Endothelial Progenitor Cells (EPCs) are thought to participate in the healing of injured vascular endothelium by incorporating into the defect sites to mediate endothelial recovery. Recently, osteopontin (OPN) was shown to be fundamental in accelerating estrogen-dependent healing of injured blood vessels. Here, we are investigating the effect OPN has on EPC behavior. Late outgrowth human EPCs (LEPCs) were derived from circulating monocytes isolated by leukophoresis, and grown in culture until passage six. L-EPCs were then assayed for adhesion, spreading, chemotaxis, and haptotaxis, as well as resistance to detachment by flow electric cellsubstrate impedance sensing (ECIS). The results of standard and ECIS methods showed both dose and time dependent responses in cell adhesion and spreading. In addition, OPN promoted haptotactic migration of EPCs in Boyden chamber assays. LEPCs seeded onto 10μM OPN substrates and exposed to laminar flow had grater survival and higher resistance to detachment than OPN/static and flow only conditions. CD44 and !1 integrins were only responsible for approximately 50% of LEPCs adhesion to OPN compared to the unblocked condition. Western blots showed that Rho GTPases were activated in L-EPCs seeded on OPN. However, this activation could not be completely blocked by either CD44 or !1 integrin antagonists. These data confirm the direct effects of OPN on EPCs adhesion, and suggest that OPN works by mediating cell adhesion during vascular injury.

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The endothelium is an integral mediator of vascular homeostasis and a dysfunctional endothelium is now recognized as an early marker of atherosclerosis. Importantly, the non-invasive measurement of endothelial function by flow-mediated dilation (FMD) predicts future cardiovascular events. However, the appropriate method of its assessment and the mechanisms that govern FMD are still poorly understood. We investigated alternative parameters and methods of FMD measurement in healthy volunteers and cardiovascular disease patients. We found time to peak FMD to be highly variable both within and between individuals. Accordingly, continuous arterial diameter measurement post-cuff release was more sensitive in discriminating between health and disease compared to the measurement of diameter at 60’’ post-cuff release. Reperfusion to an ischemic tissue can paradoxically contribute to endothelial dysfunction development and further tissue damage, in a phenomenon known as ischemia and reperfusion (IR) injury. Previous exposure to sublethal ischemia (ischemic preconditioning (IPC)) can reduce sensitivity to IR injury and pharmacologic agents have since been shown to mimic this response. Using the FMD technique, we investigated various preconditioning strategies to prevent IR-induced endothelial dysfunction in the forearm vasculature of healthy volunteers. The sodium-hydrogen exchanger inhibitor amiloride and the angiotensin-converting enzyme inhibitor captopril were found not to provide endothelial protection from IR. In contrast, potent protection from IR-induced endothelial dysfunction was observed during the high-estrogen, late follicular phase of the menstrual cycle in pre-menopausal women. Finally, daily episodes of IPC were found to provide endothelial protection equipotent to an acute episode of IPC. The findings from the FMD methodological study highlight the importance of continuous arterial diameter measurement post-cuff deflation, and provide mechanistic insight that may contribute to measurement standardization and normalization. The results of the preconditioning studies improve our understanding of potential approaches to mitigate the detrimental effects of IR on the endothelium in humans.

Modular tissue engineering is a novel approach to creating scalable, self-assembling three-dimensional tissue constructs with inherent vascularisation. Under initial methods, the subcutaneous implantation of human umbilical vein endothelial cell (HUVEC)-covered collagen modules in immunocompromised mice resulted in significant host inflammation and limited HUVEC survival. Subsequently, a minimally-invasive injection technique was developed to minimize surgery-related inflammation, and cell death was attributed to extensive apoptosis within 72 hours of implantation. In confirmation of in vitro results, coating collagen modules with fibronectin (Fn) was shown in vivo to reduce short-term HUVEC apoptosis by nearly 40%, while increasing long-term HUVEC survival by 30% to 45%. Consequently, a 100% increase in the number of HUVEC-lined vessels was observed with Fn-coated modules, as compared to collagen-only modules, at 7 and 14 days post-implantation. Furthermore, vessels appeared to be perfused with host erythrocytes by day 7, and vessel maturation and stabilization was evident by day 14.

The NF-κB/Rel family of transcription factors and IκB inhibitors play a key role in regulation of gene expression in inflammation and immunity. Previous studies from our laboratory suggested that steady-state levels of p65 and other NF-κB components in the normal mouse aorta determine the magnitude of NF-κB target gene expression in response to pro-inflammatory stimuli, however, the mechanism(s) by which steady-state levels of NF-κB components are set is not clear. This study aims at elucidating the mechanisms behind NF-κB homeostasis and how that affects atherosclerosis susceptibility. In HeLa cells and HUVEC, siRNA silencing of p65 correlated with reduced steady-state expression of a subset of NF-κB/Rel and IκB genes at the transcriptional and post-transcriptional levels, respectively, in addition to reducing TNFα-induced NF-κB/Rel and IκB gene expression. This correlation was also observed in atherosclerosis-susceptible mouse aortic endothelium suggesting the role of p65 in modulating NF-κB homeostasis and affecting atherosclerosis susceptibility.

The objective of this project was to evaluate the effects of local endothelial progenitor cell (EPC) therapy on bone regeneration in a segmental defect in the rat femur. Animals from the EPC-treated (N=28) and control (N=28) groups were sacrificed at 1, 2, 3, and 10 weeks post-operatively. Bone healing was evaluated with radiographic, histological, and micro computed tomography (micro-CT) scans. Radiographically; mean scores of the EPC group at 1, 2, and 3 weeks were significantly higher compared to control group. At 10 weeks, all the animals in the EPC group had complete union (7/7), but in the control group none achieved union (0/7). Histologically, specimens from EPC-treated animals had abundant new bone formation compared to controls. Micro-CT assessment showed significantly improved parameters of bone healing for the EPC group compared to control group. In conclusion, local EPC therapy significantly enhanced bone regeneration in a segmental bone defect in rat femur.

The objective of this project was to evaluate the effects of local endothelial progenitor cell (EPC) therapy on bone regeneration in a segmental defect in the rat femur. Animals from the EPC-treated (N=28) and control (N=28) groups were sacrificed at 1, 2, 3, and 10 weeks post-operatively. Bone healing was evaluated with radiographic, histological, and micro computed tomography (micro-CT) scans. Radiographically; mean scores of the EPC group at 1, 2, and 3 weeks were significantly higher compared to control group. At 10 weeks, all the animals in the EPC group had complete union (7/7), but in the control group none achieved union (0/7). Histologically, specimens from EPC-treated animals had abundant new bone formation compared to controls. Micro-CT assessment showed significantly improved parameters of bone healing for the EPC group compared to control group. In conclusion, local EPC therapy significantly enhanced bone regeneration in a segmental bone defect in rat femur.

The NF-κB/Rel family of transcription factors and IκB inhibitors play a key role in regulation of gene expression in inflammation and immunity. Previous studies from our laboratory suggested that steady-state levels of p65 and other NF-κB components in the normal mouse aorta determine the magnitude of NF-κB target gene expression in response to pro-inflammatory stimuli, however, the mechanism(s) by which steady-state levels of NF-κB components are set is not clear. This study aims at elucidating the mechanisms behind NF-κB homeostasis and how that affects atherosclerosis susceptibility. In HeLa cells and HUVEC, siRNA silencing of p65 correlated with reduced steady-state expression of a subset of NF-κB/Rel and IκB genes at the transcriptional and post-transcriptional levels, respectively, in addition to reducing TNFα-induced NF-κB/Rel and IκB gene expression. This correlation was also observed in atherosclerosis-susceptible mouse aortic endothelium suggesting the role of p65 in modulating NF-κB homeostasis and affecting atherosclerosis susceptibility.

B. henselae, B. bacilliformis and B. quintana are capable of causing vasoproliferative diseases in humans by modulating apoptosis and proliferation of endothelial cells. Bartonella clarridgeiae, a close relative of the pathogenic Bartonellae, has been implicated in human disease but has not yet been isolated from a human patient. Both B. bacilliformis and B. clarridgeiae have flagella and a flagellar type 3 secretion system, while B. henselae and B. quintana do not. We created 2 non-motile mutants of B. clarridgeiae by interrupting the flagellin gene, flaA, or the flagellar motor genes, motBC. We investigated whether B. clarridgeiae could invade human endothelial cells (HMECs) and if functional flagella were important for invasion. The non-motile mutants and the wild-type strain were capable of entering HMECs in vitro. The flaA mutant was deficient in attachment, but the HMECs in culture with the flaA mutant demonstrated increased proliferation. The motBC mutant showed enhanced invasion. Differential secretion of proteins was revealed by 2-D electrophoresis and MALDI-TOF analysis of secretomes from the co-cultures compared to uninfected HMECs. HMECS infected with wild-type B. clarridgeiae secreted proteins indicative of proliferation. The flaA mutant induced the secretion of proteins involved in cytoskeletal rearrangement, cell migration, and proliferation. The motBC-infected HMECs showed signs of hypoxia. The co-chaperonin GroES was found in higher concentration in the supernatant of the hyper-invasive motBC strain/HMEC co-culture than the wild-type co-culture and was found at a very low concentration in the flaA culture supernatant. Cross-talk between secretion systems is suggested.

The number of people having diabetes mellitus is increasing worldwide at an alarming rate. An unbalanced diet rich in carbohydrates and saturated fats, obesity and lack of physical activity, are being blamed. The worldwide prevalence of diabetes for all age-groups has been estimated to be 2.8% in 2000 and projected to be 4.4% by the year 2030. The pathogenesis of diabetes, especially the recent epidemic increase in type 2 diabetes, is still far from clear. Endothelial dysfunction, commonly defined as reduced endothelium-dependent relaxation due to reduced availability of the vasodilator mediator nitric oxide (NO), is a hallmark of diabetes mellitus. Methylglyoxal (MG) is a highly reactive dicarbonyl compound mainly formed as an intermediate during glycolysis. MG is also formed to a lesser extent from protein and amino acid metabolism. However, the relative contribution of various metabolic precursors to MG formation is not known. Levels of MG have been found to be elevated in diabetic and hypertensive conditions but it is not known whether MG is the cause or the effect of these pathological conditions. The aim of my project was (i) to quantify the amount of MG and oxidative stress produced from various substrates in cultured A10 vascular smooth muscle cells (VSMCs), (ii) to investigate the acute in vivo effects of a single dose of MG on glucose tolerance in male Sprague-Dawley (SD) rats, (iii) to investigate the effects of MG on endothelial function and (iv) to investigate the effects, and the underlying molecular mechanisms, of chronic administration of MG on glucose homeostasis in male SD rats. The results show that aminoacetone, a protein metabolism intermediate, is the most potent substrate for MG formation on a molar basis, whereas D-glucose and fructose are equipotent. I also established optimum sample preparation protocols for reproducible measurement of MG in biological samples by high performance liquid chromatography (HPLC). In normal SD rats a single acute dose of MG induced glucose intolerance, reduced adipose tissue glucose uptake and impaired the insulin signalling pathway, which was prevented by the MG scavenger and advanced glycation end product (AGE) breaking compound, alagebrium (ALT-711). MG and high glucose (25 mM) induced endothelial dysfunction in rat aortic rings and cultured endothelial cells by reducing endothelial nitric oxide synthase (eNOS) phosphorylation at Ser-1177, activity and NO production. MG and high glucose also increased oxidative stress and further reduced NO availability in rat aortic rings and cultured endothelial cells. Chronic administration of MG in normal SD rats by continuous infusion with a subcutaneously implanted minipump for 28 days (60 mg/kg/day), induced metabolic and biochemical abnormalities of glucose homeostasis and insulin regulation that are characteristic of type II diabetes. In MG treated rats, insulin stimulated glucose uptake in adipose tissue, and glucose stimulated insulin release from freshly isolated pancreas, were significantly reduced as compared to saline treated control rats. At a molecular level, insulin gene transcription was significantly impaired and apoptosis and DNA fragmentation were more prevalent in the pancreas of MG treated rats as compared to untreated control rats. All of these in vivo effects of MG were attenuated by the MG scavenger, alagebrium. Our data strongly indicate that MG is a causative factor in the pathogenesis of endothelial dysfunction and type 2 diabetes mellitus.