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No description available.

Abdominal aortic aneurysm (AAA) is a major cardiovascular disease and involves enhancement of renin-angiotensin system and recruitment/activation of inflammatory factors such as matrix metalloproteases (MMP's). Caveolae has been shown to play a role in a number of different cardiovascular diseases through different mechanisms including regulation of oxidative stress, inflammation and degradation of extracellular matrix components through MMP's. In addition, endothelial cell caveolae are known to localize the Ang-II (AT1) receptor and regulate renin-angiotensin signaling. Based on these findings, we evaluated the role of caveolae in AAA formation in the murine model. Here, eight week old mice were co-infused with Ang-II and BAPN, a lysyl oxidase inhibitor, to induce AAA. We found that mice lacking the main structural protein of caveolae, caveolin-1, did not develop AAA compared to WT animals in spite of hypertensive blood pressures measured by telemetry in both groups. This finding suggests that intact Ang-II signaling remains in place in caveolin-1 knockout mice. To begin to address the underlying mechanism by which caveolae contributes to AAA, we measured the level of oxidative stress and MMP's in aneurysms. We found an increased expression of MMP-2 and MMP-9 in vessels of WT mice displaying aneurysms. This increase in expression was not observed in Cav-1 knockout mice. Furthermore, KO mice showed less oxidative stress then their WT counterparts as assessed by anti-nitrotyrosine staining. Next we examined the characteristics of early AAA formation in wild-type mice. We found caveolae associated proteins, endothelial nitric oxide synthase (eNOS) and NADPH oxidase 2 (Nox2), were upregulated in early AAA formation, particularly in the endothelium. Also, Vascular Cell Adhesion Molecule (VCAM) was upregulated in the endothelium. However, macrophage infiltration and MMP-2 activation was not observed in early AAA development. In order to elucidate the role of endothelial caveolae in the formation of AAA, we induced AAA, as previously described, in endothelial specific cav-1 knockout mice. Preliminarily findings show endothelial specific knockout mice do not form AAA as compared to their WT littermates. In conclusion, caveolae appears to play a critical role in the formation of AAA in mice via oxidative stress, and recruitment and/or activation of MMPs, specifically MMP-2 and MMP-9. Early markers of AAA formation include VCAM, NOX2, eNOS, and protein nitration. Also, preliminary results indicate that endothelial specific knockout mice do not develop AAA. / Cell Biology

Altered fluid flow, which is found in branches and curvatures of arteries, results in abnormal forces on the endothelial cells (EC). These forces have been shown to alter EC gene expression and phenotype and to activate several cellular structures including G-proteins, ion channels, adhesion molecules, and caveolae. Recently, PECAM-1 has been implicated as the primary sensor of hemodynamic forces in EC. Shear stress rapidly induces tyrosine phosphorylation of PECAM-1 and the recruitment of SHP-2. These events appear to contribute to shear-activation of ERK1/2. Additionally, PECAM-1 has been shown to form a mechanosensory signaling complex with VE-cadherin, VEGFR2, and βcatenin which plays a role in adhesion molecule expression and regulation of NF-κB. Past work has shown that caveolae membrane domains also serve as mechanotransduction sites that regulate many of these same second messengers. Based on these novel observations, we hypothesize that the PECAM-1 mediated mechanotransduction requires caveolar membrane domains to effectively propagate mechano-signals. In this study, we intended to specifically test this hypothesis by 1) evaluating the role of caveolae in shear stress-induced PECAM-1 tyrosine phosphorylation, recruitment of SHP-2, and formation of a signaling complex with VE-cadherin, VEGFR2, and βcatenin and 2) determining the functional significance of PECAM-1 compartmentalization within caveolae with regard to changes in endothelial cell phenotype induced by atherogenic patterns of flow. Here, we have identified a pool of PECAM-1 which localizes within lipid rafts and caveolar membranes. This pool of PECAM-1 was shown to be activated by tyrosine phosphorylation and recruitment of mechanosignaling complex members in response to shear stress. We were also able to demonstrate complex formation in an in vivo model of disturbed blood flow. The significance of PECAM-1 compartmentalization to these membrane microdomains was demonstrated in endothelial cells treated with raft/caveolae disrupting compounds where shear stress-induced PECAM-1 tyrosine phosphorylation was markedly attenuated. Finally, we attempted to generate an adenovirus expressing a mutant form of PECAM-1 which was unable to target to lipid rafts in order to determine the importance of PECAM-1 localization in lipid rafts and caveolae on its downstream signaling in response to shear stress. Results from these studies provide new knowledge as to how endothelial cells respond to changing hemodynamic parameters, which could provide greater insight into how flow influences vascular homeostasis. / Physiology

Pathological cardiac stressors, including persistent hypertension or damage from ischemic heart disease, induce a chronic demand for enhanced contractile performance of the heart. The cytosolic calcium (Ca2+) transient that regulates myocyte contraction must be persistently increased in disease states in order to maintain cardiac output to sustain the metabolic requirements of the body. Associated with this enhanced intracellular Ca2+ ([Ca2+]i) state is pathological cardiac myocyte hypertrophy, which results in large part from the activation of Ca2+-dependent activation of calcineurin (Cn)-nuclear factor of activated T cells (NFAT) signaling. The puzzling feature of this hypertrophic signaling is that the cytosolic [Ca2+] that controls contractility appears to be separate from the [Ca2+] which activates Cn-NFAT signaling. The overarching theme of this dissertation is to explore the source and spatial constraints of pathological hypertrophic signaling Ca2+ and to investigate how it is possible that sensitive and finely tuned Ca2+-dependent signaling pathways are regulated in the background of massive Ca2+ fluctuations that oscillate between 100nM and upwards of 1-2μM during each cardiac contractile cycle. L-type Ca2+ channels (LTCCs) are a major source of Ca2+ entry in cardiac myocytes and are known to play an integral role in the initiation of myocyte excitation contraction-coupling (EC-coupling). We performed a number of experiments to show that a small population of LTCCs reside outside of EC-coupling domains within caveolin (Cav-3) signaling microdomains where they provide a local source of Ca2+ to activate Cn-NFAT signaling. We designed a Cav-targeted LTCC blocker that could eliminate Cn-NFAT activation but did not reduce myocyte contractility. The activity of Cav-targeted LTCCs could also be upregulated to enhance hypertrophic signaling without affecting contractility. Therefore, we believe that caveolae-localized LTCCs do not participate in EC-coupling, but instead act locally to control the coordinated activation of Cn-NFAT signaling that drives pathological remodeling. Transient Receptor Potential (TRP) channels are also thought to provide a source of Ca2+ for activation of hypertrophic signaling. The canonical family of TRP channels (TRPC) is expressed at low levels in normal adult cardiac tissue, but these channels are upregulated in disease conditions which implicates them as stress response molecules that could potentially provide a platform for hypertrophic Ca2+ signaling. We show evidence that TRPC channel abundance and function increases in cardiac stress conditions, such as myocardial infarction (MI), and that these channels are associated with hypertrophic responses, likely through a Ca2+ microdomain effect. While we found that TRPC channels housed in caveolae membrane microdomains provides a source of [Ca2+] for induction of cardiac hypertrophy, this effect also requires interplay with LTCCs. We also found that TRPC channels have negative effects on cardiac contractility, which we believe are due to local activation of Ca2+/calmodulin-dependent protein kinase (CaMKII) and subsequent modulation of ryanodine receptors (RyRs). Further, we found that inhibiting TRPC channels in a mouse model of MI led to increased basal myocyte contractility and reduced hypertrophy and cardiac structural and functional remodeling, as well as increased survival. Collectively, the data presented in this dissertation provides comprehensive evidence that Ca2+ regulation of Cn-NFAT signaling and resultant pathological hypertrophy can be coordinated by spatially localized and regulated Ca2+ channels. The compartmentalization of LTCCs and TRPC channels in caveolae membrane microdomains along with pathological hypertrophy signaling effectors makes for an attractive explanation for how Ca2+-dependent signaling pathways are regulated under conditions of continual Ca2+ transients that mediate cardiac contraction during each heart beat. Elucidation of additional Ca2+ signaling microdomains in adult cardiac myocytes will be important in more comprehensively resolving how myocytes differentiate between signaling versus contractile Ca2+. / Molecular and Cellular Physiology

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Cardiovascular disease is the leading cause of mortality in Western societies and is linked to multiple modifiable risk factors including lifestyle choices. Emerging evidence implicates exposure to persistent environmental pollutants, such as polychlorinated biphenyls (PCBs), as a risk factor for the development or progression of cardiovascular disease. To reduce disease risks, it is critical to identify sensible means of biomedically reducing the toxicity of persistent organic pollutants and related environmental stressors. First, we tested a hypothesis that endothelial cell inflammation and subsequent cardiovascular toxicity initiated by coplanar PCBs is modulated by the crosstalk between caveolae and Nuclear factor (erythroid-derived 2)-like 2(Nrf2) related proteins. Caveolae are lipid-enriched organelles found abundantly in endothelial cells and are important mediators of endocytosis and signal transduction. Caveolin-1 (Cav-1), the major structural protein of caveolae, is known to bind and concentrate multiple proteins related to cardiovascular disease and PCB toxicity. Downregulation of Cav-1 protects against PCB-induced vascular toxicity, but possible mechanisms of this defense remain elusive. Studies using endothelial cells isolated from mice deficient in Cav-1 as well as in vitro silencing assays demonstrated that loss of Cav-1 increases available antioxidant enzymes by upregulating the antioxidant master controller Nrf2. Nutritional interventions focused on diets high in bioactive food components, such as polyphenols or certain fatty acids, may prove to be effective at decreasing environmental pollutant induced diseases. To test the hypothesis that dietary intervention can sensitize Nrf2 and/or caveolae signaling pathways, leading to a more effective anti-inflammatory defense against PCB insults, mice were fed a green tea polyphenol enriched diet and challenged with coplanar PCB 126. Mice fed an enriched diet and exposed to PCBs exhibited lower levels of oxidative stress and higher levels of multiple Nrf2 target antioxidant enzymes. Also, in separate in vitro studies, pretreatment of endothelial cells with the endogenously formed nutrient metabolite, nitro-linoleic acid, altered caveolae and Nrf2 related proteins, resulting in a modified response to PCB exposure. Together, these data support the paradigm that nutritional modulation may be a sensible means of reducing disease risks associated with exposure to environmental pollutants.

La sarcopénie est une maladie dégénérative liée à l’âge qui se caractérise par une perte progressive et involontaire de la masse et de la force musculaire. Elle s’accompagne d’une atteinte de la régénération musculaire et d’une accumulation des espèces réactives de l’oxygène. Les cavéoles sont des invaginations de la membrane plasmique. Dans le muscle, elles jouent un rôle dans la différenciation des cellules satellites et dans le maintien de l’unité contractile dans le muscle différencié. Certaines formes de myopathies sont consécutives à l’absence de cavéoles dans le muscle. Elles sont également impliquées dans la médiation de signaux liés à la régulation du stress oxydant. Afin de mieux comprendre les mécanismes régulant la mise en place de la sarcopénie, nous avons étudié ici les relations existant entre le stress oxydant et les cavéoles. Des cellules musculaires de souris ont été traitées par l’H2O2 et une diminution du taux des cavéolines-1et -3 a été mise en évidence dans des myoblastes et les myotubes, respectivement. Il apparaît donc que les protéines constitutives des cavéoles sont effectivement sensibles au stress oxydant dans les cellules musculaires. En présence d’H2O2, la fonction des cavéoles (endocytose et résistance au stress mécanique) était également significativement altérée dans des myoblastes. L’ensemble des résultats obtenus suggère que le stress oxydant aurait un effet sur les cavéoles, ce qui pourrait entraîner des conséquences sur la régénération et le maintien de l’intégrité musculaire au cours du vieillissement. / Sarcopenia is an age-related degenerative disease which is characterized by a progressive and involuntary loss of muscle mass and strength. It is accompanied by an impairment of muscle regeneration and accumulation of reactive oxygen species. Caveolae are invaginations of the plasma membrane. In muscle, they play a role in the differentiation of satellite cells and in maintaining the contractile unit of the differentiated skeletal muscle. Some myopathies are resulting from the absence of caveolae in muscle. Caveolae are also involved in mediating signals related to the regulation of oxidative stress. To better understand the mechanisms involved in the development of sarcopenia, we investigated here the relationship between oxidative stress and caveolae. Mouse muscle cells were treated with H2O2 and decreased levels of caveolin-1 and -3 were demonstrated in myoblasts and myotubes, respectively. It therefore appears that caveolae constituent proteins are actually sensitive to oxidative stress in muscle cells. In the presence of H2O2, caveolae functions (endocytosis and resistance to mechanical stress) were also significantly degraded in myoblasts. Altogether, these data suggest that oxidative stress would affect caveolae, which could have consequences on regeneration and maintenance of muscle integrity during aging.

Le peptide antimicrobien LL-37 a été retrouvé surexprimé dans différents types de cancer et plus particulièrement dans le cancer du sein dans lequel il est associé au développement des métastases. Nous avons observé, in vitro, que la migration de trois lignées cancéreuses mammaires est augmentée par le peptide LL-37 et son énantiomère (D)-LL-37, excluant la fixation du peptide à un récepteur protéique. Sur les cellules cancéreuses mammaires MDA-MB-435s, le peptide se fixe à la membrane plasmique et diminue sa fluidité. La microscopie électronique localise LL-37 dans les cavéoles et à la surface de structures impliquées dans la migration cellulaire, les pseudopodes. LL-37 induit une entrée de calcium via le canal TRPV2 dont l’activité est augmentée par son recrutement dans les pseudopodes. Ce recrutement est dépendant de l’activation de la voie de signalisation PI3K/AKT induite par LL-37. L’entrée de calcium via TRPV2 est potentialisée par l’activation du canal potassique BKCa, localisé aussi dans les pseudopodes. Des ARN interférents contre TRPV2 inhibent à 70% la migration induite par LL-37, donnant un rôle prépondérant à ce canal dans les effets pro-migratoire du peptide. La fixation du peptide LL-37 aux membranes des cellules cancéreuses et l’activation de canaux ioniques constituent un nouvel axe de recherche pour comprendre le rôle du peptide dans la progression tumorale. / The antimicrobial peptide LL-37 is overexpressed in several types of cancer, among which breast cancer were it is associated with metastasis development. Our experiments on three mammary cancer cell lines have shown that LL-37 increases cell migration. Both its natural (L)-form and its (D)-enantiomer are equally active, excluding a specific binding to a protein receptor. On the MDA-MB-435s cell line, LL-37 attaches to plasma membrane and reduces its fluidity. Electron microscopy localized LL-37 on the surface of pseudopodia, structures implicated in cell migration, and in caveolae. LL-37 induces calcium entry via the TRPV2 channel, which is recruited to pseudopodia. Recruitment depends on activation of PI3K/AKT signaling induced by LL-37. Calcium entry via TRPV2 is potentiated by activation of the BKCa potassium channel also located in pseudopodia. TRPV2 suppression by RNA interference results in 70% reduction of cell migration induced by LL-37, attributing a crucial role of this channel to the promigratory effects of the peptide. Binding of LL-37 to cancer cell membranes and in consequence the activation of ion channels constitutes a novel research field to understand its role in tumor progression.

Les cavéoles sont des invaginations de la membrane plasmique qui nécessitent les cavéolines pour leur biogénèse. Récemment, mon laboratoire d’accueil a décrit un nouveau rôle pour les cavéoles dans la réponse au stress mécanique (Sinha et al, Cell, 2011). Des mutations de la Cavéoline-3 (Cav3), isoforme spécifique du muscle, qui mènent à la rétention de la protéine dans l’appareil de Golgi, ont été décrites dans certaines dystrophies musculaires (DM). Mon projet consiste en l’identification du lien fonctionnel entre les mutations de la Cavéoline-3 et les dystrophies musculaires, qui ont comme phénotype principal un défaut d’intégrité et de réparation membranaire et des dérégulations dans l’homéostasie du muscle.Dans des myotubes humains provenant d’un patient portant la mutation Cav3-P28L ou Cav3-R26Q, j’ai pu montré une diminution de la quantité de cavéoles à la membrane plasmique. En conséquence, les myotubes mutants ne sont plus capables de tamponner l’augmentation de la tension membranaire provoquée par un stress mécanique, ce qui conduit à un défaut d’intégrité membranaire. J’ai aussi montré que la voie de l’interleukin-6 (IL6), importante pour l’homéostasie du muscle, est hyperactivée dans les myotubes mutants, révélant un rôle de régulateur négatif de la voie IL6 par Cav3. De plus, cette voie n’est plus régulée négativement quand un stress mécanique est appliqué comme c’est le cas dans les myotubes sauvages (WT). De manière intéressante, les myotubes mutés phénocopient une déplétion de Cav3 et ce phénotype est réversible lorsque l’on reforme des cavéoles à la membrane plasmiques des myotubes mutés en exprimant la forme WT de Cav3. Ceci confirme un lien direct entre les mutations de Cav3 induisant l’absence de cavéoles et le défaut de mécano-protection et mécano-signalisation de la voie IL6. / Caveolae are plasma membrane invaginations that require caveolin proteins for their biogenesis. Recently, our laboratory reported a new role for caveolae in the cell response to mechanical stress (Sinha et al, Cell, 2011). Mutations in the CAV3 gene (muscle isoform), which lead to Cav3 retention in the Golgi apparatus, are associated with muscular dystrophies (MD). My project consists in identifying the functional link between Cav3 mutations and MDs, which exhibit defects in membrane integrity and repair, and in muscle homeostasis.In Cav3-P28L and Cav3-R26Q mutated human myotubes, I showed a lack of caveolae structures at the plasma membrane. This results in a failed buffering of membrane tension increase upon mechanical stress, which leads to membrane integrity defects. I also showed that the interleukin-6 (IL6) pathway, important for muscle homeostasis, is overactivated in mutant myotubes, showing evidence of a negative regulation of the pathway by Cav3. Furthermore, the IL6 pathway is no longer negatively regulated upon mechanical stress, as it is the case in wild-type (WT) myotubes. Interestingly, mutated myotubes phenocopy Cav3 depletion, and the phenotype is reversible with caveolae reformation upon expression of the WT form of Cav3. This confirms the direct link between Cav3 mutations and the absence of caveolae with failed mechano-protection and IL6/STAT3 mechano-signaling.