Introdução: A hipotermia tópica (HT) e o pré-condicionamento isquêmico (PCI) são métodos utilizados para diminuir a lesão de isquemia/reperfusão (I/R). A eficácia do uso concomitante da HT e PCI (HT+PCI) no fígado em relação à inflamação e à citoproteção antioxidante não está elucidada. Objetivo: Avaliar o processo inflamatório e os mecanismos de segunda linha de defesa antioxidante na lesão de I/R hepática em ratos em relação à utilização das técnicas de HT e PCI de forma isolada ou associada. Métodos: Ratos Wistar (n=32) foram submetidos à isquemia hepática parcial (70%) durante 90 minutos seguida por 120 minutos de reperfusão. Os animais foram alocados nos grupos sham (n=4), isquemia normotérmica (IN, n=7), PCI (n=7), HT (n=7) e HT+PCI (n=7). O PCI consistiu na aplicação consecutiva de 10 minutos de isquemia e reperfusão antes do insulto isquêmico. A HT foi induzida pela superfusão de solução salina a 26°C sobre os lobos isquêmicos. A eutanásia foi realizada ao término do experimento e as amostras foram coletadas para a realização das análises moleculares utilizando as técnicas de ELISA e Western Blot, com o objetivo de comparar os perfis pró-inflamatório, anti-inflamatório e antioxidante. Resultados: O grupo HT comparado ao grupo IN apresentou diminuição da concentração do fator de necrose tumoral (TNF)-α, interleucina (IL)-1β, IL-6 e IL-12 e um aumento dos níveis de IL-10. O grupo HT apresentou menor expressão da óxido nítrico sintase induzível (iNOS) e um aumento da expressão da óxido nítrico sintase endotelial (eNOS). A expressão da NAD(P)H quinone oxidoreductase-1 (NQO1) foi menor no grupo HT. O PCI não demonstrou diferença significativa em relação a esses marcadores quando comparado ao grupo IN. O grupo HT+PCI apresentou menor concentração de IL-12 e menor expressão da iNOS e NQO1, mas em relação a estas moléculas a utilização de HT isolada demonstrou um comportamento semelhante. O grupo HT+PCI apresentou maior expressão da Kelch-like ECH-associated protein (Keap)-1 e menor expressão do nuclear erythroid 2-related factor 2 (Nrf2) nuclear e citoplasmático em relação ao grupo IN. Conclusão: O método de HT foi eficaz na proteção contra a lesão inicial de I/R. O uso de PCI isolado desencadeou a ativação da segunda linha de defesa antioxidante. A aplicação combinada de HT+PCI não confere benefício adicional em relação ao processo inflamatório quando comparado ao grupo HT, mas apresenta a vantagem de evitar a ativação da segunda linha de defesa antioxidante. / Background: Topical hypothermia (TH) and ischemic preconditioning (IPC) are used to decrease ischemia/reperfusion (I/R) injury. The effectiveness of using concomitantly TH and IPC (TH+IPC) in liver, regarding inflammation and antioxidant cytoprotection, is lacking. Aim: To evaluate the process inflammatory and second-line antioxidant defense mechanisms in hepatic I/R injury in rats in relation to the use of techniques TH and IPC isolate or associated. Methods: Wistar rats (n=32) subjected to partial (70%) hepatic ischemia during 90 minutes followed by 120 minutes of reperfusion. Livers from the animals allocated in sham (n=4), normothermic ischemia (NI, n=7), IPC (n=7), TH (n=7) and TH+IPC (n=7) groups. IPC consisted of consecutive 10-minute periods of ischemia and reperfusion before the ischemic insult. TH was induced by the superfusion of cooled saline at 26oC onto the ischemic lobes. Euthanasia was undertaken exactly at the end of the experiment and samples were collected for molecular analyses by ELISA and Western Blot assays, aiming to compare pro-inflammatory, anti-inflammatory and antioxidant profiles. Results: Compared with NI, TH presented decreased tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-12 concentrations and increased IL-10 levels. TH displayed lower inducible nitric oxide synthase (iNOS), higher endothelial nitric oxide synthase (eNOS) expressions. NAD(P)H-quinone oxidoreductase-1(NQO1) expression was also lower in TH. Isolate IPC showed no differences regarding all these markers compared to NI. TH+IPC showed decreased IL-12 concentration and reduced iNOS and NQO1 expressions, but regarding these molecules isolate TH behaved similarly. TH+IPC showed higher Kelch-like ECH-associated protein (Keap)-1 and diminished nuclear and cytosolic nuclear erythroid 2-related factor 2 (Nrf2) expressions than NI. Conclusion: TH was the effective method of protection against early I/R injury. Isolated IPC entailed triggering of second-line antioxidant defense enzymes. Combined TH+IPC seemed to confer no additional advantage over isolated TH in relation to the inflammatory process, but had the advantage of avoid activation second-line antioxidant defense enzymes.

A gravidade da síndrome isquemia/reperfusão determina o prognóstico da lesão renal aguda (LRA). Agentes como o citrato de sildenafil (Sil) e a N-acetilcisteína (NAC) tem demonstrado efeito renoprotetor na LRA isquêmica com resultados ainda inconclusivos. Esse estudo investigou o efeito do Sil e da NAC na LRA com diferentes tempos de isquemia. Foram utilizados grupos de ratos Wistar, adultos e machos: SHAM; Isquemia 30 min (clampeamento dos pedículos renais por 30 min); Isquemia 30 + Sil (Sil 0,25 mg/kg 60 min antes da isquemia renal); Isquemia 30 + NAC (NAC 150 mg/kg antes e após a isquemia renal); Isquemia 45 (clampeamento dos pedículos renais por 45 min); Isquemia 45 + Sil e Isquemia 45 + NAC. Foram avaliadas a função renal (clearance de creatinina e fração de excreção de sódio-FENa); a lesão oxidativa (peróxidos urinários; substâncias reativas ao ácido tiobarbitúrico - TBARS; óxido nítrico - NO e tióis no tecido renal); a síntese protéica da óxido nítrico sintase induzível iNOS e da heme oxigenase-1 HO-1 (western blotting) e análise histológica renal (área intersticial relativa - AIR e lesão tubulointersticial). Os grupos 30 min tratados com Sil e NAC demonstraram melhora da função renal, redução dos índices oxidantes e NO, ausência de iNOS e presença de HO-1. Nos grupos 45 min, o Sil manteve a função renal, porém demonstrou proteção tubular e oxidante; a NAC não demonstrou efeito protetor em nenhum dos parâmetros avaliados. Quanto à histologia, apenas o Sil induziu redução da AIR e da lesão tubulointersticial nos tempos 30 e 45 min. O estudo confirmou que as características funcionais e histológicas induzidas pelo tempo de isquemia na LRA determinam as respostas às manobras farmacológicas de prevenção. A expressão HO-1 pode ser considerada um mediador de proteção renal. / Severity of ischemic/reperfusion injury syndrome determines the prognosis of acute kidney injury (AKI). Agents such as sildenafil citrate (Sil) and N-acetylcysteine (NAC) have demonstrated renoprotective effect on ischemic AKI which data is still inconclusive. This study investigated the protective action of Sil and NAC in the AKI with different time of ischemia. Adult, male, Wistar rats were divided: SHAM, Ischemia 30 min (renal pedicles clamping for 30 min), Ischemia 30 + Sil (Sil 0,25 mg/kg 60 min before renal ischemia), Ischemia 30 + NAC (NAC 150 mg/kg before and after renal ischemia), Ischemia 45 min (renal pedicles clamping for 45 min), Ischemia 45 + Sil, Ischemia 45 + NAC. Renal function (creatinine clearance and urine sodium fractional excretion - FENa); oxidative injury (urinary peroxides, thiobarbituric acid reactive substances - TBARS, nitric oxide - NO and thiols in renal tissue); expression of inducible nitric oxide synthase - iNOS and heme oxygenase-1 HO-1 (western blotting) and kidney histological analysis (fractional interstitial area - FIA and tubuleinterstitial injury) were evaluated. Sil and NAC treatment in 30 min renal ischemia induced increase in renal function, decrease in the rate of oxidation and NO levels, iNOS absence and HO-1 expression. In the Ischemia 45 groups, Sil it maintained renal function, however demonstrated tubular and oxidative protection; NAC produced no renoprotective effect on any of the parameters evaluated. Histology studies showed that, only Sil induced reduction in FIA and tubuleinterstitial injury at 30 and 45 min ischemic time. The study confirmed that the functional and histological characteristics induced by time of ischemia determine the renoprotective pharmacological agent action in the AKI. HO-1 expression can be a renal protection mediator.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Cardiovascular disease is a major health problem and remains the number one cause of death worldwide. For centuries, medicinal plants have been used in different cultures as medicines for the treatment and control of various diseases. Olea africana, also known as the wild olive, is amongst the herbal plants used by people to treat many ailments.Recently, scientific studies on the hypotensive, vasodilatory and antidysarrhythmic effects of O. africana have been reported. Triterpenoids isolated from the O. africana leaves, for example, have antioxidant properties. The aqueous extract from the leaves of O. africana also have angiotensin-converting enzyme (ACE) inhibitory effects. ACE inhibitors and antioxidants protect the heart against ischemic-reperfusion injury. The serine / threonine protein kinase B (PKB) also known as Akt is activated downstream of phosphoinositide 3- (PI-3) kinase (PI-3-Kinase) and is involved in cardioprotection against ischemia-reperfusion injury. Angiotensin II (AII) decreases the intrinsic PI-3-kinase activity. In this study, we hypothesized that ACE inhibitors increase PI-3-kinase activity and thus activates PKB. The aims of this study were: 1) to determine whether treatment with the crude aqueous extract of leaves of O. africana protect the heart against ischemic-reperfusion injury and 2) if so, to determine whether the protection is mediated via the PKB signaling mechanism. Hearts isolated from male Wistar rats were perfused with different concentrations of the plant extract. In one set of experiments, male Wistar rats were treated with the plant extract (1000 mg/kg/day) for 5 weeks for the evaluation of cardiac function before and after ischemia. At the end of the experiments, hearts were freeze-clamped and kept for PKB / Akt determination. In another set of experiments, we determined the effect of O. africana extract (1000 mg/kg/day) or captopril (50 mg/kg/day) on infarct size. Rats fed jelly served as controls for captopril. In a subset of experiments, hearts were frozen immediately after treatment with O. africana extract (1000 mg/kg/day) or captopril (50mg/kg/day) and PKB were determined.Perfusion with the plant extract significantly decreased coronary flow (p<0.05). The heart function was decreased as evidenced by observed decreases in the force of contraction and heart rate, although these were not measured. Chronic treatment with the crude aqueous plant extract had no effect on cardiac function before ischemia, functional recovery (% left ventricular developed pressure and % rate pressure product) and PKB /Akt phosphorylation (p>0.05). Both the aqueous extract of O. africana leaves and captopril had no effect on infarct size compared to the control group (p>0.05). Captopril,however, improved the recovery of the left ventricular developed pressure. Non-perfused hearts isolated from rats treated with O. africana extract and captopril did not show any response to both captopril and the O. africana extract treatment as measured by PKB /Akt phosphorylation. The results of the present study suggest that the crude aqueous extract of O. africana is not cardioprotective against ischemia-reperfusion injury in this system of the isolated perfused rat heart.

Ischemia and reperfusion are natural steps during kidney transplantation, and IRI is considered one of the most important nonspecific factors affecting allograft dysfunction. Transplanted organs experience several episodes of ischemia, in which cold ischemia occurs during allograft storage in preservation solutions. Even though cold ischemia has been studied extensively, all of the studies have been carried out in vitro and ex vivo models. There is no in vivo model available to examine renal IRI induced solely by cold ischemia. In the present study, we developed an in vivo mouse model to study renal IRI induced exclusively by cold ischemia through clamping the renal pedicle for 1 to 5 hours. During the ischemic phase, blood was flushed from the kidney with cold saline through a small opening on the renal vein. The kidney was kept cold in a kidney cup with circulating cooled saline, while the body temperature was maintained at 37℃ during the experiment. The level of kidney injury was evaluated by plasma creatinine, KIM-1, NAGL, GFR, and histology. Plasma creatinine was significantly increased from 0.15±0.04 mg/dl in the sham group to 1.14±0.21 and 2.65±0.14 mg/dl in 4 and 5-hours ischemia groups at 24 hours after cold IRI. The plasma creatinine in mice with ischemic time <3 hours demonstrated no significant increase compared with sham mice. Changes in KIM-1, NAGL, GFR and histology were similar to plasma creatinine. 65 In summary, we developed and characterized a novel in vivo IRI-induced AKI mouse model exclusively produced by cold ischemia.

Ischemic injury in the heart is followed by an increase in SNS activity and the higher this activity, the poorer patient outcomes. An index of SNS activity in models of ischemia can be achieved by measuring NE, β-AR, and perhaps indirectly PDE4 to give an intracellular aspect on SNS signaling. A 20 minute ischemia-reperfusion was induced in a rat model with physiological measurements at 2-5 weeks post IR. At 3 weeks post IR, rats displayed increased PDE4 expression, decreased β 1-AR expression, increased plasma NE, decreased tissue NE storage, increased Doppler E/A ratio and unchanged LV ejection fraction. PET analysis with FDG revealed no infarct at 2 weeks, while analysis with [13N]NH3 displayed no resting flow defect but revealed trends in flow reserve impairment as early as 2.5 weeks with recovery at 5 weeks post-surgery. Applications of this model could be non-invasive imaging of PDE4 with (R)-[11C]Rolipram PET at early time points for development towards prognostic and therapy guidance in humans.

Dans le cadre du développement d’une stratégie clinique de diminution de l’invasivité de l’acte de Chirurgie cardiaque, axée à la fois sur la réduction du traumatisme de la paroi thoracique, de l’ischémie myocardique peropératoire, et de l’agressivité de la CEC, une étude prospective randomisée a été réalisée pour comparer l’impact sur le métabolisme myocardique en peropératoire de l’utilisation de la cardioplégie cristalloïde Custodiol® versus la solution de cardioplégie de St Thomas au cours de la chirurgie coronarienne. L’objectif de cette étude est de comparer les modifications periopératoire de la concentration dans l’espace interstitiel de lactate, pyruvate, glycérol et glucose dans les deux groupes de cardioplégie et ceci depuis le déclampage jusqu'à 24h en post-opératoire. Matériels et méthodes. Vingt-huit patients ont pu être inclus dans l’étude. Le monitorage a été pratiqué avec la technique de microdialyse (cathéter CMA 70, Analyseur CMA 600, CMA Microdialysis,Sweden), avec une mesure toutes les 10 minutes pendant le temps du clampage et la première heure post déclampage, puis toutes les heures, des concentrations interstitielles des métabolites. Les concentrations plasmatiques des troponines à la sortie du bloc opératoire et à H +12 ont été également évaluées dans les deux groupes. Résultats : Des 28 patients inclus et randomisés, 22 ont pu bénéficier d’un monitorage complet (12 pour le groupe Custodiol® et 10 pour le groupe St Thomas). Six ont été exclus pour des raisons techniques (1 arrachement, 3 plicatures, 1 chute du cathéter et 1 dysfonctionnement de l’analyseur). Une analyse comparative entre les patients inclus et exclus de l’étude ne montre pas de différences significatives pour les facteurs de risque cardiovasculaires, la FEVG, l’âge, le genre. Les valeurs moyennes des concentrations +/- écart type de lactate, pyruvate, glucose et glycérol au déclampage (T0,) sont les suivants : groupe Custodiol® : 2.77+/-1.81 mmol l-1 ; 13.74+/-20.87 μmol l-1 ; 0.46+/-0.84 mmol l-1 ; 196.99+/-122.22 mmol l-1 ; groupe St Thomas : 0.89+/-0.64 mmol l-1 ; 6.49+/-9.10 μmol l-1 ; 0.19+/-0.18 mmol l-1 ; 73.17+/-72.11 mmol l-1. Les temps de CEC et de clampage ont été respectivement dans le groupe Custodiol® de : 94.2+/-14 min et 59.8+/-15 min, et, dans le groupe St Thomas de 82.6+/-15.9 min et 55.8+/-16.29 et min (p=ns). Les concentrations post-opératoires en troponine T (sortie de bloc et H+12) ont été respectivement de 2.8+/-1.8 et 7.4+/-5.3 μmol/L pour le groupe Custodiol® et de 3.3+/-4.0 et 5.0+/-3.6 μmol/L (p=ns) pour le groupe Saint Thomas. Aucun évènement clinique ou électrocardiographique n’a eu lieu en post opératoire dans les deux groupes. Conclusion. Le monitorage de l’état redox myocardique interstitiel a été possible dans les deux groupes de façon sûre et efficace et a permis de déceler des variations des concentrations en métabolites dans les deux groupes en l’absence d’évènements cliniques. Les résultats de ces analyses retrouvent, au déclampage, des concentrations significativement plus hautes de lactate et glycérol dans le groupe Custodiol®. Ces différences s’effacent rapidement pendant la phase de reperfusion avec une tendance (non significative) à une concentration de lactates plus basse dans le groupe de patients du groupe Custodiol®. Des études multicentriques ciblées sur des clampages longs supérieurs à 90 min nous semblent nécessaires pour définir si une différence à la fois métabolique et clinique peut exister entre les différentes solutions de protection cardiaque / In our unit, the challenge is to develop a clinical strategy of reduction of the invasiveness of the “On pump procedure” of cardiac surgery: that means a reduction of the chest wall trauma, of the cross clamping perioperative myocardial ischemia, and of the invasiveness of the extra-corporeal circulation. In this background, we organized a randomized perspective study in order to assess the impact of the perioperative myocardial redox metabolism during the on pump coronary surgery protected with Custodiol® versus St Thomas crystalloid cardioplegias. Objectives: To assess the presence and the severity of the perioperative myocardial ischemia in the Custodiol® versus St Thomas group, defined as the interstitial myocardial concentrations of lactate, pyruvate, glycerol and glucose, at the time of the removal of the aortic clamp. Materials and methods : Twenty height patients could be enrolled in the study and were randomized in the Custodiol® and in the St-Thomas group. Monitoring was assessed with the technique of the cardiac microdialysis (CMA 70 probe, CMA 600 analyzer, CMA Microdialysis, Sweden), by dosing every ten minutes during the aortic cross clamping period and every hour out of the operating room, up to 24 hours, the interstitial myocardial concentrations of Lactate, pyruvate, glycerol and glucose. The Lactate/pyruvate ratio and glucose/lactate ratios and 12 hours post-operative troponin plasmatic concentrations were also assessed. Statistical analysis comparing the Custodiol® versus ST Thomas group were performed via a t-test. Results: Out of the 28 enrolled patients, twenty-two (12 of the Custodiol® group and 10 of the St Thomas group) could be successfully monitored with the microdialysis technique. Six were excluded because of technical reasons (one intempestive ablation, 3 iatrogenic plication of the tube, 1 felled out of the table, one due to a dysfunction of the analyzer). The comparative analysis between included and excluded patients did not prove any statistical result in terms of cardiovascular risk factors, EF, age and gender. At declamping time (T0), mean values of concentrations of lactate, pyruvate, glucose and glycerol were the following: Custodiol® group: 2.77+-1.81 mmol l-1;13.74+-20.87 μmol l-1;0.46+-0. mmol l-1;196.99+-122.22 mmol l-1 ; St Thomas : 0.89+-0.64 mmol l-1 ; 6.49+-9.10 μmol l-1; 0.19+-0.18 mmol l-1; 73.17+-72.11 mmol l-1. Cross clamping and CPB times were respectively 94.2+/-14 et 59.8+/-15 min (Custodiol®), and 82.6+/-15.9 et 55.8+/-16.29 et minutes (St-Thomas) (p=ns) . Post operative plasmatic levels of Troponin (arrival in the ICU and 12 H+12) were respectively de 2.8+/-1.8 and 7.4+/-5.3 (pour le groupe Custodiol®) et de 3.3+/-4.0 et 5.0+/-3.6 μmol/L (Saint Thomas) (ns). Conclusion: Monitoring of the interstitial myocardial redox state was safely possible in both groups and allowed to assess metabolic different findings in the two cardioprotective methods that were not enhanced by perioperative clinical ischemic events. Microdialysis assessed, at the time of aorta declamping, significantly higher concentrations of lactate and Glycerol in the Custodiol® group. That difference regressed during the reperfusion phase with a tendency for a lower lactate level in the Custodiol® group. Multicentric studies focused on cross clamping time longer than 90 minutes seem necessary to enhance metabolic interstitial and clinical superiority between cardioprotective solutions

Le foie et le poumon sont deux organes intimement liés et les atteintes pulmonaires sont fréquentes dans les pathologies hépatiques. En transplantation hépatique, les atteintes pulmonaires, les complications et la morbi-mortalité qu’elles engendrent, peuvent être en lien avec la pathologie hépatique du receveur mais aussi avec le donneur via le greffon, sa qualité et sa préservation. L’objectif de cette thèse était d’étudier deux aspects de cette interaction : 1- l’impact de l’insuffisance respiratoire aigüe (définie par le recours à la ventilation mécanique) sur le devenir post-transplantation ii) les conséquences des phénomènes d’ischémie-reperfusion du greffon hépatique sur les paramètres cardio-pulmonaires du receveur.Dans le premier travail, nous avons étudié, sur une cohorte de patients transplantés hépatiques à l’Hopital Henri Mondor (n=350 patients), le devenir post-opératoire en fonction de la présence ou non d’une ou plusieurs défaillances d’organe (neurologique, respiratoire, rénale, hémodynamique, hépatique et de la coagulation). Les patients avec au moins une défaillance d’organe ont une diminution significative de la survie post-LT à 90 jours (79% contre 96%) et 1 an (70% contre 91%) comparés aux patients cirrhotiques sans défaillance. Dans un deuxième travail issu de la même cohorte, nous développons un modèle permettant de prédire la mortalité à court terme et composé de 6 facteurs dont l’existence d’une défaillance d’organe. Le 3e travail, avec un effectif plus important (cohorte de l’Agence de la Biomédecine, PHRC « Optimatch »), a cherché à confirmer nos premiers résultats et à étudier le poids de chacune des défaillances d’organe et en particulier de la défaillance pulmonaire. Non seulement la présence d’une défaillance mais le nombre de défaillances au moment de la TH influence négativement le devenir à 3 mois des transplantés hépatiques. De plus, les défaillances pulmonaire et rénale sont des facteurs de risque indépendant de mortalité à 3 mois post-transplantation. Ces travaux montrent aussi que l’influence de ces défaillances d’organe peut être modulée en fonction du type de greffon, i.e. des critères du donneur.En plus de la qualité du greffon hépatique, la conservation de celui-ci a un impact sur la fonction du greffon et sur les paramètres cardio-pulmonaires chez le receveur. En effet dans un modèle de transplantation hépatique chez le gros animal (cochon), nous avons montré que les lésions d’ischémie/reperfusion du greffon engendrées entre le prélèvement et l’implantation ont des conséquences sur la fonction du greffon, le myocarde et le poumon. Ces lésions sont modulables selon la technique de préservation, notamment via l’utilisation de machine de perfusion.Ces travaux démontrent qu’au cours de la transplantation hépatique l’atteinte du parenchyme pulmonaire a un rôle clinique majeur chez le receveur et est médiée en partie par des phénomènes d’ischémie-reperfusion du greffon potentiellement modifiables par des améliorations des pratiques médicales. / Liver and lung are two closely related organs. In liver transplantation, lung damage, complications, morbidity and mortality can be related to the liver disease of the recipient but also to the donor via the graft, its quality and its preservation. The aim of this manuscript was to study two aspects of these interactions: 1- the impact of acute respiratory failure (defined by the use of mechanical ventilation) on the post-transplantation outcome ii) the consequences of the ischemia-reperfusion injury of the graft on cardiopulmonary parameters in the recipient.In the first study, we investigated the impact of one or more organ failure (cerebral, lungs, kidney, circulation, hepatic and coagulation) on the 90-day mortality post LT. Patients with at least one organ failure had a significant decrease in post LT survival at 90-day (79% versus 96%) and 1 year (70% versus 91%) compared with cirrhotic patients without failure. In a second study, from the same cohort, we developed a model to predict short-term mortality. This model is composed of 6 factors including the existence of organ failure. In the third study, with a largest cohort (Agence de Biomedicine, PHRC “Optimatch”), we have confirmed these first results and we observe that the number at the LT influences the outcome. Decision tree-modeling identified 6 subgroups further classified in 4 increasing risk classes, highlighting the prognostic importance of respiratory failure and renal failure at the LT as well as complex interactions between donor and recipient features.In addition to the quality of the graft, its preservation has an impact on the graft function and on the cardiopulmonary parameters in the recipient. Indeed, in a model of LT in the large animal (pig) we show that the ischemia / reperfusion injuries, generated between the sampling and the implantation, have consequences on graft function, myocardium and lung. These lesions could be subdued according to the preservation technique.These studies demonstrate that during LT the recipient's pulmonary complications and its morbidity are related to the recipient’s pre-existing hepatic disease and to the donor via the graft through ischemia-reperfusion phenomena of the graft.

Notre équipe s’intéresse à la relation structure-fonction d’une protéine, l’antithrombine (AT), un inhibiteur physiologique de la coagulation, en vue d’un développement thérapeutique. Cette protéine anticoagulante, capable de lier un motif pentasaccharidique sur les dérivés hépariniques, possède en outre, à fortes concentrations (500%), des propriétés anti-inflammatoires médiées par sa liaison aux héparan-sulfates cellulaires. Ce profil a mené à l’évaluation de l’AT dans des situations associant un emballement de la coagulation et de l’inflammation, comme c’est le cas au cours du sepsis sévère et d’autres situations d’ischémie-reperfusion (I/R). Cependant, les fortes concentrations utilisées dans les études précliniques nécessiteraient d’administrer des doses d’AT incompatibles avec le profil de sécurité de cette protéine anticoagulante.Dans ce contexte, nous avons, au cours de ce travail, caractérisé un variant d’AT (AT-N135Q-Pro394) dépourvu d’activité anticoagulante et doué d’une affinité augmentée pour l’héparine. Ce variant est capable de piéger des dérivés hépariniques et apparait comme un candidat idéal pour une utilisation comme antidote en cas de surdosage en héparine non fractionnée (HNF), héparines de bas poids moléculaire (HBPM) ou fondaparinux. Par ailleurs, ce variant pourrait être utilisé à des doses cytoprotectrices, sans risque hémorragique.Afin de tester cette dernière hypothèse, nous avons développé un modèle d’I/R rénale chez la souris, qui s’accompagne d’une augmentation significative de marqueurs de dysfonction rénale (Urée, Créatinine, Kim-1) et de l’inflammation (expression tissulaire de cxcl-1, il-6). L’AT avait déjà été montrée protectrice (Mizutani et al, Blood 2003) dans un modèle murin comparable. De façon surprenante, nous n’avons observé aucun des effets protecteurs décrits, ni sur l’inflammation ni sur la fonction rénale, et que ce soit avec de l’AT plasmatique, de l’AT recombinante ou encore avec un mélange équimolaire d’AT latente et native. Ce même modèle nous a pourtant permis de mettre en évidence les effets nephroprotecteurs et anti-inflammatoires d’une autre protéine anticoagulante, la protéine C activée. Ces résultats décevants font écho à la rétractation pour fraude de l’article de Mizutani et al. en 2013. Le travail approfondi que nous avons mené nous permet de clarifier la littérature et d’affirmer que l’AT, d’origine plasmatique ou recombinante, ne possèdent pas d’effet protecteur dans l’I/R rénale chez la souris. Dans ces conditions, le variant AT-N135Q-Pro394 n’a pas été testé.Concernant la seconde indication, l’AT-N135Q-Pro394 avait déjà été évaluée in vivo comme antidote aux dérivés hépariniques, HNF, HBPM et fondaparinux, avec d’excellents résultats. Néanmoins, cet effet antidote a été exploré spécifiquement par mesure de l’activité anti-facteur Xa alors que l’AT inhibe plusieurs enzymes de la cascade de coagulation tel que les facteurs VIIa, IXa et IIa. Nous avons donc exploré cet effet antidote dans un test plus global de la coagulation, le test de génération de thrombine (TGT) pour pouvoir le comparer aux autres stratégies non spécifiques utilisées pour antagoniser les dérivés hépariniques (facteur VII activé recombinant, concentré de complexes prothrombiques activés ou protamine). De façon intéressante, dans un plasma mimant un surdosage, notre variant présente un effet antidote supérieur aux agents hémostatiques et au sulfate de protamine vis-à-vis du fondaparinux et des HBPMs, respectivement, et équivalent au sulfate de protamine vis-à-vis de l’HNF. Enfin, dans du plasma en l’absence d’anticoagulant, l’AT-N135Q-Pro394 ne montre aucun effet sur la génération de thrombine contrairement aux agents hémostatiques et au sulfate de protamine qui, ajoutés seuls dans du plasma, modifient significativement le profil des TGT / Our team topic focuses on the structural-function relationship of a natural anticoagulant, antithrombin (AT), in order to develop potential therapeutic agents. AT inhibits several serine proteases of the coagulation cascade and its inhibitory activity is increased when AT binds to a pentasaccharidic motif contained within in the heparin derivatives. At high concentrations (500%), AT also exerts anti-inflammatory and cytoprotective properties through its binding to heparan sulfate proteoglycans, making it a good candidate for supportive therapy in clinical settings associating inflammation and coagulation activation. Indeed, AT has already been evaluated in vivo in various models of ischemia-reperfusion injury (IRI) and AT even reached a large-scale clinical trial in severe sepsis. However, the high concentrations of AT that are needed to exert anti-inflammatory properties are inconsistent with the safety profile of this anticoagulant protein.In this context we have further characterized an AT variant (AT-N135Q-Pro394) with increased affinity to heparin but devoid of anticoagulant activity. Indeed, this variant was described to be able to trap heparin derivatives and our work was to pursue the characterization of this variant as an antidote toward heparin derivatives in clinical situations of overdosing. In addition, this AT variant binds to heparan sulfate proteoglycans with higher affinity, as compared to native AT, and appears as a promising cytoprotective agent whose administration would not be associated with any bleeding risk.To test the latter hypothesis, we developed a murine model of renal IRI in which the renal function was severely impaired, as attested by increased kidney injury markers (urea, creatinine, kim-1) and local kidney inflammation (renal gene expression of il-6 and cxcl-1). Indeed, in 2003, Mizutani et al. reported a protective effect of AT in a similar murine model of renal IRI. Surprisingly, we observed none of the described protective effects, neither on inflammation nor renal function, with plasma AT, recombinant AT and an equimolar mixture of native and latent AT. Nevertheless, the same model enabled us to highlight the nephroprotective and anti-inflammatory properties of another anticoagulant protein, activated protein C (APC), as previously reported. These disappointing results coincided with the withdrawal in 2013 of the study of Mizutani et al., and our work allowed us to clarify the literature and to claim that neither recombinant nor plasma-derived native nor latent forms of AT exhibit a protective effect in renal IRI in mice. Under these conditions, AT-N135Q-Pro394 variant has not been tested in our model.AT-N135Q-Pro394 has also been previously shown to efficiently neutralise the anticoagulant activity of heparin derivatives, including unfractionated heparin (UFH), low molecular weight heparins (LMWH) and fondaparinux in vivo. Nevertheless, this reversal effect was only explored by anti-factor Xa assays whereas AT inhibits a number of coagulation proteases, including factors VIIa, IXa and IIa. Therefore, we explored AT-N135Q-Pro394 variant in a more global coagulation assay, the thrombin-generation assay (TGA), in order to compare its activity with non-specific reversal agents used toward heparin derivative overdose (recombinant-activated factor VII, activated prothrombin-complex concentrate or protamine). Interestingly, in plasma mimicking an overdose, our variant demonstrated greater reversal efficiency as compared to hemostatic agents and protamine sulfate toward fondaparinux and LMWH, respectively, and was as efficient as protamine sulfate toward UFH. Finally, when added to native plasma (in the absence of heparin derivative), AT-N135Q-Pro394 showed no effect on thrombin generation unlike hemostatics and protamine sulfate that all significantly affect the TGA profile

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22362号 / 医博第4603号 / 新制||医||1043(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 稲垣 暢也, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22696号 / 医博第4640号 / 新制||医||1045(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 羽賀 博典, 教授 妹尾 浩, 教授 木村 剛 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM