Cardiovascular disease is the leading cause of mortality in the western world. The pro-inflammatory and pro-proliferative etiology of vascular proliferative diseases is well characterized, while much less is known about the mechanisms of anti-inflammatory and anti-proliferative processes. Interleukin-19 (IL-19) is a newly described member of the IL-10 family of anti-inflammatory interleukins, and our group was the first to discover IL-19 expression in activated, synthetic, but not quiescent, contractile human vascular smooth muscle cells (hVSMC). We also found that IL-19 is anti-inflammatory and anti-proliferative for hVSMC. IL-19 is able to reduce the abundance of COX-2, IL-1β, IL-8, and Cyclin D1 transcripts which contain AU-rich elements (ARE) in their 3'-untranslated regions (3'-UTR). IL-19 is able to reduce the abundance of HuR, a stabilizing RNA-binding protein, which we feel provides a mechanism for these effects. The overall goal of this study is to elucidate IL-19's anti-inflammatory and anti-proliferative mechanism(s) in hVSMC in the context of vascular proliferative diseases. This goal has directed our overall hypothesis: IL-19's anti-proliferative and anti-inflammatory effects in hVSMC are mediated, at least in part, by modulation of HuR abundance and translocation, resulting in decreased stability of mRNA transcripts. HuR functions through a translocation mechanism, and IL-19 is able to reduce HuR cytoplasmic abundance. IL-19 also reduces HuR phosphorylation, which is a pre-requisite for HuR translocation, possibly through a PKCα-dependent mechanism. The stability of ARE-containing transcripts is reduced with IL-19 treatment, and reducing HuR expression by siRNA has the same inhibitory effect. VSMC are important mediators in the initiation of atherosclerosis. Oxidized low-density lipoprotein (ox-LDL) is able to induce IL-19 expression in these cells. VSMC are known to express scavenger receptors that take up ox-LDL. IL-19 is able to reduce the uptake of ox-LDL and the abundance of ox-LDL induced LOX-1 and CX-CL16 scavenger receptors. Interestingly, these scavenger receptors also have ARE in their 3'-UTR. IL-19 is able to reduce ox-LDL induced HuR cytoplasmic abundance. HuR knockdown by siRNA reduces the uptake of ox-LDL by hVSMC. These data suggest that IL-19 reduced scavenger receptor abundance may be due to decreased total and cytoplasmic HuR abundance. IL-19 reduces the abundance of ox-LDL induced COX-2 expression. Taken together, these results demonstrate that IL-19 down-regulates vital steps in vascular proliferative disease processes through an HuR-dependent mechanism. / Molecular and Cellular Physiology

Cardiovascular disease is the leading cause of death in the United States, and coronary artery disease (CAD) kills over 370,000 people annually. There are available therapies that offer a temporary solution; however, only a heart transplant can fully resolve heart failure, and donor organ shortages severely limit this therapy. C-kit+ human cardiac stem cells (hCSCs) offers a viable alternative therapy to treat cardiovascular disease by replacing damaged cardiac tissue; however, low cell viability, low retention/engraftment, and uncontrollable in vivo differentiation after transplantation has limited the efficacy of stem cell therapy. Tissue engineering solutions offer potential tools to overcome current limitations of stem cell therapy. Materials derived from natural sources such as keratin from human hair offers innate cellular compatibility, bioactivity, and low immunogenicity. Keratin proteins extracted using oxidative chemistry known as keratose (KOS) have shown therapeutic potential in a wide range of applications including cardiac regeneration. My studies utilize KOS hydrogels to modulate c-kit+ hCSC differentiation, and explore the capability of differentiated cells to regenerate vascular tissue. In the first Chapter, we reviewed literature relevant to keratin-based biomaterials and their biomedical applications, the use of stem cells in cardiovascular research, and the differentiation of vascular smooth muscle cells (VSMCs). The section on biomedical applications of keratin biomaterials focuses on the oxidized form of keratin known as keratose (KOS), because this was the material used for our research. Since we planned to use this material in conjunction with c-kit+ hCSCs, we also briefly explored the use of stem cells in cardiovascular research. Additionally, we examined some key signaling pathways, developmental origins, and the cell phenotype of VSMCs for reasons that will become clear after observing results from chapters 2 and 3. Based upon our review of the literature, KOS biomaterials and c-kit+ hCSCs were determined to be promising as a combined therapeutic for the regeneration of cardiac tissue. Research in Chapter 2 focused on characterizing the effects of KOS hydrogel on c-kit+ hCSC cell viability, proliferation, morphology, and differentiation. Results demonstrated that KOS hydrogels could maintain hCSC viability without any observable toxic effects, but it modulated cell size, proliferation, and differentiation compared to standard tissue culture polystyrene cell culture (TCPS). KOS hydrogel produced gene and protein expression consistent with a VSMC phenotype. Further, we also observed novel "endothelial cell tube-like" microstructures formed by differentiated VSMCs only on KOS hydrogel, suggesting a potential capability of the hCSC-derived VSMCs for in vitro angiogenesis. Results from this study lead us to question what signaling pathways might be responsible for the apparent VSMC differentiation pattern we observed on KOS hydrogels. Research in Chapter 3 explored the time course of VSMC differentiation, cell contractility, inhibition of VSMC differentiation, and measured protein expression of transforming growth factor beta 1 (TGF-β1) and its associated peptides for hCSCs cultured on KOS hydrogels, tissue culture polystyrene, and collagen hydrogels. A review of VSMC differentiation signaling pathways informed our decision to investigate the role of TGF-β1 in VSMC differentiation. Results demonstrated that KOS hydrogel differentiated hCSCs significantly increased expression for all three vascular smooth muscle (VSM) markers compared to TCPS differentiated cells. Additionally, KOS differentiated hCSCs were significantly more contractile than cells differentiated on TCPS. Recombinant human (rh) TGF-β1 was able to induce VSM differentiation on TCPS. VSM differentiation was successfully inhibited using TGF-β NABs and A83-01. Enzyme-Linked Immunosorbent Assay (ELISA) analysis revealed that both TCPS and KOS hydrogel differentiated cells produced TGF-β1, with higher levels being measured at early time points on TCPS and later time points on KOS hydrogels. Results from supplementing rhTGF-β1 to TCPS and KOS hydrogels revealed that KOS seems to interact with TGF-β to a greater extent than TCPS. Western blot results revealed that latency TGFβ binding protein (LTBP-1) and latency associated peptide (LAP) had elevated levels early during differentiation. Further, the levels of LTBP-1 and LAP were higher on KOS differentiated hCSCs than TCPS hCSCs. This study reaffirms previous results of a VSM phenotype observed on KOS hydrogels, and provides convincing evidence for TGF-β1 inducing VSM differentiation on KOS hydrogels. Additionally, results from ELISA and western blot provide evidence that KOS plays a direct role in this pathway via interactions with TGF-β]1 and its associated proteins LTBP-1 and LAP. Results from chapter 2 and 3 offered significant evidence that our cells exhibited a VSMC phenotype, and that TGF-β1 signaling was a key contributor for the observed phenotype, but we still needed an animal model to explore the therapeutic potential of our putative VSMCs. Research in Chapter 4 investigated a disease model to test the ability of KOS hydrogel differentiated cells to regenerate vascular tissue. To measure vascular regenerative capability, we selected a murine model of critical limb ischemia (CLI). CLI was induced in 3 groups (n=15/group) of adult mixed gender NSG mice by excising the femoral artery and vein, and then treated the mice with either PBS (termed as PBS-treated), Cells differentiated on TCPS (termed as Cells from TCPS), or KOS hydrogel-derived VSMCs (termed as Cells from KOS). Blood perfusion of the hind limbs was measured immediately before and after surgery, then 14, and 28 days after surgery using Laser Doppler analysis. Tissue vascularization, cell engraftment, and skeletal muscle regeneration were measured using immunohistochemistry, 1,1'-Dioctadecyl3,3,3',3'-Tetramethylindocarbocyanine Perchlorate (DiL) vessel painting, and hematoxylin and eosin (HandE) pathohistological staining. During the 4-week period, both cell treatment groups showed significant increases in blood perfusion compared to the PBS-treated control, and at day 28 the Cells from KOS group had significantly better blood flow than the Cells from TCPS group. Additionally, the Cells from KOS group demonstrated a significant increase in the ratio of DiL positive vessels, capillary density, and a greater density of small diameter arterioles compared to the PBS-treated group. Further, both cell-treated groups had similar levels of engraftment into the host tissue. We conclude that Cells from KOS therapy increases blood perfusion in an NSG model of CLI, but does not lead to increased cell engraftment compared to other cell based therapies. Overall, the results from this dissertation demonstrated that KOS hydrogels produce VSMC differentiation from c-kit+ hCSCs mediated by TGF-β1 signaling, and that the differentiated cells are able to increase blood perfusion in a CLI model by increasing capillary density, suggesting enhanced angiogenesis. Future studies should explore potential protein-protein interactions between KOS, TGF-β1 and its associated proteins. Additionally, we should plan animal studies that examine the efficacy of our cells to regenerate cardiac tissue following an acute myocardial infarction (AMI). / PHD

The apoptosis and dysfunction of vascular smooth muscle cells in the human descending thoracic aorta is often associated with cardiovascular diseases like aortic dissection and aneurysms. Knowledge of the mechanical effects of contractile smooth muscle cells plays a crucial role in the understanding these potentially lethal conditions. Located in the medial layer, vascular smooth muscle cells are arranged in the so-called herringbone pattern. In regards to the mechanics of the aorta, the consequences of this type of anisotropy have not been fully discussed in literature so far. In this end, a novel hyperelastic constitutive law is proposed which accounts for the dispersive, transmural alignment of vascular smooth muscle cells and their characteristic length-tension behaviour. The model is calibrated with experimental data and is applied to the simulation of an aortic ring under in vivo conditions. By approximating the geometry of the aorta as a layered, thick-walled cylinder, the corresponding quasistatic, mechanical boundary value problem is solved semi-analytically. It is shown that the herringbone pattern induces shear deformation and equalises the normal stress gradients in the aortic wall. Since arterial vessels are able to actively adapt and alter the alignment and activity of smooth muscle cells, the existence of the herringbone pattern is in accordance with Fung's principle of optimal operation.

Au cours des maladies cardiovasculaires (MCV), il peut se produire divers problèmes de santé, telle que l’insuffisance cardiaque ou encore l’HTA. Ces phénomènes se caractérisent, entre autres, par une augmentation de synthèse d’endotheline-1 (ET-1), un neuropeptide synthétisé par les cellules endothéliales ayant un effet vasoconstricteur sur les cellules musculaires lisses vasculaires (CMLV). Ainsi, la surexpression de ce vasopeptide, mène à terme, au maintien de l’HTA aggravée des sujets, précédée ou concomitante à l’athérosclérose ou à la resténose, cliniquement illustrées par une prolifération et une migration anormale des CMLV de la media vers l’intima des vaisseaux sanguins. Parallèlement, il a été observé que la protéine sirtuine-1 (Sirt-1), membre de la famille des protéines histones déacétylases (HDAC), présente des propriétés anti-athérosclérotiques par sa capacité d’atténuer la prolifération et la migration des CMLV. Des travaux récents ont aussi montré qu’au cours de l’HTA la protéine Sirt-1 est faiblement exprimée dans les CMLV. Son implication dans le développement des pathologies vasculaires semble apparente, mais des études demeurent nécessaires pour décrire son rôle exact dans la pathogenèse des MCV. Dans cette optique, l’objectif de cette étude a été d’observer la variation d’expression de Sirt-1 dans les CMLV, isolées de l’aorte ascendante de rat, en réponse à l’ET-1. On a remarqué qu’une heure de stimulation des CMLV avec l’ET-1 induit une diminution de l’expression de Sirt-1 via l’activation des récepteurs ETA. Ces résultats suggèrent que la capacité d’ET-1 à atténuer l’expression de Sirt-1 serait un éventuel mécanisme d’action avec des effets favorisant les MCV. / Cardiovascular diseases (CVD) are associated with several vascular dysfunctions such as heart failure and hypertension. These phenomena cause increased synthesis of endothelin-1 (ET-1), a neuropeptide, synthesized by endothelial cells which has vasoconstrictor action on vascular smooth muscle cells (VSMC). Overexpression of this vasopeptide leads eventually to hypertension (HTA). This usually happen after atherosclerosis or restenosis, leading to proliferation and migration of VSMC from media to intima. It was shown that during atherosclerosis, the protein sirtuin-1 (Sirt-1), a member of protein histone deacetylases (HDAC), has an anti-atherosclerotic effect due to its ability to diminish proliferation and migration of VSMC. It has also been observed that during hypertension, Sirt-1 was poorly expressed in VSMC. Its role in vascular pathophysiology remains sparsely studied, therefore it’s essential to explore it. In the present study we investigated the expression of Sirt-1 in VSMC isolated from the ascending aorta of rats, in response to ET-1 stimulation. We observed that Sirt-1 expression decreases after 1 hour of stimulation by ET-1 via ETA receptors. In summary, these results suggest that the ability of ET-1 to attenuate Sirt-1 expression in VSMC, may be a potential mechanism for promoting CVD.

L’athérosclérose est étroitement liée au diabète de type 2. De fortes concentrations plasmatiques en acides gras libres (AGL) et en insuline sont des caractéristiques retrouvées chez les patients souffrant de ces deux pathologies. Les AGL, présents dans notre alimentation, font partie de l’environnement auquel les cellules sont exposées. Leurs effets dépendent de leur nature, les acides gras saturés (AGS) étant néfastes et les acides gras monoinsaturés (AGMI) plus protecteurs. Ils ont donc des effets variés sur les cellules musculaires lisses vasculaires (CMLV) impliquées dans la pathogénèse de l’athérosclérose. Ainsi, l’objectif principal de ce projet de maîtrise était d’évaluer l’impact de deux combinaisons d’AGL sur la viabilité des CMLV, en condition hyperinsulinémique ou non. Les deux combinaisons renfermaient les mêmes AGL mais en proportions différentes, l’une étant plus riche en AGS et l’autre en AGMI. Nos résultats ont montré que les combinaisons d’AGL ont un effet pro-apoptotique principalement dû aux AGS. L’acide oléique présent dans les combinaisons atténue cependant cet effet. Il diminue même plus fortement l’apoptose des CMLV lorsqu’associé à un AGS que lorsqu’utilisé seul. Cet impact est significatif uniquement dans certaines proportions de ces AGL et est plus efficace en présence d’insuline. Ces résultats mettent en lumière la présence d’une compétition entre mécanismes anti- et pro-apoptotiques en fonction des proportions d’AGS versus AGMI et de l’insulinémie chez les CMLV. Ils soulignent également l’importance de la présence des AGMI dans les diètes riches en AGS et pourraient être utiles pour l’élaboration de nouvelles diètes adaptées aux patients athérosclérotiques et diabétiques. / Atherosclerosis is closely linked to type 2 diabetes. High plasmatic concentrations of free fatty acids (FFA) and insulin are common features in patients suffering from both diseases. FFA, present in our diet, are part of the environment to which body cells are exposed. Their effects are dependent of their nature, being harmful for saturated fatty acids (SFA) and more protective for monounsaturated fatty acids (MUFA). They can have therefore various effects on vascular smooth muscle cells (VSMC) implicated throughout the development of atherosclerosis. Thus, this study aimed to assess the impact of two FFA combinations on VSMC viability, whether or not in a hyperinsulinemic condition. Both combinations contained the same FFA but in different proportions, one being richer in SFA and the other in MUFA. Our results showed that FFA combinations have a pro-apoptotic impact, mainly due to SFA. However, the presence of oleic acid in the combinations attenuated this effect. Furthermore, oleic acid had the capacity to reduce more strongly VSMC apoptosis when combined with a SFA than when used alone, although only under specific FFA ratios. This impact is even more effective in presence of insulin. These results highlight the presence of a competition between anti- and pro-apoptotic mechanisms dependent of FFA ratios (SFA vs. MUFA) and insulinemia to which are exposed VSMC. They also underline the importance of the presence of MUFA such as oleic acid in diets rich in SFA and could be useful for the development of new diets adapted to atherosclerotic and diabetic patients.

Les Erythropoietin-producing hepatocyte (EPH) sont la plus grande famille de récepteurs tyrosine kinase. Leurs ligands, les éphrines (EFNs), sont aussi des molécules exprimées à la surface cellulaire. Les EPH/EFNs sont impliqués dans de nombreux processus biologiques. L'hypertension artérielle (PA) est une maladie chronique qui, aujourd'hui, est devenue un problème médical critique dans le monde entier et un enjeu de santé publique. La découverte de nouvelles thérapeutiques de l'hypertension sont d'une grande importance pour la santé publique. Jusqu’à tout récemment, il existe seulement quelques études concernant le rôle de l’axe EPH/EFNs sur la fonction des cellules musculaires lisses vasculaires (CMLV). Dans nos études précédentes, nous avons montré qu'EPHB6 et EFNB1, de concert avec les hormones sexuelles, régulent la PA. Dans la présente étude, nous avons constaté que les différents membres de la famille EPH/EFN peuvent réguler soit positivement, soit négativement, la contractilité des CMLV et la PA: tandis que EPHB4 et EFNB2 appartiennent à la première catégorie, EFNB1, EFNB3 et EPHB6 appartiennent à la deuxième. In vivo, des souris males, mais non pas des femelles, porteuses d’une mutation EPHB4 (KO) spécifique du muscle lisse présentent une PA diminuée, comparée aux souris témoins (WT). Les CMLV de souris EPHB4 KO, en présence de testostérone, ont montré une contractilité réduite lors de la stimulation par la phényléphrine (PE). Au niveau moléculaire, la phosphorylation de la protéine kinase II dépendante de Ca2+/calmoduline et de la kinase de la chaine légère de la myosine (CLM) est augmentée, tandis que la phosphorylation de la kinase de la CLM est réduite dans les CMLV KO lors de la stimulation par PE, par rapport au WT CMLV. Cela fournit une base moléculaire à la réduction de la PA et de la contractilité des CMLV chez les souris EPHB4 KO. EFNB2 est le ligand majeur de l’EPHB4. Comme attendu, les souris EFNB2 KO spécifique du muscle lisse avaient un phénotype de PA semblable, quoique non identique, aux souris EPHB4 KO. Les souris mâles EFNB2 KO, mais pas femelles, sous régime régulier ou riche en sel, présentent une PA réduite, par rapport à leurs homologues WT. Au niveau cellulaire, les CMLV des souris KO ont montré une contractilité réduite lors de la stimulation par PE par rapport aux témoins WT. Une région de l’acide aminé (aa) 313 à l’aa 331 dans la partie intracellulaire d’EFNB2 est essentielle pour la signalisation inverse qui régule la contractilité des CMLV, selon des études de mutation-délétion. Dans une étude de génétique humaine, nous avons identifié, dans le gène EFNB2, six SNP qui étaient associées significativement au risque d'hypertension artérielle, de façon dépendante du sexe, ce qui corrobore nos résultats chez les souris. En revanche, la délétion du gène EFNB3 (KO) chez les souris femelles aboutit à une PA élevée et à une augmentation des résistances des petites artères in vivo, améliore la contractilité des petites artères ex-vivo et augmente la contractilité des CMLV in vitro. Les souris mâles KO ont une PA normale, mais la castration conduit à une augmentation significative de la PA dans les souris KO, mais pas dans les souris WT. Les CMLV des souris KO femelles ont montré une phosphorylation accrue de la CLM et une phosphorylation réduite de la kinase de la CLM, ce qui fournit à nouveau une base moléculaire aux phénotypes de PA et de contractilité des CMLV observés. Ce changement de signalisation est attribuable à une protéine adaptatrice Grip1. En effet, dans une étude d'association pan génomique par le Consortium International pour la Pression Sanguine, un SNP dans le gène GRIP1 a approché le seuil de significativité de la valeur p pour son association avec la pression diastolique. Nos recherches, pour la première fois, ont révélé que EPH/EFNs sont de nouveaux composants dans le système de régulation de la PA. Les membres de la famille EPH/EFN peuvent agir comme des forces Yin et Yang pour régler finement le tonus des vaisseaux pour assurer l'homéostasie de la PA et de sa régulation. Ces effets de EPH/EFNs dépendent du sexe et des niveaux d’hormones sexuelles. À partir de ces nouvelles connaissances, nous pourrions développer une nouvelle thérapie personnalisée pour l’hypertension artérielle, utilisant des antagonistes d'hormones sexuelles ou des thérapies de remplacement d'hormones sexuelles, selon les niveaux d'hormones sexuelles des patients et les mutations dans les gènes de l'EPH/EFN. / Erythropoietin-producing hepatocyte (EPH) kinases are the largest family of receptor tyrosine kinases. Their ligands, ephrins (EFNs), are also cell surface molecules. Ephs/EFNs are implicated in many biological processes. Hypertension is a chronic medical condition of high arterial blood pressure (BP). New hypertension therapeutic treatments are of great importance for public health. Until recently, there are only a few studies related to the role of EPHs/EFNs in vascular smooth muscle cell (VSMC) function. In our previous studies, we have found that EPHB6 and EFNB1 function in concert with sex hormones to regulate BP. In the present investigation, we found that different EPH/EFN family members can either positively or negatively regulate the VSMC contractility and BP: while EPHB4 and EFNB2 belong to the former category, EFNB1, EFNB3 and EPHB6, the latter. In vivo, male but not female smooth muscle-specific EPHB4 knockout (KO) mice presented decreased BP, compared to WT controls. VSMCs from EPHB4 KO mice in the presence of testosterone showed reduced contractility. EFNB2 is the major ligand of EPHB4. As expected, smooth muscle-specific EFNB2 KO mice had a similar although not identical BP phenotype as EPHB4 KO mice. Male but not female EFNB2 KO mice on regular or high-salt diet presented reduced BP, compared to WT counterparts. At the cellular level, the KO VSMCs showed reduced contractility compared to WT controls. In a human genetic study, we identified in the EFNB2 gene six SNPs that were significantly associated with hypertension risk in a sex-dependent way, corroborating our findings in mice. On the other hand, EFNB3 gene KO in female mice resulted in elevated BP and small artery resistance in vivo, enhanced small arterial contractility ex vivo, and augmented VSMC contractility in vitro. Male KO mice had normal BP, but castration led to significant BP elevation in KO but not in WT mice. VSMCs from female KO mice showed heightened MLC phosphorylation and reduced MLC kinase phosphorylation. This signaling change was mediated through an adaptor protein Grip1. Indeed, in a genome-wide association study by the International Consortium for Blood Pressure, an SNP in the GRIP1 gene approached the significant threshold p-value for its association with diastolic BP. Our research for the first time revealed that EPHs/EFNs are novel components in the BP regulation system. Members of the EPH/EFN family may act as Yin and Yang forces to finely tune the vessel tone for BP homeostasis and regulation. Such effects of EPHs/EFNs depend on sex and sex-hormone levels. Based on this new knowledge, we could develop novel personalized hypertension therapy using sex hormone antagonists or sex hormone replacement therapy, depending on the sex hormone levels of the patients and mutations in EPH/EFN genes.

A vasodilatação promovida pela triiodotironina (T3) ocorre por sua ação direta sobre o relaxamento das células musculares lisas vasculares (CMLV), porém os mecanismos envolvidos são desconhecidos. Neste estudo mostramos que o T3 rapidamente relaxa as CMLV através da geração de óxido nítrico (NO), via óxido nítrico sintase neuronal e induzível (nNOS e iNOS), efeitos mediados pela sinalização PI3K/Akt. Ensaios funcionais em aortas sem endotélio, incubados com T3, mostraram menor resposta contrátil a Fenilefrina (FE), efeito este revertido pelo L-NAME, inibidor da NOS. Aortas de ratos hipertiroideos apresentaram aumento do receptor de Angiotensina II (AngII) do tipo 2 (AT2), acompanhado de diminuição de proteínas contráteis. In vitro o T3 diminui estas proteínas contráteis via AT2. Aortas sem endotélio dos ratos hipertiroideos apresentaram menor reatividade a AngII e maior relaxamento ao nitroprussiato de sódio (NPS), efeitos estes mediados via AT2. Por fim, observamos que o T3 é capaz de induzir produção de NO nas CMLV via PI3K/Akt, a qual é ativada pelo AT2 / 3,3\',5-triiodo-l-thyronine (T3) has been shown to induce vasodilation by its direct effect on vascular smooth muscle cells (VSMC). However, the mechanism by which T3 causes VSMC relaxation is still unknown. Here, we have shown that T3 causes rapid relaxation of VSMC via increased NO production from inducible and neuronal nitric oxide synthase (NOS). We further showed that these effects were mediated by PI3K/Akt signaling pathway. Vascular reactivity studies showed that endothelium-denuded aortas treated with T3 had a decreased response to phenylephrine which was reserved by L-NAME, NOS inhibitors. Aortas from hyperthyroid rats showed an upregulation of AT2 accompanied by decreased of contractile proteins. In vitro we observed that T3 decreases contractile proteins via AT2. Furthermore, endothelium-denuded aortas from hyperthyroid rats showed a decreased response to angiotensinII and augmented relaxation to sodium nitroprusside (SNP) via AT2 participation. Our data also suggests that PI3K/Akt signaling pathway is involved in T3-induced NO production in VSMC via AT2.

INTRODUÇÃO: O polimorfismo dentro do gene TCF7L2, rs7903146, é, até o momento, o marcador genético mais significantemente associado ao risco de diabetes mellitus tipo 2, sendo também associado à doença arterial coronariana. Contudo, pouco ainda se conhece sobre o papel funcional desse polimorfismo na patologia dessas doenças. O objetivo desse projeto foi investigar esse papel funcional, no fenótipo de células vasculares de músculo liso de 92 indivíduos, usando abordagens de comparação de níveis de expressão gênica e de comparação de correlações de expressão gênica, de modo que tais comparações fossem representadas visualmente como redes de interação gênica. MÉTODOS: Inicialmente, foram comparados os níveis de expressão de 41 genes (genes que possuem ou estão perto de variantes genéticas associadas ao diabetes mellitus tipo 2 e outros genes relacionados às vias de sinalização de diabetes mellitus tipo 2 ou às vias de proliferação celular) entre indivíduos com o alelo associado ao risco de diabetes mellitus tipo 2 (CT e TT) e indivíduos sem o alelo de risco (CC) do rs7903146. Com a finalidade de se observar se os genes estavam se relacionando de modo diferente entre os grupos genotípicos, foram comparados os padrões de correlação de expressão dos 41 genes. RESULTADOS: Quanto às comparações de níveis de expressão entre os grupos, cinco formas de splicing do gene TCF7L2 e os genes CDKAL1, IGF2BP2, JAZF1, CDKN2B, CAMK1D, JUN, CDK4, ATP2A2, e FKBP1A apresentaram níveis de expressão significativamente diferentes. Quanto às comparações de correlação de expressão entre os grupos, os genes RXR?, CALM1, CALR e IGF2BP2 foram os que mostraram os mais diferentes padrões de correlação com os outros genes. CONCLUSÃO: Deste modo, o alelo de risco analisado é apontado como tendo influência em cis na regulação da expressão de determinadas formas de splicing do gene TCF7L2 em células vasculares de músculo liso; além de parecer influenciar nas expressões e nas interações de genes relacionados à homeostase glicolítica e/ou proliferação celular. Sendo assim, através de nossas análises identificaram-se possíveis candidatos-alvos no tratamento de redução do risco em indivíduos com alto risco de desenvolvimento de diabetes mellitus tipo 2 e de doença arterial coronariana, especialmente os indivíduos que possuem os genótipos de risco analisados do gene TCF7L2 / INTRODUCTION: The SNP within the TCF7L2 gene, rs7903146, is, to date, the most significant genetic marker associated with type 2 diabetes mellitus risk, well as being associated with coronary artery disease. Nonetheless, its functional role in these diseases pathology is poorly understood. The aim of the present study was to investigate this role, in vascular smooth muscle cells from 92 patients undergoing aortocoronary bypass surgery, using expression levels and expression correlation comparison approaches, which were visually represented as gene interaction networks. METHODS: Initially, the expression levels of 41 genes (seven TCF7L2 splice forms and other 40 relevant genes) were compared between rs7903146 wild-type (CC) and type 2 diabetes mellitus risk (CT + TT) genotype groups. Next, the expression correlation patterns of the 41 genes were compared between genotypic groups in order to observe if the relationships between genes were different. RESULTS: Five TCF7L2 splice forms and CDKAL1, IGF2BP2, JAZF1, CDKN2B, CAMK1D, JUN, CDK4, ATP2A2 and FKBP1A genes showed significant expression differences between groups. RXR?, CALM1, CALR and IGF2BP2 genes were pinpointed as showing the most different expression correlation pattern with other genes. CONCLUSION: Therefore, type 2 diabetes mellitus risk alleles appear to be influencing TCF7L2 splice form\'s expression in vascular smooth muscle cells; besides it can be influencing expression and interactions of genes related to glucose homeostasis and/or cellular proliferation. Thereby, through our analysis were identified possible treatment target candidates for risk reduction in individuals with high-risk of developing type 2 diabetes mellitus and coronary artery disease, especially individuals harboring TCF7L2 risk genotypes

Estudos caracterizaram o envolvimento da PDI na modulação da geração de EROs pela Nox1 como moduladores da migração de células do músculo liso vascular (VSMC) mediados por fatores de crescimento derivados de plaqueta (PDGF). Outros estudos vêm demonstrando o envolvimento do fator de crescimento epidermal (EGFR) no remodelamento vascular, após a transativação via Angiotensina II. Entretanto o papel da PDI na ativação do EGFR via Nox1 na hipertensão arterial ainda permanece desconhecido. Objetivo foi caracterizar o papel da PDI na expressão de Nox1 dependente do EGFR durante o desenvolvimento da hipertensão arterial. Resultados demonstram um aumento da expressão de HB-EGF e ativação de ERK 1/2 na aorta de animais SHR com 8 semanas e 12 semanas de idade, e no plasma de animais SHR com 12 semanas. Ainda, a OvxPDI acarretou em um aumento na expressão gênica de Nox-1 tanto na OVXPDI quanto na forma OvxPDIMUT. Resultados mostram um novo papel da PDI na expressão gênica de Nox-1 via EGFR e a participação desta tiol oxido redutase na gênese da hipertensão arterial. / Studies characterizing the involvement of PDI in the modulation of ROS by Nox1 as modulators of cell migration of vascular smooth muscle (VSMC) mediated by growth factors derived from platelets (PDGF). Other studies have demonstrated the involvement of the epidermal growth factor receptor (EGFR) on vascular remodeling after transactivation via Angiotensin II. However the role of PDI in the activation of EGFR via Nox1 in hypertension remains unknown. Objective was to characterize the role of PDI in Nox1 dependent EGFR expression during the development of hypertension. Results show an increase of HB-EGF expression and ERK 1/2 activation in the aortic SHR at 8 weeks and 12 weeks of age, and plasma SHR at 12 weeks. Still, the OvxPDI resulted in an increase in gene expression of Nox-1 both in OVXPDI and in OvxPDIMUT way. Results show a new role of PDI in gene expression of Nox-1 via EGFR and the participation of this thiol reductase oxide in the pathogenesis of hypertension.

L'objectif principal de ce travail était de comparer l'implication (i) de cellules vasculaires, cellules musculaires lisses vasculaires (CML) et cellules endothéliales (CE), ou des cellules circulantes, les plaquettes, et (ii) des microparticules (MP) issues de ces différentes cellules dans la génération de la thrombine mais également dans son inhibition par les systèmes anticoagulants de la protéine C activée (PCa) et de l'inhibiteur de la voie du facteur tissulaire (TFPI), et d'identifier les mécanismes et les déterminants responsables des différences observées entre ces supports cellulaires pour la coagulation. Nous avons démontré que l'intégrine [alpha]v[gamma]3 qui est le récepteur pour la prothrombine sur les surfaces vasculaires était impliquée dans la génération de thrombine à la surface des CML soumises ou non à des déformations mécaniques cycliques. A l'état de base, les CML et les CE ont un potentiel thrombinique similaire, mais moins important que celui des plaquettes. Nous avons montré un rôle synergique du TFPI avec la PCa dans l'inhibition de la génération de thrombine à la surface de ces cellules plus importante avec les CML qu'avec les CE. L'ensemble de nos résultats suggère que les CML pourraient exercer des effets procoagulants comparables aux CE mais avec des régulations différentes en réponse aux facteurs pro- et anticoagulants, et que les MP issues de cellules vasculaires ont un pouvoir thrombogène très supérieur à leurs cellules d'origine / The main objective of this study was to compare the implication (i) of vascular cells, smooth muscle cells (SMC) and endothelial cells (EC), or circulating cells, platelets, and (ii) microparticles (MP) derived from these different cells in the generation of thrombin but also in its inhibition by the activated protein C (APC) and the tissue factor pathway inhibitor (TFPI), and to identify the mechanisms and determinants responsibles for observed differences between these different cell supports for coagulation. We have demonstrated that [alpha]v[gamma]3 integrin, the prothrombin receptor on the vascular surfaces, was involved in the generation of thrombin on the surface of these cells subjected or not subjected to cyclic mechanical deformations. At baseline, SMC and EC, have equivalent thrombin generating capacities, but less than that of platelets. We have shown a synergistic role of TFPI with APC in the inhibition of thrombin generation at the surface of these cells, more important with SMC than with EC. Taken together, our results suggest that SMC may exert procoagulant effects comparable to EC but with different regulations in response to pro-and anticoagulant factors, and that MP derived from vascular cells have a very higher thrombogenic activity compared to their parent cells