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Les antagonistes des récepteurs aux minéralocorticoïdes (MR) appartiennent à l'arsenal thérapeutique pour le traitement de diverses maladies cardiovasculaires, mais les mécanismes conférant leurs effets bénéfiques sont encore mal compris. Une partie de ces effets peuvent être liée à la régulation de l'expression du canal Ca2+ de type L Cav1.2, largement impliqué dans l'insuffisance cardiaque et l'hypertension. Nous montrons que MR fonctionne comme un facteur de transcription transformant le signal de l'aldostérone dans l'utilisation du 'cardiaque' promoteur alternatif P1, dirigeant l'expression du long N-terminal transcrit (Cav1.2-LNT. L'aldostérone augmente de façon concentration- et de temps dépendente l'expression de Cav1.2-LNT dans les cardiomyocytes en raison de l'activation du promoteur P1, par interactions des MR avec des séquences spécifiques de l'ADN sur le promoter P1. Ce mécanisme de cis-régulation induit l'activation de promoteur P1 dans les cellules vasculaires conduisant à une nouvelle signature moléculaire de Cav1.2-LNT associé à une sensibilité réduite aux bloqueurs des canaux Ca2+. Ces résultats révèlent Cav1.2-LNT comme une cible minéralocorticoïde spécifique qui pourrait influencer sur l'éfficacité thérapeutique dans les maladies cardiovasculaires. / The mineralocorticoid receptor (MR) antagonists belong to the current therapeutic armamentarium for the management of cardiovascular diseases, but the mechanisms conferring their beneficial effects are still poorly understood. Part of these MR effects might be related to the L-type Cav1.2 Ca2+ channel expression regulation, critically involved in heart failure and hypertension. Here, we show that MR acts as a transcription factor triggering aldosterone signal into specific alternative 'cardiac' P1-promoter usage, given rise to long (Cav1.2-LNT) N-terminal transcripts. Aldosterone increases Cav1.2-LNT expression in cardiomyocytes in a time- and dose-dependent manner due to MR-dependent P1-promoter activity, through specific DNA sequence-MR interactions. This cis-regulatory mechanism induced a MR-dependent P1-promoter switch in vascular cells leading to a new Cav1.2-LNT molecular signature with reduced Ca2+ channel blocker sensitivity. These findings uncover Cav1.2-LNT as a specific mineralocorticoid target that might influence the therapeutic outcome of cardiovascular diseases.

Spiral-artery (SA) remodeling is a fundamental process during pregnancy that involves the action of cells of the initial vessel, such as vascular smooth-muscle cells (VSMCs) and endothelial cells, but also maternal immune cells and fetal extravillous trophoblast cells (EVTs). Mast cells (MCs), and specifically chymase-expressing cells, have been identified as key to a sufficient SA remodeling process in vivo. However, the mechanisms are still unclear. The purpose of this study is to evaluate the effects of the MC line HMC-1 and recombinant human chymase (rhuCMA1) on human primary uterine vascular smooth-muscle cells (HUtSMCs), a human trophoblast cell line (HTR8/SV-neo), and human umbilical-vein endothelial cells (HUVEC) in vitro. Both HMC-1 and rhuCMA1 stimulated migration, proliferation, and changed protein expression in HUtSMCs. HMC-1 increased proliferation, migration, and changed gene expression of HTR8/SVneo cells, while rhuCMA treatment led to increased migration and decreased expression of tissue inhibitors of matrix metalloproteinases. Additionally, rhuCMA1 enhanced endothelial-cell-tube formation. Collectively, we identified possible mechanisms by which MCs/rhuCMA1 promote SA remodeling. Our findings are relevant to the understanding of this crucial step in pregnancy and thus of the dysregulated pathways that can lead to pregnancy complications such as fetal growth restriction and preeclampsia.

Atherosclerosis is a macrophage-dominated nonresolving inflammatory disease of the arterial wall. Macrophage processes, including apoptosis, influence lesion development in atherosclerosis. Cannabinoids, compounds structurally related to Δ9-tetrahydrocannabinol (THC), the active ingredient in marijuana, exert their effects through cannabinoid receptors, CB1 and CB2. Cannabinoid treatment, THC or Win55,212-2, reduces atherosclerosis in ApoE-null mice by a mechanism thought to involve CB2. However, the exact role of CB2 in atherosclerosis remains unclear. We found that CB2-null macrophages are resistant to oxysterol/oxLDL-induced apoptosis leading us to hypothesize that CB2 may modulate macrophage apoptosis in atherosclerosis. To determine the functions of CB2 in atherosclerosis, we fed low density lipoprotein receptor-null (Ldlr-/-) and Ldlr-/- mice genetically deficient in CB2, an atherogenic diet for 8 and 12 weeks. CB2 deficiency did not significantly affect aortic root lesion area after 8 or 12 weeks; however, after 12 weeks, CB2-deficient lesions displayed increased lesional macrophage and smooth muscle cell (SMC) content and a ~2-fold reduction in lesional apoptosis. CB2-deficienct lesions also displayed reduced collagen content and elevated elastin fiber fragmentation that was associated with elevated levels of the extracellular matrix degrading enzyme, matrix metalloproteinase 9 (MMP9). These results demonstrate that although CB2 signaling does not affect atherosclerotic lesion size it does modulate lesional apoptosis, cellularity and ECM composition. Ldlr-/- and CB2-deficient Ldlr-/- mice were also subjected to daily treatments with Win55,212-2, a synthetic cannabinoid, over the last 2 weeks of an 8 week atherogenic diet to identify CB2-dependent and CB2-independent effects of cannabinoid receptor stimulation on atherosclerosis. Win55,212-2 did not affect hypercholesterolemia, aortic root lesion area, lesional macrophage infiltration, or ECM composition in either genotype but did significantly reduce total plasma triglyceride levels and lesional SMC content, independent of CB2. Surprisingly, lesional apoptosis was dose-dependently repressed by Win55,212-2 in Ldlr-/- mice by a CB2-dependent mechanism. All together, these results support the suggestion that CB2 may be a target for novel therapies aimed at modulating lesional apoptosis and cellularity to increase lesion stability and reduce the vulnerability to rupture.

Peripheral Artery Disease is a very serious disease characterized by an arterial occlusion due to atherosclerotic plaques. In response to an arterial occlusion, arteriogenesis occurs, causing smooth muscle cells to transition from a contractile to synthetic state. Also following an arterial occlusion, functional impairment was seen in the collateral circuit. An immunofluorescence protocol was developed in order to assess the impact of collateral enlargement (arteriogenesis) on smooth muscle phenotype at various time points. Smooth muscle α-actin was used to mark all smooth muscle cells, Ki-67 was used to label proliferating smooth muscle cells, and a fluorescent nuclear stain was used to quantify the number of cells present. Samples of the profunda femoris and gracilis were dissected from each mouse hind limb (one ligated, one sham) at three different time points: 3 days, 7 days, and 14 days after a femoral artery ligation surgery. Smooth muscle cell phenotype and luminal cross-sectional area were assessed in the profunda femoris and the midzone of the gracilis collaterals. Smooth muscle cells were proliferating at 3 and 7 days following the occlusion in the gracilis collaterals and significant collateral vessel growth was observed over the two week period. No proliferation was observed in the profunda femoris and although there was an increasing trend in vessel size over the two week period, the averages were not significantly different. The phenotypic transition of the smooth muscle cells was not the cause of vascular impairment in the collateral circuit. This shows that further research is needed to characterize impairment in the collateral circuit.

No description available.

No description available.

<p>Atherosclerosis is a disease affecting the blood vessels that is inflammatory in nature, and plays an important role in cardiovascular disease (CVD), one of the leading causes of morbidity and mortality worldwide. Oncostatin M (OSM), a member of the IL-6/gp130 cytokine family, has been implicated in atherosclerosis both in mouse models and in humans. OSM synergizes with other stimuli in various systems to regulate cells. Infectious pathogens as well as danger associated host molecules stimulate members of the innate immune system, including Toll-like Receptors (TLRs), to respond in a pro-inflammatory manner to cause cell activation and cytokine release. Experiments were performed to determine whether OSM and LPS (a TLR-4 ligand) synergize in regulation of vascular wall cells <em>in vitro</em>.</p> <p>Upon stimulation of Aortic Adventitial Fibroblasts from mice (MAAFs) and humans (HAoAFs) as well as Human Aortic Smooth Muscle Cells (HAoSMCs) with LPS in combination with OSM, it was determined that there was a synergistic increase in IL-6 and VEGF levels in the cell supernatants as measured by ELISA compared to either treatment alone. MAAFs were also able to synergistically express KC upon stimulation with LPS and OSM, while in HAoAFs and HAoSMCs, LPS induced IL-8 levels were supressed by OSM. These effects were unique to OSM among gp130 cytokine members, as treatment of these cells with LPS in combination with LIF, IL-6, IL-31, or IL-11 had no marked effects compared to LPS alone. Furthermore, MCP-1 steady state mRNA levels were elevated 6 hours post stimulation with LPS and OSM compared to either treatment alone in HAoAFs and HAoSMCs.</p> <p>While OSM did not appear to modulate TLR-4 expression, OSM treatment resulted in an increased phosphorylation signal in STAT-1,-3, and -5, as well as Akt in MAAFs and HAoAFs. In addition, combined LPS and OSM stimulation resulted in an increased phosphorylation signal of the MAPK p38 compared to either treatment alone. Furthermore, a neutralizing antibody to the OSMr-β was able to inhibit HAoAF IL-6 responses to PBMC conditioned medium. Together, these findings indicate that OSM and LPS can synergize <em>in vitro </em>to induce the expression of inflammatory factors in vascular wall cells, emphasizing the potential role of OSM, TLR-4 ligands, and adventitial fibroblasts in vascular inflammation.</p> / Master of Science (MSc)

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-1&beta;, 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 PKC&alpha;-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