Thesis (Ph. D.)--Marshall University, 2002. / Title from document title page. Document formatted into pages; contains ix, 128 p. Includes abstract. Bibliographical references are at the end of each chapter.

Thesis (Ph. D.)--Marshall University, 2002. / Title from document title page. Document formatted into pages; contains xi, 136 p. with illustrations. Includes abstract. Includes bibliographical references (p. 120-136).

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FOXO3a is a pro-apoptotic transcription factor which shows increased activation in vascular smooth muscle cells (VSMCs) of advanced atherosclerotic plaques, specifically within the intimal layer. Since VSMC apoptosis plays a crucial role in the pathophysiology of atherosclerosis, we investigated the mechanisms underlying FOXO3a-mediated cell death in this particular cell type. We aimed to characterise a novel VSMC system (FOXO3aA3ERTM) and use these cells to validate MMP-13 and TIMP3 as new FOXO3a target genes. Also, we sought to determine the mechanisms of FOXO3aA3ERTM-mediated VSMC apoptosis, particularly regarding MMP-13 and TIMP3, potential MMP-13 substrates in the extracellular matrix and the precise apoptotic signalling involved. Furthermore, we aimed to investigate whether VSMC-specific activation of FOXO3aA3ERTM in mouse affects vascular remodelling during injury and whether this is reliant on MMP-13. Lastly, we aimed to address if endogenous FOXO3a upregulates MMP-13 in mouse and human VSMCs. Our laboratory has created a transgenic rat VSMC line which stably expresses an inducible FOXO3a mutant allele known as FOXO3aA3ERTM and previous microarray experiments identified matrix metalloproteinase 13 (MMP-13) as a potential novel FOXO3a target gene. Initially, we described several key features of the FOXO3aA3ERTM VSMCs used throughout this thesis, and subsequently demonstrated that MMP-13 is a bona fide target whose expression is rapidly upregulated upon FOXO3a activation, leading to markedly higher levels of protein, cleavage and proteolytic capacity. This induction of MMP-13 was responsible for the vast majority of FOXO3a-mediated apoptosis which was accompanied by prominent degradation of fibronectin, a glycoprotein found in the extracellular matrix. However, we could not identify a terminal apoptotic pathway. FOXO3a also downregulated the endogenous MMP inhibitor TIMP3, the recombinant protein of which reduced both MMP-13 proteolysis and FOXO3a-mediated apoptosis. Activation of FOXO3aA3ERTM in the VSMCs of medium and large arteries in mice resulted in heightened expression of MMP-13 in the vessel wall, which contributed to enhanced neointimal formation during carotid ligation. Finally, endogenous FOXO3a activation leads to increased MMP-13 expression in human VSMCs, but not mouse. Overall, we have shown that FOXO3a promotes VSMC apoptosis through MMP-13 both in vitro and in vivo, a novel pathway that has important implications for the pathogenesis and treatment of vascular disease.

Atherosclerosis is the leading cause of death in the Western world. Although mitochondrial DNA (mtDNA) damage has been implicated in atherosclerosis, it is unclear whether the damage is sufficient to impair mitochondrial respiration, and mitochondrial dysfunction has not been demonstrated. Treatment of vascular smooth muscle cells (VSMCs) with an atherogenic lipid, oxidised low-density lipoprotein (OxLDL), dose dependently decreased basal and maximal respiration and fat-feeding of apolipoprotein E deficient (ApoE-/-) mice reduced mitochondrial DNA copy number relative to nuclear DNA in aortas. Mitochondrial respiration of ApoE-/- mouse aortas, assessed through a 24-well Seahorse extracellular flux analyser, was not affected prior to the development of atherosclerotic plaques. Developed human carotid atherosclerotic plaques were dissected into defined regions including healthy media, shoulder region, fibrous cap and core and their respiration was investigated. The respiratory reserve capacity (RRC) of the shoulder region was similar to the media. However, the cap RRC was significantly reduced compared to healthy media. In contrast, the extracellular acidification rates (ECAR) of the media, shoulder, cap and core regions were similar. In addition, mtDNA copy number was significantly reduced in tissues derived from human plaques compared to healthy arteries and expression of complexes I and II of the electron transfer chain (ETC) were significantly reduced in plaque VSMCs. OxLDL induced mitophagy in human VSMCs and plaque VSMCs demonstrated increased levels of mitophagy without compensatory upregulation of proteins involved in mitochondrial biogenesis. Understanding the role of mitochondrial metabolism and signalling is important for our understanding of disease progression and may lead to future therapeutic targets.

Aberrant phenotypes of airway smooth muscle cells are central to the pathophysiology of asthma. The hypercontractile nature of these cells and hypertrophy are the key reasons for the excessive narrowing of the airways observed in allergic asthma. Although previous studies have indicated a role of enhanced content of smMLCK in modulating the contractile reactivity, as well as an indication of hypertrophy of HASM cells in asthmatic conditions, the effect of IgE on the expression of smMLCK in HASM cells is not fully understood. In this study, we demonstrate that IgE augments the expression of smMLCK at the mRNA and protein level. Inhibition of IgE binding with anti-FcεRI blocking antibody, Syk silencing, pharmacological inhibitors to MAPK (ERK1/2, p38, and JNK) and PI3K significantly diminished the IgE-mediated smMLCK expression in HASM cells. Finally, we found that IgE, similar to metacholine induces the contraction of HASM cells grown on collagen gel matrix. Our data suggest that IgE stimulates the phosphorylation of ERK, P38, STAT3 and induces the dephosphorylation of smMLCK to phosphorylate myosin regulatory light chain in HASM cells. Taken together, our data suggest a modulatory role of IgE in regulating the contractile machinery and hypertrophic phenotype of HASM cells.

Aberrant phenotypes of airway smooth muscle cells are central to the pathophysiology of asthma. The hypercontractile nature of these cells and hypertrophy are the key reasons for the excessive narrowing of the airways observed in allergic asthma. Although previous studies have indicated a role of enhanced content of smMLCK in modulating the contractile reactivity, as well as an indication of hypertrophy of HASM cells in asthmatic conditions, the effect of IgE on the expression of smMLCK in HASM cells is not fully understood. In this study, we demonstrate that IgE augments the expression of smMLCK at the mRNA and protein level. Inhibition of IgE binding with anti-FcεRI blocking antibody, Syk silencing, pharmacological inhibitors to MAPK (ERK1/2, p38, and JNK) and PI3K significantly diminished the IgE-mediated smMLCK expression in HASM cells. Finally, we found that IgE, similar to metacholine induces the contraction of HASM cells grown on collagen gel matrix. Our data suggest that IgE stimulates the phosphorylation of ERK, P38, STAT3 and induces the dephosphorylation of smMLCK to phosphorylate myosin regulatory light chain in HASM cells. Taken together, our data suggest a modulatory role of IgE in regulating the contractile machinery and hypertrophic phenotype of HASM cells.

Obesity is a major risk factor for insulin resistance, type 2 diabetes, cardiovascular disease (CVD), and vascular calcification. Vascular calcification is correlated with advanced CVD and a significant predictor of cardiovascular events. Obese individuals tend to have increased levels of circulating leptin, an adipocytokine that is a significant independent predictor of cardiovascular disease. We have shown that daily intraperitoneal injections of exogenous leptin (125 μg/mouse/d) can promote vascular calcification in an ApoE-/- mouse model of atherosclerosis. This increase in calcification is associated with an increase in the expression of several osteoblast-specific markers and is independent of any affect on atherosclerotic lesion size. Our studies suggest that leptin mediates the osteogenic differentiation of vascular smooth muscle cells (VSMCs) to promote vascular calcification via a pathway involving the inhibition of glycogen synthase kinase (GSK)-3 activity. Other studies have suggested that endoplasmic reticulum (ER) stress-induced GSK-3 activity promotes the development of atherosclerosis. Therefore, we hypothesized that during the progression of vascular disease, GSK-3 functions as a checkpoint for VSMCs at which cells can commit to: i) de-differentiation, thereby contributing to atherosclerosis, or ii) osteogenic differentiation, thereby contributing to vascular calcification. We investigated the effects of modulating GSK-3 activity on the differentiation of VSMCs in vitro. We found that many of the molecular tools that are typically used to modulate ER stress can promote the expression of osteoblast-specific markers and the osteogenic differentiation of MOVAS cells. However, because many of these interventions affect multiple pathways in MOVAS cells, the specific role of the ER stress – GSK-3 pathway is difficult to discern. Future studies are required to determine the effects of direct modulation of GSK-3 on vascular calcification and to delineate the mechanisms/effects of various ER stressors in the osteogenic differentiation of VSMCs. / Thesis / Doctor of Philosophy (Medical Science)