Les maladies cardiovasculaires sont une des principales causes de morbidité et de mortalité au monde. La plus courante est l’infarctus du myocarde définit pathologiquement par la mortalité cellulaire dû à une ischémie prolongée d’une partie du ventricule gauche. L'ischémie est caractérisée par un apport sanguin insuffisant causé par une obstruction d’une artère coronaire. La restauration, en clinique, du flux sanguin, appelée reperfusion, est considérée comme la méthode la plus efficace contre les dommages ischémiques. Paradoxalement, cette restauration du flux sanguin est associée à une exacerbation de la lésion tissulaire, entraînant alors des lésions d'ischémie-reperfusion (I/R). Dans le but de limiter ces lésions, le conditionnement ischémique myocardique est une avancée majeure dans le domaine de la cardioprotection. Ce protocole confère ses effets cardioprotecteurs via le recrutement de divers mécanismes endogènes suivant l’activation de deux voies intracellulaires : la voie RISK (Reperfusion Injury Salvage Kinase) et/ou la voie SAFE (Survivor Activator Factor Enhancer). Ces voies impliquent l'activation de différentes cascades de signalisation et de protéines kinases. En particulier, concernant la voie SAFE, le transducteur de signal et l'activateur de transcription-3 STAT3, a été identifié comme un acteur clé dans le postconditionnement ischémique (PostCI). Il est suggéré que les effets cardioprotecteurs attribués à STAT3 soient liés à ses effets en tant que facteur de transcription et en tant que régulateur de l’activité mitochondriale, mais tout n’est pas encore connu. En revanche, il est admis que STAT3 est activé par la phosphorylation ciblant les résidus tyrosine 705 et sérine 727. Dans nos travaux actuels, nous avions initialement pour objectif d’étudier les rôles cardioprotecteurs mitochondriaux de STAT3 après une I/R et un PostCI. Cependant, nous n'avons pas été en mesure de détecter STAT3 dans les mitochondries de cardiomyocytes adultes de souris, dans des conditions basales et de stress, en utilisant différentes approches. Fait intéressant, nous avons montré une localisation exclusive de STAT3 dans les myocytes cardiaques adultes, le long des tubules T, et nous avons mis en évidence les inconvénients des techniques précédemment utilisées.Outre les rôles putatifs de STAT3 dans les mitochondries, nous avons ciblé ses effets dans la signalisation et la génomique au cours de l'I/R et du PostCI. Nous avons tout d’abord cherché à déterminer, pendant l’I/R et le PostCI, la cinétique temporelle d’activation de STAT3 et des autres kinases de la voie RISK, notamment Akt et les MAPK ERK1 / 2, JNK et p38. En outre, nous avions pour objectif d’étudier les liens entre les voies SAFE et RISK en déchiffrant les liens entre STAT3 et les kinases RISK au cours du PostCI. Nous avons montré qu’après une ischémie et un temps court de reperfusion, STAT3 et ERK1/2 sont activés, et que l’utilisation d’un PostCI active d’autant plus STAT3 en induisant exclusivement la phosphorylation de sa tyrosine. Nous avons également montré que l’interconnexion entre les voies SAFE et RISK, dans le protocole PostCI utilisé, se fait par STAT3 et ERK1/2. À partir de ces résultats, nous nous sommes dirigés vers la génomique grâce à laquelle nous avons étudié l'activité de STAT3 au cours de l'IPoC. À cet égard, nous avons montré que STAT3 est impliqué dans la régulation de la réponse inflammatoire au cours de la PostCI. Dans l’ensemble, cette étude présente une approche globale des fonctions mitochondriales, de signalisation et génomiques de STAT3 dans le contexte de la protection cardiaque / Cardiovascular diseases are leading causes of morbidity and mortality worldwide. Among the mostly prevailing cardiovascular diseases is myocardial infarction, which is pathologically defined as myocardial death due to a prolonged ischemia. Ischemia is an insufficient supply of blood caused by a blockade in the coronary arteries. The early restoration of blood flow is considered the most effective method against the ischemic lesions. Paradoxically, this blood flow restoration is associated with an exacerbation of the tissue injury, leading to the ischemia-reperfusion (I/R) injury. To avoid this injury, the myocardial ischemic conditioning protocol has rejuvenated the field of cardioprotection. This protocol confers its cardioprotective effects via recruiting various endogenous mechanisms following the activation of two intracellular pathways: the reperfusion injury salvage kinase (RISK) or survivor activator factor enhancer (SAFE) pathways. These pathways involve the activation of different signaling cascades and protein kinases. Zooming in through the SAFE pathway, the signal transducer and activator of transcription-3, STAT3, has been identified as a prominent key player in ischemic postconditioning (IPoC). The cardioprotective effects attributed to STAT3 are suggested to be linked to its roles as a transcription factor and as a regulator of the mitochondrial activity, but these are not well studied and elaborated. STAT3 is activated by phosphorylation, which targets the tyrosine 705 and serine 727 residues. In our current work, we initially aimed to investigate the mitochondrial cardioprotective roles of STAT3 following I/R and IPoC. However, we were not able to detect STAT3 in the mitochondria of adult mouse cardiomyocytes under variousbasal and stress conditions using different approaches. Interestingly, we showed an exclusive STAT3 pattern in adult cardiac myocytes, along the T-tubules, and highlighted drawbacks of previously used techniques. Aside from the mitochondrial roles of STAT3, we targeted its signaling and genomic roles during I/R and IPoC. We first aimed to determine, during I/R and IPoC, the temporal kinetics of activation of STAT3 and the other kinases of the RISK pathway including Akt and the MAPKs ERK1/2, JNK and p38. In addition, we aimed to decipher the interlink between the SAFE and RISK pathways through deciphering the interlink between STAT3 and the RISK kinases following IPoC. We showed that a short reperfusion time activates STAT3 and ERK1/2 following ischemia, and that the application of IPoC further activates STAT3 through inducing its tyrosine phosphorylation. We also showed that the interlink between SAFE and RISK pathways, in the IPoC protocol we used, is through STAT3 and ERK1/2. From this signaling level, we moved toward the genomic level whereby we investigated the genomic activity of STAT3 during IPoC. In this regard, we have shown that STAT3 is involved in the regulation of the inflammatory response during IPoC. Overall, this study presents a global approach of STAT3’s mitochondrial, signaling and genomic functions in the context of cardiac protection

L’ostéopontine (OPN) est une protéine synthétisée et sécrétée par de nombreux types cellulaires. Elle joue un rôle important dans la régulation de la réponse inflammatoire et immune. Elle est également pro-fibrogénique, et présente des propriétés anti-apoptiques. Les NAFLD et ALD sont les premières causes d’ hépatopathies en France. Le spectre de ces complications va de la stéatose à la stéatohépatite, la fibrose, la cirrhose voire le carcinome hépatocellulaire. Le tissu adipeux joue un rôle important dans la survenue et l’évolution des NAFLD. Nous montrons que l'OPN favorise l'inflammation du foie et du tissu adipeux dans les NAFLD, en favorisant le recrutement de macrophages, de cellules dendritiques et de lymphocytes T et en modulant la polarisation de ces cellules immunes. Chez les patients alcooliques, nous rapportons que l’OPN constitue un marqueur prédictif de la fibrose hépatique. Les lésions induites par l'IR hépatique sont la principale cause de dommages survenant au cours des chirurgies du foie. Le rôle de l'OPN lors de l’IR n’a pas été étudié dans le foie. Mes études ont mis en évidence que l'OPN pourrait jouer un rôle protecteur. Son invalidation (OPN-/-) aggrave les lésions hépatiques (inflammation, souffrance et nécrose hépatocytaire) induites par l’IR chez la souris. Ce rôle protecteur de l’OPN pourrait être dû à sa capacité à prévenir la mort hépatocytaire et à limiter la production toxique de NO dans les macrophages. L’ensemble de ces travaux a permis de mettre en évidence de nouveaux rôles de l'OPN dans les lésions induites par l'IR hépatique et pourrait constituer une cible thérapeutique pour les maladies chroniques du foie. / Osteopontin (OPN) is a protein synthesized and secreted by many different types of cells. It plays an important part in the regulation of the inflammatory and immune response. OPN is also pro-fibrogenic, and has anti-apoptotic properties. The nonalcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD) are the leading causes of liver disease in France. The range of these complications goes from steatosis to steatohepatitis, fibrosis, cirrhosis and even hepatocellular carcinoma. Adipose tissue plays a significant part on the occurrence and evolution of the NAFLD. We show that OPN facilitates liver’s and adipose tissue’s inflammation in the NAFLD, by facilitating the intake of macrophages, dendritic cells and T cells, and by modulating the polarization of these immune cells. For alcoholic patients, we show that OPN is one of the predictive markers of liver fibrosis. The lesions induced by ischemia-reperfusion (IR) are the main cause of damages occurring during liver’s surgery. The role of OPN in hepatic injury induced by IR has not yet been investigated. My studies demonstrate that OPN could have a protecting role. OPN deficiency in mice (OPN-/-) increases hepatic lesions caused by IR (inflammation, and cell death). OPN could thus partially prevent hepatic injury and inflammation induced by IR. This could be due to its ability to prevent hepatocyte death and production of toxic NO by macrophages. OPN could thus be an important actor in the pathogenesis of chronic liver disease.

L'arrêt cardiaque (AC), responsable de 50000 morts par an en France, est un problème de santé publique. La majorité des décès survenant chez les patients réanimés avec succès est liée à une défaillance multiviscérale associée à une réponse inflammatoire systémique définissant le syndrome post-AC. L'AC réanimé est un modèle extrême d'ischémie-reperfusion (I/R) globale à l'origine de dysfonctions mitochondriales. L'ouverture du pore de transition de perméabilité mitochondrial (mPTP), inhibée par la ciclosporine A (CsA), joue un rôle déterminant dans les lésions d'I/R focales. Nous avons fait l'hypothèse que des interventions thérapeutiques prévenant l'ouverture du mPTP pourraient limiter le syndrome post-AC. Au cours de ce travail de thèse, nous avons mis au point un modèle d'AC chez le lapin et utilisé des préparations de mitochondries isolées à partir des organes vitaux pour étudier le rôle du mPTP dans la physiopathologie des défaillances d'organes post-AC. Dans un premier travail, nous avons montré que l'administration in vivo de CsA prévenait, en inhibant le mPTP au niveau du myocarde, la dysfonction cardiovasculaire post-AC. Nous avons étendu cette démonstration aux autres organes vitaux et montré ainsi des effets protecteurs mitochondriaux ubiquitaires de la CsA. Par la suite, nos travaux se sont attachés à étudier l'influence de la température corporelle sur les dysfonctions mitochondriales impliquées dans le syndrome post-AC. Nos résultats ouvrent de nouvelles perspectives dans le traitement de l'AC chez l'Homme / Cardiac arrest (CA) is a public health with more than 50,000 sudden deaths annually in France. A majority of immediate survivors die of multiple organ failure combined with systemic inflammatory response known as the post-CA syndrome. Resuscitated CA represents a model of whole body ischemia-reperfusion (I/R) leading to mitochondrial dysfunctions. Opening of the mitochondrial permeability transition pore (mPTP), which can be inhibited by cyclosporine A (CsA), play a key role in reperfusion injury after focal ischemia. We hypothesized that therapeutic intervention targeting the mPTP could prevent the post-CA syndrome. In the present work, we developed a model of CA in rabbits and we used mitochondria preparations from vital organs to study the role of the permeability transition in the pathophysiology of the post-CA syndrome. In our first study, we have determined that CsA, by inhibiting mPTP opening (in heart), prevented CA-induced myocardial dysfunction. We extended this demonstration to the other vital organs and therefore reported a ubiquitous mitochondrial protective effect of CsA. Subsequently, we have focused our research on the influence of temperature on mitochondrial dysfunction involved in the post-CA syndrome. Our experimental findings open up new therapeutic perspectives in the treatment of CA in Humans

Includes bibliographical references (leaves 292-352) Determines the role of matrix metalloproteinases, MMP-2 and MMP-9 in reperfusion injury following skeletal muscle ischaemia; and, whether inhibition of MMPs by doxycycline protects against tissue damage.

Bibliography: leaves 140-190.

[Truncated abstract] Clinical therapeutic agents to directly inhibit ischaemic neuronal death are presently unavailable. One approach to developing therapeutics is based upon the identification of proteins up-regulated by 'preconditioning', a natural adaptive response utilised by the neural cells to counter damaging insults, such as ischaemia. Thus, my project aimed to firstly identify proteins differentially expressed following erythropoietin (EPO) mediated neuronal preconditioning and secondly to assess whether any of these proteins possessed neuroprotective activity using in vitro ischaemia like models. To achieve the first aim, it was shown that in vitro neuronal EPO preconditioning could: (i) induce cell signal changes in neuronal cultures, (ii) protect neurons against in vitro ischaemia and (iii) induce differential protein expression. Overall, 40 differentially expressed proteins were identified in cortical neuronal cultures following EPO preconditioning. In order to investigate the neuroprotective or neurodamaging activity of proteins induced by EPO preconditioning I developed an adenoviral expression system for use in neuronal cultures. To this end, I assessed the suitability of four promoters (cytomegalovirus [CMV], rous sarcoma virus [RSV], human synapsin 1 [hSYN1], rat synapsin 1 [rSYN1]) previously used to express proteins in neuronal cultures and demonstrated the superiority of the RSV promoter for this purpose. ... Finally, in order to validate this adenoviral expression system, I over-expressed the anti-apoptotic protein Bcl-XL in neuronal cultures and subsequently confirmed its neuroprotective activity in the in vitro ischaemia and oxidative stress models used in my project. Using this adenoviral vector system and the in vitro oxidative stress model I assessed a number of proteins up-regulated by EPO preconditioning. The results of this preliminary study indicated that cyclophilin A (CyPA), peroxiredoxin 2 (PRDX2) and superoxide dismutase 1 (SOD1) over-expression were neuroprotective. It was subsequently verified that adenoviral mediated over-expression of CyPA and PRDX2, v but not SOD1 in cortical neuronal cultures could protect neurons from in vitro ischaemia. I also confirmed that CyPA mRNA increased in the rat hippocampus in response to 3 minutes of global cerebral ischaemia. Interestingly, an increase in CyPA, PRDX2 or SOD1 protein was not observed in the same experimental paradigm. To investigate CyPA's mode of action I confirmed that cultured neurons, but not astrocytes, express the CyPA receptor, CD147. It was also demonstrated that administration of exogenous CyPA protein to neuronal cultures could protect neurons against oxidative and ischaemic injury. I further demonstrated that exogenous administration of CyPA induces a rapid and transient activation of the extracellular signal-regulated kinase (ERK) 1/2 pathway in neuronal cultures. From this observation, I have proposed that the extracellular mediated neuroprotective activity of CyPA occurs via CD147 receptor signalling and activation of ERK1/2 pro-survival pathways. Based on the findings reported in this thesis, the neuroprotective activities of PRDX2 and CyPA warrant further investigation as targets for the development of new therapies to treat cerebral ischaemia.

Stroke is the 3rd leading cause of death and the leading cause of major disability worldwide. Currently, there are no neuroprotective drugs approved for the acute treatment of ischaemic stroke. The vast majority of stroke therapeutics failed in clinical trials due to toxic side effects and/or a clinically irrelevant therapeutic window. This thesis is focused on exploiting the delayed neurodegeneration that occurs in the compromised penumbra, as these cells may be capable of being saved by therapeutic intervention in a clinically obtainable window. In order to investigate the ischaemic cascade and be able to draw conclusions that are applicable to humans, the international gold standard animal model for cerebral ischaemia, the filament insertion middle cerebral artery occlusion (MCAO) model, was established at the University of Otago. This model was validated under new laboratory conditions and employed adult male Sprague Dawley rats. After testing multiple occlusion lengths, it was concluded that a 2hr ischaemic period was sufficient to produce a consistent infarct of optimal size. It has been well documented that neuroinflammation contributes to much of the delayed progression of neural injury post-stroke. Therefore, the catechin (-)-epigallocatechin gallate (EGCG), which is an anti-inflammatory, anti-oxidant and free-radical scavenging agent was investigated in the MCAO model of stroke. 50mg/kg i.p. of EGCG or saline was administered immediately post-MCAO and animals were sacrificed at 72hr post-filament insertion. The results confirmed that treatment with EGCG was neuroprotective and non-toxic. However, EGCG also induced an over 50% increase in the risk of haemorrhagic conversions. The anti-platelet effects of EGCG and lack of toxicity suggests that the catechin may prove to be an efficacious prophylactic for stroke. The contrary findings for EGCG treatment led to the re-evaluation of the neuroinflammatory pathway for alternate mechanisms to target therapeutic interventions. The temporal profile of the primary inducible enzymes nitric oxide synthase (NOS), cyclooxygenase (COX) and arginase (and their isoforms) were quantified 0, 3 and 7 days post-stroke. In both hemispheres, total NOS activity exhibited a significant and sustained up-regulation to 7 days post-occlusion. In the ipsilateral hemisphere at least half of the total increase was accounted for by inducible NOS (iNOS) expression. Arginase, which competes with NOS for L-arginine, demonstrated a delayed but significant increase in activity by day 7 in the infarcted hemisphere, thereby correlating well with the downward slope of NOS activity (illustrating the switch in the conversion pathway). COX activity was observably increased in the ipsilateral hemisphere, but the up-regulation did not reach significance by day 7. Alternately, the contralateral hemisphere displayed a significant decrease in activity by day 3. These results give conclusive evidence that the contralateral hemisphere is NOT an appropriate internal control and imply that NOS and COX inhibitors may prove to be efficacious for a much longer therapeutic window than current treatments. However, the delayed induction of COX activity may also indicate that this enzyme has a finite therapeutic window, as it may also stimulate remodelling of surviving neural networks. The prolonged up-regulation of inflammatory mediators implies that there may be an induction of an autoimmune component to the response. Therefore, the thymus (T) lymphocyte activation was quantified up to 14 days post-stroke. Cluster of differentiation (CD) 3⁺ T lymphocytes (equally contributed to by CD4⁺ and CD8⁺ T cells) exhibited a significant and sustained up-regulation in the infarcted region from day 3 up to at least day 14 post-ischaemia. Quantitative analysis of all cells present post-stroke determined that immune cells make up an average of 73% of all cells present in the 'peak' ischaemic areas. The CD4⁺ T helper cell response was delineated by double immunohistochemical labelling. Interferon-γ positively labelled with CD4⁺ T cells at days 3, 7 and 14 post-insult detailing a Th1-driven pro-inflammatory response. This evidence indicates that the autoimmune response is critical post-ischaemia and that it may be highly susceptible to modification by anti-inflammatory therapeutic intervention. The primary downstream effect of the pro-inflammatory/immune cascade is apoptosis. The main organelle responsible for the 'go, no go' response to apoptotic factors is the mitochondria. In order to distinguish whether mitochondrial dysfunction was initiated shortly after ischaemia induction or if it was delayed, like the inflammatory/immune response, to a clinically relevant window, the temporal profile of mitochondrial complex inactivation was studied. It was found that mitochondrial membrane viability was impaired by day 3, followed by a significant decrease in respiratory complex activation and an increase in tissue injury by oxidative stress by 7 days post-ischaemia. These results indicate that targeting the early decrease in membrane viability or mitochondrial permeability transition pore opening combined with anti-apoptotic therapeutics, may attenuate the proceeding mitochondrial impairment in oxidative phosphorylation, reactive oxygen species generation and subsequent cell death cascades. The current investigations into the temporal profile and quantitative contributions of the inflammatory, immune and apoptotic mechanisms post-stroke highlight potential strategies for modulation by acute stroke therapeutics. Furthermore, the general knowledge amassed from these studies dictates the necessity of a new approach to therapeutic intervention. The acknowledgement of so many contributing systems suggests that in addition to a thrombolytic, a combination therapy involving multiple neuroprotectants should be employed to account for the multifaceted nature of the sequelae of ischaemic stroke.

Bibliography: leaves 140-190 / xiv, 190, [10] leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--University of Adelaide, 1987

Includes bibliographical references (leaves 292-352) / xvi, 352 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Determines the role of matrix metalloproteinases, MMP-2 and MMP-9 in reperfusion injury following skeletal muscle ischaemia; and, whether inhibition of MMPs by doxycycline protects against tissue damage. / Thesis (M.D.)--University of Adelaide, Dept. of Surgery, 2002

Perinatal hypoxic ischemic (HI) injury is a leading cause of long-term neurological complications in newborn babies. Matrix metalloproteinases (MMPs) are a family of endopeptidases that are capable of degrading the extracellular matrix (ECM) components. They are considered to be integral in many physiological processes. However, recently it has been demonstrated that the inappropriate activity of these proteases, particularly MMP-2 and 9, contribute to the pathogenesis of cerebral ischemia in the adult brain. Given that ECM disruption is frequently observed following injury to the developing brain, it is possible that MMPs play an important role in HI injury processes in the developing brain. Therefore, this thesis evaluated the hypothesis that MMP-2 and 9 participate in the pathophysiology of HI injury to the developing brain. Since ECM remodelling is a fundamental process during brain development it was important to first characterise the MMP-2 and 9 profiles in the normal developing forebrain. We demonstrated that MMP-2, which mainly was observed in cortical plate neurons, declined with age, thus indicating a potential role in the development and differentiation of the cortical plate. Conversely, MMP-9 was increased with age, particularly during active myelination, indicating that it may contribute in myelination. Secondly, we showed an upregulation of MMP-9 within the ischemic core during the early hours following HI injury, suggesting that MMP-9 may be involved in the development of delayed injury processes following hypoxic ischemia. On the contrary, MMP-2 was strongly upregulated during a later stage following injury surrounding the ischemic core possibly suggesting that it plays a role in wound repair processes. Thirdly, the profiles of tissue (tPA) and urokinase (uPA) plasminogen activators were characterised following HI injury since they are known to be major upstream activators of MMPs. uPA upregulation paralleled that of MMP-2 suggesting a function for uPA in wound repair processes following HI injury to the developing brain through activation of MMP-2. In contrast with uPA, tPA activity remained unaffected following injury at both ages. Finally, MMP-9 activity was inhibited using a very specific MMP-2/9 inhibitor, SB-3CT, to determine if the MMP-9 deficiency protects the developing brain from HI injury. The elevated MMP-9 activity following HI injury was attenuated by the SB-3CT treatment. Although SB-3CT failed to confer any significant neuroprotection, we recommend that further investigations are needed before discounting the role of MMP-9 during HI injury to the developing brain. In conclusion, we suggest that MMP-9 is induced following an insult to the developing brain potentially contributing to the delayed neuronal death whilst MMP-2 is involved in essential developmental, differentiation and wound repair processes.