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CARDIOVASCULARSCIENCES 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)

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Alexandre C. Hueb (Brazil), Antônio S. Martins (Brazil)

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Cardiovasc. Sci. Forum - Jul./ Sep. 2006 - Vol. 1/ Number 3

CARDIOVASCULARSCIENCES FORUM

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:: 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

7 :: INSTRUCTIONS FOR AUTHORS

Page 49 :: UPCOMING MEETINGS SESSION

Page 50 :: PEER REVIEW

Cardiovascular Sciences ForumCardiovasc Sci Forum Jul./Sep. 2006 - Vol. 1 / Number 3

Contents

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Evolution and Challenges in thePhisiopatology of the

Ischemia and Reperfusion

Carlos Henrique Marques dos Santos*

In 1968, McCORD(1) proposed thatenzyme xanthine-oxidase, precursory ofsuperoxide radical and present in many tissues,was able to be related to damage tissuessubmitted to the ischemia.

In 1986, PARKS & GRANGER(2) proposedthat free radicals produced in reperfusion phasewould be the principal patogenes inductors oftissue injury in ischemia and reperfusion, beingthis the main historical mark in the evolution oftissue ischemia and reperfusion.

The ischemia is a condition of decreaseor interruption in the blood supply of oxygenand nutrients to certain area, during a period.With the deficiency of blood supply, it canhappen the tissue death(3). However, thereperfusion has a fundamental participation indevelopment of tissue injury.

PARKS & GRANGER(2) demonstratedthat three hours of ischemia following for onehour of reperfusion determined larger lesionthat four hours of ischemia exclusive.The reperfusion can hurt the organ separatelylike in reperfusion of myocardium after a acutemyocardial infarct(4). However, can to hurtdistant organs too, like in the lung edema afterischemia and reperfusion of extremity (3) .

The ischemia determine a role ofalterations in the celular level that can culminatein celular death. The absence of oxygen impedesthe oxidative fosforilation in mitochondrion, thatis the more efficient way of energy production.

Thus, the anaerobic glicolise be the main sourceof energy, and, being less efficient, is notappropriate ATP consumed reposition. Thedecrease of ATP harm the transport assets ofíons through the membrane, taking theaccumulation of sodium, and, by diffusion,water inside the cell, with consequent edema(5).

The ischemia still determines an increaseof calcium permeability, promoting his entrancein the cell. The increase of the intracellularcalcium, potentiated by the decrease of histransport assets for the extracellular space, ATPdependent, presents several harmful effects:alteration in the form of the cell by contractionof the skeleton; fosfolipases ativation, withconsequent metabolites liberation of aracdonicacid starting from the cellular membrane andof 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, alterationsthat will intensify in the reperfusion suffering.When exposed to the ischemia, the cells alterhis citoeskeleton and his forms, generatingsmall intercellular pores, determining anincrease of the endothelial permeability, couldtake to the formation of tecidual edema(7) .

The endothelial cell, when exposed to ischemia,increase his interleucine-1 and tumoral factor ofnecrosis production, increasing his adhesiveness forleukocytes, although that phenomenon is more evidentafter the reoxygenation (8) .

* Mato Grosso do Sul Federal University - Surgical Department (Prof. Dr. José Carlos D. V. Pontes) and São Francisco de Assis CardiovascularFoundation. Address: Rua Aluízio de Azevedo, 606 - Jardim São Bento - Campo Grande – Mato Grosso do Sul – Brasil - CEP: 79004050,E-mail: [email protected]

EDITORIAL

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The degradation of ATP stocks for theenergy production during the ischemia takesto an elevation of the AMP concentration. Thisis catabolized in adenosine, inosine, and so,hypoxanthine. The hypoxanthine serves as apurine oxidizable substratum to xanthine-dehydrogenase or oxidase, and, parallel, thereis the conversion of xanthine-dehydrogenasein xanthine-oxidase. This conversion is madein an irreversible way, through proteasesactivated by the calcium, or in an reversibleway, for the oxidation of sulfidrile groups. So,during the ischemia we have the formationmainly of hypoxanthine and xanthine-oxidase,that they have fundamental paper during thereperfusion unchaining the process of freeradicals production(5, 9, 10).

The free radicals of oxygen are chemicalspecies characterized by the presence of oneno duplicated electron in the last orbit, actedgraphically by a point. This characteristic cheksit those substances a great capacity of to reactwith others, turning them importants oxidantor reducers agents(11).

The free radicals of oxygen implicated inthe reperfusion lesion are: superoxide anion, thehidroxile radical na the hydrogen peroxide. Theperoxide hydrogen not constituted in a radical,because it doesn’t present free electron in hisorbit. This way, the most correct way to refer thethose substances is denominating them speciesreactivate poisonous of the oxygen (SRPO)(12).

The xanthine-oxidase depends on theoxygen for the metabolization of hypoxanthinein xanthine and superoxide radical. The enzymesuperoxide-dismutase catalyzes the convertionof the superoxide in peroxide of hydrogen, whilethe enzime catalase converts the peroxide ofhydrogen in water and oxygen. In the presenceof iron ions can have the conversion ofperoxide of hydrogen in hidroxile radical(11, 12) .

The SRPO can harm any biochemicalcomponent of the cell, but the fats, proteins (somuch structural as enzymatic) and nucleicsacids are their main objective. As they presentgreat reactivity, the SRPO interact with the first

structures that find, in general the fosfolipidesof cellular membrane or of the organellesmembranes. The reaction of SRPO with thepolinsatured fat acids from cellular membranetakes to the formation of several lipidic radicals(peroxide lipidics, hidroperoxilipidics,malondiadehyde), in a chain of reactions thatculminate with the dysfunction of membraneand cellular damage. That lipidic peroxidationalso promotes fosfolipase A2 ativation that,acting on the fosfolipides of the cellularmembrane, liberates fat acids that aremetabolized by ciclooxigenase, generatingprostaglandins and tromboxane, or bylipoxigenase, generating the leukotrienes(12) .

Besides the direct lesions on the cells, theSRPO participate, with other mediators, suchas leukotrienes, tromboxane A2 and factor ofactivation of interactions among leukocites andendothelium, that provoke increase of capilarpermeability and tecidual damage(13) .

The participation of the leukocites in thereperfusion damage happens for the liberationof substances trhough own degradation. Enterthese substances, some are free radicals. Thepolimorfonuclears posses nicotinamida adeninefosfate oxidase capable to reduce the moleculeof oxygen, generating the superoxide anion. Theleukocites produces still proteolitics enzimes,including elastase, colagenase and gelatinase,that participate in the tecidual lesion (14).

The compression of the capillary bed bytecidual and endothelium cells and also for theinterstice, all edemaciate during the ischemia,can take to the bankruptcy of reperfusion ofcertain segments of microcirculation, with focaltecidual hypoxia, being this another mechanismof tecidual lesion after the reperfusion(15) .

With the knowledge of this sequence ofevents, it is believed that can have forms ofacting in some level of chain reaction ofprocess of ischemia and reperfusion, in wayto inhibit the tecidual injury. This is themotivation of countless works that for objectiveto find one or more substances capable to blockthe formation or action of the free radicals.

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BURNS DK, GOLDSTEIN A, STERN D.Hypoxia-mediated induction of endothelial cellinterleukin-1. An autocrine mechanismpromoting expression of leukocyte adhesionmolecules on the vessel surface. J Clin Invest1992; 90: 2333-9.

9. SCHANAIDER A, PERROTTA U, MADI K.Importância dos radicais livres derivados dooxigênio na fisiopatologia das afecçõesisquêmicas 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 inpostischemic tissue injury. N Engl J Med 1985;312(3): 159-63.

12. HALLIWELL B. Reactive oxygen species in livingsystems: source, biochemistry, and role in humandisease. Am J Med 1991; 91: 14-22.

13. TEDDER TF, STEEBER DA, CHEN A, ENGELP. The selectins: vascular adhesion molecules.FASEB J 1995; 9: 866-73.

14. ROOS D. The involvement of oxygen radicalsin microbicidal mechanisms of leukocytes andmacrophages. Klin Wochenschr 1991; 69:975-80.

15. MENGER MD. Microcirculatory disturbancessecondary to ischemia-reperfusion. TransplProc 1995; 27: 2863-5.

BIBLIOGRAPHICAL REFERENCES

1. McCORD JM. Oxygen derived free radicals inpost-ischemic tissue injury. E Engl J Med 1985;312: 159-63.

2. PARKS DA , GRANGER DN. Contributions ofischemia and reperfusion to mucosal lesionformation. 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. Myocardialconsequences of reperfusion. Prog CardiovascDis 1987; 30: 23-44.

5. PARKS DA, GRANGER DN. Xanthineoxidase:biochemistry, distribuition andphysiology. Acta Physiol Scand 1986; 548:87-99.

6. OKUDA M, LEE HC, CHANCE B, KUMAR C.Role of extracellular Ca 2+ in ischemia-reperfusion injury in the isolated perfused ratliver. Circ Shock 1992; 37: 209-19.

7. OGAWA S, GERLACH H, ESPOSITO C, PASAGIAN-MACAULAY A, BRETT J, STERN D. Hypoxiamodulates the barrier and coagulant function ofcultured bovine endothelium. J Clin Invest 1990;85: 1090-8.

8. SHREENIWAS R, KOGA S, KARAKURUM M,PINSKY D, KAISER E, BRETT J, WOLITZKY BA,NORTON C, PLOCINSKI J, BENJAMIN W,

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Adult Human Vascular Endothelial CellsSeeded onto No-React® Treated Bovine

Internal Mammary Arteries: An in Vitro Study

P. M. Dohmen*, M. Stein-Konertz, S. Posner, W. Erdbrügger, W. Konertz

ORIGINAL ARTICLES

ABSTRACT ----------------------------------------------------------------------------------------------------

Background: Alternative grafts are under investigation as the number of patients with re-operations and insufficient autologous bypass material increases. This study was performed to compareendothelial 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 pre-coating. Similar the polytetrafluoroethylene graft were divided into two groups, group IIIendothelial cell seeded and group IV endothelial cell seeded with fibrin glue pre-coating. Graftswere mounted during seeding and rotated for up to 3 hours. During the conditioning phase, acontinuous surveillance of the circulating medium was performed and adjusted to maintainoptimal cell viability.

Results: Two million endothelial cells were inserted for each grafts. Seeding endothelialcell 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 x105 cells/cm². After coating the grafts with fibrin glue, cell density significantly increased in groupII 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 endothelialcell loss after the conditioning phase.

Conclusions: It seems to be possible to seed endothelial cells onto bovine internalmammary arteries. Endothelial cell density almost doubled as compared to polytetrafluoroethylenegrafts and seems to favor biological graft matrices.

Key Words: internal bovine mammary artery, anti-calcification, coronary bypass surgeryendothelial 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]

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Introduction

In 1967, Favaloro performed the firstsaphenous vein graft implantation in a patientsuffering from coronary heart disease(1). As thelong-term patency rate of this graft is limited(2),several autologous vessels have been studied.Since the eighties, the internal mammary arterybecame the golden standard for coronary arterybypass grafting, showing most favourablepatency rates(3). Although results improved byusing different autologous graft material, thenumber of patients with previous operation,extended varicosis, previous varicosis-stripping,or a history of thrombophlebitis increases(4) andso alternative non-autologous grafts sources areneeded. Polytetrafluoroethylene (PTFE) graftshave been used in coronary heart disease,however the patency rates of these smalldiameter grafts are extremely poor(5).Alternatively to these prosthetic grafts smalldiameter bovine mammary arteries were used,however patency rate was only 15.8% after 23months of implantation(6). Our group(7) was ableto increase the patency rate of 4 mm diameterPTFE grafts by seeding the grafts with autologousendothelial cells. Follow-up showed a patencyrate of up to 81 % at five years. Although theseresults are encouraging the no-touchimplantation technique and the handling of stiffPTFE material makes surgery extremelydemanding. Biological tissue, which can bemanipulated more easily during surgery, withoutdestroying the seeded vascular endothelial cells(VEC) layer at the inner surface, may overcomeimplantation difficulties of PTFE grafts.

This in vitro study evaluates thepossibility to seed VEC onto small diameterbovine internal mammary arteries (SIMA),treated with glutaraldehyde and afterwardsneutralized with the No-React® treatment(8).

Materials and Methods

This study was approved by the EthicalCommittee of the Charité. Adult human VEC

were harvested out of leftovers from saphenousveins, which otherwise would have beendiscard.

The 4.0 mm SIMA’s were treated withglutaraldehyde and afterwards detoxified by theNo React® treatment (Shelhigh Ltd., Newark,USA)(8). The internal diameter of the used PTFEprostheses (Medino GmbH, Gehrden,Germany) was also 4.0 mm.

Endothelial cellharvesting and cultivation

Human pieces of great saphenous vein,with a length of 4-8 cm, were transported tothe cell culture laboratory. VEC were harvestedas previously described(9,10). In brief, separationof the VEC was performed by usingCollagenase II 0.1% (Boehringer IngelheimPharmaceuticals, Inc., Ridgefield, Conn) for 15minutes under cell culture conditions in ahumidified incubator (37°C, 5% CO2, and 98%air saturation). A suspension of VEC werecollected and centrifuged at 500g for 10minutes. Total culturing time was 2 to 3 weeksusing 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 growthfactor (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 VECgrowth was evaluated by daily microscopicexamination. The Casy 1 cell-counter (SchaeferSystem GmbH, Reutlingen, Germany) wasused for VEC cell counting.

Graft preparationGraft coating

Graft coating was performed by the useof Tissuecol Duo S (Immuno, Baxter,Unterschießheim, Germany) to increasebinding capacity of VEC to both graft-types of

11


group II and IV. Grafts were cannulated at bothsides and the fibrin component was injectedfirst. Afterwards the thrombin component wasinjected into the graft and a 4mm diameterFogarty catheter was used to assure a smoothinner surface. Residual clumps were carefullyflushed with physiological solution. Grafts werekept in medium.

Static graft seeding

The grafts were fixed at both sides witha running 5-0 Prolene suture-line (Ethicon Inc,Sommerville, NJ) in a special developed bio-inherent bioreactor. The bioreactor was filled

Figure 1 - Schematic drawing of the static seeding phase of VEC onto the No-React® treated bovineinternal mammary artery. 1. Bioreactor. 2 - Vascular graft prosthesis. 3 - Rotating unit. 4 - Humidifiedincubator. 5 - Driver unit. 6 - Filtersystem

with VEC and placed into a biostrabilisator(Biegler Medizinelektronik GmbH, Mauerbach,Austria) to turn the graft in a calculated wayduring a period of three hours at cell cultureconditions (37°C, 5% CO2 and 98% airsaturation) (figure 1). A sedimentationtechnique was used, allowing VEC to bind atthe inner surface of the grafts. The final seededgraft was stored in a humidified incubator(37°C, 5% CO2 and 98% air saturation) foranother 7 to 10 days, to improve VECconfluence. The cell seeding density wascalculated by counting the total number of VECprovided into the graft minus the remaining VECin solution after seeding.

Graft conditioning

After the seeding, the bioreactor includingthe grafts were placed into a circulatory systemusing a 10 ml disposable pump (Medos AG,Aachen, Germany). During the conditioningphase the flow was increased until a maximumflow of 0.2 L/min was achieved. The totalduration of the conditioning phase wascompleted after 1 hour. Cell density at the grafts

was again calculated as well as the endothelialcell viability. Finally the seeded grafts werefixated in 10% formalaldehyde for histologicalexamination.

Histological follow up

Immunohistochemical staining wasperformed with factor VIII-related antigen(DAKO, Hamburg, Germany) at the VEC, to

12

show that the cultured cells were exclusivelyendothelial cells, without contamination ofinterstitial cells.

Giemsa (Sigma Chemical Co, St.Louis,Mo )and hematoxylin and eosin (HE) stainingwas routinely performed in four micrometerthickness longitudinal sections. After seeding,the first samples were taken. Next samples weretaken after the conditioning phase in bothgroups. Also there was a documentation of theseeding density of the SIMA and the PTFEgrafts after the conditioning phase, to documentthe findings of the VEC counting.

Statistics

Quantitative data were expressed asmean and standard deviation. Comparisonsbetween the groups were made with the t-test.The level for statistical significance was set ata p-value < 0.05. Data management andstatistical analysis was performed with SPSS10.0 (SPSS Inc., Chicago, USA).

Results

Endothelial cellharvesting and cultivation

After a period of 2 to 3 weeks at least 2 x106 endothelial cells were available, which wasfound to be a sufficient number to seed a 4.0cm grafts with a diameter of 4.0 mm. Medianendothelial cell viability was 95.5% (range 93.4to 97.7%).

Graft preparationGraft seeding

The VEC binding capacity after seedingwas in group I 1.29 ± 0.09 x 105 cells/cm² andin group II 2.27 ± 0.17 x 105 cells/cm². The useof fibrin glue significantly increased the bindingcapacity of endothelial cells in the BIMA grafts(p<0.003). In group, endothelial bindingcapacity III was 0.84 ± 0.11 x 105 cells/cm²

and in group IV 1.35 ± 0.08 x 105 cells/cm².The use of fibrin glue increased endothelial celldensity at the inner surface of the graft(p<0.002).

Graft conditioning

The VEC binding capacity after theconditioning phase was for group I 1.08 ± 0.13x 105 cells/cm² and in group II 2.22 ± 0.16 x105 cells/cm². The decrease of endothelial celldensity after the conditioning phase in group I16.3% and in group II 2.2 %. In group III, theVEC 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 theconditioning phase in group III was 14.3 % andin group IV was 3.0 %. There was a significantdecrease of endothelial cell binding betweengroup I and III (p< 0.016) and between groupII and IV (p<0.008) of VEC binding after theconditioning phase as using different matricesto bind cells onto, however within the groupsthe decrease was never statistical significant.

Histology

Immunohistochemical staining showedthat the cells which were cultured at the tissuelaboratory were a monoculture of endothelial,confirming the absence of contamination withinterstitial cells (figure 2).

Figure 2. Factor VIII staining of the VEC cell culture.

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With Giemsa staining, histologicalvisualization of the different cell density afterseeding could be documented in all groups. Ingroup I, VEC density binding to the graft washigh, as there was a monolayer of endothelialcells seen at the inner surface. However therewere free spots at the graft inner surface whichdidn’t show a confluent covering endothelialcell layer. After the conditioning phase therewas a certain cell loss seen due to the shearstress of the flow. With the use of fibrin glue,the number of endothelial cells seeded onto theBIMA graft increased 1.7 times and there wasa confluent monolayer of endothelial cells atthe inner surface. Even after giving shear stressto the endothelial cells there was only aminimum loss of VEC and a confluentmonolayer could still be seen in group II (figure3). In group IV, there was also a monolayer ofVEC seen after the seeding, however the cell-free spots were more frequent, especially if nofibrin glue was used (group III) prior to theendothelial cell seeding. After the conditioningphase the number of cells even furtherdecreased, and consequently cell-free areasincreased.

Using HE staining it was possible toshow that in both groups not only theendothelial cells were closely attached to eachother, but also to the graft (figure 4).

Figure 3. HE staining of the VEC seeded SIMAafter the conditioning phase, which shows aconfluent monolayer.

Figure 4. Giemsa staining of the VEC seeded SIMAafter the conditioning phase, which shows aconfluent monolayer.

Discussion

Alternative graft material is needed insituations of absence of sufficient autologousgraft material. Xenogenic graft materials havebeen introduced experimentally as well asclinically (11,12,13,14). Major problems rose bytissue failure, due to aneurysm formation of thebiological tissue. Dardik et al(15) startedumbilical vein graft fixation with the use ofglutaraldehyde which should overcome tissuedegeneration. During the same period of timebovine internal mammary arteries, afterglutaraldehyde fixation, were implanted intopatients. Unfortually these grafts showed a highincidence of biodegeneration, calcification andthrombosis(16,17). It has been shown thatglutaraldehyde treatment leads to tissuedegeneration after years of implantation, whichis well known in bioprosthetic heart valvereplacement(18,19). Gabbay et al(8) developed ananit-mineralization technique forglutaraldehyde fixed material, so called No-Reactâ procedure. This treatment shouldovercome calcification of material and soprolong the functionality of tissue valveprostheses and bovine internal mammaryarteries. There are several papers publishedabout the successful elimination of tissuecalcification by the use of No-Reactâ

14

treatment(20), including a recent paperdescribing the use of bioprosthetic heart valvesin children with good hemodynamic results hasbeen reported(21).

Another disadvantage of the use of bovinemammary arteries, before anti-mineralizationtreatment, is high thrombogenicity and sodecrease of patency rate after implantation. Thenatural barrier of vessels are viable endothelialcells, which have showen antithromboticproperties. Our group(22) showed in a clinicaltrial the use of small diameter PTFE grafts duringcoronary bypass surgery in patients who hadno suitable graft material. Through seeding withautologous endothelial cells we were able toincrease the patency rate up to 90.5 % at 4 yearsof follow up.

This feasibility study was performed toinvestigate the possibility to seed endothelialcells on No-Reactâ detoxified glutaraldehydetreated bovine mammary arteries. The medianviability of the seeded endothelial cells was95.5% which shows that No-Reactâ treatmentnot only is able to overcome calcification ofglutaraldehyde tissue, but also detoxifiesglutaraldehyde treated tissue. In vitro it seemsthat this treatment is highly efficient as theabsolute number of VEC binding to the BIMAmatrix was significantly higher as comparedto the group with PTFE grafts. This wasindependent from pre-coating. The number ofVEC binding to the SIMA group was 1.5 timeshigher compared to similar treated PTFE grafts.Even if the PTFE grafts were pre-coated the

absolute number of VEC have been almostsimilar to the SIMA matrix. On the other handif the grafts of both groups were pre-coated theabsolute number of VEC at the SIMA matrixwas 1.7 times higher then the PTFE grafts.This in vitro study showed also that the absolutenumber of VEC seeding onto a graft is moredepending on the graft material, the matrix hasbeen attached to. More important seems thefibrin glue pre-coating during the conditioningphase. The number of VEC drops in group I16.3% without pre-coating performed. Pre-coating increased the binding density of VECand only 2.2% were eliminated by flow. Whenthe PTFE grafts of group II has evaluatedsimilarly, the number of VEC decreased 14.3%without pre-coating. Coating the PTFE matrixwith fibrin glue, also improved the attachmentof VEC with a loss of 3.0%.

In summary, this in vitro study showedthat the use of fibrin glue pre-coating is able todecrease the VEC loss after the conditioningphase, however the absolute number of VECseems to be more depending of the materialthe matrix has been made of. The No-Reactâ

treatment of glutaraldehyde fixed tissue seemsto be efficient to allow endothelial cells to coverthis graft as a monolayer.

Acknowledgements:

We would like to thank Mrs. Krüger forher excellent work in the laboratory and in theculturing of endothelial cells.

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REFERENCES

1. Cooley DA. In Memoriam: Tribute to RenéFavaloro, Pioneer of Coronary Bypass. TexHeart Inst J 2000;27(3):231-232.

2. Lytle BW, Loop FD, Taylor PC, SimpfendorferC, Kramer JR, Ratliff NB, Goormastic M,Cosgrove DM. Vein graft disease: The clinicalimpact of stenoses in saphenous vein bypassgrafts to coronary arteries. J Thorac CardiovascSurg 1992;103:831-40.

3. Bergsma TM, Grandjean JG, Voors AA,Boonstra PW, den Heyer P, Ebels T. Lowrecurrence of angina pectoris after coronaryartery bypass graft surgery with bilateralinternal thoracic and right gastoepiploicarteries. Circulation 1998;97:2402-5.

4. Deaton DW, Stephens JK, Karp RB, GamlielH, Rocco F, Perelman MJ, Liddicoat JR, GlickDB, Watkins CW. Evaluation of cryopreservedallograft venous conduit in dogs. J ThoracCardiovasc Surg 1992;103:153-62.

5. Chard RB, Johnson DC, Nunn GR, Cartmill TB.Aorta-coronary bypass grafting withpolytetrafluoroethylen conduits: Early and lateroutcome in eight patients. J Thorac CardiovascSurg 1987;94:132-4.

6. Mitchell IM, Essop AR, Scott PJ, Martin PG,Gupta NK, Saunders NR, Nair RU, WilliamsGJ. Bovine internal mammary artery as aconduit for coronary revascularization: long-term results. Ann Thor Surg 1993;55(1):120-2.

7. Konertz W, Koch C, Dohmen PM, Laube H,Rutsch W. Five year follow up of patientsreceiving tissue engineered coronary arterybypass grafts. Circulation 2001;104(17): SupplII:362.

8. Abolhoda A, Yu S, Oyarzun R, Allen K,McCormick J, Han S, Kemp F, Bogden J, Lu Q,Gabbay S. No-react detoxification process: asuperior anticalcification method forbioprostheses. Ann thor Surg 1996;62:1724-1730.

9. Dohmen PM, Meuris B, Flameng W, KonertzW. Influence of ischemic time and temperatureon endothelial cell growth after transport. IntJ Artif Organs 2001;24(10):281-285.

10. Dohmen PM, Ozaki S, Verbeken E, YpermannJ, Flameng W, Konertz W. Tissue

engineering of a pulmonary xenograft heart valve. Asian Cardiovasc Thoracic Surg 2002; 10:25-30.

11. Dale WA, Lewis MR. Modified bovineheterografts for arterial replacement. Ann Surg1969;169:927-46.

12. Slovin Ja. Arterial below the knee bypass grafts.Experience with the modified bovineheterograft. Am J Surg 1974;128:58-64.

13. Cutler BS, Thompson JE, Patman RD, PerssonAV, Manfredi PD. The modified bovine arterialgraft: a clinical study. Surgery 1974;76:963-973.

14. Keshishain JM, Smith NP, Adkins PC, Camp F,Yahr WZ, Hill L. Clinical experience with themodified bovine arterial heterograft. JCardiovasc 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 ofexperience with glutaraldehyde-tanned humanumbilical cord vein graft for lower limbrevascularization: An analysis of 1275 cases. JVasc Surg 2002;35:64-71.

16. Rosenberg N. The bovine arterial graft and itsseveral applications. Surg Gyn and Obstr1976;142:104-108.

17. Dale WA, Lewis MR. Further experiences withbovine arterial grafts. Surgery 1976;80:711-721.

18. Riddle JM, Magilligan DJ, Stein PD. Surfacemorphology of degeneration of porcinebioprosthetic valves four to seven yearsfollowing implantation. J Thorac Cardiovasc Surg1981;81:279-87.

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19. Bengtsson L, Radegran K, Haegerstrand A. Invitro endothelialization of commerciallyavailable heart valve bioprotheses with culturedadult human cells. Eur J Cardio-Thorac Surg1993;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.

21. Marianeschi SM, Iacona GM, Seddio F, AbellaRF, Conduluci C, Cipriani A, Iorio FS, GabbayS, Marcelletti CF. Shelhigh No-react porcinepulmonary valve conduit: a new alternative tothe homograft. Ann Thorac Surg 2001;71:619-623.

22. Laube HR, Duwe J, Rutsch W, Konertz W.Clinical experience with autologous endothelialcell-seeded polytertafluoroethylene coronaryartery bypass grafts. J Thorac Cardiovasc Surg2000;120:134-141.

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Pravastatina e Síndrome daPravastatina e Síndrome daPravastatina e Síndrome daPravastatina e Síndrome daPravastatina e Síndrome daRRRRResposta Inflamatória Sistêmica poresposta Inflamatória Sistêmica poresposta Inflamatória Sistêmica poresposta Inflamatória Sistêmica poresposta Inflamatória Sistêmica por

Circulação ExtracorpóreaCirculação ExtracorpóreaCirculação ExtracorpóreaCirculação ExtracorpóreaCirculação Extracorpórea

G. F. Teixeira Filho, J.R.M. Sant´Anna, P.R.Prates,R.A.K. Kalil, A.H. Neto, M. Santos, I. Nesralla

ORIGINAL ARTICLES

RESUMO ------------------------------------------------------------------------------------------------------

Objetivo - Avaliar a possível ação antiflamatória da pravastatina em um modelo bemdefinido de inflamação que é a síndrome de resposta inflamatória decorrente da CEC. Por tantoforam dosados mediadores pró-inflamatórios interleucina 6, interleucina 8, TNF-a e proteína Creativa antes e após a CEC e a drenagem mediastinal pós-operatória.

Material e Métodos - Foram selecionados 20 pacientes portadores de cardiopatiaisquêmica e candidatos a cirurgia de revascularização do miocárdio. Dez pacientes receberam80 mg de Pravastatina 36 e 12 horas antes da cirurgia (grupo P) e dez pacientes foram alocadoscomo grupo controle (grupoC). As amostras foram coletadas antes, logo após a CEC, 6, 12 e 24horas após. O teste Mann-Whitney foi empregado para testar diferenças entre grupos em cadatempo de coleta da amostra. Para testar a diferença no mesmo grupo de paciente foi empregadoo 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 maisbaixos do ue o grupo controle p=0,004. Em relação aos níveis plasmáticos de TNF-a e interleucina6, não houve significância estatística entre os dois grupos. O grupo “P” mostrou diminuiçãosignificativa 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 comparadoao grupo controle p=0,019.

Conclusão - Os resultados encontrados em nosso trabalho sugerem que a pravastatinaapresenta atividade antiflamatória devido a redução dos níveis plasmáticos de proteína C-reativae interleucina 8 e, que provavelmente a sua ação seja a nível da ativação endotelial expressapelos níveis reduzidos de interleucina 8 principal citocina envolvida na ativação depolimorfonucleares.----------------------------------------------------------------------------------------------------------------------

Instituto de Cardiologia do Rio Grande do Sul - Unidade de Pesquisa - Dr. Guaracy F. Teixeira FºAv. Princesa Isabel, 395 - Santana - Porto Alegre Zip 90.620-001Phone/Fax.: 00-55-51-230.3600 Ext.3777 e-mail:[email protected]

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Introdução

Circulação extracorpórea (CEC) éessencial em grande número de cirurgiascardíacas. Está associada com reaçãoinflamatória que pode resultar em disfunçãode órgãos, retardo na recuperação ou mesmoóbito do paciente(1-3). Esta resposta inflamatóriacomplexa inclui a ativação de complemento,liberação de endotoxina, liberação de cininas,ativação de leucócitos bem como a expressãode moléculas de adesão e a produção de váriassubstâncias, incluindo-se radicais queconvertem as células endoteliais a um estadoativo. A ativação imediata da célula endotelialé devida a degradação do complementocirculante, sendo o evento mais significativoda interação do sangue com o circuito deCEC(1). Posteriormente, as células endoteliaissão ativadas pelos mediadores inflamatórios,como as citocinas ou lipopolissacarídeos(4).

A ativação do endotélio vascular tem umpapel determinante na resposta sistêmica quese segue a CEC(5). Nos anos recentes, dadosexperimentais e de observação demonstrandoque a terapêutica com pravastatina reduz onúmero e a atividade de células inflamatóriaspresentes nas placas ateroscleróticas permiteminferir que esta substância pode mostrar açõesantinflamatória importante(6).

Nossa hipótese é que a pravastatinareduza a resposta inflamatória da CEC, sendoobjetivo deste estudo prospectivo randomizadoinvestigar se a pravastatina afeta a liberaçãode mediadores pró-inflamatórios em pacientessubmetidos a cirurgia cardíaca com CEC.

Pacientes e Métodos

Pacientes

Após aprovação pelo Comitê de Ética dainstituição, 20 doentes com indicação de cirurgiade revascularização miocárdica com CEC foramconsiderados no estudo, seguindo-se a obtençãode consentimento pós-informação. Pacientes

com infecção ativa ou recente, transfusãosanguínea, infarto do miocárdio prévio, cirurgiacardíaca prévia ou que utilizaram drogasredutoras de lipídeos nos últimos 3 meses foramexcluídos. Dez pacientes receberam 80 mg V.Ode pravastatina 36 horas e 12 horas antes dacirurgia (grupo P) e 10 pacientes foramconsiderados como grupo controle, nãorecebendo a medicação (grupo C).

Técnica operatória

Todos os pacientes foram pré-medicados com sulfato de protamina (0.2mg/Kg IM) e sulfato de atropina (0.5 mg IM).A anestesia foi induzida com citrato defentanil (10 mg/Kg EV) e tiopetal sódico (3mg/Kg EV). Relaxamento muscular foiinduzido com brometo de pancurônio (0.10mg/Kg EV). Foi iniciada ventilação mecânicae a anestesia suplementada pela inalação dehalotano a 0.4 %.

Monitorização operatória (ECG, pressãoarterial, pressão venosa central, débiotourinário, temperatura nasofaringea e retal) foiidentica em todos os pacientees. Cefalotina foiusada como antibiótico profilático antes da daesternotomia (2.0 g EV e 1.0 g EV antes doinício da CEC). Hidrocortizona (500 mg EV)foi administrada em todos os pacientes apósindução da anestesia.

Os componentes do sistema de CECconsistiram de um oxigenador de membranacapilar composta de fibras resistentes aoplasma (Maxima For te; Medtronic, Inc.Anaheim, California), reservatório decardiotomia, reservatório de cardioplegia efiltro arterial (Macchi Biomedical Eng. SãoPaulo, SP). Estes componentes foramconectados por tubos de cloreto de polivinila(Macchi Biomedical Eng. São Paulo, SP).O volume de enchimento consistiu de 2 L desolução eletrolítica, sendo administradoconcentrado de hemácias quando ohematócrito era inferior a 20%. Antes do inícioda CEC, heparina (4 mg/Kg EV) era

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administrada para prolongar o tempo decoagulação ativado (TCA) acima de 600segundos. CEC não pulsátil foi estabelecidacom fluxo de 2,4 L/m2 e hipotermia moderada(32ºC nasopharyngeal) .

Preservação miocárdica foi obtidamediante infusão de solução cardioplégicacristalóide hipotérmica (St. Thomas II, 4ºC)na raiz aórtica, após pinçamento deste vaso,em dose única (300 mL/m2). Aquecimento foiiniciado durante a conclusão das anastomosesdistais e a pinça aórtica removida a seguir.Anastomoses proximais foram realizadas comoclusão parcial da aorta ascendente. CEC foisuspensa, o sangue remanescente no oxigenarfoi transfundido e a heparina revertida comsulfato de protamina EV. Não foi empregadaultrafi l tração ou outro tipo dehemoconcentração.

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 foramretiradas do cateter venoso central antes deCEC (após indução da anestesia|), após CEC(10 minutos depois da reversão daanticoagulação pela protamina) e 6, 12 e 24 hapós CEC. Apenas CRP foi dosada com 24após CEC e as demais avaliações efetuadasaté 12 h pós CEC.

As amostras foram coletadas em tubosde ácido tetraceticodiaminoetileno (EDTA),imediatamente centrifugadas a 1000 xg por 10minutos e guardadas a –20 ºC até que asavaliações fossem efetuadas. Imunoensaiospara TNF-a, IL-6 e IL-8 foram realizados comkits disponíveis no comércio (R&D Systems,Mineapolis, MN), de acorco com as instruçõesdo fabricante. Concentrações de proteína C-reactiva (CRP) foram medidas usando-se kitscomerciais (Turbiquant CRP, Dade Behring,Marburg, Germany).As variáveis observadas para avaliar evolução

clínica dos pacientes incluíram a drenagemmediastinal e o tempo de internação hospitalar.

Análise Estatística

Os resultados são apresentados comomediana e os quartils superior e inferiorindicados em parênteses. A apresentação deresultados diferentes do acima são indicados.O teste U de Mann-Whitney foi usado paraidentificar diferenças entre grupos em cadaintervalo observado. O testes de Wilcoxon foiusado para identificar diferenças dentro decada grupo. Em todos os casos, um valor de pinferior a 0,05 foi considerado significativo.

Resultados

As características intraoperatórias dosdois grupos de pacientes estão mostradas natabela 1.

Por razões técnicas, CRP não foiavaliada em 2 pacientes no intervalo de 24 hapós CEC. Em um paciente, pelo mesmomotivo, CRP, TNFa, IL-6 e IL-8 não foi medida6h após CEC.

Mediadores inflamatórios

PCR– Proteína C-reactiva teve valormediano antes da CEC de 5,0 (5,0 – 9,3) nogrupo C e de 9,9 (7,0 – 15,6) no grupo P. Estadiferença é estatisticamente significativa(p=0.015).

Níveis plasmáticos de PCR nãomostraram diferenças significativas entreambos os grupos imediatamente, 6 e 12h apósCEC. Níveis plasmáticos foram 5.0 (5.0/5.6),7.7 (5.8-10.2) e 31.9 (25.3-37.5) mg/dl nogrupo 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 Papresentou um valor significativamente inferiordo que o grupo C: (38.7-73.6) contra 109.0(104.0-112.0) mg/dL (p = 0.004),respectivamente (figura 1).

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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.

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TNFa - Níveis plasmáticos do fator-alfade necrose tumoral não mostrarammodificações significativas nos períodosavaliados, para ambos os grupos. Nãoocorreram variações significativas nacomparação entre grupos. Níveis plasmáticosmédio 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 paraantes de CEC, imediatamente, 6 e 12h apósCEC. 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 domesmo grupo e entre grupo).

IL-6 - Em ambos os grupos, os níveisde inter leucina 6 levels aumentaramsignificativamente, se comprados comvalores prévios à CEC. Níveis plasmáticosde IL-6 foram 18.9 (8.2-27.5), 51.4 (40.6-74), 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, imediatamenteapós e 6 e 12 h após CEC. Não foramencontradas diferenças significativas entregrupos em qualquer dos períodos deavaliação (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 foramde 330 (280-730), 770 (560-1505), 600 (380-730) and 705 (510-1290) mg/dL ino grupo Ce de 240 (107.5-430), 277.5 (167.5-510) 144(122.5-200) e 295 (195-1129) mg/dL no grupoP, para avaliações antes, imediatamente após,

6 e 12 após CEC. Não foram encontradasdiferenças significativas nas avaliações pós-CEC se comparadas com controle prévio parao grupo P. Mas no grupo C ocorreu umadiferença significativa para o valor de 12 h, secomparado ao valor prévio a CEC (p=0.007).

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elementos inflamatórios circulantes sãocomplementos, citocinas e proteínas de faseaguda(3,7). Existe uma variedade equipamentosutilizados em cirurgia cardíaca que conduzema ativação sistêmica do complemento.

O evento precoce da ativação docomplemento, que está baseado em umacascata enzimática comparável a vista nacoagulação sangüínea, pode ser deflagradoem duas rotas(4). A rota clássica é a ativaçãopelos complexos antígeno-anticorpo e a rotaalternativa é a ativação pelas paredescelulares bacterianas e por superfíciesestranhas.

A exposição do sangue ao CECrepresenta a rota de ativação alternativa,enquanto que a reversão de heparina pelaprotamina é uma rota clássica(8). Liberação deendotoxina na circulação pode ativar ambasas rotas clássica e alternativa(9).

Com 6 h após CEC, o valor registradopara o grupo P foi significativamente inferiorao registrado para o grupo C : 144 (122,5-200) vs. 600 (380-730) mg/dL p=0,017(figuras 3 e 4).

Variáveis clínicas - Não foramencontradas diferenças no período deinternação em ambos os grupos. A média dehospitalização foi de 8.0 (7.0-11.0) para ogrupo C e de 7.5 (7.0-8.5) dias para o grupoP; p = 0.247. Drenagem mediastinal foisignificativamente inferior para o grupo P doque para o grupo C : 600 (395.0-835.0) ml vs990 (800.0-1070.0) ml; p = 0.019.

Discussão

Diversos estudos registraram oenvolvimento de mediadores solúveis naresposta inflamatória patológica da CEC. Estes

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.

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Quando o complemento é ativado,fatores solúveis, como C5a e C3a são liberadosna circulação. C5a determina a liberação demediadores inflamatórios à partir demastócitos (mast cells) e atua como poderosoatrativo químico para neutrófilos. C5a tambémcausa ruptura capilar, perda de granulaçãopelos neutrófilos e a expressão da molécula P-seletina dos neutrófilos na superfície deplaquetas e do endotélio(7). C5a ativa plaquetase monócitos, resultando na liberação decitocinas e de outros mediadores inflamatóriosque amplificam a adesão entre neutrófilos e acélula endotelial.

Em resposta aos sinais inflamatórios(produtos de ativação do complemento,citocinas, radicais livre derivados da hipóxiaou oxigenação), células endoteliais sãoconvertidas ao estado ativado. Isto resulta emmodificações profundas na expressão genéticae função celular de células endoteliais.

Parecem existir duas fases da ativaçãocelular endotelial durante CEC:

A primeira ocorre porque produtos dedegradação do complemento circulantes

iniciam uma resposta adesiva pelos neutrófilosimediata e de curta duração. A Segunda fasese deve a cascata de cinina que amplifica aadesão neutrófilo-endotelial(4).

Estudos recentes mostram que citocinassão mensageiros intracelulares e os mediadoresmais importantes da injúria vascular e dadisfunção de órgãos(7,10). A liberação de TNF-a, IL-6, IL-8 e CRP são marcadores deprocesso inflamatório intenso que ocorradurante CEC. Se demonstrou consistentementeque níveis de IL-6 e IL-8 estão elevados duranteCEC(10,11). Também existem registros, emborainconsistentes, de níveis plasmáticos TNF-aneste processo(7,11). O fenômeno chave daresposta inflamatória que se segue a CEC é odano agudo da célula endotelial(4).

Aterosclerose resulta da resposta dacélula endotelial a injúria crônica que se seguea adesão e migração no sub endotélio deneutrófilos, linfócitos, plaquetas e macrófagos.Formas crônicas de injúria celular endotelialpodem resultar na prolongada expressão demoléculas de adesão de leucócitos, que atraemneutrófilos para a área. A presença de proteases

Figura 4 - Sangramento pós-operatório no grupo P e C.*p=0.019 comparado entre o grupo P e C.

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de radicais livres e citocinases que determinamruptura do sub endotélio causam a proliferaçãode células musculares lisas e a formação deuma placa fibrosa.

Assim, a progressão da lesão ateroscleróticaé marcada pelo acúmulo de camadas alternadasde células musculares lisas e de macrófagosligados a lipídios. As camadas de tecido fibroso ede células musculares lisas cobrem um núcleo delipídeos e produtos necróticos. Estas placas têmtendência a ruptura, que conduz ao infarto domiocárdio agudo pela oclusão coronária comtrombo plaquetário e a morte súbita

A ativação do sistema complementodesempenha um papel importante napatogenese da aterosclerose, provavelmentepor ativar células endoteliais(13). Exposição decélulas endoteliais aos complementos deflagraa indução de citocinas pró-inflamatórias,como IL6 e IL-8. Componentes docomplemento estimula a ativação de célulasendoteliais, resultando em uma expressãoaumentada da proteína-1 quimioatrativa demonócitos (MCP-1) e de outras citocinas queativam a adesão firme de monócitos aoendotélio, um evento chave para iniciar apatogênese da aterosclerose. Assim, aresposta inflamatória da CEC e daaterosclerose têm em comum a ativação decélulas endoteliais devida a estimulo decomponentes de complemento e a indução decitocinas pró-inflamatórias, como ainterleucina 8.

Em nosso estudo, o grupo controle (C)mostrou um nível significativamente inferior deCRP quando comparado ao grupo tratado (P),antes de CEC. É possível que isto decorra dapresença de lesões ateroscleróticas mais ativasneste grupo do que no grupo C(14).

No presente estudo, nossos resultadospermitem algumas inferências. Primeiro, o pré-tratamento com pravastatina antes de CECpossibilita uma resposta inflamatória sistêmicareduzida. Os níveis reduzidos de proteína C-reativa no grupo tratado com pravastatina 24horas antes de CEC confirmou a ação anti-

inflamatória desta substância. Isto écorroborado pelos reduzidos níveis de IL-8 noseu pico de liberação (6h), fato consistente comreduzida resposta inflamatória.

Em segundo, efeitos da administraçãode pravastatina são imediatos. Isto édemonstrado pelo curto tempo de resposta daação anti-inflamatória. Esta observação sugereque a ação anti-inflamatória da pravastatinanão é imediata pela redução de lipídeo.

Finalmente, a pravastatina reduziusignificativamente o sangramento mediastinalpós-operatório, conforme observado no grupotratado (P).

Nossas observações concordam comalgumas pesquisas prévias que mostramreduzido ou nenhum aumento nos níveisplasmáticos de INF-a(9). OS resultados sugeremainda que, em pacientes submetidos a cirurgiacardíaca, os níveis plasmáticos de IL-6 sãoelevados durante CEC, confirmandoobservações prévias da literatura(7).

Observamos um aumento marcado nonível plasmático de IL-6 nos dois gruposavaliados, mas no grupo tratado compravastatina este fato não correspondeu a umaelevação de CRP. Esta observação podesignificar que a pravastatina tenha uma açãoindependente da liberação de IL-6. Éreconhecido que IL-6 tem propriedades pró-inflamatória e anti-inflamatória(15). Emboranão se possa definir o exato mecanismo deação, as observações apóiam a hipótese deque a pravastatina tenha ação anti-inflamatória(16).

As observações confirmam os achadosde Weber et al, quanto a que a redutase HMG-CoA interfira diretamente com mecanismos-chave para que os leucócitos desempenhemesta resposta inflamatória(16).

A ativação do complemento por si sópode conduzir a ativação de neutrófilos. Seacredita ainda que o grau de inflamaçãoinduzida pela ativação de neutrófilos érelacionado aos níveis séricos de IL-8. Isto foiconfirmado in vitro por Urbich et al(17). Em

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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 wasdesigned to evaluate action of pravastatin on inflammatory reaction after extracorporeal circulation.

Methods – In a prospective, randomized study, 20 patients undergoing eletive coronaryartery bypass grafting were investigated. Ten patients received 80mg p.o. of pravastatin 36 and12h before surgery, and a control group of 10 did not. Plasma levels of C-reactive protein, tumornecrosis factor-alfa, interleukin-6, interleukin-8 and postoperative blood loss were analysed beforeand after cardiopulmonary bypass.

Results – Tumor necrosis factor-alfa did not change significantly in each of the momentsmeasured in either group. Interleukin-6 in both groups significantly increased after CPB whencomparing to the measures pre bypass and there was no significant diferences between the twogroups. Interleukin-8 increased (p=0.017) in group control at 6h after CPB compared with groupP. C-reative protein was increased (p=0.015) in group pravastatin before CPB compared withcontrol. 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). Medianlevels 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 reducedsystemic inflammatory response. The effects of administration are immediate and antinflammatoryaction is not mediated by lipid lowering. Pravastatin also reduced mediastinal postoperative bleeding.----------------------------------------------------------------------------------------------------------------------

nosso estudo, não avaliamos os produtos daativação do complemento, mas os reduzidosníveis plasmáticos de IL-8 parecem expressarreduzidos valores de produtos de ativação docomplemento. Previamente, IL-8 foi observadoem lesões ateroscleróticas(18).

Kiener e associados demonstraram queas estatinas podem ser diferenciadas quantoao seu efeito pró-inflamatório nos leucócitos.O tratamento de monócitos isolados porelutriação com lovostatina, sinvastatina ouatorvastatina aumentou marcadamente aprodução TNF-a, IL-8 e IL-1 b quando ascélulas foram subseqüentemente tratadas comLPS, complexos imunitários ou super-antígenos. Em contrate, pré-tratamento compravastatina não elevou estas citocinasinflamatórias(19).

Achados recentes de Simoni eassociados sugerem que na aterosclerosehuman, IL-8 representa um importantemediador da angiogênese e pode contribuir

para a formação de placas devido a suaspropriedades angiogênicas(20).

Assim, nossos dados sugerem que apravastatina tenha uma ação anti-inflamatória durante CEC. É possível que apravastatina reduza os produtos da ativaçãodo complemento e/ou IL-8. O estudo mostraainda um sangramento mediastinal reduzidono grupo tratado. Isto poderia ser explicadopela redução na ativação de célulasendoteliais pela pravastatina, fonte principalde fator tecidual durante CEC. Isto resulta emum consumo aumentado de fatores decoagulação(21).

Investigações futuras são necessáriaspara elucidar o papel exato da pravastatinana resposta inflamatória que se segue a CECMas nossos dados sugerem que o pro-tratamento com pravastatina reduzsignificativamente níveis plasmáticos de CRPe IL-8 e a resposta inflamatória em pacientessubmetidos a circulação extra-corpórea.

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REFERÊNCIAS

1. Kirklin JK, Westaby S, Blackstone EH, KirklinJW, Chenoweth DE, Pacífico AD. Complementand damaging effects of cardiopulmonarybypass J Thorac Cardiovasc Surg1983;86:845-57.

2. Tennenberg SD, Clardy CW, Bailey WW,Solomkin JS. Complement activation and lungpermeability during cardiopulmonary bypass.Ann Thorac Surg 1990;50:597-601.

3. Butler J, Rocker GM, Westaby S. Inflammatoryresponse to cardiopulmonary bypass. AnnThorac Surg 1993;55:552-59.

4. Edmunds Jr LH. Inflammatory response tocardiopulmonary bypass. Ann Thorac Surg1998;66:S12-16.

5. Verrier ED. The vascular endothelium: friendor foe? Ann Thorac Surg 1993;55:818-19.

6. Williams JK, Sukhova GK, Hemington DM,Libby P. Pravastatin has cholesterol-loweringindependent effects on the artery wall ofatherosclerotic monkeys. J Am Coll Cardiol1998;31:684-91.

7. Steimberg JB, Kajelansk DP, Olson JD, WeilerJM. Cytokine and complement levels inpatients undergoing cardiopulmonary bypass.J Thorac Cardiovasc Surg 1993;106:1008-16.

8. Utley JR. Pathophysiology of cardiopulmonarybypass: current issues. J Cardiovasc Surg1990;5:177-89.

9. Jansen NJG, van Oeveren W, Gu YJ, et al.Endotoxin release and tumor necrosis factorformation during cardiopulmonary bypass. AnnThorac Cardiovasc Surg 1992;54:744-48.

10. Kawamura T, Wakusawa R, Okada K, ImadaS. Elevation of cytokines during open heartsurgery with cardiopulmonary bypass:participation of interleukin-8 and 6 inreperfusion injury. Can J Anaesth1993;40:1016-21.

11. Kalfin RE, Engelman RM, Rouson JA, et al.Induction of interleukin-8 expression duringcardiopulmonary bypass. Circulation1993;88:401-16.

12. Ross R, Fuster V. The pathogenesis ofatherosclerosis. In: Fuster V, Ross R, Topol EJ,eds. Atherosclerosis and coronary arterydisease. Philadelphia: Lippicott-Raven,1996:441-60.

13. Bhakadi S. Complement and atherogenesis:the unknown connection. Ann Med1998;30:503-07.

14. Liuzzo G, Biasucci LM, Gallimore JR, et al. Theprognostic value of C-reactive protein andserum amyloid A protein in severe unstableangina. N Engl J Med 1994;331:417-24.

15. Opal SM, DePalo VA. Anti-inflammatorycytokines. Chest 2000;117:1162-72.

16. Weber C, Erl W, Weber KSC, Weber PC.HMG-CoA reductase inhibitors decreaseCDIIb expression and CDIIb-dependentadhesion of monocytes to endothelium andreduce increased adhesiveness of monoctesisolated from patients withhypercholesterolemia. J Am Coll Cardiol1997;30:1212-17.

17. Urbich C, Fritzenwanger M, Zeiher AM,Dimmeler S. Laminar shear stress upregulatesthe complement inhibitory protein clusterin. Anovel potent defense mechanism againstcomplement-Induced endothelial cellactivation. Circulation 2000;101:352-55.

18. Koch AE, Kunkel SC, Pearce WH, et al.Enhanced production of the chemotacticcytokines interleukin-8 and monocytechemoattractant protein-1 in humanaddominal aortic aneuryms. Am J Pathol1993;142:1423-30.

19. Kiener P, Davis PM, Murray JL, Renkin BM.Characterization of the pro-inflammatoryeffects of HMG-CoA reductase inhibitors. JMoll Cell Cardiol 1998;30:738.

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20. Simonini A, Moscucci M, Muller DWM, et al.IL-8 is an angiogenic factor in human coronaryatherectomy tissue. Circulation2000;101:1519-26.

21. Boyle Jr EM, Vernier ED, Spiess BD. Endothelialcell injury in cardiovascular surgery: theprocoagulant response. Ann Thorac Surg1996;77:1080-4.

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Cardiovascular Imaging: Nine Years Patencyof a Small Caliber Vascular ProsthesisSeeded with Autogous Endothelial Cells

P. M. Dohmen*, A. Lembcke, D. Gabbieri, W. Konertz.

ORIGINAL CARDIOVASCULAR IMAGING

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 forrevascularization. As there was no sufficient autologous grafts available, a 4 mm expandedpolytetrafluoroethylene graft was seeded with autologous vascular endothelial cells (AVEC). Atnine years, multi-slice computed tomography showed a patent AVEC seeded graft (Figure 1,2).

* Address reprint requests: Dr. P. M. Dohmen MD PhD, Department of Cardiovascular Surgery, Charité Hospital, HumboldtUniversity Berlin, Luisenstraße 13, D-10117 Berlin. Telephone +49 30 450 522092 Fax +49 30 450 522921 E-mail :[email protected]

Figure 1. Three dimensional cardiac reconstruction shows a patent internal mammary artery grafted tothe left anterior descending artery (arrows) and a patent graft to the first marginal branch (arrows).

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Figure 2. The sagital sections show heavy calcification ( black arrows) of the first marginal branch. Theseeded graft showed to be patent, with absence of any narrowing over the total length of the graft (redarrows). Notice that the run off in this area is limited.

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Fisiología Aplicada de La Vasculogénesis

Alberto J. Crottogini, Gustavo L.V. Javanel.

UPDATING ARTICLES

Departamento de Ciencias Fisiológicas, Farmacológicas y Bioquímicas, Universidad Favaloro, Buenos Aires, Argentina

Introducción

Los vasos sanguíneos son conductosespecializados en transportar la sangre y enmediar las interacciones entre el contenido dela luz vascular y el tejido circundante. Elfuncionamiento normal de los tejidos dependedel adecuado abastecimiento de oxígeno ynutrientes, y del lavado de los desechos pormedio de esta función de transporte vascular.En los últimos años el entendimiento de cómose forman los vasos sanguíneos ha pasado aser un objetivo primordial y desafiante en laactividad científica, ya que muchas terapiaspodrían basarse en el control localizado de sucrecimiento. En Cardiología la inducción de laproliferación vascular ha cobrado gran interéscomo alternativa para la enfermedadaterosclerótica coronaria y periférica. A pesarde los grandes avances logrados en laprevención y el tratamiento, la cardiopatíaisquémica es la principal causa de muerte enpaíses desarrollados y subdesarrollados. Laenfermedad vascular periférica, por su parte,es una condición progresivamente invalidantey mutiladora que provoca un deterioro graveen la calidad de vida. Es por esto que el estímulodel crecimiento de vasos sanguíneos es unobjetivo prioritario de la investigación actual.

Vasculogénesis,Angiogénesis y Arteriogénesis

La proliferación vascular es unfenómeno complejo y altamente regulado, enel que están involucrados diversos mediadoresbioquímicos, algunos inhibidores y otrosestimuladores.(1) El balance entre estosmediadores regula el proceso.(2) Existensituaciones fisiológicas (ciclo endometrial,cicatrización de heridas, etc.) en las que elbalance se inclina transitoriamente hacia elestímulo y luego retorna al estado basal dequiescencia. Cuando la regulación no es laadecuada, la proliferación vascular exageradao insuficiente contribuye a la patogénesis demuchas enfermedades, por ejemplo el cáncer,la retinopatía proliferativa, las enfermedadesisquémicas o neurodegenerativas, la pre-eclampsia, etc.(3)

Se han definido ciertos términos quedistinguen los distintos tipos de proliferaciónvascular. Se designa vasculogénesis aldesarrollo de un plexo vascular primitivo apartir de células con alta potencialidadevolutiva (por ejemplo stem cells).(4)

Inicialmente, este término era reservado parala formación de nuevos vasos sanguíneos enla etapa embrionaria, a partir de angioblastoso hemangioblastos. Sin embargo, actualmentese conoce la participación de célulasprogenitoras y precursoras provenientes de la

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médula ósea en el desarrollo de plexossanguíneos durante la vida adulta. Esteproceso es conocido como vasculogénesispost-natal.(3,5)

El término angiogénesis se ha reservadopara referirse a la formación de capilares (ovasos sanguíneos de mayor diámetro peroformados sólo por endotelio) a partir deconductos pre-existentes formados por célulasadultas (capilares o vénulas post-capilares). Elproceso de expansión y remodelamiento deplexos vasculares endoteliales, generadosinicialmente mediante vasculogénesis, ha sidotambién llamado angiogénesis.(6)

En cambio, se denomina arteriogénesisal crecimiento y formación de arterias yarteriolas (es decir conductos más importantes,constituidos no sólo por endotelio sino tambiénpor músculo liso vascular) a partir de otrasarterias. Este es el mecanismo involucrado enel desarrollo de la circulación colateral, quetiene un rol importantísimo en la adaptaciónde los tejidos a obstrucciones vascularesprogresivas. Clásicamente la arteriogénesis serefirió a la expansión de pequeñas colateralesinnatas y su remodelamiento en arterias másgrandes. Actualmente se considera que lageneración de vasos arteriales completamentenuevos también puede ocurrir (formación denovo de arterias colaterales).7 Incluso existeevidencia de que el crecimiento de arteriolaspuede resultar del reclutamiento de célulasmusculares lisas a partir de vasos capilarespreexistentes.(8)

Fisiología de la Angiogénesis

Los mecanismos de la proliferaciónvascular no están aún totalmente comprendidos.Si bien resulta lógico pensar que haysubstancias y pasos comunes a todos losprocesos, se sabe que la angiogénesis ocurrecomo consecuencia de la isquemia, la cualestimula la expresión del factor de transcripciónHIF-1a (hypoxia inducible factor 1a).(9) Estefactor de transcripción a su vez “enciende” genes

que codifican para proteínas vinculadas a lahipoxia, tales como la eritropoyetina, el VEGFy sus receptores. El VEGF es un mitógeno decélulas endoteliales y el factor de crecimientoparadigmático de la angiogénesis,(10) aunquerecientemente se han descrito nuevos efectos delVEGF. Este factor angiogénico estimula laproliferación y migración de células endotelialesy su organización tubular. Otros factores decrecimiento involucrados en la angiogénesis sonel PlGF (placental growth factor, un análogo delVEGF), el HGF (hepatocyte growth factor, oscatter factor), los FGF (factores de crecimientofibroblástico) tipo 1, 2, 4 y 5, las efrinas y lasangiopoietinas.(1) El PlGF y el HGF sonmitógenos de células endoteliales y promuevenla proliferación de capilares. En cambio losFGFs son mitógenos de otras células ademásde los endoteliocitos, aunque también handemostrado tener una potente actividadangiogénica. Las efrinas están involucradas enel establecimiento de la identidad arterial ovenosa del endotelio vascular,(1) mientras quelas angiopoietinas están directamenterelacionadas con la desestabilización del vasosanguíneo (el pasaje a un estado más plásticoque permite la proliferación celular y elcrecimiento de neovasos) y con la ulteriormaduración o re-estabilización del plexovascular.(11) En ausencia de ciertos estímulos (porejemplo VEGF) los vasos desestabilizadosterminan desapareciendo (regresión vascular).La regresión vascular y el “podado” (pruning)de los vasos excedentes son procesos muyimportantes para eliminar los vasosinnecesarios, ya que la arquitectura final de lared vascular no debe ser insuficiente perotampoco excesiva para las demandas deltejido.(4,12)

Se han descrito dos mecanismos deangiogénesis: 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 yluego comienza a crecer hacia el lugar de donde

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proviene el estímulo angiogénico. Laintususcepción se refiere a la formación depuentes o pilares transluminales de matrizextracelular y endotelio que dividen el vaso

Fisiología de la Arteriogénesis

La arteriogénesis depende principal-mente de otros estímulos diferentes a lahipoxia, tales como la tensión de cizallamiento(“shear stress”) y la activación de losmonocitos. Ante una obstrucción arterial, elflujo se desvía hacia las incipientes colateralesde pequeño diámetro.(6,7) Sobre las paredesde estas colaterales el shear stress es alto, locual estimula la secreción endotelial de MCP-1 (monocyte chemoattractant protein 1). LaMCP-1 actúa sobre el receptor CC de losmonocitos, activándolos y ejerciendo unefecto quimiotáctico sobre estas células, quese acumulan en el endotelio y en el espaciosubintimal vascular y secretan distintosfactores de crecimiento, como el VEGF, FGF-

preexistente generando nuevos espaciosintervasculares de tejido intersticial yconsecuentemente nuevos vasos máspequeños.(13)

Figura 1: Mecanismos de la angiogénesis. A: por brote (“sprouting” angiogenesis); B: porintususcepción (“non-sprouting” angiogenesis). VEGF: factor de crecimiento de endotelio vascular.PlGF: factor de crecimiento placentario. VEGFR: receptor para el VEGF. TIE: receptor paraangiopoietinas.

2 (fibroblast growth factor 2), TGF-b1(transforming growth factor b1), y enzimas,como colagenasas, metaloproteinasas yactivadores del plasminógeno.Consecuentemente, la membrana basal esdegradada, las células musculares lisascambian del fenotipo contráctil al fenotipoproliferativo y comienzan a dividirse junto conlas otras células de todas las capas del vaso.(14)

Al mismo tiempo, la matriz extracelular vasiendo degradada para permitir el crecimientoexpansivo de la arteria o para permitir eldesarrollo de los neovasos arteriales.Finalizada la proliferación, la matrizextracelular y la membrana basal sonresintetizadas, las células musculares lisas yendoteliales retornan a su fenotipo quiescentey el vaso es por último estabilizado.

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Angiogénesis yArteriogénesis Terapéuticas

La inducción terapéutica de laproliferación vascular puede lograrse dediversas maneras. Si bien la formación denuevos capilares funcionantes contribuye amejorar la perfusión tisular, el objetivo debeincluir la generación de nuevas arterias yarteriolas.(15 )Los capilares distales sonimprescindibles para la distribución del flujosanguíneo en los tejidos, pero las arteriasproximales son las encargadas de hacer llegarese caudal y abastecer el lecho capilar. Segúnla ley de Pouseuille, el caudal depende del radiodel conducto elevado a la cuarta potencia. Poreso, las arterias de conductancia, con su radioimportante, son de enorme relevancia en lacirculación colateral ya que transportangrandes caudales de sangre, mientras que lasarteriolas son las encargadas de regular quéproporción del caudal es derivado a cadatejido. La gran diferencia con respecto a loscapilares radica en que las arterias y arteriolasposeen, además de mayor diámetro, elastinay músculo liso vascular en su túnica media.La túnica media así constituida les confierepropiedades elásticas, la capacidad deresponder a los estímulos fisiológicos y másestabilidad y resistencia a la compresiónoriginada por la contracción sistólica.

La inducción terapéutica de laproliferación vascular puede lograrse mediantela administración de factores angiogénicos, esdecir proteínas capaces de gatillar el proceso(terapia proteica),(16) o de los genes quecodifican para estas proteínas (terapiagénica).(17) Una tercera alternativa ha surgidorecientemente y es la administración de célulascon alta potencialidad evolutiva, capaces dedar origen a las células adultas que formaránnuevos vasos y de secretar diversos factoresangiogénicos que regularán este proceso(terapia celular o vasculogénesisterapéutica).(3,18) Aún más, estás células puedenser transfectadas con genes codificantes para

factores de crecimiento antes de ser injertadas(transferencia génica ex vivo).(19) Acontinuación discutiremos brevemente las trestécnicas y citaremos los estudios más recientes.

Terapia Celular

La terapia por implante celular ha sidoinvestigada con diversos tipos de células, desdemédula ósea fresca hasta células clasificadassegún marcadores de membrana, obtenidas dela médula ósea (células madre hematopoyéticaso mesenquimáticas) o de la sangre periférica(células precursoras endoteliales).(18) Estascélulas pueden ser modificadas genéticamenteantes de ser implantadas, para que secretenintensamente algún factor angiogénico. Ciertasproteínas movilizan células totipotentes oprecursoras a partir de la medula ósea, porejemplo factores angiogénicos, como el VEGF,o factores hematopoyéticos como el GM-CSF(granulocyte-macrophage colony-stimulatingfactor). Orlic y col. observaron que lamovilización de células de la médula óseamediante G-CSF (granulocyte colony-stimulating factor) y SCF (stem cell factor) enratones con infarto de miocardio inducía laproliferación de capilares y arteriolas en el tejidomiocárdico.(20) En mamíferos superiores coninfarto agudo de miocardio los resultados de estatécnica han sido controvertidos: en babuinoshubo una mejoría en la perfusión miocárdica(21)

pero en monos rhesus no se encontródiferenciación celular e inclusive hubo mayormortalidad.(22) En pacientes con enfermedadcoronaria, el GM-CSF intracoronario y luegosubcutáneo mejoró, en el corto plazo, lacirculación colateral.(23) Sin embargo, no sedemostró cómo actuó el GM-CSF en estospacientes.

Terapia Proteica

Los factores de crecimiento sonproteínas, generalmente de pequeño tamañoy corta vida media, capaces de regular, tanto

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parácrina como autócrinamente, la migración,proliferación, diferenciación y crecimiento celular.Algunos pueden inducir o potenciar laproliferación vascular ya que estimulan al menosuno de los pasos descritos más arriba. Los másestudiados en modelos animales de isquemiamiocárdica crónica y periférica fueron el FGF-2y el VEGF.(16) En pacientes con enfermedadvascular periférica, el FGF-2 demostró resultadospositivos a 90 días.(24) En pacientes coronarios,en cambio, no hubo resultados concluyentementepositivos,(25,26) fundamentalmente por el marcadoefecto placebo observado en los grupos control,que dificulta objetivar diferencias con los grupostratados. Otras desventajas fueron la corta vidamedia y la dificultad en administrar grandes dosisde VEGF debido a su potente efectovasodilatador.

Terapia Génica

La terapia génica se refiere a laadministración o transferencia de materialgenético a un paciente con fines terapéuticos.Cuando el objetivo terapéutico es la inducciónde proliferación vascular, el gen empleado seráel que codifica para una proteína angiogénicao arteriogénica.(27)

El material genético puede seradministrado unido a una cadena circular de

ADN desnudo (plásmido) o asociado acompuestos que facilitan la transfección (ingresodel material genético a la célula) llamados“vectores” (virus o liposomas). La principalventaja de los virus frente a los plásmidos es lamayor la eficiencia de transfección, aunque estacaracterística se asocia a una respuestainflamatoria en el paciente y al riesgo derespuesta inmune adversa. Esto ademásdificulta la administración repetida de genestransportados en vectores virales. Los plásmidos,en cambio, son menos eficientes pero másseguros. Nuevas técnicas de transferenciagénica (virus adenoasociados, nuevosliposomas) están siendo estudiadas paramejorar la eficiencia de la transfección.(17)

Diversos autores (entre ellos nuestrogrupo) demostraron que la transferencia génicade factores de crecimiento es segura e induceangiogénesis, redundando en una mejoría delflujo, la perfusión, la función miocárdica, eincluso la proliferación de arteriolas (figura 2) ycardiomiocitos (miocardiogénesis).(17,27-30)

Actualmente, ensayos clínicos fase I y IIhan demostrado la seguridad y sugerido laeficacia de la transferencia génica de factoresangiogénicos en la isquemia miocárdica(31,32)yperiférica.(33) Sin embargo, aún se necesitanestudios con mayor número de pacientes parapoder obtener resultados más confiables.

Figura 2: Microfotografía de miocardio porcino conneoformación arteriolar inducida por transferenciade un plásmido codificante para vascularendothelial growth factor (VEGF165). Obsérvese lapresencia 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): ¿algomás que un mitógeno de células endoteliales?.Revista Argentina de Hemodinamia, Angiografía yTerapéutica por Cateterismo 2004 (in press), conpermiso del Editor).

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Comentario Final

En la vida adulta el ser humano tiene lapotencialidad de formar nuevos vasossanguíneos. Desentrañar la fisiología de esteproceso es fundamental para usar laangiogénesis y la arteriogénesis comoterapéuticas de la enfermedad isquémicacoronaria y periférica, o para inhibirla, comoen el caso del cáncer. A pesar de los grandes

avances producidos en la última década, esmucho más lo que se ignora que lo que sesabe. Mientras la ciencia nos sigue aportandoinformación, la medicina ya ha comenzadoa intentar, con los conocimientos disponibles,la angiogénesis y la arteriogénesisterapéuticas en el hombre. Los resultadosiniciales no sen espectaculares, pero elcamino a recorrer es largo y el desafío siguevigente.

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5. Rafii S, Meeus S, Dias S, Hattori K, Heissig B,Shmelkov S, Rafii D, Lyden D. -Contributionof marrow-derived progenitors to vascularand cardiac regeneration. Semin Cell Dev Biol2002; 13: 61-67.

6. Carmeliet P. - Mechanisms of angiogenesis andarteriogenesis. Nat Med 2000; 6: 389-395.

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11. Ramsauer M, D’Amore PA. - Getting Tie(2)dup in angiogenesis. J Clin Invest 2002; 110:1615-1617.

12. Dimmeler S, Zeiher AM. - Endothelial cellapoptosis in angiogenesis and vesselregression. Circ Res 2000; 87: 434-439.

13. Burri PH, Djonov V. - Intussusceptiveangiogenesis - the alternative to capillarysprouting. Mol Aspects Med 2002; 23: S1-S27.

14. Cai WJ, Koltai S, Kocsis E, Scholz D, KostinS, Luo X, Schaper W, Schaper J. - Remodelingof the adventitia during coronaryarteriogenesis. Am J Physiol Heart CircPhysiol 2003; 284: H31-40.

15. Chiu RC-J. - Therapeutic cardiac angiogenesisand myogenesis: the promises and challengeson a new frontier. J Thorac Cardiovasc Surg2001; 122: 851-852.

16. Post MJ, Laham R, Sellke FW, Simons M. -Therapeutic angiogenesis in cardiology usingprotein formulations. Cardiovasc Res 2001;49: 522-531.

17. Khan TA, Sellke FW, Laham RJ. - Gene therapyprogress and prospects: therapeuticangiogenesis for limb and myocardialischemia. Gene Ther 2003;10: 285-291.

18. Abbott JD, Giordano FJ. - Stem cells andcardiovascular disease. J Nucl Cardiol2003;10: 403-412.

19. Iwaguro H, Yamaguchi J, Kalka C, MurasawaS, Masuda H, Hayashi S, Silver M, Li T, IsnerJM, Asahara T. - Endothelial progenitor cellvascular endothelial growth factor genetransfer for vascular regeneration. Circulation2002;105: 732-738.

20. Orlic D, Kajstura J, Chimenti S, Limana F,Jakoniuk I, Quaini F, Nadal-Ginard B, BodineDM, Leri A, Anversa P. - Mobilized bonemarrow cells repair the infarcted heart,improving function and survival. Proc NatlAcad Sci USA 2001; 98: 10344-10349.

21. Norol F, Merlet P, Isnard R, Sebillon P, BonnetN, Cailliot C, Carrion C, Ribeiro M, CharlotteF, Pradeau P, Mayol JF, Peinnequin A, DrouetM, Safsafi K, Vernant JP, Herodin F. - Influenceof mobilized stem cells on myocardial infarctrepair in a nonhuman primate model. Blood2003;102:4361-4368.

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22. Orlic D, Arai AE, Sheikh FH, Agyeman KO,McGhee J, HoytRF, Sachdev V, Yu Z-X, San H,Metzger ME, Dunbar CE. - Cytokine mobilizedCD34+ cells do not benefit rhesus monkeysfollowing induced myocardial infarction. Blood2002; 100(11): Abstract #94.

23. Seiler C, Pohl T, Wustmann K, Hutter D,Nicolet PA, Windecker S, Eberli FR, MeierB. - Promotion of collateral growth bygranulocyte-macrophage colony-stimulatingfactor in patients with coronary arterydisease. A randomized, double-blind,placebo-controlled study. Circulation2001;104:2012-2017.

24. Lederman RJ, Mendelsohn FO, AndersonRD, Saucedo JF, Tenaglia AN, Hermiller JB,Hillegass WB, Rocha-Singh K, Moon TE,Whitehouse MJ, Annex BH; TRAFFICInvestigators. Therapeutic angiogenesis withrecombinant fibroblast growth factor-2 forintermittent claudication (the TRAFFICstudy): a randomised trial. Lancet 2002; 359:2053-2058.

25. Henry TD, Annex BH, McKendall GR, AzrinMA, Lopez JJ, Giordano FJ, Shah PK,Willerson JT, Benza RL, Berman DS, GibsonCM, Bajamonde A, Rundle AC, Fine J,McCluskey ER; VIVA Investigators. - The VIVAtrial: Vascular endothelial growth factor inIschemia for Vascular Angiogenesis.Circulation 2003; 107: 1359-1365.

26. Simons M, Annex BH, Laham RJ, Kleiman N,Henry T, Dauerman H, Udelson JE, GervinoEV, Pike M, Whitehouse MJ, Moon T, ChronosNA. - Pharmacological treatment of coronaryartery disease with recombinant fibroblastgrowth factor-2: double-blind, randomized,controlled clinical trial. Circulation 2002; 105:788-793.

27. Ylä-Herttuala S, Alitalo K. - Gene transfer asa tool to induce therapeutic vascular growth.Nat Med 2003; 9: 694-701.

28. Crottogini A, Meckert PC, Vera Janavel G,Lascano E, Negroni J, Del Valle H, DulbeccoE, Werba P, Cuniberti L, Martinez V, DeLorenzi A, Telayna J, Mele A, Fernandez JL,

Marangunich L, Criscuolo M, Capogrossi MC,Laguens R. - Arteriogenesis induced byintramyocardial vascular endothelial growthfactor 165 gene transfer in chronicallyischemic pigs. Hum Gene Ther 2003; 14:1307-1318.

29. Laguens R, Cabeza Meckert P, Vera JanavelG, Del Valle H, Lascano E, Negroni J, WerbaP, Cuniberti L, Martinez V, Melo C,Papouchado M, Ojeda R, Criscuolo M,Crottogini A. - Entrance in mitosis of adultcardiomyocytes in ischemic pig hearts afterplasmid-mediated rhVEGF165 gene transfer.Gene Ther 2002; 9: 1676-1681.

30. Laguens R, Cabeza Meckert P, Vera JanavelG, De Lorenzi A, Lascano E, Negroni J, DelValle H, Cuniberti L, Martinez V, DulbeccoE, Melo C, Fernandez N, Criscuolo M,Crottogini A. - Cardiomyocyte hyperplasiaafter plasmid-mediated VEGF gene transferin pigs with chronic myocardial ischemia. JGene Med 2004;6:222-227.

31. Grines C, Rubanyi GM, Kleiman NS, MarrottP, Watkins MW. - Angiogenic gene therapywith adenovirus 5 fibroblast growth factor-4 (Ad5FGF-4): a new option for thetreatment of coronary artery disease. Am JCardiol 2003; 92: 24N-31N.

32. Hedman M, Hartikainen J, Syvanne M, StjernvallJ, Hedman A, Kivela A, Vanninen E, Mussalo H,Kauppila E, Simula S, Narvanen O, Rantala A,Peuhkurinen K, Nieminen MS, Laakso M, Ylä-Herttuala S. - Safety and feasibility of catheter-based local intracoronary vascular endothelialgrowth factor gene transfer in the preventionof postangioplasty and in-stent restenosis andin the treatment of chronic myocardialischemia: phase II results of the KuopioAngiogenesis Trial (KAT). Circulation 2003;107: 2677-2683.

33. Makinen K, Manninen H, Hedman M, Matsi P,Mussalo H, Alhava E, Ylä-Herttuala S. - Increasedvascularity detected by digital subtractionangiography after VEGF gene transfer to humanlower limb artery: a randomized, placebo-controlled, double-blinded phase II study. MolTher 2002; 6: 127-133.

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Bases Fisiológicas de La Variabilidadde La Frecuencia Cardíaca

Eduardo R. Migliaro y Paola Contreras

UPDATING ARTICLES

Departamento de Fisiología. Facultad de Medicina. Montevideo. URUGUAY

Introducción

Las ciencias de la vida hanexperimentado en el último siglo un avancesustancial, a partir del desarrollo de técnicasanalíticas que ampliaron el conocimiento demecanismos celulares y moleculares. Ejemplode ello son los avances en el conocimiento de laactividad eléctrica celular y los canales iónicos,la expresión de proteínas mensajeras, el papeldel óxido nítrico, la descripción del genomahumano y otros que han impactado fuertementeen el campo de la fisiología y de la medicina.

Algunos autores sostienen que estosavances han alejado a los fisiólogos del estudiode la función de los órganos en formaintegrada,(1) que es un campo tradicional de lafisiología.(2) Sin embargo, este campo no debeser abandonado, porque la comprensión de lasfunciones del ser humano necesita integrar laactividad de cada órgano en un sistema únicoy coordinado.(3) Este sistema integrado es unsistema complejo, que como tal, da lugar a laaparición de un orden emergente diferente ala suma de las partes.(4) Se pueden considerara los órganos como osciladores biológicos quefuncionan en forma acoplada y cuyo desacoplegenera trastornos de la función del todo, sinque necesariamente estén afectadas laspartes.(5,6)

El estudio del ritmo cardíaco ha

interesado a los investigadores desde hacevarios siglos,(7) en el siglo XVIII Spthen Haleshizo la primera descripción de los cambioscíclicos de la actividad cardíaca y la presiónarterial.

Las modificaciones en estos ciclosvienen siendo estudiadas como indicadores dela regulación cardíaca, se ha postulado ademásque su estudio es una forma de analizar elacople entre órganos y por lo tanto puedeconsiderarse como un índice del nivel de eseacople.(9, 10,11)

La Variabilidad de laFrecuencia Cardíaca.

Los intervalos entre los latidos de uncorazón normal, muestran entre sí levesdiferencias de duración que se traducen encambios del ritmo cardíaco. Estos cambios enel ritmo siguen ciertos patrones de repetición,por lo que las prolongaciones y acortamientosde los intervalos se repiten de manera cíclica.Uno de los ejemplos más conocidos es laarritmia sinusal respiratoria. Esta modifica losintervalos siguiendo el patrón de la respiración,lo que impone una frecuencia de variaciónrelativamente alta si la comparamos con otrasinfluencias.Los métodos informáticos han facilitado la

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medición y almacenamiento de los intervalosentre latidos, por lo que resulta sencillo estudiarsu variación. Este tipo de análisis es el que seconoce como Variabilidad de la FrecuenciaCardíaca (VFC) y se ha convertido en unaherramienta muy útil para la investigación y eldiagnóstico clínico. (12,13,14,15,16)

Su utilidad deriva de la sencillez de suregistro y de las correlaciones fisiológicas ypatológicas que se han encontrado. En esteúltimo terreno, la VFC ha demostrado ser unbuen predictor de morbimortalidad,(17) enparticular en pacientes que han sufrido infartode miocardio,(18,19) pero también en ladiabetes,(20,21) la insuficiencia cardíaca,(22) laenfermedad de Chagas(23) y la enfermedadcoronaria.(24) Recientemente nuestro grupo hademostrado que la VFC tiene capacidadpredictiva, en pacientes críticos que puedenevolucionar a la disfunción orgánicamúltiple.(11)

Formas de Medir la VFC

La VFC puede ser calculada a partir decualquier señal que identifique una fase dadadel ciclo cardíaco, por ejemplo: ruidos,imágenes ecocardiográficas, doppler y otrasformas de registro de la actividad cardíaca. Sinembargo, el electrocardiograma (ECG) es laherramienta más utilizada en virtud de sudifusión y por proveer registros con referenciasmuy exactas en el tiempo como lo son lasondas del complejo ventricular QRS. Por estarazón es muy frecuente que se identifiquen losintervalos entre latidos como intervalos R-R, otambién como intervalos N-N (por normal-normal), lo que señala que para calcular la VFCse usan ondas R “normales” entendiendo comotales sólo aquellas de origen sinusal. Disponiendo en un gráfico la duración de losintervalos N-N en función del tiempo se obtieneel tacograma que es la base del análisis de laVFC (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 intervaloo su equivalencia en minutos.

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los métodos de registro pueden ser de pocosminutos (5 a 10) o de varias horas. Muchos delos análisis de la VFC se basan en el ECG de24h (Holter),(16) que es el método másadecuado para el análisis de VFC en funciónde ritmos circadianos, o para la comparaciónde la VFC entre la noche y el día. Sin embargo,cabe consignar que para el diagnóstico de VFCdisminuida en estados patológicos el Holter noparece tener ventajas frente a métodos demenor duración.(25, 26,27)

En nuestros estudios utilizamos undispositivo que consta de un electrocardiógrafoconvencional, que se conecta a un conversoranalógico digital (A/D) y permite almacenar elECG en el disco duro de una computadora(esquema en Figura 2). Posteriormenteanalizamos el registro con un softwareespecialmente diseñado, que detecta las ondasR, permite su validación visual, mide losintervalos entre ellas y finalmente calcula losíndices de VFC.

Figura 2 : Esquema para registro de la VFC usado por los autores.

Índices de VFC. Para la evaluaciónnumérica de la VFC se han ensayado una largaserie de índices que se agrupan según la forma deanálisis de la VFC (por revisiones ver citas 8 y15), a la fecha ninguno de ellos satisface todas lasnecesidades. Aludiremos brevemente a algunosíndices útiles para los fines de este capítulo.

1) Índices Estadísticos

a. SDNN: Es un índice muy usado y desimple definición (el desvío estándar de todoslo intervalos N-N en la muestra).

b. rMSSD: Muy similar al anterior encuanto a la fórmula para calcularlo, perosustituye la resta de cada intervalo de la media,por la resta de dos intervalos adyacentes. Esohace que sea un índice muy útil para evaluarcambios rápidos de la VFC.

2) Índices en el Ámbito de laFrecuencia (Análisis Espectral)

Para realizar el estudio espectral, el perfildel tacograma se trata como una señalcompuesta por múltiples ondas de diferentesfrecuencias. Se aplican luego métodos comola transformada rápida de Fourier (FFT),modelado autoregresivo (ARMA) o métodoshíbridos que generan un espectro de potenciasdonde se dispone la potencia (varianza) decada onda en función de su frecuencia(Figura 3).

El espectro se divide en bandas defrecuencia (ver también Tabla I) y sobre estabase se estima la densidad espectral de cadabanda. Existen numerosos estudios quecorrelacionan las bandas del espectro confenómenos fisiológicos. (15)

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Figura 3: El análisis del tacograma como una señal compleja permite derivar de él un espectro defrecuencias. En la parte derecha de la figura se observa un espectro típico de la VFC donde se destacandos bandas. La de baja frecuencia (Low Frequency, LF) que abarca el espectro de 0.04-0.15 Hz y las dealta 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.

Factores FisiológicosInvolucrados en la VFC

Las células del nódulo sinusal seinfluyen mutuamente de modo que generanun ri tmo único pero necesariamente

Tabla I. Nombre y unidades de índices espectrales.

variable.(28) Esta interacción entre célulasmarcapaso, es responsable de una primeraforma de variabilidad, muy pequeña si se lacompara con los grandes cambios que seintroducen por la vía de la regulaciónextracardíaca.

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El principal regulador extracardíaco es elSistema Nervioso Autónomo (SNA). Elbalance entre la rama simpática y laparasimpática incrementa la variabilidadpropia del nódulo sinusal. Vistos por separado,el parasimpático tiene el conocido efecto deincremento de la duración de los intervalos,mientras que el simpático los disminuye.Debido a que el parasimpático tiene unalatencia de respuesta menor que la delsimpático(29) su influencia es dominante en lasmodificaciones rápidas de la VFC como lasinducidas por la respiración.

Esta dependencia de la VFC con el SNA,ha llevado a que varios autores consideren queel análisis de la VFC es una buena medida dela función autónoma. Es así que los cambiosen la postura,(30) los fenómenos vasomotoresligados al control baroreflejo de la presiónarterial,(31) o la reacción de alarma(32) tienenun correlato muy claro en la VFC. También seha establecido claramente que la VFCdisminuye con la edad(17,33). Se supone que elenvejecimiento del SNA y de las estructurascardíacas pueden estar en la base de estecomportamiento.(34) La figura 4 ilustra larelación entre los valores de VFC, la edad y lafrecuencia cardíaca.

Además de los neurotransmisoresautónomos más estudiados, acetilcolina ynoradrenalina, existen otras sustancias que puedenactuar sobre efectores propios o sobre lasterminaciones presinápticas. Entre dichassustancias destacamos las purinas y el oxido nítricoque juegan un papel relevante en la modulaciónautonómica.(35,36) También cabe consignar lasrelaciones entre SNA y procesos inflamatorios(37)

que seguramente habrán de abrir interesantes víasde estudio en el futuro inmediato.

Otros autores han puesto en duda esepapel de “evaluador autonómico” que se leatribuye a la VFC.(38) Es claro que otrasinfluencias pueden modificar la función delnódulo sinusal, entre ellas: la temperaturaactuando en forma directa sobre las células delnódulo, factores endócrinos y metabólicos yfenómenos mecánicos.(39)

Uno de los mecanismos de modificaciónde la VFC más evidentes y más intensamenteestudiados son los cambios ligados al ciclorespiratorio. Como ya se mencionó la respiraciónimpone al ritmo cardíaco un ritmo propio(arritmia sinusal respiratoria) que tiene un ciclorelativamente rápido (0.2 Hz aprox.), por lotanto se dispone en la zona HF del espectro defrecuencias (ver Tabla I y Figura 3).

Figura 4: Efecto sobre la VFC (medida por el rMSSD) del incremento de la frecuencia cardíaca en dosgrupos de individuos de edades diferentes. Se puede apreciar la disminución de la VFC al aumentar lafrecuencia cardíaca y además se verifica que los valores de rMSSD son mayores en el grupo más joven.(49)

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Esta correlación se hace más evidentecuando la respiración se hace rítmica, comose observa en la Figura 5. También se expresaclaramente durante la vocalización de algunostextos religiosos,(40) o en rutinas de relajacióny meditación,(41, 42) situaciones en las que secontrola voluntaria o involuntariamente larespiración.

En principio se ha sostenido que lainfluencia de la respiración está mediada porel parasimpático que se estimula en laespiración y se inhibe durante la inspiración.Al respecto hay estudios que demuestran lainhibición que ejercen las neuronasinspiratorias sobre las vagales,(43) el efecto delos baroreceptores en este terreno también hasido profusamente estudiado.(44) Sin embargo,en los últimos tiempos han cobrado nuevoimpulso los mecanismos relacionados con losgases respiratorios(45, 46) y con factores

mecánicos, sean estos a partir de receptorespulmonares(47) o aquellos que responden al flujode sangre en la aurícula derecha disparandoel reflejo de Bainbridge.(48)

Conclusiones

Las modificaciones del ritmo cardíacohan interesado a los investigadores desdehace siglos. En los últimos tiempos se hapuesto especial atención al significado de laVFC como expresión de mecanismosreguladores que actúan sobre el corazón y elorganismo en general. Las bases fisiológicasde la VFC aún no han sido esclarecidas entodos sus detalles, sin embargo se siguetrabajando intensamente en este terreno paraensanchar los horizontes del conocimiento yafianzar el uso de esta herramienta en elterreno médico.

Figura 5: Registro simultáneo de respiración e intervalos R-R en un individuo normal respirando en formarítmica (metrónomo). En color gris se observa el registro del flujo aéreo y en negro se observan lasmodificaciones de los intervalos R-R. Se puede apreciar la estrecha correlación entre respiración y VFC(Migliaro y col. no publicado).

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Instruction for Authors

1 - Objectives: The Cardiovascular Sciences Forum aims to serve all the Cardiovascular Sciencesfields of investigation to hold together multiprofessional experience to optimize the generation ofnew ideas, improving mankind resources in the prevention and treatment of cardiovasculardiseases.

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Upcoming Meetings Session

- IACS INTERNATIONAL ACADEMY OF CARDIOVASCULAR SCIENCESThe 2nd World Congress in JapanKouseinenkin (Wel City Sapporo), Sapporo - JapanJuly 14 - 16, 2006

- 16th WORLD CONGRESS (WSCTS 2006)Ottawa Congress Centre, Ottawa, CanadaAugust, 17 - 20, 2006

- V INTERNATIONAL SYMPOSIUM ON MYOCARDIAL CYTOPROTECTIONWill be held un Pecs, HungarySeptember, 28 - 30, 2006

- GLOBAL SYMPOSIUM ON THE HEART HEALTH & DISEASEWinnipeg, MB. Canada - October 12 - 15, 2006

- BRAZILIAN SOCIETY OF CARDIOLOGY61st Brazilian Congress of CardiologyXXII South American Congress of CardiologyOctober, 21 - 25, Recife-PE

- INTERNATIONAL CONGRESS OF CARDIOVASCULAR SCIENCESScientific 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- X SOUTH-AMERICAN SYMPOSIUM INTERNATIONAL ACADEMY OF CARDIOVASCULAR SCIENCE- IV BRAZILIAN SYMPOSIUM OF THE INTERNATIONAL SOCIETY FOR HEART RESEARCH- VI INTERNATIONAL FORUM ON APPLIED CARDIOVASCULAR PHYSIOLOGY- III STUDENT’S BRAZILIAN CONGRESS ON CARDIOLOGY- AÇÃO DE GRAÇAS /THANKSGIVING

December, 7 – 10, Rio Othon Palace Hotel, Copacabana, Rio de Janeiro-RJ

- 34st CONGRESS OF THE BRAZILIAN SOCIETY OF CARDIOVASCULAR SURGERYApril 12 – 14, 2007Costal do Santinho Resort – Florianópolis /SC

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I - Outline of the Peer Review Routine:

Days:

1 - 2. Confirmation to the authors of manuscript reception

2 - 7. Evaluation of the attendance to the norms of the Instruction to Authors and sendingof copies to three judges, among members of the Archives Editorial Board or to the ScientificCouncil of Referees according to the specific field of the article.

15 - 30. Author’s information regarding peer review exigences.

II - Statements of the Revision Conclusion:

Date: ___/ ___/ ___

Reviewer Code : _____________________________________________________Reviewer Name : _____________________________________________________First Author : _____________________________________________________Title : _____________________________________________________Evaluation : _____________________________________________________

Advice:( ) Excellent ( ) Acceptable( ) Accept ( ) Major Revision( ) Good ( ) Weak( ) Minor Revision ( ) Reject

Comments for the Authors : ______________________________________________________________________________________________________________________________________________________________________________________________________

Peer Review

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