Background: Heart transplantation remains the gold standard for end-stage heart failure, with current ex vivo organ storage times limited to 4 to 6 h before critical tissue damage occurs. Many preservation solutions exist in an attempt to limit both ischemic and reperfusion damage. In order to compare the effects of various storage solutions, mitochondrial function can be used to provide a sensitive analysis of cellular metabolic function. Methods: Experimental plates were seeded with cardiac myoblasts and kept in suspended animation for either 4 or 8 h at either 4(o) or 21 degrees C, in Celsior (R), Perfadex (R), or Somah storage solutions. Cells were then reanimated for 1 h at 37 degrees C to simulate a reperfusion or clinical transplant scenario. Cellular bioenergetics were measured immediately thereafter to examine biochemical differences between preservation solutions and their effectiveness on preserving metabolic function. Results: The oxygen consumption rates of Somah solution were significantly higher than Celsior (R) and Perfadex (R) at 4 degrees C, with the exception of Perfadex (R) at 4(o) for 4 h. This effect was sustained up to 8 h. At 21 degrees C, oxygen consumption rates of Somah solution are significantly higher than Celsior (R) and Perfadex (R) at basal conditions after 4 h, but this effect is not sustained after 8 h. Conclusions: The purpose of this experiment was to study the efficacy of various preservation solutions on a mitochondrial level. The significantly higher oxygen consumption rates of Somah at 4 degrees C suggests that Somah solution may have the ability to protect cellular mitochondrial integrity, improve transplanted organ function by reducing ischemic-reperfusion injury, and thereby improve transplant outcomes. Given that Somah offers benefits over Celsior (R) and Perfadex (R) at 4 degrees C, it should be a target in future organ preservation solution research.

La préservation statique à froid (SCS) est l’étalon-or de la préservation des organes après une greffe. En raison de la pénurie d’organes et de l’augmentation du nombre de patients figurant sur la liste d’attente, le recours aux organes provenant des donneurs à critères élargis, lesquels sont très sensibles au syndrome d’ischémie-reperfusion (IRS), ce qui entraîne une non-fonction primaire (PNF) ou un dysfonctionnement précoce (EAD), est de plus en plus fréquent.Cette recherche avait pour but d’étudier et d’identifier de nouvelles stratégies pour améliorer la qualité de la préservation des organes - d'atténuer les séquelles de l'IRS en utilisant la machine de perfusion hépatique à différentes températures et à différentes périodes d'utilisation après le prélèvement de l'organe ou en ajoutant une hémoglobine extracellulaire en tant que transporteur d'oxygène pendant le SCS.Deux modèles différents ex-vivo ont été analysés : L’un chez le petit animal avec des foies de rats normaux et stéatosiques, pour la perfusion hypothermique (HMP) et SCS avec le transporteur d'oxygène et au niveau préclinique, des foies humains stéatosiques récusés, pour la perfusion normothermique (NMP).Les résultats ont confirmé de manière significative l'intérêt de l’HMP dans la phase pré-ischémique du SCS et celui de l'utilisation de l'hémoglobine extracellulaire en améliorant la fonction hépatique, le maintien de l'anatomie des hépatocytes et en réduisant des marqueurs du stress oxydatif, de l'apoptose et de l'inflammation. Egalement, l'utilisation de NMP a permis d'analyser les foies sévèrement stéatosiques pouvant être récupérés pour une transplantation dans un avenir très proche.Cette recherche met en évidence de nouvelles approches en matière de préservation d'organes susceptibles d'augmenter le pool d'organes et d'améliorer les résultats en transplantation hépatique.Mots-clés : greffe de foie, stockage froid dans le froid, perfusion dans une machine à foie, lésion de reperfusion par ischémie, transporteur d'oxygène. / Static cold storage (SCS) is the gold standard of organ preservation after being procured for transplantation. Due to the organ shortage and the increase of number of patients in the waiting list have pushed the use organs from extended criteria donors which are very susceptible to the ischemia reperfusion syndrome (IRS) leading to primary non-function (PNF) or to early allograft dysfunction (EAD).This research was aimed to study and identify new strategies to improve the quality of organ preservation -liver, to attenuate the IRS sequels by using the liver perfusion machine (LPM) at different temperatures and times of usage after the organ procurement or by adding an extracellular hemoglobin as an oxygen carrier during SCS.Two different ex-vivo models were analyzed: small animal -normal and steatotic rat livers, for hypothermic perfusion (HMP) and SCS with the oxygen carrier and preclinical -steatotic discarded human livers, for normothermic perfusion (NMP).The results significantly confirmed the benefit of the HMP in the preischemic phase of SCS and that of the use of the extracellular hemoglobin by improving the liver function, maintenance of the hepatocytes anatomy and by a reduction of the oxidative stress, apoptosis and inflammation markers. Also, the use of NMP permitted to analyze the severely steatotic livers that can be rescued for transplantation in the very near future.This investigation unveils new approaches in organ preservation that could increase the pool of organs and improve the results in liver transplantation. Key words: liver transplantation, static cold storage, liver machine perfusion, ischemia reperfusion injury, oxygen carrier.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23107号 / 医博第4734号 / 新制||医||1051(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 平井 豊博, 教授 湊谷 謙司, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Introdução: Para realizar-se transplante de órgãos, é necessário que se preserve o enxerto durante a fase de isquemia fria. Entretanto, as soluções de preservação atuais não apresentam capacidade de oxigenar o tecido, o que causa, inevitavelmente, lesão celular. Em vista disso, busca-se associar substâncias antiinflamatórias, vasodilatadoras e antioxidantes à solução de preservação, tentando, assim, melhorar a qualidade da preservação hepática durante a fase de isquemia fria. Animais e métodos: Realizou-se a hepatectomia do doador em 36 ratos Wistar, divididos em 3 grupos de 12 animais. Os fígados desses ratos foram perfundidos e preservados durante 36 horas. No grupo 1, considerado grupo controle, foi utilizada a Solução da Universidade de Wisconsin (UW); o grupo 2 teve a UW acrescida de prostaglandina E1; o grupo 3 teve a UW acrescida de N-acetilcisteína. Realizaram-se biópsias hepáticas e coletaramse amostras da solução de preservação nos tempos de 12, 24 e 36 horas. Resultados: O estudo bioquímico da solução de preservação demonstrou que os níveis de transaminases se elevam com o passar do tempo, mas isso ocorreu em menor nível quando a UW foi acrescida de N-acetilcisteína. A análise histopatológica das lâminas das biópsias revelaram um infiltrado inflamatório portal menor quando a UW foi acrescida de prostaglandina E1. Conclusão: Assim, entendemos que a prostaglandina E1, considerada um potente antiinflamatório e vasodilatador, e a N-acetilcisteína, tida como um excelente antioxidante, acarretam efeito protetor ao enxerto hepático quando associadas à solução de preservação. / Introduction: In order to transplant an organ, it is necessary to preserve the graft during the cold ischemia period. The current preservation solutions are not able to oxygenate the tissue, which inevitably causes cell damage. We intend to associate non-inflammatory, vessel dilating and non-oxidative substances to the preservation solution to try to improve the quality of the liver preservation during the cold ischemia.Animals and methods: We performed the donor’s hepatectomy in 36 Wistar mice divided into 3 groups of 12 mice. The animals’ livers were perfused and preserved for 36 hours. In group 1, the control group, University of Wisconsin solution (UW) was used. In group 2, the UW was used together with prostaglandin 1 (0.5mcg/ml) and in group 3 UW was used together with N-acetylcysteine (0.3mg/ml). Hepatic biopsies were carried out, and samples of the preservation solution were collected in the preservation periods of 12, 24 and 36 hours. Results: The biochemical study of the preservation solution showed that the levels of transaminases increase over time, but this occurred less when UW was added to N-acetylcysteine (p>0,05). The hystopathological analysis of the biopsies showed a smaller portal inflammatory injury when UW was added to prostaglandin E1 (p<0,05). Conclusion: We therefore understand that prostaglandin E1, which is considered to be powerful non-inflammatory and vessel dilator, and Nacetylcysteine, which is an excellent non-oxidative substance, have a protective effect on the liver graft when associated with the preservation solution.

Introdução: Para realizar-se transplante de órgãos, é necessário que se preserve o enxerto durante a fase de isquemia fria. Entretanto, as soluções de preservação atuais não apresentam capacidade de oxigenar o tecido, o que causa, inevitavelmente, lesão celular. Em vista disso, busca-se associar substâncias antiinflamatórias, vasodilatadoras e antioxidantes à solução de preservação, tentando, assim, melhorar a qualidade da preservação hepática durante a fase de isquemia fria. Animais e métodos: Realizou-se a hepatectomia do doador em 36 ratos Wistar, divididos em 3 grupos de 12 animais. Os fígados desses ratos foram perfundidos e preservados durante 36 horas. No grupo 1, considerado grupo controle, foi utilizada a Solução da Universidade de Wisconsin (UW); o grupo 2 teve a UW acrescida de prostaglandina E1; o grupo 3 teve a UW acrescida de N-acetilcisteína. Realizaram-se biópsias hepáticas e coletaramse amostras da solução de preservação nos tempos de 12, 24 e 36 horas. Resultados: O estudo bioquímico da solução de preservação demonstrou que os níveis de transaminases se elevam com o passar do tempo, mas isso ocorreu em menor nível quando a UW foi acrescida de N-acetilcisteína. A análise histopatológica das lâminas das biópsias revelaram um infiltrado inflamatório portal menor quando a UW foi acrescida de prostaglandina E1. Conclusão: Assim, entendemos que a prostaglandina E1, considerada um potente antiinflamatório e vasodilatador, e a N-acetilcisteína, tida como um excelente antioxidante, acarretam efeito protetor ao enxerto hepático quando associadas à solução de preservação. / Introduction: In order to transplant an organ, it is necessary to preserve the graft during the cold ischemia period. The current preservation solutions are not able to oxygenate the tissue, which inevitably causes cell damage. We intend to associate non-inflammatory, vessel dilating and non-oxidative substances to the preservation solution to try to improve the quality of the liver preservation during the cold ischemia.Animals and methods: We performed the donor’s hepatectomy in 36 Wistar mice divided into 3 groups of 12 mice. The animals’ livers were perfused and preserved for 36 hours. In group 1, the control group, University of Wisconsin solution (UW) was used. In group 2, the UW was used together with prostaglandin 1 (0.5mcg/ml) and in group 3 UW was used together with N-acetylcysteine (0.3mg/ml). Hepatic biopsies were carried out, and samples of the preservation solution were collected in the preservation periods of 12, 24 and 36 hours. Results: The biochemical study of the preservation solution showed that the levels of transaminases increase over time, but this occurred less when UW was added to N-acetylcysteine (p>0,05). The hystopathological analysis of the biopsies showed a smaller portal inflammatory injury when UW was added to prostaglandin E1 (p<0,05). Conclusion: We therefore understand that prostaglandin E1, which is considered to be powerful non-inflammatory and vessel dilator, and Nacetylcysteine, which is an excellent non-oxidative substance, have a protective effect on the liver graft when associated with the preservation solution.

Introdução: Para realizar-se transplante de órgãos, é necessário que se preserve o enxerto durante a fase de isquemia fria. Entretanto, as soluções de preservação atuais não apresentam capacidade de oxigenar o tecido, o que causa, inevitavelmente, lesão celular. Em vista disso, busca-se associar substâncias antiinflamatórias, vasodilatadoras e antioxidantes à solução de preservação, tentando, assim, melhorar a qualidade da preservação hepática durante a fase de isquemia fria. Animais e métodos: Realizou-se a hepatectomia do doador em 36 ratos Wistar, divididos em 3 grupos de 12 animais. Os fígados desses ratos foram perfundidos e preservados durante 36 horas. No grupo 1, considerado grupo controle, foi utilizada a Solução da Universidade de Wisconsin (UW); o grupo 2 teve a UW acrescida de prostaglandina E1; o grupo 3 teve a UW acrescida de N-acetilcisteína. Realizaram-se biópsias hepáticas e coletaramse amostras da solução de preservação nos tempos de 12, 24 e 36 horas. Resultados: O estudo bioquímico da solução de preservação demonstrou que os níveis de transaminases se elevam com o passar do tempo, mas isso ocorreu em menor nível quando a UW foi acrescida de N-acetilcisteína. A análise histopatológica das lâminas das biópsias revelaram um infiltrado inflamatório portal menor quando a UW foi acrescida de prostaglandina E1. Conclusão: Assim, entendemos que a prostaglandina E1, considerada um potente antiinflamatório e vasodilatador, e a N-acetilcisteína, tida como um excelente antioxidante, acarretam efeito protetor ao enxerto hepático quando associadas à solução de preservação. / Introduction: In order to transplant an organ, it is necessary to preserve the graft during the cold ischemia period. The current preservation solutions are not able to oxygenate the tissue, which inevitably causes cell damage. We intend to associate non-inflammatory, vessel dilating and non-oxidative substances to the preservation solution to try to improve the quality of the liver preservation during the cold ischemia.Animals and methods: We performed the donor’s hepatectomy in 36 Wistar mice divided into 3 groups of 12 mice. The animals’ livers were perfused and preserved for 36 hours. In group 1, the control group, University of Wisconsin solution (UW) was used. In group 2, the UW was used together with prostaglandin 1 (0.5mcg/ml) and in group 3 UW was used together with N-acetylcysteine (0.3mg/ml). Hepatic biopsies were carried out, and samples of the preservation solution were collected in the preservation periods of 12, 24 and 36 hours. Results: The biochemical study of the preservation solution showed that the levels of transaminases increase over time, but this occurred less when UW was added to N-acetylcysteine (p>0,05). The hystopathological analysis of the biopsies showed a smaller portal inflammatory injury when UW was added to prostaglandin E1 (p<0,05). Conclusion: We therefore understand that prostaglandin E1, which is considered to be powerful non-inflammatory and vessel dilator, and Nacetylcysteine, which is an excellent non-oxidative substance, have a protective effect on the liver graft when associated with the preservation solution.

Seit der Einführung der University of Wisconsin (UW)-Lösung ist für die humane Lebertransplantation eine sichere Kaltkonservierung von 24 Stunden möglich. Ungeachtet dieser Verbesserung kann es postoperativ weiterhin zu schweren Leberfunktionsstörungen bis hin zum Transplantatversagen kommen. Die Leberfunktionsstörungen stehen in engem Zusammenhang mit der Qualität und der Dauer der Organkonservierung. Eine verbesserte Organkonservierung sollte deshalb eine höhere Funktionsrate nach Implantation und eine Verlängerung der sicheren Konservierungsdauer ermöglichen. Mit der Untersuchung sollte die Frage beantwortet werden, ob eine kontinuierliche Perfusion von Schweinelebern mit der UW-Lösung anstelle einer Standardkaltlagerung in UW-Lösung einen geringeren Konservierungsschaden nach sich zieht. Weiterhin sollte mit dem Ziel der Etablierung eines in-vitro-Modells geklärt werden, ob mit dem modifizierten Modell der extrakorporalen Perfusion nach P. Neuhaus neue Konservierungslösungen unter Einsparung von Versuchstieren getestet werden können. Vor diesem Hintergrund wurde in zwei weiteren Versuchsgruppen die kontinuierliche Kaltperfusion mit der Freie Universität (FU)- bzw. der HTK-Lösung durchgeführt. Der Konservierungsschaden der Leber wurde während der sich an die Kaltkonservierung anschließenden Warmperfusion mit Schweineblut in-vitro quantifiziert. Die Unterschiede zwischen der kontinuierlichen Perfusion und der Kaltlagerung waren signifikant, wobei eine geringere Serumkonzentration der Transaminasen in der UW-Gruppe mit einer höheren Gallesekretion, einem geringeren arteriellen Widerstand und einer geringeren Zunahme des Lebergewichts einherging. Die Ergebnisse der FU-Gruppe lassen im Vergleich mit der kontinuierlichen Perfusion mit UW-Lösung eine ähnliche Konservierungsgüte annehmen, während der Konservierungsschaden in der HTK-Gruppe signifikant stärker ausgeprägt war. Zusammenfassend erwies sich die kontinuierliche Kaltperfusion mit der UW-Lösung als das besser geeignete Konservierungsverfahren, nahelegend, daß ein Einsatz eine sinnvolle Alternative zur Standardkaltlagerung sein könnte. Langjährige Erfahrungen mit der kontinuierlichen Perfusion bei der Nierentransplantation beim Menschen zeigen, daß im klinischen Alltag eine kontinuierliche Organperfusion technisch möglich ist. / COMPARISON OF DIFFERENT PRESERVATION SOLUTIONS FOR LONG-TERM CONTINUOUS PERFUSION OF THE LIVER The introduction of the University of Wisconsin (UW) solution for liver preservation in 1988 allowed for the first time the extension of the safe cold storage time up to 24 hours. Nevertheless, severe organ dysfunction of the liver may still occur postoperatively, depending on the quality and the duration of organ preservation. Therefore, improved organ preservation should make possible a higher rate of immediate organ function after transplantation and the extension of the safe cold storage time avoiding any wastage of organs due to liver dysfunction. Our study aimed to investigate whether continuous perfusion of pig livers in comparison to simple cold storage with the UW solution results in improved preservation. Furthermore, we examined if our extracorporal perfusion system, modified by P. Neuhaus, would be suitable to test new preservation solutions before clinical use allowing the establishment of a new in-vitro model. Thus, livers were perfused continuously using the Free University (FU) solution and the Histidine Tryptophan Ketoglutarate (HTK) solution respectively and preservation was compared to the UW solution. The preservation injury was measured in-vitro during a subsequent warm perfusion of the organ with pig blood. Differences between continuous perfusion and simple cold storage were significant. Lower concentrations of the transaminases in the UW group were associated with a higher bile secretion, a lower arterial pressure and a lower increase of the liver weight. Results obtained for the FU group suggest a comparable quality of preservation compared to livers continuously perfused with UW solution, whereas the extent of the preservation injury was significantly higher in the HTK group. In conclusion, in our experimental design continuous perfusion with UW solution seems to be a better method for organ preservation suggesting that the clinical use of this technique may be beneficial. Large experience with continuous perfusion for human renal procurement has proven its technical feasibility.

Made available in DSpace on 2016-01-26T12:51:22Z (GMT). No. of bitstreams: 1 wilsonjoseduca_tese.pdf: 1506382 bytes, checksum: 16d07e2101b80e16a2d850b2111fb461 (MD5) Previous issue date: 2009-06-10 / liver transplantation (OLT) is today the gold standard for the treatment of the end-stage liver disease. The preservation of graft is the cornerstone for the OLT with cadaveric donor. In this context, it is important to evaluate the effectiveness of different solutions used for it. Our objective was to compare the results of OLT, carried out with cadaveric donor, preserved with the solutions of preservation ViaSpan® or Celsior®. Casuistic and Method: we evaluated retrospectively 72 recipients of the OLT. Of these, 36 had their graft preserved with ViaSpanâ solution (Group A) and 36 with Celsiorâ solution (Group B) as follows: the donor was perfused in situ of 1000 ml in the portal vein of ViaSpanâ or Celsiorâ and 3000 ml of Euro-Collins in aortic artery; in the table managed ViaSpanâ or Celsiorâ 500 ml in the portal vein, 250 ml in hepatic artery and 250 ml in the biliary duct. The following variables in groups A and B were evaluated: cost of the solutions, characteristics of the donors, characteristics of the recipients, intraoperative details, reperfusion injury and esteatose of graft with biopsy carried out after the reperfusion. As results of the OLT were evaluated: grafts with primary nonfunction (PNF), initial poor function (IPF), rejection, complications of the biliary duct, complications of the hepatic artery, retransplantation, follow up of the receiver in the first year after the OLT. Results: bigger warm ischemia and duration of surgery in group A (p= 0,002 and 0.001 respectively). The cost of the Celsior solution was lower (less than U$ 266.00 per litre). The remaining: characteristics of the donors, characteristics of the recipients, reperfusion injury, steatosis, PNF, PDF, rejection, retransplantation and recipients survival had not shown significant difference in statistics. Stenosis of the biliary duct was 3 cases (8.3%) in group A and 8 (22.2%) in group B (p= 0.19) and thrombosis of the hepatic artery were 4 cases (11.1%) in group B and none in group A (p= 0.11). Conclusion: the result of OLT, performed with cadaveric donor, preserved with ViaSpanâ or Celsiorâ solutions was similar. However we observe a trend of bigger number of stenosis of the biliary duct and thrombosis of the hepatic artery in the recipients of agencies preserved with the Celsiorâ solution. Thus, we believe that more research is necessary to clarify this relation. / O transplante de fígado (TxF) é hoje o padrão ouro para o tratamento da doença hepática terminal. A preservação do enxerto é a pedra fundamental para o TxF com doador cadáver. Nesse contexto, é importante avaliar a eficácia das diferentes soluções de preservação. Nosso objetivo foi comparar os resultados de TxF, realizados com órgãos de doadores cadáver, preservados com as soluções de preservação ViaSpan® ou Celsior®. Casuística e Método: Avaliamos retrospectivamente 72 pacientes submetidos a TxF. Desses, 36 tiveram seus enxertos preservados com a solução ViaSpan&#61666; (Grupo A) e 36 com Celsior&#61666; (Grupo B) da seguinte forma: perfusão in situ de 1000 ml na veia porta de ViaSpan&#61666; ou Celsior&#61666; e 3000 ml de Euro-Collins na aorta; e na mesa administrou-se 500 ml de ViaSpan&#61666; ou Celsior&#61666; na veia porta, 250 ml na artéria hepática e 250 ml na via biliar. Avaliamos as seguintes variáveis nos grupos A e B: custo das soluções, dados dos doadores, dados dos receptores, dados do intra-operatório, lesão de preservação e esteatose do enxerto com biópsia realizada após a reperfusão. Como resultado do TxF avaliamos: falência primária (FPE) e disfunção primária do enxerto (DPE), rejeição, complicações da via biliar, complicações da artéria hepática, retransplante, sobrevida do receptor no primeiro ano de pós-transplante. Resultados: O tempo de isquemia quente e tempo cirúrgico maiores no grupo A (p= 0,002 e 0,001 respectivamente). O custo da solução Celsior&#61666; foi menor (R$ 400,00 a menos por litro). O restante dos dados dos doadores, dados dos receptores, lesão de preservação e esteatose do enxerto com biópsia realizada após a reperfusão, FPE e DPE, retransplante, sobrevida do receptor não mostraram diferença estatística. A estenose da via biliar foi de 3 (8,3%) casos no grupo A e 8 (22,2%) no grupo B (p= 0,19) e a trombose da artéria hepática foi 4 (11,1%) casos no grupo B e ausente no grupo A (p= 0,11). Conclusão: O resultado de TxF, realizado com doador cadáver, preservado com as soluções ViaSpan® ou Celsior® foi similar. Contudo observamos um maior número de estenose de via biliar e trombose arterial nos receptores de órgãos preservados com a solução Celsior&#61666;. Assim, acreditamos que sejam necessários novos trabalhos para esclarecer esta relação.

Introduction: Coronary artery bypass grafts are most commonly performed using saphenous vein grafts to complement the internal thoracic artery. The saphenous vein will remain popular despite its lower patency rate because it is easily accessible and lengthy enough to perform multiple bypasses. Therefore, several approaches have been studied, with the common goal of finding the optimal conditions that reduce graft failure. They include novel harvest techniques, new preservation preparations, innovative genetic therapies and experimental drugs. We believe a pharmacological pre-conditioning with an anti-oxidative and anti-inflammatory drug during the crucial time of harvest may spark beneficial survival response from the endothelial cells. One particular compound is Celastrol, an HSP90 inhibitor, which displays those antioxidant and anti-inflammatory properties. Methods: Human umbilical vein endothelial cells (HUVEC) were pretreated with various concentrations of Celastrol (10-10M, 10-8M and 10-6M). In order to reproduce oxidative stress found in ischemia/reperfusion, cells were exposed to hydrogen peroxide for a short and extended period (1h and 24h). To mimic storage condition encountered in clinical settings, cells were also exposed in heparinized normal saline. The viability was assessed by LIVE/DEAD assay. As for migrative and proliferative properties, scratch tests were performed. Finally, various protective intracellular pathways were evaluated by Western blot. Results: This study shows that pre-treatment with Celastrol promotes survival in HUVEC submitted to oxidative stress. Notable improvement in cellular viability was detected as early as 1 hour after oxidative stress (H2O2 4 mM), 76.6% vs 66.1% (p=0.005). Significant survival benefits are also reported after prolonged oxidative stress (H2O2 0.5 mM for 24 hours); viability was 93.7% vs 76.9% (p=0.001) for Cel 10- 8 M and 96.6% vs 76.9% (p=0.002) for Celastrol 10-10M when compared to the vehicle. Celastrol, however, did not significantly affect viability of HUVEC stored in heparinized normal saline. Celastrol at 10-6 M promotes faster and more complete wound closure compared to the vehicle or to lower dosages. Celastrol triggers early activation of the RISK pathway, inducing activation of both Akt and ERK1/2 within the first 15 minutes of treatment. Celastrol also induces the expression of HSP70 and HO-1, effectors of the Heat Shock Response and the anti-oxidative response respectively. Conclusion: Pre-treatment by Celastrol provides survival benefits in endothelial cells under oxidative stress. It also stimulates endothelial cell proliferation and migration, promoting faster and more complete re-endothelialisation. Celastrol can potentially be used as an additive to storage solutions to limit endothelial injury and promote graft protection. / Introduction: La chirurgie de pontage coronarien requiert, dans la grande majorité des cas, l’utilisation de l’artère mammaire interne en combinaison avec un ou des greffons provenant de la grande veine saphène. Malgré le taux de perméabilité inférieur aux artères, la veine saphène reste un choix populaire de conduit en raison de son accessibilité et de sa longueur. De ce fait, le greffon veineux devient la cible de multiples approches et le sujet de nombreuses études visant à optimiser sa perméabilité. Celles-ci incluent le raffinement des techniques de prélèvement, les solutions de préservations, les agents pharmacologiques ainsi que la thérapie génique. Il est davantage intéressant de combiner les approches afin de joindre leurs bénéfices, comme, par exemple, ajouter un agent pharmacologique à une solution de préservation. Un agent potentiel serait le Celastrol, connu pour être un inhibiteur du HSP90 et possède des propriétés antioxydantes et anti-inflammatoires. Méthodologie: Des cellules endothéliales humaines provenant de la veine ombilicale (HUVEC) sont pré-conditionnées à de multiples concentrations de Celastrol (10-10M, 10-8M and 10-6M) pendant une heure avant d’être soumises aux conditions de stress. Pour reproduire les conditions per-opératoires de prélèvement, les cellules endothéliales ont été préservées dans du salin (NS) héparinisé. Pour mimer le stress secondaire à l’ischémie/reperfusion, les cellules ont aussi été soumises à diverses concentrations de H2O2. Une analyse de la viabilité cellulaire fut conduite par le test de LIVE/DEAD. La capacité de ré-endothélialisation est étudiée grâce à l’épreuve de scratch test. Les voies intracellulaires de survie telles que le RISK pathway (Akt, ERK1/2), le Heat shock response (HSP70) et la réponse anti-oxydante (via l’activité de HO-1) ont été examinées par immunoblot. Résultats: Les résultats démontrent que la préservation des cellules endothéliales dans du NS héparinisé est associée à une augmentation de la mortalité comparativement au milieu de culture (20.4% vs 1.9%, p=0.004). Toutefois, un traitement au Celastrol n’affecte pas significativement la survie des cellules endothéliales dans le NS héparinisé. Le stress oxydatif induit aussi une augmentation de la mortalité, et ce à dose-dépendante. Suivant un court stress 6 oxydatif (H2O2 4 mM), un pré-traitement au Celastrol 10-10M est associé à une meilleure viabilité comparativement au véhicule (76.6% vs 66.1%, p=0.005). Lorsque soumises à un stress oxydatif prolongé (H2O2 0.5 mM pendant 24h), les HUVEC pré-traitées au Celastrol à 10-8M et 10-10M démontrent une amélioration significative de la viabilité, 93.7% vs 76.9% (p=0.001) et 93.6% vs 76.9% (p=0.002) respectivement. Quant à la ré-endothélialisation, un traitement au Celastrol 10- 6M est associé à une fermeture plus rapide et complète comparativement au véhicule. Un court traitement au Celastrol active précocement les kinases de la voie de RISK (Akt et ERK). Le traitement induit aussi l’expression de HSP70 et HO-1 qui reste soutenue jusqu’à 48 heures posttraitement. Conclusion: Le Celastrol active plusieurs voies de protection intracellulaire tels que le RISK pathway, le Heat Shock Response et la réponse antioxydante via l’activité de HO-1. En corrélation avec cette réponse, il améliore la survie des cellules endothéliales dans un milieu oxydatif. Le Celastrol promeut aussi une ré-endothélialisation plus complète et rapide. Cette étude met en valeur les bénéfices potentiels du Celastrol sur les cellules endothéliales. Afin d’optimiser la protection du greffon, le Celastrol pourrait donc être considéré comme agent adjuvant à une solution de préservation.

INTRODUCTION : Les greffons de veine saphène interne (GVS) sont encore régulièrement utilisés comme conduits en chirurgie de pontage aorto-coronarien (PAC). Les dommages subis par les segments de veine saphène pendant le prélèvement et le stockage favorisent une dysfonction endothéliale qui se manifeste par une diminution de la production d'oxyde nitrique et/ou par une augmentation du niveau de stress oxydant pouvant entraîner une défaillance du greffon veineux se traduisant par une occlusion. La solution saline héparinée est la solution de préservation de référence malgré plusieurs études démontrant ses effets néfastes sur les GVS. GALA est une solution de préservation de greffons autologues vasculaires spécialement développée pour préserver l'intégrité structurale et fonctionnelle de la couche endothéliale des greffons utilisés en chirurgie de pontages aorto-coronariens. OBJECTIF : Comparer la préservation de l'intégrité des fonctions endothéliales des greffons de veine saphène après le stockage dans la solution GALA versus dans la solution saline héparinée dans le cadre d’une étude contrôlée et randomisée en étudiant la réactivité vasculaire en chambres d’organes. RÉSULTATS : Les segments de GVS d'un total de quinze patients ont été obtenus et divisés en anneaux de 3 mm de largeur. Il n'y avait pas de différences significatives dans les niveaux de contraction en réponse au chlorure de potassium, à la phényléphrine, ni dans les concentrations de phényléphrine nécessaires pour atteindre le niveau de contraction cible entre les anneaux du groupe GALA versus le groupe de saline héparinée. Les courbes dose-réponse du groupe solution GALA ont démontré une amélioration significative des relaxations dépendantes de l'endothélium par rapport au groupe solution saline héparinée. Les contractions et relaxations indépendantes de l'endothélium induites respectivement par la phényléphrine et le nitroprussiate de sodium étaient similaires dans les anneaux de GVS des deux groupes. CONCLUSION : L’utilisation intra-opératoire d'une solution développée spécifiquement pour la préservation de l’intégrité endothéliales présente un potentiel d’avantages cliniques chez les patients qui subissent une chirurgie de PAC. Les observations précédentes suggèrent que la solution GALA pourrait réduire la dysfonction endothéliale associée à la défaillance des greffons veineux et incite des évaluations à long terme plus approfondies dans le cadre d’essais cliniques. / INTRODUCTION: Saphenous vein grafts (SVGs) are still commonly used as conduits for coronary artery bypass grafting (CABG). Injury to SVGs during harvesting and storage promotes endothelial dysfunction, which is attributed to a decrease in production of nitric oxide and/or increased level of oxidative stress that can lead to vein graft failure (VGF). Heparinized saline is still the standard of care intraoperative preservation solution despite several studies demonstrating its detrimental effects on SVGs. GALA is an innovative one-time intraoperative graft storage solution developed to preserve endothelial integrity. OBJECTIVE: To investigate, in a randomized controlled study, endothelial functional integrity of saphenous vein grafts following storage in GALA vs heparinized saline using ex vivo vascular reactivity studies in organ chamber experiments. RESULTS: Segments of saphenous vein grafts from a total of fifteen patients were obtained and divided into 3 mm wide rings for evaluation. There were no significant differences in the levels of contraction in response to potassium chloride and to phenylephrine between groups, nor in the concentrations of phenylephrine needed to achieve the target level of contraction in saphenous vein graft rings. Concentration-response curves of the GALA group demonstrated a significant improvement in endothelium-dependent relaxations compared to the heparinized saline group. Endothelium-independent contractions and relaxations induced by phenylephrine and sodium nitroprusside, respectively, were not altered in saphenous vein graft rings from both groups. CONCLUSIONS: Intraoperative application of a solution developed for graft preservation demonstrated a potential benefit to protect endothelial and vascular functional integrity in saphenous vein grafts of patients undergoing CABG. These data suggest that the GALA solution may reduce endothelial dysfunction associated with vein graft failure and warrant further long-term evaluation in clinical trials.