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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)

Yes / MicroRNAs are short, non-coding RNAs that target messenger RNAs for degradation. miR-145 is a vascular-enriched microRNA that is important for smooth muscle cell (SMC) differentiation. Under healthy circumstances, SMC exist in a contractile, differentiated phenotype promoted by miR-145. In cases of disease or injury, SMC can undergo reversible dedifferentiation into a synthetic phenotype, accompanied by inhibition of miR-145 expression. Vascular disorders such as atherosclerosis and neointimal hyperplasia are characterised by aberrant phenotypic switching in SMC. This review will summarise the physiological roles of miR-145 in vascular SMC, including the molecular regulation of differentiation, proliferation and migration. Furthermore, it will discuss the different ways in which miR-145 can be dysregulated and the downstream impact this has on the progression of vascular pathologies. Finally, it will discuss whether miR-145 may be suitable for use as a biomarker of vascular disease.

Widespread changes in gene expression accompany vascular smooth muscle cell (VSMC) phenotypic switching, a hallmark of vascular disease. Upon insult, VSMCs downregulate contractile proteins and upregulate genes linked to vascular remodelling, such as matrix metalloproteinases (MMPs) and pro-inflammatory cytokines. However, the epigenetic mechanisms which regulate VSMC phenotypic switching remain unclear. This thesis explores the role of histone 3 lysine 9 dimethylation (H3K9me2), a repressive epigenetic mark, in regulating the expression of disease-associated VSMC genes. Intriguingly, murine models of VSMC phenotypic switching revealed reduced levels of H3K9me2 upon loss of the contractile state while chromatin immunoprecipitation (ChIP) identified a subset of IL-1α/injury-responsive VSMC gene promoters enriched for H3K9me2. To test the functional importance of H3K9me2 for VSMC gene regulation the methyltransferase G9A/GLP was pharmacologically inhibited in vitro and in vivo. The resulting loss of H3K9me2 attenuated the expression of contractile VSMC markers and significantly potentiated IL-1α/injury-induced expression of MMP and pro-inflammatory genes. H3K9me2-mediated regulation of contractile and IL-1α-responsive VSMC gene expression was confirmed in cultured human VSMCs (hVSMCs). This prompted the use of hVSMCs to investigate the mechanism underlying H3K9me2-dependent regulation of IL-1α-mediated VSMC genes. Interestingly, G9A/GLP inhibition did not influence the level of IL-1α-induced nuclear localisation of the NFkB transcription factor p65 but significantly increased IL-1α-induced p65 binding to the IL6 promoter, correlating with reduced H3K9me2 levels. In contrast, enrichment of p65 was not observed at reported NFkB sites within the MMP3 promoter after IL-1α stimulation. Rather, IL-1α-induced MMP3 expression was dependent on JNK activity and G9A/GLP inhibition potentiated IL-1α-induced binding of the AP-1 transcription factor cJUN to the MMP3 promoter. Collectively, these findings suggest that H3K9me2 plays a role in maintaining the contractile VSMC state and prevents binding of both NFkB and AP-1 transcription factors at specific IL-1α-regulated genes to possibly block spurious induction of a pro-inflammatory state.

published_or_final_version / abstract / Pharmacology / Doctoral / Doctor of Philosophy

Determination of the physiologic roles of serotonin (5-HT) has long been hampered by the lack of compounds specific for certain of the 5-HT receptor subtypes. The objective of this dissertation was to characterize vascular serotonin receptors in certain arteries and to develop functional assays for the putative 5-HT₁(A) and 5-HT₂ receptors in vascular tissue to test novel compounds. Although 5-HT₁(A) receptor involvement in the 5-HT contraction of the canine basilar artery was previously reported, the 8-OH-DPAT (5-HT₁(A) specific agonist) EC₅₀ values in the canine, rabbit, guinea pig, and bovine basilar arteries studies were not consistent with the presence of 5-HT₁(A) receptors. Studies examining the 5-HT₂ selective antagonist ketanserin, several novel aryltryptamines with a range of affinities, and enantiomers of spiroxatrine, in the 5-HT-contracted rat aorta showed a good correlation between the aorta affinities and the affinities of these compounds at the [³H] ketanserin binding site (defined as 5-HT₂) in the rat frontal cortex. Comparison of the affinities of several known and novel compounds in the rat aorta and the rabbit femoral artery to the [³H] ketanserin site affinities in the frontal cortices of both species showed that the rabbit femoral artery 5-HT₂-like receptor was similar but not identical to either the rat aorta or the CNS sites from either species. The rabbit aorta and the rat femoral artery were then examined to determine if the 5-HT₂ receptor heterogeneity was species or vascular bed specific. The results from all four vascular tissues showed that no two tissues had identical responses to the compounds studied. The rat aorta appeared unique in the lack of agonist activity of RU24969 and the non-competitive antagonism of 5-HT by methysergide, but correlated to the CNS site for the affinities of all compounds. The major finding of the dissertation was the definitive evidence for vascular 5-HT₂ receptor heterogeneity; this subtype was previously thought to be homogeneous. Development of more selective compounds for 5-HT receptor subtypes may lead to greater understanding of the physiological roles of serotonin.

Endothelial cells (ECs) and smooth muscle cell (SMC) play a key part during development of fibrosis in the intima being partly responsible for synthesis of matrix metalloproteinase (MMPs) and various regulators and substrates of these enzymes. Omega-3 (n-3) polyunsaturated fatty acids (PUFA) consumption, mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), has beneficial effects on atherosclerosis but its effect on the development of fibrosis is relatively unknown. <i>Objective:</i> Determine the effects of EPA and DHA, alone or in combination, on fibrosis-related factors in aortic SMCs (AoSMCs) and human umbilical vein ECs (HUVECs) and human aortic ECs (HAECs). <i>Results:</i> Treatment of cells with/without 10 μM DHA, EPA, oleic acid (OA) or vehicle control (VC) altered expression of MMPs, regulators and substrates of MMPs and inflammatory cytokines. EPA increased the α-actin:β-actin ratio indicative of a more contractile SMC phenotype and gelatinase (MMP-2 and -9) activity in HUVECs. In aortic cells, EPA and DHA decreased uPAR mRNA and protein expressions. DHA, EPA and DHA: EPA (at 3:1 and 1:1) decreased SMC migration, this did not involve uPA/plasmin activity. <i>Conclusion:</i> EPA and DHA could decrease inflammatory cytokines and the fibrogenic environment in atherosclerotic lesions by decreasing MMP expression and activity. These fatty acids may also reduce SMC migration and proliferation, independently of uPA/plasmin activity, potentially reducing SMC build up in the intima. This could possibly prevent and/or show plaque progression and increase the stability of advanced plaques.

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.