Sphingosine-1-phosphate receptor type 1 (S1PR1), one of the five S1PRs that signals in response to bioactive lysosphingolipid S1P, regulates several fundamental processes in distinct cell types and is implicated in atherosclerosis. Using the cre-lox recombination system, previous studies identified that knocking out S1PR1 in myeloid and endothelial cells promotes plaque development in atherogenic mouse models. In the process of generating S1pr1lox/lox; ApoEKO/KO control mice, we unexpectedly noticed that S1pr1lox/lox mutation alone, in the absence of cre recombinase, reduces high-fat (HF) diet-induced atherosclerosis in S1pr1lox/lox; ApoEKO/KO mice compared to S1pr1WT/WT; ApoEKO/KO mice. Although S1pr1lox/lox allele partially suppressed S1pr1 levels in macrophages and vascular smooth muscle cells (VSMC), the presence of this mutation in a non-BM derived cell type was responsible for this reduced atherosclerosis in S1pr1lox/lox; ApoEKO/KO mice. We speculated that it could be VSMCs due to their abundance in the vascular wall and their role in foam cell formation. In this thesis, we directly tested the effects of inactivating S1PR1 in smooth muscle cells (Tagln-creTG; S1pr1lox/lox; ApoEKO/KO mice) on atherosclerosis. Our results demonstrated that deleting S1PR1 in smooth muscle cells drastically reduces atherosclerosis in apoE-deficient mice. The aortic SMCs isolated from these mice also exhibited reduced cell proliferation and lipid droplet formation in response to S1PR1 agonist SEW2871 compared to S1PR1-WT VSMCs. Furthermore, we also tested the effects of directly inhibiting S1PR1 with S1PR1 selective antagonist Ex26 at a dosage of 0.1 mg/kg/hr in S1pr1WT/WT; ApoEKO/KO mice and Tagln-creTG; S1pr1lox/lox; ApoEKO/KO mice. The prolonged exposure to Ex26 substantially reduced atherosclerotic plaque development in apoE KO mice on an HFD compared to DMSO-treated apoE KO mice. However, this protection was completely lost in mice that lack the S1pr1 gene in VSMCs. Overall, our results suggest that knocking out S1PR1 in VSMCs results in atheroprotection that surpasses the effects of inactivating S1PR1 in macrophages and endothelial cells which are known to promote atherosclerosis. / Dissertation / Doctor of Philosophy (PhD)

MicroRNAs play an important role in biological regulation. Recently miR-34a has been reported to regulate tumour cell cycle progression and apoptosis. However, the functional role of miR-34a in smooth muscle cell (SMC) differentiation from stem cells is yet unclear. Main objectives of this PhD project are to determine the functional role of miR-34a and its target genes in SMC differentiation and underlying mechanisms. Mouse embryonic stem (ES) cells were seeded on collagen coated flasks in differentiation medium to allow SMC differentiation. Upon analysis, miR-34a was significantly up-regulated during SMC differentiation. Results demonstrated that overexpression of miR-34a significantly promoted SMC-specific gene expression, while knockdown of miR-34a inhibited expression of SMC specific gene. Enforced expression and knockdown of miR-34a in differentiating ES cells up-regulated and down-regulated, respectively, several SMC transcription factors in a similar manner. It was also found that miR-34a overexpression in stem cells promoted SMC differentiation in vivo. Furthermore, deacetylase sirtuin 1 (Sirt1) was identified as one of the top targets of miR-34a. Surprisingly, Sirt1 was demonstrated to be positively regulated by miR-34a during SMC differentiation in a cellular context and RNA sequence dependent manner. VIII Mechanistically, the data suggested that miR-34a promoted differentiating stem cells arrest at G0/G1 phase, and a significant decreased incorporation of miR-34a and SirT1 RNA into Ago2-RISC complex was observed upon SMC differentiation. The results demonstrated that Sirt1 acted as a transcriptional activator in the regulation of SMC gene during ES cell differentiation. Finally, H3K9 tri-methylation around the promoter regions of the SMαA and SM22α genes was also found to be significantly inhibited by SirT1 overexpression. These findings suggest that miR-34a plays an important role in SMC differentiation from ES cells. Meanwhile, Sirt1 can be regulated by miR-34a through an unexpected pathway and it was identified as a functional modulating target in miR-34a mediated SMC differentiation.

Pulmonary arterial hypertension (PAH) is a disease in which pulmonary vascular resistance increases. The cell membrane of pulmonary artery smooth muscle cells (PASMC) in PAH patients is depolarised, resulting in disrupted Ca2+ signalling leading to smooth muscle constriction and PASMC proliferation and migration. In rat pulmonary artery (PA) smooth muscle the KV7 K+ channels, encoded by the KCNQ genes, have been proposed to contribute to the resting K+ current, promoting low resting tone by maintaining a negative membrane potential and low intracellular Ca2+. KV7 channel activating drugs have the potential to counteract the dysfunctional signalling during PAH by causing hyperpolarisation. This study set out to determine if the KV7 channels are expressed in human PA and if so whether they can alter vascular tone, PASMC proliferation and/or migration due to their ability to reduce intracellular Ca2+ indirectly. The effects of KV7 K+ channel modulators on human PA tone were measured using myography, while KCNQ gene expression was examined with quantitative PCR. Markers of proliferation (5-bromo-2'-deoxy-uridine (BrdU) and Ki67 antigen), were used to measure PASMC proliferation, while migration was assessed using the scratch-wound assay. Human PASMCs express all KCNQ genes, except KCNQ2. The KV7 channel blockers XE991, linopirdine and (-)chromanol 293B, constricted PAs. The KV7 channel activators retigabine and zinc pyrithione (ZnPy) relaxed PAs pre-constricted with agonists. The retigabine response was enhanced in PAs constricted with Bay K 8644, abolished in ionomycin constricted PAs and reduced in the presence of 90 mM K+, suggesting inhibition of voltage-gated Ca2+ influx. Similar experiments on rat PAs suggest that only part of the ZnPy-induced relaxation can be attributed to KV7 channel activation. The KCNQ5 gene remained in cultured PASMCs while no KV7 channel modulator altered proliferation or migration. Thus KV7.5 channels could possibly be a marker of differentiated PASMCs and/or be involved in the regulation of cell phenotype. The results imply that KV7 channels play a role in regulating PA tone and Ca2+ signalling in PA smooth. It is concluded that although KCNQ5 transcripts are preserved in proliferating PASMC, it is unlikely they play a role in PASMC proliferation or migration. In summary, KV7 channel activators may be useful in the treatment of PAH since they can prevent vasoconstriction.

TTAGGG repeat factor 2 (TRF2) is a protein that plays an important role in capping telomere ends from DNA damage responses. Telomere DNA consists of double strand repeats of the TTAGGG sequence ending with a 3'single-stranded overhang of the guanine strand (the G-strand overhang). TRF2 protects telomeres from being recognized as double-stranded breaks. It is thought that this protection is performed through the formation of T-loop structures and recruitment of proteins into a complex called shelterin. The exact mechanism of T-loop formation is unknown. I show with in vitro biochemical studies that TRF2 specifically interacts with telomeric ss/ds DNA junctions and binding is sensitive to the sequence of the G-strand overhang and double-stranded DNA sequence at the junction. Binding assays with TRF2 truncation mutants suggest that TRF2 interacts with both the double-stranded DNA through the C-terminal DNA binding domain and the G-strand overhang through the N-terminus. Mobility shifts and atomic force microscopy with truncation mutants bound to telomeric DNA also show that a previously uncharacterized "linker" region within TRF2 is involved in DNA-specific TRF2 oligomerization. From these observations, I suggest that TRF2 forms protective loops by oligomerizing through both a previously characterized dimerization domain and the linker region. I propose that loop formation involving the telomere ends is accomplished through direct interactions between TRF2 and the G-strand overhang. In addition to DNA protection, a new role has emerged for TRF2 in sensing DNA damage. TRF2 can be phosphorylated within its dimerization domain by ATM and recruited to DNA damage foci in cells. The inhibition of TRF2 function alone has been shown to induce senescence and apoptosis in vascular endothelial cells. Since the common stimuli for a senescence phenotype is activation of a DNA damage response, I studied the relationship between DNA damage and TRF2 phosphorylation. Ex-vivo characterization of DNA damage-induced changes in vascular smooth muscle cells (VSMC) was undertaken. VSMC treated with H202 induced an increase in reactive oxygen species (ROS), and 8-oxo-guanine accumulation resulting in cell cycle arrest, chromatin condensation and a senescent phenotype. Interestingly phosphorylated TRF2 and ATM were also up regulated. Balloon injury was used to test the connection between phosphorylated TRF2 and senescence during vascular remodeling in rat arteries. Vascular remodeling as judged by neointima formation was associated with accumulation of 8-oxo-guanine, DNA damage signaling, including phosphorylated TRF2, an increase in cell cycle inhibitors and senescence. These events were exaggerated in aged animals and are consistent with a role in telomere dysfunction, and age related diseases.

Chronic oxidative injury by allylamine induces proliferative vascular smooth muscle cell (vSMC) phenotypes in the rat aorta similar to those seen in rodent and human atherosclerotic lesions. In this study, we evaluate the potential role of cyclin dependent kinase inhibitors, p21 and p27, and extracellular regulated kinases (ERK1/2) to mediate the proliferative advantage of oxidatively stressed (i.e. allylamine injured) vSMC. Isolated rat aortic SMC from allylamine treated and control rats were cultured on different extracellular matrix (ECM) proteins. Following mitogen restriction, cultures were stimulated with serum with or without inhibitors of NF-kB or MEK. Western blot analysis was performed to identify protein differences between treatment groups. Basal levels of p21 were 1.6 fold higher in randomly cycling allylamine cells than control counterparts seeded on a plastic substrate, a difference lost when cells were seeded on collagen. p27 levels were comparable in both cell types irrespective of substrate. Basal levels of p21 and p27 were 1.4 fold higher in G0 synchronized allylamine cells compared with G0 synchronized control cells seeded on a plastic substrate. Following cell cycle progression, differences in protein levels were not detected. Treatment with 100 nM pyrollidine dithiocarbamate (PDTC) resulted in significant decreases in p21 and p27 in allylamine cells versus control cells following serum stimulation for 9 hours. This decrease was even greater for p21 in allylamine cells when grown on collagen relative to control cells. Alterations in peak and temporal activation of ERK1/2 were observed in allylamine cells seeded on a plastic substrate as compared to control cells, following serum stimulation. Seeding on collagen decreased the enhanced peak phosphorylation of ERK1/2 and increased the sustained activity in allylamine cells compared with control counterparts. Inhibition of ERK1/2 activity resulted in reduced p21 expression in both cells types, but the response was markedly enhanced in allylamine cells, and preferentially observed on a restrictive collagen substrate. We conclude that induction of proliferative (i.e. atherogenic) phenotypes following repeated cycles of oxidative injury involves ERK1/2 activity and modulation of the cyclin dependent kinase inhibitors, p21 and p27, in a matrix-dependent manner.

NF-κB activity is critical in the regulation of atherosclerotic vascular smooth muscle cell (vSMC) phenotypes induced following oxidative injury by allylamine. The present studies were designed to detail dysregulation of NF-κB activity in these altered phenotypes, and to assess the importance of NF-κB in the regulation of osteopontin, a cytokine which modulates atherosclerosis. Increased degradation of IκBα was observed in allylamine-induced atherosclerotic vSMC phenotypes (henceforth referred to as allylamine cells). Enhanced phosphorylation of I-κ-kinases was observed by Western immunoblotting. NF-κB DNA binding activity as assessed by electrophoretic mobility shift assay demonstrated changes in the kinetics and magnitude of induction of binding. Enhancement of NF-κB binding activity was evident in allylamine cells compared to controls when seeded on plastic, fibronectin, and laminin, but not collagen I. Posttranscriptional alterations in Rel protein expression and nuclear localization partly account for changes in NF-κB DNA binding activity. Promoter-specific NF-κB binding profiles suggest altered dimer prevalence as a consequence of the changes in Rel protein expression. The expression of NF-κB regulated genes osteopontin and MMP-2 was enhanced in allylamine-treated aortas, while cyclin D1 and MMP-9 were unchanged. As the importance of osteopontin in atherosclerosis has been described in several models, subsequent studies were designed to assess osteopontin promoter activity. Activity of the osteopontin promoter was significantly reduced in allylamine cells compared to controls as assessed using a luciferase reporter. Deletion analysis suggested the presence of inhibitory cis-acting elements in the regulatory region of the gene. Mutation of these elements, including VDRE, AP-1, NF-κB, and USF1, indicated that NF-κB and USF1 mediate suppression of osteopontin promoter activity in allylamine cells. Decreased serine phosphorylation of immunoprecipitated RelA/p65 was observed in allylamine cells, indicating decreased ability of this protein to transactive gene promoters. NF-κB was found to play a role in suppression of osteopontin promoter activity by collagen I-mediated integrin signaling. These findings suggest that enhancements in NF-κB activity suppress osteopontin promoter activity in oxidant-activated vSMC cultures. Dysregulation of NF-κB activity occurs as a result of altered matrix and intracellular signaling upstream of the nucleus and possibly differential dimer assembly leading to cell-specific profiles of NF-κB-dependent gene regulation.

The research presented here deals with synthetic materials for application in tissue engineering, primarily poly(ethylene glycol) (PEG) and poly(dimethyl siloxane)star (PDMS)star. Tissue engineering seeks to repair or replace damaged tissue through implantation of cell encapsulated in an artificial scaffold. Cell differentiation and extracellular matrix (ECM) deposition can be influenced through a wide variety of in vitro culture techniques including biochemical stimuli, cell-cell interactions, mechanical conditioning and scaffold physical properties. In order to systematically optimize in vitro conditions for tissue engineering experiments, the individual effects of these different components must be studied. PEG hydrogels are a suitable scaffold for this because of their biocompatibility and biological "blank slate" nature. This dissertation presents data investigating: the effects of glycosaminoglycans (GAGs) as biochemical stimuli on pig vocal fold fibroblasts (PVFfs); the effects of mechanical conditioning and cell-cell interactions on smooth muscle cells (SMCs); and the effects of scaffold physical properties on SMCs. Results show that GAGs influence PVFf behavior and are an important component in scaffold design. Hyaluronic acid (HA) formulations showed similar production in collagen I and III as well as reduced levels of smooth muscle a-actin (SMa-actin), while chondroitin sulfate (CSC) and heparin sulfate showed enriched collagen III environments with enhanced expression of SMa-actin. A physiological flow system was developed to give comprehensive control over in vitro mechanical conditioning on TEVGs. Experiments performed on SMCs involved creating multi-layered TEVGs to mimic natural vascular tissue. Constructs subjected to mechanical conditioning with an endothelial cell (EC) layer showed enhanced expression of SMC differentiation markers calponin h1 and myocardin and enhanced deposition of elastin. Consistent with other studies, EC presence diminished overall collagen production and collagen I, specifically. Novel PDMSstar-PEG hydrogels were studied to investigate the effects of inorganic content on mesenchymal stem cell differentiation for use in TEVGs. Results agree with previous observations showing that a ratio of 5:95 PDMSstar: PEG by weight enhances SMC differentiation markers; however, statistically significant conclusions could not be made. By studying and optimizing in vitro culture conditions including scaffold properties, mechanical conditioning and multi-layered cell-cell interactions, TEVGs can be designed to maximize SMC differentiation and ECM production.

Chronic oxidative injury by allylamine induces proliferative vascular smooth muscle cell (vSMC) phenotypes in the rat aorta similar to those seen in rodent and human atherosclerotic lesions. In this study, we evaluate the potential role of cyclin dependent kinase inhibitors, p21 and p27, and extracellular regulated kinases (ERK1/2) to mediate the proliferative advantage of oxidatively stressed (i.e. allylamine injured) vSMC. Isolated rat aortic SMC from allylamine treated and control rats were cultured on different extracellular matrix (ECM) proteins. Following mitogen restriction, cultures were stimulated with serum with or without inhibitors of NF-kB or MEK. Western blot analysis was performed to identify protein differences between treatment groups. Basal levels of p21 were 1.6 fold higher in randomly cycling allylamine cells than control counterparts seeded on a plastic substrate, a difference lost when cells were seeded on collagen. p27 levels were comparable in both cell types irrespective of substrate. Basal levels of p21 and p27 were 1.4 fold higher in G0 synchronized allylamine cells compared with G0 synchronized control cells seeded on a plastic substrate. Following cell cycle progression, differences in protein levels were not detected. Treatment with 100 nM pyrollidine dithiocarbamate (PDTC) resulted in significant decreases in p21 and p27 in allylamine cells versus control cells following serum stimulation for 9 hours. This decrease was even greater for p21 in allylamine cells when grown on collagen relative to control cells. Alterations in peak and temporal activation of ERK1/2 were observed in allylamine cells seeded on a plastic substrate as compared to control cells, following serum stimulation. Seeding on collagen decreased the enhanced peak phosphorylation of ERK1/2 and increased the sustained activity in allylamine cells compared with control counterparts. Inhibition of ERK1/2 activity resulted in reduced p21 expression in both cells types, but the response was markedly enhanced in allylamine cells, and preferentially observed on a restrictive collagen substrate. We conclude that induction of proliferative (i.e. atherogenic) phenotypes following repeated cycles of oxidative injury involves ERK1/2 activity and modulation of the cyclin dependent kinase inhibitors, p21 and p27, in a matrix-dependent manner.

NF-κB activity is critical in the regulation of atherosclerotic vascular smooth muscle cell (vSMC) phenotypes induced following oxidative injury by allylamine. The present studies were designed to detail dysregulation of NF-κB activity in these altered phenotypes, and to assess the importance of NF-κB in the regulation of osteopontin, a cytokine which modulates atherosclerosis. Increased degradation of IκBα was observed in allylamine-induced atherosclerotic vSMC phenotypes (henceforth referred to as allylamine cells). Enhanced phosphorylation of I-κ-kinases was observed by Western immunoblotting. NF-κB DNA binding activity as assessed by electrophoretic mobility shift assay demonstrated changes in the kinetics and magnitude of induction of binding. Enhancement of NF-κB binding activity was evident in allylamine cells compared to controls when seeded on plastic, fibronectin, and laminin, but not collagen I. Posttranscriptional alterations in Rel protein expression and nuclear localization partly account for changes in NF-κB DNA binding activity. Promoter-specific NF-κB binding profiles suggest altered dimer prevalence as a consequence of the changes in Rel protein expression. The expression of NF-κB regulated genes osteopontin and MMP-2 was enhanced in allylamine-treated aortas, while cyclin D1 and MMP-9 were unchanged. As the importance of osteopontin in atherosclerosis has been described in several models, subsequent studies were designed to assess osteopontin promoter activity. Activity of the osteopontin promoter was significantly reduced in allylamine cells compared to controls as assessed using a luciferase reporter. Deletion analysis suggested the presence of inhibitory cis-acting elements in the regulatory region of the gene. Mutation of these elements, including VDRE, AP-1, NF-κB, and USF1, indicated that NF-κB and USF1 mediate suppression of osteopontin promoter activity in allylamine cells. Decreased serine phosphorylation of immunoprecipitated RelA/p65 was observed in allylamine cells, indicating decreased ability of this protein to transactive gene promoters. NF-κB was found to play a role in suppression of osteopontin promoter activity by collagen I-mediated integrin signaling. These findings suggest that enhancements in NF-κB activity suppress osteopontin promoter activity in oxidant-activated vSMC cultures. Dysregulation of NF-κB activity occurs as a result of altered matrix and intracellular signaling upstream of the nucleus and possibly differential dimer assembly leading to cell-specific profiles of NF-κB-dependent gene regulation.

An important part of repair processes activated by vascular injury is the recruitment of vascular smooth muscle cells (SMC) from the existing contractile coat. Phenotypic modulation of SMCs enables these cells to proliferate and migrate into the vessel intima. Despite its importance in vessel repair, this plasticity of SMCs can also promote both the pathogenesis of atherosclerosis as well as neointimal formation following revascularization- induced injury. Vascular growth factors are major contributors to the migratory and proliferative responses to injury. IGF-1 is one such growth factor that elicits a response via its receptor, the IGF-1R, a classical tyrosine kinase receptor. However, it has been suggested that the IGF-1R may also be coupled to a heterotrimeric G protein and can thus initiate cellular responses via this alternate pathway. The objective of this study was to investigate the structural aspects of IGR-1R coupling to a heterotrimeric G protein in SMCs, as well as the contribution of this pathway to the cellular responses. In a porcine primary SMC culture model, IGF-1R co-precipitated with both the α- and β-subunits of a G protein, with the latter demonstrating activation dependent precipitation. The specific Gα class activated by IGF-1R was Gαi, in a manner that was independent of the activity of the tyrosine kinase. Both Gαi1 and Gαi2 directly interacted with the receptor. Gβγ mediated the activation of MAPK and its inhibition was sufficient to attenuate both the proliferation and migration of SMCs in vitro. In contrast, the contribution of Gαi was related to regulation of protein translation and histone modification. The data supports the conclusion that IGF-1 regulates the phenotype of vascular SMCs at least partially via a non-classical G protein-coupled receptor. Investigation into the individual subunits of the G protein complex led to the elucidation of a model in which both components play an integral role in the IGF-1 response, independent of the receptor tyrosine kinase activity. In one case, an interplay of specific Gαi-subunits leads to modulation of the VSMC translational and transcriptional responses, while in the other, release of the Gβγ-subunit activated the MAPK response in a manner that significantly contributes to both the migration and proliferation of SMCs.