L’hème oxygénase-1 (HO-1), enzyme antioxydante, est capable de prévenir l’initiation tumorale tandis qu’elle promeut la progression de certaines tumeurs et l’angiogenèse. Ce travail a recherché si HO-1 peut moduler les microARNs et régule le développmemnt du carcinome pulmonaire humain non à petites cellules (NSCLC). La surexpression stable de HO-1 dans les cellules du NSCLC NCI-H292 accroit la production globale des miARNs et diminue significativement l’expression des oncomirs et angiomirs, tandis qu’elle augmente les miARNs suppresseurs de tumeurs. Le plus amplement diminué est le miR-378. Dans les cellules surexprimant HO-1 la p53 est aussi augmentée, Ang-1 et MUC5AC diminuées, prolifération migration et potentiel angiogéniques réduits. Les effets de HO-1 sur la prolifération tumorale, la migration et et l’expression de miR-378 sont modulées par CO. Au contraire, la surexpression stable de miR-378 décroit celle de HO-1 et de p53 tandis qu’elle accroît celle de MUC5AC, VEGF, IL-8 et Ang-1 et en conséquence accroit la prolifération, migration la stimulation des cellules endothéliales. L’ajout de HO-1 à des cellules surexprimant miR-378 réverse l’effet de miR-378 sur la prolifération et la migration des cellules cancéreuses. In vivo, les tumeurs surexprimant HO-1 sont de taille réduite, moins vascularisées et oxygénées et moins métastatiques tandis que la surexpression de miR-378 produit les effets inverses. Conformément, chez les patients NSCLC, l’expression de HO-1est réduite dans les métastases lymphatiques par rapport à la tumeur primaire tandis que miR-378 n’est pas modifié de manière significative. En conclusion, les résultats in vitro et in vivo indiquent que l’action coordonnée entre HO-1 et miR-378 module de manière significative la progression et l’angiogenèse du carcinome humain pulmonaire non à petites cellules. / Heme oxygenase-1 (HO-1), an antioxidant enzyme can prevent tumor initiation while it has been demonstrated to promote various tumors progression and angiogenesis. Here it was investigated whether HO-1 can modulate microRNAs and regulate human non-small cell lung cancer (NSCLC) development. Stable HO-1 overexpression in NSCLC NCI-H292 cells enhanced global production of miRNAs and significantly diminished expression of oncomirs and angiomirs, whereas upregulated tumor suppressive miRNAs. The most potently downregulated was miR-378. HO-1 overexpressing cells displayed also upregulated p53, downregulated Ang-1 and MUC5AC, reduced proliferation, migration and diminished angiogenic potential. CO was a mediator of HO-1 effects on tumor cells proliferation, migration and miR-378 expression. In contrast, stable miR-378 overexpression decreased HO-1 and p53 while enhanced expression of MUC5AC, VEGF, IL-8 and Ang-1 and consequently increased proliferation, migration and stimulation of endothelial cells. Adenoviral delivery of HO-1 to miR-378 overexpressing cells reversed miR-378 effect on proliferation and migration of cancer cells. In vivo, HO-1 overexpressing tumors were smaller, less vascularized and oxygenated and less metastatic, whereas miR-378 overexpression exerted the opposite effects. Accordingly, in patients with NSCLC, HO-1 expression was lower in metastases to lymph nodes than in primary tumors while miR-378 did not differ significantly. To conclude, in vitro and in vivo data indicate that interplay between HO-1 and miR-378 significantly modulates NSCLC progression and angiogenesis.

No description available.

O gás monóxido de carbono (CO) possui diversas funções no sistema nervoso central (SNC) funcionando como neuromodulador, como por exemplo da regulação da temperatura corporal, da nocicepção e mais recentemente, do comportamento emocional. Este neuromodulador gasoso é produzido pela ação da enzima heme oxigenase (HO), a qual é encontrada em diferentes áreas do SNC. Com destaque, esta enzima tem alta expressão no locus coeruleus (LC), o que sugere o envolvimento do CO na modulação de funções desempenhadas por esta estrutura. O LC localiza-se na ponte, sendo a maior origem da inervação noradrenérgica do SNC. Esta estrutura tem participação ativa na modulação das respostas relacionadas ao estresse, em particular, na modulação do comportamento emocional, desde que integra o Sistema de Inibição Comportamental (SIC), o qual inclui ainda o sistema septo-hipocampal e os núcleos da rafe. O SIC é responsável por comandar respostas defensivas de avaliação de risco, alerta e atenção, as quais podem ser eliciadas pela ansiedade. Dentro desta perspectiva, o presente estudo teve como objetivo avaliar se a ativação sistêmica da via HO-CO pode modular o comportamento emocional de ratos, e se há participação do LC. Assim, este trabalho avaliou se o tratamento sistêmico via intraperitoneal (i.p.) agudo (3 h antes) ou crônico (10 dias/2 vezes ao dia) com um liberador de monóxido de carbono (CORM-2), ou com indutor da enzima HO (CoPP), altera as respostas comportamentais no teste do labirinto em cruz elevado (LCE) e no teste claro-escuro (TCE) em ratos, bem como a expressão da enzima HO no LC. Em uma segunda etapa foi avaliado se a administração aguda de CORM-2 ou CoPP altera o comportamento avaliado no LCE e no TCE de ratos submetidos ao estresse crônico variado (ECV) por 10 dias. Os resultados mostraram que o CO induzido pela administração aguda ou crônica de CORM-2 ou CoPP possui efeito ansiolítico. Ainda, o tratamento com CORM-2 ou CoPP aumentou a expressão da enzima HO-1 em células localizadas no LC, sem alterar a imunorreatividade à enzima HO-2. Considerando os grupos submetidos ao estresse ECV, nem a ativação da via HO-CO ou o ECV apresentaram efeitos significativos nos comportamentos avaliados nos testes LCE e TCE. Os resultados do presente estudo sugerem que o tratamento sistêmico com drogas que modulam a liberação de CO possui claro efeito ansiolítico. Assim, é possível que o CO possa ser uma droga com potencial terapêutico para o tratamento de desordens neuropsiquiátricas. / The carbon monoxide gas (CO) has several functions in the central nervous system acting as a neuromodulator, such as in the body temperature regulation, nociception and more recently, in the emotional behavior modulation. This gas is produced by the action of the heme oxigenase enzyme (HO), which is found in different areas of the central nervous system (CNS). It is important to note that this enzyme has high expression in the locus coeruleus (LC), suggesting the involvement of CO in the modulation functions performed by this midbrain structure. LC is located in the pons, being the source of majority of the noradrenergic innervation of the CNS. This structure is intimately involved in the stress modulation responses, particularly in the emotional behavior regulation, since it integrates the Behavioral Inhibition System (BIS), which also includes septum-hippocampal system and raphe nucleus. The BIS is responsible for defensive responses like the risk assessment and alertness trigged by anxiety. Within this perspective, the present study was designed to evaluate whether the systemic HO-CO pathway can modulate emotional behavior of rats, and if the HO enzyme of the neurons located into LC is involved in this response. Thus, this study evaluated whether the acute systemic i.p. treatment (3 hours before) or chronic (10 days / 2 times a day) with a carbon monoxide releaser (CORM-2) or inducer of heme enzyme oxygenase (CoPP), is able to alter the behavioral responses in the elevated plus maze (EPM) and in the light-dark box test (LDB) in rats, and the HO enzyme expression in the LC. Furthermore, the effect of the acute administration of CORM-2 or CoPP was evaluated in the emotional behavior assessed in the EPM and LDB by rats submitted to unpredictable chronic stress (during 10 days). The results showed that the CO induced by acute or chronic administration of CORM-2 or CoPP has an anxiolytic-like effect. Furthermore, treatment with CORM-2 or CoPP promoted an increase of HO-1 enzyme expression in cells located in the LC without altering the immunoreactivity of HO-2 enzyme. Still, considering the rats subjected to stress UCS neither the activation of HO-CO pathway nor the UCS protocol altered the emotional behavior evaluated in the EPM and LDB tests. The results of this study suggest that systemic treatment with drugs that modulate the CO release has anxiolytic effect. Thus, it is possible that CO can be a potential drug therapeutic target for neuropsychiatric disorders.

O gás monóxido de carbono (CO) possui diversas funções no sistema nervoso central (SNC) funcionando como neuromodulador, como por exemplo da regulação da temperatura corporal, da nocicepção e mais recentemente, do comportamento emocional. Este neuromodulador gasoso é produzido pela ação da enzima heme oxigenase (HO), a qual é encontrada em diferentes áreas do SNC. Com destaque, esta enzima tem alta expressão no locus coeruleus (LC), o que sugere o envolvimento do CO na modulação de funções desempenhadas por esta estrutura. O LC localiza-se na ponte, sendo a maior origem da inervação noradrenérgica do SNC. Esta estrutura tem participação ativa na modulação das respostas relacionadas ao estresse, em particular, na modulação do comportamento emocional, desde que integra o Sistema de Inibição Comportamental (SIC), o qual inclui ainda o sistema septo-hipocampal e os núcleos da rafe. O SIC é responsável por comandar respostas defensivas de avaliação de risco, alerta e atenção, as quais podem ser eliciadas pela ansiedade. Dentro desta perspectiva, o presente estudo teve como objetivo avaliar se a ativação sistêmica da via HO-CO pode modular o comportamento emocional de ratos, e se há participação do LC. Assim, este trabalho avaliou se o tratamento sistêmico via intraperitoneal (i.p.) agudo (3 h antes) ou crônico (10 dias/2 vezes ao dia) com um liberador de monóxido de carbono (CORM-2), ou com indutor da enzima HO (CoPP), altera as respostas comportamentais no teste do labirinto em cruz elevado (LCE) e no teste claro-escuro (TCE) em ratos, bem como a expressão da enzima HO no LC. Em uma segunda etapa foi avaliado se a administração aguda de CORM-2 ou CoPP altera o comportamento avaliado no LCE e no TCE de ratos submetidos ao estresse crônico variado (ECV) por 10 dias. Os resultados mostraram que o CO induzido pela administração aguda ou crônica de CORM-2 ou CoPP possui efeito ansiolítico. Ainda, o tratamento com CORM-2 ou CoPP aumentou a expressão da enzima HO-1 em células localizadas no LC, sem alterar a imunorreatividade à enzima HO-2. Considerando os grupos submetidos ao estresse ECV, nem a ativação da via HO-CO ou o ECV apresentaram efeitos significativos nos comportamentos avaliados nos testes LCE e TCE. Os resultados do presente estudo sugerem que o tratamento sistêmico com drogas que modulam a liberação de CO possui claro efeito ansiolítico. Assim, é possível que o CO possa ser uma droga com potencial terapêutico para o tratamento de desordens neuropsiquiátricas. / The carbon monoxide gas (CO) has several functions in the central nervous system acting as a neuromodulator, such as in the body temperature regulation, nociception and more recently, in the emotional behavior modulation. This gas is produced by the action of the heme oxigenase enzyme (HO), which is found in different areas of the central nervous system (CNS). It is important to note that this enzyme has high expression in the locus coeruleus (LC), suggesting the involvement of CO in the modulation functions performed by this midbrain structure. LC is located in the pons, being the source of majority of the noradrenergic innervation of the CNS. This structure is intimately involved in the stress modulation responses, particularly in the emotional behavior regulation, since it integrates the Behavioral Inhibition System (BIS), which also includes septum-hippocampal system and raphe nucleus. The BIS is responsible for defensive responses like the risk assessment and alertness trigged by anxiety. Within this perspective, the present study was designed to evaluate whether the systemic HO-CO pathway can modulate emotional behavior of rats, and if the HO enzyme of the neurons located into LC is involved in this response. Thus, this study evaluated whether the acute systemic i.p. treatment (3 hours before) or chronic (10 days / 2 times a day) with a carbon monoxide releaser (CORM-2) or inducer of heme enzyme oxygenase (CoPP), is able to alter the behavioral responses in the elevated plus maze (EPM) and in the light-dark box test (LDB) in rats, and the HO enzyme expression in the LC. Furthermore, the effect of the acute administration of CORM-2 or CoPP was evaluated in the emotional behavior assessed in the EPM and LDB by rats submitted to unpredictable chronic stress (during 10 days). The results showed that the CO induced by acute or chronic administration of CORM-2 or CoPP has an anxiolytic-like effect. Furthermore, treatment with CORM-2 or CoPP promoted an increase of HO-1 enzyme expression in cells located in the LC without altering the immunoreactivity of HO-2 enzyme. Still, considering the rats subjected to stress UCS neither the activation of HO-CO pathway nor the UCS protocol altered the emotional behavior evaluated in the EPM and LDB tests. The results of this study suggest that systemic treatment with drugs that modulate the CO release has anxiolytic effect. Thus, it is possible that CO can be a potential drug therapeutic target for neuropsychiatric disorders.

T cells chronically stimulated by their antigen often become dysfunctional and lose effector functions and proliferative capacity. This state of unresponsiveness is referred as T cell exhaustion. In order to investigate this, we developed a laboratory model, which allowed us to stimulate chronically in vivo a monoclonal population of CD4+ T cells. This model is based on the adoptive transfer of TCR transgenic CD4+ T cells specific for the male mHAg into male recipient mice. We found that systemic exposure to the male antigen modified deeply anti-male TcR-transgenic CD4+ T cells, plunging them into a state of functional unresponsiveness. Microarray analysis revealed that, in comparison with naive T cells, transferred T cells displayed a gene expression profile very similar to that of virus-specific exhausted CD8+ T cells. Moreover, like exhausted CD8+ T cells, exhausted CD4+ T cells lost their capacity to secrete IFN-ã as well as to proliferate in response to antigen stimulation, and T cell unresponsiveness was controlled by the engagement of programmed death receptor 1 (PD-1) present at the surface of T cells. <p>MicroRNAs are key molecules in shaping T cell function. In order to explore the possibility that chronic antigenic stimulation could shape the pool of microRNAs in exhausted anti-male CD4+ T cells that would account for specific changes in protein synthesis, we compared by microarray analysis the specific expression of microRNAs in naive CD4+ T cells and exhausted CD4+ T cells. Ninety five of them were found differentially expressed, among which, microRNA-155 (miR-155) displayed one of the highest changes. To identify the importance of miR-155 in T cell exhaustion, we analyzed miR-155-deficient CD4+ T cells after chronic exposure to systemic antigen. We found that, chronically-stimulated miR-155-/- CD4+ T cells were retained in a deeper state of unresponsiveness than miR-155+/+ CD4+ T cells. Furthermore, inhibition of PD-1/PD-L1 interaction did not promote antigen-dependent expansion of miR-155-deficient CD4+ T cells, nor did it stimulate T cell inflammation of several organs, contrary to what was observed in mice that received miR-155-sufficient CD4+ T cells. Thus, our observations demonstrated that miR-155 deficiency played a dominant role over PD-1-mediated inhibition of T cells and that miR-155 was required for restoring function in exhausted CD4+ T cells.<p>Next, we explored the mechanism by which exhausted miR-155-/- CD4+ T cells were kept in a deeper unresponsiveness state than miR-155+/+ counterparts. By comparative microarray analysis of gene expression between exhausted miR-155+/+ CD4+ T cells and miR-155-/- CD4+ T cells, heme oxygenase 1 (HO-1) was identified as a specific target of miR-155. Finally, inhibition of HO-1 activity restored the capacity of exhausted miR-155-/- CD4+ T cells to promote autoimmune inflammation in adoptively-transferred recipients. <p>Taken together, our study identified miR-155-mediated regulation of protein expression as a critical factor for restoring function in exhausted CD4+ T cells. Our results also present regulation of HO-1 expression in T cells as one of the mechanisms by which miR-155 promote T cell-driven inflammation. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

No description available.

L’athérosclérose est une maladie vasculaire inflammatoire chronique qui se développe progressivement au cours de la vie. Les mécanismes impliqués sont complexes et la recherche de nouveaux candidats impliqués dans l'athérogénèse est toujours d'actualité. L’Angiopoietine-like 2 (Angptl2) est une protéine relativement peu connue, aux propriétés pro-angiogéniques et pro-inflammatoires, qui appartient par homologie à la grande famille des angiopoietines, mais dont le récepteur n'est pas encore clairement identifié. Les situations pathologiques dans lesquelles l’Angptl2 jouerait un rôle crucial sont diverses, mais sa contribution moléculaire dans le développement de l’athérosclérose est inconnue. Par differential display, nous avons initialement identifié l'Angptl2 comme étant surexprimée dans des cellules endothéliales sénescentes, isolées et cultivées à partir d'artères mammaires internes de patients athérosclérotiques ayant subi un pontage coronarien. Cette découverte a été la à base de mon projet, et mes objectifs ont été 1) de déterminer l'implication de l’Angptl2 vasculaire en présence de facteurs de risques tels que le tabagisme et la dyslipidémie, 2) de produire et de purifier une protéine recombinante fonctionnelle de l’Angptl2 afin d'identifier in vitro de nouvelles propriétés cellulaires de l'Angptl2 et 3) d'étudier in vivo le potentiel pro-athérogénique de l'Angptl2 recombinante dans un modèle murin de dyslipidémie sévère. Nous avons montré que l’Angptl2 est sécrétée préférentiellement dans des conditions pro-oxydantes et pro-inflammatoires, avec une augmentation de son expression endothéliale de l’ordre de 6 fois chez des patients coronariens fumeurs atteints de maladie pulmonaire obstructive chronique. Suite à ces résultats, nous avons émis l’hypothèse que l’Angptl2, en plus de ses fonctions pro-inflammatoires connues, possède des propriétés pro-oxydantes. Nous avons démontré que l’Angptl2 recombinante stimule en effet la production de radicaux libres dans des HUVEC en culture, via l’inhibition partielle de la voie cytoprotectrice antioxydante Nrf2/HO-1 et potentiellement via l'activation de kinase intracellulaire de type p38. A l'aide de souris dyslipidémiques LDLr-/-; hApoB-100+/+, nous avons démontré que le niveau d’Angptl2 plasmatique, vasculaire et dans les plaques athéromateuses, augmente parallèlement avec le développement de l’athérosclérose. De plus, une stimulation avec l’Angptl2 recombinante engendre chez ces souris une réponse inflammatoire évaluée par l’expression endothéliale de cytokines et de molécules d'adhésion et par l’infiltration de leucocytes sur l’endothélium vasculaire. Finalement, l’administration intraveineuse de la protéine recombinante d’Angptl2 pendant quatre semaines à des souris LDLr-/-; hApoB-100+/+ augmente de 10 fois l'expansion de la plaque athérosclérotique et double leur taux de cholestérol circulant. Nous avons aussi montré que chez des patients athérosclérotiques, l'Angptl2 plasmatique est 6 fois plus élevée que chez des sujets sains du même âge. Nos études semblent donc définir l’Angptl2 comme un facteur contribuant directement au développement de l'athérosclérose en favorisant la sénescence, l’inflammation et l’oxydation des cellules endothéliales. Ces propriétés pourraient globalement définir l'Angptl2, non seulement comme un nouveau biomarqueur circulant de l’athérosclérose, mais également comme l'un de ses promoteurs. / Atherosclerosis is a chronic vascular inflammatory disease that develops gradually during life. While the control mechanisms of this disease are complex and variable, research continues to identify new protein candidates involved in atherogenesis. Angiopoietin-like2 (Angptl2) is a relatively unknown protein, recently shown to display angiogenic and pro-inflammatory properties. Based upon structural homology, Angptl2 is a member of the angiopoietin family; however, the Angptl2 receptor has not yet been clearly identified. The reported pathological situations in which Angptl2 may play a crucial role are multiple, but its molecular contribution in the development of atherosclerosis remains unknown. By differential display, we initially identified Angptl2 as being overexpressed in senescent endothelial cells, isolated and cultivated from internal mammary arteries of atherosclerotic patients undergoing coronary bypass. This observation was at the basis of my project. My specific objectives were 1) to determine the abundance of vascular Angptl2 in the presence of risk factors such as smoking and dyslipidemia, 2) to produce and purify a functional recombinant human Angptl2 protein in order to examine its effects on cellular function in vitro, and 3) to study the pro-atherogenic potential of Angptl2 in vivo using a mouse model of severe dyslipidemia. We showed that Angptl2 is preferentially secreted under pro-oxidant and pro-inflammatory conditions, with a 6-fold increase in endothelial Angptl2 expression in smoker coronary patients with chronic obstructive pulmonary disease. Based on these results, we hypothesized that, in addition to its known pro-inflammatory functions, Angptl2 has pro-oxidant properties. Accordingly, we demonstrated that recombinant Angptl2 stimulates the production of free radicals by HUVEC, an action exerted, at least in part, by the inhibition of the cytoprotective antioxidant pathway, Nrf2/HO-1, and potentially via the activation of the intracellular p38 MAPK pathway. In dyslipidemic LDLr-/-; hApoB-100+/+ mice, we showed that the levels of endogenous Angptl2 in plasma, vascular tissue and atherosclerotic lesions increase in parallel with the development of atherosclerosis. In addition, stimulation with recombinant Angptl2 induces an inflammatory response, as assessed by the expression of cytokines and adhesion molecules and by infiltration of leukocytes into the vascular endothelium. Furthermore, intravenous infusion of purified recombinant Angptl2 for four weeks promoted a 10-fold increase in the formation of atherosclerotic plaques in LDLr-/-; hApoB-100+/+ mice and doubled their circulating cholesterol levels. Finally, we also demonstrated that plasma Angptl2 is 6-fold higher in atherosclerotic patients than in age-matched healthy subjects. These studies therefore strongly suggest that Angptl2 could directly contribute to the development of atherosclerosis by promoting senescence, inflammation and oxidation in endothelial cells. Such properties indicate that Angptl2 may be both a new biomarker of atherosclerosis, as well as one of its contributors.

L’athérosclérose est une maladie vasculaire inflammatoire chronique qui se développe progressivement au cours de la vie. Les mécanismes impliqués sont complexes et la recherche de nouveaux candidats impliqués dans l'athérogénèse est toujours d'actualité. L’Angiopoietine-like 2 (Angptl2) est une protéine relativement peu connue, aux propriétés pro-angiogéniques et pro-inflammatoires, qui appartient par homologie à la grande famille des angiopoietines, mais dont le récepteur n'est pas encore clairement identifié. Les situations pathologiques dans lesquelles l’Angptl2 jouerait un rôle crucial sont diverses, mais sa contribution moléculaire dans le développement de l’athérosclérose est inconnue. Par differential display, nous avons initialement identifié l'Angptl2 comme étant surexprimée dans des cellules endothéliales sénescentes, isolées et cultivées à partir d'artères mammaires internes de patients athérosclérotiques ayant subi un pontage coronarien. Cette découverte a été la à base de mon projet, et mes objectifs ont été 1) de déterminer l'implication de l’Angptl2 vasculaire en présence de facteurs de risques tels que le tabagisme et la dyslipidémie, 2) de produire et de purifier une protéine recombinante fonctionnelle de l’Angptl2 afin d'identifier in vitro de nouvelles propriétés cellulaires de l'Angptl2 et 3) d'étudier in vivo le potentiel pro-athérogénique de l'Angptl2 recombinante dans un modèle murin de dyslipidémie sévère. Nous avons montré que l’Angptl2 est sécrétée préférentiellement dans des conditions pro-oxydantes et pro-inflammatoires, avec une augmentation de son expression endothéliale de l’ordre de 6 fois chez des patients coronariens fumeurs atteints de maladie pulmonaire obstructive chronique. Suite à ces résultats, nous avons émis l’hypothèse que l’Angptl2, en plus de ses fonctions pro-inflammatoires connues, possède des propriétés pro-oxydantes. Nous avons démontré que l’Angptl2 recombinante stimule en effet la production de radicaux libres dans des HUVEC en culture, via l’inhibition partielle de la voie cytoprotectrice antioxydante Nrf2/HO-1 et potentiellement via l'activation de kinase intracellulaire de type p38. A l'aide de souris dyslipidémiques LDLr-/-; hApoB-100+/+, nous avons démontré que le niveau d’Angptl2 plasmatique, vasculaire et dans les plaques athéromateuses, augmente parallèlement avec le développement de l’athérosclérose. De plus, une stimulation avec l’Angptl2 recombinante engendre chez ces souris une réponse inflammatoire évaluée par l’expression endothéliale de cytokines et de molécules d'adhésion et par l’infiltration de leucocytes sur l’endothélium vasculaire. Finalement, l’administration intraveineuse de la protéine recombinante d’Angptl2 pendant quatre semaines à des souris LDLr-/-; hApoB-100+/+ augmente de 10 fois l'expansion de la plaque athérosclérotique et double leur taux de cholestérol circulant. Nous avons aussi montré que chez des patients athérosclérotiques, l'Angptl2 plasmatique est 6 fois plus élevée que chez des sujets sains du même âge. Nos études semblent donc définir l’Angptl2 comme un facteur contribuant directement au développement de l'athérosclérose en favorisant la sénescence, l’inflammation et l’oxydation des cellules endothéliales. Ces propriétés pourraient globalement définir l'Angptl2, non seulement comme un nouveau biomarqueur circulant de l’athérosclérose, mais également comme l'un de ses promoteurs. / Atherosclerosis is a chronic vascular inflammatory disease that develops gradually during life. While the control mechanisms of this disease are complex and variable, research continues to identify new protein candidates involved in atherogenesis. Angiopoietin-like2 (Angptl2) is a relatively unknown protein, recently shown to display angiogenic and pro-inflammatory properties. Based upon structural homology, Angptl2 is a member of the angiopoietin family; however, the Angptl2 receptor has not yet been clearly identified. The reported pathological situations in which Angptl2 may play a crucial role are multiple, but its molecular contribution in the development of atherosclerosis remains unknown. By differential display, we initially identified Angptl2 as being overexpressed in senescent endothelial cells, isolated and cultivated from internal mammary arteries of atherosclerotic patients undergoing coronary bypass. This observation was at the basis of my project. My specific objectives were 1) to determine the abundance of vascular Angptl2 in the presence of risk factors such as smoking and dyslipidemia, 2) to produce and purify a functional recombinant human Angptl2 protein in order to examine its effects on cellular function in vitro, and 3) to study the pro-atherogenic potential of Angptl2 in vivo using a mouse model of severe dyslipidemia. We showed that Angptl2 is preferentially secreted under pro-oxidant and pro-inflammatory conditions, with a 6-fold increase in endothelial Angptl2 expression in smoker coronary patients with chronic obstructive pulmonary disease. Based on these results, we hypothesized that, in addition to its known pro-inflammatory functions, Angptl2 has pro-oxidant properties. Accordingly, we demonstrated that recombinant Angptl2 stimulates the production of free radicals by HUVEC, an action exerted, at least in part, by the inhibition of the cytoprotective antioxidant pathway, Nrf2/HO-1, and potentially via the activation of the intracellular p38 MAPK pathway. In dyslipidemic LDLr-/-; hApoB-100+/+ mice, we showed that the levels of endogenous Angptl2 in plasma, vascular tissue and atherosclerotic lesions increase in parallel with the development of atherosclerosis. In addition, stimulation with recombinant Angptl2 induces an inflammatory response, as assessed by the expression of cytokines and adhesion molecules and by infiltration of leukocytes into the vascular endothelium. Furthermore, intravenous infusion of purified recombinant Angptl2 for four weeks promoted a 10-fold increase in the formation of atherosclerotic plaques in LDLr-/-; hApoB-100+/+ mice and doubled their circulating cholesterol levels. Finally, we also demonstrated that plasma Angptl2 is 6-fold higher in atherosclerotic patients than in age-matched healthy subjects. These studies therefore strongly suggest that Angptl2 could directly contribute to the development of atherosclerosis by promoting senescence, inflammation and oxidation in endothelial cells. Such properties indicate that Angptl2 may be both a new biomarker of atherosclerosis, as well as one of its contributors.

L’expression ‘programmation périnatale’ est employée pour décrire les effets à long terme d’un environnement gestationel néfaste observés chez la progéniture. Ce concept est aujourd’hui bien reconnu. Notre laboratoire a déjà démontré l’impact de l’hyperglycémie maternelle sur le développement rénal des embryons à l’aide des souris HoxB7-GFP transgéniques (Tg) et qui se traduit par une augmentation des espèces réactives de l’oxygène (ROS) et une néphrogenèse perturbée. Les rejetons affectés présentent ainsi des reins plus petits et possédant un nombre inférieur de néphrons à la naissance, et développent une hypertension et des dommages rénaux à l’âge adulte (20 semaines). Dans la première étude, nous avons tenté de réduire la production excessive de ROS dans les reins en développement par la surexpression de la catalase (CAT). Pour ce faire, nous avons croisé les souris CAT-Tg qui surexpriment la CAT dans les cellules des tubules proximaux rénaux (RPTCs) aux souris HoxB7-GFP-Tg afin de générer les souris HoxB7/CAT-GFP-Tg. Nous espérons observer la normalisation du nombre de néphrons et la prévention de l’hypertension et des dommages rénaux observés chez la progéniture issue d’un environnement gestational hyperglycémique. Nous avons observé que la surexpression de CAT dans les RPTCs permet de normaliser la dysmorphogenèse rénale présente chez les embryons de mères diabétiques. À l’âge adulte, la surexpression de CAT dans les RPTCs permet également de réduire la génération des ROS et l’hypertension, tout en améliorant la morphologie et la fonction rénale. Afin de définir les mécanismes impliqués dans ce processus, nous avons étudié le rôle potentiel de Nrf2 (‘nuclear factor-erythroid 2p45 (NF-E2) related factor-2’; un facteur de transcription des gènes antioxidants) et HO-1 (hème oxygénase-1’; une enzyme antioxidante). À la fois Nrf2 et HO-1 sont de forts antioxidants et ont été rapportés comme protecteurs pour le rein. Nous avons observé une surexpression des gènes et protéines Nrf2 et HO-1, en plus d’une translocation nucléaire accrue de Nrf2, dans les RPTCs de la progéniture des mères diabétiques, indiquant que chez les souris surexprimant CAT, Nrf2 et HO-1 sont tous deux bien activés et fonctionnels. En conclusion, nos études suggèrent que la surexpression de CAT dans les RPTCs permet de prévenir la programmation de l’hypertension et les dommages rénaux observés à l’âge adulte chez la progéniture issue de mères diabétiques, en partie suite à l’activation du système de défense Nrf2-HO-1 dans leurs reins. Il a déjà été démontré que l’activation du système rénine-angiotensine (RAS) intrarénal induit l’hypertension en augmentant la constriction des artérioles et la réabsorption du sodium par les tubules rénaux. Une activation du récepteur AT1R et de ses voies de signalisation induit également les dommages rénaux observés dans plusieurs pathologies. Dans le cadre de mon second article, nous avons identifié un nouveau mécanisme par lequel l’angiotensine (Agt) intrarénale induit l’hypertension et des dommages rénaux en réduisant l’expression de l’aquaporine 1 (AQP1, le canal pour l’eau le plus important dans les RPTCs). Des souris transgéniques surexprimant l’Agt de rat (rAgt-Tg) dans leurs RPTCs et des clones stables de cellules immortalisées de tubule proximal de rein de rat (IRPTCs) surexprimant le rAgt (pRSV/rAgt-IRPTC) ont été étudiés. Lorsque comparés aux souris non-transgéniques, les souris rAgt-Tg développent de l’hypertension et des dommages rénaux. Ces changements sont atténués par le traitement avec une double inhibition du RAS (losartan et perindopril). L’expression des protéines AQP1 et HO-1 est réduite dans les RPTCs, tandis que Nrf2 et le transporteur sodique NHE3 sont augmentés, à la fois in vivo et in vitro. Ces changements sont renversés par la double inhibition du RAS chez les animaux expérimentaux. Même si les niveaux de Nrf2 sont élevés, une accumulation cytosolique causée par une augmentation de l’export nucléaire induit par GSK3β se produit et ne parvient donc pas à induire l’expression des gènes en aval comme HO-1, ni à réduire l’expression de l’AQP1. En conclusion, nos résultats suggèrent qu’une déficience en Nrf2 nucléaire mène à une diminution de l’expression de HO-1 et une régulation négative de l’AQP1, jouant un role dans l’hypertension et les dommages rénaux induits par l’Agt intrarénal. L’hypertension et les dommages rénaux sont des maladies très hétérogènes et multifactorielles qui impliquent l’interaction de diverses molécules et voies de signalisations, et sont influencées par plusieurs facteurs environnementaux tels la diète ou la programmation périnatale. Tous ces différents facteurs contribuent à la progression de l’hypertension et des dommages rénaux, rendant les stratégies de traitement d’autant plus complexes. Dans notre étude, nous avons évalué le développement de l’hypertension dans deux circonstances : l’hypertension de la progéniture programmée par le diabète maternel et l’hypertension induite par l’activation du RAS intrarénal. Nous avons démontré que la génération des ROS dans les reins constitue un facteur majeur commun dans nos deux modèles d’hypertension chez la souris. De plus, le gène/facteur de transcription antioxydant Nrf2, sensible aux ROS, joue un rôle important dans le processus. Grâce à une meilleure compréhension des diverses voies qui mènent à la progression de l’hypertension, nous espérons qu’il sera possible de développer de meilleurs traitements pour faire face à l’hypertension. / The term ‘perinatal programming’ is used to describe the phenomenon that maternal adverse environment during pregnancies which have profound influences to their offspring later in life. And this concept is well accepted. Previously, we successfully created an in vivo murine model and demonstrated that maternal diabetes constitutes an adverse in utero environment that may fundamentally impair nephrogenesis and subsequently program of the offspring to develop hypertension and kidney injury in adulthood. It appears that enhanced reactive oxygen species (ROS) generation, activation of the nuclear factor-kappa B (NF-kB), intrarenal renin- angiotensin system (RAS) and p53 pathways were involved in the underlying mechanisms. In our first study, we investigated whether overexpression of catalase (CAT) in renal proximal tubular cells (RPTCs) could prevent the perinatal programming of hypertension and kidney injury in male offspring of diabetic dams and examined the potential underlying mechanisms both in vivo and in vitro. Our data demonstrate that CAT overexpression in RPTCs exert a direct effect on nephrogenesis in utero and ameliorate maternal diabetes- induced dysnephrogenesis. And further consequently, CAT overexpression in RPTCs preventing maternal diabetes-induced perinatal programming, mediated at least in part, via the nuclear factor-erythroid 2p45 (NF-E2) related factor-2 (Nrf2)- heme oxygenase (HO)- 1 defense system. Intrarenal RAS activation has attracted more attention in recent years due to studies have been reported that activation of the intrarenal RAS can elicit hypertension and kidney injury independently from the systemic RAS. Previously, we established a murine model (Agt-Tg) that specifically overexpress rat angiotensinogen (Agt) in their RPTCs and develops hypertension and nephropathy. Aquaporin 1 (AQP1) is the major water channel within renal RPTCs, but whether it has a regulatory role in the development of hypertension and nephropathy remains elusive. Our second study aimed to examine the regulation of AQP1 expression in an intrarenal RAS-induced hypertension and kidney injury, focusing on underlying molecular mechanisms. We believe that both our in vivo and in vitro studies identified a novel mechanism(s) in which Agt overexpression in RPTCs enhances cytosolic accumulation of Nrf2 via the phosphorylation of pGSK3β Y216. Consequently, less intranuclear Nrf2 is available to trigger HO-1 expression as a defense mechanism and subsequently diminishes AQP1 expression in RPTCs. In conclusion, our data suggest that Agt mediated-downregulation of AQP1 and Nrf2 signaling may play an important role in intrarenal RAS-induced hypertension and kidney injury. Hypertension and kidney injury is a heterogeneous and multifactorial disease that involves the interaction of various molecules/pathways and the influence of environmental factors, for instance, diet and perinatal programming. Such diverse causes contribute to the progression of hypertension and kidney disease, making the strategy of treatment even more complex. In our present study, we evaluated the development of hypertension under two circumstances: maternal diabetes-programmed hypertension in offspring and intrarenal RAS activation-induced hypertension. We found that ROS generation in the kidneys is a major and common factor in both hypertensive mice model. Also, the ROS-sensitive antioxidant gene/transcription factor – Nrf2, plays an important role in the process. By understanding the pathways that lead to hypertension progression, we can hopefully develop more effective treatments to cope with the disease.

Many neurodegenerative diseases are characterized by the accumulation of misfolded proteins that often share common morphological and biochemical features, and can similarly co-localize with several other proteins, including various chaperone proteins. Chaperone proteins, like heat shock protein 27 (HSP27), heme oxygenase 1 (HO-1) and clusterin, have been implicated as potent modulators of misfolded proteins, thus may play important roles in the pathogenesis of neurodegenerative diseases. The present study aims to investigate their roles in the pathogenesis of Frontotemporal lobar degeneration (FTLD), Alzheimer's disease (AD), Parkinson's disease (PD), and Motor neuron disease (MND) by determining their distribution and amount via immunohistochemical staining and western blotting in diseased and control subjects.There were distinct patterns of HSP27 and clusterin immunostaining in different brain regions. For HSP27, patients with AD and FTLD were in general more severely affected than were patients with MND and control subjects. For clusterin, patients with AD and FTLD were more severely affected than control subjects where neurons and glial cells were concerned, while patients with AD and control subjects were more severely affected than those with FTLD where diffuse and cored plaques were concerned. However, there were no obvious differences in the pattern of HO-1 immunostaining in various brain regions in patients with AD or FTLD relative to control subjects. Moreover, there was no association between HSP27, HO-1 and clusterin with disease or histological type, and the ‘classic’ neuropathological changes in FTLD, AD and MND were not immunoreactive to any of these proteins. There were significant correlations between the degrees of HO-1 and clusterin immunostaining in many brain areas for both AD and FTLD cases, and for all cases overall, but none between HSP27 and clusterin or HSP27 and HO-1. Present results suggest an involvement with ongoing cellular stress, misfolded or unfolded protein accumulation or the deficits/failure of other relevant protein quality control systems, in the pathogenesis of these neurodegenerative diseases. Present work may therefore have implications for the further development of ideas concerning the cause or treatment of neurodegenerative diseases where there is aberrant accumulation of misfolded, aggregated protein, and perhaps for conformational diseases in general. However, there are still many issues remain to be elucidated. Further research aimed at understanding the function and mechanisms of the chaperone system, and other protein quality control mechanisms, in the pathogenesis of neurodegenerative diseases is still needed.