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.

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

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.

Tortuosity of eye fundus blood vessels is one of parameters that describe state of the blood vessels. It can be detected from fundus images. The increase in vessel tortuosity was observed in eyes of patients with advanced background diabetic retinopathy, papilloedema, arterial hypertension, even in some completely healthy eyes (in this case tortuosity does not change in time). Thus the feature of the line – tortousity – could be used as the diagnostic feature in medical applications. Unfortunately, completely reliable definition and numerical estimation of tortuosity of line (blood vessel) does not exist, although there were some more or less successful attempts to define it. This work presents a new way to estimate the tortuosity using the integral of square derivative of curvature. It is compared with the existing methods both theoretically and experimentally. Three types of lines have been used for experimental comparison: theoretical models of retinal vessels (straight line, parabola, sinusoid and their combinations), lines extracted from real fundus images and lines extracted from optometric scale for retinal vessel evaluation. To ease the extraction of the lines from these images a new version of one of the methods for vessel tracking has been created and implemented.

Objective. To assess the influence of diabetes mellitus on early morbidity and mortality following open vascular surgical interventions. Methods. Clinical data on patients subjected to open vascular surgical procedures over a 5 year period at the Durban Metropolitan Vascular Service was culled from a prospectively maintained computerized database. They were divided according to the type of surgical procedure performed. These were open abdominal aortic surgery, peripheral bypass surgery, lower extremity major amputation and carotid endarterectomy. They were further subdivided into 2 groups, diabetic and non-diabetic. Results. 1104 charts were analysed. There were no significant differences in demographics and risk factors between the two groups. 273 patients had open abdominal aortic surgery. 217 (79%) were non-diabetic. diabetic patients had significantly higher incidence of myocardial infarction (p=0.00001) (6 of 6 patients), graft sepsis (p=0.000001) (7 of 7 patients) and mortality rate (p=0.0335) (5 of 10 patients). 337 patients had peripheral bypass procedures. 204 (60%) of these were non-diabetic. There was a high prevalence of smokers among non-diabetics and of hypertension among diabetics. Diabetic patients had a preponderance of graft infection (p=0.0015) (15 of 20 patients) and cardiovascular complications (p=0.0072) (7 of 8 patients). 230 patients had lower extremity major amputations, 81 (35%) were diabetic and 149 (65%) non-diabetic. Myocardial infarction and death (6 of 8 patients each) were significantly higher among diabetics (p =0.04). 264 patients had carotid endarterectomy, 170 (64%) being non-diabetic. The surgical outcome was similar between the two groups. Conclusions. This is retrospective study and as such it has some its limitations. Not all patients might have been included in the study and some of the information might have been lost. The numbers in this study are large and these limitations would appear not to have influenced the outcome of this study. This study has shown that diabetes mellitus had diverse influence on the early outcome following different vascular surgical procedures. Diabetes mellitus significantly increased the incidence of graft sepsis among those who had aorto-bifemoral bypass and peripheral bypass procedures. The incidence of peri-operative cardiovascular morbidity was significantly increased among diabetics who had peripheral bypass procedures, open abdominal aortic surgery and lower extremity major amputations. Diabetes mellitus had no influence on the surgical outcome following carotid endarterectomy. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2012.