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

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Errata slip inserted. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Errata slip inserted. Includes bibliographical references.

Vascular smooth muscle cells (SMCs) regulate vessel contraction but during diseases including atherosclerosis, SMCs undergo functional changes that contribute to pathology. Chronic inflammation in the vasculature exacerbates disease progression. Acute phase serum amyloid A (SAA) is up-regulated during inflammation and expressed in atherosclerotic lesions. Previous work in our laboratory demonstrated that SAA activates secretory phospholipase A2 group IIA (sPLA2), whose products impact cellular cholesterol homeostasis. It was hypothesized that SAA promotes cholesterol trafficking from the plasma membrane to the endoplasmic reticulum (ER) in an sPLA2-dependent manner. SAA induced SMC cholesterol accumulation in the ER. Levels of plasma membrane cholesterol decreased, confirming that cholesterol moved from the plasma membrane to the ER. Another family member, (cytosolic phospholipase A2, group IV), was also required for SAA-induced sPLA2 activation and cholesterol mobilization. SAA activated neutral sphingomyelinase and blocking this activity inhibited cholesterol trafficking. These studies show that SAA activated sPLA2 which activated neutral sphingomyelinase. As a result, sphingomyelin was cleaved, which liberated cholesterol for movement to the ER. Additional studies demonstrated that SAA repressed expression of SMC contractile markers including Acta2 and Myh11. Toll-like receptor 2 (TLR2) is an SAA receptor implicated in atherogenesis and it was hypothesized that TLR2 plays a role in SAA-mediated phenotype/gene changes. The TLR2 ligands, FSL and Pam3CSK4, down-regulated SMC contractile marker expression. Knockdown of TLR2 demonstrated that SAA-mediated phenotype modulation was TLR2-dependent. SAA, FSL, and Pam3CSK4 also induced mRNA expression of pro-inflammatory and adhesion genes, changes inhibited by TLR2 knockdown. SAA repressed activity of the αSMA promoter, demonstrating transcriptional regulation. Myocardin, a transcription factor required to drive expression of SMC contractile genes, was down-regulated by SAA and FSL. Myocardin overexpression abrogated SAA- and FSL-mediated repression of the αSMA and SM22α promoters. These studies demonstrate that SAA promoted a phenotypic switch through activation of TLR2 and down-regulation of myocardin expression. Taken together, novel SAA- and TLR2-mediated mechanisms of cholesterol trafficking and phenotypic modulation in SMCs are shown. Importantly, this work uncovers previously unknown effects of TLR2 signaling on vascular SMCs and provides a context by which TLR2 activation and lesion-associated SAA may promote atherosclerosis. / 2017-12-01T00:00:00Z

Indiana University-Purdue University Indianapolis (IUPUI) / Under normal conditions, smooth muscle cells do not replicate, or proliferate, and provide a means of contraction for many internal organs, including blood vessels and the gut. However, under abnormal or disease conditions, such as congenital heart disease and cancer, smooth muscle cells acquire the ability to replicate, to make extracellular matrix proteins and to migrate. Thus, determining how smooth muscle cells regulate these processes is crucial to understanding how the cells can switch between normal and diseased states. Serum response factor (SRF) is a widely expressed protein that plays a key role in the regulation of smooth muscle differentiation, proliferation and migration. It is generally accepted that one way that SRF can distinguish between these functions is through pathway-specific co-factor interactions. A novel SRF co-factor, chromodomain helicase DNA binding protein 8 (CHD8), was originally isolated from a yeast two-hybrid assay. CHD8 is widely expressed in adult tissues including smooth muscle. Data from in vitro binding assays indicate that the N-terminus of CHD8 can interact directly with the MADS domain of SRF. Co-immunoprecipitation assays verified the ability of these two proteins to interact within cells. Adenoviral-mediated shRNA knockdown of CHD8 in smooth muscle cells resulted in statistically significant 10-20% attenuation of expression of SRF-dependent, smooth muscle-specific genes. Similar experiments revealed that knockdown of CHD8 did not affect the SRF-dependent induction of immediate early genes required to promote proliferation. In contrast, knockdown of CHD8 in A10 vascular smooth muscle cells resulted in a marked induction in of apoptosis, characterized by increases in apoptotic markers such as phospho-H2A.X, cleaved PARP and activated caspase-3. These data suggest that CHD8 may play a specific role in modulating SRF’s activity toward anti-apoptotic genes, thereby regulating smooth muscle cell survival.

No description available.

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.

Increased bulk of smooth muscle mass around the airways is a typical feature of asthma. Several mediators act in concert or antagonistically to regulate airway smooth muscle (ASM) cell proliferation. This thesis focuses on fibroblast growth factor (FGF)2 and transforming growth factor (TGF)[béta]1 which are known to be sequentially upregulated in the lung following allergic challenge and have recently been shown to synergize together in ASM cell proliferation. Emphasis is put toward the conflicting studies documenting the mitogenic effect of TGF[béta]1 in vitro and to its seemingly potent effect in vivo. Thereafter, different asthma mediators, such as IL-4 and IL-13, are introduced and how their mitogenic potential toward ASM cells could be altered by FGF2 is presented. Finally, how the controversial issue between in vitro and in vivo data regarding the mitogenic effect of leukotrienes could be reconciliated and how it could be related to FGF2 and TGF[béta]1 proliferative synergism is discussed.