published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Cardiovascular diseases are a major cause of death worldwide. Aneurysmal rupture in cerebral arteries or loss of endothelial integrity in the course of atherosclerosis or therapeutic angioplasty lead to exposure of vascular smooth muscle cells (SMC) to blood components such as sphingolipids. Sphingosylphosphorylcholine (SPC) and sphingosine 1-phosphate (S1P) are two naturally occurring sphingolipids, which are vasoprotective in the healthy endothelium-lined vessel, but may promote vascular disease by causing functional changes of SMC. Vascular inflammation is an important factor in various pathologies. SPC can activate pro-inflammatory signalling pathways in rat cerebral artery. Here these observations are extended by showing that SPC elicits monocyte chemoattractant protein-1 production in rat cerebral artery SMC ex vivo. Thus, in addition to being a vasoconstrictor, SPC may promote the development of life-threatening prolonged cerebral vasospasm following subarachnoid haemorrhage by supporting vascular inflammation. It is also demonstrated that SPC prevents tumour necrosis factor-a (TNF)-stimulated adhesion of macrophages to rat aortic SMC in vitro by interfering with adhesive properties of SMC, but not macrophages. While this effect appeared to be mediated by the S1P receptor S1P2, S1P itself did not reduce macrophage adhesion. The anti-adhesive action of SPC also depended on lipid rafts. However, SPC did neither prevent TNF-induced nuclear factor kB activation nor cell adhesion molecule expression in SMC. SPC-induced cyclooxygenase 2 expression in aortic SMC was dispensable for its anti-adhesive effect. In contrast, the inhibitory effect of SPC on TNFinduced expression of inducible nitric oxide synthase is probably involved in its anti-adhesive effect because it was mimicked by respective pharmacological blockade. The results also demonstrate that nitric oxide promotes leukocyte adhesion to vascular SMC, while it has the opposite effect on endothelial cells. These findings may help understand cardiovascular diseases and define novel treatment approaches.

No description available.

Subarachnoid hemorrhage (SAH) following cerebral aneurysm rupture is associated with substantial morbidity and mortality. The ability of SAH to induce vasospasm in large diameter pial arteries has been extensively studied, although the contribution of this phenomenon to patient outcome is unclear. Conversely, little is known regarding the impact of SAH on intracerebral (parenchymal) arterioles, which are critical for regulation of cerebral blood flow. To assess the function of parenchymal arterioles following SAH, measurements of diameter, intracellular Ca2+ ([Ca2+]i) and membrane potential were performed in intact arterioles from unoperated (control), sham-operated and SAH model rats. At physiological intravascular pressure, parenchymal arterioles from SAH animals exhibited significantly elevated [Ca2+]i and enhanced constriction compared with arterioles from control and sham-operated animals. Elevated [Ca2+]i and enhanced tone following SAH were observed in the absence of vascular endothelium and were abolished by the L-type voltage-dependent Ca2+ channel (VDCC) inhibitor nimodipine. Molecular assessment of the L-type VDCC CaV1.2 indicated unchanged mRNA and protein expression in arterioles from SAH animals. Increased CaV1.2 activity following SAH may also reflect enhanced pressure-induced membrane potential depolarization of arteriolar smooth muscle. Membrane potential measurements in arteriolar myocytes using intracellular microelectrodes revealed approximately 7 mV depolarization at 40 mmHg in myocytes from SAH animals. Further, when membrane potential was adjusted to similar values, arteriolar [Ca2+]i and tone were similar between groups. These results demonstrate that greater pressure-dependent membrane potential depolarization results in increased activity of CaV1.2 channels, elevated [Ca2+]i and enhanced constriction of parenchymal arterioles from SAH animals. Thus, impaired regulation of parenchymal arteriolar [Ca2+]i and diameter may restrict cerebral blood flow in SAH patients. Although nimodipine is used clinically to prevent delayed neurological deficits in SAH patients, the use of this drug has been limited by hypotension and treatment options remain inadequate. Therefore, our next objective was to explore strategies to selectively suppress CaV1.2 channels in the cerebral vasculature. To do so, we examined the physiological role of smooth muscle CaV1.2 splice variants containing the alternatively-spliced exon 9* in cerebral artery constriction. Using antisense oligonucleotides, we demonstrate that suppression of exon 9*-containing CaV1.2 splice variants results in substantially reduced cerebral artery constriction to elevated extracellular [K+]. In addition, no further reduction in constriction was observed following suppression of all Cav1.2 splice variants, suggesting that exon 9* splice variants are functionally dominant in cerebral artery constriction. In summary, results shown in this dissertation demonstrate that increased CaV1.2 activity following SAH results in enhanced constriction of parenchymal arterioles. Furthermore, evidence is provided supporting the concept that CaV1.2 splice variants with exon 9* are critical for cerebral artery constriction and may provide a novel target for the prevention of delayed ischemic deficits in SAH patients.

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.

No description available.

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.

Neointimal proliferation is a key process underlying many cardiovascular diseases such as atherosclerosis and angioplasty-induced restenosis. Vascular smooth muscle cells (SMC) are significant contributors to the development and stability of the neointimal lesion. This is due, in part, to their capacity to be phenotypically modulated, facilitating SMC proliferation in response to mechanical injury, their subsequent migration, and deposition of extracellular matrix. The aim of this thesis was to characterize the function of two genes identified in our laboratory to be upregulated shortly after mechanical injury of vascular SMC and their exposure to fibroblast growth factor (FGF)-2, an injury-induced cytokine. The first is activation transcription factor (ATF) 4, which is upregulated by FGF-2 and mechanical injury in vascular SMC in vitro, and by balloon-injury in the artery wall. The induction of ATF4 by FGF-2 was shown to be mediated through the PI3K pathway, and preceded by phoshorylation of eIF2alpha, a known upstream effector of ATF4 activation. Knock-down of ATF4 expression inhibited balloon-injury induced neointimal hyperplasia, suggesting that ATF4 is a key player in the SMC response to injury. Furthermore, microarray analysis identified several genes whose transcription in response to FGF-2 may be regulated by ATF4. In particular, this work demonstrates that ATF4 is necessary for VEGF-A upregulation in SMC in response to FGF-2 and mechanical injury in vitro and in the artery wall following balloon-injury. The second is a translation factor, YrdC203. Using confocal fluorescence microscopy, YrdC203 was found to localize partially to the ER, and with RPL12, a component of the 60S ribosomal subunit. Immunoprecipitation studies demonstrate that YrdC203 also interacts with an initiation factor, eIF5B. Mutation of an initiation factor’s signature on the exterior of YrdC203 perturbed its interaction with RPL12 and eIF5B, and inhibited the increase in protein synthesis observed with overexpression of YrdC203. This implicates YrdC203 as a translation factor responsible for ensuring protein synthesis in vascular SMC in response to injury. The present work provides evidence for new molecular mechanisms, transcriptional and translational, regulating the response of vascular SMC to injury. This would provide leads for future therapeutic targets.

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 134-145).

Thesis (Ph. D.)--University of Kentucky, 2003. / Title from document title page. Document formatted into pages; contains x, 192p. : ill. Includes abstract. Includes bibliographical references (p. 165-189).