Vascular smooth muscle cell (VSMC) accumulation is a hallmark of atherosclerosis and vascular injury. However, fundamental aspects of proliferation and the phenotypic changes within individual VSMCs, which underlie vascular disease remain unresolved. In particular, it is not known if all VSMCs proliferate and display plasticity, or whether individual cells can switch to multiple phenotypes. To assess whether proliferation and plasticity in disease is a general characteristic of VSMCs or a feature of a subset of cells, multi-colour lineage labelling is used to demonstrate that VSMCs in injury-induced neointimal lesions and in atherosclerotic plaques are oligo-clonal, derived from few expanding cells, within mice. Lineage tracing also revealed that the progeny of individual VSMCs contribute to both alpha Smooth muscle actin (aSma)-positive fibrous cap and Mac-3-expressing macrophage-like plaque core cells. Co-staining for phenotypic markers further identified a double-positive aSma+ Mac3+ cell population, which is specific to VSMC-derived plaque cells. In contrast, VSMC-derived cells generating the neointima after vascular injury generally retained expression of VSMC markers and upregulation of Mac3 was less pronounced. Monochromatic regions in atherosclerotic plaques and injury-induced neointima did not contain VSMC-derived cells expressing a different fluorescent reporter protein, suggesting that proliferation-independent VSMC migration does not make a major contribution to VSMC accumulation in vascular disease. Similarly, VSMC proliferation was examined in an Angiotensin II perfusion model of aortic aneurysm in mice, oligo-clonal proliferation was observed in remodelling regions of the vasculature, however phenotypic changes were observed in a large proportion of VSMCs, suggesting that the majority of VSMCs have some potential to modulate their phenotype. To understand the mechanisms behind the inherent VSMC heterogeneity and observed functionality, the single cell transcriptomic techniques Smart-seq2 and the Chromium 10X system were optimized for use on VSMCs. The work within this thesis suggests that extensive proliferation of a low proportion of highly plastic VSMCs results in the observed VSMC accumulation after injury, and the atherosclerotic and aortic aneurysm models of cardiovascular disease.

Rationnel : L’artérite à cellules géantes (ACG) est une vascularite primitive des gros vaisseaux dont le diagnostic repose sur la mise en évidence d’un infiltrat inflammatoire et de cellules géantes à la biopsie d’artère temporale (BAT). On note également un remodelage de la paroi vasculaire lié à une prolifération des cellules musculaires lisses vasculaires (CMLV) pouvant aboutir à une occlusion artérielle. Objectif : Caractériser les auto-anticorps dirigés contre les cellules endothéliales (CE) et les CMLV au cours de l’ACG et préciser le rôle des CMLV dans le remodelage pariétal. Méthodes : La recherche d’auto-anticorps a reposé sur un immunoblot 2D couplé à la spectrométrie de masse. Les protéomes des CMLV d’artère ombilicale, d’artère pulmonaire et d’aorte humaines normales a été comparés par protéomique différentielle (2D-DIGE). Nous avons utilisé la 2D-DIGE et des puces d’expression pan-génomiques pour comparer les CMLV issues de BAT de patients suspects d’ACG (avec un diagnostic final d’ACG ou non), afin d’identifier les mécanismes contribuant à la prolifération des CMLV. Résultats : Chez 15 patients atteints d’ACG, nous avons notamment identifié la lamine, la vinculine et l’annexine A5 comme cible des auto-anticorps anti-CMLV. Les antigènes cibles identifiés sont liés à Grb2, une protéine adaptatrice impliquée dans la prolifération des CMLV. Nous avons mis en évidence des protéomes différents au sein des CMLV humaines normales selon leur origine vasculaire et avons principalement identifié des protéines du cytosquelette et du métabolisme énergétique.A partir des CMLV isolées des BAT et à l’aide d’Ingenuity®, nous avons identifié l’endothéline 1 (ET-1) et la paxilline comme des molécules impliquées dans le remodelage vasculaire. En immunohistochimie et par qPCR, nous avons confirmé l’expression de l’ET-1 et de ses récepteurs ETAR et ETBR au sein des artères temporales de patients atteints d’ACG. Enfin, nous avons inhibé la prolifération des CMLV avec du macitentan, un inhibiteur d’ETAR et en particulier avec son métabolite actif, mais pas avec d’autres inhibiteurs des récepteurs de l’ET-1. Conclusion : Nous avons identifié chez les patients atteints d’ACG des anticorps anti-CMLV dont le rôle pathogéne potentiel reste à définir. Les différences protéiques observées à partir des CMLV humaines normales pourraient correspondre à des phénotypes différents. A partir d’un matériel biologique unique, nous avons pu montrer que la prolifération excessive des CMLV au cours de l’ACG pouvait être inhibée par le macitentan ce qui permet d’envisager un usage thérapeutique de cette molécule. / Background : Giant cell arteritis (GCA) is a large vessel vasculitis and its diagnosis usually relies on the identification of an inflammatory infiltrate made of mononuclear cells and giant cells upon temporal artery biopsy. There is also a remodeling process in the arterial wall due to an excessive proliferation of vascular smooth muscle cells (VSMC) which can sometimes lead to arterial occlusion. Purpose: Identify auto-antibodies targeting either endothelial cells (EC) and/or VSMC during GCA and better understand the role of VSMC in the remodeling process. Methods : Auto-antibodies were detected by a 2-dimensionnal immunoblot and their target antigens were identified by mass spectrometry. Proteoms of umbilical artery, pulmonary artery and aorta VSMC were compared by 2 dimension differential in gel electrophoresis (2D-DIGE). In order to identify mechanisms involved in VSMC proliferation in GCA, we used both 2D-DIGE and pan genomic chips in order to compare VSMC isolated at the time of temporal artery biopsy (TAB) from patients with a final diagnosis of GCA or another diagnosis. Results : In 15 patients with GCA, we identified lamin, vinculin and Annexin A5 as target antigens of anti-VSMC antibodies. Target antigens were linked with Grb2, an adaptator protein involved in VSMC proliferation. Normal VSMC originating from different vascular beds have differ in protein contents with differential expression of cytoskeleton and energy metabolism proteins. We compared VSMC from TAB with Ingenuity software and identified endothelin-1 (ET-1) and paxillin as proteins involved in vessel remodeling. We confirmed by immunohistichemistry and qPCR that ET-1 and its receptor ETAR and ETBR were expressed in temporal arteries from patients with GCA. Last, we reduced VSMC proliferation with Macitentan, an ETAR and ETBR antagonist and significantly inhibited VSMC proliferation with its active metabolite whereas other ET-1 inhibitors had no effect. Conclusion : We identified anti-VSMC auto-antibodies in patients with GCA. Their pathogenic role remains to be determined. Normal VSMC from different vascular locations differ in protein conten which might reflect different phenotypes and different properties. The escessive proliferation of VSMC from patients with GCA was inhibited by Macitentan. This drug might constitute a future therapeutic option.

Une augmentation de la concentration de l’angiotensine II (Ang II) contribue à la prolifération, la migration et l’hypertrophie des cellules musculaires lisses vasculaires (CMLVs) par l’activation des voies des mitogen-activated protein kinases (MAPK) et de la phosphoinositide 3-kinase (PI3K)/protéine kinase B (PKB/Akt). L’Ang II induit l’activation du facteur de transcription early growth response protein-1 (Egr-1) et sa suractivation est remarquée dans les lésions athérosclérotiques et les modèles animaux de lésions vasculaires. La régulation des facteurs de transcription est effectuée par des histones désacétylases (HDACs) qui désacétylent les lysines des histones et protéines non-histones. L’Ang II induit la phosphorylation et l’export nucléaire de la classe IIa des HDACs, particulièrement les HDAC5, et une augmentation de celles-ci est observée dans les maladies vasculaires. L’Ang II est un puissant activateur des voies des MAPK et de la PI3K/Akt, toutefois l’implication de ces voies dans la phosphorylation des HDAC5 et l’expression de l’Egr-1 dans les CMLVs reste inexplorée. Dans cette étude, l’Ang II a induit la phosphorylation des HDAC5 sur la sérine 498 dans les A10 CMLVs. Un blocage pharmacologique de l’extracellular signal-regulated kinase 1/2 (ERK1/2) par U0126 n’a montré aucun effet significatif sur la phosphorylation et l’exclusion nucléaire des HDAC5 induite par l’Ang II. Par contre, l’inhibition de la voie PI3K par wortmannin, de l’Akt par SC66 ou le knockdown de l’Akt par des petits ARN interférents (siRNA) a atténué la phosphorylation et l’export nucléaire des HDAC5 induits par l’Ang II. Par ailleurs, l’inhibition de l’Akt ou le knockdown de cette kinase a diminué l’expression de l’Egr-1 induite dans les CMLVs stimulées par l’Ang II. L’inhibition des HDACs de la classe IIa par MC1568 ou TMP-195 ou bien le knockdown des HDAC5 a diminué l’expression de l’Egr-1 induite par l’Ang II. De plus, le blocage de l’export nucléaire des HDAC5 par la leptomycine B ou la KPT-330 a empêché la localisation cytoplasmique des HDAC5 et a atténué l’expression de l’Egr-1 en réponse à une stimulation de l’Ang II. L’hypertrophie vasculaire induite par l’Ang II a pu être inhibée par la suppression de l’HDAC5 et l’Egr-1. En conclusion, l’Ang II induit la phosphorylation et l’exclusion nucléaire des HDAC5 par la voie PI3K/Akt et non celle de ERK1/2; de plus, l’Ang II induit l’expression de l’Egr-1 à l’aide des HDAC5 via la voie Akt contribuant ainsi à l’hypertrophie des CMLVs. / Elevated concentration of angiotensin II (Ang II) contributes to vascular smooth muscle cells (VSMCs) proliferation, migration and hypertrophy by the activation of the mitogen-activated protein kinases (MAPK) and phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB/Akt) pathways. Ang II induced the expression of early growth response protein-1 (Egr-1), which is a transcription factor that is upregulated in atherosclerosis lesions and in animal models of vascular injuries. The activation or derepression of gene transcription is mediated by histone deacetylases (HDACs), which deacetylate lysine residues from histone and non-histones proteins. Ang II-induced the phosphorylation and nuclear export of class IIa HDACs, notably HDAC5, and its elevated activation is observed in vascular pathologies. Ang II is a potent activator of the MAPK and PI3K/Akt pathways, however their implication in the phosphorylation of HDAC5 and Egr-1 expression in VSMCs remain unexplored. In this study, Ang II-induced HDAC5 phosphorylation at serine 498 in A10 VSMCs and pharmacological blockade of the extracellular signal-regulated kinase 1/2 (ERK1/2) by U0126 did not affect the phosphorylation and nuclear exclusion of HDAC5 in response to Ang II. Whereas, pharmacological inhibition of the PI3K by wortmannin, Akt by SC66 or small interfering RNA (siRNA)-induced silencing of Akt attenuated Ang II-induced HDAC5 phosphorylation and its nuclear export. Furthermore, inhibition or knockdown of Akt suppressed Ang II-induced Egr-1 expression. In addition, the inhibition of class IIa HDAC5 by MC1568, TMP-195 or HDAC5 knockdown by siRNA reduced Ang II-induced Egr-1 expression. The blockade of the nuclear export of HDAC5 by leptomycin B or KPT-330 prevented the cytoplasmic localization of HDAC5 and attenuated the expression of Egr-1 by Ang II in VSMCs. Moreover, HDAC5 or Egr-1 depletion prevented Ang II-induced cell hypertrophy. In summary, Ang II-induced HDAC5 phosphorylation and its nuclear export is mediated by the PI3K/Akt and not the ERK1/2 pathway, in addition, Ang II-induced Egr-1 expression involves the implication of HDAC5 via the Akt pathway which subsequently leads to VSMC hypertrophy.

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.

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.

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