Revista jul_sep 2006_PDF - Cardiovascular Sciences Forum

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CARDIOVASCULAR
SCIENCES FORUM
Cardiovasc Sci Forum Jul./Sep. 2006 - Vol. 1 / Number 3
EDITORIAL COORDINATION
Otoni M. Gomes (Brazil), Alfredo I. Fiorelli (Brazil),
José Carlos Dorsa V. Pontes (Brazil), Pascal Dohmen (Germany)
Tomas A. Salerno (USA)
ASSOCIATED EDITORS
Alexandre C. Hueb (Brazil), Antônio S. Martins (Brazil)
Bruno Botelho Pinheiro (Brazil), Domingo M. Braile (Brazil),
Domingos Sávio Souza (Sweden), Elias Kallás (Brazil),
Michael Dashwood (England), Ricardo Gelpi (Argentina)
Sponsored by: Fundação Cardiovascular São Francisco de Assis – ServCor (MG - Brazil)
Fundação Cardiovascular S. Francisco de Assis / ServCor - Thruth is Jesus . St John 14.6
President: Elaine Maria Gomes (OAB)
Scientific Coordination: Otoni M. Gomes
Clinic Director: Eros Silva Gomes
Events Administration: Elton S. Gomes
Scientific Council :
Prof. Dr. Alan Tonassi Paschoal
Prof. Dr. Alcino Lázaro da Silva
Prof. Dr. Alexandre Ciappina Hueb
Prof. Dr. Alfredo I. Fiorelli
Prof. Dr. Arnaldo A. Elian
Prof. Dr. Carlos Henrique V. Andrade
Prof. Dr. Cristina Kallás Hueb
Prof. Dr. Elias Kallás,
Prof. Dr. Eduardo S. Bastos
Prof. Dr. Evandro César V. Osterne
Prof. Dr. Fábio B. Jatene
Prof. Ivan Berkowitz – MBA. Harvard (Canadá)
Prof. Dr. José Carlos D. V. Pontes
Prof. Dr. José Teles de Mendonça
Prof. Dr. Noedir A.G. Stolf
Prof. Dr. Sérgio Nunes Pereira
Prof. Dr. Tofy Mussivand (Canadá)
Prof. Dr. Tomas A. Salerno (USA)
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Scientific Co-sponsorship by: South American Section of the International Academy of Cardiovascular Sciences (IACS-SAS),
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(DECAM-SBCCV), SBCCV Departament of Cardiology (SBCCV-DECARDIO, SBCEC-Brazilian Society of Extracorporeal
Circulation.
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Cardiovasc. Sci. Forum - Jul./ Sep. 2006 - Vol. 1/ Number 3
CARDIOVASCULAR
SCIENCES FORUM
Cardiovasc Sci Forum Jul./Sep. 2006 - V
ol. 1 / Number 3
Vol.
SCIENTIFIC BOARD - BRAZIL
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Cardiovascular Sciences Forum
Cardiovasc Sci Forum Jul./Sep. 2006 - Vol. 1 / Number 3
Contents
:: EDITORIAL
Page 06
- Evolution and Challenges in the
Phisiopatology of the Ischemia and Reperfusion (English Text)
Carlos Henrique Marques dos Santos
:: ORIGINAL ARTICLES
Page 09
- Adult Human Vascular Endothelial Cells Seeded onto No-React®
Treated Bovine Internal Mammary Arteries: An in Vitro Study (English Text)
P. M. Dohmen, M. Stein-Konertz, S. Posner, W. Erdbrügger, W. Konertz
Page 17
- Pravastatin and Sistemic Inflamatory Response
Syndrome by Extracorporeal Circulation (Portuguese Text)
G. F. Teixeira Filho, J. R. M. Sant´Anna, P. R. Prates, R. A.K. Kalil, A.H. Neto, M. Santos,I. Nesralla
:: ORIGINAL CARDIOVASCULAR IMAGING
Page 28
- Cardiovascular Imaging: Nine Year Patency of a Small Caliber
Vascular Prosthesis Seeded With Autologous Endothelial Cells (English Text)
Dohmen P. M.,Lembcke A., Gabbieri D., Konertz W.
:: UPDATING ARTICLES
Page 30
- Vasculogenesis Applied Physiology (Spanish Text)
Alberto J. Crottogini, Gustavo L. Vera Janavel
Page 38
- Physiology Basis of the Heart Rate Variability (Spanish Text)
Eduardo R. Migliaro y Paola Contreras
Page 47 :: INSTRUCTIONS FOR AUTHORS
Page 49 :: UPCOMING MEETINGS SESSION
Page 50 :: PEER REVIEW
5
EDITORIAL
Evolution and Challenges in the
Phisiopatology of the
Ischemia and Reperfusion
Carlos Henrique Marques dos Santos*
In 1968, McCORD (1) proposed that
enzyme xanthine-oxidase, precursory of
superoxide radical and present in many tissues,
was able to be related to damage tissues
submitted to the ischemia.
In 1986, PARKS & GRANGER(2) proposed
that free radicals produced in reperfusion phase
would be the principal patogenes inductors of
tissue injury in ischemia and reperfusion, being
this the main historical mark in the evolution of
tissue ischemia and reperfusion.
The ischemia is a condition of decrease
or interruption in the blood supply of oxygen
and nutrients to certain area, during a period.
With the deficiency of blood supply, it can
happen the tissue death (3) . However, the
reperfusion has a fundamental participation in
development of tissue injury.
PARKS & GRANGER(2) demonstrated
that three hours of ischemia following for one
hour of reperfusion determined larger lesion
that four hours of ischemia exclusive.
The reperfusion can hurt the organ separately
like in reperfusion of myocardium after a acute
myocardial infarct(4). However, can to hurt
distant organs too, like in the lung edema after
ischemia and reperfusion of extremity (3) .
The ischemia determine a role of
alterations in the celular level that can culminate
in celular death. The absence of oxygen impedes
the oxidative fosforilation in mitochondrion, that
is the more efficient way of energy production.
Thus, the anaerobic glicolise be the main source
of energy, and, being less efficient, is not
appropriate ATP consumed reposition. The
decrease of ATP harm the transport assets of
íons through the membrane, taking the
accumulation of sodium, and, by diffusion,
water inside the cell, with consequent edema(5).
The ischemia still determines an increase
of calcium permeability, promoting his entrance
in the cell. The increase of the intracellular
calcium, potentiated by the decrease of his
transport assets for the extracellular space, ATP
dependent, presents several harmful effects:
alteration in the form of the cell by contraction
of the skeleton; fosfolipases ativation, with
consequent metabolites liberation of aracdonic
acid starting from the cellular membrane and
of the organels and free radicals production(6).
The endothelial cells and the leucocites,
fundamental elements in the reperfusion lesion,
are affected already in the ischemia, alterations
that will intensify in the reperfusion suffering.
When exposed to the ischemia, the cells alter
his citoeskeleton and his forms, generating
small intercellular pores, determining an
increase of the endothelial permeability, could
take to the formation of tecidual edema(7) .
The endothelial cell, when exposed to ischemia,
increase his interleucine-1 and tumoral factor of
necrosis production, increasing his adhesiveness for
leukocytes, although that phenomenon is more evident
after the reoxygenation (8) .
* Mato Grosso do Sul Federal University - Surgical Department (Prof. Dr. José Carlos D. V. Pontes) and São Francisco de Assis Cardiovascular
Foundation. Address: Rua Aluízio de Azevedo, 606 - Jardim São Bento - Campo Grande – Mato Grosso do Sul – Brasil - CEP: 79004050,
E-mail: [email protected]
6
The degradation of ATP stocks for the
energy production during the ischemia takes
to an elevation of the AMP concentration. This
is catabolized in adenosine, inosine, and so,
hypoxanthine. The hypoxanthine serves as a
purine oxidizable substratum to xanthinedehydrogenase or oxidase, and, parallel, there
is the conversion of xanthine-dehydrogenase
in xanthine-oxidase. This conversion is made
in an irreversible way, through proteases
activated by the calcium, or in an reversible
way, for the oxidation of sulfidrile groups. So,
during the ischemia we have the formation
mainly of hypoxanthine and xanthine-oxidase,
that they have fundamental paper during the
reperfusion unchaining the process of free
radicals production(5, 9, 10).
The free radicals of oxygen are chemical
species characterized by the presence of one
no duplicated electron in the last orbit, acted
graphically by a point. This characteristic cheks
it those substances a great capacity of to react
with others, turning them importants oxidant
or reducers agents(11).
The free radicals of oxygen implicated in
the reperfusion lesion are: superoxide anion, the
hidroxile radical na the hydrogen peroxide. The
peroxide hydrogen not constituted in a radical,
because it doesn’t present free electron in his
orbit. This way, the most correct way to refer the
those substances is denominating them species
reactivate poisonous of the oxygen (SRPO)(12).
The xanthine-oxidase depends on the
oxygen for the metabolization of hypoxanthine
in xanthine and superoxide radical. The enzyme
superoxide-dismutase catalyzes the convertion
of the superoxide in peroxide of hydrogen, while
the enzime catalase converts the peroxide of
hydrogen in water and oxygen. In the presence
of iron ions can have the conversion of
peroxide of hydrogen in hidroxile radical(11, 12) .
The SRPO can harm any biochemical
component of the cell, but the fats, proteins (so
much structural as enzymatic) and nucleics
acids are their main objective. As they present
great reactivity, the SRPO interact with the first
structures that find, in general the fosfolipides
of cellular membrane or of the organelles
membranes. The reaction of SRPO with the
polinsatured fat acids from cellular membrane
takes to the formation of several lipidic radicals
(peroxide lipidics, hidroperoxilipidics,
malondiadehyde), in a chain of reactions that
culminate with the dysfunction of membrane
and cellular damage. That lipidic peroxidation
also promotes fosfolipase A2 ativation that,
acting on the fosfolipides of the cellular
membrane, liberates fat acids that are
metabolized by ciclooxigenase, generating
prostaglandins and tromboxane, or by
lipoxigenase, generating the leukotrienes(12) .
Besides the direct lesions on the cells, the
SRPO participate, with other mediators, such
as leukotrienes, tromboxane A2 and factor of
activation of interactions among leukocites and
endothelium, that provoke increase of capilar
permeability and tecidual damage(13) .
The participation of the leukocites in the
reperfusion damage happens for the liberation
of substances trhough own degradation. Enter
these substances, some are free radicals. The
polimorfonuclears posses nicotinamida adenine
fosfate oxidase capable to reduce the molecule
of oxygen, generating the superoxide anion. The
leukocites produces still proteolitics enzimes,
including elastase, colagenase and gelatinase,
that participate in the tecidual lesion (14).
The compression of the capillary bed by
tecidual and endothelium cells and also for the
interstice, all edemaciate during the ischemia,
can take to the bankruptcy of reperfusion of
certain segments of microcirculation, with focal
tecidual hypoxia, being this another mechanism
of tecidual lesion after the reperfusion(15) .
With the knowledge of this sequence of
events, it is believed that can have forms of
acting in some level of chain reaction of
process of ischemia and reperfusion, in way
to inhibit the tecidual injury. This is the
motivation of countless works that for objective
to find one or more substances capable to block
the formation or action of the free radicals.
7
BIBLIOGRAPHICAL REFERENCES
1. McCORD JM. Oxygen derived free radicals in
post-ischemic tissue injury. E Engl J Med 1985;
312: 159-63.
2. PARKS DA , GRANGER DN. Contributions of
ischemia and reperfusion to mucosal lesion
formation. Am J Physiol 1986; 13: 749-53.
3. PINHEIRO BV, HOLANDA MA, ARAÚJO FG,
ROMALDINI H. Lesão pulmonar de reperfusão.
J Pneumol 1999; 25(2):124-36.
4. BECKER LC, AMBROSIO G. Myocardial
consequences of reperfusion. Prog Cardiovasc
Dis 1987; 30: 23-44.
5. PARKS DA, GRANGER DN. Xanthine
oxidase:biochemistry, distribuition and
physiology. Acta Physiol Scand 1986; 548:
87-99.
6. OKUDA M, LEE HC, CHANCE B, KUMAR C.
Role of extracellular Ca 2+ in ischemiareperfusion injury in the isolated perfused rat
liver. Circ Shock 1992; 37: 209-19.
BURNS DK, GOLDSTEIN A, STERN D.
Hypoxia-mediated induction of endothelial cell
interleukin-1. An autocrine mechanism
promoting expression of leukocyte adhesion
molecules on the vessel surface. J Clin Invest
1992; 90: 2333-9.
9. SCHANAIDER A, PERROTTA U, MADI K.
Importância dos radicais livres derivados do
oxigênio na fisiopatologia das afecções
isquêmicas intestinais. Folha Med 1991;
103(2): 53-8.
10. HANGLUND U, BULKLEY GB, GRANGER DN.
On the pathophysiology of intestinal ischemic injury.
Acta Chir Scand 1987; 153: 321-4.
11. McCORD JM. Oxygen-derived free radicals in
postischemic tissue injury. N Engl J Med 1985;
312(3): 159-63.
12. HALLIWELL B. Reactive oxygen species in living
systems: source, biochemistry, and role in human
disease. Am J Med 1991; 91: 14-22.
13. TEDDER TF, STEEBER DA, CHEN A, ENGEL
P. The selectins: vascular adhesion molecules.
FASEB J 1995; 9: 866-73.
7. OGAWA S, GERLACH H, ESPOSITO C, PASAGIANMACAULAY A, BRETT J, STERN D. Hypoxia
modulates the barrier and coagulant function of
cultured bovine endothelium. J Clin Invest 1990;
85: 1090-8.
14. ROOS D. The involvement of oxygen radicals
in microbicidal mechanisms of leukocytes and
macrophages. Klin Wochenschr 1991; 69:
975-80.
8. SHREENIWAS R, KOGA S, KARAKURUM M,
PINSKY D, KAISER E, BRETT J, WOLITZKY BA,
NORTON C, PLOCINSKI J, BENJAMIN W,
15. MENGER MD. Microcirculatory disturbances
secondary to ischemia-reperfusion. Transpl
Proc 1995; 27: 2863-5.
8
Cardiovasc. Sci. Forum -ORIGINAL
Jul./ Sep. 2006 - Vol.
1/ Number 3
ARTICLES
Adult Human Vascular Endothelial Cells
Seeded onto No-React® Treated Bovine
Internal Mammary Arteries: An in Vitro Study
P. M. Dohmen*, M. Stein-Konertz, S. Posner, W. Erdbrügger, W. Konertz
ABSTRACT ---------------------------------------------------------------------------------------------------Background: Alternative grafts are under investigation as the number of patients with reoperations and insufficient autologous bypass material increases. This study was performed to compare
endothelial cell seeding on bovine internal mammary arteries and polytetrafluoroethylene grafts.
Methods: Twelve seeded bovine mammary internal arteries were divided into two groups
(n=6 each); group I endothelial cell seeded, group II endothelial cell seeded with fibrin glue precoating. Similar the polytetrafluoroethylene graft were divided into two groups, group III
endothelial cell seeded and group IV endothelial cell seeded with fibrin glue pre-coating. Grafts
were mounted during seeding and rotated for up to 3 hours. During the conditioning phase, a
continuous surveillance of the circulating medium was performed and adjusted to maintain
optimal cell viability.
Results: Two million endothelial cells were inserted for each grafts. Seeding endothelial
cell density was in group I 1.29 x 105 ± 0.09 x 105 cells/cm² in group III and 0.84 x 105 ± 0.11 x
105 cells/cm². After coating the grafts with fibrin glue, cell density significantly increased in group
II 2.27 x 105 ± 0.17 x 105 cells/cm² and group IV 1.35 x 105 ± 0.08 x 105 cells/cm², respectively
(p<0.003) and (p<0.002). In both graft-types there was a non-significant number of endothelial
cell loss after the conditioning phase.
Conclusions: It seems to be possible to seed endothelial cells onto bovine internal
mammary arteries. Endothelial cell density almost doubled as compared to polytetrafluoroethylene
grafts and seems to favor biological graft matrices.
Key Words: internal bovine mammary artery, anti-calcification, coronary bypass surgery
endothelial cells, PTFE grafts.
Short Title: Endothelial cell seeding of bovine mammary arteries
----------------------------------------------------------------------------------------------------------------------
Address reprint requests dr. P.M. Dohmen MD, Department of Cardiovascular Surgery, Charité, Humboldt University Berlin, Luisenstraße 13,
D-10117 Berlin. Telephone +49 30 450 522092 Fax: +49 30 450 522921 E-mail : [email protected]
9
Introduction
In 1967, Favaloro performed the first
saphenous vein graft implantation in a patient
suffering from coronary heart disease(1). As the
long-term patency rate of this graft is limited(2),
several autologous vessels have been studied.
Since the eighties, the internal mammary artery
became the golden standard for coronary artery
bypass grafting, showing most favourable
patency rates(3). Although results improved by
using different autologous graft material, the
number of patients with previous operation,
extended varicosis, previous varicosis-stripping,
or a history of thrombophlebitis increases(4) and
so alternative non-autologous grafts sources are
needed. Polytetrafluoroethylene (PTFE) grafts
have been used in coronary heart disease,
however the patency rates of these small
diameter grafts are extremely poor (5) .
Alternatively to these prosthetic grafts small
diameter bovine mammary arteries were used,
however patency rate was only 15.8% after 23
months of implantation(6). Our group(7) was able
to increase the patency rate of 4 mm diameter
PTFE grafts by seeding the grafts with autologous
endothelial cells. Follow-up showed a patency
rate of up to 81 % at five years. Although these
results are encouraging the no-touch
implantation technique and the handling of stiff
PTFE material makes surgery extremely
demanding. Biological tissue, which can be
manipulated more easily during surgery, without
destroying the seeded vascular endothelial cells
(VEC) layer at the inner surface, may overcome
implantation difficulties of PTFE grafts.
This in vitro study evaluates the
possibility to seed VEC onto small diameter
bovine internal mammary arteries (SIMA),
treated with glutaraldehyde and afterwards
neutralized with the No-React® treatment(8).
Materials and Methods
This study was approved by the Ethical
Committee of the Charité. Adult human VEC
10
were harvested out of leftovers from saphenous
veins, which otherwise would have been
discard.
The 4.0 mm SIMA’s were treated with
glutaraldehyde and afterwards detoxified by the
No React® treatment (Shelhigh Ltd., Newark,
USA)(8). The internal diameter of the used PTFE
prostheses (Medino GmbH, Gehrden,
Germany) was also 4.0 mm.
Endothelial cell
harvesting and cultivation
Human pieces of great saphenous vein,
with a length of 4-8 cm, were transported to
the cell culture laboratory. VEC were harvested
as previously described(9,10). In brief, separation
of the VEC was performed by using
Collagenase II 0.1% (Boehringer Ingelheim
Pharmaceuticals, Inc., Ridgefield, Conn) for 15
minutes under cell culture conditions in a
humidified incubator (37°C, 5% CO2, and 98%
air saturation). A suspension of VEC were
collected and centrifuged at 500g for 10
minutes. Total culturing time was 2 to 3 weeks
using DelBecco’s modified Eagle’s Medium
(DMEM, Sigma Chemical Co, St.Louis, Mo)
with 20% fetal calve serum ( PAA, Colbe,
Germany), 10 µg/ml basic fibroblast growth
factor (Boehringer Ingelheim Pharmaceuticals,
Inc., Ridgefield, Conn) and antibiotics
(Penicillin 100 U/ml and Streptomycin 100 mg/
ml, Sigma Chemical Co, St.Louis, Mo).
Medium was changed every 2nd day and VEC
growth was evaluated by daily microscopic
examination. The Casy 1 cell-counter (Schaefer
System GmbH, Reutlingen, Germany) was
used for VEC cell counting.
Graft preparation
Graft coating
Graft coating was performed by the use
of Tissuecol Duo S (Immuno, Baxter,
Unterschießheim, Germany) to increase
binding capacity of VEC to both graft-types of
group II and IV. Grafts were cannulated at both
sides and the fibrin component was injected
first. Afterwards the thrombin component was
injected into the graft and a 4mm diameter
Fogarty catheter was used to assure a smooth
inner surface. Residual clumps were carefully
flushed with physiological solution. Grafts were
kept in medium.
Static graft seeding
The grafts were fixed at both sides with
a running 5-0 Prolene suture-line (Ethicon Inc,
Sommerville, NJ) in a special developed bioinherent bioreactor. The bioreactor was filled
with VEC and placed into a biostrabilisator
(Biegler Medizinelektronik GmbH, Mauerbach,
Austria) to turn the graft in a calculated way
during a period of three hours at cell culture
conditions (37°C, 5% CO 2 and 98% air
saturation) (figure 1). A sedimentation
technique was used, allowing VEC to bind at
the inner surface of the grafts. The final seeded
graft was stored in a humidified incubator
(37°C, 5% CO2 and 98% air saturation) for
another 7 to 10 days, to improve VEC
confluence. The cell seeding density was
calculated by counting the total number of VEC
provided into the graft minus the remaining VEC
in solution after seeding.
Figure 1 - Schematic drawing of the static seeding phase of VEC onto the No-React® treated bovine
internal mammary artery. 1. Bioreactor. 2 - Vascular graft prosthesis. 3 - Rotating unit. 4 - Humidified
incubator. 5 - Driver unit. 6 - Filtersystem
Graft conditioning
After the seeding, the bioreactor including
the grafts were placed into a circulatory system
using a 10 ml disposable pump (Medos AG,
Aachen, Germany). During the conditioning
phase the flow was increased until a maximum
flow of 0.2 L/min was achieved. The total
duration of the conditioning phase was
completed after 1 hour. Cell density at the grafts
was again calculated as well as the endothelial
cell viability. Finally the seeded grafts were
fixated in 10% formalaldehyde for histological
examination.
Histological follow up
Immunohistochemical staining was
performed with factor VIII-related antigen
(DAKO, Hamburg, Germany) at the VEC, to
11
show that the cultured cells were exclusively
endothelial cells, without contamination of
interstitial cells.
Giemsa (Sigma Chemical Co, St.Louis,
Mo )and hematoxylin and eosin (HE) staining
was routinely performed in four micrometer
thickness longitudinal sections. After seeding,
the first samples were taken. Next samples were
taken after the conditioning phase in both
groups. Also there was a documentation of the
seeding density of the SIMA and the PTFE
grafts after the conditioning phase, to document
the findings of the VEC counting.
Statistics
Quantitative data were expressed as
mean and standard deviation. Comparisons
between the groups were made with the t-test.
The level for statistical significance was set at
a p-value < 0.05. Data management and
statistical analysis was performed with SPSS
10.0 (SPSS Inc., Chicago, USA).
and in group IV 1.35 ± 0.08 x 105 cells/cm².
The use of fibrin glue increased endothelial cell
density at the inner surface of the graft
(p<0.002).
Graft conditioning
The VEC binding capacity after the
conditioning phase was for group I 1.08 ± 0.13
x 105 cells/cm² and in group II 2.22 ± 0.16 x
105 cells/cm². The decrease of endothelial cell
density after the conditioning phase in group I
16.3% and in group II 2.2 %. In group III, the
VEC binding capacity was 0.72 ± 0.11 x 105
cells/cm² and in group IV 1.31 ± 0.07 x 105
cells/cm². The decrease of the VEC after the
conditioning phase in group III was 14.3 % and
in group IV was 3.0 %. There was a significant
decrease of endothelial cell binding between
group I and III (p< 0.016) and between group
II and IV (p<0.008) of VEC binding after the
conditioning phase as using different matrices
to bind cells onto, however within the groups
the decrease was never statistical significant.
Results
Histology
Endothelial cell
harvesting and cultivation
After a period of 2 to 3 weeks at least 2 x
10 endothelial cells were available, which was
found to be a sufficient number to seed a 4.0
cm grafts with a diameter of 4.0 mm. Median
endothelial cell viability was 95.5% (range 93.4
to 97.7%).
6
Immunohistochemical staining showed
that the cells which were cultured at the tissue
laboratory were a monoculture of endothelial,
confirming the absence of contamination with
interstitial cells (figure 2).
Graft preparation
Graft seeding
The VEC binding capacity after seeding
was in group I 1.29 ± 0.09 x 105 cells/cm² and
in group II 2.27 ± 0.17 x 105 cells/cm². The use
of fibrin glue significantly increased the binding
capacity of endothelial cells in the BIMA grafts
(p<0.003). In group, endothelial binding
capacity III was 0.84 ± 0.11 x 105 cells/cm²
12
Figure 2. Factor VIII staining of the VEC cell culture.
With Giemsa staining, histological
visualization of the different cell density after
seeding could be documented in all groups. In
group I, VEC density binding to the graft was
high, as there was a monolayer of endothelial
cells seen at the inner surface. However there
were free spots at the graft inner surface which
didn’t show a confluent covering endothelial
cell layer. After the conditioning phase there
was a certain cell loss seen due to the shear
stress of the flow. With the use of fibrin glue,
the number of endothelial cells seeded onto the
BIMA graft increased 1.7 times and there was
a confluent monolayer of endothelial cells at
the inner surface. Even after giving shear stress
to the endothelial cells there was only a
minimum loss of VEC and a confluent
monolayer could still be seen in group II (figure
3). In group IV, there was also a monolayer of
VEC seen after the seeding, however the cellfree spots were more frequent, especially if no
fibrin glue was used (group III) prior to the
endothelial cell seeding. After the conditioning
phase the number of cells even further
decreased, and consequently cell-free areas
increased.
Using HE staining it was possible to
show that in both groups not only the
endothelial cells were closely attached to each
other, but also to the graft (figure 4).
Figure 3. HE staining of the VEC seeded SIMA
after the conditioning phase, which shows a
confluent monolayer.
Figure 4. Giemsa staining of the VEC seeded SIMA
after the conditioning phase, which shows a
confluent monolayer.
Discussion
Alternative graft material is needed in
situations of absence of sufficient autologous
graft material. Xenogenic graft materials have
been introduced experimentally as well as
clinically (11,12,13,14). Major problems rose by
tissue failure, due to aneurysm formation of the
biological tissue. Dardik et al (15) started
umbilical vein graft fixation with the use of
glutaraldehyde which should overcome tissue
degeneration. During the same period of time
bovine internal mammary arteries, after
glutaraldehyde fixation, were implanted into
patients. Unfortually these grafts showed a high
incidence of biodegeneration, calcification and
thrombosis (16,17) . It has been shown that
glutaraldehyde treatment leads to tissue
degeneration after years of implantation, which
is well known in bioprosthetic heart valve
replacement(18,19). Gabbay et al(8) developed an
anit-mineralization
technique
for
glutaraldehyde fixed material, so called NoReact â procedure. This treatment should
overcome calcification of material and so
prolong the functionality of tissue valve
prostheses and bovine internal mammary
arteries. There are several papers published
about the successful elimination of tissue
calcification by the use of No-React â
13
treatment (20) , including a recent paper
describing the use of bioprosthetic heart valves
in children with good hemodynamic results has
been reported(21).
Another disadvantage of the use of bovine
mammary arteries, before anti-mineralization
treatment, is high thrombogenicity and so
decrease of patency rate after implantation. The
natural barrier of vessels are viable endothelial
cells, which have showen antithrombotic
properties. Our group(22) showed in a clinical
trial the use of small diameter PTFE grafts during
coronary bypass surgery in patients who had
no suitable graft material. Through seeding with
autologous endothelial cells we were able to
increase the patency rate up to 90.5 % at 4 years
of follow up.
This feasibility study was performed to
investigate the possibility to seed endothelial
cells on No-Reactâ detoxified glutaraldehyde
treated bovine mammary arteries. The median
viability of the seeded endothelial cells was
95.5% which shows that No-Reactâ treatment
not only is able to overcome calcification of
glutaraldehyde tissue, but also detoxifies
glutaraldehyde treated tissue. In vitro it seems
that this treatment is highly efficient as the
absolute number of VEC binding to the BIMA
matrix was significantly higher as compared
to the group with PTFE grafts. This was
independent from pre-coating. The number of
VEC binding to the SIMA group was 1.5 times
higher compared to similar treated PTFE grafts.
Even if the PTFE grafts were pre-coated the
14
absolute number of VEC have been almost
similar to the SIMA matrix. On the other hand
if the grafts of both groups were pre-coated the
absolute number of VEC at the SIMA matrix
was 1.7 times higher then the PTFE grafts.
This in vitro study showed also that the absolute
number of VEC seeding onto a graft is more
depending on the graft material, the matrix has
been attached to. More important seems the
fibrin glue pre-coating during the conditioning
phase. The number of VEC drops in group I
16.3% without pre-coating performed. Precoating increased the binding density of VEC
and only 2.2% were eliminated by flow. When
the PTFE grafts of group II has evaluated
similarly, the number of VEC decreased 14.3%
without pre-coating. Coating the PTFE matrix
with fibrin glue, also improved the attachment
of VEC with a loss of 3.0%.
In summary, this in vitro study showed
that the use of fibrin glue pre-coating is able to
decrease the VEC loss after the conditioning
phase, however the absolute number of VEC
seems to be more depending of the material
the matrix has been made of. The No-Reactâ
treatment of glutaraldehyde fixed tissue seems
to be efficient to allow endothelial cells to cover
this graft as a monolayer.
Acknowledgements:
We would like to thank Mrs. Krüger for
her excellent work in the laboratory and in the
culturing of endothelial cells.
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J, Flameng W, Konertz W. Tissue
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12. Slovin Ja. Arterial below the knee bypass grafts.
Experience with the modified bovine
heterograft. Am J Surg 1974;128:58-64.
13. Cutler BS, Thompson JE, Patman RD, Persson
AV, Manfredi PD. The modified bovine arterial
graft: a clinical study. Surgery 1974;76:963973.
14. Keshishain JM, Smith NP, Adkins PC, Camp F,
Yahr WZ, Hill L. Clinical experience with the
modified bovine arterial heterograft. J
Cardiovasc Surg Torino 1971;12:433-440.
15. Dardik H, Wengerter K, Qin F, Pangililan A,
Silvestri F, Wolodiger F, Kahn M, Sussman B,
Ibrahim IM. Compasrative decades of
experience with glutaraldehyde-tanned human
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revascularization: An analysis of 1275 cases. J
Vasc Surg 2002;35:64-71.
16. Rosenberg N. The bovine arterial graft and its
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17. Dale WA, Lewis MR. Further experiences with
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18. Riddle JM, Magilligan DJ, Stein PD. Surface
morphology of degeneration of porcine
bioprosthetic valves four to seven years
following implantation. J Thorac Cardiovasc Surg
1981;81:279-87.
15
19. Bengtsson L, Radegran K, Haegerstrand A. In
vitro endothelialization of commercially
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adult human cells. Eur J Cardio-Thorac Surg
1993;7:393-398.
20. Abolhoda A, Yu S, Oyarzun R, Allen K,
McCormick J, Bogden J, Gabbay S.
Calcification of bovine pericardium:
gluteraldehyde versus no-react biomodification.
Ann Thor Surg 1996;62:169-174.
16
21. Marianeschi SM, Iacona GM, Seddio F, Abella
RF, Conduluci C, Cipriani A, Iorio FS, Gabbay
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Cardiovasc. Sci. Forum -ORIGINAL
Jul./ Sep. 2006 - Vol.
1/ Number 3
ARTICLES
Pravastatina e Síndrome da
Resposta Inflamatória Sistêmica por
Circulação Extracorpórea
G. F. Teixeira Filho, J.R.M. Sant´Anna, P.R.Prates,
R.A.K. Kalil, A.H. Neto, M. Santos, I. Nesralla
RESUMO -----------------------------------------------------------------------------------------------------Objetivo - Avaliar a possível ação antiflamatória da pravastatina em um modelo bem
definido de inflamação que é a síndrome de resposta inflamatória decorrente da CEC. Por tanto
foram dosados mediadores pró-inflamatórios interleucina 6, interleucina 8, TNF-a e proteína C
reativa antes e após a CEC e a drenagem mediastinal pós-operatória.
Material e Métodos - Foram selecionados 20 pacientes portadores de cardiopatia
isquêmica e candidatos a cirurgia de revascularização do miocárdio. Dez pacientes receberam
80 mg de Pravastatina 36 e 12 horas antes da cirurgia (grupo P) e dez pacientes foram alocados
como grupo controle (grupoC). As amostras foram coletadas antes, logo após a CEC, 6, 12 e 24
horas após. O teste Mann-Whitney foi empregado para testar diferenças entre grupos em cada
tempo de coleta da amostra. Para testar a diferença no mesmo grupo de paciente foi empregado
o teste de Wilcoxon. Em todos os casos valor de p<0,05 foi considerado significante.
Resultados - O grupo P apresentou níveis de proteína C reativa significativamente mais
baixos do ue o grupo controle p=0,004. Em relação aos níveis plasmáticos de TNF-a e interleucina
6, não houve significância estatística entre os dois grupos. O grupo “P” mostrou diminuição
significativa dos níveis de interleucina 8 comparado com o grupo controle 6 horas após a CEC.
Diminuição significativa do sangramento mediastinal ocorreu no grupo “P” quando comparado
ao grupo controle p=0,019.
Conclusão - Os resultados encontrados em nosso trabalho sugerem que a pravastatina
apresenta atividade antiflamatória devido a redução dos níveis plasmáticos de proteína C-reativa
e interleucina 8 e, que provavelmente a sua ação seja a nível da ativação endotelial expressa
pelos níveis reduzidos de interleucina 8 principal citocina envolvida na ativação de
polimorfonucleares.
----------------------------------------------------------------------------------------------------------------------
Instituto de Cardiologia do Rio Grande do Sul - Unidade de Pesquisa - Dr. Guaracy F. Teixeira Fº
Av. Princesa Isabel, 395 - Santana - Porto Alegre Zip 90.620-001
Phone/Fax.: 00-55-51-230.3600 Ext.3777 e-mail:[email protected]
17
Introdução
Circulação extracorpórea (CEC) é
essencial em grande número de cirurgias
cardíacas. Está associada com reação
inflamatória que pode resultar em disfunção
de órgãos, retardo na recuperação ou mesmo
óbito do paciente(1-3). Esta resposta inflamatória
complexa inclui a ativação de complemento,
liberação de endotoxina, liberação de cininas,
ativação de leucócitos bem como a expressão
de moléculas de adesão e a produção de várias
substâncias, incluindo-se radicais que
convertem as células endoteliais a um estado
ativo. A ativação imediata da célula endotelial
é devida a degradação do complemento
circulante, sendo o evento mais significativo
da interação do sangue com o circuito de
CEC(1). Posteriormente, as células endoteliais
são ativadas pelos mediadores inflamatórios,
como as citocinas ou lipopolissacarídeos(4).
A ativação do endotélio vascular tem um
papel determinante na resposta sistêmica que
se segue a CEC(5). Nos anos recentes, dados
experimentais e de observação demonstrando
que a terapêutica com pravastatina reduz o
número e a atividade de células inflamatórias
presentes nas placas ateroscleróticas permitem
inferir que esta substância pode mostrar ações
antinflamatória importante(6).
Nossa hipótese é que a pravastatina
reduza a resposta inflamatória da CEC, sendo
objetivo deste estudo prospectivo randomizado
investigar se a pravastatina afeta a liberação
de mediadores pró-inflamatórios em pacientes
submetidos a cirurgia cardíaca com CEC.
Pacientes e Métodos
Pacientes
Após aprovação pelo Comitê de Ética da
instituição, 20 doentes com indicação de cirurgia
de revascularização miocárdica com CEC foram
considerados no estudo, seguindo-se a obtenção
de consentimento pós-informação. Pacientes
18
com infecção ativa ou recente, transfusão
sanguínea, infarto do miocárdio prévio, cirurgia
cardíaca prévia ou que utilizaram drogas
redutoras de lipídeos nos últimos 3 meses foram
excluídos. Dez pacientes receberam 80 mg V.O
de pravastatina 36 horas e 12 horas antes da
cirurgia (grupo P) e 10 pacientes foram
considerados como grupo controle, não
recebendo a medicação (grupo C).
Técnica operatória
Todos os pacientes foram prémedicados com sulfato de protamina (0.2
mg/Kg IM) e sulfato de atropina (0.5 mg IM).
A anestesia foi induzida com citrato de
fentanil (10 mg/Kg EV) e tiopetal sódico (3
mg/Kg EV). Relaxamento muscular foi
induzido com brometo de pancurônio (0.10
mg/Kg EV). Foi iniciada ventilação mecânica
e a anestesia suplementada pela inalação de
halotano a 0.4 %.
Monitorização operatória (ECG, pressão
arterial, pressão venosa central, débioto
urinário, temperatura nasofaringea e retal) foi
identica em todos os pacientees. Cefalotina foi
usada como antibiótico profilático antes da da
esternotomia (2.0 g EV e 1.0 g EV antes do
início da CEC). Hidrocortizona (500 mg EV)
foi administrada em todos os pacientes após
indução da anestesia.
Os componentes do sistema de CEC
consistiram de um oxigenador de membrana
capilar composta de fibras resistentes ao
plasma (Maxima For te; Medtronic, Inc.
Anaheim, California), reservatório de
cardiotomia, reservatório de cardioplegia e
filtro arterial (Macchi Biomedical Eng. São
Paulo, SP). Estes componentes foram
conectados por tubos de cloreto de polivinila
(Macchi Biomedical Eng. São Paulo, SP).
O volume de enchimento consistiu de 2 L de
solução eletrolítica, sendo administrado
concentrado de hemácias quando o
hematócrito era inferior a 20%. Antes do início
da CEC, heparina (4 mg/Kg EV) era
administrada para prolongar o tempo de
coagulação ativado (TCA) acima de 600
segundos. CEC não pulsátil foi estabelecida
com fluxo de 2,4 L/m2 e hipotermia moderada
(32ºC nasopharyngeal) .
Preservação miocárdica foi obtida
mediante infusão de solução cardioplégica
cristalóide hipotérmica (St. Thomas II, 4ºC)
na raiz aórtica, após pinçamento deste vaso,
em dose única (300 mL/m2). Aquecimento foi
iniciado durante a conclusão das anastomoses
distais e a pinça aórtica removida a seguir.
Anastomoses proximais foram realizadas com
oclusão parcial da aorta ascendente. CEC foi
suspensa, o sangue remanescente no oxigenar
foi transfundido e a heparina revertida com
sulfato de protamina EV. Não foi empregada
ultrafiltração
ou
outro
tipo
de
hemoconcentração.
Avaliações laboratoriais
Liberação de proteína C-reativa (CRP),
fator-alfa de necrose tumoral (TNF-a),
interleucina-6 (IL-6), interleucina-8 (IL-8)
foram medidos..Amostras sangüíneas foram
retiradas do cateter venoso central antes de
CEC (após indução da anestesia|), após CEC
(10 minutos depois da reversão da
anticoagulação pela protamina) e 6, 12 e 24 h
após CEC. Apenas CRP foi dosada com 24
após CEC e as demais avaliações efetuadas
até 12 h pós CEC.
As amostras foram coletadas em tubos
de ácido tetraceticodiaminoetileno (EDTA),
imediatamente centrifugadas a 1000 xg por 10
minutos e guardadas a –20 ºC até que as
avaliações fossem efetuadas. Imunoensaios
para TNF-a, IL-6 e IL-8 foram realizados com
kits disponíveis no comércio (R&D Systems,
Mineapolis, MN), de acorco com as instruções
do fabricante. Concentrações de proteína Creactiva (CRP) foram medidas usando-se kits
comerciais (Turbiquant CRP, Dade Behring,
Marburg, Germany).
As variáveis observadas para avaliar evolução
clínica dos pacientes incluíram a drenagem
mediastinal e o tempo de internação hospitalar.
Análise Estatística
Os resultados são apresentados como
mediana e os quartils superior e inferior
indicados em parênteses. A apresentação de
resultados diferentes do acima são indicados.
O teste U de Mann-Whitney foi usado para
identificar diferenças entre grupos em cada
intervalo observado. O testes de Wilcoxon foi
usado para identificar diferenças dentro de
cada grupo. Em todos os casos, um valor de p
inferior a 0,05 foi considerado significativo.
Resultados
As características intraoperatórias dos
dois grupos de pacientes estão mostradas na
tabela 1.
Por razões técnicas, CRP não foi
avaliada em 2 pacientes no intervalo de 24 h
após CEC. Em um paciente, pelo mesmo
motivo, CRP, TNFa, IL-6 e IL-8 não foi medida
6h após CEC.
Mediadores inflamatórios
PCR– Proteína C-reactiva teve valor
mediano antes da CEC de 5,0 (5,0 – 9,3) no
grupo C e de 9,9 (7,0 – 15,6) no grupo P. Esta
diferença é estatisticamente significativa
(p=0.015).
Níveis plasmáticos de PCR não
mostraram diferenças significativas entre
ambos os grupos imediatamente, 6 e 12h após
CEC. Níveis plasmáticos foram 5.0 (5.0/5.6),
7.7 (5.8-10.2) e 31.9 (25.3-37.5) mg/dl no
grupo C e 6.4 (5.5-8.8), 13.0 (9.3-24.0) e 39.1
(28.8-47.7) mg/dl no grupo P.
Com 24h após CEC, o grupo P
apresentou um valor significativamente inferior
do que o grupo C: (38.7-73.6) contra 109.0
(104.0-112.0) mg/dL (p = 0.004),
respectivamente (figura 1).
19
Tabela 1. Características dos pacientes avaliados
Figura 1 - Variação da PCR nos grupos P e C.
*p=0.015 Comparação do grupo P e C antes CEC.
**P=0.004 Comparação do grupo P com grupo C 24h após a CEC.
20
TNFa - Níveis plasmáticos do fator-alfa
de necrose tumoral não mostraram
modificações significativas nos períodos
avaliados, para ambos os grupos. Não
ocorreram variações significativas na
comparação entre grupos. Níveis plasmáticos
médio de TNFa foram 11.0 (9.2-13.6), 13.4
(9.6-24.2), 9.6 (6.8-11.7) and 10.2 (9.4-11.4)
pg/ml para o grupo P, respectivamente para
antes de CEC, imediatamente, 6 e 12h após
CEC. Para o grupo P, os valores foram de 11.9
(8.8-14.0), 11.4 (10.0-22.3), 10.4 (8.8-17.6)
and 11.1 (8.9-16.1) (NS para avaliações do
mesmo grupo e entre grupo).
IL-6 - Em ambos os grupos, os níveis
de interleucina 6 levels aumentaram
significativamente, se comprados com
valores prévios à CEC. Níveis plasmáticos
de IL-6 foram 18.9 (8.2-27.5), 51.4 (40.674), 86.4 (82.8-103) e 135.4 (114.1-157.1)
mg/dL no grupo P e de 13.8 (4.9-30.5), 48.4
(30.5-69.8), 97.8 (89.4-114.8) e 160.2
(124.2-207.3) mg/dL no grupo C,
respectivamente para antes, imediatamente
após e 6 e 12 h após CEC. Não foram
encontradas diferenças significativas entre
grupos em qualquer dos períodos de
avaliação (figura 2).
Figura 2 - Liberação de IL-6 nos grupos P e C.
*p<0.05 comparação antes da CEC em cada grupo. NS intergrupos em nenhum tempo.
IL-8 - Níveis plasmáticos de IL-8 foram
de 330 (280-730), 770 (560-1505), 600 (380730) and 705 (510-1290) mg/dL ino grupo C
e de 240 (107.5-430), 277.5 (167.5-510) 144
(122.5-200) e 295 (195-1129) mg/dL no grupo
P, para avaliações antes, imediatamente após,
6 e 12 após CEC. Não foram encontradas
diferenças significativas nas avaliações pósCEC se comparadas com controle prévio para
o grupo P. Mas no grupo C ocorreu uma
diferença significativa para o valor de 12 h, se
comparado ao valor prévio a CEC (p=0.007).
21
Com 6 h após CEC, o valor registrado
para o grupo P foi significativamente inferior
ao registrado para o grupo C : 144 (122,5200) vs. 600 (380-730) mg/dL p=0,017
(figuras 3 e 4).
Variáveis clínicas - Não foram
encontradas diferenças no período de
internação em ambos os grupos. A média de
hospitalização foi de 8.0 (7.0-11.0) para o
grupo C e de 7.5 (7.0-8.5) dias para o grupo
P; p = 0.247. Drenagem mediastinal foi
significativamente inferior para o grupo P do
que para o grupo C : 600 (395.0-835.0) ml vs
990 (800.0-1070.0) ml; p = 0.019.
Discussão
Diversos estudos registraram o
envolvimento de mediadores solúveis na
resposta inflamatória patológica da CEC. Estes
elementos inflamatórios circulantes são
complementos, citocinas e proteínas de fase
aguda(3,7). Existe uma variedade equipamentos
utilizados em cirurgia cardíaca que conduzem
a ativação sistêmica do complemento.
O evento precoce da ativação do
complemento, que está baseado em uma
cascata enzimática comparável a vista na
coagulação sangüínea, pode ser deflagrado
em duas rotas(4). A rota clássica é a ativação
pelos complexos antígeno-anticorpo e a rota
alternativa é a ativação pelas paredes
celulares bacterianas e por superfícies
estranhas.
A exposição do sangue ao CEC
representa a rota de ativação alternativa,
enquanto que a reversão de heparina pela
protamina é uma rota clássica(8). Liberação de
endotoxina na circulação pode ativar ambas
as rotas clássica e alternativa(9).
Figura 3 - Variação dos níveis plasmáticos de IL-8 dos grupos P e C.
*p=0.017 comparado entre os grupos P e C 6h após a CEC.
22
Figura 4 - Sangramento pós-operatório no grupo P e C.
*p=0.019 comparado entre o grupo P e C.
Quando o complemento é ativado,
fatores solúveis, como C5a e C3a são liberados
na circulação. C5a determina a liberação de
mediadores inflamatórios à partir de
mastócitos (mast cells) e atua como poderoso
atrativo químico para neutrófilos. C5a também
causa ruptura capilar, perda de granulação
pelos neutrófilos e a expressão da molécula Pseletina dos neutrófilos na superfície de
plaquetas e do endotélio(7). C5a ativa plaquetas
e monócitos, resultando na liberação de
citocinas e de outros mediadores inflamatórios
que amplificam a adesão entre neutrófilos e a
célula endotelial.
Em resposta aos sinais inflamatórios
(produtos de ativação do complemento,
citocinas, radicais livre derivados da hipóxia
ou oxigenação), células endoteliais são
convertidas ao estado ativado. Isto resulta em
modificações profundas na expressão genética
e função celular de células endoteliais.
Parecem existir duas fases da ativação
celular endotelial durante CEC:
A primeira ocorre porque produtos de
degradação do complemento circulantes
iniciam uma resposta adesiva pelos neutrófilos
imediata e de curta duração. A Segunda fase
se deve a cascata de cinina que amplifica a
adesão neutrófilo-endotelial(4).
Estudos recentes mostram que citocinas
são mensageiros intracelulares e os mediadores
mais importantes da injúria vascular e da
disfunção de órgãos(7,10). A liberação de TNFa, IL-6, IL-8 e CRP são marcadores de
processo inflamatório intenso que ocorra
durante CEC. Se demonstrou consistentemente
que níveis de IL-6 e IL-8 estão elevados durante
CEC(10,11). Também existem registros, embora
inconsistentes, de níveis plasmáticos TNF-a
neste processo (7,11). O fenômeno chave da
resposta inflamatória que se segue a CEC é o
dano agudo da célula endotelial(4).
Aterosclerose resulta da resposta da
célula endotelial a injúria crônica que se segue
a adesão e migração no sub endotélio de
neutrófilos, linfócitos, plaquetas e macrófagos.
Formas crônicas de injúria celular endotelial
podem resultar na prolongada expressão de
moléculas de adesão de leucócitos, que atraem
neutrófilos para a área. A presença de proteases
23
de radicais livres e citocinases que determinam
ruptura do sub endotélio causam a proliferação
de células musculares lisas e a formação de
uma placa fibrosa.
Assim, a progressão da lesão aterosclerótica
é marcada pelo acúmulo de camadas alternadas
de células musculares lisas e de macrófagos
ligados a lipídios. As camadas de tecido fibroso e
de células musculares lisas cobrem um núcleo de
lipídeos e produtos necróticos. Estas placas têm
tendência a ruptura, que conduz ao infarto do
miocárdio agudo pela oclusão coronária com
trombo plaquetário e a morte súbita
A ativação do sistema complemento
desempenha um papel importante na
patogenese da aterosclerose, provavelmente
por ativar células endoteliais(13). Exposição de
células endoteliais aos complementos deflagra
a indução de citocinas pró-inflamatórias,
como IL6 e IL-8. Componentes do
complemento estimula a ativação de células
endoteliais, resultando em uma expressão
aumentada da proteína-1 quimioatrativa de
monócitos (MCP-1) e de outras citocinas que
ativam a adesão firme de monócitos ao
endotélio, um evento chave para iniciar a
patogênese da aterosclerose. Assim, a
resposta inflamatória da CEC e da
aterosclerose têm em comum a ativação de
células endoteliais devida a estimulo de
componentes de complemento e a indução de
citocinas pró-inflamatórias, como a
interleucina 8.
Em nosso estudo, o grupo controle (C)
mostrou um nível significativamente inferior de
CRP quando comparado ao grupo tratado (P),
antes de CEC. É possível que isto decorra da
presença de lesões ateroscleróticas mais ativas
neste grupo do que no grupo C(14).
No presente estudo, nossos resultados
permitem algumas inferências. Primeiro, o prétratamento com pravastatina antes de CEC
possibilita uma resposta inflamatória sistêmica
reduzida. Os níveis reduzidos de proteína Creativa no grupo tratado com pravastatina 24
horas antes de CEC confirmou a ação anti24
inflamatória desta substância. Isto é
corroborado pelos reduzidos níveis de IL-8 no
seu pico de liberação (6h), fato consistente com
reduzida resposta inflamatória.
Em segundo, efeitos da administração
de pravastatina são imediatos. Isto é
demonstrado pelo curto tempo de resposta da
ação anti-inflamatória. Esta observação sugere
que a ação anti-inflamatória da pravastatina
não é imediata pela redução de lipídeo.
Finalmente, a pravastatina reduziu
significativamente o sangramento mediastinal
pós-operatório, conforme observado no grupo
tratado (P).
Nossas observações concordam com
algumas pesquisas prévias que mostram
reduzido ou nenhum aumento nos níveis
plasmáticos de INF-a(9). OS resultados sugerem
ainda que, em pacientes submetidos a cirurgia
cardíaca, os níveis plasmáticos de IL-6 são
elevados durante CEC, confirmando
observações prévias da literatura(7).
Observamos um aumento marcado no
nível plasmático de IL-6 nos dois grupos
avaliados, mas no grupo tratado com
pravastatina este fato não correspondeu a uma
elevação de CRP. Esta observação pode
significar que a pravastatina tenha uma ação
independente da liberação de IL-6. É
reconhecido que IL-6 tem propriedades próinflamatória e anti-inflamatória(15). Embora
não se possa definir o exato mecanismo de
ação, as observações apóiam a hipótese de
que a pravastatina tenha ação antiinflamatória(16).
As observações confirmam os achados
de Weber et al, quanto a que a redutase HMGCoA interfira diretamente com mecanismoschave para que os leucócitos desempenhem
esta resposta inflamatória(16).
A ativação do complemento por si só
pode conduzir a ativação de neutrófilos. Se
acredita ainda que o grau de inflamação
induzida pela ativação de neutrófilos é
relacionado aos níveis séricos de IL-8. Isto foi
confirmado in vitro por Urbich et al(17). Em
nosso estudo, não avaliamos os produtos da
ativação do complemento, mas os reduzidos
níveis plasmáticos de IL-8 parecem expressar
reduzidos valores de produtos de ativação do
complemento. Previamente, IL-8 foi observado
em lesões ateroscleróticas(18).
Kiener e associados demonstraram que
as estatinas podem ser diferenciadas quanto
ao seu efeito pró-inflamatório nos leucócitos.
O tratamento de monócitos isolados por
elutriação com lovostatina, sinvastatina ou
atorvastatina aumentou marcadamente a
produção TNF-a, IL-8 e IL-1 b quando as
células foram subseqüentemente tratadas com
LPS, complexos imunitários ou superantígenos. Em contrate, pré-tratamento com
pravastatina não elevou estas citocinas
inflamatórias(19).
Achados recentes de Simoni e
associados sugerem que na aterosclerose
human, IL-8 representa um importante
mediador da angiogênese e pode contribuir
para a formação de placas devido a suas
propriedades angiogênicas(20).
Assim, nossos dados sugerem que a
pravastatina tenha uma ação antiinflamatória durante CEC. É possível que a
pravastatina reduza os produtos da ativação
do complemento e/ou IL-8. O estudo mostra
ainda um sangramento mediastinal reduzido
no grupo tratado. Isto poderia ser explicado
pela redução na ativação de células
endoteliais pela pravastatina, fonte principal
de fator tecidual durante CEC. Isto resulta em
um consumo aumentado de fatores de
coagulação(21).
Investigações futuras são necessárias
para elucidar o papel exato da pravastatina
na resposta inflamatória que se segue a CEC
Mas nossos dados sugerem que o protratamento com pravastatina reduz
significativamente níveis plasmáticos de CRP
e IL-8 e a resposta inflamatória em pacientes
submetidos a circulação extra-corpórea.
ABSTRACT ---------------------------------------------------------------------------------------------------Objetive – The effects of pravastatin have been documented in reducing LDL levels. In constrast,
the effect of pravastatin in inflammatory function has not yet been demonstrated. This study was
designed to evaluate action of pravastatin on inflammatory reaction after extracorporeal circulation.
Methods – In a prospective, randomized study, 20 patients undergoing eletive coronary
artery bypass grafting were investigated. Ten patients received 80mg p.o. of pravastatin 36 and
12h before surgery, and a control group of 10 did not. Plasma levels of C-reactive protein, tumor
necrosis factor-alfa, interleukin-6, interleukin-8 and postoperative blood loss were analysed before
and after cardiopulmonary bypass.
Results – Tumor necrosis factor-alfa did not change significantly in each of the moments
measured in either group. Interleukin-6 in both groups significantly increased after CPB when
comparing to the measures pre bypass and there was no significant diferences between the two
groups. Interleukin-8 increased (p=0.017) in group control at 6h after CPB compared with group
P. C-reative protein was increased (p=0.015) in group pravastatin before CPB compared with
control. Median levels are 9.9 (7.0-15.6) and 5.0 (5.0-9.3) mg/dL. Despite this previous elevation,
at 24h after CPB group P showed significantly lower levels than group control (p=0.004). Median
levels are 62 (38.7-73.6) and 109.0 (104.0-112.0) mg/dL in groups P and C, respectively.
Postoperative blood loss was significantly lower in group pravastatin than in group control
(p=0.019).
Conclusions – Our data suggest that pravastatin pre-treatment preceding CPB reduced
systemic inflammatory response. The effects of administration are immediate and antinflammatory
action is not mediated by lipid lowering. Pravastatin also reduced mediastinal postoperative bleeding.
---------------------------------------------------------------------------------------------------------------------25
REFERÊNCIAS
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7. Steimberg JB, Kajelansk DP, Olson JD, Weiler
JM. Cytokine and complement levels in
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Endotoxin release and tumor necrosis factor
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10. Kawamura T, Wakusawa R, Okada K, Imada
S. Elevation of cytokines during open heart
surgery with cardiopulmonary bypass:
participation of interleukin-8 and 6 in
reperfusion injury. Can J Anaesth
1993;40:1016-21.
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11. Kalfin RE, Engelman RM, Rouson JA, et al.
Induction of interleukin-8 expression during
cardiopulmonary bypass. Circulation
1993;88:401-16.
12. Ross R, Fuster V. The pathogenesis of
atherosclerosis. In: Fuster V, Ross R, Topol EJ,
eds. Atherosclerosis and coronary artery
disease. Philadelphia: Lippicott-Raven,
1996:441-60.
13. Bhakadi S. Complement and atherogenesis:
the unknown connection. Ann Med
1998;30:503-07.
14. Liuzzo G, Biasucci LM, Gallimore JR, et al. The
prognostic value of C-reactive protein and
serum amyloid A protein in severe unstable
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cytokines. Chest 2000;117:1162-72.
16. Weber C, Erl W, Weber KSC, Weber PC.
HMG-CoA reductase inhibitors decrease
CDIIb expression and CDIIb-dependent
adhesion of monocytes to endothelium and
reduce increased adhesiveness of monoctes
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1997;30:1212-17.
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Dimmeler S. Laminar shear stress upregulates
the complement inhibitory protein clusterin. A
novel potent defense mechanism against
complement-Induced endothelial cell
activation. Circulation 2000;101:352-55.
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27
ORIGINAL CARDIOVASCULAR IMAGING
Cardiovascular Imaging: Nine Years Patency
of a Small Caliber Vascular Prosthesis
Seeded with Autogous Endothelial Cells
P. M. Dohmen*, A. Lembcke, D. Gabbieri, W. Konertz.
Short Title: Patency of seeded grafts
Key Words: Multi-slice CT, alternative graft material, coronary bypass surgery cell seeding
Word Count: 49
A 79-year-old man suffering from severe coronary artery disease was submitted for
revascularization. As there was no sufficient autologous grafts available, a 4 mm expanded
polytetrafluoroethylene graft was seeded with autologous vascular endothelial cells (AVEC). At
nine years, multi-slice computed tomography showed a patent AVEC seeded graft (Figure 1,2).
Figure 1. Three dimensional cardiac reconstruction shows a patent internal mammary artery grafted to
the left anterior descending artery (arrows) and a patent graft to the first marginal branch (arrows).
* Address reprint requests: Dr. P. M. Dohmen MD PhD, Department of Cardiovascular Surgery, Charité Hospital, Humboldt
University Berlin, Luisenstraße 13, D-10117 Berlin. Telephone +49 30 450 522092 Fax +49 30 450 522921 E-mail :
[email protected]
28
Figure 2. The sagital sections show heavy calcification ( black arrows) of the first marginal branch. The
seeded graft showed to be patent, with absence of any narrowing over the total length of the graft (red
arrows). Notice that the run off in this area is limited.
29
UPDATING ARTICLES
Fisiología Aplicada de La Vasculogénesis
Alberto J. Crottogini, Gustavo L.V. Javanel.
Introducción
Los vasos sanguíneos son conductos
especializados en transportar la sangre y en
mediar las interacciones entre el contenido de
la luz vascular y el tejido circundante. El
funcionamiento normal de los tejidos depende
del adecuado abastecimiento de oxígeno y
nutrientes, y del lavado de los desechos por
medio de esta función de transporte vascular.
En los últimos años el entendimiento de cómo
se forman los vasos sanguíneos ha pasado a
ser un objetivo primordial y desafiante en la
actividad científica, ya que muchas terapias
podrían basarse en el control localizado de su
crecimiento. En Cardiología la inducción de la
proliferación vascular ha cobrado gran interés
como alternativa para la enfermedad
aterosclerótica coronaria y periférica. A pesar
de los grandes avances logrados en la
prevención y el tratamiento, la cardiopatía
isquémica es la principal causa de muerte en
países desarrollados y subdesarrollados. La
enfermedad vascular periférica, por su parte,
es una condición progresivamente invalidante
y mutiladora que provoca un deterioro grave
en la calidad de vida. Es por esto que el estímulo
del crecimiento de vasos sanguíneos es un
objetivo prioritario de la investigación actual.
Vasculogénesis,
Angiogénesis y Arteriogénesis
La proliferación vascular es un
fenómeno complejo y altamente regulado, en
el que están involucrados diversos mediadores
bioquímicos, algunos inhibidores y otros
estimuladores. (1) El balance entre estos
mediadores regula el proceso. (2) Existen
situaciones fisiológicas (ciclo endometrial,
cicatrización de heridas, etc.) en las que el
balance se inclina transitoriamente hacia el
estímulo y luego retorna al estado basal de
quiescencia. Cuando la regulación no es la
adecuada, la proliferación vascular exagerada
o insuficiente contribuye a la patogénesis de
muchas enfermedades, por ejemplo el cáncer,
la retinopatía proliferativa, las enfermedades
isquémicas o neurodegenerativas, la preeclampsia, etc.(3)
Se han definido ciertos términos que
distinguen los distintos tipos de proliferación
vascular. Se designa vasculogénesis al
desarrollo de un plexo vascular primitivo a
partir de células con alta potencialidad
evolutiva (por ejemplo stem cells). (4)
Inicialmente, este término era reservado para
la formación de nuevos vasos sanguíneos en
la etapa embrionaria, a partir de angioblastos
o hemangioblastos. Sin embargo, actualmente
se conoce la participación de células
progenitoras y precursoras provenientes de la
Departamento de Ciencias Fisiológicas, Farmacológicas y Bioquímicas, Universidad Favaloro, Buenos Aires, Argentina
30
médula ósea en el desarrollo de plexos
sanguíneos durante la vida adulta. Este
proceso es conocido como vasculogénesis
post-natal.(3,5)
El término angiogénesis se ha reservado
para referirse a la formación de capilares (o
vasos sanguíneos de mayor diámetro pero
formados sólo por endotelio) a partir de
conductos pre-existentes formados por células
adultas (capilares o vénulas post-capilares). El
proceso de expansión y remodelamiento de
plexos vasculares endoteliales, generados
inicialmente mediante vasculogénesis, ha sido
también llamado angiogénesis.(6)
En cambio, se denomina arteriogénesis
al crecimiento y formación de arterias y
arteriolas (es decir conductos más importantes,
constituidos no sólo por endotelio sino también
por músculo liso vascular) a partir de otras
arterias. Este es el mecanismo involucrado en
el desarrollo de la circulación colateral, que
tiene un rol importantísimo en la adaptación
de los tejidos a obstrucciones vasculares
progresivas. Clásicamente la arteriogénesis se
refirió a la expansión de pequeñas colaterales
innatas y su remodelamiento en arterias más
grandes. Actualmente se considera que la
generación de vasos arteriales completamente
nuevos también puede ocurrir (formación de
novo de arterias colaterales).7 Incluso existe
evidencia de que el crecimiento de arteriolas
puede resultar del reclutamiento de células
musculares lisas a partir de vasos capilares
preexistentes.(8)
Fisiología de la Angiogénesis
Los mecanismos de la proliferación
vascular no están aún totalmente comprendidos.
Si bien resulta lógico pensar que hay
substancias y pasos comunes a todos los
procesos, se sabe que la angiogénesis ocurre
como consecuencia de la isquemia, la cual
estimula la expresión del factor de transcripción
HIF-1a (hypoxia inducible factor 1a).(9) Este
factor de transcripción a su vez “enciende” genes
que codifican para proteínas vinculadas a la
hipoxia, tales como la eritropoyetina, el VEGF
y sus receptores. El VEGF es un mitógeno de
células endoteliales y el factor de crecimiento
paradigmático de la angiogénesis,(10) aunque
recientemente se han descrito nuevos efectos del
VEGF. Este factor angiogénico estimula la
proliferación y migración de células endoteliales
y su organización tubular. Otros factores de
crecimiento involucrados en la angiogénesis son
el PlGF (placental growth factor, un análogo del
VEGF), el HGF (hepatocyte growth factor, o
scatter factor), los FGF (factores de crecimiento
fibroblástico) tipo 1, 2, 4 y 5, las efrinas y las
angiopoietinas. (1) El PlGF y el HGF son
mitógenos de células endoteliales y promueven
la proliferación de capilares. En cambio los
FGFs son mitógenos de otras células además
de los endoteliocitos, aunque también han
demostrado tener una potente actividad
angiogénica. Las efrinas están involucradas en
el establecimiento de la identidad arterial o
venosa del endotelio vascular,(1) mientras que
las angiopoietinas están directamente
relacionadas con la desestabilización del vaso
sanguíneo (el pasaje a un estado más plástico
que permite la proliferación celular y el
crecimiento de neovasos) y con la ulterior
maduración o re-estabilización del plexo
vascular.(11) En ausencia de ciertos estímulos (por
ejemplo VEGF) los vasos desestabilizados
terminan desapareciendo (regresión vascular).
La regresión vascular y el “podado” (pruning)
de los vasos excedentes son procesos muy
importantes para eliminar los vasos
innecesarios, ya que la arquitectura final de la
red vascular no debe ser insuficiente pero
tampoco excesiva para las demandas del
tejido.(4,12)
Se han descrito dos mecanismos de
angiogénesis: la formación de brotes vasculares
(“sprouting angiogenesis”) y la intususcepción
(“non-sprouting angiogenesis”).(4) Ver figura 1.
En el primer caso el vaso nace en forma de
“brote” en la pared de otro vaso preexistente y
luego comienza a crecer hacia el lugar de donde
31
proviene el estímulo angiogénico. La
intususcepción se refiere a la formación de
puentes o pilares transluminales de matriz
extracelular y endotelio que dividen el vaso
preexistente generando nuevos espacios
intervasculares de tejido intersticial y
consecuentemente nuevos vasos más
pequeños.(13)
Figura 1: Mecanismos de la angiogénesis. A: por brote (“sprouting” angiogenesis); B: por
intususcepción (“non-sprouting” angiogenesis). VEGF: factor de crecimiento de endotelio vascular.
PlGF: factor de crecimiento placentario. VEGFR: receptor para el VEGF. TIE: receptor para
angiopoietinas.
Fisiología de la Arteriogénesis
La arteriogénesis depende principalmente de otros estímulos diferentes a la
hipoxia, tales como la tensión de cizallamiento
(“shear stress”) y la activación de los
monocitos. Ante una obstrucción arterial, el
flujo se desvía hacia las incipientes colaterales
de pequeño diámetro.(6,7) Sobre las paredes
de estas colaterales el shear stress es alto, lo
cual estimula la secreción endotelial de MCP1 (monocyte chemoattractant protein 1). La
MCP-1 actúa sobre el receptor CC de los
monocitos, activándolos y ejerciendo un
efecto quimiotáctico sobre estas células, que
se acumulan en el endotelio y en el espacio
subintimal vascular y secretan distintos
factores de crecimiento, como el VEGF, FGF32
2 (fibroblast growth factor 2), TGF-b1
(transforming growth factor b1), y enzimas,
como colagenasas, metaloproteinasas y
activadores
del
plasminógeno.
Consecuentemente, la membrana basal es
degradada, las células musculares lisas
cambian del fenotipo contráctil al fenotipo
proliferativo y comienzan a dividirse junto con
las otras células de todas las capas del vaso.(14)
Al mismo tiempo, la matriz extracelular va
siendo degradada para permitir el crecimiento
expansivo de la arteria o para permitir el
desarrollo de los neovasos arteriales.
Finalizada la proliferación, la matriz
extracelular y la membrana basal son
resintetizadas, las células musculares lisas y
endoteliales retornan a su fenotipo quiescente
y el vaso es por último estabilizado.
Angiogénesis y
Arteriogénesis Terapéuticas
La inducción terapéutica de la
proliferación vascular puede lograrse de
diversas maneras. Si bien la formación de
nuevos capilares funcionantes contribuye a
mejorar la perfusión tisular, el objetivo debe
incluir la generación de nuevas arterias y
arteriolas. (15 ) Los capilares distales son
imprescindibles para la distribución del flujo
sanguíneo en los tejidos, pero las arterias
proximales son las encargadas de hacer llegar
ese caudal y abastecer el lecho capilar. Según
la ley de Pouseuille, el caudal depende del radio
del conducto elevado a la cuarta potencia. Por
eso, las arterias de conductancia, con su radio
importante, son de enorme relevancia en la
circulación colateral ya que transportan
grandes caudales de sangre, mientras que las
arteriolas son las encargadas de regular qué
proporción del caudal es derivado a cada
tejido. La gran diferencia con respecto a los
capilares radica en que las arterias y arteriolas
poseen, además de mayor diámetro, elastina
y músculo liso vascular en su túnica media.
La túnica media así constituida les confiere
propiedades elásticas, la capacidad de
responder a los estímulos fisiológicos y más
estabilidad y resistencia a la compresión
originada por la contracción sistólica.
La inducción terapéutica de la
proliferación vascular puede lograrse mediante
la administración de factores angiogénicos, es
decir proteínas capaces de gatillar el proceso
(terapia proteica), (16) o de los genes que
codifican para estas proteínas (terapia
génica).(17) Una tercera alternativa ha surgido
recientemente y es la administración de células
con alta potencialidad evolutiva, capaces de
dar origen a las células adultas que formarán
nuevos vasos y de secretar diversos factores
angiogénicos que regularán este proceso
(terapia
celular
o
vasculogénesis
(3,18)
terapéutica).
Aún más, estás células pueden
ser transfectadas con genes codificantes para
factores de crecimiento antes de ser injertadas
(transferencia génica ex vivo). (19) A
continuación discutiremos brevemente las tres
técnicas y citaremos los estudios más recientes.
Terapia Celular
La terapia por implante celular ha sido
investigada con diversos tipos de células, desde
médula ósea fresca hasta células clasificadas
según marcadores de membrana, obtenidas de
la médula ósea (células madre hematopoyéticas
o mesenquimáticas) o de la sangre periférica
(células precursoras endoteliales). (18) Estas
células pueden ser modificadas genéticamente
antes de ser implantadas, para que secreten
intensamente algún factor angiogénico. Ciertas
proteínas movilizan células totipotentes o
precursoras a partir de la medula ósea, por
ejemplo factores angiogénicos, como el VEGF,
o factores hematopoyéticos como el GM-CSF
(granulocyte-macrophage colony-stimulating
factor). Orlic y col. observaron que la
movilización de células de la médula ósea
mediante G-CSF (granulocyte colonystimulating factor) y SCF (stem cell factor) en
ratones con infarto de miocardio inducía la
proliferación de capilares y arteriolas en el tejido
miocárdico.(20) En mamíferos superiores con
infarto agudo de miocardio los resultados de esta
técnica han sido controvertidos: en babuinos
hubo una mejoría en la perfusión miocárdica(21)
pero en monos rhesus no se encontró
diferenciación celular e inclusive hubo mayor
mortalidad.(22) En pacientes con enfermedad
coronaria, el GM-CSF intracoronario y luego
subcutáneo mejoró, en el corto plazo, la
circulación colateral.(23) Sin embargo, no se
demostró cómo actuó el GM-CSF en estos
pacientes.
Terapia Proteica
Los factores de crecimiento son
proteínas, generalmente de pequeño tamaño
y corta vida media, capaces de regular, tanto
33
parácrina como autócrinamente, la migración,
proliferación, diferenciación y crecimiento celular.
Algunos pueden inducir o potenciar la
proliferación vascular ya que estimulan al menos
uno de los pasos descritos más arriba. Los más
estudiados en modelos animales de isquemia
miocárdica crónica y periférica fueron el FGF-2
y el VEGF.(16) En pacientes con enfermedad
vascular periférica, el FGF-2 demostró resultados
positivos a 90 días.(24) En pacientes coronarios,
en cambio, no hubo resultados concluyentemente
positivos,(25,26) fundamentalmente por el marcado
efecto placebo observado en los grupos control,
que dificulta objetivar diferencias con los grupos
tratados. Otras desventajas fueron la corta vida
media y la dificultad en administrar grandes dosis
de VEGF debido a su potente efecto
vasodilatador.
Terapia Génica
La terapia génica se refiere a la
administración o transferencia de material
genético a un paciente con fines terapéuticos.
Cuando el objetivo terapéutico es la inducción
de proliferación vascular, el gen empleado será
el que codifica para una proteína angiogénica
o arteriogénica.(27)
El material genético puede ser
administrado unido a una cadena circular de
ADN desnudo (plásmido) o asociado a
compuestos que facilitan la transfección (ingreso
del material genético a la célula) llamados
“vectores” (virus o liposomas). La principal
ventaja de los virus frente a los plásmidos es la
mayor la eficiencia de transfección, aunque esta
característica se asocia a una respuesta
inflamatoria en el paciente y al riesgo de
respuesta inmune adversa. Esto además
dificulta la administración repetida de genes
transportados en vectores virales. Los plásmidos,
en cambio, son menos eficientes pero más
seguros. Nuevas técnicas de transferencia
génica (virus adenoasociados, nuevos
liposomas) están siendo estudiadas para
mejorar la eficiencia de la transfección.(17)
Diversos autores (entre ellos nuestro
grupo) demostraron que la transferencia génica
de factores de crecimiento es segura e induce
angiogénesis, redundando en una mejoría del
flujo, la perfusión, la función miocárdica, e
incluso la proliferación de arteriolas (figura 2) y
cardiomiocitos (miocardiogénesis).(17,27-30)
Actualmente, ensayos clínicos fase I y II
han demostrado la seguridad y sugerido la
eficacia de la transferencia génica de factores
angiogénicos en la isquemia miocárdica(31,32)y
periférica.(33) Sin embargo, aún se necesitan
estudios con mayor número de pacientes para
poder obtener resultados más confiables.
Figura 2: Microfotografía de miocardio porcino con
neoformación arteriolar inducida por transferencia
de un plásmido codificante para vascular
endothelial growth factor (VEGF165). Obsérvese la
presencia de glóbulos rojos dentro de las arteriolas,
indicando la funcionalidad de estos neovasos.
Barra=20 µm (Reproducido de Crottogini et al.
Vascular endothelial growth factor (VEGF): ¿algo
más que un mitógeno de células endoteliales?.
Revista Argentina de Hemodinamia, Angiografía y
Terapéutica por Cateterismo 2004 (in press), con
permiso del Editor).
34
Comentario Final
En la vida adulta el ser humano tiene la
potencialidad de formar nuevos vasos
sanguíneos. Desentrañar la fisiología de este
proceso es fundamental para usar la
angiogénesis y la arteriogénesis como
terapéuticas de la enfermedad isquémica
coronaria y periférica, o para inhibirla, como
en el caso del cáncer. A pesar de los grandes
avances producidos en la última década, es
mucho más lo que se ignora que lo que se
sabe. Mientras la ciencia nos sigue aportando
información, la medicina ya ha comenzado
a intentar, con los conocimientos disponibles,
la angiogénesis y la arteriogénesis
terapéuticas en el hombre. Los resultados
iniciales no sen espectaculares, pero el
camino a recorrer es largo y el desafío sigue
vigente.
35
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37
UPDATING ARTICLES
Bases Fisiológicas de La Variabilidad
de La Frecuencia Cardíaca
Eduardo R. Migliaro y Paola Contreras
Introducción
Las ciencias de la vida han
experimentado en el último siglo un avance
sustancial, a partir del desarrollo de técnicas
analíticas que ampliaron el conocimiento de
mecanismos celulares y moleculares. Ejemplo
de ello son los avances en el conocimiento de la
actividad eléctrica celular y los canales iónicos,
la expresión de proteínas mensajeras, el papel
del óxido nítrico, la descripción del genoma
humano y otros que han impactado fuertemente
en el campo de la fisiología y de la medicina.
Algunos autores sostienen que estos
avances han alejado a los fisiólogos del estudio
de la función de los órganos en forma
integrada,(1) que es un campo tradicional de la
fisiología.(2) Sin embargo, este campo no debe
ser abandonado, porque la comprensión de las
funciones del ser humano necesita integrar la
actividad de cada órgano en un sistema único
y coordinado.(3) Este sistema integrado es un
sistema complejo, que como tal, da lugar a la
aparición de un orden emergente diferente a
la suma de las partes.(4) Se pueden considerar
a los órganos como osciladores biológicos que
funcionan en forma acoplada y cuyo desacople
genera trastornos de la función del todo, sin
que necesariamente estén afectadas las
partes.(5,6)
El estudio del ritmo cardíaco ha
interesado a los investigadores desde hace
varios siglos,(7) en el siglo XVIII Spthen Hales
hizo la primera descripción de los cambios
cíclicos de la actividad cardíaca y la presión
arterial.
Las modificaciones en estos ciclos
vienen siendo estudiadas como indicadores de
la regulación cardíaca, se ha postulado además
que su estudio es una forma de analizar el
acople entre órganos y por lo tanto puede
considerarse como un índice del nivel de ese
acople.(9, 10,11)
La Variabilidad de la
Frecuencia Cardíaca.
Los intervalos entre los latidos de un
corazón normal, muestran entre sí leves
diferencias de duración que se traducen en
cambios del ritmo cardíaco. Estos cambios en
el ritmo siguen ciertos patrones de repetición,
por lo que las prolongaciones y acortamientos
de los intervalos se repiten de manera cíclica.
Uno de los ejemplos más conocidos es la
arritmia sinusal respiratoria. Esta modifica los
intervalos siguiendo el patrón de la respiración,
lo que impone una frecuencia de variación
relativamente alta si la comparamos con otras
influencias.
Los métodos informáticos han facilitado la
Departamento de Fisiología. Facultad de Medicina. Montevideo. URUGUAY
38
medición y almacenamiento de los intervalos
entre latidos, por lo que resulta sencillo estudiar
su variación. Este tipo de análisis es el que se
conoce como Variabilidad de la Frecuencia
Cardíaca (VFC) y se ha convertido en una
herramienta muy útil para la investigación y el
diagnóstico clínico. (12,13,14,15,16)
Su utilidad deriva de la sencillez de su
registro y de las correlaciones fisiológicas y
patológicas que se han encontrado. En este
último terreno, la VFC ha demostrado ser un
buen predictor de morbimortalidad, (17) en
particular en pacientes que han sufrido infarto
de miocardio, (18,19) pero también en la
diabetes,(20,21) la insuficiencia cardíaca,(22) la
enfermedad de Chagas(23) y la enfermedad
coronaria.(24) Recientemente nuestro grupo ha
demostrado que la VFC tiene capacidad
predictiva, en pacientes críticos que pueden
evolucionar a la disfunción orgánica
múltiple.(11)
Formas de Medir la VFC
La VFC puede ser calculada a partir de
cualquier señal que identifique una fase dada
del ciclo cardíaco, por ejemplo: ruidos,
imágenes ecocardiográficas, doppler y otras
formas de registro de la actividad cardíaca. Sin
embargo, el electrocardiograma (ECG) es la
herramienta más utilizada en virtud de su
difusión y por proveer registros con referencias
muy exactas en el tiempo como lo son las
ondas del complejo ventricular QRS. Por esta
razón es muy frecuente que se identifiquen los
intervalos entre latidos como intervalos R-R, o
también como intervalos N-N (por normalnormal), lo que señala que para calcular la VFC
se usan ondas R “normales” entendiendo como
tales sólo aquellas de origen sinusal.
Disponiendo en un gráfico la duración de los
intervalos N-N en función del tiempo se obtiene
el tacograma que es la base del análisis de la
VFC (Figura 1).
Según la duración del período de estudio
Figura 1: Tacograma formado por la disposición de los intervalos R-R en función del número de intervalo
o su equivalencia en minutos.
39
los métodos de registro pueden ser de pocos
minutos (5 a 10) o de varias horas. Muchos de
los análisis de la VFC se basan en el ECG de
24h (Holter), (16) que es el método más
adecuado para el análisis de VFC en función
de ritmos circadianos, o para la comparación
de la VFC entre la noche y el día. Sin embargo,
cabe consignar que para el diagnóstico de VFC
disminuida en estados patológicos el Holter no
parece tener ventajas frente a métodos de
menor duración.(25, 26,27)
En nuestros estudios utilizamos un
dispositivo que consta de un electrocardiógrafo
convencional, que se conecta a un conversor
analógico digital (A/D) y permite almacenar el
ECG en el disco duro de una computadora
(esquema en Figura 2). Posteriormente
analizamos el registro con un software
especialmente diseñado, que detecta las ondas
R, permite su validación visual, mide los
intervalos entre ellas y finalmente calcula los
índices de VFC.
Figura 2 : Esquema para registro de la VFC usado por los autores.
Índices de VFC. Para la evaluación
numérica de la VFC se han ensayado una larga
serie de índices que se agrupan según la forma de
análisis de la VFC (por revisiones ver citas 8 y
15), a la fecha ninguno de ellos satisface todas las
necesidades. Aludiremos brevemente a algunos
índices útiles para los fines de este capítulo.
1) Índices Estadísticos
a. SDNN: Es un índice muy usado y de
simple definición (el desvío estándar de todos
lo intervalos N-N en la muestra).
b. rMSSD: Muy similar al anterior en
cuanto a la fórmula para calcularlo, pero
sustituye la resta de cada intervalo de la media,
por la resta de dos intervalos adyacentes. Eso
hace que sea un índice muy útil para evaluar
cambios rápidos de la VFC.
40
2) Índices en el Ámbito de la
Frecuencia (Análisis Espectral)
Para realizar el estudio espectral, el perfil
del tacograma se trata como una señal
compuesta por múltiples ondas de diferentes
frecuencias. Se aplican luego métodos como
la transformada rápida de Fourier (FFT),
modelado autoregresivo (ARMA) o métodos
híbridos que generan un espectro de potencias
donde se dispone la potencia (varianza) de
cada onda en función de su frecuencia
(Figura 3).
El espectro se divide en bandas de
frecuencia (ver también Tabla I) y sobre esta
base se estima la densidad espectral de cada
banda. Existen numerosos estudios que
correlacionan las bandas del espectro con
fenómenos fisiológicos. (15)
Figura 3: El análisis del tacograma como una señal compleja permite derivar de él un espectro de
frecuencias. En la parte derecha de la figura se observa un espectro típico de la VFC donde se destacan
dos bandas. La de baja frecuencia (Low Frequency, LF) que abarca el espectro de 0.04-0.15 Hz y las de
alta frecuencia (High Frequency, HF) que abarca el espectro de 0.15-0.40 Hz.
Esta última banda es la que se relaciona con los movimientos respiratorios.
Tabla I. Nombre y unidades de índices espectrales.
Factores Fisiológicos
Involucrados en la VFC
Las células del nódulo sinusal se
influyen mutuamente de modo que generan
un ritmo único pero necesariamente
variable. (28) Esta interacción entre células
marcapaso, es responsable de una primera
forma de variabilidad, muy pequeña si se la
compara con los grandes cambios que se
introducen por la vía de la regulación
extracardíaca.
41
El principal regulador extracardíaco es el
Sistema Nervioso Autónomo (SNA). El
balance entre la rama simpática y la
parasimpática incrementa la variabilidad
propia del nódulo sinusal. Vistos por separado,
el parasimpático tiene el conocido efecto de
incremento de la duración de los intervalos,
mientras que el simpático los disminuye.
Debido a que el parasimpático tiene una
latencia de respuesta menor que la del
simpático(29) su influencia es dominante en las
modificaciones rápidas de la VFC como las
inducidas por la respiración.
Esta dependencia de la VFC con el SNA,
ha llevado a que varios autores consideren que
el análisis de la VFC es una buena medida de
la función autónoma. Es así que los cambios
en la postura,(30) los fenómenos vasomotores
ligados al control baroreflejo de la presión
arterial,(31) o la reacción de alarma(32) tienen
un correlato muy claro en la VFC. También se
ha establecido claramente que la VFC
disminuye con la edad(17,33). Se supone que el
envejecimiento del SNA y de las estructuras
cardíacas pueden estar en la base de este
comportamiento. (34) La figura 4 ilustra la
relación entre los valores de VFC, la edad y la
frecuencia cardíaca.
Además de los neurotransmisores
autónomos más estudiados, acetilcolina y
noradrenalina, existen otras sustancias que pueden
actuar sobre efectores propios o sobre las
terminaciones presinápticas. Entre dichas
sustancias destacamos las purinas y el oxido nítrico
que juegan un papel relevante en la modulación
autonómica.(35,36) También cabe consignar las
relaciones entre SNA y procesos inflamatorios(37)
que seguramente habrán de abrir interesantes vías
de estudio en el futuro inmediato.
Otros autores han puesto en duda ese
papel de “evaluador autonómico” que se le
atribuye a la VFC. (38) Es claro que otras
influencias pueden modificar la función del
nódulo sinusal, entre ellas: la temperatura
actuando en forma directa sobre las células del
nódulo, factores endócrinos y metabólicos y
fenómenos mecánicos.(39)
Uno de los mecanismos de modificación
de la VFC más evidentes y más intensamente
estudiados son los cambios ligados al ciclo
respiratorio. Como ya se mencionó la respiración
impone al ritmo cardíaco un ritmo propio
(arritmia sinusal respiratoria) que tiene un ciclo
relativamente rápido (0.2 Hz aprox.), por lo
tanto se dispone en la zona HF del espectro de
frecuencias (ver Tabla I y Figura 3).
Figura 4: Efecto sobre la VFC (medida por el rMSSD) del incremento de la frecuencia cardíaca en dos
grupos de individuos de edades diferentes. Se puede apreciar la disminución de la VFC al aumentar la
frecuencia cardíaca y además se verifica que los valores de rMSSD son mayores en el grupo más joven.(49)
42
Esta correlación se hace más evidente
cuando la respiración se hace rítmica, como
se observa en la Figura 5. También se expresa
claramente durante la vocalización de algunos
textos religiosos,(40) o en rutinas de relajación
y meditación,(41, 42) situaciones en las que se
controla voluntaria o involuntariamente la
respiración.
En principio se ha sostenido que la
influencia de la respiración está mediada por
el parasimpático que se estimula en la
espiración y se inhibe durante la inspiración.
Al respecto hay estudios que demuestran la
inhibición que ejercen las neuronas
inspiratorias sobre las vagales,(43) el efecto de
los baroreceptores en este terreno también ha
sido profusamente estudiado.(44) Sin embargo,
en los últimos tiempos han cobrado nuevo
impulso los mecanismos relacionados con los
gases respiratorios (45, 46) y con factores
mecánicos, sean estos a partir de receptores
pulmonares(47) o aquellos que responden al flujo
de sangre en la aurícula derecha disparando
el reflejo de Bainbridge.(48)
Conclusiones
Las modificaciones del ritmo cardíaco
han interesado a los investigadores desde
hace siglos. En los últimos tiempos se ha
puesto especial atención al significado de la
VFC como expresión de mecanismos
reguladores que actúan sobre el corazón y el
organismo en general. Las bases fisiológicas
de la VFC aún no han sido esclarecidas en
todos sus detalles, sin embargo se sigue
trabajando intensamente en este terreno para
ensanchar los horizontes del conocimiento y
afianzar el uso de esta herramienta en el
terreno médico.
Figura 5: Registro simultáneo de respiración e intervalos R-R en un individuo normal respirando en forma
rítmica (metrónomo). En color gris se observa el registro del flujo aéreo y en negro se observan las
modificaciones de los intervalos R-R. Se puede apreciar la estrecha correlación entre respiración y VFC
(Migliaro y col. no publicado).
43
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46
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Upcoming Meetings Session
- IACS INTERNATIONAL ACADEMY OF CARDIOVASCULAR SCIENCES
The 2nd World Congress in Japan
Kouseinenkin (Wel City Sapporo), Sapporo - Japan
July 14 - 16, 2006
- 16th WORLD CONGRESS (WSCTS 2006)
Ottawa Congress Centre, Ottawa, Canada
August, 17 - 20, 2006
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Will be held un Pecs, Hungary
September, 28 - 30, 2006
- GLOBAL SYMPOSIUM ON THE HEART HEALTH & DISEASE
Winnipeg, MB. Canada - October 12 - 15, 2006
- BRAZILIAN SOCIETY OF CARDIOLOGY
61st Brazilian Congress of Cardiology
XXII South American Congress of Cardiology
October, 21 - 25, Recife-PE
- INTERNATIONAL CONGRESS OF CARDIOVASCULAR SCIENCES
Scientific Forum XVI - Linked Events:
- INTERNATIONAL CONGRESS OF EXTRACORPOREAL CIRCULATION
- 26th ACCERJ CARDIOVASCULAR SURGERY CONGRESS
- XXIV BRAZILIAN CONGRESS ON EXTRACORPOREAL CIRCULATION
- FORUM ECUMÊNICO VIII / ECUMENIC FORUM VIII
- AMERICAN SOCIETY OF ANGIOLOGY BRAZILIAN CHAPTER - II INTERNATIONAL MEETING
- PROF. DR. NARANJAN S. DHALLA FORUM ON APPLIED CARDIOVASCULAR RESEARCH
- XXII MEETING OF PROF. DR. E. J. ZERBINI DISCIPLES
- V MEETING OF PROF. DR. DOMINGOS J. MORAES DISCIPLES
- I I SCIENTIFIC MEETING OF PROF. DOMINGO M. BRAILE FRIENDS
- VII SYMPOSIUM PROF.DR. TOFY MUSSIVAND
- SYMPOSIUM PROF. DR. PAWAN K. SINGAL
- SYMPOSIUM PROF. DR. RICARDO GELPI
- SYMPOSIUM PROF. DR. BORUT GERSAK
- SYMPOSIUM ON CARDIOLOGY FOR THE FAMILLY
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- IV BRAZILIAN SYMPOSIUM OF THE INTERNATIONAL SOCIETY FOR HEART RESEARCH
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December, 7 – 10, Rio Othon Palace Hotel, Copacabana, Rio de Janeiro-RJ
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49
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50
51
52

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