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.

Previous investigations on pulmonary artery smooth muscle cells have shown that nicotinic acid adenine dinucleotide diphosphate (NAADP) evokes highly localised intracellular Ca²⁺ bursts by mobilising thapsigargin-insensitive Ca²⁺ stores. Such localised Ca²⁺ signals may initiate global Ca²⁺ waves and contraction of the myocytes through the recruitment of ryanodine receptors (RyR) located on the sarcoplasmic reticulum (SR) via Ca²⁺-induced Ca²⁺-release (CICR). In this thesis I have shown that NAADP evokes localised Ca²⁺ signals through the mobilisation of a bafilomycin A1-sensitive, lysosome-related Ca²⁺ store. Lysosomal Ca²⁺ stores facilitate this process by colocalising with a subpopulation of RyRs on the surface of the SR to comprise a highly specialised trigger zone for Ca²⁺ signalling by NAADP. I have also shown that the proposed trigger zone for NAADP-dependent Ca²⁺ signalling may be formed between lysosomes and clusters of RyR subtype 3 (RyR3) located in close proximity to one another in the perinuclear region of cells. Localised Ca²⁺ bursts generated by NAADP-dependent Ca²⁺ release from acidic Ca²⁺ stores and subsequent CICR via RyR3 on the SR may then amplify Ca²⁺ bursts into a propagating Ca²⁺ signal by recruiting clusters of RyR subtype 2 (RyR2) located in the perinuclear and extra-perinuclear regions of the cell. The presence of this trigger zone may explain, in part, why Ca²⁺ bursts by NAADP induce, in an all-or-none manner, global Ca²⁺ signals by CICR via RyRs on the SR. Consistent with a role for NAADP and lysosomes as a discrete and agonist-specific Ca²⁺ signalling pathway utilised by vasoconstrictors, I have shown that endothelin-1 (ET-1), but not phenylephrine or prostaglandin-F2α, mobilises Ca²⁺ stores by NAADP, and that ET-1 initiates Ca²⁺ signalling by NAADP in a receptor subtype-specific manner through the activation of ETB receptors. These findings further advance our understanding of how that spatial organisation of discrete, organellar Ca²⁺ stores underpin the generation of differential Ca²⁺ signalling patterns by different Ca²⁺-mobilising messengers.

published_or_final_version / abstract / Pharmacology / Doctoral / Doctor of Philosophy

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.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on January 5, 2010). Thesis advisor: William P. Fay. Includes bibliographical references

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 6 uppsatser.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.