Since integrity and functions of vascular endothelial cells are greatly affected by shear stress, a variety of in vitro systems to subject endothelial cells under precisely controlled fluid conditions has been developed. Complicated designs of the conventional flow devices, however, have impeded such implementation. In the present study, we designed and developed a novel parallel-plate flow chamber (PPFC). It consists of multiple layers of different materials to adjust the required geometries of the chamber and provide a wide span of biomedical research applications. Because the chamber stacks separate layers to constitute the flow channel, different pieces can be easily removed or replaced. Moreover, the multilayer design only requires 2D cutting, which is easier and faster to manufacture. It is also capable of accepting up to four glass slides facing each other so that the flow within the channel is exclusively formed by endothelial cells. Furthermore, it minimizes the pressure loss across the chamber while maximizing the effective area of endothelial cells up to 96 cm2. Results from mathematical analysis and dye injection experiments showed that a uniform magnitude of shear stress is applied throughout the entire surface of endothelial cells. In addition, the morphological changes and attenuated gene expression of pro-inflammatory mediators were observed in endothelial cells exposed to the physiologically relevant shear stress. These findings indicate that our newly designed PPFC can provide a better in vitro system for versatile applications of biomedical research. The reperfusion of blood flow occurred in a number of conditions such as stroke and organ transplantation immensely augments tissue injury and can cause more severe damage than prolonged ischemia. The injuries caused by cessation and reperfusion of blood flow are closely related to the inflammatory reactions involving in endothelium-leukocyte cascade responding to a shear stress exerted by the flow. Shear stress is also known to play an important role in human chronic diseases including atherosclerosis, neurological disorders, and cancer metastasis. Therefore, it is important to investigate the transmission of mechanical stimuli such as shear stress to various complex endothelial cell signaling pathways which process as a whole is often referred as mechanotransduction. Shear stress-mediated signaling pathways have been known to trigger endothelial cell responses and contribute to the pathophysiology of human vascular diseases. The present study was designed to apply the novel PPFC to biomedical research, especially ischemia/reperfusion injury. The changes in mRNA and protein expression of inflammatory mediators in endothelial cells were analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. RBE4 and HMEC-1 cells were either maintained in continuous laminar flow condition (Normal Flow) or subjected to 1 h of flow cessation followed by reperfusion of flow (Ischemia/Reperfusion) for 24 h. Ischemia/Reperfusion significantly up-regulated expression of inflammatory mediators, such as IL-6, MCP-1, ICAM-1, VCAM-1, and E-selectin, in microvascular endothelial cells. Furthermore, antioxidant pyrrolidine dithiocarbamate (PDTC) significantly attenuated ischemia/reperfusion-induced overexpression of pro-inflammatory mediators. These data indicates that our newly designed PPFC provide a better in vitro system for versatile applications of biomedical research. / Master of Science

Thesis (MSc)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: The ultimate consequence of the interruption of blood flow to the myocardium is necrosis. In view of the prevalence of coronary artery disease in the general population, and the deleterious effects of myocardial ischaemia on myocardial tissue, it is important to develop new strategies to protect the myocardium against ischaemia. Necrosis of myocardial tissue has for a long time been considered to be the main component of the damage incurred by myocardial infarction. Recently the importance of the contribution of apoptotic cell death in the context of myocardial ischaemia/reperfusion injury has become apparent. There is a general agreement that early reperfusion is necessary to salvage myocardial tissue from cell death. Preconditioning is the phenomenon whereby brief episodes of ischaemia and reperfusion protect the heart against a subsequent longer period of ischaemia. This endogenous mechanism is the strongest form of protection against myocardial infarction that has yet been described. Apart from ischaemie preconditioning (IPC), protection can also be elicited with pharmacologic agents, such as activation of the beta-adrenergic receptor with isoproterenol. Ischaemie preconditioning protects the myocardium against necrosis, arrhythmias and apoptosis, and increases functional recovery upon reperfusion. Betaadrenergic receptor stimulated preconditioning (PPC) has been shown to improve post-ischaemie functional recovery, but it is not known whether it also protects against myocardial infarction and apoptosis. The signaling pathways involved in preconditioning have been extensively studied. A distinction is usually made between factors that act as triggers, or as mediators of protection. Triggers activate cellular responses before the onset of sustained ischaemia, and its involvement is demonstrated by showing that inhibitors of the trigger bracketing the preconditioning protocol can block its protective effect, or that transient administration with washout before sustained ischaemia can activate a protective effect. A mediator operates during sustained ischaemia, and its involvement is demonstrated by showing that infusion of an inhibitor of its action immediately prior to sustained ischaemia (without washout) can block its protective effect. Another approach to demonstrate a mediator role is to attempt to activate signal transduction pathways during sustained ischaemia. As it is not possible to infuse substances during ischaemia, activators are infused immediately prior to ischaemia without washout of the agent and subsequently its effect on protection is observed. It is clear that the evolutionary conserved stress activated pathways are involved in preconditioning. There are three pathways i.e., the extracellular receptor activated pathways (ERK), c-jun terminal activated kinases (JNK) and p38 mitogen-activated protein kinases (MAPK). The precise role of the p38 MAPK pathway has not been elucidated. Experimental evidence has suggested a role for the activation of p38 MAPK as a trigger, as well as a mediator of the protective effect of preconditioning. There is however also strong evidence that the attenuation of p38 MAPK activation during sustained ischaemia, rather than its activation, is responsible for the protection that is observed. Furthermore, the role of p38 MAPK has only been investigated in relation to its protection against necrosis, but not apoptosis. AIMS: The aim of this study was to: (I) Establish a model of preconditioning in neonatal cardiomyocyte cell culture. The reason was that such a model could potentially enable one to rapidly elucidate the signal transduction pathways in an environment without the influence of non-cardiac cells. (II) Investigate whether IPC and ~PC protect against necrosis and apoptosis. (III) Elucidate the role of the stress-activated kinase, p38 MAPK, in preconditioning. METHODS: 1. Neonatal rat cardiomyocyte cell culture model A viability assay with 3-[4,5- Dimethylthaizol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) was first developed using different concentrations - a concentration of 0.25% was found to be optimal to determine viability. Neonatal cardiomyocyte cell cultures were subjected to sustained simulated "ischaemia" by using either 5 mM KCN plus deoxyglucose (DOG) for 5 min or potassium cyanide (KCN) for 45 min. Some cell cultures were preconditioned with either chemical ischaemia (5 mM KCN for 5 min) or isoproterenol (10-7 M) for 5 min and 60 min reoxygenation before being exposed to sustained simulated ischaemia. 2. Isolated adult rat cardiomyocyte model Isolated cardiac myocytes were exposed to 2 hours of hypoxia, which was induced by pelletting the cells by centrifugation, and covering them with a thin layer of mineral oil. Some groups were preconditioned with either hypoxia for 10 min at 37° C or isoproterenol (10-7 M) for 5 min, followed by reoxygenation for 20 minutes. The trypan blue exclusion method and MTT method developed in the neonatal cardiomyocytes were used to assess viability. 3. Isolated perfused rat heart model 3.1 Infarct size was determined in a model of regional ischaemia by using tetrazolium staining and determining the area of necrosis (exclusion of tetrazolium) as a percentage of area at risk. These hearts were subjected to 35 min global ischaemia and 30 min reperfusion. Some groups were preconditioned by three cycles of 5 min global ischaemia or addition of isoproterenol (10-7 M) for 5 min, followed by 5 min reperfusion before the onset of sustained regional ischaemia. 3.2 p38 MAPK activation and markers of apoptosis: p38 MAPK activation was determined using antibodies against dual phosphorylated p38 MAPK (i.e. activated p38 MAPK). Apoptosis was measured by using antibodies against activated caspase-3, and against a fragment of PARP (PARP cleavage). For these experiments isolated rat hearts were exposed to global ischaemia for 25 min followed by 30 min reperfusion. Some groups were preconditioned with three cycles of 5 min global ischaemia. A global ischaemia model was used in order to have sufficient tissue available for the Western blot determinations. This necessitated a shorter period of sustained ischaemia, as the globally ischaemie heart does not recover sufficiently after a longer period of ischaemia such as is necessary in regional ischaemia experiments. 3.3 The role of p38 MAPK in ischaemie preconditioning was investigated by administration of SB 203580 (1IJM),a selective inhibitor of p38 MAPK, either bracketing the preconditioning (i.e. to determine its role as a trigger) or for 10 min immediately prior to sustained ischaemia (i.e. to determine its role as a mediator). The second approach was to use anisomycin, an activator of p38 MAPK, as a trigger (infusion for 10 min followed by wash out) or as a mediator (10 min immediately prior to sustained ischaemia) in the same model as used for determination of p38 MAPK activity. The infusion of anisomycin for 10 min has been shown to elicit activation of p38 MAPK to a similar extent as has been observed with an ischaemie preconditioning protocol. The endpoints used were infarct size and markers of apoptosis. RESULTS: 1. Neonatal rat cardiomyocyte cell culture model It was not possible to establish a model of preconditioning of neonatal cardiomyocytes that was consistently successful. It was therefore decided to abandon the attempts and to use a different cell model. 2. Isolated adult rat cardiomyocyte model Isolated adult cardiomyocytes were preconditioned successfully, but produced too little material to perform simultaneous determinations of cell viability and Western blots (p38 MAPK activation and markers of apoptosis). It was therefore decided to use the isolated perfused adult rat heart. 3. Isolated perfused adult rat heart model 3.1 Both IPC and PPCprotect against infarction and apoptosis: Using two models of preconditioning i.e., IPC and PPC, the protective effects of preconditioning were demonstrated convincingly against infarction (necrosis). IPC and PPC both caused a significant reduction in infarct size (12.2±1.4 and 15.2±2.6%) versus Non-PC hearts (29.6±2.9%) (p < 0.001). Both forms of preconditioning also protected against apoptosis, by significantly reducing the markers of apoptosis, caspase-3 activation and PARP cleavage. The protection afforded by both forms of preconditioning was accompanied by a marked decrease in activation of p38 MAPK upon reperfusion. The relationship between p38 MAPK and the protection that was elicited by preconditioning was then investigated, namely whether p38 MAPK acted as a trigger, or as a mediator of protection. To investigate the role of p38 MAPK as a mediator or a trigger in preconditioning, use was made of (i) a specific inhibitor of p38 MAPK activation i.e., SB 203580 and (ii) a known activator of p38 MAPK i.e., anisomycin. 3.2 p38 MAPK as a trigger of protection: Administration of SB 203580 during the IPC protocol and washed out before sustained ischaemia did not abolish the protective effect of ischaemie preconditioning, and resulted in a small, but significant increase in caspase-3 activation and PARP cleavage. On the other hand, activation of p38 MAPK with anisomycin for 10 min followed by washout also resulted in a significant reduction in necrosis (infarct size 14.9±2.2 versus 29.6±2.9% in Non-PC hearts) (p < 0.001) and both markers of apoptosis. The latter results suggested that p38 MAPK was a trigger of preconditioning. If this was the case, why didn't SB 203580 abolish the protection of IPC? The most likely explanation was that multiple protective mechanisms were activated during a multi-cycle protocol of ischaemic preconditioning, of which activation of p38 MAPK was only one. Inhibition of p38 MAPK with SB 203580 would therefore not be expected to block the activation of those mechanisms that were independent of p38 MAPK, but were still capable of protecting against necrosis or apoptosis. It is very interesting that a small increase in apoptosis was observed when SB 203580 was used in this situation, as it may indicate that the protection against apoptosis was more dependent on the activation of p38 MAPK than the protection against necrosis, as no effect was seen on infarct size. Another explanation could be that infarct size determination was not sensitive enough to detect such small effects. 3.3 p38 MAPK as a mediator of protection: Inhibition of p38 MAPK activation with SB 203580 administered 10 min before sustained ischaemia caused a significant decrease in infarct size compared to Non-PC hearts (12.6±1.9 vs 29.6±2.9%) (p < 0.001) equivalent to that of hearts preconditioned with ischaemia. This was accompanied by a similar pattern of protection against apoptosis, with significantly reduced activation of caspase-3 activation and PARP cleavage. These results strongly supported a role for the attenuation of p38 MAPK activation as a mediator of preconditioning against ischaemia/reperfusion-mediated necrosis and apoptosis. However, the results of the experiments with anisomycin were at first glance not compatible with such a conclusion. The administration of the activator of p38 MAPK, anisomycin, for 10 min immediately prior to sustained ischaemia resulted in significant protection against necrosis (infarct size 16.6±2.4% vs 29.6±2.9% in Non-PC hearts) (p < 0.01) and reduced caspase-3 activation and PARP cleavage indicating less apoptosis. The reason for these findings were probably that this method of administration of anisomycin did in fact not activate p38 MAPK during sustained ischaemia, but actually served as a trigger to protect against ischaemia - similarly as if it had been infused with washout of the drug. Support for this notion was found in the fact that p38 MAPK activation was decreased upon reperfusion. These results suggested that the logistical problem of not being able to infuse a drug into the myocardium during ischaemia could not be overcome by immediate prior infusion, and that the administration of anisomycin in this way had activated downstream effectors of the p38 MAPK signal transduction pathway. An important contender for such an effector would be heat shock protein 27 (HSP27), which has been shown to play an important role in protection against apoptosis, and stabilisation of actin, and thus the cytoskeleton. Another possibility was that anisomycin had activated the JNK stress activated kinases. The elucidation of a role of this signal transduction pathway would necessitate the use of anisomycin in the presence of an agent such as curcumin, an inhibitor of JNK. Final conclusion: The work in this thesis showed that the stress activated kinase, p38 MAPK, was involved in the protective effect of ischaemie preconditioning. The results suggested a role for the activation of p38 MAPK as a trigger of protection, and the attenuation of p38 MAPK as a mediator of protection, which was observed in the reduction of both necrosis (infarct size) and apoptosis as determined with caspase- 3 activation and PARP cleavage. / AFRIKAANSE OPSOMMING: Die afsluiting van bloedvloei na die miokardium gee aanleiding tot nekrose. In die lig van die voorkoms van koronêre bloedvatsiekte onder die algemene populasie, en die nadelige effekte van miokardiale isgemie op miokardiale weefsel, is dit belangrik om nuwe strategieë te ontwikkel wat die miokardium teen isgemie beskerm. Nekrose van miokardiale weefsel word tradisioneel as die belangrikste komponent van die skade aangerig deur miokardiale infarksie beskou. Die belang van apoptotiese seldood in die konteks van miokardiale isgemie/herperfusie (I/R) het onlangs na vore getree. Dit word algeneem aanvaar dat vroeë vroegtydige herperfusie noodsaaklik is om miokardiale weefsel te beskerm teen seldood. Prekondisionering is 'n verskynsel waartydens kort episodes van IIR die hart teen 'n daaropvolgende langer periode van isgemie beskerm. Hierdie endogene meganisme is die kragtigste vorm van beskerming teen miokardiale infarksie tot dusver beskryf. Afgesien van isgemiese prekondisionering (IPC), kan beskerming ook deur farmakologiese middels, soos byvoorbeeld die aktivering van die beta-adrenerge reseptore met isoproterenol, ontlok word. IPC beskerm die miokardium teen nekrose, arritmieë en apoptose, en verhoog funksionele herstel na herperfusie. Daar is reeds aangetoon dat betaadrenerge prekonsionering (~PC) post-isgemiese funksionele herstel verbeter, maar dit is nog onbekend of beskerming ook teen miokardiale infarksie en apoptose verleen word. Die seintransduksie paaie betrokke tydens prekondisionering is reeds in detail bestudeer. Daar word gewoonlik tussen faktore wat optree as snellers, of as mediators van beskerming, onderskei. Snellers aktiveer sellulêre response voor die aanvang van volgehoue isgemie, en hul betrokkenheid word aangetoon deurdat inhibisie van snellers tydens die prekondisionering protokol, beskerming ophef. Snellers se effekete kan ook ontlok word deur hulle tydelike toe te dien en dan net voor volgehoue isgemie weer uit te was. Mediators oefen hulle effek tydens volgehoue isgemie uit, en hulle betrokkenheid word gedemonstreer deurdat toediening van inhibitors net voor volgehoue isgemie (sonder uitwas) hulle beskermende effekte ophef. Mediators se rol kan ook aangetoon word deur te poog om seintransduksie paaie tydens volgehoue isgemie te aktiveer. Aangesien dit ontmoontlik is om middels tydens isgemie te infuseer, word aktiveerders onmiddelik voor die aanvang van isgemie toegedien sonder om hulle uit te was, sodat hulle effekte op beskerming vervolgens bestudeer kan word. Dit is duidelik dat die evolusionêr-behoue stres geaktiveerde paaie tydens prekondisionering betrokke is. Daar is drie paaie nl. die ekstrasellulêre reseptor geaktiveerde pad (ERK), c-jun terminaal geaktiveerde kinases (JNK) en p38 mitogeen geaktiveerde proteïen kinases (MAPK). Die spesifieke rol van die p38 MAPK pad is nog nie ontrafel nie. Eksperimentele bewyse stel 'n rol vir die aktivering van p38 MAPK as 'n sneller, sowel as 'n mediator van die beskermende effek van prekondisionering, voor. Daar is egter ook sterk bewyse dat 'n afname in p38 MAPK aktivering tydens volgehoue isgemie, eerder as sy aktivering, verantwoordelik is vir die waargenome beskermende effek. Verder is die rol van p38 MAPK slegs in die konteks van beskerming teen nekrose, maar nie teen apoptose nie, bestudeer. DOELWITTE: Die doelwit van hierdie studie was: (I) Die vestiging van 'n prekondisionering model in neonatale kardiomiosiet in selkultuur. Hierdie model sou potensieel 'n spoedige ontrafeling van die seintransduksie paaie sonder die invloed van nie-kardiale selle bewerkstellig. (II Om ondersoek in te stelof IPC en PPCteen nekrose en apoptose beskerm. (III) Die ontrafeling van die rol van die stres geaktiveerde kinase, p38 MAPK, tydens prekondisionering. METODES: 1. Neonatale rot kardiomiosiet weefselkultuur model 'n Lewensvatbaarheids essai is ontwikkel deur van verskillende konsentrasies van 3-[4,5-dimetielthiazol-2-yl]-2,5-difeniel-tetrazolium bromied (MTT) gebruik te maak - 'n konsentrasie van 0.25% was optimaalom lewensvatbaarheid te bepaal. Neonatale kardiomiosiet weefselkulture is onderwerp aan volgehoue gesimuleerde "isgemie" deur gebruik te maak van 5 mM KCN plus deoksiglukose (DOG) vir 5 minute of 45 min KCN. Sommige weefselkulture is geprekondisioneer deur middel van chemiese isgemie (5 mM KCN vir 5 min) of van isoproterenol (10-7 M) vir 5 minute en 60 minute reoksigenasie alvorens dit bloot gestel is aan volgehoue gesimuleerde isgemie. 2. Geïsoleerde volwasse rot kardiomiosiet model Geïsoleerde kardiomiosiete is aan twee uur hipoksie blootgestel deur selle in 'n pellet te sentrifugeer en met 'n dun lagie mineraalolie te bedek. Sommige groepe is geprekondisioneer deur middel van 10 minute hipoksie by 37°C, of toediening van isoproterenol (10-7 M) vir 5 minute gevolg deur 20 minute reoksigenasie. Die tripaanblou uitsluitings metode en MTT metode soos ontwikkel in die neonatale kardiomiosiet model is gebruik om lewensvatbaarheid te bepaal. 3. Geïsoleerde geperfuseerde volwasse rot hart model 3.1 Infarkgrootte is bepaal met 'n model van streeks isgemie deur van tetrazolium kleuring gebruik te maak, waarna die area van nekrose (uitsluiting van tetrazolium) as 'n presentasie van die risiko area bepaal is. Hierdie harte was onderwerp aan 35 minute globale isgemie en 30 minute herperfusie. Sommige groepe is geprekondisioneer met 3 siklusse van 5 minute globale isgemie, of die toevoeging van isoproterenol (10-7 M) vir 5 minute, gevolg deur 5 minute herperfusie voor die aanvang van volgehoue streeks isgemie. 3.2 p38 MAPK aktivering en merkers van apoptose: p38 MAPK aktivering is bepaal deur gebruik te maak van anti-liggame teen tweeledige gefosforileerde p38 MAPK (d.w.s. geaktiveerde p38 MAPK). Apoptose is bepaal deur gebruik te maak van anti-liggame teen geaktiveerde kaspase-3, en teen 'n fragment van PARP (PARP kliewing). Tydens hierdie eksperimente is geïsoleerde rotharte bloot gestel aan 25 minute globale isgemie gevolg deur 30 minute herperfusie. Sommige groepe is geprekondisioneer met drie siklusse van 5 minute globale isgemie. Om voldoende weefsel vir Westerse klad tegnieke te verkry, is gebruik gemaak van 'n globale isgemie model. As gevolg hiervan was 'n kort periode van volgehoue isgemie genoodsaak, aangesien die globale isgemiese hart nie voldoende herstel na 'n langer periode van isgemie nie, soos wat benodig word in streeks isgemiese eksperimente. 3.3 Die rol van p38 MAPK tydens IPC is bepaal deur die toediening van 'n 1IJM konsentrasie van SB 203580, 'n selektiewe inhibitor van p38 MAPK, hetsy tydens prekondisionering (d.w.s. om die rol as 'n sneller te bepaal), óf vir 10 minute direk voor die aanvang van volgehoue isgemie (d.w.s. om dus sy rol as mediator te bepaal). Die tweede benadering was om anisomisien, 'n aktiveerder van p38 MAPK, as sneller (toediening vir 10 minute gevolg deur uitwassing) of as mediator (10 minute direk voor aanvang van volgehoue isgemie) in dieselfde model as in die geval van p38 MAPK aktiviteit bepaling, te gebuik. Die toediening van anisomisien vir 10 minute het aangetoon dat dit p38 MAPK aktivering kan ontlok tot dieselfde maate as die IPC protokol. Die eindpunte was infarkgrootte en merkers van apoptose. RESULTATE: 1. Neonatale rot kardiomiosiet weefselkultuur model Dit was nie moontlik om 'n suksesvolle model met konsekwente resultate vir die prekondisionering van neonatale kardiomiosiete te vestig nie. Daar is dus besluit om af te sien van hierdie pogings en eerder 'n alternatiewe selmodel te gebruik. 2. Geïsoleerde volwasse rot kardiomiosiet model Geïsoleerde volwasse kardiomiosiete is suksesvol geprekondisioneer, maar het te min materiaalopgelewer vir die gelyktydige bepaling van sellewensvatbaarheid, p38 MAPK aktivering en merkers vir apoptose. Daar is dus besluit om die geïsoleerde geperfuseerde volwasse rothart te gebruik. 3. Geïsoleerde geperfuseerde volwasse rothart model 3.1 Beide IPC en PPCbeskerm teen infarksie en apoptose: Deur gebruik te maak van twee prekondisionering modelle d.w.s. IPC en PPC, is die beskermende effekte van prekondisionering teen infraksie (nekrose) oortuigend gedemonstreer. Beide IPC en PPC het In betekenisvolle afname in infarkgrootle veroorsaak (12.2 ± 1.4 en 15.2 ± 2.6% respektiewelik), vs Nie-PC harte (29.6 ± 2.9%)(p < 0.001). Beide vorme van prekondisionering het ook teen apoptose beskerm deur die apoptose merkers, kaspase-3 aktivering en PARP kliewing te verlaag. Die beskerming verkry deur beide vorms van prekondisionering is geassosieer met In merkbare afname in die aktivering van p38 MAPK na herperfusie. Die verband tussen p38 MAPK en die beskerming ontlok deur prekondisionering is gevolglik ondersoek, naamlik of p38 MAPK optree as 'n sneller of as 'n mediator van beskerming. Om die rol van p38 MAPK as 'n mediator of sneller tydens prekondisionering te ondersoek is daar gebruik gemaak van (I) 'n spesifieke inhibitor van p38 MAPK aktivering nl. SB 203580 en (II) 'n bekende aktiveerder van p38 MAPK nl. anisomisien. 3.2 p38 MAPK as 'n sneller vir beskerming: Toediening van SB 203580 tydens die IPC protokol en uitwassing daarvan voor die aanvang van volgehoue isgemie het nie die beskermende effek van IPC opgehef nie, en het gelei tot 'n klein maar betekenisvolle verhoging in kaspase-3 aktivering en PARP kliewing. Andersins het die aktivering van p38 MAPK met anisomisien vir 10 minute gevolg deur In uitwas ook tot In betekenisvolle afname in nekrose (infarkgrootte 14.9 ± 2.2 vs 29.6 ± 2.9% in Nie-PC harte) (p < 0.001) in beide merkers van apoptose gelei. Laasgenoemde resultate dui daarop dat p38 MAPK inderdaad 'n mediator van prekondisionering is. Indien dit die geval is, waarom het SB 203580 nie die beskermende effek van IPC opgehef nie? Die mees waarskynlike verklaring is dat veelvuldige beskermingsmeganismes tydens 'n multi-siklus protokol van IPC geaktiveer word, waarvan p38 MAPK aktivering slegs een is. Dit is dus onwaarskynlik dat die inhibisie van p38 MAPK met SB 203580 die aktivering van daardie meganismes onafhanklik van p38 MAPK sal blokkeer en steeds in staat sal wees tot beskerming teen nekrose en apoptose. Dit is interessant dat In klein verhoging in apoptose waargeneem is toe SB 203580 gebruik is onder hierdie toestande, aangesien dit daarop kan dui dat die beskerming teen apoptose meer afhanklik was van die aktivering van p38 MAPK as die beskerming teen nekrose, siende dat geen effek op infarkgrootte waargeneem is nie. 'n Verdere verklaring kan wees dat die bepaling van infarkgrootte nie sensitief genoeg is om sulke klein effekte waar te neem nie. 3.3 p38 MAPK as 'n mediator vir beskerming: Inhibisie van p38 MAPK aktivering deur SB 203580 toediening 10 minute voor volgehoue isgemie het 'n betekenisvolle verlaging in infarkgrootte in vergelyking met Nie-PC harte veroorsaak (12.6 ± 1.9 vs 29.6 ± 2.9%) (p < 0.001) soortgelyk aan dié van harte geprekondisioneer met isgemie. Dit is geassosieer met In soortgelyke patroon van beskerming teen apoptose, met betekenisvolle verlaagde kaspase-3 aktivering en PARP kliewing. Hierdie resultate ondersteun die rol van die afname van p38 MAPK aktivering as 'n mediator van prekondisionering teen I/R-gemedieerde nekrose en apoptose. Die resultate van die anisomisien eksperimente was met die eerste oogopslag nie in oorstemming met hierdie gevolgtrekking nie. Die toedienning van die p38 MAPK aktiveerder, anisomisien, vir 10 minute voor volgehoue isgemie het tot 'n betekenisvolle beskerming teen nekrose aanleiding gegee (infarkgrootte 16.6 ± 2.4 vs 29.6 ± 2.9% in Nie-PC harte) (p < 0.01) en verlaagde kaspase-3 aktivering en PARP kliewing wat dui op verlaagde apoptose. Die rede vir hierdie bevindings is moontlik dat die metode van anisomisien toediening nie p38 MAPK geaktiveer het tydens volgehoue isgemie nie, maar eintlik gedien het as 'n sneller vir beskerming teen isgemie - amper asof dit toegedien sou word sonder om uitgewas te word. Ondersteuning vir hierdie aanname word gevind in die feit dat p38 MAPK aktivering verlaag is na herperfusie. Hierdie resultate stel voor dat die logistiese probleem dat In middel nie tydens isgemie toegedien kan word nie, nie oorkom kan word deur onmiddelike voortydige infusie nie, en dat die toediening van anisomisien op hierdie manier gelei het tot die aktivering van stroom-af effektors van die p38 MAPK seintransduksie pad. 'n Belangrike kandidaat vir so 'n effektor is "heat shock protein 27" (HSP27), wat reeds aangetoon is om 'n belangrike rol in die beskerming teen apoptose en destabilisering, en dus die sitoskelet, te speel. 'n Ander moontlikheid is dat anisomisien die JNK stres geaktiveerde kinases geaktiveer het. Die ontrafeling van die rol van hierdie seintransduksie pad noodsaak die gebruik van anisomisien in die teenwoordigheid van 'n agent soos curcumin, 'n JNK inhibitor. Finale gevolgtrekking: Die werk soos vervat in hierdie tesis toon aan dat die stres geaktiveerde kinase, p38 MAPK, betrokke is in die beskermings effek van isgemiese prekondisionering. Die resultate dui op 'n rol vir die aktivering van p38 MAPK as 'n sneller vir beskerming, en die afname in p38 MAPK as 'n mediator vir beskerming, soos waargeneem in die vermindering van veranderlikes van beide nekrose (infarkgrootte) en apoptose soos bepaal deur kaspase-3 aktivering en PARP kliewing.

Cette étude a été conçue afin d’évaluer l’effet d’un pré-traitement à long terme au célécoxib sur la taille d’infarctus suite à un infarctus du myocarde. Sachant que le célécoxib est un anti-inflammatoire et que des dommages myocardiques peuvent découler des processus inflammatoires, l’inhibition de l’inflammation devrait hypothétiquement réduire la taille d’un éventuel infarctus. Pour ce faire, un traitement au célécoxib (3 mg/kg/jour i.p.) ou au véhicule (DMSO 50% ; EtOH 15% ; eau distillée) a été administré chroniquement pendant 28 jours à des rats mâles Sprague-Dawley (n=18 par groupe) par pompes osmotiques ALZET. Après avoir été anesthésiés, les animaux ont été sujets à l’occlusion de l’artère coronaire gauche descendante, suivie d’une période de reperfusion de 24 heures. Les résultats démontrent que la taille de l’infarctus des animaux traités au célécoxib est significativement réduite comparativement à celle du groupe témoin (37,5±2,5% versus 48,0±2,6% de la zone à risque, p < 0,05). Par la suite, l’accumulation de neutrophiles indique une hausse de ces leucocytes pour la zone ischémique, sans toutefois discriminer entre les groupes traité et non-traité, qui contenaient aussi les couches sub-endocardique et sous-épicardique. Cependant, aucune différence significative est notée entre les groupes traité et témoin au niveau de l’expression de la prostaglandine E2 plasmatique et du facteur de nécrose tumorale alpha. D’un autre côté, l’apoptose, déterminée par le ratio de Bax/Bcl2 et par un essai TUNEL est significativement réduite pour la couche sub-endocardique de la zone à risque des animaux traités au célécoxib. Enfin, l’agrégation plaquettaire, induite à l’adénosine diphosphate et analysée dans le sang complet, suggère que le célécoxib diminue l’agrégation plaquettaire. Cette étude indique alors qu’un pré-traitement au célécoxib peut réduire la taille d’infarctus par un mécanisme impliquant l’apoptose. / This study was designed to evaluate the effect of long-term pre-treatment with celecoxib, a cyclooxygenase-2 inhibitor, on myocardial infarct size. Since celecoxib is an anti-inflammatory and that myocardial damages can be present in the occurrence of inflammatory processes, inhibition of inflammation should hypothetically reduce the size of an eventual infarct. Celecoxib (3 mg/kg/day i.p.) or vehicle (DMSO 50%; EtOH 15%; distilled water) was administered chronically to male Sprague-Dawley rats (n=18 per group) through ALZET osmotic pumps for 28 days. Under anaesthesia, the animals were then subjected to left anterior descending coronary artery occlusion for 40 minutes, followed by 24-hour reperfusion. The results show that myocardial infarct size in celecoxib-treated rats was significantly reduced compared to the control group (37.5±2.5% versus 48.0±2.6% of the area at risk, p < 0.05). Accumulation of neutrophils, estimated by myeloperoxidase levels, indicated an increase in the ischemic area without any significant difference between groups. No significant difference was observed between the treated and vehicle groups in terms of plasma prostaglandin E2 and tumour necrosis factor-alpha. Apoptosis, evaluated by Bax/Bcl-2 and terminal dUTP nick-end labelled-positive cells, was significantly decreased in the subendocardial layer of the ischemic area in celecoxib-treated rats. Adenosine diphosphate-induced platelet aggregation in whole blood suggested that celecoxib diminished platelet aggregation. This study indicates that pre-treatment with celecoxib can reduce infarct size by a mechanism which may involve apoptosis.

Le récepteur éboueur CD36 facilite l’internalisation des acides gras libres non estérifiés (AGNE) au niveau des tissus cardiaque et périphériques. Lors d’une ischémie-reperfusion du myocarde (MI/R), les dommages produits sont en partie liés à l’internalisation des AGNE et à la production d’espèces réactives de l’oxygène, contrairement à ce qui est observé chez des souris déficientes en CD36 (CD36-/-). Nous avons émis l’hypothèse selon laquelle le CP-3(iv), un ligand synthétique du récepteur CD36, exercerait un effet cardioprotecteur en réduisant la taille de la zone myocardique infarcie lors d’une ischémie transitoire du myocarde. Nos objectifs étaient 1) de déterminer l’effet cardioprotecteur du CP-3(iv) et 2) de définir son mécanisme. Pour cela, des études in vivo et ex vivo ont été faites. Des souris de type sauvage ont été traitées avec le CP-3(iv) (289 nmol/kg) par voie sous-cutanée pendant 14 jours avant d’être soumises à 30 minutes d’ischémie suivant la ligature de l’artère coronaire gauche descendante et de sa reperfusion pendant une période de 6 ou 48 heures. De plus, des coeurs isolés de souris ont été perfusés 30 minutes, suivi de 40 minutes à faible débit (10%) et de 30 minutes de reperfusion pendant laquelle le coeur est perfusé avec le CP-3(iv) à une concentration de 10-6 M. Nos travaux ont montré que l’effet cardioprotecteur d’un traitement préventif par le CP-3(iv) permet de diminuer la taille de l’infarctus et préserve l’hémodynamie cardiaque de façon dépendante du CD36 puisque cet effet est non visible chez les souris CD36-/-. De plus, le CP-3(iv) exerce non seulement un effet systémique, mais aussi un effet cardioprotecteur direct sur le coeur isolé. / The scavenger receptor CD36 facilitates the internalization of non-esterified fatty acids (NEFA) on cardiac and peripheral tissues. During myocardial ischemia and reperfusion (MI/R), the damage induced is in part related to the internalization of NEFA and the production of reactive oxygen species, in opposition to what is observed in CD36-deficient mice (CD36-/-). We hypothesized that CP-3(iv), a synthetic ligand of the CD36 receptor, provides a cardioprotective effect by reducing the infarct area during a transient myocardial ischemia. Our objectives were 1) to determine the cardioprotective effect of CP-3(iv) and 2) to define its mechanism. For this, in vivo and ex vivo studies have been done. Wild-type mice were treated with CP-3(iv) (289 nmol/kg) subcutaneously during 14 days before being submitted to 30 minutes of ischemia following left anterior descending coronary artery ligature and reperfusion for a period of 6 to 48 hours. In addition, isolated mouse hearts were perfused 30 minutes, followed by 40 minutes with low flow (10%) and 30 minutes of reperfusion during which the heart is perfused with CP-3(iv) at a concentration of 10-6 M. Our work has shown that the cardioprotective effect of preventive treatment with CP-3(iv) reduces the infarct size and preserves cardiac hemodynamics in a CD36-dependent manner because this effect is not visible in CD36-/- mice. In addition, CP-3(iv) not only exerts a systemic effect, but also a direct cardioprotective effect on the isolated heart.

L'ischémie se caractérise par l'obstruction d'une artère coronaire qui prive le tissu d'un apport en oxygène et nutriments. Bien que nécessaire, la reperfusion, c'est-à-dire la réouverture de l'artère, s'accompagne de lésions tissulaires, appelées lésions de reperfusion. Au cours de l'I/R, le TNF-α, cytokine pro-inflammatoire, augmente. Sa liaison sur son récepteur TNFR1 induit le recrutement des protéines FADD et procaspase-8 formant le complexe DISC qui permet l'activation de la caspase-8. La caspase-8 clive une protéine pro-apoptotique, Bid, qui induit une perméabilisation de la membrane mitochondriale entrainant une production excessive de radicaux libres et une libération de cytochrome c. Cette dernière associée à Apaf-1 et procaspase-9 sert de plateforme d'activation à la caspase-9, qui, une fois activée, clive et active la caspase-3. La caspase-2 est une caspase initiatrice, tout comme la caspase-8. Pourtant, son rôle dans l'I/R cardiaque est peu connu. La production de ROS via la voie TNF-α/caspase-8 provoque des dommages à l'ADN. Ceci entraine l'activation de PARP-1, une enzyme impliquée dans la réparation de l'ADN. En fonctionnant, PARP-1 produit de l'ADP-ribose qui peut se fixer sur le canal TRPM2 et ainsi l'activer. L'ouverture de ce canal cationique provoque une entrée de Ca2+ qui contribue à la mort cellulaire et aux lésions de reperfusion. L'objectif de ce travail est de déterminer les mécanismes de mort cellulaire faisant intervenir la caspase-8, la caspase-2 et TRPM2 et d'évaluer les effets d'une inhibition de ces protéines sur les lésions de reperfusion. Un modèle de rat I/R met en évidence une augmentation de TNF-α après seulement 1h de reperfusion suivie d'une activation de la caspase-8. Cette activation entraine une production de ROS qui altère la structure et la fonction du canal RyR2, favorisant la fuite de Ca2+ du reticulum sarcoplasmique vers le cytosol. La caspase-2, exprimée dans le ventricule gauche, est activée avant la caspase-8 et induit une voie apoptotique de type intrinsèque. L'inhibition de la caspase-8 ou de la caspase-2 diminue les lésions de reperfusion. Parallèlement, le TNF-α induit un courant de type TRPM2 via l'activation de la caspase-8 et la production de ROS. In vivo, l'inhibition de TRPM2 par le clotrimazole diminue les lésions de reperfusion chez un modèle de souris I/R. La caspase-8, la caspase-2 et TRPM2 contribuent aux lésions de reperfusion et apparaissent comme de bonnes cibles dans la cardioprotection. / Myocardial ischemia and reperfusion (I/R) lead to repefusion injury. TNF-α, a pro-inflammatory cytokine, increases during reperfusion and contributes to this injury. The binding TNF/TNFR1 leads to the recruitment of FADD, TRADD and procaspase-8 and form a complexe named DISC. This complexe activates caspase-8, which cleaves Bid, a pro-apoptotic member of Bcl-2 family. tBid disrupts the mitochondrial membrane and induces a ROS production and a release of cytochome c, localized in intermembrane space. In cytosol, a complexe named apoptosome is formed with cytochrome c, Apaf-1 and procaspase-9 to activate caspase-9, which cleaves and activates caspase-3. Like caspase-8, caspase-2 is an initiator caspase. But little data exists on the role of this caspase in myocardial I/R.The disruption of mitochondria induces a ROS production which causes DNA damage. The enzyme PARP-1, involved in DNA repair, is then activated. By operating, PARP-1 produces ADP-ribose which can bind on TRPM2, a non selective cationic channel of TRP family. The opening of TRPM2 causes an increase of cytosolic calcium promoting cell death and reperfusion injury. The goal of this study was to determine the mechanisms of cell death after I/R involving caspase-8, caspase-2 and TRPM2 and to test an inhibitor of each protein on reperfusion injury. With a model of rat I/R, we demonstrated that TNF-α increases after only 1h of reperfusion following by a caspase-8 activation and a ROS production. Oxidative stress causes a modification of RyR2 with a leak of calcium in cytosol. Caspase-2, also expressed in ventricles, is activated before caspase-8 and induces an intrinsic apoptotic pathway until caspase-3 activation. An inhibition of caspase-8 or caspase-2 decreases the reperfusion injury.In mouse cardiomyocytes, TNF-α induces a TRPM2-like current through caspase-8 activation and ROS production. TRPM2 inhibition by clotrimazole decreases cell death and reperfusion injury in vivo.In conclusion, caspase-2, caspase-8 and TRPM2 play an important role in cell death pathway ans should be good therapeutical tools.

L’ischémie/reperfusion (I/R) induit des lésions impliquées dans la physiopathologie de la transplantation cardiaque où elles contribuent à augmenter le rejet de greffe. Le stress induit par l’ischémie entraîne la libération d’ATP conduisant à l’activation de récepteurs purinergiques (P2R) dont l’expression est établie au niveau cardiaque et immunitaire. L’objectif de ce travail a été d’explorer l’effet de la signalisation P2R sur le phénotype des cellules dendritiques (DCs) et la réponse des cardiomyocytes (CM) à l’I/R. Nous avons montré que la récepteur P2Y11 (P2Y11R) avait une action immunomodulatrice sur les DCs en diminuant la sécrétion d’IL-6 et IL-12 et en inhibant la polarisation de la réponse adaptative vers Th1. Le post-conditionnement pharmacologique ciblant P2Y11R a apporté une protection efficace sur les CM en limitant le stress oxydant et en activant la PKCe connue pour inhiber l’ouverture du mPTP. Les effets protecteurs et immunomodulateurs de P2Y11R se sont confirmés in vivo en diminuant le rejet allogénique dans un modèle murin de transplantation cardiaque hétérotopique. Nos résultats suggèrent que P2Y11R pourrait être une cible thérapeutique apportant des effets bénéfiques en transplantation cardiaque. / Ischemia/reperfusion (I/R) injuries are involved in the pathophysiology of heart transplantation where they will increase graft rejection. Ischemia generates cellular stress leading to ATP release in the extracellular medium that may activate purinergic receptors (P2R) expressed by cardiomyocytes and immune cells. Therefore, these receptors may play important regulatory roles. The aim of this study was to investigate the effect of P2R signaling on dendritic cells phenotype (DCs) and cardiomyocyte (CM) response to I/R. We showed that P2Y11 receptor (P2Y11R) exhibited an immunomodulatory role in DCs by decreasing release of IL-6 and IL-12 and inhibiting polarization of the adaptive response towards Th1. Pharmacological post-conditioning targeting P2Y11R provided effective protection to CM by limiting oxidative stress and activating PKCe known to inhibit the opening of the mPTP. The protective and immunomodulatory effects of P2Y11R stimulation were confirmed in vivo by the decrease of allogeneic acute rejection in a murine model of heterotopic heart transplantation. In conclusion, our results strongly suggest that P2Y11R may be a promising therapeutic target providing beneficial effects in cardiac transplantation.

Kardialen Strukturen, wie dem koronaren oder endokardialen Endothel, dem Myokard und auch dem Perikard werden unter physiologischen und pathophysiologischen Bedingungen zunehmend autokrine oder parakrine Funktionen zugesprochen. Es ist gut belegt, dass das Herz durch Freisetzung von löslichen Mediatoren nach Myokardischämie einen entscheidenden Anteil an der postischämischen Regulation der Vasomotion hat. Allerdings weniger bekannt ist die Bedeutung einer Mediator-vermittelten kardialen Autoregulation bei postischämischen Veränderungen der Myokardkontraktilität. In dieser Arbeit wird eine neue negativ inotrope Substanz(en) (NIS) beschrieben, die nach myokardialer Ischämie aus isolierten Herzen freigesetzt wird und die an sequentiell perfundierten Herzen, die als Bioassay eingesetzt werden, einen deutlichen kardiodepressiven Effekt hervorruft. In isolierten Feld-stimulierten Rattenkardiomyozyten reduziert NIS dosisabhängig die systolische Zellverkürzung und den Ca2+-Transienten (Konfokale Laser Scan Mikroskopie). Der negativ inotrope Effekt setzt sowohl in isolierten Herzen als auch Kardiomyozyten schnell ein und ist reversibel. Katecholamine maskieren und überspielen den negativ inotropen Effekt in Abhängigkeit von der Ischämiedauer. Voltage clamp Untersuchungen auf Einzelzellebene zeigten, dass NIS den Ca2+-Einstrom Ica über die L-Typ Ca2+-Kanäle reduziert. Somit scheint NIS die Myokardkontraktilität und Zellverkürzung über eine Verminderung der intrazellulären systolischen Ca2+-Konzentrationen durch Blockade der L-Typ Ca2+-Kanäle zu reduzieren und nicht etwa über eine Ca2+-Desensitivierung. Derzeit ist noch nicht geklärt, über welchen Mechanismus NIS den Ca2+-Einstrom blockiert. Da weder die Gewebsspiegel von cGMP und cAMP noch die PKA Aktivität durch NIS moduliert werden, ist es unwahrscheinlich, dass eine Dephosphorylierung von Untereinheiten des L-Typ Ca2+-Kanals der Funktionsweise von NIS zu Grunde liegt. Die Ergebnisse legen nahe, dass NIS direkt mit dem Ca2+-Kanal interagiert, z.B. durch Bindung an ein Kanalprotein. Die chemische Struktur von NIS ist derzeit noch ungeklärt, allerdings gibt es Hinweise, dass es sich nicht um ein Protein handelt. Die Substanz(en) ist stabil, Hitze-resistent (56°) und ein dialysierbares Molekül mit einem geringen Molekulargewicht (< 0.5 kDa). Die kardiodepressorische Substanz(en) wird nicht vom Koronarendothel freigesetzt. Eine abschließende Bewertung, ob NIS durch Aggravation der kontraktilen Dysfunktion myokardschädigend oder durch Senkung des myokardialen Sauerstoffverbrauches kardioprotektiv wirkt, ist derzeit noch nicht möglich. / Autocrine and paracrine functions are increasingly being attributed under physiological and pathophysiological conditions to cardiac structures such as the coronary or endocardial endothelium, the myocardium, and the pericardium as well. Reliable evidence exists to confirm that the heart, through the release of soluble mediators after myocardial ischemia, plays a decisive role in post-ischemic regulation of vasomotion. Less well-known, however, is the significance of mediator-effected cardiac autoregulation in cases of post-ischemic changes of myocardial contractility. This study describes a new negative inotropic substance or substances, NIS, released from isolated hearts after myocardial ischemia. NIS elicits marked cardiodepressive effects on sequentially perfused hearts used as bioassays. In isolated field-stimulated rat cardiomyocytes, NIS reduces, as a function of dose, systolic cell shortening and Ca2+ transients (as detected by confocal laser-scan microscopy). The negative inotropic effect occurs quickly both in isolated hearts as well as in cardiomyocytes, and is reversible. Catecholamines counteract the negative inotropic effect, as a function of ischemia duration. Voltage-clamp investigations on the single-cell level have disclosed that NIS reduces Ca2+ inflow Ica via L-type Ca2+ channels. NIS appears to decrease myocardial contractility and cell shortening through reduction of intracellular systolic Ca2+ concentration, by blockade of L-type Ca2+ channels: and not, say, by Ca2+ desensitization. It has not yet been definitely established by which mechanism NIS blocks Ca2+ inflow. Since NIS modulates neither the tissue level of cGMP and cAMP, nor PKA activity, it is improbable that NIS acts by dephosphorization of sub-units of the L-type Ca2+ channel. The results of this study imply that NIS directly interacts with the Ca2+ channel, perhaps by binding to a channel protein. Although the chemical structure of NIS has not yet been elucidated, there are indications that it is not a protein. The substance(s) is/are stable, heat resistant (up to 56°C), and dialyzable as a molecule with low molecular weight (< 0.5 kDa). It is not yet possible to provide conclusive evaluation of whether NIS acts to damage the myocardium by aggravation of the contractile dysfunction, or whether it exerts cardioprotective action by diminishing myocardial oxygen consumption.

Les cardiofibroblastes (CF) possèdent des rôles clés dans la régulation de la structure et du fonctionnement myocardique. Leurs implications physiopathologiques, notamment dans le remodelage et la fibrose, ont été largement décrites dans les maladies cardiovasculaires chroniques. Cependant, leurs rôles au cours de la phase aigüe d'ischémie-reperfusion (l/R) restent encore à élucider. Nous avons donc émis l'hypothèse que les CF pouvaient participer à la protection des cardiomyocytes (CM) face aux lésions d'l/R. Le but de ce travail a donc consisté en l'exploration et l'identification des mécanismes de cette protection. Un modèle cellulaire de CM et CF de rats nouveau-nés in vitro et un modèle d'l/R in vivo chez la souris ont été utilisés. Nos résultats montrent que la présence des CF, en co-culture avec les CM, augmente de façon paracrine leur viabilité, face aux lésions d'l/R. Cette action paracrine a été confirmée par l'utilisation du sécrétome de CF hypoxiques capable, à lui seul, d'augmenter la viabilité des CM. Ces résultats ont été corroborés par des expériences d'l/R in vivo, dans lesquelles les souris traitées avec le sécrétome de CF présentent une diminution de la taille d'infarctus. De plus, nous avons montré que TlMP-1, un facteur fortement détecté dans le sécrétome de CF, est capable de diminuer à la fois la mortalité cellulaire in vitro des CM et la taille de l'infarctus in vivo. L'utilisation d'inhibiteurs pharmacologiques nous a permis de mettre en évidence que cette protection paracrine était médiée en partie par l'activation des voies de signalisation Pl3K/Akt et ERK1/2. En conclusion cette étude démontre pour la première fois que les CF participent, de façon paracrine, à la protection des CM au cours la phase aigüe d'ischémie reperfusion. TlMP-1 semble être un des facteurs clé de cette cardioprotection par les CF. En parallèle de ce travail, plusieurs études collaboratives ont été réalisées, sur une cible majeure d'investigation dans la cardioprotection : le pore de transition de perméabilité mitochondriale et notamment sa régulation par le complexe l de la chaîne respiratoire et les échanges calciques, ainsi que son implication dans la défaillance multi-organe face à l'arrêt cardiaque / Roles of cardiac fibroblasts (CF) in the regulation of myocardial structure and function have been emphasized in the last decade. Their implications in pathophysiological aspects of chronic heart diseases such as myocardial remodelling and fibrosis is now well established. However their contribution to the acute phase of ischemia reperfusion injury still remains elusive. We hypothesized that CF may contribute to cardiomyocytes (CM) protection against ischemia reperfusion (l/R) injuries. This study was designed to investigate this protection and identify some of its mechanisms. Experiments were performed both on isolated neonatal rat CF and CM in vitro and in vivo mice model of myocardial infarction. We demonstrated that the presence of CF increases CM viability in co-cultures and that CF protect CM against l/R injuries in a paracrine manner. lt was confirmed by a similar effect of hypoxic CF secretome alone on CM viability. These findings were corroborated by in vivo experiments in which an infarct size reduction was observed in CF secretome treated mice. Furthermore, experiments with Tissue lnhibitor of Metalloproteinases-1 (TlMP-1), abundantly detected in CF secretome, was able to both decrease CM cell death and infarct size. Experiments with pharmacological inhibitors provided more evidence that this paracrine protection is partly mediated by Pl3K/Akt and ERK signalling pathways. Our data demonstrated for the first time that CF participate in cardioprotection during the acute phase of ischemia reperfusion, via a paracrine pathway, involving TlMP-1. Besides this first work, other collaborative studies have been performed, to investigate a major target in cardioprotection research : the mitochondrial permeability transition pore and its regulation by chain respiratory complex l and Ca2+ transfers and finally its implication in multiple organ failure in cardiac arrest

L'ischémie-reperfusion (IR) rénale entraîne des lésions de nécrose tubulaire aigue, nécessitant parfois une épuration extra rénale transitoire voir définitive. La mitochondrie joue un rôle important dans la physiopathologie de ces lésions d'IR en entrainant la mort cellulaire. L'étude de l'IR dans la cellule cardiaque a permis de mettre en évidence le rôle central du pore de transition de perméabilité mitochondriale (mPTP) dans le déclenchement de cette mort cellulaire. La ciclosporine (CsA) a été proposée comme thérapeutique pour protéger la cellule des lésions d'IR en retardant l'ouverture de ce mPTP. Cependant la CsA a des effets rénaux vasoconstricteurs aigus, nécessitant une validation expérimentale de sa protection dans l'IR rénale. Au cours de ce travail de thèse, nous avons mis au point un modèle murin d'IR rénale. Ensuite nous avons montré que le post-conditionnement à la CsA, ainsi que le post-conditionnement ischémique, permettent d'améliorer la fonction rénale avec un retard à l'ouverture du mPTP. Dans un deuxième travail, nous montrons que le pré conditionnement à la CsA est dose et temps dépendant, et médié en partie par l'augmentation d'expression d'une protéine chaperonne, l'Heat Shock Protéine 70 (HSP70). L'injection en bolus de CsA permet également d'améliorer la fonction rénale dans ce modèle d'IR avec un retard à l'ouverture du mPTP. Nos résultats ouvrent de nouvelles perspectives dans la protection rénale, notamment dans la réduction des épisodes d'insuffisance rénale aigue après chirurgie aortique ou en transplantation rénale / Ischemia-reperfusion (IR) is a situation encountered in transplantation or during aortic surgery, which can result in renal damages, requiring sometimes transient or definitive dialysis. Mitochondria play a crucial role in the pathophysiology of IR causing cell death. Previous studies of cardiac IR highlighted the role of mitochondrial permeability transition pore (mPTP). Cyclosporin A (CsA) has been proposed as a treatment to protect the kidney from IR by the delay of the opening of the mPTP. However, CsA has acute renal hemodynamic effects and a long-term toxicity, requiring an experimental validation of its protection in the renal IR. In this work, we developed a mouse model of renal IR. In a first study, we showed that the post-conditioning with CsA and ischemic postconditioning improve renal function with a delay of the opening of the mPTP. In a second study, we showed that a high dose of CsA injected just before the ischemia improves renal function and leads to the delay of the opening of mPTP mediated by an increase of HSP70. Our results open new perspectives in renal protection, especially for reducing episodes of acute renal failure in aortic surgery or in renal transplantation

L'infarctus du myocarde est la première cause de mortalité dans le monde. La reperfusion précoce est le traitement central de la prise en charge thérapeutique. Mais bien que salvatrice, la reperfusion s'accompagne elle-même de lésion dite de reperfusion. La mitochondrie et l'ouverture du pore de transition de perméabilité mitochondriale (mPTP) sont au centre de ces lésions de reperfusion. Le dysfonctionnement mitochondrial et la nécrose entraine par ailleurs une intense réponse inflammatoire locale et systémique. Le post-conditionnement ischémique et pharmacologique (par la cyclosporine A, CsA) constitue une voie de recherche importante afin de limiter les lésions de reperfusion. Mes travaux de thèse se sont attachés à étudier le rôle de la Sirtuine 3 dans la cardioprotection ainsi que d'étudier la réponse inflammatoire locale et systémique induite par l'ischémie/reperfusion (I/R) myocardique.Nous avons précisé le mécanisme d'action du post-conditionnement, qui semble médié par l'activation de la sirtuine 3 et la désacétylation de la cyclophiline D qui contribue à inhiber l'ouverture du mPTP. Nous avons ensuite montré que l'I/R myocardique induisait une intense réponse inflammatoire chez l'homme avec un rôle particulier d'IL-17A, IL-6, IL-8 et IL-10. Cependant, cette réponse inflammatoire n'était pas modifiée par l'utilisation de CsA. Enfin, nous avons pu montrer que le facteur induit par l'hypoxie (HIF-1a), qui est surexprimé lors de l'I/R est un important activateur la réponse inflammatoire, notamment sur l'inflammasome, et que le Nicotinamide Mononucléotide possède un rôle anti-inflammatoire en empêchant la stabilisation de HIF-1a. Tous ces éléments sont autant de cibles thérapeutiques potentielles à développer avec de nouvelles études / Myocardial infarction (MI) is the first cause of death in the world. Reperfusion is the key treatment of MI. However, reperfusion can cause reperfusion injuries. Mitochondria and mitochondrial permeablility transition pore are the target of reperfusion injuries. Mitochondrial dysfonction and necrosis lead to an intense local and systemic inflammation. Ischemic post-conditioning (PC) and pharmacologic PC (with cyclosporine A, CsA) are used to limit reperfusion injuries. During my thesis, I worked on cardioprotective effet of sirtuin 3 and I studied inflammation induced by myocardial ischemia/reperfusion (I/R). I have shown that ischemic PC involve sirtuin 3 and deacetylation of cyclophilin D. I demonstrated that myocardial I/R induce an intense inflammatory response in Human with a key role of IL-17A, IL-6, IL-8 and IL-10. However, this inflammatory response is not modulated by the administration of CsA. A least, we studied the role of HIF-1a that is over expressed during I/R. We showed that HIF-1a activate inflammasome and the secretion of IL-1beta and IL-18. Furthermore, Nicotinamied Mononucleotide has anti-inflammatory effets with an action of HIF-1a. Taken together, these data contribute to develop new target for cardioprotection