Endothelial Progenitor Cells (EPCs) were first identified from human blood samples as a population of circulating mononuclear cells capable of displaying a mature endothelial cell phenotype in culture. Subsequent studies have established that EPCs arise from the bone marrow (BM) and incorporate into the endothelium at sites of blood vessel growth, suggesting a potential role for these cells in neovascularization. Furthermore, a decline in EPC count has been correlated to multiple vascular pathologies, indicating that EPC number could serve as a biomarker of cardiovascular disease. Unfortunately, due to the variability in techniques used for EPC isolation and identification, considerable heterogeneity exists within the population of cells commonly defined as EPCs. In order for the clinical potential of EPCs to be fully realized, thorough characterization of the BM-derived cell populations involved in neovascularization is required. The objective of our study was to determine the functional significance of circulating EPCs in postnatal vascular growth and repair. Two separate strategies were employed to achieve this objective. In the first, we attempted to generate a novel mouse model where the pool of bone marrow-derived endothelial precursors was drastically reduced or eliminated. Our overall approach was to deliver a "suicide" gene, under control of an endothelial cell-specific promoter, to bone marrow cells for use in bone marrow transplantation (BMT) experiments. Mice receiving BMTs would therefore lack the ability to deliver viable BM-derived EPCs to sites of neovascularization. Our central hypothesis for this study was that a reduction in EPC viability would hinder endogenous vascular repair mechanisms, thereby exacerbating cardiovascular disease. In the second strategy, we attempted to identify novel progenitor cell populations based on the transcriptional regulation of pro-angiogenic genes. Our overall approach was to transduce BM with a retrovirus containing a fluorescent reporter gene under control of pro-angiogenic promoters for use in transplantation experiments. Our central hypothesis for this study was that unique populations of BM-derived cells could be identified by expression of the fluorescent reporter gene directed by the Vascular Endothelial Growth Factor (VEGF), endothelial Nitric Oxide Synthase (eNOS) and Vascular Endothelial (VE) Cadherin promoters. The BMT strategy utilized to address our first hypothesis was unsuccessful due to the use of a truncated form of the pro-apoptotic Bax as our suicide gene target. A plasmid encoding GFP fused to the truncated Bax fragment (ΔN-Bax, consisting of amino acids 112-192 of the full length protein) was used in transfection experiments to assess ΔN-Bax function. The GFP:ΔN-Bax fusion protein formed distinct extranuclear aggregates (presumably due to mitochondrial translocation) but did not induce apoptosis in transfected cells. The ΔN-Bax fragment also did not induce cell death when targeted to endothelial cells with retoviral-mediated gene delivery or in a transgenic mouse setting. To address our second hypothesis, we generated retroviral vectors containing the fluorescent tdTomato reporter under control of the VEGF, eNOS and VE Cadherin promoters. Significant fluorescence was detected in cultured endothelial cells and ex vivo-expanded BM cells. Following transplantation of transduced BM cells into lethally irradiated recipient mice, we were able to identify circulating populations of tdTomato-positive cells using flow cytometry. With these results we have identified novel subpopulations of circulating BM-derived cells which may play a significant role in post-natal neovascularization in mice. Therefore, results acquired from these studies could lead to improved cell therapy techniques for treatment of vascular disease.

Thesis (Ph. D.)--University of Oregon, 2003. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 137-145). Also available for download via the World Wide Web; free to University of Oregon users.

published_or_final_version / Medicine / Master / Master of Research in Medicine

published_or_final_version / Pharmacology and Pharmacy / Master / Master of Philosophy

Despite the medical advances in recent decades, cardiovascular disease (CVD) remains one of the leading causes of mortality in most developing countries. Ongoing efforts have been focused on evaluating new strategies targeting on novel risk factors. Vitamin D deficiency, a previously neglected condition, has recently attracted much attention from the scientific community with its potential extra-skeletal effects. There is accumulating evidence from epidemiological studies that a suboptimal 25-hydroxyvitamin D [25(OH)D] level is associated with all-cause and cardiovascular mortality, increased risk of coronary heart disease, stroke and peripheral vascular disease, and various traditional CVD risk factors including hypertension, diabetes mellitus (DM) and metabolic syndrome. Several theories have been proposed to explain these relationships but none receive universal recognition. There is recent laboratory evidence that vitamin D may exert specific effects in patients with DM. However, relationships between vitamin D deficiency and supplementation on vascular function in this group of patients are unclear. In this dissertation, I sought to explore the effects of vitamin D deficiency on vascular function in patients with type II DM in a cross-sectional study. In the later part, the results of a randomized controlled trial investigating the effects of daily vitamin D supplementation in type II DM patients are presented and discussed. The cross-sectional study (Chapter 3) investigated the association of vitamin D status with endothelial function as measured by brachial flow-mediated dilation (FMD) and circulating endothelial progenitor cell (EPC) numbers in 280 patients with type II DM. The results showed that suboptimal vitamin D status was more common among patients with DM. Furthermore, patients with vitamin D deficiency had significantly lower brachial FMD (mean difference = -1.43%, 95% CI: -2.31 to -0.55, P = 0.001) and CD133/KDR+ EPC counts (mean difference = -0.12%, 95% CI: -0.21 to -0.02, P = 0.022) than those with sufficient vitamin D after adjustment for age, sex and cardiovascular risk factors, including HbA1c levels. Based on these positive results, the objectives of the randomized controlled trial (Chapter 4) were to study and confirm the effects of daily oral vitamin D supplementation on the vascular function in this group of patients. Over a 12-week period, 100 DM patients with suboptimal vitamin D status were randomized to receive 5,000 IU/day vitamin D or placebo. There were no reported adverse events including hypercalcemia, although a slight increase in serum ionized calcium (treatment effect 0.037 mmol/L, P = 0.018) was recorded in the vitamin D group. Despite a significant improvement in serum 25(OH)D in the treatment group, supplementation of vitamin D did not result in any significant improvement in vascular function as determined by FMD, circulating EPC count or arterial stiffness (all P > 0.05). Furthermore, the serum level of high-sensitivity C-reactive protein, oxidative stress markers, low- and high-density lipoprotein and glycated haemoglobin were also similar between two groups (all P > 0.05). The results of this study did not support a therapeutic role of supplementation with vitamin D for cardiovascular benefits. In conclusion, the results of these studies demonstrated that deficiency of vitamin D was associated with worse vascular function in patients with type II DM. However, vitamin D supplementation did not result in any significant benefits on vascular function or improvement in traditional CVD risk factors in DM patients. Further large clinical trials on vitamin D supplementation in patients with DM using clinical outcomes rather than surrogate CVD markers are necessary to confirm its benefits. / published_or_final_version / Medicine / Master / Master of Research in Medicine

Liver kinase B1 (LKB1), a serine/threonine kinase, is responsible for the activation of AMP-activated protein kinase (AMPK), the master regulator of energy metabolism. LKB1/AMPK signaling pathway possesses a wide range of biological functions in regulating cell cycle progression, cell polarity, senescence and inflammation. In cultured endothelial cells, the pro-senescence function of LKB1/AMPK signaling pathway has been observed. However, the mechanisms by which LKB1 is regulated in endothelial cells remain largely uncharacterized. Furthermore, little is known about the effects of activated endothelial LKB1/AMPK signaling pathway on vascular and energy homeostasis. The present study aimed to investigate the upstream molecular mechanisms regulating LKB1 protein stability during endothelial senescence and the downstream pathophysiological effects of hyperactivated AMPK signaling in endothelial cells. In cultured model of cellular senescence, the lysine (K) 64 residue of LKB1 was found to be crucial for mediating its pro-senescence activities. The protein stability and intracellular localization of LKB1 mutant with K64 replaced by arginine (R) was different from the wild type protein. K64R exhibited enhanced effects on promoting endothelial senescence. Moreover, mutation of this residue attenuated the binding to HERC2, a newly identified E3 ubiquitin ligase for LKB1, in turn preventing its ubiquitination and degradation. Using a transgenic mouse model that selectively over-expresses a constitutively active AMPK α1 subunit (EC-AMPK) in endothelial cells, the influence of hyperactivated AMPK signaling on metabolic and vascular functions was investigated. Under standard chow condition, the metabolic phenotypes were similar between wild type and EC-AMPK mice; under high fat diet condition, EC-AMPK mice showed more severe obesity-induced fatty liver injury. Selective activation of AMPK in endothelial cells caused vascular and hepatic inflammation. Cyclooxygenase-2 (COX-2) was found to be the mediator for the pro-inflammatory functions of AMPK in vascular endothelial cells and facilitated to the development of obesity-induced fatty liver injury in EC-AMPK mice. Evaluation using isolated arteries revealed an increased systolic blood pressure and abnormal endothelial function in EC-AMPK miceunder high fat diet. AMPK activation in endothelium of the blood vessel could not block vascular remodeling associated with dietary obesity. Taken in conjunction, the above findings suggest that continuous activation of LKB1/AMPK signaling elicits adverse effects on both energy and vascular homeostasis. / published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

published_or_final_version / Surgery / Master / Master of Surgery

published_or_final_version / abstract / toc / Surgery / Master / Master of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

No description available.