Nicholls, StephenDi Bartolo, BelindaBursill, ChristinaMulangala, Jocelyne2021-11-042021-11-042020https://hdl.handle.net/2440/132934Peripheral arterial disease (PAD) refers to the obstruction or blockage of arteries of the lower extremity vessels. PAD is characterised by accelerated arterial calcification and impairment in angiogenesis, both of which associate with adverse cardiovascular outcomes. The pathogenesis of lower extremity PAD remains poorly described but is regarded secondary to atherosclerosis due to fatty plaque build-up within the arterial wall. Despite the many clinical advances available to treat PAD, impairment in microcirculation continues to put patients at constant risk of ischemia; increasing their risk of developing limb amputations which subsequently affects their quality of life. Therapies for PAD are centred around alleviating stenosis and promoting tissue revascularization following ischemia, and while atherosclerosis is recognised as the main cause of PAD, acute or chronic limb ischemia may be the result of various other cardiovascular risk factors. Calcification is implicated as a contributor to PAD, but the mechanisms underlying this are not fully elucidated. Firstly, I sought to determine the effect of elevated calcium on ischemia-driven angiogenesis. Human coronary artery endothelial cells were cultured and incubated with calcification medium (CM) (CaCl₂ 2.7mM, Na₂PO₄ 2.0mM) for 24h and conditioned to either normoxia or hypoxia. The Hindlimb ischemia model was used to assess the role of high calcium on ischemia-driven angiogenesis in vivo, on 8-week-old and 24-week-old mice, with high calcium levels (OPG(-/-)) and wildtype (C57BL6/J, control) mice. Blood flow reperfusion was assessed by Laser Doppler Perfusion Imaging (LDPI). In vitro, CM upregulated mRNA expression of calcification inductive genes, Bmp2 and Runx2. CM significantly decreased tubule formation in normoxia, with further reduction in hypoxia (13%); and reduced cell migration in normoxia alone. CM increased the expression of key angiogenic markers Hif-1α and Vegfa mRNA levels in normoxic conditions, which coincided with an increase in their protein expression in normoxia; and reduction in hypoxia. Incubation with CM reduced cell viability and induced cell death in EC. 8-week-old OPG(-/-) mice showed elevated serum calcium levels and ALP activity, an important diagnostic marker of calcification. LDPI showed striking reductions in blood-flow reperfusion of 8-week OPG(-/-) mice compared to controls at day 14; which was further impaired in an aged cohort of mice, with a significant reduction in blood flow at day 10. Mechanistically, ischemic limbs of 8-week old OPG(-/-) mice showed significant upregulation HIF-1α and reduced CD31 neovessels. Clinical scoring revealed significantly worse outcomes for both limb function and tissue ischemia in the OPG(-/-) mice versus controls, assessed by Tarlov scores, with higher values indicating a worse outcome. Furthermore, to assess the effect of calcification milieu on inflammatory-driven angiogenesis, cells were stimulated with both CM and TNF-α in vitro to induce inflammation. Overall, CM had no additive effect in driving inflammatory-driven angiogenesis, with no effects on HIF- 1α and VEGFA expression. CM however induced the expression of inflammatory markers ICAM, VCAM, MCP1 and NF- κB (P65) expression in treated cells compared to controls. When this was modelled in vivo using the peri-arterial femoral cuff placement, OPG(-/-) mice with high calcification milieu displayed no changes in angiogenic marker expression though displayed significantly elevated inflammatory-marker expression. This is the first demonstration that high-levels of calcium negatively regulates ischemia-driven angiogenesis. These findings have implications for the development of therapies that can suppress calcification in PAD.enCalcificationangiogenesisPeripheral Arterial Diseaseendothelial cellsischemiaThesis