512 89 160
[email protected]
01.08.200831.12.2012
Cell biology,
translational
pharmacology,
cardiovascularneuronal biology
Prof. Dr.
Willem
van Son
01.01.200431.12.2006
Dept. of Molecular Pharmacology; Centre of
Pharmacy, UMCG, University of Groningen; Antonius
Deusinglaan 1,9713 AV Groningen
+31-50-363-3322 +31-50-363-6908
[email protected]
http://www.rug.nl/farmacie/onderzoek/onderzoeks
Clinical
Div. Nephrology - Dept. Internal Medicine; UMCG;
transplantation
University of Groningen, Hanzeplein 1; 9713 GZ
role complement Groningen;
in brain
+31-50-3616161/3614877; +31-50-3619310
death/ischemia
[email protected]
reperfusion/
http://www.rug.nl/staff/w.j.van.son/index
chronic allograft
failure
9
Name
Time Period
Research Area
Affiliation
Prof. Dr.
Hauw
The
(retired)
Prof. Dr.
Elisabeth
de Vries
01.01.200431.12.2006
Clinical
immunology
01.01.200431.12.2006
Brain tumour
Prof. Dr.
Dick
de Zeeuw
01.01.200431.12.2006
Clinical
nephrology
Dr.
Jan
Zijlstra
01.01.200431.12.2006
Sepsis
Past address: Dept. Clinical Immunology; UMCG;
University of Groningen, Hanzeplein 1; 9713 GZ
Groningen; +31-50-3614339; +31-50-3121576
[email protected]
Dept. Medical Oncology; UMCG; University of
Groningen, Hanzeplein 1; 9713 GZ Groningen;
+31-50-3612821/3611847; +31-50-3614862
[email protected]
http://www.rug.nl/umcg/faculteit/disciplinegroepen/on
cologie/index
Dept. Clinical Pharamcology; UMCG; University of
+31-50-3632810; +31-50-3632812
[email protected]
http://www.rug.nl/umcg/faculteit/disciplinegroepen/kli
nischefarmacologie/index
Dept. of Intensive Care; UMCG; University of
+31-50-3612315 [email protected]
http://www.rug.nl/staff/j.g.zijlstra/index
10
2. Research Results
Vascular medicine is considered an important and interesting research area that only
recently has received the attention that it deserves. At the transition from GRK880-1 to
GRK880-2 and -3, the focus of the graduate school changed from four topics
(immunologically mediated endothelial damage, endothelial damage by hypxia or metabolic
disease, tumor angiogenesis, and stress, hormones and the vascular system) to a focus on
mechanisms that mediate vascular damage and repair.
The multidisciplinary expertise in the
participating
academic
institutions
allowed to touch upon research
approaches in clinical areas as diverse
as hypertension, vasculitis, tumor
angiogenesis and diabetic microvascular
complications. New discoveries were
made in the areas of endothelial
signaling, vascular cell communication,
inflammation, metabolic and tumor
associated endothelial damage.
More specifically, in the area of
endothelial signalling, novel FOXO
dependent transcriptional regulators of
endothelial cell survival were identified
which display an orchestrated role in endothelial cell survival (Czymai, T et al., J Biol Chem
2010). In continuation, the role of functionally relevant mediators oft he MEK5/Erk5 pathway
were elucidated, and the revealed P21-activated kinase 1 (PAK) 1 as novel flow-repressed
gene and migration-relevant target of the MEK5/ERK5/KLF2 pathway. This previously
unknown pathway may limit undesired cell migration in unperturbed endothelium and reduce
the sensitivity to migration cues under pathological conditions.
The role of the tissue microenvironment on endothelial homeostasis and plasticity was
defined as it determines a set of factors which can induce transdifferentiation, in this case, of
liver sinusoidal endothelial cells (Géraud C., et al, Hepatology 2010).
RhoGEF 17 was identified as a novel complex partner required for the formation of
adherence junctions in endothelial and smooth muscle cells, linking junctional proteins to
Rho-dependent alterations of the cytoskeleton, migration and viability (Lutz S, et al., Cell
Signal 2013). The RhoA kinases ROCKI/II with a so far undefined role in vascular
homeostasis was studied in context of angiogenesis. It was found that ROCKI/II act as
negative regulators of angiogenesis, and may serve as therapeutic targets of inhibit hypoxia
induced angiogenesis such as proliferative retinopathy.
Adaptive remodelling processes play an important role in diseases affecting the general
vascular tone, and in particular responses to increased shear stress. Increased wall tension
drives maladaptive inward remodeling through factors that maintain the SMCs in a contractile
state. Myocardin was identified as a remodeling factor, and a novel mechanism by which
increased wall tension inactivates myocardin leading to a switch to the synthetic SMC
phenotype was detected (Pfisterer L, et al., Cardiovasc Res 2012).
The consortium was able to contribute important novel aspects relevant to diabetic
microvascular damage. Pericyte migration was defined as a major factor relevant in early
pericyte loss, relevant for the development of diabetic retinopathy which is the most common
microvascular damage in diabetes (Pfister et al., Diabetes 2008). Decorin and soluble
biglycan were identified as a crucial trigger of albuminuria which represents the most
important incipient clinical biomarker of diabetic nephropathy (Merline R, Lazaroski S, et al.,
J Physiol Pharmacol 2009). In a combined model of diabetes and hyperlipidemia, the effects
of the lipid regulated nuclear receptors LXRa and b on diabetic nephropathy were studied. It
was shown that the selective activation of LXRa in monocytes/macrophages was sufficient to
prevent diabetic nephropathy (Kiss E, et al., AJP 2013).
As a particular achievement, a novel non-hyperglycemic model of retinopathy, was
characterized, and pathways involved in secondary vasoregression were established.
Antigen processing and the specific role of CD 74 in capillary damage were delineated,
11
identifying the similarities to both diabetic retinopathy, and Alzheimer’s disease (Feng Y,
Wang Y, et al. PlosOne 2009 and 2011, Busch et al. Cell Physiol Biochem 2012, Busch et
al., PlosOne 2014).
As a major genetic determinant of diabetic kidney disease, the role of the CNDP1 gene
coding for the human serum carnosinase was further elucidated. The polymorphism of this
gene was identified as a gain of function mutation associated with reduced serum carnosine
levels, hyperglycemia was specified as a determinant of enzyme activity, and the
pharmacological supplementation of L-Carnosine, the substrate of CNDP1 revealed
antifibrotic and antiapoptotic functions in the diabetic kidney, and preserved retinal
microvessels (Riedl et al., Diabetes 2007, Diabetes 2010, Cell Physiolog Biochem 2011,
Pfister et al., Cell Physiol Biochem 2011).
In the microvasculature, the interaction with blood cells such as leucocytes and
macrophages is particularly important, since both the waste removal function and the
potential to induce endothelial damage via inflammatory signals is clinically highly relevant,
but also quite enigmatic. Stabilin-1 is expressed on scavenging cells and sinusoidal
endothelial cells with relevance for handling of waste products and secretion of newly
synthesized mediators of cell-cell crosstalk. Therefore, the identification of binding partners
and the mechanisms of intracellular trafficking is important. A new molecular mechanism was
discovered that controls intracellular transport essential for scavenging and secretory
function of stabilin-1. It was demonstrated that the intracellular sorting adaptors GGAs
interact with novel binding motifs of stabilin-1 and that both GGA interacting sites are
required for the cooperative action during stabilin-1 medaited secretion of cytokine-like
proteins and clearance of soluble components of extracellular matrix. Both functions are
essential for the control of inflammatory activation of the endothelium by macrophages
(Zhang J, et al., Mol Cell Biol 2009).
Cell-cell communication was also studied at sites of experimental atherosclerosis. It was
found that atherosclerosis-prone sites present the ligand ephrinB2 which may interact with
the EphB2 receptor on monocytes to promote inflammatory activation (Korff T, Braun J, et
al., Blood 2008; Braun J et al., ATVB 2011). Conversely, the functional consequences of the
ephrinB2-EphB2 interactions in endothelial cells were studied. Inflammation was found to
upregulate the ligands ephrinB1 and B2 in endothelial cells, and forward signaling promoted
the activation of EphB receptor bearing monocytes. EphrinB reverse signaling induced the
surface deposition of vWf and the overexpression of molecules which are known to indicate
endothelial dysfunction and increased monocyte-endothelial attachment. Combined analysis
of four different models revealed that the EphB/ephrinB system contributes to the
inflammatory response of endothelial cells whereby reverse signaling determines monocyte
behaviour (adhesion and transmigration) and endothelial permeability (Liu H, et al., Throm
Haemost 2014).
Similarly, the role of the Delta-Notch signaling in vascular damage was studied. Apart from
its dominant role in regulation angiogenesis, it was identified that Notch signaling in
endothelial cells also regulates glucose and fatty acid metabolism, suggesting that Notch
signaling in the adult vasculature is a critical regulator of tissue homeostasis and metabolism
(Berger C., et al, manuscript in revision). Ongoing work suggests that the metabolic
phenotype of mice with a conditional inactivation of endothelial Notch represented by lower
blood glucose in the presence of unaltered insulin levels (= improved insulin sensitivity) may
result from improved trans-endothelial insulin transport through caveolae.
Another cell population with a major role in vascular homeostasis is the platelet. A novel
model to study platelet protein expression and cell trafficking (using MEG-01 cells treated
with valproic acid) identified the serotonin and the dopamine transporter as critical regulators
of megakaryopoiesis and platelet-like particle formation (Schweinfurth N, et al., Platelet
2010). Moreover, to identify key mechanisms involved in platelet serotonin metabolism, the
novel model was further tested using a novel differentiation protocol to analyze serotonin
transporter densities in cultured blood cells, and to measure changes in platelet activity
markers associated with mental stress in humans.
The role of the (microvascular) endothelium was studied in relation to small vessel systemic
vasculitis, as it is believed that binding of ANCA to their putative antigens and FC receptors
on white blood cells is critical in the pathogenesis. ANCA binding to TNF stimulated
12
neutrophils leads to ROS overproduction that can damage endothelial cells, but endothelial
cells can defend themselves via inhibition of the oxidative burst. Using neutrophils from
antibody-associated vasculitis (AAV) patients, it was found that endothelial cells inhibit ROS
production, but not degranulation, ruling out that endothelial cells insufficiently inhibit ROS
production of neutrophils. Degranulation may serve as a biomarker of active AAV patients (Al
Laham F et al., Clin & Exp Immunol
2010).
AAV can become aggravated with
immunosenescence rendering age a risk
factor for autoimmune diseases. This
important
clinical
question
was
addressed using AAV-related fatigue as
a determinant. In a large clinical cohort
study, the role of age as a major
determinant of fatigue could be excluded
(Tuin J, Sanders JS, Buhl BM, et al.
Arthritis Res Ther 2013).
Donor catecholamine pretreatment has
been demonstrated to reduce the need
for dialysis after kidney transplantation
(Schnülle P, Göttmann, U, Höger S, et al., JAMA 2009) and improve heart allograft survival
and function (Benck U et al., 2011). As dopamine may result in hypertension and
tachycardia, compounds lacking these side effects, while maintaining the benefits were
developed. It was identified that the catecholamine analogue N-octanoyl dopamine (NOD)
lacks hemodynamic effects while it limits the expression and regulation of proinflammatory
transcription factors which may play a critical role in endothelial barrier function after cold
preservation in lung allografts (Hottenrott M, Wedel J, et al., PlosOne 2013). These studies
are extended to heart transplantation (Vettel C, Hottenrott M, et al., J Pharmacol Exp Ther
2014) with similar beneficial effects. NOD can also mitigate transplant vasculopathy in an
allogeneic rat model by mechanisms involving proliferation of immune and vascular cells
(Stamellou E, et al., PlosOne 2014).
In turn, monocytes/macrophages are important determinants of the microvasculature in
response to inflammation and in tumors. The transcription factor FoxQ1 was studied in
human monocytes during inflammatory conditions and it was found that this transcription
factor is critical for monocyte extravasation and deep migration at sites of inflammation and
in tumors.
The microvascular integrity and tightness of the blood-tissue barrier is of utmost importance
in two organs in which edema can have fatal consequence, one being the eye the other one
being the brain. In the brain, the kallikrein-kinin system is a major determinant of the bloodbrain-barrier. Using a model of focal ischemia, the KKS was characterized with de novo
expression of the B1 receptor, an dann upregulation of the B2 receptor in brain tissue, brain
arteries, and in the MV compartment suggesting that the KKS is an early inflammatory
response in this model (Seiffge, DJ, Lapina N, et al., Experimental Neurology 2012).
Consecutively, a model of permanent focal brain ischemia in rats mimicking malignant stroke
in humans was successfully developed (NC Shanbhag et a., unpublished) and
neuroprotective concepts are tested.
The advent of novel imaging technologies provides a highly relevant technical platform for
clinical education and research. The combined expertise of radiology and cardiology
addressed the question whether coronary plaque composition of patients with NSTEMI can
be distinguished from stable coronary patients by intravascular ultrasound (IVUS) and Dual
Source CT. The results suggest that plaque quality cannot be differentiated between the two
groups by sophisticated imaging, but that with soluble CD40 ligand, there is a biomarker
candidate that can predict NSTEMI (Sudarski S, et al., Acad Radiol 2013).
This clinical study was supported by an experienced graduated physician and radiologist.
Supervision was performed during the scientific, experimental, technical and administrative
steps as requested by the DFG reviewers.
13
In summary (see Figure left), GRK 880 Vascular Medicine has met its first aim, i.e. to enable
young talented graduates to understand and solve complex problems of vascular medicine
with a high potential of medical translation. The excellent result was only possible because of
the complementary and joint expertise of both universities. As proof of its trend-setting
potential, selected areas of research have been chosen to serve as the teaching focus in an
EU-funded program between the universities of Groningen and Heidelberg and southamerican medical research institutions in Mexico and Chile (EULAMDIMA;
www.healthcompetence.eu/converis). Furthermore, 20 % of the project results have been
propagated to the newly established IRTG 1874 DIAMICOM in which diabetic microvascular
complications is the focus (see http://www.umm.uni-heidelberg.de/ag/grk1874). On record,
50% of the projects in GRK880/2-3 have shared publications. Four joint projects were
developed with a focus on vascular medicine as associated projects that resulted in joint
publications. Overall, GRK 880 Vascular Medicine has accomplished aim 1 more than
predicted.
3. Qualification, Supervision, Cooperations
The second aim of GRK 880 Vascular Medicine was the allocation of a co-educational
framework for young graduate students with biomedical and medical background, and the
implementation of the GRK education in the scientific track of the reformed medical
curriculum in Mannheim. The review board at second on-site visit appreciated that the
international MD/PhD degree is unique in Germany, and as such, its integration into the
available doctoral degrees at the University of Heidelberg is one of the most important
achievements during GRK 880. Currently twelve candidates have passed or are in the
process of completing this degree. It was expected, that four MD/PhD degrees would be
completed per period. With currently twelve candidates that either have completed their
MD/PhD r are in the process of completing, GRK880 has clearly lived-up to this expectation.
This achievement is obviously the most relevant value of the cooperation between the two
universities, and unique in the European science area. On a local level, a testimony of the
qualification standards of GRK 880 is that the Faculty of Life Sciences at the Heidelberg
University accepted the GRK teaching program as blueprint for the program for Dr. rer. nat.
(Cooption via PI Prof. Wieland).
During GRK 880/2-3, the education program was continuously developed further as multidisciplinary approach to teach vascular medicine involving courses, workshops and
seminars. An emphasis was put on state-of-the-art lectures presented by guest professors,
on technical workshops, and on seminars teaching soft-skills. The Groningen University
Institute for Drug Exploration (GUIDE) provided an ORPHEUS (Organisation for PhD
Education in Biomedicine and health Sciences in the European System) compatible program
of workshops and tutorials. The networking was greatly endorsed by the acquisition of a
videoconference system that allowed for the participation of the three locations (Mannheim,
Heidelberg, Groningen) of GRK 880 in the regular seminar program. Furthermore, the GRK
880 teaching program benefited from the interaction with the Transregio SFB 23. As a
response to the reviews from GRK 880-1, a structured series of basic lectures was
established during year one of GRK 880-2 and -3.
With the Groningen partner, we shared a lively season school program that did not only
cover our focus of vascular medicine, but also implemented novel faculty programs such as
Vascular Aging in Groningen, and the interaction with the Transregio SFB 23 in
Mannheim/Heidelberg/Frankfurt (Vascular Differentiation and Remodelling). With the
videoconference system, the opportunity was provided to participate in the regular seminar
program given by SFB PIs and by invitees which represented the field of vascular biology
and medicine on a state of the art level.
GRK 880 also started a season school program with other international graduate schools
with compatible subjects (GRK 1302 Tübingen-Dundee, GRK 1508 „EUCAR“ AachenMaastricht, GRK 1566 „PROMISE“ Giessen-Barcelona) that finally culminated in a meeting
of four international graduate schools in Rauischholzhausen Castle, organized by GRK 1566
in 2013.
Based on the success of this meeting in 2013, the international graduate schools decided to
persue these meetings in the future and a follow up meeting of GRK 1508, 1566, and the
14
new 1874 was held in March 2014 in Heidelberg, organized by GRK 1874. Beyond these
meetings, all graduates of GRK880 actively participated in national and international
meetings, including the meetings of the SFB/TR23, SFB 834, keystone meetings, meetings
of the German Cardiac Society, German Society for Microcirculation and Vascular Biology
and of the German Diabetes Association.
With the exception of two projects, each project was supervised by two investigators in
Mannheim/Heidelberg. The cooperative supervision was particularly diligent in the area of
vasculitis, transplantation medicine, vascular biology and physiology, and diabetic
nephropathy. All graduates enjoyed the presentation rounds (progress reports) as fixed part
of each season school. The supervision concept (A – PI level – daily-weekly contact; B – PI
progress report; C – external review – progress report) proofed effective, but stimulated
discussions as to whether to establish formerly a thesis advisory committee to increase the
success rates.
During the fourth year in the MD/PhD program, supervision was provided from the Dutch
partner who also guided the promotion defence in Groningen. During GRK 880-2, a
studentship was established with great success. It allowed not only to support the graduates
in their research work, but also allowed for intensifying of the exchange between labs,
particularly at the technology level.
Local input also came from the graduate school of the University of Heidelberg, and from the
Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology
by providing specific support in the development of the teaching program, the allocation of
graduates, and in supervision strategies.
In medicine, the GRK 880 program is the first of its kind at the Mannheim Medical Faculty.
On several occasions, including the review of the progress report, and during the review of
the new GRK 1874 DIAMICOM, it was noted that the continuum of structured scientific
education for the future clinical researcher (Junior Scientific Master Class, GRK, International
PhD Groningen) is unique in Germany and should set an example. Compared with the
standard supervision of medical doctoral thesis, the GRK training differs in three important
aspects: a. it provides a structured doctoral education platform, b. it increases the time of
scientific education to 3 years and one optional year
(3+1 years vs 1 year) and c. it increases the scientific output of medical students (1-2 first
and up to 16 co-author papers). This training enables medical graduates to become
independent clinical researchers and prepares them to apply for external funding. We are
certain that this is most significant change in medical doctoral education. However, since the
reformed curriculum in Medicine at the Mannheim (and also at the Heidelberg) Medical
faculty gives strategic room for this scientific education, the time to thesis is not critically
prolonged while allowing for a significant increase in the quality of the medical doctoral
thesis.
Gender issues did not develop during GRK880-2-3. The majority of graduate candidates
were female with no visible discrimination during the funding period. Support letters and
references were broadly distributed, and post-doctoral positions were available for female
GRK alumni. Habilitations of both, female and male co-PIs were supported and successfully
completed (Susanne Lutz, Yuxi Feng, Simone Höger, Uwe Göttmann, Marc Schmidt, Sigrid
Hoffmann, Thomas Korff, Rainer Birck, Wilhelm Schmitt, Benito Yard, Jens Kroll). GRK880
also provided a working platform for graduates to develop into PIs of a subsequent graduate
school (Guido Krenning, Sibylle Hauske).
The education in GRK880 also aimed at developing the graduates into future independent
researchers. This included the submission of grant applications, and travel and research
awards. A considerable number of prices were awarded to graduates of the school, including
invitations to the Nobel Laureate Meetings, organized by the DFG, to which only top
graduates are invited.
Cooperations with Groningen PIs and within Heidelberg University, and with SFB Transregio
23 was fostered during GRK 880 for the benefit of both, graduates and PIs. Development of
novel projects, joint publications, and a steep increase of experimental expertise through the
application of complementary techniques resulted. Although not applicable to all partners, the
broad spectrum of available expertise within GRK 880 caused a substantial added value in
the field of vascular medicine. It may be mentioned at this point that the “Wissenschaftsrat”
15
accepted that vascular medicine is one “Forschungsschwerpunkt” of the Mannheim Medical
Faculty to which GRK 880 contributed (http://www.wissenschaftsrat.de/download/archive/pm
_0314.pdf).
In summary, the second aim of GRK 880 – providing a joint educational framework for young
graduates
from
medicine
and
life
sciences
–
was
also
achieved.
16
Appendix 2: Individual Reports of the Graduates
2a) Graduates (GRK 880/2)
Project number: GRK 880/2, Project 1
First and last name of doctoral researcher: Tobias Czymai
(Working) title of doctoral project: FoxO forkhead transcription factors in endothelial
activation, vascular damage and repair
Names of supervisors: M.Goebeler, M. Schmidt, G. Molema
1. General information
Duration of Research Training Group membership:
01 March 2007 - 28 February 2010
Funding (type and duration):
IRTG fellowship (01 March 2007 - 28 February 2010)
Start of doctoral training and (anticipated) date of doctoral degree:
Start 01 March 2007; End: 25 November 2010; Dr. Sc. hum.
Commencement of studies; subject; university and date of degree (diploma, MA, etc.):
01.10.2002; Biotechnology; University of Applied Sciences Bingen;
22.11.2006; Diploma-Engineer Biotechnology
Occupation after leaving the IRTG, if applicable:
01 August 2010 until today: Product manager at R-Biopharm AG in Darmstadt
2. Description of doctoral project and research results achieved to date
The FoxO subfamily of forkhead transcription factors comprises four related proteins,
FoxO1/FKHR, FoxO3/FKHR-L1, FoxO4/AFX, and FoxO6 that represent key phosphorylation
targets of a growth factor-activated signaling pathway composed of PI3K
(phosphatidylinositol-3OH-kinase) and PKB/AKT, which antagonizes FoxO activity through
direct phosphorylation at three conserved residues. In analogy to their C. elegans
counterpart, DAF16, FoxOs play crucial roles in important biological processes including lifespan regulation, glucose metabolism, stress responses and cellular survival. Remarkably,
expression of PKB-insensitive FoxO mutants bearing alanine replacements at the three PKB
phosphorylation sites (FoxO.A3) can counteract many functions of active PI3K/PKB and
induces either a G1/G0 cell cycle arrest or apoptosis, depending on the cell type studied.
While early cell culture experiments supported the view that FoxOs act functionally
redundant, recent studies have challenged this concept suggesting that in certain tissues
FoxOs may also have divergent targets and fulfill alternative functions. This includes
endothelial cells (ECs) where FoxO 1 and 3 have been reported to regulate non-redundant
but overlapping sets of angiogenesis- and vascular remodeling genes as well as
inflammation-associated genes.
Interestingly, earlier data of our group suggested that FoxOs can regulate gene expression
both through direct DNA binding to a conserved FoxO responsive element (FRE) and by
influencing the transactivation capacity of other transcription factors, raising the possibility
that cell- and isoform-specific FoxO functions may rely on indirect mechanisms of gene
regulation.
17
Goals
Major goal of this study was to analyse the relative contribution of FRE-dependent
(=classical/direct) and -independent (=alternative/indirect) transcriptional mechanisms to
vascular functions by comparing the transcriptional and cellular responses to conditional
activation of FoxO3 and a corresponding FRE-mutant. Considering the important role of
FoxOs in the regulation of cellular survival particular emphasis was laid on the role of these
mechanisms in apoptosis regulation as this represents a key process determining vascular
damage in the context of inflammation.
Summary of the results:
a) Conditional activation of FoxO3 in ECs results in apoptosis irrespective of FRE-binding
To study the impact of FoxO3 on ECs we used a conditionally active FoxO3.A3 mutant,
fused to a mutant form of the hormone-binding domain of the murine estrogen receptor
(FoxO3.A3.ER), which renders this protein conditionally active by addition of 4 hydroxytamoxifen (4OHT) to the culture medium. Additionally, we generated a corresponding FREbinding mutant of FoxO3 by site-directed mutagenesis of histidine 212 in the third DNAbinding helix of the forkhead box to arginine (FoxO3.A3.H212R-ER). Surprisingly, 4OHTinduced activation of both mutants resulted in a substantial induction of apoptosis as
indicated by the presence of cleaved effector Caspase-3 in Western blots and by subdiploidy
analysis of propidium-iodine stained cells. Although the pro-apoptotic response was less
prominent with the FRE-binding mutant these data provided first evidence that FoxO3 can
regulate important endothelial functions by indirect mechanisms.
b) FoxO3 regulates vascular remodelling genes in an FRE-dependent manner but proapoptotic, pro-inflammatory and cell-cycle-related genes by indirect mechanisms
To analyse which other functions might be regulated by alternative mechanisms we
retrovirally infected human primary ECs with either empty vector, FoxO3.A3.ER or
FoxO3.A3.ER.H212R and compared the gene expression patterns of the differently
tranduced cells upon treatment with 4OHT or medium by gene chip microarray. Interestingly,
bioinformatical functional annotation cluster analysis of the 4OHT-regulated genes revealed
a unique overrepresentation of genes associated with vascular remodelling/migration in the
class of FRE-dependent transcripts whereas cell-cycle-relevant gene clusters were primarily
found in the group of genes co-regulated by both mutants. Remarkably, we also noticed a
statistically significant overrepresentation of genes related to leucocyte differentiation in the
co-regulated group and an enrichment of apoptosis-releated clusters in both experimental
groups. However, consistent with the less pronounced apoptotic response triggered by the
FRE-binding mutant the total enrichment score of apoptosis-related clusters was lower for
the co-regulated group than for the group of direct FoxO3 targets. These results from our
bioinformatical analysis were also validated by additional qRT-PCR experiments performed
for selected FoxO3 targets representative for different enriched clusters. Using BrdU
labelling, DNA profiling and qRT-PCR we furthermore could confirm that FoxO3-induced cell
cycle arrest and regulation of important G1-S genes such as Cyclin D1 and PCNA occurred
independently of FRE-binding. This result corroborates our earlier findings that FoxOs can
effectively repress D-type cyclins in various cell types despite of the absence of conserved
FRE-binding sites in their promoter sequences.
c) FoxO3-induced apoptosis and occurs indendently of the extrinsic death-receptor pathway
Our microarray analysis revealed regulation of multiple apoptosis-associated genes. These
included both genes involved in the mitochondrial/intrinsic apoptosis pathway (such as BIM
and NOXA) as well as transcripts involved in extrinsic (death-receptor dependent) apoptosis
(e.g. TRAIL). However, further elucidation of the mechanism of FoxO3-induced apoptosis,
demonstrated that conditional FoxO3-activation failed to activate Caspase 8 cleavage that
typifies death receptor activation. Moreover, overexpression of the anti-apoptotic Caspase 8
antagonist cFLIP, which inhibits death-receptor signalling was incapable to restore FoxO3induced apoptosis in ECs, excluding an extrinsic apoptosis mechanism.
18
d) FoxO3-induced apoptosis is mediated by BIM and NOXA
In conflict with earlier reports that implicated the prominent pro-apoptotic Bcl2 family member
BIM as direct FoxO target, our microarray data suggested FoxO3-induced BIM expression to
occur in an FRE-independent fashion. This was confirmed by chromatin immune precipitation
(CHIP) experiments, which provided evidence that the FRE-binding mutant was effectively
recruited to the BIM promoter but failed to bind to an established FRE-element in the IGFBP1
promoter. Consistent with the presumed key role of BIM in mediating the FRE-independent
portion of FoxO3-induced apoptosis, BIM knockdown largely prevented apoptosis induction
by FoxO3.A3.H212R-ER but failed to abolish the pro-apoptotic response to activation of the
FRE-binding proficient FoxO3.A3 mutant. This indicated the existence of at least one
additional direct FoxO3 target that contributed to FoxO3-induced apoptosis. siRNA
experiments proved the proapoptotic Bcl2 family member NOXA to exert this function since
knockdown of NOXA significantly decreased apoptosis induction by FoxO3.A3 but failed to
affect apoptosis triggered by its FRE-binding mutant.
Conclusion
Our data show that FoxO transcription factors regulate endothelial gene expression both by
direct binding to a conserved FoxO responsive element (FRE) and via alternative
mechanisms. In addition they reveal NOXA and BIM as key direct and indirect FoxO3 targets
relevant for its proapoptotic role in ECs. Intriguingly, the two tumor suppressive functions
attributed to FoxOs (i.e. cell cycle suppression and apoptosis induction) both did not depend
on FRE-binding, suggesting that indirect mechanisms importantly contribute to the tumor
suppressive role of FoxOs. Another unexpected result of our experiments was that cellspecific functions such as vascular remodelling strictly required direct DNA binding. Thus,
our experiments suggest that the outcome of FoxO activation is likely dependent on tissuespecific expression of the different FoxOs as well as on presence and activation status of
cofactors and posttranslational modification enzymes, which together determine the cell typespecific responses to FoxO factors.
Status: Study completed
3. Comments on the qualification programme and supervision strategy
The qualification programme offered an excellent mixture of lectures, methodical laboratory
courses, soft-skill trainings and schools.
During my research the regular lectures offered state-of-the-art knowledge from key
researchers and the lab courses helped me to extend my methodical possibilies for research.
The schools offered great opportunities for an intensive exchange and discussion with other
researchers and created a lot ideas for my own research project.
I still benefit from the soft skill courses in my job outside of academia.
In combination with the intensive supervision of my PI and direct supervisor, the qualification
programme was one of the key factors for the excellent and fast accomplishment of my PhDthesis
Participation in the teaching program of GRK 880:
attended lectures: 41
attended labcourses: 7
attended workshops: 6
attended schools: 6
attended symposia: 1
Research stays or internships at other research institutions both at home and abroad,
for IRTGs: stays at the partner university:
06.10.2008 - 17.10.2008: Design and data analysis of microarray expression studies; GUIDE
AIO Course UMCG, Groningen, The Netherlands
19
Participation in schools, conferences, congresses, etc., at home and abroad:
17.07. - 19.07.2008: International Symposium: Vascular Differentiation and Remodeling,
Frankfurt, Germany
02.09. - 05.09.2009: EC7, Vienna, Austria
25.06. - 26.06.2009: Apoptosis and Cancer Conference, Dartmouth College, USA
4. Own publications
1.
Czymai T, Viemann D, Sticht C, Molema G, Goebeler M, Schmidt M. Foxo3
modulates endothelial gene expression and function by classical and alternative
mechanisms. J Biol Chem. 2010;285:10163-10178
2.
Ohnesorge N, Viemann D, Schmidt N, Czymai T, Spiering D, Schmolke M, Ludwig S,
Roth J, Goebeler M, Schmidt M. Erk5 activation elicits a vasoprotective endothelial
phenotype via induction of kruppel-like factor 4 (klf4). Journal of Biological Chemistry.
2010;285:26199-26210
3.
Bergstraesser C, Hoeger S, Song H, Ermantraut L, Hottenrot M, Czymai T, Schmidt
M, Goebeler M, Ponelies N, Stich C, Loesel R, Molema G, Seelen M, van Son W, Yard BA,
Rafat N. Inhibition of vcam-1 expression in endothelial cells by corm-3: The role of the
ubiquitin-proteasome system, p38, and mitochondrial respiration. Free radical biology &
medicine. 2012;52:794-802
20
First and last name of doctoral researcher: Francis Peyre
(Working) title of doctoral project: Tumor induced endothelial differentiation in vitro
Names of supervisors: S. Goerdt, K. Schledzewski, J. Kamps, H. Moshage
01 January 2007 - 31 December 2009
IRTG fellowship (01 January 2007 - 31 December 2009)
01 January 2007- 23 June 2011
Master of Immunology Level 2 and Master of Science Degree Level 1, University of the
Mediterranean Sea, Marseilles, France, 13.04.2006
Clinisciences as Scientific Advisor (Nanterre)
Endothelial cells compose the inner part of every blood vessel. The vascular endothelium
plays the role of barrier between the vessel lumen and the surrounding tissue, controlling the
transit of substances solved in the blood as well as the movement of leukocytes into and out
of the blood stream. Endothelial cells are involved in many aspects of vascular biology as
control of blood pressure, haemostasis, and angiogenesis which is the neoformation of blood
vessels from a pre-existing one and a crucial event in metastatic development. According to
their localisation, endothelial cells display great differences in shape and also in function. In
the present work, we focused on lung microvascular endothelial cells (LMECs) and on liver
sinusoidal endothelial cells (LSECs). LMECs treated with TS/A (a murine breast cancer
strain) -conditioned medium presented a change in their morphology and underwent an
increase of tube formation in vitro. Affymetrix gene profiling of both LMECs and LSECs
treated with TS/A-conditioned medium was performed and several gene families were found
to be over expressed. Different matrix metalloproteinases (MMPs); CC and CXC
inflammatory cytokines; as well as Ptges1, coding for the enzyme responsible of the
production of the prostaglandin E2 (PGE2), displayed an increase in their transcription after
treatment. PGE2 is a lipid principally implicated in inflammation but also in metastasis spread
and angiogenesis, and it was chosen as a candidate for TS/A-conditioned medium effects.
LMECs treated with PGE2 showed the same morphologic change than that produced by
TS/A-conditioned medium. However the cells did not display any stimulation in the set of
genes induced by TS/A-conditioned medium, and on functional level the tube formation in
vitro was increased but did not reach significance. The data exposed in this dissertation
show that ECs isolated ex vivo from preferential target organs of metastasis can undergo
transdifferentiation and angiogenic activation induced by tumor-derived soluble factors, in
vitro. TS/A-conditioned medium can trigger a kind of “edothelial-mesenchymal transition”
morphological change in LMECs, comparable to epithelial-mesenchymal transition, and
enhance their angiogenic potential in tube forming assay in vitro. Moreover, in response to
this stimulus, LMECs can over-express molecules which could be relevant for promotion and
treatment of tumor metastases in vivo.
21
No comment provided by the graduate
Participation in the teaching programme of GRK 880:
attended schools: 6
Not applicable
Participation in conferences, congresses, etc., at home and abroad:
14th International Symposium of the Cells of the Hepatic Sinusoid (ISCHS) Aug 31 - Sep 4,
2008, Tromso, Norway, aktive Teilnahme (Poster)
EC7, 7th international symposium on the biology of endothelial cells, Wien, 02-05 September
2009 (poster presentation).
4. Own publications
1.
Geraud C, Schledzewski K, Demory A, Klein D, Kaus M, Peyre F, Sticht C,
Evdokimov K, Lu S, Schmieder A, Goerdt S. Liver sinusoidal endothelium: a
microenvironment-dependent differentiation program in rat including the novel junctional
protein liver endothelial differentiation-associated protein-1. Hepatology. 2010; 52: 313-26.
2.
Klein D, Demory A, Peyre F, Kroll J, Augustin HG, Helfrich W, Kzhyshkowska J,
Schledzewski K, Arnold B, Goerdt S. Wnt2 acts as a cell type-specific, autocrine growth
factor in rat hepatic sinusoidal endothelial cells cross-stimulating the VEGF pathway.
Hepatology. 2008; 47: 1018-31.
3.
Klein D, Demory A, Peyre F, Kroll J, Geraud C, Ohnesorge N, Schledzewski K,
Arnold B, Goerdt S. Wnt2 acts as an angiogenic growth factor for non-sinusoidal endothelial
cells and inhibits expression of stanniocalcin-1. Angiogenesis. 2009; 12: 251-65.
22
First and last name of doctoral researcher: Jingjing Zhang
Title of doctoral project: Mechanism of stabilin-1-mediated intracellular trafficking
Names of supervisors: J. Kzhyshkowska, K. Schledzewski, S. Goerdt, M. Harmsen
01.06.2006 start of PhD project,
Final PhD examination: 02.11.2011 Dr. sc. hum, summa cum laude
09.1993 – 07.1997: Beijing University of Traditional Chinese Medicine (Beijing).Degree:
Bachelor of Science
09.2002-09.2005: Mannheim University of Applied Sciences. Degree: Master of Science in
Biotechnology
Maternity leave
Background of the project
Stabilin-1 was discovered on our department and specific marker expressed on scavenging
cells, such as alternatively activated macrophages and sinusoidal endothelial cells that are
important for the clearance of non-self and unwanted components from blood, lymph and
extracellular spaces in tissues. Stabilin-1 has been described as a scavenger receptor
internalizing acLDL, the matricellular glycoprotein SPARC, and growth hormone family
member PL. Besides its endocytic function, stabilin-1 also delivers the endogenously
synthesized chitinase-like protein SI-CLP from the biosynthetic compartment to the
endosomal/lysosomal system (Kzhyshkowska te al, Blood, 2006). In alternatively activated
macrophages, SI-CLP is expressed in Lamp1-positive classic lysosomes and also found
colocalized with CD63, which is specifically associated with a subpopulation of secretory
lysosomes. SI-CLP is partially expressed in p62lck-positive late endosomes, but not found in
EEA1-positive stabilin-1-containing endosomes. Immunoprecipitation experiments performed
in the liver sinusoidal endothelial cell extracts showed that stabilin-1 can be detected in
complex with clathrin and internalization adaptor AP-2 (Hansen et al, Exp. Cell Research,
2005). The authors concluded that stabilin-1 is constitutively internalized via the clathrindependent endocytic pathway, which is the classical pathway for the internalization of
scavenger receptors. However, the mechanism of endocytosis was still unknown and direct
interaction of stabilin-1 with AP-2 was not confirmed. Moreover, cytoplasmic tail of stabilin-1
does not contain the motifs for AP-2 binding suggesting that other than AP-2 sorting adaptors
mediate stabilin-1 internalization. GGA proteins play an important role in intracellular protein
sorting processes. Previous work of our group has demonstrated that stabilin-1 is colocalized
with GGA2 and GGA3 in the trans-Golgi Netwrok (TGN) in human macrophages. Inhibition of
the GGAs-mediated trafficking with brefeldin A caused accumulation of stabilin-1 in the TGN
(Kzhyshkowska et al, J Leuk Biol, 2004). The DXXLL-type motif, DDSLL, has been identified
in the cytoplasmic tail of stabilin-1 as a GGA-binding motif in our laboratory. DXXLL signal
has been found in the cytoplasmic tail of several receptors and other proteins that shuttle
23
between the TGN and endosomes. In our laboratory characterization of the interaction of
stabilin-1 cytoplasmic tail with various GGAs has been performed using GST pull down
assay. Kzhyshkowska et al showed that the interaction between stabilin-1 and GGA2 was
stronger than interaction of stabilin-1 with GGA1 and GGA3. Deletion of the DDSLL in the
cytoplasmic tail of stabilin-1 decreased the binding of GGAs the stabilin-1 cytoplasmic tail,
but did not completely abrogate the interaction TGN (Kzhyshkowska et al, J Leuk Biol,
2004).. Next, the second binding site of GGAs, an acidic cluster EDDADDD, was identified
using a set of deletion and truncation mutants within the 70-amino-acid cytoplasmic tail of
stabilin-1. GST pull down assay revealed that both DDSLL and EDDADDD are required for
the interaction of stabilin-1 cytoplastic tail with GGAs in vitro. The major goal of the project
was to identify binding partners and molecular mechanism regulating intracellular localization
of stabilin-1.
Specific objectives of the PhD project included:
1. Generation of CHO cells stably expressing recombinant full length stabilin-1 with
deleted GGA binding sties. These cells were required for functional analysis of
stabilin-1//GGA interactions during stabilin-1-meidated endocytosis.
2. Analysis of the intracellular and surface localization of stabilin-1 mutants in the stably
transfected CHO cells.
3. Analysis of the role of GGA-binding motifs during stabilin-1 mediated endocytosis.
4. Functional consequence of stabilin-1 mediated endocytosis of SPARC
5. Analysis of the role of GGA-binding motifs during stabilin-1 mediated intracellular
sorting of newly synthesized SI-CLP.
The results of the PhD project
In the present work, the role of GGA-binding sites within cytoplamic tail of stabilin-1 in its
endocytic and intracellular sorting function was investigated. The first GGA-binding site was
identified as a classical dileucine binding motif, DDSLL, at the stabilin-1 cytoplasmic tail. But
deletion of DDSLL did not abolish its interaction with GGA proteins. The second binding site
of GGAs, EDDADDD, was then also identified at the stabilin-1 cytoplasmic tail. Deletion of
both DDSLL and EDDADDD abrogates the interaction of stabilin-1 and all three GGAs as
shown by the GST-pull-down assay. According to these results, deletions were introduced
into the cytoplasmic tail of stabilin-1 in order to remove each GGA binding site separately
and both GGA binding sites simultaneously. Expression constructs for stabilin-1wt and
stabilin- with deleted GGA binding sites DSSLL, EDDADDD as well as double deletion for
both sites were generated and used for the development of the model systems for analysis of
the mechanism of stabilin-1 mediated endocytosis and intracellular sorting.
Effect of the deletions on the endocytic function of stabilion-1 was examined in CHO cells
stably transfected with wt stabilin1-as well as stabilin-1 mutants. Using flow cytometry and
confocal microscopy it was demonstrated that deletion of GGA binding sites does not affect
either stabilin-1 surface expression or endocytosis of stabilin-1 ligands acLDL, SPARC, PL
as well as a-stabilin-1 mAb MS1. However, deletion of EDDADDD but not DDSLL resulted in
impaired transport of SPARC to the late endosomal compartment. In addition, it was found
that SPARC can be targeted not only to the lysosomal degradation, but also to the
degradation proteasomes in stabilin-1-positive macrophages.
Second model system was used in order to examine the role of GGAs in intracellular taffikicn
of stabilin-1. The system was based on the stable expression of SI-CLP in H1299 cells
(H1299-SI-CLP-FLAG cell line). H1299-SI-CLP-FLAG cells lack endogenous SI-CLP and
express the recombinant FLAG-tagged SI-CLP. In these cells SI-CLP-FLAG is missorted into
the globular nuclear structures or localizes to the nuclear rim. Transient overexpression of
stabilin-1 causes complete redistribution of SI-CLP-FLAG into stabilin-1-positive cytoplasmic
structures, partially colocalized with TGN. Using this model system for the stabilin-1
mediated intracellular sorting of SI-CLP, the ability of stabilin-1 with deleted of GGA-binding
sites to sort SI-CLP was investigated. Double immunofluorescence revealed that not only a
single deletion of the classical DXXLL-type motif (DDSLL) but also a single deletion of the
novel GGA-binding acidic cluster EDDADDD led to the abrogation of stabilin-1 mediated
translocation of SI-CLP. Therefore, cooperative action of both the DDSLL and the EDDADDD
GGA-binding site is necessary for efficient stabilin-1-mediated intracellular sorting.
24
At the same time, investigation of the role of GGA binding sites on the late steps of stabilin-1meidated ligand transport along the endocytic pathway revealed that DDSLL and EDDADDD
motifs have distinct roles in this process. Thus, deletion of the DDSLL site had no effect on
the trafficking of stabilin-1 endocytic ligand SPARC into the late endocytic pathway. In
contrast, deletion of the EDDADDD site alone or in combination with DDSLL site resulted in
the impaired trafficking of SPARC along the endocytic pathway. The ligand was partially
arrested in the enlarged early/sorting endosomes. This difference in function of two distinct
GGA binding sites can be explained by the site-specific assembling of the additional
components of sorting protein complexes after GGA binds to the specific motif of stabilin-1.
Alternative explanation is that the GGA binding sites are used by other adaptors that remain
to be identified in future studies.
In additional, the investigation of the role of GGA binding sites in the degradation of SPARC
in the CHO-based model system allowed making an unexpected observation suggesting that
SPARC can undergo degradation in the alternative, non-lysosomal system. This assumption
was tested in the stabilin-1 positive primary human macrophages. Indeed, it was found that
SPARC can be degraded in proteasomes. This finding opens a perspective to investigate in
future the role of stabilin-1-mediated endocytosis in the proteasomal targeting of the
internalized ligands.
In conclusion, the results of the present study demonstrate for the first time that both GGA
interacting sites are required for the cooperative action during intracellular sorting of SI-CLP
performed by stabilin-1. Moreover, GGA-binding EDDADDD site is specifically required for
the efficient stabilin-1-mediated targeting of SPARC to the late endocytic pathway.
The generated models systems with CHO cells stably transfected with stabilin-1wt and
mutant deficient in specific transport abilities in future can be used for the analysis of the role
of surface expressed stabili1 for the interaction with its counter-ligand CD63. Expressed on
the surface of other cell types, including tumor cells. In frames of the project PhD student has
analysed several human melanoma cell lines, both available commercially and unique lines
generated in the Department of Dermatology, Medical faculty Mannheim for the expression
of CD63. Two cell lines, MEWO and MV-3 were found to have high level of CD63 expression
both in intracellular secretory lysosomes and on the cell surface. These cell lines will be
investigated for their ability to adhere to stabilin-1 expressed on the surface of CHO cells.
3. Comments on the qualification programm and supervision strategy
During my PhD program in frames of GRK880/2 I have mastered number of techniques in
protein biochemistry, cell and molecular biology, immunology and advanced microcopy
including confocal microcopy. I have generated several cellular model systems and leaned
how to design experiments and how to evaluate the results. During the educational program
of GRK880/2 I have acquired a lot of theoretical knowledge in endothelial cell biology and
vascular medicine. I have attended several practical courses and learned complementary
techniques in vascular biology. I have attended several joint Mannheim/Groningen spring
and autumn schools and enjoyed international scientific atmosphere and knowledge
exchange between different research groups. I had excellent opportunity to present my
results regularly and discuss the data with other students and principal investigators. I have
significantly improved my presentation skills. I have also attended classes on scientific
writing and learned how to write research papers. Supervision of my thesis by principal
investigator from Mannheim and Groningen significantly improved quality of my research.
attended schools: 4
25
n.a. (due to heart insufficiency)
Participation in conferences, congresses, etc., at home and abroad (as presenting
author):
n.a. (due to heart insufficiency)
4. Own publications
1.
Zhang J, Gratchev A, Riabov V, Mamidi S, Schmuttermaier C, Krusell L, Kremmer E,
Workman G, Sage EH, Jalkanen S, Goerdt S, Kzhyshkowska J. Novel GGA-binding site is
required for intracellular sorting mediated by stabilin-1. Mol Cell Biol, 2009 Nov;29(22):6097105.
2.
Kzhyshkowska J*, Gratchev A*, Schmuttermaier C, Brundiers H, Krusell L, Mamidi S,
Zhang J, Workman G, Sage EH, Anderle C, Sedlmayr P, and Goerdt S. Alternatively
activated macrophages regulate extracellular levels of the hormone placental lactogen via
receptor-mediated uptake and transcytosis. J Immunol, 2008 Mar 1;180(5):3028-37(*equal
contribution).
3.
Kzhyshkowska J, Zhang J. GGA1 Targeted Proteins Database 1 (2008), [22660],
10.2970/tpdb.2009.222.
26
First and last name of doctoral researcher: Sandra Lazaroski
(Working) title of doctoral project: Innate Immune Receptors: Toll-like Receptors in
Degenerative Renal Vascular Disease
Names of supervisors: H.-J. Gröne, L. Schaefer, J. van den Born
11 June 2007 - 10 June 2010
IRTG scholarship (11 June 2007 - 10 June 2010)
06/2007 Start of doctorate, completion of doctorate: 10/10/2011
Diploma in molecular biology and physiology 28 December 2005. Belgrade, Serbia
31 October 2011 Postdoctoral researcher at the Centre for Chronic Immunodeficiency,
University Clinic Freiburg, Albert-Ludwigs University Freiburg
Biglycan is a ubiquitously expressed member of the class I family of small leucine- rich
proteoglycans (SLRPs). One or two chondroitin-/dermatan-sulfate glycosaminoglycan (GAG)
side chains are covalently bound to leucine-rich repeats (LRRs) of a 42 kDa protein core of
biglycan. Biglycan is sequestered in the extracellular matrix (ECM) where it interacts with its
components including collagens and elastin. In its soluble form, which results from its
proteolytic release from the ECM or secretion by activated macrophages, biglycan acts as
danger signal (damage-associated molecular pattern, DAMP) and constitutes an
endogenous ligand of the innate immune receptors Toll-like Receptor (TLR) 2 and 4. This
binding induces downstream signaling events that lead to the activation of p38, ERK and NFkB, with subsequent generation of TNF- and macrophage inflammatory protein-2 (MIP-2).
In addition, by clustering TLR2/4 and purinergic P2X7/P2X4 receptors, biglycan induces the
formation of a multireceptor signaling complex that activates the NLR family, pyrin domain
containing 3 (NLRP3) inflammasome in a ROS- and heat-shock protein 90–dependent
manner, driving the apoptosis-associated speck-like protein containing caspase activation
and recruitment domain (ASC)/caspase-1– mediated maturation and secretion of IL-1 .
Despite its ubiquitous expression, biglycan is not uniformly distributed throughout the tissues
and organs. In murine lung, liver, spleen and bone, biglycan is expressed abundantly;
opposite to its expression in kidney, heart and skin. Biglycan is synthesized by a variety of
cells of the kidney. Under pathological conditions, as has been demonstrated in the context
of various renal diseases, biglycan expression pattern is altered. Indeed, numerous studies
have indicated a marked correlation between biglycan expression and the severity of renal
inflammatory diseases, suggesting that biglycan may initiate kidney injury during
inflammation. Nonetheless, the underlying mechanisms of biglycan actions in the pathology
of kidney diseases remain poorly understood.
Glomerular diseases constitute the major causes of end-stage renal disease (ESRD) that
requires dialysis or, ideally, kidney transplantation. These include focal segmental
glomerulosclerosis
(FSGS),
minimal
change
disease
(MCD),
membranous
27
glomerulonephropathy (MN) and diabetic nephropathy (DN), which are characterized by
podocyte injury and albuminuria. Podocytes are essential for normal kidney function because
they constitute the outer layer of the glomerular filtration barrier (GFB), thereby preventing
albumin leakage into the urine or microalbuminuria. Podocytes are highly specialized cells
that form foot processes (FP), which interdigitate between neighboring cells, building filtration
slits. FPs are rich in filamentous (F)-actin, which is important to maintain their intact structure,
critical for normal podocyte morphology and function. The disturbance of the podocyte actin
skeleton and/or its associated proteins is one of the main causes of FP retraction and
proteinuria in the course of glomerular disease. FP effacement leads to podocyte apoptosis
and/or detachment from the glomerular basement membrane (GBM) and a reduction in
podocyte numbers or podocytopenia.
Small Rho-GTPases (Rac1, RhoA and Cdc42) are the key players in the regulation of
podocyte migration and its actin dynamics. Moreover, Rac1 is a component of the
nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex and is involved in
the production of reactive oxygen species (ROS). ROS itself is known to contribute to
podocyte injury including FP effacement, apoptosis and subsequent albuminuria. Previous
studies also indicate that the activation of Rac1 leads to enhanced podocyte motility, FP
effacement and proteinuria, and more importantly, the specific inhibition of Rac1 was shown
to prevent albuminuria, demonstrating a direct role for Rac1 in proteinuric renal diseases.
In this study, we aimed to investigate the role of biglycan in the pathology of glomerular
diseases, and its mechanisms of actions leading to podocyte injury and subsequent
albuminuria. We used biglycan deficient (Bgn-/0) mice to investigate the role of biglycan in the
cause of podocyte injury and albuminuria. We observed that biglycan deficiency attenuates
albuminuria in the streptozotocin-induced model of diabetes. Moreover, increased biglycan
expression in podocytes of patients with diabetic nephropathy (DN) and minimal change
disease (MCD) was observed. These observations suggested that biglycan mediates the
podocytic changes that take place during the early stages of kidney pathophysiology, like in
diabetic nephropathy, in which the potential involvement of biglycan has been previously
addressed. To obtain deeper insights into the mechanisms of biglycan-induced albuminuria,
we used a model of LPS-induced albuminuria. Our data demonstrated that biglycan-deficient
mice treated with LPS had a significantly lower albuminuria compared to wild type
counterparts, whereas TLR2/4-deficient animals did not develop albuminuria, indicating that
endogenous biglycan is able to stimulate albuminuria via TLR2 and 4 in mice under
pathological conditions. in vivo overexpression of exogenous human soluble biglycan
(hBGN) significantly increases albuminuria. The ability of soluble biglycan to aggravate
albuminuria was also demonstrated in a model of LPS-induced proteinuria. We show that
biglycan is overexpressed in glomeruli, mostly in podocytes, inducing Rac1 activation via
TLR2/4 and the production of ROS. These effects lead to podocyte cytoskeleton remodeling,
apoptosis and excretion in the urine. More importantly, biglycan expression is enhanced in
podocytes of patients with diabetic nephropathy, with the biglycan expression levels
correlating with the severity of the patients' disease progress, proving the existence of a
direct link between biglycan expression in the kidney and the extent of albuminuria.
Altogether, our results demonstrate that soluble biglycan is a danger signal that aggravates
proteinuria under various pathological conditions, worsening considerably the disease
outcomes. Our study further highlights the needs for therapeutic approaches that block
soluble biglycan-induced-signaling under pathological conditions.
3. Comments on the qualification program and supervision strategy
Graduate school and the qualification program were of extremely high quality. There were
balanced amounts of lectures, practical workshops and scientific meetings (autumn schools)
organized by the graduate school itself.
28
attended lectures: 26 + (22 In Frankfurt)
attended schools: 4
n.a.
Annual Meeting of the German Connective Tissue Society, Cologne, March 31st-April 2nd,
2011
4. Own publications
1.
Merline R, Lazaroski S, Babelova A, Tsalastra-Greul W, Pfeilschifter J, Schluter KD,
Gunther A, Iozzo RV, Schaefer RM, Schaefer L: Decorin deficiency in diabetic mice:
aggravation of nephropathy due to overexpression of profibrotic factors, enhanced apoptosis
and mononuclear cell infiltration, J Physiol Pharmacol 2009, 60 Suppl 4:5-13.
2.
Dreieicher E, Beck KF, Lazaroski S, Boosen M, Tsalastra-Greul W, Beck M, Fleming
I, Schaefer L, Pfeilschifter J: Nitric oxide inhibits glomerular TGF-beta signaling via SMOC-1,
J Am Soc Nephrol. 2009 Sep; 20(9):1963-74
29
First and last name of doctoral researcher: Frederick Pfister
(Working) title of doctoral project: Vasoregression in incipient diabetic retinopathy
Names of supervisors: H.-P. Hammes, I. Molema, M. Harmsen
01 February 2007 - 31 January 2010
MD position IRTG GRK 880/2 (01 February 2007 – 31 January 2010), PhD position GUIDE
in Groningen (01 February 2010 - 31 October 2010)
July 2005 start of practical work, 31 October 2010 end of practical work, thesis submitted
09/2011, Doctoral degree (PhD) awarded 2011
MD submitted 2014
10/02 -10/09 Medicine, University of Heidelberg
Assistant physician, Institute of Pathology, UMM
Assistant physician, Institute of Pathology, University of Erlangen/Nürnberg
DR is a clinically well-defined, sight-threatening, chronic microvascular complication that
affects virtually all patients with diabetes mellitus. DR is characterized by gradually
progressive damage in the retinal microvasculature, leading to areas of retinal nonperfusion,
increased vasopermeability, and in response to retinal non-perfusion, pathologic intraocular
proliferation of retinal vessels. Mechanisms underlying the progressive alterations in retinal
microvessels which precede and stimulate neovascularization are not well understood.
The primary and predominant pathological changes in the diabetic retinal microvasculature
are the loss of pericytes and endothelial cells and the progressive occlusion of capillaries.
The mechanisms causing vasoregression in the diabetic retina are complex. The specific
biochemical changes that occur under diabetic conditions include apoptosis and destructive
signaling pathways in pericytes and endothelial cells. However, growing evidence suggests
that alternative mechanisms including activations of ligand-receptor systems that determine
the fate of retinal capillaries during vascular development and maturation are also involved in
pericyte loss during incipient DR. Ang-2 is among the relevant growth factors induced by
hypoxia and hyperglycemia and acts dependent on the VEGF environment either
proangiogenic or vasoregressive. Ang-2 is upregulated at the angiogenic front of a growing
vasculature and induces destabilization of endothelial cells via down-regulation of
intercellular junction molecules and promotes endothelial cell proliferation in vitro. It is not
known, however, what the precise role of Ang-2 is in physiological and pathological
angiogenesis. We show that overexpression of Ang-2 in the retina enhances retinal vascular
outgrowth and capillary density during physiological angiogenesis and reduced pericyte
coverage. Similarly, intraretinal vascular regrowth and preretinal neovascularization are
increased in the model of oxygen-induced retinopathy, and newly formed vessels were also
pericyte-deficient. In line with the notion that capillaries with reduced pericyte coverage are
more susceptible to angiogenic signals, Ang-2 overexpressing mice revealed endothelial cell
proliferation and enhanded vascular sprouting. Together, Ang-2 is an important regulator of
pericyte recruitment and investment to the growing vasculature under physiological and
pathological conditions, and pericyte deficient capillaries show enhanced angiogenesis and
30
endothelial cell proliferation under Ang-2 overexpression in a hypoxic environment.
Furthermore, our data indicate that in the mature retinal vasculature pericytes are in
particular sensitive to changes in Ang-2 expression.
Pericyte loss and vasoregression are the typical hallmarks of incipient DR. It is clear,
however, that these vascular lesions do not only occur in the diabetic retina. Such lesions
can also be found in retinae from humans that are over 50 years of age. That the lesions in
aged rat retinas have striking anatomical and histological similarities to those found in
diabetic rat retinas has recently been demonstrated by a comparative study between nondiabetic and diabetic rats. It is therefore conceivable that the mechanisms underlying agerelated and diabetic retinal vasoregression bear similarities. It has been suggested that the
age-related imbalance of pro- and anti-angiogenic factors may contribute to the vascular
changes observed in aging and diabetic retinas. We evaluated age dependent retinal
vascular changes and alterations in gene expression of growth factors in healthy and in
heterozygous Ang-2 deficient mice. Our data show that Ang-2 deficiency protects from agerelated decrease of the number of endothelial cells and pericytes and significantly
decelerated the formation of acellular capillaries. Moreover, gene expression analysis
revealed that Ang-2 deficiency prevented the decrease of VEGF and Ang-1 protein observed
in aged wild-type mice. These data suggest that the vasoprotective effect of Ang-2 gene
dose reduction results from consequently higher levels of survival factors, such as VEGF and
Ang-1 and further illustrates that the ratio of these factors is crucial for capillary cell survival
in the mature vasculature. Our previous work uncovered that Ang-2 is linked to pericyte loss,
thus plays an important role in diabetic retinopathy. Overexpression of human Ang-2 in the
mouse retina during development resulted in reduced pericyte coverage and accelerated
retinal angiogenesis. However, the further fate of retinal capillaries in this gain-of-function
model is unknown, and a final proof for the causal role of Ang-2 in DR was still missing. We
showed that overexpression of Ang-2 in retinas of healthy causes pericyte loss and the
formation of acellular capillaries equivalent to the vasoregression found in diabetic mice. If
the Ang-2 overexpressing mice are diabetic, a significantly worse vascular pathology is
present compared to wild type counterparts. These results provide further evidence that Ang2 plays a critical role in retinal vascular maintenance and in retinal vascular damage of early
diabetic retinopathy by regulating pericyte coverage. Nevertheless, the mechanisms by
which Ang-2 causes pericyte drop-out remained unclear.
Data from human and animal studies suggested that diabetic pericyte loss is the result of
apoptosis induced by activation of NFkB. However, the frequency of apoptotic pericytes
detected in retinal digest specimens is too low to explain the amount of pericytes lost after
several months of diabetes Thus, other mechanisms than apoptosis must also be involved.
The developmental origin and the morphological diversity of pericytes in retinal capillaries
indicate that pericytes consist of a heterogeneous population. We determined the pattern of
pericyte loss in the diabetic retina by performing quantitative retinal morphology in healthy
and diabetic mice. Moreover, to investigate the impact of Ang-2 on pericytes, we examined
healthy and diabetic retinas from mice with different levels of Ang-2 expression. We
categorized pericytes into three classes, one being located at vessel branches („saddle
pericytes“), another being located on straight parts of capillaries, and a third one showing
different degrees of detachment from adjacent endothelial cells („migrating pericytes“). We
show that saddle pericytes remained unaffected by diabetes, while pericytes on straight parts
of capillaries were reduced in diabetic retinas. The decrease of pericytes on straight
capillaries is paralleled by an increased number of migrating pericytes, exclusively detaching
from straight capillary parts. Migrating pericytes from the saddle position was never pbserved
suggesting that these pericytes might represent a distinct population from a different origin.
In Ang-2 overexpressing non-diabetic animals, the numbers of migrating pericytes are also
increased whereas in Ang-2 deficient non-diabetic mice, the numbers are reduced. Together,
the data from chapter 5 strongly support that migration of pericytes is an alternative
mechanism contributing to the loss of pericytes in the diabetic retina, that involves the AngTie ligand-receptor system. Avoiding early damage in the diabetic retina, such as pericyte
loss and vasoregression, is the goal of primary prevention. We studied two promising
therapeutic agents, Epo and carnosine, which are characterized by their vaso- and
neuroprotective function. Recently, it has been reported that Epo plays an important role in
31
the pathogenesis of proliferative diabetic retinopathy and experimental inhibition of EPO is as
effective as inhibition of VEGF in the ROP model. However, the impact of Epo on
vasoregression in experimental diabetic retinopathy has not been assessed yet. We show
that low-dose Epo treatment of diabetic rats over 3 months, reduces oxidative stress, inhibits
methylglyoxal-modification of retinal proteins and ameliorates pro-survival signals in the
diabetic retinas. Furthermore, Epo treatment prevents Ang-2 upregulation paralleled by
reduced pericyte loss and reduced glial activation. The formation of acellular capillaries and
the loss of pericytes were significantly reduced in treated diabetic animals after 6 months of
hyperglycemia, demonstrating that low-dose Epo treatment could sufficiently prevent the
development of vasoregression in diabetic retinopathy. Of note, the increased levels of
leukostasis, recently proposed to be involved in the pathogenesis of vasoregression in
diabetes, were not affected by Epo treatment, suggesting that vasoregression in the diabetic
retina can successfully be prevented without correcting leukostasis. A number of reports
demonstrated that the endogenous dipeptide carnosine suppresses the progression of
secondary complications of diabetes, such as diabetic cataract, nephropathy and
neuropathy. However, the therapeutic potential of carnosine in the treatment of DR has not
been tested. We supplemented diabetic rats with carnosine and examined the effect on
biochemical, growth factor, vascular and neuroglial changes. We show that carnosine
treatment prevented diabetic pericyte loss and acellular capillary formation. In contrast to the
effects observed in Epo treated animals, the protective effect of carnosine is independent of
ROS- and AGE-inhibition. However, vascular protection by carnosine was associated with
normalized Ang-2 levels and induction of Hsp-27 in activated glial cells. Therefore, the study
emphasizes the importance Ang-2 and other factors in the development of DR and
introduces the neuroglial retina as a target for intervention of retinal vasoregression. In
summary, the results achieved in this thesis emphasize the importance of the Ang-2 in
diabetic vasoregression. Beside direct hyperglycemic toxicity, the initiation and propagation
of diabetic retinopathy strongly depends on firm pericyte coverage and the complex interplay
between survival factors, such as VEGF, NGF and Hsp and other growth factors like the
angiopoietins. Furthermore, recent evidence and data generated in this thesis introduced the
neuroglial retina as a major target of hyperglycemic damage and a key component in the
pathogenesis of incipient DR. Of note, neuroglial cells contribute substantially to the overall
production of the above mentioned key factors of DR. Understanding of the interaction
between neuroglial and vascular cells and the importance of factors released by it is of
utmost importance for the development of novel and specific molecular drug targets with the
potential to prevent or arrest the progression of intraretinal vasoregression in DR.
From my personal experiences with the ITRG 880 I can really recommend this fantastic
education program to every medical student and young researcher who wants to push his
scientific education and career forward. As a member of the GRK 880/2 from 2007 to 2011, I
attended numerous high quality lecture, several scientific training courses and international
scientific meetings/congresses and it was possible for me to generate data for first- or coauthorships of 13 peer review publications, 2 reviews and 3 book chapters and the german
doctoral degree (MD) and to award the international doctoral degree (PhD). In my opinion,
these numbers speak for themselves and highlight the quality of program and the supervision
of my projects by my supervisor. Beside, this great scientific success, I got in touch and
familiar with colleges and friends from germany, the Netherlands, France, China and Russia
and with cooperating partners from the Netherlands. Some of these connections still exist
and are the basis for my current and future scientific work.
attended schools: 5
32
University of Groningen 01.022010-12.05 2010, Faculty of Medical Sciences, Pathology and
Medical Biology, UMCG, Groningen, the Netherlands
2009 International Symposium of the PREDICTIONS project, Karl-Franzens Medical
University of Graz, Graz, Austria, oral presentation
2009 Angiopoietin-2 Modulates Pericyte Migration in Experimental Diabetic Retinopathy;
69th Scientific Sessions of the American Diabetes Association, San Francisco, United
States; poster presentation
2009 Carnosine protects against glomerular Apoptosis and Podocyte loss, 44. Jahrestagung
der Deutschen Diabetes-Gesellschaft, Leipzig, Germany; oral presentation
2008 Angiopoietin-2 modulates Pericyte Migration in Experimental Diabetic Retinopathy,
Angiogenesis: Molecular Mechanisms and Functional Interactions, Kloster Seeon, Germany;
poster presentation
2008 Decrease of hypoxia-induced neovascularisation in angiopoietin-2 deficiency by
reduced MMP activity, European Association for the Study of Diabetic Eye Complications,
Amsterdam, Netherlands; poster presentation
2008 Influence of the Carnosine/Carnosinase-1-system on the development of Diabetischen
Retinopathy, 43. Jahrestagung der Deutschen Diabetes-Gesellschaft, Munich, Germany,
oral presentation
2007 The Carnosine/Carnosinase-1 system influences the development of incipient diabetic
retinopathy, Joint Meeting of the Society for Microcirculation and Vascular Biology and the
6th International Symposium on the Biology of Endothelial Cells, Heidelberg, Germany;
poster presentation
2007 Identification of Pericyte subpopulations in a spontaneous diabetic mouse model, 42.
Jahrestagung der Deutschen Diabetes-Gesellschaft in Hamburg, Deutschland, Hamburg,
Germany; oral presentation
Awards
2007 Deutschen Diabetes-Gesellschaft Project Grant: The effect of insulin and analogues
on angiogenesis in a model of hypoxia-induced proliferative retinopathy
2008 Deutschen Diabetes-Gesellschaft Project Grant: Wirkung von Angiopoietin-2 auf die
Migration von Perizyten in der diabetischen Retinopathie
2009 Deutschen Diabetes-Gesellschaft Project Grant, Untersuchungen zur
Behandlungsmöglichkeit der experimentellen diabetischen Retinopathie mit dem Dipeptid
Carnosin
2011 Einladung als Nachwuchswissenschaftler zum Lindau Nobel Laureate Meeting in
Lindau am Bodensee
4. Own publications
1.
Pfister F, Riedl E, Wang Q, vom Hagen F, Deinzer M, Harmsen MC, Molema G, Yard
B, Feng Y, Hammes HP. Oral carnosine supplementation prevents vascular damage in
experimental diabetic retinopathy. Cell Physiol Biochem. 2011;28(1):125-36
33
2.
Riedl E, Pfister F, Braunagel M, Brinkkötter P, Sternik P, Deinzer M, Bakker SJ,
Henning RH, van den Born J, Krämer BK, Navis G, Hammes HP, Yard B, Koeppel H.
Carnosine prevents apoptosis of glomerular cells and podocyte loss in STZ diabetic rats. Cell
Physiol Biochem. 2011;28(2):279-88
3.
Wang Q, Gorbey S, Pfister F, Höger S, Dorn-Beineke A, Krügel K, Berrone E, Wu L,
Korff T, Lin J, Busch S, Reichenbach A, Feng Y, Hammes HP. Long-term treatment with
suberythropoietic Epo is vaso- and neuroprotective in experimental diabetic retinopathy. Cell
4.
Riedl E, Koeppel H, Pfister F, Peters V, Sauerhoefer S, Sternik P, Brinkkoetter P,
Zentgraf H, Navis G, Henning RH, van den Born J, Bakker SJ, Janssen B, van der Woude
FJ, Yard BA. N-glycosylation of carnosinase influences protein secretion and enzyme
activity: implications for hyperglycemia. Diabetes. 2010 Aug;59(8):1984-90
5.
Wang Q, Pfister F, Dorn-Beineke A, vom Hagen F, Lin J, Feng Y, Hammes HP. Lowdose erythropoietin inhibits oxidative stress and early vascular changes in the experimental
diabetic retina. Diabetologia. 2010 Jun;53(6):1227-38
6.
Pfister F, Wang Y, Schreiter K, vom Hagen F, Altvater K, Hoffmann S, Deutsch U,
Hammes HP, Feng Y. Retinal overexpression of Angiopoietin-2 mimics diabetic retinopathy
and enhances vascular damages in hyperglycemia. Acta Diabetol. 2010 Mar;47(1):59-64
7.
Feng Y, Wang Y, Stock O, Pfister F, Tanimoto N, Seeliger MW, Hillebrands JL,
Hoffmann S, Wolburg H, Gretz N, Hammes HP. Vasoregression linked to neuronal damage
in the rat with defect of polycystin-2. PLoS One. 2009 Oct 6;4(10):e7328
8.
Feng Y, Vom Hagen F, Wang Y, Beck S, Schreiter K, Pfister F, Hoffmann S, Wagner
P, Seeliger M, Molema G, Deutsch U, Hammes HP. The absence of angiopoietin-2 leads to
abnormal vascular maturation and persistent proliferative retinopathy. Thromb Haemost.
2009 Jul;102(1):120-30
9.
Feng Y, Wang Y, Pfister F, Hillebrands JL, Deutsch U, Hammes HP. Decreased
Hypoxia-Induced Neovascularization in Angiopoietin-2 Heterozygous Knockout Mouse
through Reduced MMP Activity. Cell Physiol Biochem. 2009;23(4-6):277-284
10.
Pfister F, Feng Y, vom Hagen F, Hoffmann S, Molema G, Hillebrands JL, Shani M,
Deutsch U, Hammes HP. Pericyte migration: a novel mechanism of pericyte loss in
experimental diabetic retinopathy. Diabetes. 2008 Sep;57(9):2495-502
11.
Wang Y, Vom Hagen F, Pfister F, Bierhaus A, Feng Y, Gans R, Hammes HP.
Receptor for advanced glycation end product expression in experimental diabetic
retinopathy. Ann N Y Acad Sci. 2008 Apr;1126:42-5
12.
Feng Y, Pfister F, Schreiter K, Wang Y, Stock O, Vom Hagen F, Wolburg H,
Hoffmann S, Deutsch U, Hammes HP. Angiopoietin-2 deficiency decelerates age-dependent
vascular changes in the mouse retina. Cell Physiol Biochem. 2008;21(1-3):129-36
13.
Feng Y, vom Hagen F, Pfister F, Djokic S, Hoffmann S, Back W, Wagner P, Lin J,
Deutsch U, Hammes HP. Impaired pericyte recruitment and abnormal retinal angiogenesis
as a result of angiopoietin-2 overexpression. Thromb Haemost. 2007 Jan;97(1):99-108
Reviews
1.
Pfister F, Przybyt E, Harmsen MC, Hammes HP. Pericytes in the eye. Pflugers Arch Eur J Physiol. 2013. 465:789-796
2.
Hammes HP, Feng Y, Pfister F, Brownlee M. Diabetic retinopathy: targeting
vasoregression. Diabetes. 2011 Jan;60(1):9-16.
34
First and last name of doctoral researcher: Yumei Wang
(Working) title of doctoral project: Characterization of a new animal model for retinal
vasoregression
Names of supervisors: H.-P. Hammes, Y. Feng, J.- L. Hillebrands
01 April 2007 – 31 March 2010
IRTG fellowship (01 April 2007 – 31 March 2010)
Start of doctoral training and date of doctoral degree:
04/2007 start of practical work, 03/2010 end of practical work, thesis submitted 23
November 2009, Doctoral degree (PhD) awarded 22 November 2011
Bachelor degree/ China, Henan Province, June 1998
Associate Chief Physician Wuhan, China
Retinal vasoregressioncharacterized by theformation of acellular capillaries, is the hallmark
of vascular eye diseases, such as diabetic retinopathy.Vasoregression is a complex
processes entailing activation of neuroglia and vascular damage. There is emerging
evidence indicating that neuroglial changes can precede vascular alterations. Recently, we
described a new transgenic rat (PKD TGR) that contains a c-terminal truncated human
polycystin-2 cDNA under control of CMV promoter and develops extensive retinal
vasoregression. Our data suggest that PKD TGR retinasexhibit primary neuronal
degeneration and secondary vasoregression. These data suggest some similarities between
this model and the changes found in DR. However, little is known about the molecular
mechanisms of retinal vasoregression. In order to identify genes and pathway
involved in the transition from neurodegeneration to vasoregression, we performed gene
microarray analysis, TaqMan real-time PCR, western blot analysis and immunofluorescence
staining of 1 month- and 3 month-old PKD TGR and SD retinas, i.e. before and after the
initiation of vasoregression. Our study demonstrated that predominantly genes involved in
immune/inflammatory pathways were strongly upregulated, CD74 being thestrongest
upregulated one. Glial cells including astrocytes and Müller cells became activated prior to
vasoregression. CD74 was predominantly expressed in microglial cells in proximity of the
deep capillary layers in which vasoregression ensues. HSP27 was upregulated in glial cells
around degenerating vessels in PKD rats. Up-regulated expression of FGF2 and CNTF prior
to vasoregression likely reflects a response to photoreceptor damage, while NGF
upregulated after the onset of vasoregression may have an association to vascular
degeneration.Our findings indicate that genes involved in immune/inflammatory pathways
may act as predominant regulators in the development of vasoregression in the degenerating
retina and a marked activation of glia cells may closely link to the vasoregression in the PKD
retina. Taken this together, the mechanisms underlying vasoregression in PKD TGR seem to
be a sequential cascade starting from neuronal damage.Defects in cilia of the photoreceptor
cells lead to apoptotic changes in these cells, releasing molecules and cell debris that can
35
contribute to activation of glial and microglial cells. Glial cells produce, among many others,
small heat shock proteins and neurotrophins to protection the retina from further injury, and
phagocytotic microglia may eliminate harmful molecules, but also release inflammatory
cytokines, which may cause or aggravate blood vessel damage. Vasoregression may occur
when pericytes and endothelial cells are exposed to glial activation and lose their function in
maintaining vascular integrity.
Our study suggests that CD74 and immune/inflammatory pathways together with activated
glia cells can cause retinal vasoregressionas exemplified in this animal model.Further
analysis addressing functional modification may provide insights of the relative importance of
the genetic alterations identified herein.
GRK 880 plays a pivotal role in scientific research work and training. It is nonreplaceable. As
an excellent educational platform focusing on the vascular medicine, which is particularly
important especially for me, because my major is nephropathy, i.e., a vascular disease. The
experience in GRK 880 helps me to build a bridge between basic medical knowledge and
clinical science in my field. It assists both exact design and successful implementation of
scientific research project in scientific research work in my present job.It is beneficial for
improving the levels of scientific projects and establishing cooperation relations. In talent
training,it can enrich knowledge, deepen thinking, enlighten thoughts and improve ability. For
example, the “Scientific Writing” class helps me to know how to write an excellent scientific
paper, and the “Presentation Skills” improve my ability to present myself. The events
including meetings, seminars and lectures, theoretical courses and practical courses, provide
the basic sciences which are necessary to the clinical doctors and improve the experiment
skills for a clinical doctor.
attended schools: 5
09/2010 – 12/2010 University of Groningen
n.a.
4. Own publications
1.
Feng Y, Wang Y, Yang Z, Wu L, Hoffmann S, Wieland T, Gretz N, Hammes HP.
Chronic hyperglycemia inhibits vasoregression in a transgenic model of retinal degeneration.
Acta Diabetol. 2014, 51: 211-8
2.
Wang Y, Landheer S, van Gilst WH, van Amerongen A, Hammes HP, Henning RH,
Deelman LE, Buikema H. Attenuation of renovascular damage in Zucker diabetic fatty rat by
NWT-03, an egg protein hydrolysate with ACE- and DPP4-inhibitory Activity. PLoS One.
2012;7(10):e46781
3.
Feng Y, Wang Y, Li L, Wu L, Hoffmann S, Gretz N, Hammes HP. Gene expression
profiling of vasoregression in the retina--involvement of microglial cells. PLoS One.
2011;6(2):e16865
4.
Feng Y, Wang Y, Stock O, Pfister F, Tanimoto N, Seeliger MW, Hillebrands JL,
Hoffmann S, Wolburg H, Gretz N, Hammes HP. Vasoregression linked to neuronal damage
in the rat with defect of polycystin-2. PLoS One. 2009;4(10):e7328.
36
First and last name of doctoral researcher: Jennifer Braun
(Working) title of doctoral project: The impact of the Eph/ephrin system on the interaction
between leukocytes and vascular cells
Name of supervisors: M. Hecker, T. Korff, R. Henning, I. Molema
01 January 2007- 18 June 2010
2000; Biology; 09/2006 TU Darmstadt
1)
Post-Doc, University of Heidelberg 07-08/2010
2)
Post-Doc, DKFZ 10/2010-11/2013
3)
Scientific Project Coordinator, DKTK since 12/2013
Cardiovascular diseases, arteriosclerosis in particular, are the leading causes of death in the
Western world as approximately 50% of the population suffer from these diseases.
Arteriosclerosis is a pathophysiological alteration of the vascular wall. Hence, it is of great
interest to investigate the mechanisms leading to these pathological alterations. As the
Eph/ephrin system is a crucial player of vascular remodelling processes, we hypothesized
this system may be involved in the pathomechanism of arteriosclerosis. Therefore, the aim of
this study was to elucidate a possible involvement of this system in the pathophysiological
remodelling process of arteriosclerosis. Analysis of the distribution of ephrinB2 in the arterial
system of the mouse revealed a heterogeneous expression pattern of this molecule. The
overall expression of ephrinB2 was rather weak with an up-regulation in large conduit
arteries like the aorta. Subsequent studies of the aortic arch verified a strong expression at
the inner curvatures but a weak expression at the outer curvatures of the bifurcations. This
expression pattern coincides with the so-called arteriosclerosis-prone sites in LDL-R- or
ApoE-deficient mice, respectively. Detailed analysis of these plaques indicated a correlation
between the ephrinB2 expression in the endothelial cells covering the plaque and the
number of infiltrated monocytes/macrophages and pointed towards a possible role of the
Eph/ephrin system plaque-development. Moreover, monocytes express the receptors for
ephrinB2 EphB1-4 with EphB2 being up-regulated upon trans-differentiation into
macrophages. By applying atomic force microscopy (AFM), we could demonstrate that
monocytes physically interact with ephrinB2. Furthermore, adhesion of these cells to
endothelial-intact mouse aortic segments abundantly expressing ephrinB2 was inhibited by
pre-treatment with soluble ephrinB2. As the development of arteriosclerotic plaques is not
only dependent on the adhesion of monocytes to the endothelium but also on their
transmigration into the vascular wall we analyzed if this process is enhanced by ephrinB2.
The corresponding analyses confirmed that ephrinB2 not only affects the migration as such
but also the transmigration of these cells through the endothelium. In addition, interaction
37
with ephrinB2 led to a pro-inflammatory activation of the monocytes, as evidenced by an
increased expression of the arteriosclerosis-related chemokines MCP-1 and Interleukin-8 by
these cells. Further protein arrays showed that the release of these chemokines is enhanced
as well. The specifity of this reaction was confirmed by blocking the ephrinB2-dependent
pathways. Moreover, the monocytic receptor EphB2 was phosphorylated upon interaction
with ephrinB2. Taken together, these observations establish ephrinB2 as a molecule that is
abundantly expressed at remodelling sites of the arterial tree and as new determinant of the
pro-inflammatory stimulation of monocytes and therefore is likely to contribute to the
pathogenesis of atherosclerosis. This shows a new function of ephrinB2 in cardiovascular
(patho)physiology, which clearly exceeds its role as a mere arterial marker molecule.
No comment submitted
Participation in the teaching programme of GRK 880
attended schools: 6
n.a.
Joint Meeting of the Society of Microcirculation and Vascular Biology and the 6th International
Symposium of the Biology of Endothelial Cells; Heidelberg 4.-6. Oktober 2007
DPG (87. Jahrestagung der Deutschen Physiologischen Gesellschaft); Köln, 02.-05. März
2008
International Symposium "VAscular Differentiation and Remodeling"; Frankfurt/Main 17.-19.
Juli 2008
5th European Meeting on Vascular Biology and Medicine, 14.9.-17.9. 2009, Marseille
4. Own publications
1.
Korff T, Braun J, Pfaff D, Augustin HG, Hecker M Role of ephrinB2 expression in
endothelial cells during arteriogenesis: impact on smooth muscle cell migration and
monocyte recruitment. Blood 2008; 112:73‐81.
2.
Braun J, Hoffmann S, Feldner A, Ludwig T, Henning R, Hecker M, Korff T Endothelial
cell ephrinB2‐dependent activation of monocytes in arteriosclerosis. Arter Thromb Vasc Biol
2011; 31(2):297-305.
38
First and last name of doctoral researcher: Malte Depping
Title of doctoral project: Modulation of human in vivo triosephosphate isomerase
transcription with alteration of obesity and impaired glucose tolerance
Names of supervisors: A. Bierhaus †, P. Nawroth, S. Bakker, R. Gans
01 March 2007 - 30 June 2009
IRTG fellowship (01 March 2007 - 30 June 2009)
03/2007 Start of doctoral training, date of doctoral degree: 11 December 2012.
10/2004, Medicine, Medical Faculty of Heidelberg, Heidelberg University, 16 June 2011:
Medical Degree
Please note any circumstances which could have affected your scientific work:
My project’s supervisor, Prof. Bierhaus, died from cancer during the last period of my
project.
Occupation after leaving the IRTG:
08/2011 Residency at the Center of Psychosocial Medicine, Department of General
Psychiatry, University of Heidelberg, Germany
Late diabetic complications are causally related to chronic hyperglycemia. The toxic effects
of high glucose levels are believed to result from elevated metabolic flux and consecutively
increased formation of reactive glucose metabolites. Current treatment strategies of diabetes
aim at lowering elevated glucose levels, however some patients with good glycemic control
still develop severe complications, suggesting persistent changes in cellular metabolism
despite restoration of euglycemia. Apart from hyperglycemia, susceptibility to late diabetic
complications may be related to impaired transcription of glycolytic enzymes that maintain
physiological substrate flux and reduce the formation of reactive glucose metabolites.
Triosephosphate isomerase (TPI) is a crucial glycolytic enzyme. If this enzyme’s function is
impaired, as e.g. in the rare genetic enzymopathy TPI deficiency, severe glucose-induced
cellular damage can be observed independent from elevated blood glucose levels. In
pathobiological models of diabetic complications, alterations of TPI transcription have yet not
been investigated. In this study, by means of real time quantitative PCR, we investigated TPI
transcription in erythrocytes from peripheral blood of obese patients with normal glucose
tolerance (NGT), impaired glucose tolerance (IGT), or type 2 diabetes mellitus (DM) before
and after completing a 12-week controlled lifestyle modification program. We predicted that
lower levels of TPI transcription would be found in DM compared to NGT and IGT, and that
such a reduction would be reversible in the DM group following exercise-based lifestyle
intervention. 19 subjects with NGT, 5 with IGT, and 3 with DM were enrolled. Pretreatment
TPI transcription showed a trend towards lower transcription in the DM group, which however
did not reach statistical significance. An analysis of variance revealed a main effect of group
39
for change of transcription with intervention. A significant increase in transcription with
intervention was observed in the DM subjects, but not in the NGT or in the IGT subjects.
Across the entire patient group, change in TPI transcription with intervention was related to
lowered fasting glucose and improved glucose tolerance following intervention. These results
indicate that alterations of glycolytic enzyme transcription may be associated with severity of
metabolic derangement in diabetes mellitus, potentially reflecting ’hyperglycemic memory’.
Such measures of transcription may warrant consideration as diagnostic determinants of
susceptibility to late diabetic complications.
Participation in the IRTG enabled me to be first introduced to general research principles,
laboratory problem solving, and scientific exchange among fellow colleagues. Sadly, my
project’s supervisor, Angelika Bierhaus, died during the period of my project. I will keep her
in mind as a warm-hearted person who dedicated herself to her research.
I found the IRTG’s training program to be suitable with regard to content and organization. I
would however recommend future medical students to temporarily pause their medical
teaching while engaging in the IRTG, as a compelling medical school’s curriculum may at
times be in conflict with laboratory duties and the IRTG teaching program. Since completing
the IRTG, I have engaged myself in a research area and methodology different from my
doctoral project. My initial research experience has however proven beneficial since then.
attended lab courses: 0
attended schools: 3
for IRTGs, stays at the partner university:
n.a.
Revisiting Default Mode Network Function in Major Depression, Evidence for Disrupted
Subsystem Connectivity, 2014 European Congress of Psychiatry, Munich, Oral presentation
Longitudinal task-negative network analyses in preclinical Huntington's disease, 2013 Annual
Conference of the German Association for Psychiatry, Psychotherapy and Psychosomatics,
Berlin, Poster presentation
Abnormal gray and white matter volume in delusional infestation, 2012 Annual Conference of
the German Association for Psychiatry, Psychotherapy and Psychosomatics, Berlin, Poster
presentation
Default-mode network changes in preclinical Huntington’s disease, 2012 Annual Conference
of the German Society of Biological Psychiatry, Heidelberg, Poster presentation
Local organization committee, 2012 Annual Conference of the German Society of Biological
Psychiatry, Heidelberg
4. Own publications
1.
Wolf RC, Sambataro F, Vasic N, Baldas EM, Ratheiser I, Landwehrmeyer BG,
Depping MS, Thomann PA, Sprengelmeyer R, Sussmuth SD, Orth M. Visual system
40
integrity and cognition in early Huntington's disease. The European Journal of Neuroscience.
2014; Epub ahead of print.
2.
Wolf R, Huber M, Lepping P, Sambataro F, Depping MS, Karner M, Freudenmann
RW. Source-based morphometry reveals distinct patterns of aberrant brain volume in
delusional infestation. Progress in Neuro-Psychopharmacology & Biological Psychiatry;
2014; 48, 112-116.
3.
Hirjak D, Thomann PA, Depping MS, Bienentreu SD, Wolf RC. Neuronale Korrelate
des Psychoserisikosyndroms. Funktionelles Neuroimaging bei Personen mit erhöhtem
Psychoserisiko. Nervenheilkunde. 2014; 33, 64-74.
4.
Wolf RC, Huber M, Depping MS, Thomann PA, Karner M, Lepping P, Freudenmann
RW. Abnormal gray and white matter volume in delusional infestation. Progress in NeuroPsychopharmacology & Biological Psychiatry. 2013; 46, 19-24.
5.
Hirjak D, Thomann PA, Depping MS, Wolf RC. Neuronale Korrelate des
Psychoserisikosyndroms. Strukturelles Neuroimaging bei Personen mit erhöhtem
Psychoserisiko. Nervenheilkunde. 2013; 32, 582-591.
6.
Sprengelmeyer R, Orth M, Muller HP, Wolf RC, Gron G, Depping MS, Kassubek J,
Justo D, Rees EM, Haider S, Cole JH, Hobbs NZ, Roos RA, Durr A, Tabrizi SJ, Sussmuth
SD, Landwehrmeyer BG. The neuroanatomy of subthreshold depressive symptoms in
Huntington's disease: a combined diffusion tensor imaging (DTI) and voxel-based
morphometry (VBM) study. Psychological Medicine. 2013; 1-12.
41
First and last name of doctoral researcher: Natalia Lapina
(Working) title of doctoral project: Role of the kallikrein-kinin (KK) system in the
cerebrovascular system in ischemia-reperfusion injury
Names of supervisors: L. Schilling, R. Henning
01 June 2007- 31 May 2010
IRTG fellowship (01 June 2007- 31 May 2010)
06/2007 Start of doctorate, anticipated submission of doctoral thesis on 01 July 2014,
anticipated completion of doctorate: Autumn/Winter 2014
1996-2002
Study of human medicine with specialisation medical biochemistry at the
Russian State Medical University, Moscow.
24.07.2002
Diploma “Medical biochemistry”
01 June 2010 Division of Neurosurgical Research, Medical Faculty Mannheim, Heidelberg
University
Bradykinin, the major metabolite of the kallikrein-kinin system (KKS) affects vascular function
including induction of endothelium-dependent vasodilatation in arteries and an increase of
blood-brain barrier permeability leading to brain edema and swelling. These effects are due
to interacting with constitutively expressed B2 receptors or B1 receptors following de novo
expression. A full KKS has been found in the brain tissue, but its expression in the cerebral
vasculature has not yet been studied. The KKS is considered an important component of
early inflammation under pathological conditions including ischemia-reperfusion. In the
current project (#7 of the International Research Training School GRK 880 2/3 “Vascular
Medicine”) we have addressed the expression of the KKS in brain parenchyma, brain
macrovasculature (pial arteries, PAs) and cerebral microvessels (MVs) on the level of gene
expression KKS in response to a standardised ischemia reperfusion injury.
The first step was to establish a method of high yield high purity MV extraction from the rat
hemisphere. In order to protect the mRNA as much as possible MV extraction was performed
in a purely mechanical way at < 4°C throughout. The mechanical isolation of brain MVs
includes homogenization, density gradient centrifugation, and the purification of capillaries by
filtration. Purity of MVs extraction was regularly checked by light microscopy revealing mainly
capillaries and a small fraction of pre-/postcapillary MVs. The purity was also checked on the
level of gene expression using these cell-type specific markers: synapsin (SYN; neuronal
marker), calponin (CNN; smooth muscle cell marker), platelet derived growth factor receptor
(PDGF-R; pericyte marker), glial fibrillary acidic protein (GFAP, astrocyte marker), and
endothelial nitric oxide synthetase (eNOS, endothelial cell marker). Elongation factor (EF)-1
was used as reference in reverse transcription real time quantitative PCR (RT-qPCR)
measurements. Quantification was done using the Ct methodology.
In MV extracts there was a clear-cut (~100 fold) enrichment of eNOS and PDGF-R mRNA
species over brain tissue reflecting the high purity of the MV extracts. Unexpectedly, GFAP
42
mRNA also showed an enrichment (~40fold) in MVs vs. tissue. However, even higher levels
of GFAP mRNA were measured in the extraparenchymal PA (~60fold enrichment vs. tissue).
When we extended these studies into the extracerebral internal carotid artery, the common
carotid artery, and the aorta the GFAP mRNA level decreased steadily with increasing
distance from the brain. Using confocal microscopy we found GFAP immunoreactivity (IR)
around MV resembling the structure of glial endfeet. In whole mount preparation from
extraparenchymal PAs GFAP IR was detected in the adventitial layer of the arterial wall and
in vessel free regions of the pia-arachnoid membrane. From these observations we suggest
that the sources of GFAP mRNA were glial endfeet in MVs and meningeal cells in the case
of arteries. The latter may well play an important role in the induction and maintenance of
blood-brain barrier features in the cerebral macrovasculature.
Under control conditions a complete KKS gene expression pattern including the B2-receptor,
angiotensin converting enzyme (ACE), kininogen (KIN), and kallikrein (KAL) was found in the
arterial wall as well as in the MVs. Quantitatively, marked tissue-specific differences in the
expression levels for B2, KIN, and ACE were observed. The sequence was PAs > MVs >
tissue with 100 to 1,000 fold higher expression levels in PAs over tissue.
After focal brain ischemia, both transient and permanent a de novo expression of the B1
receptor was found in brain tissue as well as in the PA and MV compartment. The B2
receptor mRNA was markedly upregulated, most notably in the MVs. The largest degree of
upregulation, however, was observed with KIN in all tissue compartments. This upregulation
was not restricted to the ischemic side but was also present in samples of tissue, PAs, and
MVs taken from the contralateral side. The functional meaning of this generalized
upregulation is not yet clear.
In conclusion, I have established a new method of brain MV extraction particularly suited for
gene expression studies. An apparent enrichment over brain tissue with GFAP mRNA is
probably due to a tight association of astrocytic endfeet with MVs and does not conflict with
the purity of the MV extraction. A complete KKS gene expression was found in brain tissue,
MVs and extraparenchymal arteries the latter exhibiting the highest expression levels for the
B2 receptor, ACE, and KIN. Thus, it is concluded that the KKS in the brain displays a
predominantly macrovascular expression pattern. After focal ischemia there is a de novo
expression of the B1 receptor and an upregulation of the B2 receptor in brain tissue, brain
arteries, and the MV compartment. The highest degree of upregulation was found for KIN in
tissue, PAs, and MVs, and it was not restricted to the ischemic side but occurred in the
contralateral side as well. These observations further substantiate the functional importance
of the KKS as an early inflammatory response. However, the pathophysiological importance
of the KIN remains to be clarified.
For me it was important to have an opportunity not only to get acquainted with the method
theoretically, but also to apply them under supervision of the skilled colleagues. The courses
I had a chance to take allowed me not only to learn about the new methods and improve my
skills, but also to familiarize myself with other laboratories. Furthermore the lectures in which
I had the honor to participate helped me learn more about the subjects which had not been
directly related to my thesis and provided me with a perspective to the vascular medicine
which was of great importance for me.
In addition I would like to mention the opportunity to go through the detailed and consecutive
discussion of all the steps of the thesis which was kindly offered to me (starting with the
planning stage and conducting experiments and ending with final results and the writing
itself) guided by my supervisor and supported by students-colleagues and professors. A
particularly friendly atmosphere at springs and autumn schools is to be mentioned. And of
course participation in the German and international conferences gave skill a presents of
results and cooperation with international research groups.
43
attended schools: 6
n.a.
44
04-06.10.2007: Join Meeting 2007 Annual Meeting of the Society for Microcirculation and
Vascular Biology and 6th International Symposium on the Biology of Endothelial Cells
Heidelberg
31.10-01.11.2008: 34 Jahrestagung der Sektion „Intrakranieller Druck, Hirnödem und
Hirndurchblutung“ der Deutschen Gesellschaft für Neurochirurgie, Mainz. Studies on the
expression of the Kallikrein - kinin system (KKS) in cerebral ischemia: comparison between
brain tissue and brain arteries. (Oral presentation)
24-27.05.2009: 60. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie Joint
Meeting mit Benelux-Länder und Bulgarien, Münster, Studies on the gene expression of the
Kallikrein - kinin system (KKS) in brain arteries and tissue in a rat model of focal cerebral
ischemia. (Poster)
08.-10.10.2009 : Gemeinsame Jahrestagung Gesellschaft für Mikrozirkulation und vaskuläre
Biologie und der „Schweizerischen Gesellschaft für Mikrozirkulation“ in Bern, Studies of the
gene expression of the Kallikrein - kinin system (KKS) in the cerebroarterial vasculature –
comparison with brain tissue in focal ischemia models. (Poster)
06.-07.11.2009 : Jahrestagung der Sektion „Intrakranieller Druck, Hirndurchblutung und
Hydrocephalus“ der deutschen Gesellschaft für Neurochirurgie, Homburg/Saar. Änderungen
der Genexpression im Kallikrein - Kinin System im Hirngewebe und in cerebralen Arterien bei
transienter und permanenter fokaler Ischämie (Oral presentation)
14-16.08.2010: Gesellschaft für Mikrozirkulation und Vaskuläre Biologie. Evidence for a
pathophysiological role of tissue hypoxia during early reperfusion after transient focal
ischemia in rats (Poster)
21-25.10.2010: Studies on the gene expression of bradykinin receptor subtypes 1 and 2 in
brain tissue following transient middle cerebral artery occlusion in rats (Poster)
05-06.11.2010: Jahrestagung der Sektion „Intrakranieller Druck, Hirndurchblutung und
Hydrocephalus“ der deutschen Gesellschaft für Neurochirurgie, Berlin. Untersuchungen zur
Rolle des Kallikrein-Kinin Systems und von VEGF/flt1 in der Entwicklung der akuten
Schwellung im Modell der fokalen cerebralen Ischämie (Oral presentation)
21-25.09.2010: Neurowoche in Mannheim Studies on the gene expression of Bradikinin
receptor subtypes 1 and 2 in brain tissue following transient middle cerebral artery occlusion
in rats (Poster)
25-28.05.2011: XXVth International Symposium on Cerebral Blood Flow and Metabolism &
The Xth International Conference on Quantification of Brain Function, Barcelona; Early
hemispheric swelling in focal brain ischemia: comparing the roles of bradykinin and vascular
endothelial growth factor (Poster)
13-16.10.2011: Joint Meeting of the European Society for Microcirculation (ESM) and the
German Society of Microcirculation and Vascular Biology (GfMVB), Münich; Alteration of gene
expression of the kallikrein-kinin system in brain tissue, arteries and microvessels after focal
cerebral ischemia (Poster)
28-29.10.2011: 37.Jahrestagung der Sektion „Intrakranieller Druck, Hirndurchblutung und
Hydrozephalus“ der Deutschen Gesellschaft für Neurochirurgie, Tübingen Änderung der
Genexpression im Kallikrein-Kinin-System in Hirngewebe, Arterien und Mikrogefässen in
einem Modell der fokalen cerebralen Ischämie (oral presentation)
26-27.10.2012: Tagung der Sektion Intrakranieller Druck, Hirndurchblutung und
Hydrozephalus der DGNC Heidelberg; Nachweis von GFAP mRNA in Mikrogefäßen und
pialen Arterien der Ratte (oral presentation)
20-23.05.2013: XXVIth International Symposium on Cerebral Blood Flow, Metabolism and
Function and the XIth International Conference on Quantification of Brain Function with PET
(Brain 2013), Shanghai; Studies on the kallikrein-kinin system in cerebral arteries,
microvessels and brain tissue after brain ischemia damage (Poster)
26.-28.09.2013: 2nd Joint Meeting of the GfMVB and the SSMVR , Dresden; High yield
microvessel extraction from rat brain: characterisation of cell-type specific marker genes and
comparison with cerebral arteries (Poster). Poster Prise.
45
4. Own publications
1.
I.Y.Petrushanko, N.B.Bogdanov, N.Lapina, A.A.Boldarev, M.Gassmann and
A.Y.Bogdanova Oxygen-induced regulation of Na/K ATPase in cerebellar granule cells, J
Gen Physiol. 2007 Oct;130(4):389-98
2.
David J. Seiffge, Natalia E. Lapina, Charalambos Tsagogiorgas, Bastian Theisinger,
Rob H. Henning, Lothar Schilling “Impact of enhanced oxygen supply on tissue damage and
hypoxia in focal ischemia” Experimental Neurology 237 (2012) 18–25
46
First and last name of doctoral researcher: Firas Al Laham
(Working) title of doctoral project: Predictive value of serum parameters for disease
activity in ANCA-associated systemic vasculitis (AASV)
Names of supervisors: B. Yard, P. Heeringa, C.G.M. Kallenberg
01 January 2007- 18 June 2010
Biotechnology, Magister rer. nat. (Österreich), 06/2006
Postdoc/ Damaskus
In anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated small vessel systemic
vasculitis, e.g. Wegener's granulomatosis, microscopic polyangiitis, Churg–Strauss
syndrome and ANCA-positive rapidly progressive glomerulonephritis (RPGN), it is believed
that concomitant binding of ANCA to their putative antigens and to Fc receptors on the cell
surface of neutrophils or monocytes is a critical event in the pathogenesis of these diseases
Binding of ANCA to tumour necrosis factor (TNF)-α-primed neutrophils leads unambiguously
to the production of reactive oxygen species (ROS), which can subsequently damage the
endothelium. Recently however, it has been reported that endothelial cells have the intrinsic
property to inhibit ROS production by neutrophils via the release of adenosine. This therefore
raises the question as to whether ROS production by ANCA-stimulated neutrophils is
important in the pathogenesis of AAV. It must be emphasized, however, that studies using
neutrophils from AAV patients have not been performed in such a setting; hence it is not
known if neutrophils from these patients behave similarly to healthy controls. If endothelial
cells do not inhibit oxidative burst or degranulation in neutrophils from AAV patients, this
could explain, at least partly, the vascular pathology observed in AAV patients. Hence, this
study was undertaken to test the hypothesis that in the interaction of endothelial cells with
neutrophils of AAV patients there is a lack in the control of oxidative burst and degranulation.
The main findings of this study are the following: first, our study confirms and extends
previous data that endothelial cells inhibit ROS production, but not degranulation, of fMLPactivated neutrophils. Endothelial cells neither inhibit ROS production nor degranulation
when neutrophils are challenged with PMA. Second, healthy controls and patients in
remission do not differ in this respect. However, in the only active patient who was studied,
degranulation was inhibited by endothelial cells before initiation of treatment but not 6 weeks
later under immunosuppressive treatment. Third, endothelial cells inhibit LPS-mediated TNFα production in co-cultures of endothelial cells and PBMC, but enhance IL-8 production in cocultures of neutrophils and endothelial cells. This was also not different between patients and
controls.
47
In conclusion, our data do not support the hypothesis that endothelial cells inhibit ROS
production of neutrophils from AAV patients inadequately. Impaired neutrophil degranulation
may exist in active patients, but this finding needs to be confirmed. It also remains to be
elucidated how and if immune modulation at the vascular interface is impaired in vasculitis
patients. Further studies are warranted to address if ATP release by activated neutrophils is
diminished in these patients. Similarly, the expression of CD39 and CD73 at vasculitic lesion
might provide further clues as to whether the lack of adenosine generation might underlie
vascular damage at these sites.
No comment provided by the graduate
attended schools: 5
n.a.
n.a.
4. Own publications
1.
Al Laham F, Kälsch AI, Heinrich L, Birck R, Kallenberg CG, Heeringa P, Yard B.
Inhibition of neutrophil-mediated production of reactive oxygen species (ROS) by endothelial
cells is not impaired in anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated
vasculitis patients. Clin Exp Immunol. 2010 Aug;161(2):268-75.
48
First and last name of doctoral researcher: Eva Pfister (maiden name: Riedl)
(Working) title of doctoral project: Carnosine as a protective factor in the glomerular
capillary loop
Names of supervisors: B. Yard, J. van den Born, G. Navis
01 February 2007 - 31 January 2010
IRTG fellowship (01 February 2007 - 31 January 2010), PhD-Position GUIDE in Groningen
(2010)
2007- 2011 (PhD in Groningen)
Medical studies; University of Heidelberg; State examination and licensure as a doctor
(10/2009 and 01/2010)
Specialist medical training for pathology
Diabetic nephropathy (DN) is the most common cause for end stage renal disease (ESRD)
and is recognized as an urgent medical problem of world wide dimension. Approximately one
third of the diabetic patients develop DN. A number of risk factors are associated with the
development of DN, including prolonged duration of diabetes, poor glycemic control, raised
blood pressure and hypercholesterolemia. It is clear however, that these risk factors alone
are not sufficient for the development of this complication, since susceptibility to developing
DN seems to be genetically determined. We have recently demonstrated the CNDP1 gene is
a susceptibility locus for susceptibility to develop DN. In the present project the role of
carnosine and CNDP1 for DN was further investigated.
The main findings of this project are the following. 1) The polymorphism in the CNDP1 gene
is a gain of function mutation that influences CNDP1 secretion. 2) Hyperglycemia influences
CNDP1 secretion in diabetic patients without renal involvement. This is most likely due to an
increased efficiency of N-glycosilytion. 3) CNDP1 in human serum is present in two
conformations. 4) Homocarnosine is a natural inhibitor of CNDP1. 5) L-carnosine inhibits
high glucose mediated matrix accumulation in human mesangial cells by interfering with
TGFß production and signaling. 6) L-carnosine prevents apoptosis of glomerular cells and
podocyte loss in STZ diabetic rats.
The interaction with Groningen was enjoyable and really helped me to pursue my personal
goals.
49
attended schools: 5
01.02.2010 -12.05.2010 stay in Groningen UMCG lab of Prof. Navis.
2007 DDG-Congress in Hamburg
2007 Podocytes in-vitro Conference in Dublin
2008 RCSG in Wales
2008 DDG-Congress in Munich
2009 DDG-Congress in Leipzig
2007 Joint Meeting of the Society of Microcirculation and Vascular Biology in Heidelberg
2009 Carnosinase Symposia in Graz
2008 ASN in San Francisco
4. Own publications
1.
Riedl E, Koeppel H, Brinkkoetter P, Sternik P, Steinbeisser H, Sauerhoefer S,
Janssen B, van der Woude FJ, Yard BA. A CTG polymorphism in the CNDP1 gene
determines the secretion of serum carnosinase in Cos-7 transfected cells.Diabetes. 2007
Sep;56(9):2410-3.
2.
Peters V, Kebbewar M, Jansen EW, Jakobs C, Riedl E, Koeppel H, Frey D,
Adelmann K, Klingbeil K, Mack M, Hoffmann GF, Janssen B, Zschocke J, Yard BA.
Relevance of allosteric conformations and homocarnosine concentration on carnosinase
activity. Amino Acids. 2010 May;38(5):1607-15
3.
Riedl E, Koeppel H, Pfister F, Peters V, Sauerhoefer S, Sternik P, Brinkkoetter P,
Zentgraf H, Navis G, Henning RH, Van Den Born J, Bakker SJ, Janssen B, van der Woude
FJ, Yard BA. N-glycosylation of carnosinase influences protein secretion and enzyme
activity: implications for hyperglycemia. Diabetes. 2010 Aug;59(8):1984-90
4
Peters V, Jansen EE, Jakobs C, Riedl E, Janssen B, Yard BA, Wedel J, Hoffmann
GF, Zschocke J, Gotthardt D, Fischer C, Köppel H. Anserine inhibits carnosine degradation
but in human serum carnosinase (CN1) is not correlated with histidine dipeptide
concentration. Clin Chim Acta. 2011 Jan 30;412(3-4):263-7
5.
Köppel H, Riedl E, Braunagel M, Sauerhoefer S, Ehnert S, Godoy P, Sternik P,
Dooley S, Yard BA. L-carnosine inhibits high-glucose-mediated matrix accumulation in
human mesangial cells by interfering with TGF-β production and signalling. Nephrol Dial
Transplant. 2011 Dec;26(12):3852-8.
6.
Riedl E, Pfister F, Braunagel M, Brinkkötter P, Sternik P, Deinzer M, Bakker SJ,
Henning RH, van den Born J, Krämer BK, Navis G, Hammes HP, Yard B, Koeppel H.
Carnosine prevents apoptosis of glomerular cells and podocyte loss in STZ diabetic rats. Cell
7.
Pfister F, Riedl E, Wang Q, vom Hagen F, Deinzer M, Harmsen MC, Molema G, Yard
B, Feng Y, Hammes HP. Oral carnosine supplementation prevents vascular damage in
experimental diabetic retinopathy Cell Physiol Biochem. 2011;28(1):125-36.
8.
Adelmann K, Frey D, Riedl E, Koeppel H, Pfister F, Peters V, Schmitt CP, Sternik P,
Hofmann S, Zentgraf HW, Navis G, van den Born J, Bakker SJ, Krämer BK, Yard BA,
Hauske SJ. Different conformational forms of serum carnosinase detected by a newly
developed sandwich ELISA for the measurements of carnosinase concentrations. Amino
Acids. 2012 Jul;43(1):143-51.
50
Graduates (GRK 880/2-3)
Project number: GRK 880/2-3, Project 16
First and last name of doctoral researcher: Caroline Berger
Title of doctoral project: Blockade of endothelial Notch signaling in cellular systems and
adult mice
Names of supervisors: A. Fischer, H. Augustin, G. Molema
01 October 2008 - 30 June 2011
IRTG Fellowship (01 October 2008 - 31 March 2011)
Start of doctoral training in October 2008; graduation 08 February 2013 (Dr. rer. nat.; magna
cum laude)
10/2003 – 8/2008: Studies of Biology (Dipl. Biol.) at University of Karlsruhe (TH)
4/2011 – 12/2012 PhD student in the same group
Since 8/2013: Regulatory Affairs Specialist, Stryker Leibinger GmbH & Co. KG, Freiburg
Angiogenesis is a fundamental process during embryogenesis, inflammation and wound
healing. The formation of new vessels is coordinated by proteins of the VEGF and the Notch
signaling. Dysfunction of the precisely balanced crosstalk between Notch and VEGF
signaling entails the formation of a non‐functional vascular network. These imbalances play a
critical role during progression of many diseases including atherosclerosis and tumor growth.
Blocking of Notch signaling, by small molecule inhibitors or DLL4‐specific antibodies,
perturbs tumor perfusion and inhibits tumor growth in animal models.
This study aimed to gain deeper insight into the complex function of Notch signaling in the
endothelium. For this purpose, small soluble Notch ligand and receptor peptides were
generated, which consist of the respective interaction domains only. Furthermore, the effects
of deleting endothelial Notch signaling in adult mice were investigated.
Application of the designed soluble DLL1, DLL4, and JAG1 ligands, as well as, the soluble
NOTCH1 receptor blocked Notch signaling in endothelial and myogenic cells. All soluble
ligands consistently exerted pro‐angiogenic effects in vitro. The effects of DLL1 and DLL4
were markedly stronger than that of the JAG1 ligand and could also evoke elevated
sprouting angiogenesis in the retina of newborn mice. Treatment with the soluble Notch
receptor reduced endothelial sprouting in vitro. However, in vivo application of soluble
NOTCH1 receptor protein resulted in increased retinal sprouting with elevated numbers of tip
cells. Thus, the soluble ligands suppressed Notch receptor activity by acting as competitors
for endogenous membrane‐bound ligands; whereas the soluble receptor acted as a decoy
for the different Notch ligands.
Genetic studies with adult mice after endothelial‐specific deletion of Notch signaling were
performed. These mice developed cardiomyopathy within a few months; whereas vascular
tumors developed after one year. In an ApoE‐deficient model of hyperlipidemia, the deletion
of endothelial Notch signaling improved the glucose tolerance of mice, but caused
51
development of steatohepatitis. Thus, Notch signaling in the adult vasculature could be
identified as a critical regulator of organ homeostasis as well as glucose and fat metabolism.
The graduate school offered various opportunities to improve my scientific and non-scientific
qualification. I took part in several workshops teaching specific techniques, which I could
successfully use for my projects afterwards. The graduate school also offered highly helpful
courses for soft skills. I also benefitted from the fruitful cooperation with other graduate
schools, which allowed me to participate in their workshops as well.
The graduate school organized several meetings and retreats with other graduate schools
dedicated to familiar topics. These events offered the possibility for exchange with other
students and allowed me to discuss my own work with PIs from other universities. I also
enjoyed the summer schools, which on a regular basis brought together the students from
Mannheim and Groningen. The large support of my graduate school allowed me to present
my research at international meetings for cell biology and angiogenesis as well. My
supervisors greatly supported my work with their expertise. They discussed my research
strategy and also helped me to overcome methodological problems.
attended schools: 5
n.a.
Development and characterisation of optimized Notch-blocking constructs to interfere in tumor
angiogenesis - 33rd Annual Meeting of the German Society for Cell Biology (DGZ). March
10–13, 2010, Regensburg
6th Swiss Zebrafish Annual Meeting, Biozentrum Basel, CH, April 13th 2012
4. Own publications
1.
Adam MG, Berger C, Feldner A, Yang WJ, Wüstehube-Lausch J, Herberich SE,
Pinder M, Gesierich S, Hammes HP, Augustin HG, Fischer A. Synaptojanin-2 binding protein
stabilizes the Notch ligands DLL1 and DLL4 and inhibits sprouting angiogenesis. Circulation
Res. 2013. 113(11):1206-18.
52
First and last name of doctoral researcher: Julia Rauch
(Working) title of doctoral project: (Patho)physiological role of NO/cGMP-induced
RhoGEF17 activation in the vasculature
Names of supervisors: T. Wieland, S. Lutz, M. Schmidt
01 July 2008 – 30 June 2011
IRTG fellowship (01 July 2008 - 31 March 2011)
07/2008 start of practical work, 31 October 2011 end of practical work, thesis submitted
01/2012,
Doctoral degree (PhD) awarded 26 April 2012
10/02 -10/07 Human Biology (diploma), University of Greifswald
Scope International AG, Mannheim
The monomeric GTPase RhoA and the second messenger cGMP play important roles in the
regulation of different processes in vascular smooth muscle cells like contraction, relaxation
and differentiation. Work in our group identified the guanine nucleotide exchange factor
RhoGEF17 as a mediator of a cGMP-induced activation of RhoA, which is expressed in
vascular smooth muscle cells. Based on this data investigated the function of RhoGEF17 in
vascular as well as in tracheal smooth muscle cells. Therefore, the endogenous expression
of RhoGEF17 in isolated rat aortic and tracheal smooth muscle cells was suppressed by an
adenovirus encoding a specific shRNA targeting RhoGEF17 mRNA. The knockdown of
RhoGEF17 resulted in a change of smooth muscle cell morphology, a loss of actin stress
fibers and a decreased expression of the GTPase RhoA. Moreover the expression level of
RhoGEF17 influenced the attachment of vascular smooth muscle cells to a surface after
detachment and affected the proliferation of tracheal smooth muscle cells. The knockdown
experiments confirmed the requirement of RhoGEF17 for the cGMP-induced activation of
RhoA in vascular smooth muscle cells. Overexpression of the cGMP-dependent kinase
cGKIα, which activates RhoGEF17 through phosphorylation, caused an additional increase
in the cGMP induced RhoA activity. A stimulation of tracheal smooth muscle cells with
cGMP, however, did not elicit an activation of RhoA. Several lines of evidence for a
physiological relevance of the cGMP/cGKIα/RhoGEF17 signaling pathway in vascular
smooth muscle cells were obtained. Knockdown of RhoGEF17 ablated cGMP-induced
relaxation in a 3D culture model of vascular smooth muscle cells whereas the contraction in
response to serum was not affected. With regard to cell adhesion, cGMP stimulated the
attachment of isolated vascular smooth muscle cells to a surface under control conditions,
but had no effect on the adhesion process of cells with a reduced expression of RhoGEF17.
In summary, the data collected indicate that RhoGEF17 is required for the regulation of
vascular as well as tracheal smooth muscle cell integrity. Especially in vascular smooth
muscle cells, it additionally plays a role as a mediator of cGMP-dependent signaling.
53
I learned a lot from my supervisors and I had received continuous supervision, input and
exchange during the time span of the graduate school as well as after my membership in the
graduate school had officially ended.
The meetings organized by the graduate school were very balanced with regard to length
and frequency. I profited from the multiple practical workshops being provided by the GRK
and partner graduate schools. Due to the support by the GRK I got the opportunity to present
my data on national and international meetings, by means of posters and oral presentations.
It was largely valuable for me to get routine in statistics, laboratory work, and constant
literature study.
attended schools: 5
09/2010 – 12/2010 University of Groningen
Analysis of the function of RhoGEF17 in isolated smooth muscle cells.
Deutsche Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie,
Jahrestagung 2010, Mainz, March 23 –25, Oral presentation
Function of RhoGEF17 in isolated smooth muscle cells. Figon Dutch Medicines Days, 2011,
Amsterdam, October 4-6, Poster presentation
RhoGEF17 mediates cGMP/cGK induced adherence and relaxation of vascular smooth
muscle cells. Deutsche Gesellschaft für Experimentelle und Klinische Pharmakologie und
Toxikologie, Jahrestagung 2012, Dresden, March 19- 22, Poster presentation
4. Own publications
1.
Lutz, S., Mohl, M., Rauch, J., Weber, P., Wieland, T. (2013) RhoGEF17, a Rhospecific guanine nucleotide exchange factor activated by phosphorylation via cyclic GMPdependent kinase Iα. Cell Signal. 25, 630-638.
54
Project number: GRK 880 2-3, Project 18
First and last name of doctoral researcher: Nina Schweinfurth
(Working) title of doctoral project: Culture-derived platelets: Impact of serotonin
metabolism on differentiation and function
Names of supervisors: F. Lederbogen, M. Deuschle, P. Schloss, M. Harmsen
01 October 2008 – 30 June 2011
IRTG fellowship (01 October 2008 – 31 March 2011)
10/2008 Start of doctorate, anticipated submission of doctoral thesis in 06/2014, anticipated
completion of doctorate: Autumn/Winter 2014
10/2006; Medical School, Medical Faculty Mannheim, University of Heidelberg; graduation
date: 03 December 2013 (last step of the German Medical Licensing Examination /
approbation as physician)
01/03/2014 residency in internal medicine at the department of hematology, oncology,
clinical immunology and palliative medicine, St. Vincentius Hospital Karlsruhe, teaching
hospital of the University of Freiburg, Germany
Platelets play a pivotal part in recruitment of CD34+ endothelial progenitor cells (EPCs)
toward vascular lesions, using glycoprotein receptors (e.g. P-selectin/PSGL-1, beta1- and
beta2-integrin) to mediate adhesion and EPC homing. Recent data indicate that serotonin
conjugation is used to augment the retention of these adhesive proteins on platelet surface.
Further details of this mechanism came from mice rendered selectively deficient in
tryptophan hydroxylase, the rate limiting enzyme in serotonin synthesis. In this animal model,
the serotonin-deficient platelets exhibited a reduced adhesion due to a blunted secretion of
adhesive -granular proteins.
Serotonin is synthesized in the enterochromaffin cells in the gastrointestinal tract and taken
up into blood platelets by the serotonin transporter (SERT). Thus, the density of cell-surface
expressed SERT proteins directly controls the concentration of serotonin in platelets.
Consequently, platelet serotonin content also can be lowered by selective serotonin
reuptake-inhibitors (SSRIs), substances used to treat depression, anxiety and other
psychiatric disorders. Interestingly, the use of these substances is associated with a lower
rate of cardiac events in depressed subjects, but rarely, also with an increased bleeding
tendency. In addition, it had been shown that SSRIs not only block SERT activity, but also
reduce the cell surface density of SERT proteins in serotonergic neurons. Our aim in this
GRK project was to analyse the impact of altered serotonin metabolism on surface adhesion
molecule expression and SERT expression in functional culture derived platelets. In order to
compare transcriptional versus posttranslational effects, we performed these studies both, in
megakaryocytes and in mature platelets. The rationale behind this strategy was that in
contrast to megakaryocytes platelets are devoid of cell nuclei and thus incompetent for
mRNA transcription.
55
To do so, we choose the megakaryoblastic cell line MEG-01. Upon induction of
differentiation, this cell line allows to follow MEG-01 megakaryoblasts all the way down to
megakaryoctes and finally to functional platelets in vitro. One obstacle, however, was that
usually production of platelets from MEG-01 derived megakaryocytes was generally induced
by treatment with the phorbolester PMA and that the cells did not survive for much longer
than 6 days in culture. Therefore we first had to develop a new, more efficient and gentle
differentiation protocol for MEG-01 cells. In this line, we have investigated the impact of alltrans-retinoic acid (ATRA) and valproic acid (VPA) on the differentiation of megakaryocytes
and platelets from the megakaryocyte progenitor cell line MEG-01. Our data revealed that
treatment with ATRA (10-11 M) and VPA (2X10-3 M) induces megakaryopoiesis of MEG-01
cells as estimated by polyploidy, formation of characteristic proplatelets and elevated
expression of the megakaryocytic markers CD41 and CD61. The resulting megakaryocytes
stayed viable for more than 3 weeks and shed platelet-like particles positive for CD41, CD61
and CD42b into the supernatant. Platelet-like particles responded to thrombin receptor
activating peptide (TRAP-6) with increased externalization of P-selectin. Thus, ATRA and
VPA proved to be efficient agents for the gentle induction of megakaryopoiesis and
thrombopoiesis of MEG-01 cells providing the possibility to study molecular events
underlying megakaryopoiesis and human platelet production over longer time periods.
As a next step, we investigated the expression of SERT on developing megakaryocytes and
platelet-like particles derived from the megakaryocyte progenitor cell line MEG-01 upon
differentiation by valproic acid (VPA) and all-trans retinoic acid (ATRA). Because we had also
characterized the expression of the dopamine transporter (DAT) on human blood platelets,
we included the characterization of DAT expression in developing megakaryocytes in this
study. Our results showed that MEG-01 cells express SERT and DAT and that VPA and
ATRA induce a significant increase of transporter expression on developing megakaryocytes
and platelets. As compared to ATRA, VPA more efficiently induced SERT expression but not
DAT expression. Comparable to naïve platelets and neurons, SERT was localized to both
the cell surface and intracellular compartments. Hence we have shown that VPA and ATRAtreated MEG-01 cells provide a model well-suited to studying platelet monoamine transporter
expression, not only during transcription and translation but also with respect to protein
trafficking to and from the cell surface.
Great balance of interdisciplinary and international supervision. Graduate school network
provided extraordinarily valuable opportunity for knowledge and methodological exchange
and progress.
attended schools: 6
Research stays in the Cardiovascular Regenerative Medicine Research Group (CAVAREM),
head: Dr. MC Harmsen, PhD, Department of Medical Biology and Pathology, University
Medical Center Groningen, the Netherlands
- 08/2009
- 08/2011 - 07/2012 (PhD research)
08 - 11/2012 research elective in psychiatric research, head: Prof. Dr. CU Correll, MD, at the
Zucker Hillside Hospital, North Shore Long Island Jewish Health Systems and Hofstra
University School of Medicine, Glen Oaks, NY, USA
56
07/2012 Donald J. Cohen Fellowship and Grant for the 20th World Congress of the
International Association for Child and Adolescent Psychiatry and Allied Professions
(IACAPAP) in Paris, France
06/ 2012 Continuous selective serotonin reuptake inhibitor (SSRI) treatment downregulates
surface serotonin transporter (SERT) expression in maturating megakaryocytes and platelet
like particles from MEG-01 cells. Poster, 7th Int. Platelet Symposium in Beverly, MA, USA.
10/ 2011 VPA and ATRA differentially impact on SERT expression in maturating MKs and
PLT-like particles from MEG-01 cells. Poster, 2nd Int. Joint SFB/GRK Meeting on Vascular
Biology, Mannheim, Germany
05/ 2010 Megakaryopoiesis and platelet-like particle formation from the megakaryoblastic cell
line MEG-01 is induced by ATRA and VPA. Poster, 6th Int. Platelets Symposium in Jerusalem,
Israel
4. Own publications
1.
Schweinfurth N, Hohmann S, Deuschle M, Lederbogen F, Schloss P. Valproic acid
and all trans retinoic acid differentially induce megakaryopoiesis and platelet-like particle
formation from the megakaryoblastic cell line MEG-01.
Platelets. 2010; 21(8):648-57.
2.
Hohmann S, Schweinfurth N, Lau T, Deuschle M, Lederbogen F, Banaschewski T,
Schloss P. Differential expression of neuronal dopamine and serotonin transporters DAT and
SERT in megakaryocytes and platelets generated from human MEG-01 megakaryoblasts.
Cell Tissue Res. 2011 Nov;346(2):151-61.
3.
Schneider P, Weber-Fahr W, Schweinfurth N, Ho YJ, Sartorius A, Spanagel R,
Pawlak CR. Central metabolite changes and activation of microglia after peripheral
interleukin-2 challenge. Brain Behav Immun. 2012 Feb;26(2):277-83.
4.
Schweinfurth N, Schloss P, Deuschle M, Lederbogen F. Manipulation of serotonin
transporters in human platelets: Option for P-Selectin control?
Response to: Transporters in human platelets: physiologic function and impact for
pharmacotherapy. [e-Letter], Blood. May 25, 2012
57
First and last name of doctoral researcher: Christina Maximilia Hottenrott
(Working) title of doctoral project: N-octanoyl dopamine preconditioning: Protection
against pro-inflammatory and kryo-induced injury during the transplantation process
Names of supervisors: B. Yard, H. G.D. Leuvenink
01 July 2008 - 30 June 2011
IRTG Fellowship (01 July 2008 - 31 March 2011), PhD Fellowship GUIDE 01 October 2012
– 01 October 2013, Department of Thoracic Surgery 01 October 2013 -01 October 2014
01/2008 Start of doctorate, submission of MD doctoral thesis planned for June 2014, PhD
defense planned 2015
Autumn/winter 2005, Human medical studies, Medical Faculty Pecs, Hungary; Reception of
approbation June 2012 Medical Faculty Mannheim, University Heidelberg
01 October 2013 Continuation of PhD thesis in the department of Thoracic Surgery
Although donor dopamine treatment has shown its salutary effect on transplantation outcome
in kidney and heart recipients, in approxiamtly 12% of treated donors dopamine treatment
has tob discontinued as a consequence of high blood pressure and tachycardia. Since the
protective effect of donor dopamine treatment is not attributed to its hemodynamic properties
we have developed a dopamine variant that is completely devoid of hemodynamic activity. In
the present project N-octanoyl was exploited to address its anti-inflammatory potential on
human endothelial cells and to study its protectective properties on cold inflicted injury of
cardiomyocytes. The main findings of this project are the following. 1) NOD down-regulates a
wide range of κB regulated pro-inflammatory mediators, e.g. chemokines and adhesion
molecules, yet not all κB regulated genes were affected by NOD. Down-regulation of
inflammatory mediators had functional consequences for the adherence of PBMC to
endothelial cells and was associated with inhibition of NF-κB. 2) Inhibition of NF-κB occurred
independently of IκBα degradation and was reflected by an overall decrease in p65
expression and a decreased phosphorylation of p65Ser276. 3) de novo protein synthesis
was not required for inhibition of NF-κB, hence excluding that up-regulation of HO-1 was
involved in the anti-inflammatory properties of NOD. In line with this, it was found that in HO1 siRNA transfected cells NOD mediated inhibition of VCAM-1 expression was not impaired.
Finally, we provide evidence that redox activity and hydrophobicity are important molecular
entities that are required for the anti-inflammatory properties of NOD.
In the present project we also sought to explore the biological plausibility of our clinical
observation that treatment of the brain-dead cardiac donor with low-dose DA is associated
with an improved clinical outcome after heart transplantation. We hypothesized that
dopamine pre-treatment increases the viability of cardiomyocytes during cold preservation
and that NOD is superior in this regard. Our data clearly substantiate this hypothesis as pretreatment with DA or NOD concentration-dependently reduces cell damage and enhances
tolerance of cardiomyocytes to withstand cold preservation in culture. A similar loss of
58
damage was seen if NOD was applied to rat hearts before explantation. In cultured
cardiomyocytes, ATP depletion was prevented, and as a consequence spontaneous
contractility as well as responsiveness to adrenergic stimuli is preserved upon re-warming.
The supervision in Germany has influenced strongly the scientific working approach and
helped me working independently. The very close collaboration between the laboratory of the
Nephrology department in Mannheim and the Department of Thoracic Surgery has led to a
very broad and sophisticated insight in transplantation medicine. As a result of our
collaborative work with N-octanoyl-dopamine, stem cells and mitochondria the PhD has been
extended and might be continued in a Postdoc position.
attended schools: 8
10/2012- 10/2014 (expected): PhD in the department of Thoracic Surgery in collaboration with
the Department of Surgery
N-octanoyl dopamine ameliorates lung function in the acute phase after transplantation.
IMIRT 2014, Poitiers, France
N- octanoyl dopamine reduses the function of the ATP Synthase, IMIRT 2014, Poitiers,
France
N- octanoyl dopamine reduses the function of the ATP Synthase;; Bootcongress 2014,
Leiden, The Netherlands
UW solution preserves organ quality significantly better than HTK solution after prolonged
cold ischemia time: a comparative experimental study;; 19. Jahrestagung der Deutschen
Transplantations Gesellschaft, Hamburg August 2010, Germany
N-Octanoyl-Dopamine reduces inflammation and compared to Dopamine improves renal
function in acute renal failure; 19. Jahrestagung der Deutschen Transplantations
Gesellschaft, Hamburg August 2010, Germany
N-octanoyl dopamine as anti-inflammatory agent for donor preconditioning;; 40th Annual
Meeting der Deutsche Gesellschaft für Immunology (DGfI), Leipzig September 2010,
Germany
N-octanoyl dopamine as anti-inflammatory agent for donor pre-conditioning; Jahrestagung
der Deutschen Gesellschaft für Nephrologie, Göttingen September 2009, Germany
N-octanoyl dopamine as anti-inflammatory agent for donor pre-conditioning; Annual Meeting
of the American Society of Nephrology (ASN), San Diego 2009, United States of Amerika
59
4. Own publications
1.
J. Krebs, C. Tsagogiorgas, P Pelosi, P. Rocco, M. Hottenrott, C. Sticht, B. Yard,
T.Luecke; Open lung approach with low tidal volume mechanical ventilation attenuates lung
injury in rats with massive brain damage;Critical Care 2014, 18:R 59
2.
C. Vettel*, M.C. Hottenrott*, R.Spindler, U. Benck, P. Schnuelle, C. Tsagogiorgas,
B.K. Krämer, S. Hoeger, A. El-Armouche, T. Wieland, B.A. Yard; Dopamine and lipophilic
derivates protect cardiomyocytes against cold preservation injury;J Pharmacol Exp Ther.
2014 Jan;348(1):77-85
3.
M.C. Hottenrott*, J. Wedel*, S. Gaertner, E. Stamellou, T. Kraaij, L. Mandel, R.
Loesel, C. Sticht, S. Hoeger, L. Ait-Hsiko, A. Schedel, M. Hafner, B. Yard, C. Tsagogiorgas;
N-octanoyl dopamine inhibits the expression of a subset of κB regulated genes: potential role
of p65 Ser276 phosphorylation;PLoS One. 2013 Sep 2;8(9)
4.
C. Tsagogiorgas, J. Wedel, M. Hottenrott, M.O. Schneider, U. Binzen, W. Greffrath,
R.D. Treede, B. Theisinger, S.Theisinger, R. Waldherr, B.K. Krämer, M. Thiel, P. Schnuelle,
B.A.Yard, S.Hoeger; N-octanoyl-dopamine is an agonist at the capsaicin receptor TRPV1
and mitigates ischemia-induced acute kidney injury in rat;PLoS One. 2012;7(8)
5.
C. Bergstraesser, S. Hoeger, H. Song , L. Ermantraut, M. Hottenrott, T. Czymai,
M. Schmidt, M. Goebeler, N. Ponelies, C.Stich, R. Loesel, G. Molema, M. Seelen, W. van
Son, B.A. Yard, N. Rafat; Inhibition of VCAM-1 expression in endothelial cells by CORM-3:
the role of the ubiquitin- proteasome system, p38, and mitochondrial respiration;Free Radic
Biol Med. 2012 Feb 15;52(4):794-802
* equally contributed
60
Project number: GRK880/2-3, project 21
First and last name of doctoral researcher: Míriel Tonja Teichmann
(Working) title of doctoral project: Role of RhoA kinases ROCK I/II in angiogenesis and
arteriogenesis
Name of supervisors: J. Kroll, T. Korff, H. Augustin, M. Hecker, R. Henning
01 December 2008 – 30 November 2011
Funding (type and duration): IRTG fellowship (1 December 2008 - 31 March 2011); PhD
student at the Department of Vascular Biology and Tumorangiogenesis; Stipend of Medical
Faculty Mannheim of Heidelberg University (01 April 2011 – 30 November 2011) PhD student at
the Department of Vascular Biology and Tumorangiogenesis
Start of doctoral training and (anticipated) date of doctoral degree: 01 December 2008;
PhD September 2012, Heidelberg University
Life Science, University of Kassel, Diploma (October 2008)
Where applicable, please note any circumstances which could have affected your scientific
work1: long-term illnesses, (about 10 month within three years)
Occupation after leaving the RTG, if applicable:
Patent Professional
Angiogenesis, the formation of blood from pre-existing one, is an essential process during
embryonic development and in several pathological conditions including tumor growth,
ischemic diseases and inflammation. Sprouting angiogenesis is stimulated by angiogenic
growth factors, such as VEGF, and shows characteristic and hierarchically organized
patterns of events. Firstly, endothelial cells secrete proteases which degrade the vessel
basement membrane, followed by migrating (called tip cells) and proliferating (called stalk
cells) endothelial cells towards an angiogenic stimulus (Adams RH and Alitalo, 2007).
Arteriogenesis is a specialized type of vessel growth that occurs during vessel occlusion in
myocardial ischemia and in arterial periphery disease and is mainly characterized by an
increase in the width of existing arterial vessels. Arteriogenesis is induced by increased
blood pressure resulting in an increase of radial wall stress and higher blood flow (Troidl K
and Schaper W, 2012).
61
The processes of angiogenesis and arteriogenesis are regulated by a large set of angiogenic
growth factors. VEGF is the pivotal growth factor in angiogenesis and regulates several
endothelial actions including migration, proliferation and permeability. Arteriogenesis is
mainly regulated by cytokines and cell adhesion receptor. For example, MCP-1 MMP, bFGF
and TNF-α show increased expression levels caused by a rise of shear stress and MCP-1
has been identified as an important factor for recruiting monocytes to active sites of
arteriogenesis (Troidl K and Schaper W, 2012). Angiogenesis and arteriogenesis are
regulated by a complex network of intracellular signal cascades. One major pathway is the
activation of the serine/theronine RhoA dependent kinases ROCK I and ROCK II. Both,
ROCK I and ROCK II, are expressed in the vascular system and both kinases consist of an
amino-terminal kinase domain, a potential coiled-coil-forming region including the Rhobinding domain (RBD) and a pleckstrin homology (PH) domain, which has an internal
cysteine-rich domain. In the inactive form, the PH domain and the RBD of ROCK bind to the
amino-terminal region of the protein, which forms an autoinhibitory loop. Activated, GTPbound Rho, binds to RBD of ROCK, which results in an open conformation of the kinase and
sets the catalytic activity free (Schofield AV and Bernard O, 2013). Based on ROCK I’s and
ROCK II’s vascular expression and its activation by several angiogenic growth factors, we
aimed to identify the function of ROCK I and ROCK II in physiological and pathophysiological
blood vessel growth. Based on a large set of biochemical, functional and in vivo experiments
we identified ROCK I and ROCK II as negative regulators of the microvasculature. In detail,
we have achieved the following data:
1. Pharmacological inhibition of Rho-dependent kinases ROCK I and ROCK II by H1152 and
Fasudil activates retinal angiogenesis in mice leading to increased neovascularization.
2. Pharmacological as well as siRNA based inhibition of ROCK I/II in endothelial cells
activates sprouting angiogenesis in vitro.
3. Pharmacological inhibition and expression silencing of ROCK I/II activates angiogenic
signaling (enhanced VEGF receptor 2 and ERK1/2 phosphorylation) in endothelial.
4. In a rat myocardial infarction (MI) model, treatment with H1152 (using an osmotic mini
pump for 7 days; sham n=8; MI + water n=16; MI + H1152 n=16) does not affect body
weight, organs weight (heart and kidney) and H1152 is well tolerated by the animals.
5. Aorta heart rate, LV heart rate, blood pressure and LV systolic blood pressure in a rat
myocardial infarction model is unaltered after H1152 treatment.
6. Anti CD31 staining to analyse blood vessel formation in the ischemic rat hearts after
H1152 treatment, did not show an increased neovascularization in the myocardium.
In conclusion, the study shows that inhibition of the ROCK I/II activity in endothelial cells
leads to an increased angiogenic response in cultured endothelial cells and in the mouse
retina. Therefore, ROCK I/II act as negative regulators of angiogenesis and thus, modulation
of ROCK I/II activity seems to be an interesting therapeutic target to enhance or inhibit
angiogenesis in the eye microvasculature. Yet, since a similar protective function after ROCK
I/II inhibition did not increase angiogenesis or arteriogenesis in a rat myocardial infarction
model, further studies are necessary to either improve therapeutic application of ROCK I/II
inhibitors, develop better ROCK I/II inhibitors and to dissect differences in aniogenesis
induction in the retina and angiogenesis/arteriogenesis induction in the rat myocardium.
The central aim of GRK880 “Vascular Medicine” was to establish a teaching platform for
medical and life science doctoral students and to create a supportive and productive
research environment. Medical students (MDs) had to combine their clinical studies with
solid, hands on training in experimental research laboratories aimed at understanding the
underlying molecular mechanisms altered in diseases. Basic scientists (PhDs) with a future
career in academia (research institutes) or industry are more competitive when they have a
sound clinical understanding. GRK880 established such an environment where MD students
and PhD students were similar trained. The GRK880 offered trainings on several levels:
courses, workshops and seminars in Vascular Medicine. For teaching purpose, GRK880 also
benefit from established teaching programs established at the Medical Faculty Mannheim
62
including the MaReCuM, the Master of Translational Medicine and other graduate schools of
the Medical Faculty Mannheim (Vascular Cell Biology, SFB/TR23) and of Heidelberg
University (HBIGS; Helmholtz International Graduate School). The training program also
offered courses for general orientation, such as project management, a course in writing a
paper in English, and presentation skills. Supervision of Miriel Teichmann’s practical work in
the research laboratory included three different levels: 1. daily individual supervision by the
PI; 2. Mrs. Teichmann met the PI on a weekly basis to discuss results, to review future
experiments and to exchange information and 3. Mrs. Teichmann met with the other PIs,
partners and with the speaker at regular seminars, schools and during progress report
meetings. During these meetings, scientific progress and difficulties were recorded.
One important aspect of GRK880 was the international collaboration with the University of
Groningen. Miriel Teichmann went there three times for the Autumn Schools. She presented
her data, discussed progress and problems with the dutch PIs, including Profs. Henning,
Moshage, Hillebrands and Buikema. In October 2010, she stayed for four weeks at the
University of Groningen in the lab of Prof. Robert Henning. During that time she tested the
ROCK I/II inhibitor H1152 in a rat myocardial infarction model to identify potential benefits of
H1152 on blood vessel formation during ischemia. Mrs. Teichman was strongly supported by
Prof. Henning’s lab, the project was well organized and all necessary equipment and tools
were offered to her. Likewise, Mrs. Teichmann was personally well integrated in the
laboratory.
attended schools: 4
University of Groningen/Netherlands (October 2010), Rat myocardial infarction model, effect of
ROCK I/II inhibitor on ischemia induced angiogenesis
International Symposium of SFB/TR23, SFB834 and GRK880 in Mannheim, Germany 2011
(Poster presentation).
4. Own publications
n.a.
63
Graduates (GRK 880/3)
First and last name of doctoral researcher: Ravi Kumar Komaravolu
(Working) title of doctoral project: Elucidation of functionally relevant mediators of the
MEK5/Erk5 pathway in endothelial cells
Names of supervisors: S.Goerdt, M.Goebeler, M. Schmidt, M. Schmidt (Groningen)
01 July 2010 - 30 June 2013
IRTG fellowship (01 July 2010 - 30 June 2013)
07/2010 Start of doctorate, anticipated submission of doctoral thesis on 01 July 2014,
anticipated completion of doctorate: Winter 2014
M.Tech: Biotechnology, JNT University, 2008, Hyderabad, India
M.Sc: Microbiology, Andhra University, 2004, Visakhapatnam, Andhra Pradesh.
B.Sc: Biochemistry, Microbiology, Appl Nutrition and Public Health 2002, ANU, Guntur.
01 August 2013 – 31 March 2014: PhD completion scholarship of the IZKF Würzburg
(Interdisziplinäres Zentrum für Klinische Forschung der Universität Würzburg).
Since April 01, 2014 unemployed and looking for Post-doctoral opportunities in the area of
vascular signaling.
Description of the project:
Extracellular-signal-regulated kinase 5 (Erk5) is a member of the mitogen-activated protein
kinase (MAPK) family. Erk5 activity has been implicated in regulating proliferation, migration
and survival associated with growth factors such as EGF, VEGF and G-CSF. Moreover, Erk5
is activated by various stress stimuli including oxidative and fluid shear stress, UV and
hyperosmolarity. Targeted germline deletion of Erk5 or its upstream activator MEK5 revealed
an essential role of the MEK5/Erk5 pathway in cardiovascular development. Erk5 knockout
mice die at the onset of blood flow at embryonic stage 10.5 and are characterized by
increased endothelial apoptosis, compromised angiogenesis and heart malformation. These
defects arise due to endothelial dysfunction since cardiomyocyte-specific knockout mice for
Erk5 were inapparent while endothelial-specific ERK5 deletion phenocopied the full
knockout. Conditional deletion of ERK5 in adult mice lead to vascular leakage, suggesting a
major role of the Erk5 pathway in the vessel maintenance. In vitro, Erk5 is potently activated
by experimental conditions imitating laminar blood flow in endothelial cells (EC), which
represents a major protective force exerting various beneficial functions including inhibition of
thrombosis, inflammation and apoptosis of ECs. While this observation implicates a role of
Erk5 in protective flow responses its exact role in ECs is still unclear and the functionally
relevant downstream effectors of the MEK5/Erk5 pathway remain elusive.
64
To elucidate the role of Erk5 activation in ECs we previously performed a transcriptome
analysis of human primary ECs expressing a constitutively active mutant of MEK5, MEK5D.
Intriguingly, this study revealed that forced Erk5 activation triggered a largely protective gene
expression pattern in human primary ECs, which closely resembled that induced by laminar
shear stress. Accordingly, this pattern was characterized by statistically significant
suppression of inflammatory genes and increased expression of vasodilatory, hemostatic
and anti-thrombotic genes. In addition we noticed that MEK5D expression resulted in several
morphological changes associated with a dramatic anti-migratory and anti-angiogenic
response that was also reflected by a significant regulation of several migration-related gene
clusters. Subsequent analysis of candidate transcriptional effectors put the mechanoreceptive Krüppel-like transcription factors KLF2 and KLF4 into focus as they importantly
contributed to Erk5-dependent protective gene expression. Forced expression of both KLF2
and KLF4 was shown to inhibit endothelial migration and angiogenesis. However, both KLFs
exhibit a substantial degree of functional and transcriptional redundancy upon
overexpression, raising questions about their individual contribution to Erk5-dependent
migration arrest. In addition, due to our focus on the identification of primary Erk5 target
genes our inital transcriptome analysis fell short in providing compelling migration-relevant
secondary targets that satisfactorily could account for the dramatic anti-migratory phenotype
of Erk5-activated cells.
Goals
This follow-up study thus had three major goals:
a) clarification of the role of KLF2 and KLF4 regarding downstream mediation of the
observed Erk5-dependent anti-migratory changes
b) identification of migration-relevant secondary targets and
c) elucidation of the physiological relevance of Erk5-dependent migration arrest.
Summary of the results:
a) Erk5-mediated inhibition of migration relies on KLF2
To unequivocally clarify the individual contribution of each KLF to the anti-migratory response
to Erk5 we performed in vitro migration assays with MEK5D-expressing ECs depleted of
KLF2, KLF4 or both KLFs in combination. Surprisingly, these experiments revealed that only
KLF2 knockdown was capable to substantially rescue the migration defect of MEK5Dtransduced cells. By contrast knockdown of KLF4 at best had a mild effect and no additional
pro-migratory influence of combined KLF2/KLF4 knockdown was observed suggesting that
the potent anti-migratory effect of Erk5 activation relied on KLF2.
b) Erk5 activation leads to repression of p21-activated kinase 1 (PAK1)
To identify functionally relevant secondary targets of Erk5 we performed qRT-PCR-based
arrays with human primary ECs infected with constitutively active MEK5D. In contrast to our
initial microarray study, we chose a late time-point (72h post infection) to identify secondary
Erk5 targets rather than primary response genes. These assays revealed a potent repression
of the established pro-migratory Rac and Cdc42 effector PAK1, which was confirmed by
additional immunoblots. Intriguingly, the reported phenotype of PAK-depleted cells was
similar to that observed with MEK5D-expressing cells and likewise characterized by
decreased focal adhesion turnover and random focal adhesion distribution suggesting that
the observed loss of PAK1 could account for the strong anti-migratory response to Erk5
activation.
c) MEK5D-dependent PAK1 repression is mediated via Erk5 and KLF2
To validate that the loss of PAK1 expression in MEK5D-transduced cells was mediated via
Erk5 we used pharmacological inhibitors for Erk5. Both the highly specific Erk5 inhibitor
XMD8-92 and the dual-specific MEK5/Erk5 inhibitor BIX02188 reverted MEK5D-induced
PAK1 repression at concentrations sufficient to block MEK5D-dependent Erk5
phosphorylation. Consistent with the expendability of KLF4 for establishment of the Erk5dependent migration arrest RNAi experiments demonstrated that only knockdown of KLF2
but not of KLF4 was sufficient to normalize PAK1 mRNA and protein levels in MEK5D cells,
suggesting that KLF2 acts as a specific, non-redundant PAK1 repressor in ECs.
d) Laminar flow represses PAK1 via KLF2
To confirm our findings in a more physiological setting we performed laminar shear stress
experiments. Exposure of human primary ECs to LSS resulted in a robust activation of Erk5,
65
which correlated with a time-dependent increase of KLF2 and KLF4 mRNA as well as an
appropriately timed PAK1 repression at both protein and mRNA level. Subsequent
knockdown experiments confirmed a dependency of flow-mediated PAK1 repression on
KLF2 since PAK1 mRNA expression was preserved in KLF2 siRNA-transfected ECs upon
flow treatment but was only slightly increased upon KLF4 siRNA transfection under these
conditions.
e) PAK1 re-expression increases migration of MEK5D-infected ECs
Since the best-established endothelial function of PAK1 is its pro-migratory action we
evaluated if PAK1 loss could account for the observed migration defect of MEK5D cells.
Indeed, reconstitution experiments revealed that re-expression of wild type but not kinasedead PAK1 from a constitutive viral promoter partially reverted the migration defect of
MEK5D cells confirming PAK1 as functionally relevant gene downstream of Erk5,
Conclusion
Our data for the first time reveal PAK1 as flow-repressed gene and migration-relevant target
of the MEK5/Erk5/KLF2 pathway. This finding may have far-reaching implications since
PAK1 not only acts as pro-migratory factor but also been reported to exert pro-inflammatory
and vessel-destabilizing functions in endothelial cells and has recently been shown to
promote atherosclerosis initiation. Intriguingly, our findings raise the possibility that via
modulation of PAK1 hemodynamic forces may importantly take influence on endothelial
migration and potentially other atherosclerosis-relevant functions. Our data thus may
encourage future efforts to target PAK1 signalling as potential strategy to interfere with
atherosclerosis progression.
I sincerely thank my supervisors and the speaker of the graduate school GRK 880/3 for their
continuous support. This study greatly helped me to understand the molecular mechanism of
MEK5/ERK5 pathway in endothelial cells and their significance in vascular diseases. I
thoroughly enjoyed regular lab meetings and the scientific meetings held by the graduate
school. Participation in both the spring and autumn schools greatly enhanced and helped me
to focus more on vascular biology. In fact, I strongly feel happy that the scientific curiosity
maintained by the graduate school was always more than my expectations. I thank the
speaker of this graduate school for giving me the opportunity to finish my doctoral studies in
time. Moreover, I would like to thank my supervisors for allowing me to participate in the joint
workshop meetings and retreats at Giessen, Groningen, Wuerzburg and Konstanz to present
my data in the forms of poster and oral presentations. Without these meetings I would have
missed a chance to meet vascular research experts and maintain my scientific network.
In addition, I would like to thank our collaborative partners at the Netherlands, where I have
largely benefited from the special techniques learned from the faculty of medical sciences at
UMCG. I believe I have completed the project with in the stipulated time and looking forward
to the publication of my work in a reputed journal.
Participation in the teaching programme of GRK 880/3:
attended lectures:
34
attended schools:
6
66
01 November 2010 to 30 December 2010
Two months research training on small GTPase pulldown assay at the Department of
Molecular Pharmacology, Prof. Dr. Martina Schmidt, UMCG, Groningen, Netherlands.
01 December 2011 to 02 December 2011
Two day training on cell migration assays at the Institute of Molecular Virology IMV and
ZMBE, PD Dr.Viktor Wixler, University of Muenster.
07.06.2013 IZKF- Retreat 2013, Kloster Banz
08.06.2013
Interdisziplinäres Zentrum für Klinische Forschung,
Julius-Maximilians-Universität Würzburg
Sprecher: Professor Dr. Thomas Hünig
4. Own publications
n.a
67
First and last name of doctoral researcher: Siladitta Biswas
(Working) title of doctoral project: Physiological mechanisms and pathological changes in
sinusoidal endothelial cell endocytosis: identification of key regulators as targets for hepatic
vascular repair
Names of supervisors: S. Goerdt, K. Schledzewski, J. A. A. M. Kamps, H. Moshage
15 November 2011 - 31 December 2013
IRTG fellowship (15 November 2011 - 31 December 2013)
15 November 2011 Start of doctorate, anticipated submission of doctoral thesis on Summer
semester 2015, anticipated completion of doctorate: Winter semester 2015/16
Winter semester 2008, MSc Biotechnology, Technical University Hamburg-Harburg, Winter
semester 2010/11: Reception of degree
n.a.
Endothelial cells (ECs) display marked heterogeneity in different organs and in different
segments of the vascular tree. Liver sinusoidal endothelial cells (LSECs) are a prime
example of uniquely differentiated microvascular EC that exert highly specialized functions
as professional endocytes and participate in induction of hepatic immune tolerance. Despite
their highly specialized microvascular differentiation, LSECs retain remarkable phenotypic
and functional plasticity. Unfortunately, not much is known about the mechanisms that
control regular LSEC differentiation and LSEC transdifferentiation during pathogenic
processes. A major setback in deciphering LSEC-specific differentiation is the fact that
LSECs are not amenable to long-term cultures in vitro. LSECs rapidly lose their characteristic
morphology as well as some of their specialized functions in culture. Hitherto, attempts to
improve LSEC culture conditions have had limited success, indicating that a better
understanding of the molecular programs underlying LSEC-specific differentiation in vivo and
dedifferentiation in vitro is urgently needed. For a comprehensive analysis of the molecular
programs mediating LSEC-specific differentiation, a two-sided, comparative gene expression
profiling approach was performed. Selection of the genes that were both overexpressed in
LSEC in comparison to LMEC (Lung Microvascular Endothelial Cells) and down-regulated in
LSEC upon short-term cultivation resulted in identification of an LSEC-specific gene
signature including genes in several functional categories. Among these molecules, liver
endothelial differentiation-associated protein (Leda)-1 was identified as a novel homolog of
adherens junction-associated protein-1 (Ajap-1/Shrew-1) involved in cell adhesion and
polarity.
Within the LSEC specific gene signature, a novel uncharacterized gene (GenBank Access.
No. 00101459.1) was identified, whose 2,456 basepair (bp) cDNA codes for a putative type-1
transmembrane protein of 282 amino acids (aa) with a predicted molecular weight of 30 kDa,
including a 27 aa n-terminal signal peptide in human. This protein was named liver
68
endothelial differentiation-associated protein (Leda)-1. The putative Leda-1 protein
sequences in mouse and human display striking identity. A Blast search using rat Leda-1 as
a query in the nonredundant National Center for Biotechnology Information (NCBI) database
identified Leda-1 genes in the vertebratum phylum and revealed a significant homology to
adherens junction-associated protein-1 (Ajap-1/Shrew1), a protein that targets adherens
junctions in polarized epithelial MDCK cells and influences cell invasion.
For further studies, a polyclonal guinea pig antibody was raised against a 22 aa c-terminal
peptide of rat Leda-1. When used in western blotting experiments, this anti-Leda-1 antibody
labeled a protein with a molecular weight of 26 kDa in lysates of freshly isolated LSEC.
Signal intensity in lysates of LSEC48h was drastically reduced and no signal was obtained
from LMEC. Leda-1 labeled both the central parts as well as the periphery of LSEC and
displayed full coverage of the vessel wall; in addition, Leda-1 was mostly located at the
abluminal/basal side of LSEC. To further analyze Leda-1 function, MDCK cells which do not
express Leda-1 endogenously were stably transfected with full-length human Leda-1. Leda-1
localized to the basolateral compartment of the membrane as demonstrated by its location
below ZO-1, a cytoplasmic protein that targets tight junctions separating the apical and
basolateral compartments in polarized cells. Furthermore, Leda-1 specifically targeted
adherens junctions in MDCK cells, as shown by colocalization with E-cadherin. These data
suggest a role for Leda-1 in cell polarity and adhesion.
In addition it has been shown that Leda-1/PANP binds to the Pilr-alpha and can activate this
receptor. Furthermore it was shown that binding to Pilr-alpha relies on its N-terminal Oglycosylation.
To further study Leda-1 processing and functions several recombinant protein fragments of
different C- and N-terminal areas of Leda-1 is generated, as it displays extensive posttranslational modification by proteolysis and glycosylation. For this purpose Mouse/ Human
full length Leda-1 or N-terminal Leda-1 or C-terminal Leda-1 fragments coding plasmids are
generated. These DNA constructs are then stably transfected in eukaryotic cell lines and
further used to study post-translational modification. In the mean time, protein fragments are
used for immunization of animals (rats and mice) to generate new poly- and monoclonal
antibodies against differing and independent epitopes as well as to investigate binding of
these fragments to potential interaction partners.
After generation of antibodies and protein fragments of Leda-1 it is planned to analyse Leda1 knock out mice, which are currently located in the animal facility of University Clinic
Mannheim. Firstly Leda-1 KO characteristic is confirmed by WB and qRT-PCR and then the
animals are expected to be characterized by assessing viability, survival and general
behavior. In addition the KO and WT mice are utilized to characterize expression of Leda-1 in
all organs of mice with a set of antibodies to further delineate Leda-1 expression and
processing patterns in different organs. Subsequently a comprehensive survey of pathologic
abnormalities and functional deficits of different organs will be carried out to scrutinize Leda1 functions in mice.
Status: Research in progress
The meetings planned with other graduate schools were really helpful for me. They were
organized in a regular interval of time. The interaction with different people performing
different research really improved my scientific thought process. Guest lecturers invited from
all over the world to speak about their projects was also an exciting process to learn different
aspects of biology. Workshops organized for basic courses (scientific writing, statistics) and
different practical courses were very beneficial for me as an aspiring graduate student.
69
attended schools: 3
n.a.
International Vascular Biology Meeting, June 2-5, 2012, Wiesbaden
4. Own publications
1.
Evdokimov, K., S. Biswas, M. Adrian, J. Weber, K. Schledzewski, M. Winkler, S.
Goerdt and C. Geraud (2013). "Proteolytic cleavage of LEDA-1/PIANP by furin-like
proprotein convertases precedes its plasma membrane localization." Biochem Biophys Res
Commun 434(1): 22-27.
70
First and last name of doctoral researcher: Ilja Ovsiy
Title of doctoral project: Analysis of the expression and function of transcription factor
Forkhead box Q1 (FoxQ1) in macrophages
Names of supervisors: J. Kzhyshkowska, K. Schledzewski, S. Goerdt, M. Harmsen
01 March 2010 - 28 February 2013
IRTG fellowship fellowship (01 March 2010 - 28 February 2013)
01 March 2010 start of PhD project
Final PhD examination: 10 March 2014, Dr. sc. hum, magna cum laude
03/2006-02/2010: University of Applied Science, Mannheim (FH Mannheim); MA:
Biotechnology
03/2010-01/2013: Medical Faculty Mannheim, University of Heidelberg, PhD in Cell and
Molecular Biology
Since 02.2013: Postdoctoral researcher at the Laboratory of Cellular Immunology, University
Clinic Frankfurt
Background and original aims of the project.
The major goal of the project was to identify novel molecular mechanisms that regulate
transmigration of different cell types through the endothelium in caner and in inflammatory
conditions. The original process that was in focus of our investigation was analysis of
Stabilin-1/CD63 as a novel receptor/ligand pair as a potential target to modulate sinusoidal
endothelial cell-mediated adhesion / transmigration in inflammation and metastasis. The
working hypothesis was based on our original data produced by yeast two-hybrid screening,
where we have identified CD63 as a counter-receptor for stabilin-1 and demonstrated by
affinity chromatography, we found that CD63 binds to fasciclin domains 3, 4 and 7 of stabilin1. A next step was to establish in vitro system for the analysis of CD63/stabilin-1 interaction.
According to the project plan CHO-Satbilin-1 stably transfected single-cell derived cell lines
have been generated and role of stabilin-1 in the adhesion of CD63-psotve cells and their
transmigration has been analysed. However it was found that despite interactions with CD63
in vitro, stbailin.1 does not have statistically significant effects on the adhesion of CD63+
cells. Searching for the new molecular mechanism that mediated inducible transmigration of
different cell types through the endothelium in inflammatory conditions, we focused on the IL4 stimulated macrophages. During chronic inflammation and in cancer Th2-derived cytokine
IL-4 mediates alternative activation of macrophages. Bound to its receptor IL-4 mediates
signal cascades in the cell leading to transcription initiation of various M2 associated target
genes. However, the specificity of IL-4 associated gene expression profile as well as the role
of known IL-4 target genes in transmigration of monocytes/macrophages through the
endothelium was still poorly understood.
Transcription factor FoxQ1 was identified in our laboratory as IL-4-induced gene in human
primary macrophages. Forkhead box Q1 is winged helix/forkhead transcription factor
expressed by various cancer cell lines as well as in the cells of epithelial origin. Published
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data indicated that FoxQ1 modulates natural killer cells function and regulates EMT in the
cells of epithelial origin. However, the role of FoxQ1 in monocytes /macrophage activities and
their ability to transmigrate through the endothelium in inflammatory conditions was not
investigated. Therefore, the main focus of the present work was to characterize the function
of FoxQ1 in monocytes during inflammatory conditions.
The aims and objectives of the performed PhD project included:
1. Investigation of FoxQ1 expression regulation in monocytes/macrophages by
inflammatory and cancer-related cytokines.
2. Identification of the role of FoxQ1 in monocyte/macrophage functions and
transmigration through the endothelium in inflammatory conditions.
3. Identification of the effects of FoxQ1 on the transcriptional changes in macrophages
and analysis of the mechanisms of FoxQ1-dependend macrophage functions.
The results of the PhD project
Analysis of FoxQ1 expression in different stimulated human monocyte–derived macrophages
by qRT-PCR revealed that IL-4 is primary factor needed for FoxQ1 expression. Human
monocytes respond to IL-4 stimulation by FoxQ1 expression already after 3 hours, and this
effect is increasing during monocyte to macrophage differentiation. The effect of IL-4 is
amplified by TGF-β1 in macrophages that express TGF-βRII on their surface.
Expression of FoxQ1 in epithelial cells was shown to be induced by TGF-β1 and led to
transcriptional repression E-cadherin that in turn caused increase of epithelial-mesenchymal
transition and cell motility. Elevated levels of FoxQ1 in mammary and colorectal cancer
epithelial cells led to their enhanced metastatic activity. The ability of TGF-β1 to affect FoxQ1
expression in macrophages was studied. TGF-β1 alone or in combination with
dexamethasone failed to induce FoxQ1 production in macrophages. Only after IL-4 induction
TGF-β1 starts to contribute to the FoxQ1 production. As expected, presence of
dexamethasone enhanced this effect of TGF-β1 due to the discovered earlier ability of
dexamethasone to stimulate expression of TGF-βRII receptor on the surface of
macrophages. The pro-inflammatory Th1-dervied cytokine IFNγ did not stimulate the
expression of FoxQ1 in human monocytes. These findings indicated that FoxQ1 is
overexpressed in macrophages during alternative activation, when they are displaying antiinflammatory properties. It is known, that tumour cells with elevated IL-4 and TGF-β1
production condition tumour infiltrating macrophages to support tumour growth and immune
escape. Enhanced expression of FoxQ1 by monocytes in this situation correlates to its
contribution to epithelial-mesenchymal transition that leads to suggestion about FoxQ1 as a
mediator of macrophages migration.
Using short time course experiments was demonstrated that human monocytes respond to
the stimulation with anti-inflammatory cytokine IL-4 by FoxQ1 expression already after 3
hours. This very rapid response indicates the contribution of FoxQ1 to early stages of
macrophage activation, when monocytes start to migrate to the sites of inflammation.
Interestingly, production of transcription factor FoxQ1 is not induced by inflammatory
chemotactic cytokine MCP-1 in monocytes, i.e. the cells require the first anti-inflammatory
signal by IL-4 to start expressing FoxQ1.
Cell-based model system was generated in order to identify FoxQ1-induced genes and to
analyse function of FoxQ1. Murine macrophage-like RAW 264.7 cells were stably transfected
with pEF6/V5-HisB-mFoxQ1 construct or empty vector pEF6/V5-HisB. Comparison of three
RAW-mFoxQ1 and three RAW-vector single cell-derived clones revealed, that
overexpression of recombinant FoxQ1 does not affect basic macrophage functions
endocytosis and phagocytosis, but suppresses both basic inflammatory response and
inducible inflammatory response of macrophages to LPS by secretion of TNF-α.
In order to identify possible target genes of transcription factor FoxQ1 expression profiles of
five RAW-mFoxQ1 clones and five RAW-vector clones were analyzed and compared using
Affymetrix microarray assay. Affymetrix chip assay revealed that FoxQ1 target genes can be
involved in cell motility and cytoskeletal dynamics. Downregulation of cell migration
suppressors Plexin C1, LSP1 and Claudin 11 in RAW-mFoxQ1 cells was confirmed by qRTPCR. Semaphorin 7a receptor Plexin C1 was found to be suppressed in human-derived
72
microphages after treatment with IL-4 and in monocytes of patients with atopic dermatitis,
indicating that Plexin C1 is target gene of FoxQ1 in human monocytes.
All results of previous experiments as well as published evidence are pointing that FoxQ1 is
playing an important role in motility of cells. In order to be sure that FoxQ1 has no additional
effects on the amount of cells during the analysis of migration, BrdU proliferation assay was
performed. The proliferation test revealed that FoxQ1 has no effect on monocytes
proliferation, since no difference of BrdU incorporation between stable RAW-mFoxQ1 and
RAW-vector clones was observed (Figure 28). These data correlate with report of Zhang et
al. indicating that FoxQ1 is not involved in cell proliferation. In the present work was
demonstrated that human monocytes start to express FoxQ1 in response to stimulation with
IL-4 after very short time (3 hours) suggesting FoxQ1 contributes to the early stages of the
cell differentiation. During the monocyte to macrophage differentiation process the
proliferative activity of cells is suppressed.
To analyze the migration activity of FoxQ1 overexpressing monocytes, in vitro transmigration
assay using Neuro Probe chemotaxis chamber was established. Hereby, following
parameters were optimized: amount of cells, fixation and staining of cells, gradient and
transmigration time. Migration analysis of different amounts of cells during different time
spans revealed 1.5x106 cells/ml as an optimal number of cells for migration during 2 h. The
bigger amount of cells led to production of cell-clusters so that it was impossible to count
them. Due to different transmigration speed of RAW-mFoxQ1 and RAW-vector clones
decreasing amount of cells in the assay was impossible, because transmigrating cell were
not detectable in the slowest clones. Fixation and staining procedure were optimized,
whereby fixation with 4% PFA and cell labelling with the staining solution from Cell Biolabs
proved to be very effective. During optimization of nutritional gradient the highest migration
rate of tested cells yielded 2% → 20% FCS gradient, so that it was enough cells to be
properly stained and quantified. This FCS gradient was used for further analysis of
transmigration activity of RAW-mFoxQ1 and RAW-vector clones. To investigate the effect of
inflammatory chemoattractant MCP-1 on the RAW-mFoxQ1 chemotaxis assay toward MCP1 gradient (0 ng/ml → 100 ng/ml) was performed.
Comparison of transmigration rate between RAW-mFoxQ1 clones and RAW-vector clones
revealed enhanced migratory activity of FoxQ1 overexpressing monocytes. RAW-mFoxQ1
cells migrated 19.5 times more efficient compared to RAW-vector cells in presence of MCP-1
and 15.5 times faster toward FCS gradient. These findings indicate FoxQ1 as an effective
enhancer of monocytes migratory activity.
In order to analyse whether FoxQ1 has a general effect on macrophage motility
independently on the gradient of stimuli scratch assay was performed. It was observed
already after 4 hours that RAW-mFoxQ1 cells reconstruct the monolayer more effectively
then RAW-vector clones. Although chemotactic factors were absent in the culture medium,
macrophage-like cells overexpressing FoxQ1 showed increased migration activity, however
the effect of FoxQ1 was much weaker (only approximately 3.5 times) in the absence of
stimuli gradient. These data indicate that FoxQ1 has a general effect on the macrophage
migration activity, and this FoxQ1 function can be strongly activated in response to the
nutritional stimuli or major inflammatory chemotactic cytokine MCP-1. Moreover, this effect of
MCP-1 is not related to the induction of FoxQ1 expression as it was demonstrated in human
monocyte-derived macrophages.
In addition, generated anti-FoxQ1 antibody (HFQC 1D5) were very specific in recognition of
recombinant over expressed FoxQ1, however the sensitivity of HFQC 1D5 was insufficient
for the quantitative analysis of endogenous FoxQ1. Therefore Real-time PCR remains to be
the most reliable method for the FoxQ1 quantitative analysis in monocytes/macrophages.
In conclusion, the results of the thesis project indicate that monocytes rapidly respond IL-4 by
FoxQ1 upregulation that in turn mediate efficient migration of monocytes toward MCP-1
gradient. The FoxQ1-induced cell migration can be mechanistically explained by the
suppression of Plexin C1, LSP1 and Claudin 11. TGFbeta, a multifunctional cytokine
supporting tumor growth and having multiple effects on chronic inflammation including
progression of atherosclerotic plaque, was shown by us to stimulate FoxQ1 expression in M2
macrophages. Therefore, monocytes, upon their induced migration into tissue, differentiate
into M2 macrophages and respond to TGFbeat by expression of TGFbeta. The TGFbeta-
73
induced FoxQ1+ macrophages in turn can further migrate deeply into tumor tissues or into
atherosclerotic plaque. Therefore FoxQ1 may support increased monocytes extravasation
and deep migration to the sites of chronic inflammation or into tumours.
Experimental results are being prepared for publication as follows:
Ilja Ovsiy, Ioannis Manousaridis, Vladimir Riabov, Sergij Goerdt, Elisabeth Kremmer2, Guido
Krenning3, Marco Harmsen, Alexei Gratchev, and Julia Kzhyshkowska. IL-4/FoxQ1 pathway
stimulates transmigration of monocytes. Target Journal: Journal of Immunology
3. Comments on the qualification programm and supervision strategy
During my PhD program in frames of GRK880/3 I have made a great progress as a
researcher in fundamental science. I have mastered number of techniques in cell and
molecular biology, endothelial cell biology, immunology and advanced microcopy including
confocal microcopy. I had a great chance to develop the skill of an independent investigator
and after learning functional transmigration assay in Groningen, I have transferred this
technology to my host group at the Medical Faculty Mannheim, and modified this technique
to answer experiential questions of my PhD project. During the educational program of
GRK880/3 I have obtained profound knowledge in endothelial cell biology and associated
disorders, learned a lot of soft skills, in particular the conference presentation and scientific
writing skills. Participation in the seminar and joint Mannheim/Groningen spring and autumn
schools allowed me to develop high quality presentation skills. I have also obtained
experience i writing of scientific publications. I had a great opportunity to exchange the
experience and theoretical knowledge both with different principal investigators a well as with
other MD and PhD students from GRK880/3. The quality of my thesis had benefit due to
supervision by PIs from Medical Faculty Mannheim and Groningen, since the supervisors
had complimentary expertise.
The obtained experimental and theoretical knowledge and soft skill are essential for the
continuation of my research career.
attended schools: 8
2011. Practical project on the methodology of cell transmigration at the Department of
Pathology and Laboratory Medicine, Medical Biology Section, University of Groningen
Medical Center, Groningen. Supervisor of practical project: Prof. Dr. Marco Harmsen
Participation in conferences, congresses, etc., at home and abroad (as presenting
author):
2nd International SFB Symposium (SFB Transregio23, SFB834, GRK880), Mannheim, Role
of FoxQ1 in monocyte/endothelial interactions (2011)
17th International Vascular Biology Meeting 2012, Wiesbaden, The role of FoxQ1 in
monocytes transmigration (2012)
Gesellschaft für Mikrozirkulation und Vaskuläre Biologie e.V. - Annual Meeting, Mannheim,
The Role of FoxQ1 in the transmigration of monocytes in inflammatory conditions (2012)
74
4. Own publications
1.
Gratchev A, Ovsiy I, Manousaridis I, Riabov V, Orekhov A, Kzhyshkowska J.
Novel monocyte biomarkers of atherogenic conditions. Curr Pharm Des. 2013;19(33):585964. Review.
2.
Fuchs T, Puellmann K, Hahn M, Dollt C, Pechlivanidou I, Ovsiy I, Kzhyshkowska J,
Gratchev A, Fleig J, Emmert A, Neumaier M, Beham AW, Kaminski WE. A second
combinatorial immune receptor in monocytes/macrophages is based on the TCRγδ.
Immunobiology. 2013 Jul;218(7):960-8.
75
First and last name of doctoral researcher: Katja Wagenblaß
(Working) title of doctoral project: Impact of LXR on the development of diabetic
nephropathy
Names of supervisors: H.-J. Gröne, L. Schäfer, J. van den Born
IRTG 880/3 fellowship (01 January 2010 - 31 December 2012)
01/2010 Start of doctorate, submission of doctoral thesis on 10 January 2013, completion of
doctorate: 18 March 2013
Winter semester 2004, Biology, University of Heidelberg, August 2009: Reception of
Diploma degree
Perora GmbH, Heidelberg
Diabetes mellitus is a metabolic disease with a worldwide increasing prevalence.
Approximately one third of affected patients develop a renal manifestation as defined by
diabetic nephropathy. Abnormal lipid metabolism and renal accumulation of lipids are thought
to contribute to diabetic nephropathy. The hypothesis for this PhD thesis is that activation of
the nuclear transcription factors Liver – X – receptors, as key regulators of lipid metabolism
and immune system, are able to ameliorate diabetic nephropathy. To test this hypothesis, the
effect of LXR activation by the synthetic ligand GW3965 was evaluated in animal models of
hyperglycemic and hyperlipidemic LDL receptor knockout mice (LDLR-/-) or mice deficient for
endothelial nitrogen monoxide synthase (eNOS-/-).
Treatment of diabetic and hyperlipidemic experimental animals with the LXR agonist
GW3965 reduced renal lipid accumulation and improved renal function and morphology.
Genes involved in cholesterol efflux (ABCA1, ABCG1) were upregulated with concomitant
downregulation of proinflammatory and profibrotic cytokines (TNFα, TGFβ). In addition, LXR
activation through GW3965 reduced parameter of lipid associated oxidative and nitrosative
stress (xanthin – oxidoreductase, 3 – nitrotyrosine). Complemental in vitro experiments could
confirm the results of the animal studies. GW3965 treatment in HK-2 cells stimulated with
oxLDL lead to upregulation of genes involved in cholesterol efflux and reduced lipid droplet
formation. Furthermore, it could be shown that LXR activation decreased mitochondrial
activity and the production of reactive oxygen species. These data provide insight into the
mechanisms of intracellular lipid accumulation mediated renal lesions, which can be
effectively regulated by LXRs.
These results demonstrate potent regulatory effects of LXR in preventing renal damage in
diabetes mellitus. Activation of LXR improved significantly lipid – aggravated diabetic
nephropathy in mice. Therefore, synthetic ligands of LXR are promising therapeutic agents in
preventing renal participation in diabetic patients and are focused by current research.
76
I largely profited from the PhD programme offered by the GRK, getting the opportunity to
present my data on national and international meetings, by means of posters and oral
presentations. It was very valuable for me as my direct supervisor disliked that PhD students
spent time in lectures and meetings instead of working in the lab of the research group.
Thanks to GRK I had a platform to broaden my knowledge and interact with other
researchers.
The meetings organized by the graduate school itself were in my opinion very balanced with
regard to length and frequency. I also profited from the multiple practical workshops being
provided by the GRK and partner graduate schools.
I was guided well by my supervisors and I had received continuous supervision, input and
exchange during the time span of the graduate school.
attended schools: 4
n.a.
n.a.
4. Own publications
1.
Kiss E, Kränzlin B, Wagenblaβ K, Bonrouhi M, Thiery J, Gröne E, Nordström V,
Teupser D, Gretz N, Malle E, Gröne HJ. Lipid droplet accumulation is associated with an
increase in hyperglycemia-induced renal damage: prevention by liver X receptors. Am J
Pathol. 2013 Mar;182(3):727-41. Epub 2013 Jan 12
77
First and last name of doctoral researcher: Stephanie Busch
(Working) title of doctoral project: Identification of novel cell-cell communication pathways
and genes in polycystin-associated retinal vasoregression
Names of supervisors: H.-P. Hammes, Y. Feng, N. Gretz, S. Hoffmann, J.-L. Hillebrands, J.
van den Born
01 March 2010 - 31 December 2012
IRTG fellowship (01 March 2010 - 31 December 2012)
01/2010 start of doctorate, submission of doctoral thesis in 03/2014, anticipated completion
of doctorate: autumn 2014
Autumn/winter 2007, Human medical studies, Medical Faculty Mannheim, University
Heidelberg, 15 November 2013: Reception of approbation
01 April 2014 Residency at the Institute for Oral and Maxillofacial Surgery, Klinikum der
Stadt Ludwigshafen GmbH, Ludwigshafen, Germany
The transgenic polycystic kidney disease rat (PKD) develops a photoreceptor degeneration
and secondary vasoregression. Prior to vasoregression, activation of the innate immunity
system, especially CD74-positive microglia, occurs. These microglial cells are localized in
proximity to capillaries undergoing regression, predominantly in the deep vascular layer. The
aim of this project was to identify the pathomechanism of vasoregression in the PKD rat,
used as a model for nonproliferative diabetic retinopathy.
The project was divided into three parts.
1. Identification of possible CD74 activators.
2. Study the effector system of microglia.
3. Therapeutic intervention to prevent capillaries from degeneration.
The first part was dedicated to identify potential activators of CD74 positive microglia. MIF,
Tubulin-alpha, Tubulin-beta, Glutathion S-Transferase and GP96 were excluded from the list
of possible activators due to CD74-distant localization. No precise activator had been
identified. Massive cell degeneration in the PKD model is likely to activated microglia in an
unspecific manner. Gene analysis revealed complement as one of the major upregulated
pathways. Localization of different complement activation pathways showed an activation of
the alternative pathway in the PKD model. C3 was just present in the deep capillary layer of
PKD rats. However, MAC was not present neither in PKD or in SD rats. As a consequence,
complement seems to be a signaling pathway in the PKD model, but not causal to capillary
damage. Given that Alzheimer’s disease (AD) shows broad overlap to the pathogenesis of
the PKD model - neuronal injury, activation of microglia and astrocytes and secondary vessel
degeneration – we determined the activation of astrocytes and the accumulation of amyloid-ß
in PKD retinae. Immunohistochemistry of PKD retinae for vimentin, carboxymethyllysin, betaAmyloid 1-42, High-Mobility-Group-Protein B1 (HMGB1) and amyloid protein precursor
(APP) was performed. Adjunct to astrocyte activation, accumulation of beta-Amyloid 1-42
78
and HMGB1 in astrocytes and around vessels of the superficial network was found in PKD
retinae prior to the onset of vasoregression. APP was localized adjacent to the outer
segment of photoreceptors in PKD and control rats. The parallel appearance of AD-related
peptides indicates an alarmine based response to photoreceptor degeneration and
secondary vasoregression. The model has broad overlap with AD and may be suitable to
study beneficial pharmacological concepts.
The second part brought the microglia itself in focus. Cell culture was used to test wether
microglia were influenced by surrounding medium (containing LPS or GM-CSF). Cultured
cells revealed different surface marker depending on surrounding medium. We showed that
M1-stimulated microglia had a higher adhesion to endothelial cells than M2-stimulated
microglia, indicating higher aggressivity of M1-microglia againgst endothelium and thereby
vessels than M2-microglia. Typical M1/M2 marker were localzied in PKD and SD retinae.
Interetingly typical M1 marker like Il-1ß and IL-6 were localized in a typical morpholoy of
macroglia, not microglia.
In the last part of this project we aimed to prevent capillaries form vasoregression. To target
the whole neuroglialvascular unit, neuro- and vasoprotective Erythropoietin (EPO) was
intraperitoneally injected to four weeks old male homozygous PKD rats three times a week
with a dose of 256IU/kg body weight. For comparison EPO-like peptide, missing unwanted
side effects of EPO-treatment was given five times a week with a dose of 10µg/kg body
weight. Matched EPO respectively water-injected Spraque Dawley and PKD rats were hold
as control. After four weeks of treatment the animals were sacrificed and neuronal and
vascular morphology was quantified. EPO-treatment exclusively protected thinning of the
central retina by 10% (p<0.05), consisted of an increase of the outer nuclear layer by 12%
(p<0.01) and the outer segments of photoreceptors by 26% (p<0.001). Quantification of cell
nuclei revealed no difference. The number of endothelial cells and pericytes did not change
after treatment with EPO or EPO-like peptide. There was a non significant reduction of
migrating pericytes by 23% respectively 34%. Formation of acellular capillaries was
significantly reduced by 49% (p<0,001) respectively 40% (p<0.05). In conclusion, EPO
protects the neurovascular unit this model of retinal neurodegeneration. This finding
strengthens EPO in its protective capability for the whole neuroglialvascular unit.
To test whether microglial activation is a bystander effect of photoreceptor degeneration or
causally involved in vasoregression, we depleted microglia by intravitreal injection of
clodronate-coated liposomes. Immunofluorescence, gene analysis and retinal morphology
were used to quantify the effect of clodronate-treatment. While the absolute number of
microglia was modestly affected by clodronate, the amount of activated CD74-positive
microglia in the deep capillary layer decreased by 50% upon clodronate-treatment.
Quantitative retinal morphometry showed a significant reduction of acellular capillaries by
31% (p<0.01) only in the deep capillary layers. Gene analysis of whole retinal lysate revealed
no major changes in the expression of macrophage polarity markers, but complement factor
3 and B were increased by 110% and 72%, respectively. This study shows that a reduction
of activated CD74-expressing microglia in the deep capillary layer leads to a reduction of
acellular capillaries, suggesting that microglial activation damages the vascular system
I learned a lot during my time in the graduate school. We had the chance to participate in
different schools, meetings and practical workshops. As we were working in the same field,
there was a great exchange between students, different labs and even countries.
Due to many presentations we were supposed to do during GRK meetings, we had a great
chance to practice for oral presentations or posters at congresses.
I owe the graduate school all the basic skills, being important for research, as statistic,
literature study, paper writing and so on.
79
attended schools: 5
Research Elective at the Experimental Nephrology, UMCG, Groningen, Netherlands (03/2012
and 04/2012)
Mikroglia schädigt Retinakapillare, Kongress der Deutschen Diabetes Gesellschaft (DDG)
05/2012, Berlin, oral presentation
Activated microglia promote vasoregression, Congress of the European Association for the
Study of Diabetes (EASD) 10/2012, Stuttgart, poster
Protection of the Neurovascular Unit in a Model of retinal Neurodegeneration by EPO and
EPO-like Peptide, Congress of the American Diabetes Association (ADA) 06/2013, Chicago,
poster
4. Own publications
1.
Busch S, Wu L, Feng Y, Gretz N, Hoffmann S, Hammes HP.: Alzheimer's disease
and retinal neurodegeneration share a consistent stress response of the neuro-vascular unit.
Cell Physiol Biochem. 2012;30(6):1436-43. doi: 10.1159/000343331. Epub 2012 Nov 22.
2.
Vogler S, Pannicke T, Hollborn M, Grosche A, Busch S, Hoffmann S, Wiedemann P,
Reichenbach A, Hammes HP, Bringmann A.: Müller cell reactivity in response to
photoreceptor degeneration in rats with defective polycystin-2. PLoS One. 2013 Jun
3;8(6):e61631. doi: 10.1371/journal.pone.0061631. Print 2013.
3.
Feng Y, Busch S, Gretz N, Hoffmann S, Hammes HP.: Crosstalk in the retinal
neurovascular unit - lessons for the diabetic retina. Exp Clin Endocrinol Diabetes. 2012
Apr;120(4):199-201. doi: 10.1055/s-0032-1304571. Epub 2012 Mar 8. Review.
4.
Wang Q, Gorbey S, Pfister F, Höger S, Dorn-Beineke A, Krügel K, Berrone E, Wu L,
Korff T, Lin J, Busch S, Reichenbach A, Feng Y, Hammes HP.: Long-term treatment with
suberythropoietic Epo is vaso- and neuroprotective in experimental diabetic retinopathy. Cell
Physiol Biochem. 2011;27(6):769-82. Epub 2011 Jun 17.
5.
Busch S, Kannt A, Kolibabka M, Schlotterer A, Wang Q, Feng, Y, Hoffmann S, Gretz
N, Hammes HP: Systemic treatment with Erythropoietin protects the neurovascular unit in a
rat model of retinal neurodegeneration PLOS ONE Decision: Accepted [PONE-D-1415319R1]
80
First and last name of doctoral researcher: Larissa Pfisterer
(Working) title of doctoral project: The role of myocardin and miRNA 143/145 in
biomechanically induced vascular damage
Names of supervisors: M. Hecker, T. Korff, R. Henning, I. Molema
01 January 2010- 25 June 2013
15 October 2003, Biology, University Karlsruhe and Tübingen, Diploma in April 2009
Post-Doc (scholarship granted by the DGK) at the Institute of Physiology and
Pathophysiology
Cardiovascular diseases are still the major cause of death in industrialized countries with
hypertension being the most important risk factor for these pathologies, namely for the onset
and development of arteriosclerosis. Hypertension itself typically leads to an increase in
peripheral resistance causing a rise in diastolic blood pressure initiating detrimental changes
in structure and function of arterial blood vessels. This local rise in transmural pressure in
these blood vessels causes an increase in wall tension to which the arterial smooth muscle
cells (SMCs) first respond with active vasoconstriction, the so-called myogenic response,
aimed at reducing the biomechanical strain within the arterial vessel wall. If the increase in
blood pressure hence wall tension persists, however, the smooth muscle cells within the
media will inevitably undergo hypertrophy or hyperplasia, depending on the size of the
arterial blood vessel, resulting in an inward remodeling that according to the law of Laplace
reduces wall tension at the expense of a further rise in peripheral resistance. Depending on
the intensity and duration of the increase in wall tension, therefore, the remodeling process in
the arterial vessel wall becomes maladaptive ultimately causing an irreversible fixation of
diastolic blood pressure at an elevated level. The basis of this initially adaptive remodeling
process turning into maladaptive is a switch in phenotype of the arterial SMCs from the
contractile to the synthetic state which is earmarked by their increased rate of proliferation
and loss of their contractile capacity. The reasons why vascular SMCs in general fail to
maintain their contractile state in conditions of hypertension are still elusive.
Starting from the hypothesis that increased wall tension is the driver of this maladaptive
inward remodeling through weakening factors that maintain the vascular SMCs in the
contractile state, this work addressed the putative role of such a phenotype stabilizing factor
therein, i.e. myocardin. Mainly localized to the nucleus of these cells, myocardin is a coactivator of the transcription factor serum response factor (SRF) and as such pivotal for
maintaining the expression of contractility promoting gene products in vascular SMCs.
In this context, knockdown of myocardin in human arterial SMCs in fact revealed a loss of
expression of such gene products like smooth muscle α-actin (SMA) or calponin, and
subsequently a loss of their contractile capacity while their rate of proliferation increased. In
81
line with these findings, myocardin abundance decreased in the media of remodeling arteries
from hypertensive mice. Subsequent analyses of isolated perfused murine blood vessels and
both human and mouse cultured SMCs exposed to elevated wall tension or stretch confirmed
a loss of myocardin from the nucleus of the arterial SMCs as well as a concomitant reduction
of its abundance in the cytoplasm. Stretching of the cultured SMCs also promoted the rapid
phosphorylation of myocardin at a serine residue through activated extracellular reactive
kinase (ERK) 1/2. As a result, the phosphorylated myocardin dissociated from SRF and
bound to histone deacetylase (HDAC) 4/5 followed by its chaperone 14-3-3-mediated export
from the nucleus to the cytoplasm. There, presumably the exported myocardin was
ubiquitinated by the E3-ligase CHIP and subsequently degraded by the 26S-proteasome.
Collectively these findings point towards a novel mechanism by which elevated wall tension
or stretch causes an ERK1/2-mediated serine phosphorylation of myocardin leading to its
HDAC4/5 and 14-3-3-mediated transport from the nucleus to the cytoplasm where it is
subsequently degraded through the proteasome. As such, a pivotal transcriptional coactivator in vascular SMCs controlling the gene expression program required to maintain
their contractile phenotype is effectively inactivated and removed paving the way for their
transient or chronic switching to the synthetic phenotype that is prototypic for vascular
remodeling processes owing to an increase in wall tension. In this respect, this mechanism
may offer a novel and interesting therapeutic approach to address hypertension-related
cardiovascular disorders through maintaining the contractile vascular SMC phenotype.
Further remarks:
Initial data at the beginning of the project excluded a relevant role of the miRNA-cluster
143/145 in the context of hypertension-associated remodeling and myocardin regulation.
Following, no further experiments in that context have been performed.
The insights gained by the dissertation could be translated and applied to other projects,
such as the pathogenesis of varicose-like venous malformations (Pfisterer et al, 2014,
FASEB). Furthermore, based on the above introduced data, we could design a continuative
study, which is currently funded by a scholarship of the DGK (German Society of
Cardiology).
The presented work was awarded with the young investigator award (2012) of the GfMVB
(Society for microcirculation and vascular biology), the posterprice (2013) by the DPG
(German society of physiology), the so called Promotionspreis (2013) granted by the DHL
(Deutsche Hochdruckliga) and the Friedrich-Reutner-Preis (2014) from the medical faculty
Heidelberg.
Prof. Dr. med. Hammes always welcomed ideas and suggestions coming from the students
concerning the organization and design of the GRK-lecture-program.
The practical courses met the needs of the field of vascular biology/medicine but on the other
hand also addressed experimental approaches beyond that specific scope. The offer of
lectures and courses in the third and final period of the GRK 880 was a little reduced due to
the decreased number of students. Prof Dr. Hammes elegantly solved this problem by
opening the scientific program of the topic-related SFB/TR23 for the students of the GRK880.
In my personal case, the daily/close supervision by my PIs was very good. I had a strong and
constructive support, especially by Prof. Thomas Korff. The support by Prof. Dr. Hammes
was always very enthusiastic and motivated. He did not hesitate to offer solutions and share
input from another point of view. The feedback given by all participating PIs during progressreports at spring- respectively autumn-schools in Heidelberg or Groningen was very useful,
since they all shared the same interest but with a different focus. Therefore, one can say that
the crosslink of all projects was good. In that sense, it was possible and welcomed to
address other groups with a specific question or problem besides the official practical
courses, which provides a very efficient and helpful environment.
82
attended lectures: 15 (36 SFB/TR 23)
attended schools: 6
Research stay in Groningen in May/June 2012 in the Lab of Prof. R. Henning, Prof. M
Harmsen
DPG in Regensburg (2011), EB in San Diego (2012), IVBM in Wiesbaden (2012), GfMVB in
München (2011) und Mannheim (2012), SFB Symposium in Mannheim (2011), DGK
Herbsttagung in Hamburg (2012)
4. Own publications
1.
Pfisterer L, Feldner A, Hecker M, Korff T, Hypertension impairs myocardin function –
a novel mechanism facilitating arterial remodeling, Cardiovasc Res, 96: 120-129, 2012
2.
Korff T, Pfisterer L, Schorpp-Kistner M, miR-663 and the miRaculous vascular
smooth muscle phenotypic switch, (Editorial) Circ Res., 113: 1102-1105, 2013
3.
Pfisterer L, König G, Hecker M*, Korff T*, Pathogenesis of varicose veins – lessons
from biomechanics, (Review) Vasa 2014; 43: 88 – 99
4.
Scherer C*, Pfisterer L*, Hödebeck M, Wagner A, Cattaruzza M, Hecker M, Korff T,
From osmotic to biomechanical stress - stretch controls NFAT5 activity in vascular smooth
muscle cells, J Am Heart Assoc. 2014;3:e000626, (*authors contributed equally)
5.
Pfisterer L, Meyer R, Feldner A, Drews O, Hecker M, Korff T (2014), Bortezomib
protects from varicose-like venous remodeling, FASEB, 2014 Apr 25. [Epub ahead of print]
6.
Arnold C, Feldner A, Pfisterer L, Hödebeck M, Troidl K, Genové G, Wieland T,
Hecker M, Korff T (2014) RGS5 promotes arterial growth during arteriogenesis, accepted for
publication in EMBO Mol Med
83
First and last name of doctoral researcher: Joshua Krämer
(Working) title of doctoral project: Pyruvate Kinase Expression and Activity in Hearts,
Kidneys and Livers of Mice with Experimental Diabetes
Names of supervisors: P. Nawroth, A. Bierhaus †, S. J. L. Bakker, J. van den Born
01 February 2012 - 31 December 2013
IRTG fellowship (1 February 2012- 31 December 2013)
Start of doctorate January 2012, anticipated completion of doctorate: Autumn/Winter 2015
01 October 2007; Human medical studies; Medical Faculty Heidelberg, University
Heidelberg
n.a.
Reactive oxygen species (ROS) play a central role in the development of complications in
patients suffering from diabetes mellitus type 1 and type 2. In diabetes, the formation and
release of ROS is elevated, and additionally the activity of the antioxidant defense system is
impaired. Oxidative stress leads to the formation of reactive metabolites and a series of
harmful effects in several organs. In the vasculature, both endothelial cells and smooth
muscle cells are affected by oxidative damage. The resulting endothelial dysfunction is
considered to be a key factor in the development of microvascular and macrovascular
complications.
Metabolic pathways in diabetes are altered in a complex manner, which is not yet fully
understood. Several bottlenecks in glycolysis occur, one of which is the inhibition of the
glycolytic enzyme GAPDH by ROS. As a consequence, metabolic intermediates accumulate
and are shifted into alternative pathways, which are responsible for the development of
diabetic complications. A better understanding of the metabolic alterations that underlie the
development of these complications is necessary in order to develop better treatment
strategies for their prevention.
One of the enzymes which could be also affected is pyruvate kinase, as it is one of the ratelimiting, regulatory enzymes in glycolysis. Previous studies have shown that pyruvate kinase
activity is diminished in the liver of rats after experimental induction of diabetes. Additionally,
results from genetic studies indicate that pyruvate kinase genes are associated with several
forms of diabetes. The aim of this project was to find out if pyruvate kinase expression or
activity in different organs is altered in long-term experimental diabetes of mice.
Hearts, kidneys and livers of mice with streptozotocin-induced diabetes and of untreated
healthy mice were analyzed in comparison. Seven mice were used in each group. The mice
were killed 3 month after streptozotocin treatment. Pyruvate kinase expression was
measured by real-time PCR. Because pyruvate kinase occurs in several isoforms, isoformspecific primers were used. Pyruvate kinase activity was measured by an enzymatic assay
(in which oxidation of NADH + H+ to NAD+ was detected photometrically).
84
In the diabetic mice, the heart and the kidney exhibited a pyruvate kinase expression that is
different from healthy animals: In the heart, overall pyruvate kinase expression was reduced,
and in both heart and kidney, expression was shifted from pyruvate kinase isoenzyme M1 to
the less active isoenzyme M2. This shift was based on reduced expression of isoenzyme M1
in the heart and increased expression of isoenzyme M2 in the kidney. Despite these
changes in expression, activity in both heart and kidney didn’t differ from the control animals.
In the liver, expression as well as activity was not different between the control and diabetic
mice.
The fact that pyruvate kinase activity was not different between the diabetes and control
groups in the heart and in the kidney despite the changes observed in expression, especially
the reduction of overall pyruvate kinase expression in the heart, means that a mechanism
enhancing the specific pyruvate kinase activity must be in effect to compensate for the
impaired enzyme expression. It may be that the risk to develop diabetic complications
depends on the individual ability to activate and maintain such compensatory mechanisms to
cope with deranged metabolic pathways.
As in this study organs from mice were analyzed, the results can’t be applied to humans
without caution. Additionally, it is questionable to what extent results from an experimental
approach with artificially induced diabetes are representative for the natural disease. On
these grounds, this project was conceived as a pilot study, and the results must be confirmed
with data from human patients. Isoenzyme-specific PCR primers for human pyruvate kinase
mRNA have already been developed in our lab, and work has begun to analyze the
expression of pyruvate kinase in peripheral blood mononuclear cells of patients with diabetes
type 2.
To summarize, this study shows that in experimental diabetes of mice, the heart and the
kidney, but not the liver, exhibit a pyruvate kinase expression that is different from healthy
animals. It has to be investigated with data from humans if these findings are relevant for
diabetic patients.
In my opinion, the biggest benefit of the graduate school is the exchange with other people
doing research in the same field, by personal communication and by presentations. This
provided me a constant stream of impulses for further thoughts. I discovered lots of links and
touching points between different projects and learned to look at topics from different
perspectives, ultimately developing a better understanding of the bigger picture in the field of
vascular medicine. The dialogues with other GRK members also yielded many suggestions
and ideas of practical importance.
The supervisors were also very helpful. They always took the time for personal discussions
whenever necessary, cared for individual questions, and provided precious advice.
The graduate school workshops and seminars were interesting and informative. What I
learned there was useful beyond research and medicine, for example the presentation
workshop. I appreciate that the courses were executed by alternating lecturers and
instructors, which ensures diversity in topics and opinions. The meetings I attended were
exemplarily organized and had a good work-leisure balance. In my judgement, the
frequency and duration of the events was appropriate.
I regret that obligations due to my studies made it impossible for me to attend many events. I
suggest to transmit more lectures by videoconference from Mannheim to Heidelberg, which
makes it easier for students from Heidelberg to take part.
attended schools: 2
85
n.a.
n.a.
4. Own publications
n.a.
86
First and last name of doctoral researcher: Nagesh Shanbhag
(Working) title of doctoral project: Developing a model of permanent focal cerebral
ischemia in rats mimicking malignant stroke
Names of supervisors: L. Schilling, R. Henning
01 June 2010 - 31 May 2013
IRTG fellowship (01 June 2010 - 31 May 2013)
06/2010 Start of doctorate, anticipated submission of doctoral thesis on /2014, anticipated
completion of doctorate: 12/2014
06/ 2010, Promotion studies, Medical Faculty Mannheim; 09/ 2008, M.Sc in Experimental &
Clinical Neurosciences, University of Regensburg, 11.10.2010; September 2001, M.B.B.S
(Bachelor of Medicine & Surgery, Human Medicine, Goa University, India, 14.03.2007
Currently spending final 1year (culminating in a PhD) as part of the collaborating MD/PhD
programme at the Dept. of Clinical Pharmacy and Pharmacology, UMCG, University of
Groningen, The Netherlands since 10/October 2013 under Prof. Dr. Robert Henning
Ischemic stroke accounts for around 80% of the total stroke cases resulting in severe adult
disability and poor quality of life. Except for the thrombolysis (using recombinant tissue
plasminogen activator, rtPA), there are currently no therapeutic approaches alleviating this
condition. Thrombolysis however, has a narrow therapeutic time window (upto 4.5h after
symptom onset). Although several compounds tested pre-clinically have shown promising
alsneuroprotective effects, they have failed in the clinic trials.
Around 10% of all the supratentorial stroke patients show a rapid progression of ischemic
damage (especially involving the blockage of proximal part of internal carotid artery, ICA or
proximal trunk of middle cerebral artery, MCA), along with severe brain swelling from 24-72h
from the symptom onset with a mortality upto 80%, which is referred to as malignant stroke.
Such patients undergo decompressive craniectomy procedure to relieve the edema and
thereby, reducing the raised intracranial pressure. Various pre-clinical animal models
mimicking the clinical set up of ischemic stroke followed by reperfusion have been developed
and used widely to study the pathophysiological mechanisms and as a tool to test novel
therapeutic agents. Most of the pre-clinical models (using either blood clots, microsphere
injection, photothrombosis, intravascular occluders) mimic a state of ischemia-reperfusion
injury as seen in the clinics. However, considerable number of patients are not eligible due to
the current narrow therapeutic time window. Therefore, chronic permanent cerebral ischemia
models could prove to be a valuable tool in this regard.
The widely used approach in order to create a permanent MCA (commonly involved arterial
territory) occlusion (MCAO) using intravascular occluders (4-0 nylon suture coated with
silicone rubber at its tip in case of rats, passed through external carotid, ECA or common
carotid artery,CCA) has restricted its long-term studies due to high rate of mortality
encountered in these animals as a result of severe brain swelling (24-36h post MCAO). We
set out to develop a rat model of severe brain ischemia (malignant MCA stroke) without
87
reperfusion, by modifying the occluding silicone tip shape, in such a manner, that it
selectively blocked the proximal blood flow through the MCA, without compromising the
posterior cerebral circulation, thereby, giving rise to a low mortality, moderate-severe
ischemic volume accompanied with brain swelling and a delayed progression of cerebral
infarct (as seen in patients).
We compared the results of our modified occluders (ischemic volume, brain swelling,
hemispheric infarction along the fronto-occipital axis, mortality) with that of the groups
involving conventional lab-made and a commercially available occluders. Our modified group
involved bowling pin-shaped (BP, approximately 1mm distance between 2 ball-shaped tips,
n=7) silicon tip occluders, due to their peculiar shape of silicon coating. While the other
groups involved long tubing-based (L-TB, 2-3mm length of silicone coating, n=6), short
tubing-based (S-TB, <2mm, n=6) silicone cocated occluders and Doccol (DOC, 2-3mm,
commercially available, n=6) occluders. After several modifications of the silicon coating of
the tip, did we finally succeed in creating a selective occlusion of the MCA origin (proximal)
with a significantly reduced but with a severe degree of ischemic volume and a comparable
amount of brain swelling to the other groups at 24h. However, a high rate of mortality was
evident in the L-TB (53.8%), S-TB (64.7%) in comparison to the BP group (12.5%). All the
animals in the DOC group had a worse clinical outcome at around 24h post MCAO, which
led to their euthanisation at this stage (these animals showed evidence of a higher ischemic
volume as later detected by the high contrast Silver nitrate staining to delineate the ischemic
and non-ischemic areas in the brain). Besides, the hemispheric infarction along the frontooccipital axis showed a significant increase in the area under the curve, especially in the
occipital part of the axis in the groups, which signified a compromised posterior cerebral
circulation.
The long coverage of silicone at the occluders’ tip invariably interrupts the blood flow through
the origin of the posterior cerebral artery (PCA), resulting in a state of two-vessel occlusion.
This leads to a severe degree of ischemic damage with accompanying swelling,
compromising the cerebrovascular compliance and leading to a high rate of mortality.
Nevertheless, the short silicone coating of the occluders resulted in a mechanical
displacement (anteriorly) of the MCA origin (as evident at the base of brain upon its removal)
on the side of occlusion as compared with the contralateral non-occluded hemisphere. We
proposed the hypothesis that this displacement could to some extent, compromised the
blood flow through the PCA origin, further aggravating the ischemic damage. To test this, we
performed a selective ex vivo infusion of 2% Evans blue dye via a catheter (70µl/min,2mins)
placed in the CCA without disturbing the occluder, which is already fixed in the ECA stump,
upon in situ physiological saline perfusion (via abdominal aorta). This enabled us to visualize
and thereafter, quantify the dye spectrophotometrically (expressed as micrograms per gram
of fresh tissue) in our regions of interest (dissecting the occipital pole from the rest of
hemisphere on both sides). Thus, the groups which showed a mechanical displacement
(especially S-TB), resulted in a significantly lower levels of the dye in the right occipital pole
(occlusions in our case were performed on the right side) as compared to the BP occluder
group, proving that vascular displacement did compromise the blood flow to the posterior
cerebral territory. While a high amount of dye was also evident in the residual right
hemisphere in our BP group, signifying the circulation of the dye via collaterals between
MCA-PCA and/or MCA-ACA. This could be a potential factor for a low mortality rate
encountered in these animals.
With a negligible rate of mortality in the BP group, we extended their survival time points until
day 5. While in a separate group of animals which were utilized for longitudinal MRI imaging,
the animals could survive 3 weeks post occlusion also. At day 3 post MCAO, a significant
increase in the ischemic volume was evident, which signified a delayed phase of infarct
extension as evident clinically in patients of malignant MCA stroke. However, the brain
swelling was comparable with that of the day 1- BP group, while a significant decrease was
seen on day 5, with a well-developed collateral network between the MCA-ACA and MCAPCA as evident on the dorsal surface of the brain.
We thus, could establish a permanent focal cerebral ischemic model in adult rats with
features (severe brain infarction with edema, delayed progression of ischemia from day-1 to
88
day-3) mimicking to certain extent, a clinical condition of malignant MCA stroke. This model
could prove to be a potential tool to study the pathophysiological mechanism(s) underlying
the infarct progression and to test novel neuroprotective agents including cell therapies.
As a continuation of my work in Mannheim, I have now moved to the University of
Groningen, The Netherlands to carry out relevant therapeutic studies in the area of cerebral
ischemia, focusing primarily on CNS inflammation as a potential therapeutic target.
Status: Study completed.
Manuscript is in preparation Shanbhag NC, Henning RH, Schilling L, Long-term survival in a
model of malignant stroke in rats
Various guest lectures/talks, annual Spring and Autumn schools provided a broader view for
scientific development in the field of vascular medicine. Besides, the constant support,
feedbacks and patience from my supervisor enabled me to carry out the required study in
order to finally establish a long-term surviving rat model of permanent focal cerebral ischemia
after several modifications of the intravascular occluder. Previously established models of
permanent focal ischemia have gone along with high mortality rates, and I have analyzed the
mechanisms which enabled the long-term survival of the animals with a low mortality. I also
profited from the multiple practical workshops being provided by the GRK and partner
graduate schools. Besides, based on my experimental experience I got invovlved in a
collaboration with fellow departments in the UMM (Dept. of Computer Assisted Clinical
Medicine) under Prof. Lothar Schad resulting in a joint publication (mentioned above). In this
study a newly developed cryocoil for detection of in vivo potassium levels at a 9.4T MRI
scanner was tested in rats under control conditions and following focal cerebral ischemia.
My current work as a collaboration with the University of Groningen under Prof. Robert
Henning (Dept. of Clinical Pharmacy and Pharmacology, University Medical Centre
Groningen) will be to assess relevant therapeutic compounds in the cerebral ischemia model
established and studying neuroprotective mechanisms underlying. Based on the current
work I will earn a PhD degree from the University of Groningen.
attended schools: 5
n.a.
37.Jahrestagung der Sektion Intrakranieller Druck, Hirndurchblutung und Hydrozephalus (Oct
28—29, 2011), Tubingen, Germany. A modified intravascular occluding filament to result in
long-term survival in a rat model of permanent focal cerebral ischemia (Oral presentation)
7th International Symposium on NEUROPROTECTION and NEUROREPAIR (May 2-5, 2012),
Potsdam, Germany, Intravascular filament occlusion technique to enable long-term studies in
a rat permanent focal cerebral ischemia model (Poster presentation)
Participated in th GfMVB Annual Meeting, Mannheim, Germany, 27- 29th September, 2012
4. Own publications
n.a.
89
First and last name of doctoral researcher: Birgit Maria Buhl
(Working) title of doctoral project: Age and Immunosenescence related factors in ANCAassociated vasculitis
Names of supervisors: B. Yard, C. Kallenberg
01 January 2010 - 31 March 2012
IRTG fellowship (01 January 2010 - 31 March 2012)
01/2010 Start of doctorate, MD doctoral degree 13 July 2011, anticipated defense of PhD
doctoral thesis 02/2015
Autumn/winter 2003, Human medical studies, University Frankfurt, Autumn/winter 2005
Medical Faculty Mannheim, University Heidelberg, 27 November 2009: Reception of
approbation
01 April 2012 Phycician at Nephrology Department, University Medical Center Mannheim
Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) composes a group
of auto-immune diseases characterized by inflammation and necrosis of the small to medium
sized vessels in combination with the presence of circulating ANCAs directed against either
proteinase 3 (PR3) or myeloperoxidase (MPO). Age is known to be a risk factor for many
autoimmune diseases, most likely as a consequence of immunoscenescence that
contributes to increased autoimmunity. In the present project we questioned as to whether
age-related changes of the immune system make some people more susceptible to AAV.
We first tried to identify the determinants of AAV-related fatigue and the impact of fatigue on
Health-Related Quality of Life in patients with AAV. Fatigue was assessed in cross-sectional
cohort of 187 patients with AAV in complete remission (diagnosed 1989- 2010), using the
Multidimensional Fatigue Inventory (MFI-20), health-related quality of life (SF-36) and a
visual analogue scale for night rest. For each scale of the MFI-20patients with AAV had
significantly higher scores compared with controls and national norm values. General fatigue
and physical fatigue reached the highest scores. The MFI-20 scores negatively correlated
with SF-36 scores. Fatigue is one of the main reasons for patients not to participate in
physical activities. Participation in physical activity was associated with lower MFI-20 scores.
Also the use of corticosteroids was associated with higher levels of fatigue. Age was not a
determinant of AAV-related fatigue
In addition we hypothesized that CYP450 genotypes modify cyclophosphamide metabolism
and influence response to treatment and occurrence of adverse effects in AAV. We could
demonstrate that CYP450 SNPs involved in yclophosphamide metabolism significantly
influence the occurrence of leucopenia and relapse in patients with AAV. With respect to
immunesenescence we found a significant positive correlation between the percentages of
CD56+CD4+ T cells and age in granulamatous poly-angiitis (GPA) patients in remission (r=
0, 6 p= 0,004). We also observed elevated IL-15 serum levels in patients in remission
compared to HC (p= 0,001). In vitro stimulation of PBMCs with IL-15 increased not only the
proportion of CD4+ memory cells (CD45RO+) but also the expression of CD56 and NKG2D
90
on these cells. The driving force behind the persistent expansion of a unique cytotoxic subset
of CD4+ effector memory T-cells expressing CD56 and NKG2D+ and being TCR –
independent is likely the increased IL-15 expression in AAV patients.
Beyond the published results, one manuscript has been submitted, and two others are in
preparation, as follows:
Buhl BM, Tuin J, Rutgers A, Yard BA, Krämer , Kallenberg C, Stegeman C Impact of
Fatigue on Health-Related Quality of Life in patients with ANCA-associated vasculitis.
Submitted.
Buhl BM, Tuin J, Rutgers A, Yard BA, Krämer , Kallenberg C, Stegeman C
Cytochrome P450 Polymorphism as a predictor of adverse effects and therapy
response in patients with ANCA-associated vasculitis. In preparation.
Buhl BM, Rutgers A, Yard BA, Krämer , Kallenberg C, Stegeman C Potential role of
interleukin-15 in the immunopathogenesis of AAV In preparation.
The GRK as a whole, the interaction between Mannheim and Groningen and the different
researchers involved in this project inspired me to continue research while working as a
physician.
attended workshops:1
attended schools: 1
January 2010 - May 2011 PhD-student at the UMCG lab of Prof. Dr. Kallenberg
Poster:
ANCA-Workshop, Chapel Hill, USA, 2011
ANCA-Workshop, Paris, Frankreich, 2013
Deutscher Rheumatologie Kongress, Mannheim/Heidelberg, 2013
Deutscher Nephrologie Kongress, Hamburg, 2012
Deutscher Nephrologie Kongress, Berlin, 2013
4. Own publications
1.
Tuin J, Sanders JS, Buhl BM, van Beek AP, Stegeman CA: Androgen deficiency in
male patients diagnosed with ANCA-associated vasculitis: a cause of fatigue and reduced
health-related quality of life? Arthritis Res Ther 2013, 15(5):R117.
2.
Kalsch AI, Peters A, Buhl B, Breedijk A, Prem K, Schmitt WH, Weiss C, Heeringa P,
Kallenberg C, Birck R et al: Retinoid X receptor beta polymorphisms do not explain functional
differences in vitamins D and A response in Antineutrophil cytoplasmic antibody associated
vasculitis patients. Autoimmunity 2009, 42(5):467-474.
91
First and last name of doctoral researcher: Johannes Wedel
(Working) title of doctoral project: N-octanoyl dopamine attenuates chronic allograft
vasculopathy (original project title as formulated in the GRK880/3 application: prevention of
glycosaminoglycan degradation by carnosine – possible role for endothelial dysfunction in
diabetic patients)
Names of supervisors: B. Yard, J.-L. Hillebrands
IRTG fellowship (01 January 2010 - 31 December 2012), PhD fellowship GUIDE
01 February 2013 – 31 January 2014
01/2010 Start of doctorate, submission of MD doctoral thesis 02 January 2014, anticipated
defense of PhD doctoral thesis 09/2014
01 March 2014 Post-Doc at Boston Children’s hospital, Transplant research program,
Harvard Medical school in the lab of Prof. David Briscoe.
Allograft vasculopathy is the foremost cause for chronic allograft loss in transplant recipients.
Although the molecular mechanisms that underlie its pathophysiology remain far from clear,
it seems to result from the concerted action of immunological and non-immunological factors.
In both prospective randomized multicenter and retrospective studies it has been shown that
donor dopamine treatment has a salutary effect on transplantation outcome in renal and
heart allograft recipient. The pitfall however is that donor dopamine treatment may result in
hypertension or tachycardia in a subset of donors, while its protective effect is independent of
adrenoceptor or dopaminegic receptor engagement. By conjugation of octanoic acid to
dopamine we have developed a new compound, i.e. NOD, that completely lacks
hemodynamic action while retaining its protective properties. In the present study we have
addressed 1) to what extent NOD impairs T-cell activation, which is instrumental for allograft
vasculopathy and 2) to assess if NOD can prevent neo-intima formation in a model of aorta
transplantation in the rat. The main findings of this study demonstrate that NOD transiently
inhibits T-cell proliferation in a dose dependent fashion. This was reflected by a decreased
percentage of T-cells that became CD25 positive or started to express the memory marker
CD45RO upon mitogen activation. NOD also caused G1-arrest in cell cycling, which was
paralleled by a diminished cyclin D expression. Secondly, the T-cell suppressive properties
of NOD were most likely depending on the redox-active dopamine moiety, as N-octanoyl
Tyramine did not inhibit T-cell proliferation. Thirdly, NOD did not affect early TCR signaling,
e.g. phosphorylation of Lck and ZAP70 and CD3 capping. In contrast, activation of the redoxdependent transcription factors NF-κB and AP-1 was profoundly impaired. Last but not least,
NOD and CNI display a strong synergy to suppress T-cell proliferation.
92
To verify the immunosuppressive data in vivo and to investigate NODs potentially beneficial
properties on chronic rejection, we assessed the influence of NOD on the development of
transplant vasculopathy in allogeneic aortic transplanted rats. In the NOD intervention study,
we demonstrated that NOD significantly reduced the development of transplant vasculopathy
with a trend towards a lower graft infiltration by T cells. Also the number of neointimal αsmooth muscle actin (SMA)+ cells was significantly reduced in the allograft. Although in vitro
NOD inhibited human aortic smooth muscle cell (haSMC) proliferation by G1 arrest, in vivo
no difference was found for the proliferation markers Ki67 or PCNA.
Collectively our data clearly indicate that NOD can mitigate transplant vasculopathy. Whether
this is due to its T-cell suppressive, anti-proliferative or anti-iflammatory effect remains to be
assessed in further studies.
I largely profited from the interaction between Mannheim and Groningen enabling me to gain
insight in various aspects of transplantation research. This interaction and the GRK as a
whole have substantially contributed to my decision to continue my research career as a
Post-doc in the lab of Dr. Briscoe (Harvard Medical school).
attended schools: 6
January 2011: Internship at the UMCG (6 weeks) lab of Prof. Dr. Hillebrands
February 2012 – January 2013 Ph.D.-Student at the UMCG lab of Prof. Dr. Hillebrands.
November 2013: Research stay at Boston Children’s Hospital, Harvard Medical School, lab of
Dr. David Briscoe,
March 2014: Internship at Boston Children’s Hospital, Harvard Medical School (6 months),
lab of Dr. David Briscoe.
Oral presentations:
20. Jahrestagung der Deutschen Transplantationsgesellschaft, Regensburg, 2011
European Society of Organ Transplantation, Wien 2013
Poster:
40. Jahrestagung der Deutschen Gesellschaft für Immunologie, Leipzig 2011
22. Jahrestagung der Deutschen Transplantationsgesellschaft, Frankfurt 2013
World Transplant Congress, San Fransisco, USA, 2014
4. Own publications
1.
Stamellou E., Fontana J., Wedel J., Ntasis E., Sticht C., Becker A., Pallavi P., Wolf
K., Krämer BK, Hafner M., van Son WJ, Yard BA. N-octanoyl dopamine treatment of
endothelial cells induces the unfolded protein response and results in hypometabolism and
tolerance to hypothermia. PLoS One 2014 accepted
2.
Hottenrott MC*, Wedel J*, Gaertner S, Stamellou E, Kraaij T, Mandel L, Loesel R,
Sticht C, Hoeger S, Ait-Hsiko L, Schedel A, Hafner M, Yard B, Tsagogiorgas C. N-Octanoyl
Dopamine Inhibits the Expression of a Subset of κB Regulated Genes: Potential Role of p65
Ser276 Phosphorylation. PLoS One. 2013 Sep 2;8(9):e73122.
3.
Ait-Hsiko L, Kraaij T, Wedel J, Theisinger B, Theisinger S, Yard B, Bugert P, Schedel
A. N-octanoyl-dopamine is a potent inhibitor of platelet function. Platelets. 2013;24(6):428-34
93
4.
Tsagogiorgas C, Wedel J, Hottenrott M, Schneider MO, Binzen U, Greffrath W,
Treede RD, Theisinger B, Theisinger S, Waldherr R, Krämer BK, Thiel M, Schnuelle P, Yard
BA, Hoeger S. N-octanoyl-dopamine is an agonist at the capsaicin receptor TRPV1 and
mitigates ischemia-induced acute kidney injury in rat. PLoS One. 2012;7(8):e43525
5.
Peters V, Jansen EE, Jakobs C, Riedl E, Janssen B, Yard BA, Wedel J, Hoffmann
GF, Zschocke J, Gotthardt D, Fischer C, Köppel H. Anserine inhibits carnosine degradation
but in human serum carnosinase (CN1) is not correlated with histidine dipeptide
concentration. Clin Chim Acta. 2011 Jan 30;412(3-4):263-7.
* equally contributed
94
First and last name of doctoral researcher: Markus Jabs
(Working) title of doctoral project: Notch Signaling and Endothelial Metabolism
Names of supervisors: A. Fischer, H. Augustin, H.-P. Hammes, I. Molema
01 November 2011 - 31 December 2013
Fellowship (01 November 2011 - 31 December 2013)
Start of doctoral training in November 2011; anticipated graduation in summer 2015
10/2006 – 8/2009 Studies of Biology (B.Sc.) at Leibniz University of Hannover
10/2009 – 10/2011 Studies of Molecular Biosciences (M.Sc.) at Heidelberg University
n.a.
The vasculature is one of the largest organs in vertebrates and fulfills many different
functions, among them the transport of gases and nutrients. Because of these important
tasks, blood vessels are linked to severe diseases such as cancer and cardiovascular
disorders. The latter are the primary cause of mortality in industrialized countries. Although
the vasculature is a central communication system in vertebrates, there is only little
knowledge about how it contributes to the regulation of systemic metabolism. Interestingly,
many risk factors for cardiovascular diseases and the metabolic syndrome overlap, arguing
for a connection between these conditions. However, besides its relevance, the molecular
role of blood vessels for the development of metabolic disorders is not well understood today
and requires further investigation.
Apart from its role in the generation of new blood vessels, Notch signaling seems to be
required for the maintenance of endothelial functions in the existing vasculature. In this
context, I study the effects of endothelial Notch signaling on energy metabolism. Specifically,
I am interested in the alterations of triacylglycerol and glucose metabolism at the systemic
level caused by Notch blockade in the endothelium.
For this purpose I analyze a mouse model, in which Notch signaling is abolished specifically
in the endothelium upon tamoxifen injection (VE-Cadherin-CreERT2 / flox-RBPJ; hereafter
referred to as “ko mouse”). Compared to controls, these mice have reduced blood glucose
levels and increased insulin sensitivity. Interestingly, there is no difference regarding
circulatory insulin levels, which would potentially explain this phenotype. However, loss of
Notch signaling in the endothelium improves the trans-endothelial insulin transport, which
potentially augments the amount of biologically active insulin in the interstitium. Insulin is
actively shuttled from the blood through the endothelium to extravascular tissues by vesicles
called caveolae. The increased insulin transport upon Notch blockade is triggered by up
regulation of caveolin-1, which causes more caveolae to be formed. This implies Notch
signaling regulates the transport of insulin through the endothelium by modifying the amounts
of caveolae.
95
I also observed that there are increased amounts of triacylglycerols (TAG) in the blood of ko
mice and that the activity of lipases is decreased in the blood after heparin injection. TAG are
cleaved into glycerol and fatty acids at the apical side of the endothelium, the latter are
transported through the vessel wall to surrounding tissues, in which they are metabolized. In
vitro, Notch blockade causes down regulation of several genes involved in the cleavage of
TAG (LIPG), as well as of uptake (CD36) and shuttling (FABP4) of fatty acids. This suggests
the cleavage and transport of fats from the blood to the interstitium is reduced by endothelial
Notch blockade, which potentially is the reason for the elevated blood fat levels in ko mice.
These data show that signaling pathways within blood vessels have the potential to modulate
metabolic homeostasis. Mice without functional endothelial Notch signaling have increased
transport of insulin from the blood to the interstitium, which might cause elevated glucose
uptake by skeletal muscles and other organs. This could be the reason why these mice show
decreased blood glucose levels as well. The transport of fatty acids through the vessel wall
might be impaired in mice deficient for endothelial Notch signaling, which could explain
elevated triacylglycerol levels in their blood. My experiments suggest that the peripheral
tissues of mice lacking Notch signaling in the endothelium have limited access to fatty acids
but take up more glucose in turn. This suggests that the Notch pathway in the endothelium
has a role in switching the systemic catabolic metabolism from glucose to fat oxidation.
The graduate school offered various opportunities to improve my scientific and non-scientific
qualification. I took part in several workshops teaching specific techniques, which I could
successfully use for my projects afterwards. The graduate school also offered highly helpful
courses for soft skills. I also benefitted from the fruitful cooperation with other graduate
schools, which allowed me to participate in their workshops as well.
The graduate school organized several retreats and meetings with other graduate schools
dedicated to familiar topics. These events offered the possibility for exchange with other
students and allowed me to discuss my own work with PIs from other universities. The large
support of my graduate school allowed me to present my research at an international
meeting for blood vessels and metabolism as well.
My supervisors greatly supported my work. They discussed my research strategy and also
helped me to overcome methodological problems. Apart from my supervisors, the other PIs
from the graduate school were extremely helpful, too, and shared their expertise with me.
attended schools: 2
attended symposia:1
n.a.
Notch Signaling Regulates Systemic Energy Metabolism, Keystone Symposium on
Metabolism and Angiogenesis, Whistler, British Columbia, Canada; March 16. – 21. 2014
oral presentation and poster presentation
4. Own publications
n.a.
96
First and last name of doctoral researcher: Pamina Weber
(Working) title of doctoral project: Function of RhoGEF17 in the vascular endothelium
Names of supervisors: T. Wieland, S. Lutz, M. Schmidt
01 September 2011 – 31 December 2013
IRTG fellowship (01 September 2011 – 31 December 2013)
Start of doctorate on 09/2011, anticipated submission of doctoral thesis in Autumn 2014,
anticipated completion of doctorate in Spring 2015.
Start of Biology studies (Diploma) in 09/2004, Faculty of Biology, Eberhard-Karls-University
Tuebingen, Diploma received 11/2010
Residency at the Institute for Experimental and Clinical Pharmacology and Toxicology,
Medical Faculty Mannheim, Heidelberg University (01 January 2014 – 31 September 2014)
Monomeric GTPases of the Ras-superfamily are pivotal biological switches involved in a
huge variety of cellular functions. Based on sequence homology and physiological functions,
these monomeric GTPases are classified in five families: Ras, Rho, Rab, Ran, and Arf
(Colicelli, 2004). The best characterized members of the Rho-family are the GTPases RhoA,
Rac1, and Cdc42, which are key regulators of the actin cytoskeleton dynamics. They are
essential regulators of many physiological processes, like cell migration, proliferation and
cellular contraction. In particular actin dynamics are regulated. RhoA directs the formation of
actin stress fibres (Ridley & Hall, 1992). Rac1 promotes the assembly of lamellipodia and
membrane ruffles (Ridley et al., 1992). Cdc42 regulates the formation of filopodia (Nobes &
Hall, 1995). The spatiotemporal activation of RhoGTPases is therefore complexly regulated
by a subset of accessory proteins, which are under strict control of upstream signal
mediators. All RhoGTPases are molecular switches that cycle between an inactive, GDPbound and an active, GTP- bound state. GTPase activating proteins (GAPs) reduce the
signaling of RhoGTPases by accelerating their low intrinsic GTPase activity. Thus, the
majority of the proteins reside in the GDP- bound state within the cell (Moon & Zheng, 2003;
Bernards & Settleman, 2004; Scheffzek & Ahmadian, 2005). This inactive conformation is
stabilized by guanine dissociation inhibitors (GDIs), which bind to the GTPases and inhibit
the translocation to the membrane (Lang et al., 1996; Forget et al., 2002, Sauzeau et al.,
2000; Sawada et al., 2001). The activation of RhoGTPases and downstream effector
functions is mediated by guanine nucleotide exchange factors (GEFs) catalyzing the
exchange of GDP to GTP (Schmidt & Hall, 2002; Hakoshima et al., 2003). The largest family
of RhoGEFs is the Dbl family with their characteristic and catalytically active Dbl homology
(DH) domain and an adjacent pleckstrin homology (PH) domain. RhoGEF17 is a member of
this protein family, activating specifically RhoA, B and C (Rümenapp et al, 2002). We could
show that RhoGEF17 is under autoinhibitory constraint, which is controlled by cGMPdependent kinase Iα (Lutz et al., 2013). Furthermore it is a fact, that RhoGEF17 can directly
97
associate with actin stress fibres via an actin binding domain (ABD) within the N-terminus
(Mitin et al., 2012).
Since RhoGEF17 is a RhoA-C specific GEF, which was found to be upregulated in
endothelial cells during tumor cell- induced angiogenesis – a process which is characterized
by an increase in endothelial permeability followed by endothelial cell activation, proliferation,
and migration – we studied the role of RhoGEF17 in microvascular endothelial cells.
Therefore we performed knockdown experiments by an adenoviral delivery of shRNA in
microvascular rat fat pad endothelial cells (RFPECs). The recombinant adenovirus encodes
for a RhoGEF17-specific short hairpin RNA, by which we could effectively suppress the
expression of endogenous RhoGEF17. The loss of RhoGEF17 expression was associated
with changes in cell morphology, that include cell rounding, loss of cell-cell contacts, and the
dislocation of adherence junction of proteins from the cell membrane into sub-membranous
compartments. Furthermore the cells lost their capability to adhere efficiently to extracellular
matrix substitutes. Since these changes pointed to a reorganization of the cytoskeleton, we
analyzed actin and vinculin containing filaments. Indeed, the cells that lacked RhoGEF17
expression lost their ability to form stress fibers and showed a strongly reduced formation of
focal adhesions within the cells. This finding indicates that RhoGEF17 plays a role in the
organization of the actin cytoskeleton of endothelial cells. In accordance with these
differences, the migration behavior of the RhoGEF17 depleted endothelial cell was
dramatically altered. While the sheet migration and wound closure in a classical scratch
assay was strongly suppressed, the amount of endothelial cells displaying contactindependent single cell migration was increased. In agreement with these data we exhibited
an impaired proliferation whereas the apoptosis rate was significantly reduced.
On a molecular level, the knockdown of RhoGEF17 and the loss of cell-cell contacts are
paralleled by a reduced expression of the adherence junction proteins N-cadherin, p120catenin and β-catenin. Preliminary data indicate that this loss is due to proteasomal
degradation. Interestingly, the depletion of RhoGEF17 also reduced the protein expression
level of its effector proteins RhoA, B, and C and the Rho-inactivating protein p190RhoGAP,
which is known to directly interact with p120-catenin. Using a proximity ligation assay, the colocalization between the prominent adherence junction protein N-cadherin, p190RhoGAP,
p120-catenin, β-catenin and RhoGEF17 could be demonstrated. Importantly, the colocalization of the RhoGEF17 effector RhoA an N-cadherin was dramatically impaired in
RhoGEF17-depleted endothelial cells. Therefore our data indicate that RhoGEF17 is an
essential complex partner for the functional formation of adherence junctions in endothelial
cells linking the junction proteins to Rho-dependent alterations of the cytoskeleton, migration
and viability.
To get a first indication of the function of RhoGEF17 in vivo, we performed a morpholinomediated knockdown of its orthologue in zebrafish embryos in cooperation with Prof. Dr.
Jens Kroll. The RhoGEF17-depleted larvae displayed a phenotype similar to that reported for
an acute knockdown of VE-cadherin, thus, allowing for irregular vessel formation and
increased sprouting in the subintestinal veins. Further experiments are however necessary to
confirm this in vivo observation and to link them to an endothelial loss in RhoGEF17
expression.
In my opinion, the graduate school is a great platform to get into contact with other
laboratories. During the meetings organized by the GRK, I was introduced to the studies of
the other doctoral students, which was in several ways helpful and offered me a lot of new
ideas. Because of the very friendly and relaxed atmosphere during these meetings, it was
even for me, who attended the graduate school as one of the latest ones, no problem to
make contact both with the other students and the PIs. In this context, I also profited from the
multiple practical workshops provided by the GRK or the associated graduate schools. All
this took place in a very balanced way with regard to length and frequency. There was still
enough time for realization of the newly acquired ideas and own research.
During my still ongoing doctoral research study, I learned and still learn a lot from my
supervisors. I am very glad for the input and exchange I still receive not only from my direct
supervisors but also from all the collaborators.
98
attended schools: 6
Research internship at the Medical University Goettingen, Department of Pharmacology,
Goettingen, Germany (11-12/2013; 02-03/2014)
RhoGEF17, a novel contributor to cell adherence in vascular smooth muscle and endothelial
cells.
Deutsche Gesellschaft für Kardiologie, Jahrestagung 2014, Mannheim: April 3-6, Poster
presentation
RhoGEF17 participates in regulation of angiogenesis in endothelial cells.
Deutsche Gesellschaft für Experimentelle und klinische Pharmakologie und Toxikologie,
Jahrestagung 2014, Hannover: April 1-4, oral presentation
4. Own publications
1.
Lutz, S., Mohl, M., Rauch, J., Weber, P., Wieland, T. (2013) RhoGEF17, a Rhospecific guanine nucleotide exchange factor activated by phosphorylation via cyclic GMPdependent kinase Iα. Cell Signal. 25, 630-638.
99
First and last name of doctoral researcher: Matthias Richter
(Working) title of doctoral project: Culture-derived platelets: Impact of serotonin
metabolism on differentiation and function
Names of supervisors: P. Schloss, F. Lederbogen, M. Deuschle, M. Harmsen
01 November 2011 – 31 December 2013
IRTG fellowship (01 November 2011 – 31 December 2013)
Medical Faculty Mannheim, University Heidelberg (01 January 2014 – 31 March 2014)
Start of doctorate: 11/2011
Anticipated completion of doctorate: Winter 2014
Heidelberg
Spring 2015: Reception of approbation
n.a.
Platelets play a pivotal role in thrombus formation and thus in cardiovascular risk. Antiplatelet
drugs, such as acetylsalicylic acid or clopidogrel, are efficient in preventing coronary and
cerebrovascular events in patients at risk. An important player in platelet function is
serotonin, as this molecule is involved in the activation process and in platelet reactivity.
Platelet serotonin content is regulated by the serotonin transporter (SERT), which is present
in multiple cells like platelets, mononuclear blood cells and neurons, and provides uptake of
serotonin into the cells. SERT may be manipulated by selective serotonin transporter
inhibitors (SSRI), drugs that are commonly used in patients with mental depression. There
are several observations indicating that treatment of depressed patients with SSRIs like
citalopram may result in a beneficial effect on their cardiovascular risk. Hence, we wanted to
study the effect of the SSRI citalopram on SERT activity in platelets, taking into account a
functional polymorphism of the serotonin-transporter-linked polymorphic region (5-HTTLPR),
which is located in the promoter region of this gene. It can be distinguished between the LL-,
the SL- and the SS-type (L = long, S = short). The 5-HTTLPR was found to moderate the
influence of stressful life events on depression. Furthermore, platelet activation is increased
in elderly depressed patients, especially those with the LL-genotype. These genetic
differences may influence cardiovascular mortality in depressed patients.
The first aim of the study was to analyze the influence of candidate polymorphisms (5HTTLPR polymorphism) on serotonin metabolism and differential protein expression of
SERT in platelets by comparing two CD34+ hematopoietic progenitor cell lines with different
5-HTTLPR genotypes (MEG-01 and M-07). Previously, our group established a
differentiation method for the MEG-01 cell line in order to induce megakaryopoiesis as
estimated by polyploidy, formation of characteristic proplatelets and elevated expression of
the megakaryocytic markers CD41 and CD61. The resulting megakaryocytes stayed viable
for more than 3 weeks and shed platelet-like particles positive for CD41, CD61 and CD42b
100
into the supernatant. We tried to transfer the MEG-01 differentiation protocol to the M-07 cell
line, but while we could see clear histological signs of differentiation in the MEG-01 cells, the
M-07 cells formed colonies and seemed to proliferate at a higher rate. Other differentiation
protocols for CD34+ hematopoietic progenitor cell lines described in the literature were also
not successful in inducing differentiation of the M-07 cell line.
Since the regulation of SERT in different cell types is thought to follow similar biologic
mechanisms, we next studied the influence of 5-HTTLPR on serotonin metabolism in
monocytes from peripheral blood in humans. We obtained EDTA blood samples from healthy
volunteers and produced white blood cell pellets, from which DNA was isolated. Subjects
were typified by PCR analysis to determine the 5-HTTLPR genotype. In a second step, blood
from subjects with distinct 5-HTTLPR genotypes was collected. The mononuclear blood cells
were isolated by plastic adhesion and different washing steps. After being kept in culture for
two weeks, the cells were incubated with the SSRI citalopram for four hours and stained
immunocytochemically for SERT and integrin, a cell wall marker; protein biosynthesis was
blocked by cycloheximide. SERT internalization/expression on the cell surface of the different
cells was measured by immunofluorescence (on-cell western) to clarify the impact of the 5HTTLPR genotype. This work is still in progress, and first results suggest that monocytes
with 5-HTTLPR genotype LL express display an increased SERT density on the cell surface,
while monocytes with 5-HTTLPR genotype SL or SS internalize SERT into the cell after four
hours of incubation with citalopram.
Mental stress is associated with both platelet activation and an increased incidence of
coronary events. The second aim of our studies was therefore to test the hypothesis that
there is a change in platelet activity induced by exposure to a social evaluative stress
paradigm, which was recently established by our group. In healthy volunteers, blood was
drawn immediately before and 15 minutes after stress exposure. We produced platelet rich
plasma and measured the expression of CD41, CD62P, CD63 and PAC-1 on the cell surface
with and without activation by cADP and prostaglandin PGH2 analog U46619 by flow
cytometry. In a pilot study, we did not observe any significant change in platelet reactivity
before and after stress exposure. We attributed this negative finding to the time interval
between the end of stress exposure and the blood sampling, which could not be varied due
to technical reasons. Another explanation may be that the catecholamines responsible for
changes in platelet activity and reactivity were already metabolized at the point in time when
the second blood sample was collected. Stress testing was efficient, as salivary cortisol
concentrations nearly doubled in association with the task.
There surely is a certain lack of scientific education in medical studies nowadays, thus it was
important for me to learn more about proper research by participating in the International
Graduate School GRK 880 „Vascular Medicine“. Hearing about other projects and getting in
contact with other graduates and professors really helped me a lot, since we could share our
experiences and thereby gain more knowledge in a shorter amount of time. Therefore, my
research visit at the University Medical Center in Groningen, and the contacts I made there,
were a huge gain for my continuing scientific work.
The supervision in the GRK program was very helpful regarding my scientific progress,
especially when certain throwbacks in my studies occurred. Given that not everything worked
out as expected, there was always new input by my supervisors, which helped me stay
motivated and keep on researching.
101
attended schools: 2
attended symposia:1
Research visit at the University Medical Center Groningen, Faculty of Medical Sciences,
Pathology & Medical Biology, Groningen, The Netherlands (02/2012 – 03/2012)
German Society for Microcirculation and Vascular Biology (GfMVB) Annual Meeting 2012,
Mannheim: September 27th – 29th
4. Own publications
n.a.
102
First and last name of doctoral researcher: Prama Pallavi
(Working) title of doctoral project: Identification of molecular pathways by which Noctanoyl-dopamine inhibits vascular inflammation
Names of supervisors: B. Yard, M. Harmsen
01 October 2011 - 31 December 2014
IRTG fellowship (01 October 2011 - 31 March 2013),
10/2011 Start of doctorate, anticipated defense of PhD doctoral thesis 09/2015
June 2011, M.Sc. Biotechnology, Hochschule Mannheim
n.a.
We have previously demonstrated that N-octanoyl dopamine (NOD) is inhibiting vascular
inflammation and mitigates acute kidney injury (AKI). Moreover NOD is able to activate
TRPV1 and inhibits NFκB in a redox dependent manner. In the present study we assessed
1) if due to its redox activity NOD is able to induce the unfolded protein response (UPR) in
endothelial cells, 2) if NOD affects vascular barrier function, 3) if the renoprotective effect of
NOD in AKI is mediated by TRPV1 and 4) how NOD activates TRPV1.
The main findings of this project are the following. NOD transiently activates the UPR. This
property seems to be dependent on the redox activity of these compounds. NOD did not
affect cell viability, but strongly impaired cell proliferation of HUVEC, most likely by
attenuation of cells in the S-G2/M phase. In concordance to this, mRNA expression for a
number of genes involved in S-G2/M progression was significantly down-regulated by NOD.
Long-term NOD treatment resulted in hypometabolism and thermotolerance, as suggested
by a decreased intracellular ATP concentration, activation of AMPK and increased resistance
to cold inflicted cell injury. Independent of UPR activation NOD treatment of HUVEC
increased the transendothelial electrical resistance as indicated by increased impedance.
This was paralleled by the induction of F-actin stress fibers, redistribution of focal adhesion
contacts and activation of paxillin within the focal adhesions. In vivo studies using TRPV1-/rats it was demonstrated that the renoprotective effect of NOD in AKI was completely
depending on TRPV1 activation. NOD activates TRPV1 in a similar fashion as capsaicin as
demonstrated by docking modeling studies.
The following manuscripts are in preparation:
1.
Prama Palavi, Eleni Stamellou, Bastian Theisinger, Sonja Theisinger, Bernhard K
Kraemer, Marco Harmsen, Utta Binzen, Wolfgang Grefrath, Mathias Hafner, Benito Yard.
Analyses of different synthetic N-acyl dopamine derivatives reveal different structural
requirements for their anti-inflammatory effect and TRPV1 activation.
103
2.
Prama Pallavi, Eleni Stamellou, Christian, Bernhard K Kraemer, Marco Harmsen,
Ruediger Rudolf , Mathias Hafner, Benito Yard. Restoration of endothelial barrier function
after cold preservation does not require the formation of actin stress fibers
3.
Sarah Klotz*, Prama Pallavi*, Eleni Stamellou, Bernhard K Kraemer, Charalambos
Tsagogiorgas, Marco Harmsen, Mathias Hafner, Benito Yard. The reno-protective effect of
N-octanoyl dopamine (NOD) is mediated via TRPV1 activation
The GRK research training group, has provided a very nice platform for me to not only learn
in terms of techniques and ways one can investigate a question and but also to connect
different areas and to look at broader perspective. Interactions with different researchers
organized by GRK have inspired me to continue a career in research.
attended labcourses:0
attended schools: 3
attended symposia:1
n.a.
Presented a scientific talk titled NOD improves AKI, possible role of TRPV 1 on 7th May 2014
for Bioscience club of Heidelberg.
4. Own publications:
1.
Stamellou E., Fontana J., Wedel J., Ntasis E., Sticht C., Becker A., Pallavi P., Wolf
K., Krämer BK, Hafner M., van Son WJ, Yard BA. N-octanoyl dopamine treatment of
endothelial cells induces the unfolded protein response and results in hypometabolism and
tolerance to hypothermia. PLoS One 2014 Jun 13;9(6):e99298
104
First and last name of doctoral researcher: Sonja Sudarski
(Working) title of doctoral project: Coronary plaque composition in patients with NSTEMI
and stable coronary artery disease: A comparison of virtual histology intravascular ultrasound
analysis and Dual source CT
Names of supervisors: S. Schönberg, M. Borggrefe, H.-P. Hammes, C. Fink, T. Süselbeck,
M. Oudkerk
01 March 2010 - 31 December 2012
IRTG fellowship (01 March 2010 - 31 December 2012)
01/2010 Start of doctorate, anticipated submission of doctoral thesis on 24 June 2014,
anticipated completion of doctorate: Autumn/Winter 2014
01 December 2013 Residency at the Institute for Clinical Radiology and Nuclear Medicine,
University Medical Center Mannheim, Medical Faculty Mannheim- Heidelberg University,
Germany
The propensity of a coronary atherosclerotic plaque to rupture – its so-called vulnerability - is
known to be largely dependent on inflammatory processes at the site of the plaque leading to
an instable microanatomy. In case of rupture, sudden vessel obstruction leads to ischemia
and subsequent myocardial necrosis. The clinical manifestation is the acute coronary
syndrome (ACS), which is a life-threatining acute manifestation of coronary artery disease
(CAD). Strong efforts are made to reliably risk-stratify plaques and patients suffering from
CAD.
The reference standard for the assessment of coronary plaque composition is virtual
histology intravascular ultrasound (VH-IVUS), performed in the course of invasive coronary
angiography. However, due to its invasiveness, the search for accurate non-invasive plaque
imaging modalities is ongoing. Coronary CT angiography (cCTA) is increasingly used not
only to reliably rule out CAD but also to diagnose CAD and further characterize
atherosclerotic plaques. One objective of this study was therefore to quantitatively assess
coronary atherosclerotic plaque composition in patients presenting with ACS, in more detail,
in patients with acute non-ST elevation myocardial infarction (NSTEMI) and to compare
plaque composition with that of patients with stable CAD by means of cCTA and VH-IVUS.
Another approach to determine presence of vulnerable plaques in patients with CAD is the
assessment of chemical biomarkers circulating in the patient’s blood, associated with plaque
destabilization, inflammation and rupture. Consequently, we also assessed in our patient
cohort concentrations of matrixmetalloproteinase-9 (MMP-9), myeloperoxidase (MPO),
pregnancy associated plasma protein A (PAPP-A), placental growth factor (PlGF) and
soluable CD 40 ligand (sCD40L) by performing conventionally available enzyme-linked
105
immunoassays and investigated for possible differences between patients with NSTEMI and
patients with stable CAD.
60 patients (35 with NSTEMI) were enrolled in the course of the study. 40 corresponding
plaques (22 of NSTEMI patients) of 28 patients (15 with NSTEMI) were finally assessed by a
first-generation dual-source CT scanner and 20-MHz VH-IVUS performed in the course of
invasive coronary angiography (ICA) regarding volumes and percentages of fatty, fibrous and
calcified component, overall plaque burden and maximal percent area stenosis. cCTA plaque
analysis revealed no significant differences between plaques of patients with NSTEMI and
stable CAD regarding absolute and relative amounts of any plaque component, neither did
VH-IVUS plaque analysis. The only difference with regard to plaque morphology observed in
this study was higher area stenosis in NSTEMI patients compared to patients with stable
CAD as measured with VH-IVUS. Volumes of fatty component were measured systematically
lower in cCTA, whereas calcified and fibrous volumes were measured higher. No significant
bias was observed comparing volumes of overall non-calcified component and overall plaque
burden.
Regarding the analysis of biomarker levels, in total, 47 blood samples (28 of NSTEMI
patients) were collected. NSTEMI patients had higher MMP-9, MPO and sCD40L levels and
lower PAPP-A and PlGF levels than the group of patients with stable CAD, yet only
concentrations of sCD40L were significantly differing between NSTEMI patients and patients
with stable CAD. Levels of sCD40l were negatively and significantly correlated with PAPP-A
and PlGF levels. PAPP-A and PlGF concentrations were positively correlated with each other
and these two biomarkers were also both significantly elevated after ICA procedure
independent of patient group and the performance of percutaneous interventions in the
course of ICA. Concentrations of the study biomarkers did not correlate with concentrations
of hsTnI, CRP, HDL, LDL, total cholesterol and triglycerides. The only correlation observed
between study biomarker concentrations and plaque components was a weak positive
correlation between percentage of fatty component and sCD40L levels.
In summary, these data suggest that plaques of patients with NSTEMI and of patients with
stable CAD cannot be differentiated by mere quantification of plaque components, neither by
dual-source cCTA nor with VH-IVUS. The most promising biomarker candidate observed in
this study was sCD40L, which showed significantly higher concentrations in NSTEMI patients
than in patients with stable CAD, suggesting usefulness to determine patients at risk and
blood levels were positively correlated with fatty plaque component, which is a surrogate for
plaque vulnerability.
Longitudinal studies are warranted to evaluate to which extent coronary plaque composition
imaging can add to proper risk stratification of plaques and patients and prompt treatment
strategies, and to determine specific biomarkers of coronary plaque vulnerability. Assumed
that a reliable diagnostic approach applicable in clinical routine will be established and
vulnerable plaques could be accurately distinguished from stable plaques, proof of the
usefulness of “preventive” conservative or even interventional treatment strategies of
vulnerable plaques with regard to short-term and long-term survival and morbidity of patients
with CAD is still to be made.
Ongoing collaboration projects in the field of cardiothoracic research between the Institute of
Clinical Radiology and Nuclear Medicine, University Medical Center Mannheim and the
Center for Medical Imaging – North East Netherlands, University Medical Center Groningen
including phantom studies on coronary calcium risk scores, lung nodule detection as well as
clinical studies on myocardial perfusion imaging.
I largely profited from the early time point during my studies being introduced into research,
getting the opportunity to present my data on national and international meetings, by means
of posters and oral presentations. It was largely valuable for me to get routine in statistics,
laboratory work, and constant literature study.
106
The meetings organized by the graduate school itself were in my opinion very balanced with
regard to length and frequency. I also profited from the multiple practical workshops being
provided by the GRK and partner graduate schools.
I learned a lot from my supervisors and I had received continuous supervision, input and
exchange during the time span of the graduate school as well as after my membership in the
graduate school had officially ended.
As a medical student, I would have probably profited from taking a semester/term off to
complete my thesis in full-time. I decided not to do this, but to continue and finish my studies
and start to work immediately after the final exams, which lead to a certain delay regarding
the completion of my dissertation.
attended schools: 5
Research Elective at the Heart and Vascular Center, Department of Radiology and
Radiological Science, Medical University of South Carolina, Charleston, SC, USA (08/2011
and 09/2011)
Comparison of coronary plaque morphology in patients with stable coronary artery disease
and non-ST myocardial infarction using Dual Source CT.
European Congress of Radiology 2011, Vienna: March 3 – 7, Digital poster
Determination of Plaque Composition with Dual Source Computed Tomography and Virtual
Histology Intravascular Ultrasound in Patients with Acute Coronary Syndrome and Stable
Coronary Artery Disease.
Deutsche Gesellschaft für Kardiologie, 76. Jahrestagung, 2011, Mannheim: April 27 - 30, Oral
presentation
Evaluation of Coronary Plaque Composition with Dual Source CT and Virtual Histology
Intravascular Ultrasound in Patients with ACS and Stable Coronary Artery Disease
European Society of Cardiac Radiology, Annual Scientific Meeting, 2011, Amsterdam:
October 27 – 29, Oral presentation
Workflow improvements in cardiac CT post-processing with iterative reconstruction compared
to filtered back projection
European Congress of Radiology 2012, Vienna: March 1 – 5, Digital poster
Zusammensetzung von Koronarplaques mit Dual Source CT-Angiographie und Virtual
Histology Intravascular Ultrasound in NSTEMI-Patienten und Patienten mit stabiler KHK
93. Deutscher Röntgenkongress 2012, Hamburg: May 16 – 19, oral presentation
Coronary Dual Source CT-Angiography and Intravascular Ultrasound Assessed Plaque
Composition in Patients with Stable Coronary Artery Disease and Patients with Non-ST
Elevation Myocardial Infarction
RSNA 98th Scientific Assembly and Annual Meeting 2012, Chicago: November 25-30, Oral
presentation
107
4. Own publications
1.
Sudarski S, Apfaltrer P, Nance JW, Jr., Meyer M, Fink C, Hohenberger P, Leidecker
C, Schoenberg SO, Henzler T. Objective and subjective image quality of liver parenchyma
and hepatic metastases with virtual monoenergetic dual-source dual-energy CT
reconstructions: an analysis in patients with gastrointestinal stromal tumor. Academic
radiology. 2014;21(4):514-22. Epub 2014/03/07.
2.
Schneider D, Apfaltrer P, Sudarski S, Nance JW, Jr., Haubenreisser H, Fink C,
Schoenberg SO, Henzler T. Optimization of kiloelectron volt settings in cerebral and cervical
dual-energy CT angiography determined with virtual monoenergetic imaging. Academic
radiology. 2014;21(4):431-6. Epub 2014/03/07.
3.
Apfaltrer P, Sudarski S, Schneider D, Nance JW, Jr., Haubenreisser H, Fink C,
Schoenberg SO, Henzler T. Value of monoenergetic low-kV dual energy CT datasets for
improved image quality of CT pulmonary angiography. European journal of radiology.
2014;83(2):322-8. Epub 2013/12/24.
4.
Sudarski S, Henzler T, Schoenberg SO. Post-therapeutic positron emission
tomography/computed tomography for early detection of non-small cell lung cancer
recurrence. Translational lung cancer research. 2013;2(4).
5.
Sudarski S, Fink C, Sueselbeck T, Kayed H, Schoenberg SO, Borggrefe M,
Vliegenthart R, Oudkerk M, Henzler T. Quantitative Analysis of Coronary Plaque
Composition by Dual-Source CT in Patients with Acute Non-ST-Elevation Myocardial
Infarction Compared to Patients with Stable Coronary Artery Disease Correlated with Virtual
Histology Intravascular Ultrasound. Academic radiology. 2013;20(8):995-1003. Epub
2013/07/09.
6.
Sudarski S, Apfaltrer P, W. Nance J J, Schneider D, Meyer M, Schoenberg SO, Fink
C, Henzler T. Optimization of keV-settings in abdominal and lower extremity dual-source
dual-energy CT angiography determined with virtual monoenergetic imaging. European
journal of radiology. 2013. Epub 2013/06/15.
7.
Shi J, Schmid-Bindert G, Fink C, Sudarski S, Apfaltrer P, Pilz LR, Liu B, Haberland
U, Klotz E, Zhou C, Schoenberg SO, Henzler T. Dynamic volume perfusion CT in patients
with lung cancer: Baseline perfusion characteristics of different histological subtypes.
European journal of radiology. 2013. Epub 2013/10/08.
8.
Gruettner J, Walter T, Bolte M, Haghi D, Sudarski S, Henzler T. Incidence of
pulmonary embolism in an emergency department cohort evaluated with a simple symptombased diagnostic algorithm. In vivo (Athens, Greece). 2013;27(2):215-20. Epub 2013/02/21.
9.
Ebersberger U, Sudarski S, Schoepf UJ, Bamberg F, Tricarico F, Apfaltrer P, Blanke
P, Schindler A, Makowski MR, Headden GF, Leber AW, Hoffmann E, Vliegenthart R.
Atherosclerotic plaque burden in cocaine users with acute chest pain: analysis by coronary
computed tomography angiography. Atherosclerosis. 2013;229(2):443-8. Epub 2013/07/25.
108
First and last name of doctoral researcher: Hui Liu
(Working) title of doctoral project: The impact of ephrinB2 on the proinflammatory
activation of leukocytes
Names of supervisors: M. Hecker, T. Korff, H. Augustin, J. Kroll, R. Henning
01 October 2010 - 30 September 2013
IRTG fellowship (01 October 2010 - 30 September 2013)
01 October 2010- 18 November 2013
Study of medicin (Bachelor), University of Shanxi, China; postgraduate student for additional
3 years (Master of science, 2009), University of Shanxi, China
Postdoc, Cardiovascular Research, Rochester, USA
The binding of membrane-associated ephrinB ligands to corresponding EphB receptors in
endothelial cells (ECs) is pivotal for orchestrating the development of the circulatory system.
Although the EphB/ephrinB system has been extensively studied during early embryonic
development, its function in the adult vasculature is largely unknown. EphrinB2 is apically
expressed on quiescent endothelial cells, and the level of its expression depends on local
microenvironmental cues such as hypoxia, contact with SMCs and pro-angiogenic
stimulation by VEGF. On the other hand, EphB2 is expressed on the surface of monocytes.
The pattern of EphB2/ephrinB2 expression led to the hypothesis that their interaction might
be involved in circulating monocyte adhesion to and transmigration through the endothelial
cell monolayer, which are pivotal processes during inflammation.
Consequently, the project was focused on clarifying the role of endothelial cell ephrinB2 in
monocyte diapedesis and the underlying mechanisms. Considering the unique feature of
bidirectional signaling in the Eph/ephrin system, as well as the evidence that EphB2 forward
signaling activates monocytes, within another part of this project the impact of ephrinB
reverse signaling on the pro-inflammatory differentiation of endothelial cells and monocyte
adhesion was investigated. To further explore the role of endothelial ephrinB2 in
inflammation in vivo, tamoxifen inducible endothelial cell specific ephrinB2 knockout mice
were generated. Finally, this study was aimed at delineating the individual impact of either
ephrinB1 or ephrinB2 on the observed pro-inflammatory differentiation of endothelial cells
and monocyte diapedesis.
Against this background, we observed that the ligands ephrinB1 and ephrinB2 are localized
on the surface of ECs where their expression is upregulated during inflammation, while
EphB2 receptors are located on the surface of monocytes. Consequently, we hypothesized
that these ligands bind to EphB receptors expressed in monocytes under pro-inflammatory
conditions. To study functional consequences of their ligand-receptor interactions, ECs were
exposed to EphB2 receptor bodies or EphB2-overexpressing mouse myeloma cells upon
siRNA-mediated knockdown of ephrinB1 or ephrinB2.
109
Based on this experimental setup, we analyzed the pro-inflammatory differentiation of the
ECs and the transmigration of monocytes through the EC monolayer. Whereas forward
signaling downstream of the EphB receptors promotes activation of monocytes, ephrinB
reverse signaling induces the deposition of von Willebrand factor on the ECs surface,
increases the expression of E-selectin, VCAM-1 and granulocyte-macrophage colonystimulating factor in ECs, and consequently stimulates monocyte attachment. Moreover,
ephrinB1 and ephrinB2 are crucial for the transmigration of monocytes through the
endothelial cell monolayer. Their activation by EphB2 not only promotes PECAM-1
phosphorylation through a Src-dependent mechanism while diminishing SHP-2 activity, but
also triggers displacement of VE-cadherin from interendothelial cell junctions. In addition,
EphB2 stimulation increases the permeability of endothelial cells. In vivo, endothelial cell
specific ephrinB2 ablation was induced by injecting tamoxifen in Tie2CreERT2/ephrinB2flox/flox mice. Three distinct types of inflammatory models-TPA-induced
ear edema, thioglycollate-elicited peritonitis, atherosclerosis, as well as an arteriogenesis
model were employed to analyze the impact of ephrinB2 on monocyte extravasation and
progression of pathological processes. However, all four models indicated that loss of
endothelial ephrinB2 has no impact on monocyte infiltration, inflammatory pathology, and
progression of arteriogenesis. This might be due to the limitations of the utilized models and
compensatory effects of ephrinB1. Collectively, our data indicates for the first time
that the EphB/ephrinB system contributes to inflammatory responses of ECs whereby
ephrinB reverse signaling supports adhesion and transmigration of EphB2-expressing
monocytes, and increases endothelial cell permeability.
Practical courses were suitable to expand the technical knowledge of the PhD stundents and
improves their interactions. Hui Liu attended several lectures focusing on vascular biology.
Moreover, she also reported in some meetings which offered her the opportunity to present
data and communicate with other groups. It is a good programme for the PhD students to
gain the scientific knowledge and develop the ability to be a researcher. Prof. Korff gave
some lectures and practical courses.
Prof. Korff offered a lot of advice, came up with new ideas to improve the project, helped with
trouble shootings and encouraged the PhD student all the time. In addition to the excellent
guidance of the project, he also taught a lot about how to be a good researcher, how to
collaborate with others and how to develop the career.The GRK880 supported my work by
practical courses, seminars and lectures as well as a lively network that helped much to
connect with national and international other research teams.
attended schools: 5
Groningen, May/June 2012, laboratory of Dr. Henning/Deelman
DPG-Tagung 2011, IVBM 2012, Experimental Biology meeting (USA) 2012, GFMVB-Meeting
2012, DPG-Tagung 2013
4. Own publications
1.
Liu H, Devraj K, Möller K, Liebner S, Hecker M, Korff T. EphrinB-mediated reverse
signalling controls junctional integrity and pro-inflammatory differentiation of endothelial cells.
Thromb Haemost. 2014 Feb 13;112(1).
110
2b) Postdoctoral Researcher (GRK 880/3)
First and last name of postdoctoral researcher: Hany Kayed
(Working) title of doctoral project: Coronary plaque composition in patients with NSTEMI
and stable coronary artery disease: A comparison of virtual histology intravascular ultrasound
analysis and Dual source CT
1 September 2008 - 31 August 2012
Postdoctoral at the Institute for Clinical Radiology and Nuclear Medicine, Medical Faculty
Mannheim, University of Heidelberg, funded by the IRTG (1 September 2008 - 31 August 2012);
Postdoctoral position in the IRTG (1 September 2008 - 31 August 2012)
Subject; institution; start date of doctoral programme; degree date:
“New Molecular Factors in The Pathogenesis of Pancreatic Diseases”, University Clinic of
Surgery, University of Heidelberg, Heidelberg, Germany, Feb. 2003 - 03.02.2005.
Autumn/winter 1994, Human medical studies and 2000 Master of Radiology, Medical Faculty,
University of Cairo, Egypt. 03.02.2005 Doctorate, University of Heidelberg. 10.09.2010
Reception of approbation
Occupation after leaving the RTG, if applicable:
01/09/2012 Senior Radiologist (Oberarzt), Department of Radiology, General hospital of Celle,
Celle, Germany.
2. Description of individual project and research results achieved to date
The vulnerability of a coronary atherosclerotic plaque to rupture is largely dependent on
inflammatory processes at the site of the plaque leading to an instable microanatomy.
Consequently, sudden vessel obstruction leads to acute ischemia and myocardial infarction.
This is known clinically as acute coronary syndrome (ACS), which is a life-threatining acute
manifestation of coronary artery disease (CAD). Strong efforts are made to reliably riskstratify plaques and patients suffering from CAD.
The gold standard for the assessment of coronary plaque composition is virtual histology
intravascular ultrasound (VH-IVUS), which is performed during the invasive coronary
angiography procedure. The non-invasive coronary CT angiography (cCTA) is increasingly
used to reliably rule out or diagnose CAD. More importantly, it can be also used to
characterize atherosclerotic plaques as an alternative to the invasive VH-IVUS.
Our objective in this study was to assess the composition of the coronary atherosclerotic
plaque in patients presenting with ACS in a quantitative manner. The plaque composition
was assessed in patients with acute non-ST elevation myocardial infarction (NSTEMI) and
compared to plaque composition with that of patients with stable CAD by means of cCTA and
VH-IVUS. Another approach to determine presence of vulnerable plaques in patients with
CAD is the assessment of biomarkers circulating in the patient’s coronaries, which might be
associated with plaque destabilization, inflammation and rupture. We assessed in our patient
cohort concentrations of matrixmetalloproteinase-9 (MMP-9), myeloperoxidase (MPO),
111
pregnancy associated plasma protein A (PAPP-A), placental growth factor (PlGF) and
soluable CD 40 ligand (sCD40L) by performing conventionally available enzyme-linked
immunoassays and investigated for possible differences between patients with NSTEMI and
patients with stable CAD.
60 patients (35 with NSTEMI) were enrolled in the study. 40 corresponding plaques (22 of
NSTEMI patients) of 28 patients (15 with NSTEMI) were assessed by a first-generation dualsource CT scanner as well as by the 20-MHz VH-IVUS; which was performed in the course
of invasive coronary angiography (ICA). The assessed parameters were the volumes and
percentages of fatty, fibrous and calcified component, overall plaque burden and maximal
percent area stenosis. The only observed difference related to plaque morphology was
higher area stenosis in NSTEMI patients compared to patients with stable CAD as measured
with VH-IVUS. In cCTA volumes of fatty component were measured systematically lower,
whereas calcified and fibrous volumes were measured higher.
Regarding the analysis of biomarker levels, in total, 47 blood samples (28 of NSTEMI
patients) were collected. NSTEMI patients had higher MMP-9, MPO and sCD40L levels and
lower PAPP-A and PlGF levels than the group of patients with stable CAD, yet only
concentrations of sCD40L were significantly differing between NSTEMI patients and patients
with stable CAD. Levels of sCD40l were negatively and significantly correlated with PAPP-A
and PlGF levels. PAPP-A and PlGF concentrations were positively correlated with each other
and these two biomarkers were also both significantly elevated after ICA procedure
independent of patient group and the performance of percutaneous interventions in the
course of ICA. Concentrations of the study biomarkers did not correlate with concentrations
of hsTnI, CRP, HDL, LDL, total cholesterol and triglycerides. The only correlation observed
between study biomarker concentrations and plaque components was a weak positive
correlation between percentage of fatty component and sCD40L levels.
In summary, these data suggest that plaques of patients with NSTEMI and of patients with
stable CAD cannot be differentiated by mere quantification of plaque components. The most
promising biomarker candidate observed in this study was sCD40L, which showed
significantly higher concentrations in NSTEMI patients than in patients with stable CAD. In
addition blood levels were positively correlated with fatty plaque component, which is a
surrogate for plaque vulnerability.
3. Description of individual RTG-specific tasks
As a graduated physician and radiologist my main role was to act as a study physician in the
clinical study. In this role I furthermore supervised the doctorate during all scientific,
experimental, technical and administrative steps until she completed her thesis. Furthermore,
with my scientific expertise in basic research I acted as a link for the IRTG’s basic research
and clinical studies.
Participation in or organisation of RTG-related events:
I participated regularly in the Autumn / Spring school in
Heidelberg/Mannheim. I took part in some of the seminars as well.
both
Groningen
and
Research stays at other research institutions at home and abroad, for IRTGs also
research stays at the partner university: No
Participation in conferences, congresses, etc., at home and abroad: I participated in
congresses and conferences in the field of clinical Radiology such as ECR and ACSI as well as
regular seminars of the Institute of Clinical Radiology and Nuclear Medicine at the University
Medicine Mannheim.
112
4.
Own Publications
1.
Sudarski S, Fink C, Sueselbeck T, Kayed H, Schoenberg SO, Borggrefe M,
Vliegenthart R, Oudkerk M, Henzler T. Quantitative analysis of coronary plaque composition
by dual-source CT in patients with acute non-ST-elevation myocardial infarction compared
to patients with stable coronary artery disease correlated with virtual histology intravascular
ultrasound. Acad Radiol. 2013 Aug;20(8):995-1003.
2.
Kayed H, Meyer P, He Y, Kraenzlin B, Fink C, Gretz N, Schoenberg SO, Sadick M.
Evaluation of the Metabolic Response to Cyclopamine Therapy in Pancreatic Cancer
Xenografts Using a Clinical PET-CT System. Transl Oncol. 2012 Oct;5(5):335-43. Epub 2012
Oct 1.
3.
Gazquez C, Ribal MJ, Marín-Aguilera M, Kayed H, Fernández PL, Mengual L,
Alcaraz A. Biomarkers vs conventional histological analysis to detect lymph node
micrometastases in bladder cancer: a real improvement? BJU Int. 2012 Nov;110(9):1310-6.
4.
Sadick M, Attenberger U, Kraenzlin B, Kayed H, Schoenberg SO, Gretz N, SchockKusch D. Two non-invasive GFR-estimation methods in rat models of polycystic kidney
disease: 3.0 Tesla dynamic contrast-enhanced MRI and optical imaging. Nephrol Dial
Transplant. 2011 Oct;26(10):3101-8.
5.
Henzler T, Porubsky S, Kayed H, Harder N, Krissak UR, Meyer M, Sueselbeck T,
Marx A, Michaely H, Schoepf UJ, Schoenberg SO, Fink C. Attenuation-based
characterization of coronary atherosclerotic plaque: comparison of dual source and dual
energy CT with single-source CT and histopathology. Eur J Radiol. 2011 Oct;80(1):54-9.
113
Appendix 3: see publication list
114

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