Discipline of Medicine, University of Adelaide, Adelaide, SA 5005, Australia.
Vascular Research Centre, Heart Health Theme, South Australian Health and Medical Research Institute, Adelaide, SA 5000, Australia.
Int J Mol Sci. 2022 Mar 20;23(6):3363. doi: 10.3390/ijms23063363.
Peripheral arterial disease (PAD) is characterised by accelerated arterial calcification and impairment in angiogenesis. Studies implicate vascular calcification as a contributor to PAD, but the mechanisms remain unclear. We aimed to determine the effect of calcification on ischaemia-driven angiogenesis. Human coronary artery endothelial cells (ECs) were treated with calcification medium (CM: CaCl 2.7 mM, NaPO 2.0 mM) for 24 h and exposed to normoxia (5% CO) or hypoxia (1.2% O; 5% CO balanced with N). In normoxia, CM significantly inhibited tubule formation and migration and upregulated calcification markers of ALP, BMP2, and Runx2. CM elevated levels of calcification-protective gene OPG, demonstrating a compensatory mechanism by ECs. CM failed to induce pro-angiogenic regulators VEGFA and HIF-1α in hypoxia and further suppressed the phosphorylation of endothelial nitric oxide synthase (eNOS) that is essential for vascular function. In vivo, osteoprotegerin-deficient mice (OPG), a calcification model, were subjected to hind-limb ischaemia (HLI) surgery. OPG mice displayed elevated serum alkaline phosphatase (ALP) activity compared to wild-type controls. OPG mice experienced striking reductions in blood-flow reperfusion in both 8-week-old and 6-month-old mice post-HLI. This coincided with significant impairment in tissue ischaemia and reduced limb function as assessed by clinical scoring (Tarlov). This study demonstrated for the first time that a pro-calcific environment is detrimental to ischaemia-driven angiogenesis. The degree of calcification in patients with PAD can often be a limiting factor with the use of standard therapies. These highly novel findings require further studies for full elucidation of the mechanisms involved and have implications for the development of therapies to suppress calcification in PAD.
外周动脉疾病（PAD）的特征是动脉钙化加速和血管生成受损。研究表明血管钙化是 PAD 的一个促成因素，但机制尚不清楚。我们旨在确定钙化对缺血驱动的血管生成的影响。将人冠状动脉内皮细胞（ECs）用钙化培养基（CM：CaCl 2.7 mM，NaPO 2.0 mM）处理 24 小时，然后暴露于常氧（5% CO）或缺氧（1.2% O；5% CO 与 N 平衡）。在常氧条件下，CM 显著抑制小管形成和迁移，并上调碱性磷酸酶（ALP）、骨形态发生蛋白 2（BMP2）和 runt 相关转录因子 2（Runx2）等钙化标志物。CM 升高了钙化保护基因骨保护素（OPG）的水平，表明 ECs 存在一种代偿机制。CM 未能在缺氧条件下诱导血管生成调节剂血管内皮生长因子 A（VEGFA）和缺氧诱导因子 1α（HIF-1α），并进一步抑制了对血管功能至关重要的内皮型一氧化氮合酶（eNOS）的磷酸化。在体内，用骨保护素缺陷型（OPG）小鼠（一种钙化模型）进行后肢缺血（HLI）手术。与野生型对照相比，OPG 小鼠的血清碱性磷酸酶（ALP）活性升高。OPG 小鼠在 HLI 后 8 周和 6 月龄小鼠中均出现血流再灌注显著减少。这与组织缺血和肢体功能明显受损相一致，如临床评分（Tarlov）评估所示。本研究首次证明了促钙化环境对缺血驱动的血管生成有害。PAD 患者的钙化程度往往是标准治疗的一个限制因素。这些高度新颖的发现需要进一步研究以充分阐明所涉及的机制，并对开发抑制 PAD 中钙化的治疗方法具有重要意义。
