抑制血管钙化—DNA拓扑异构酶II抑制剂抗动脉粥样硬化新机制

Formation of arterial calcification can result in increased unstability and rupture of atherosclerotic lesion plaques, and enhance occurrence of sudden cardiovascular events. The prominent part of calcified arterial wall is the osteogenic differentiated vascular smooth muscle cells (VSMCs). Inhibition of DNA topoisomerase II (Topo II) by chemical inhibitors can play an important role in cancer biology and immunology. Meanwhile, Topo II inhibitors may have pleiotropic functions in other fields. Previously, we reported that inhibition of Topo II by chemical inhibitors can, 1) activate macrophage ABCA1/G1 expression, thereby inhibiting the formation of macrophage/foam cells; 2) activate hepatic CETP expression and ameliorate reverse cholesterol transport which results in improvement of serum lipid profiles; and 3) reduce calcification in aortic rings ex vivo and SMCs in culture. Taken together, our above findings suggest that Topo II inhibitors may reduce atherosclerosis, and the inhibition of arterial calcification should be one of the important mechanisms reducing atherosclerotic lesions. In this application, we propose to determine the effects of Topo II inhibitors on vascular calcification in vivo, ex vivo and in vitro. We will determine if Topo II inhibitor treatment can reduce vascular calcification both in vivo and in vitro by regulating osteogenic differentiation of VSMCs. At the mechanical levels, we will determine if Topo II inhibitors can regulate the signaling pathways involved in vascular calcification/osteogenic differentiation of VSMCs. By completing this project, we hope that we can elucidate that inhibition of vascular calcification is one of the important mechanisms by which Topo II inhibitors reduce atherosclerosis and increase the stability of lesion plaques. We also wish that our study will provide a novel strategy for atherosclerosis treatment.

主动脉血管钙化降低动脉粥样硬化斑块稳定性，促进斑块破裂与血管急性事件发生。血管钙化主要由血管平滑肌细胞向成骨样细胞分化所致。DNA拓扑异构酶II在肿瘤、免疫中具有重要作用。我们前期研究发现DNA拓扑异构酶II抑制剂能①刺激巨噬细胞ABCA1/G1表达，抑制其泡沫化；②促进肝细胞CETP表达和胆固醇逆转运，改善血脂水平；③抑制离体主动脉环和平滑肌细胞钙化发生。以上结果预示DNA拓扑异构酶II抑制剂对动脉粥样硬化具有防治作用，通过抑制血管钙化增加斑块稳定性。为此我们拟在在体、离体和细胞水平上开展DNA拓扑异构酶II抑制剂抑制动脉钙化的研究。现象上，明确DNA拓扑异构酶II抑制剂抑制体内外钙化发生；机理上，明确DNA拓扑异构酶II抑制剂抑制平滑肌细胞钙化的信号通路，确定效应分子。我们的研究将阐明DNA拓扑异构酶II抑制剂抗血管钙化的作用机制，为临床防治动脉粥样硬化提供新策略。

血管内膜钙化，特别是点状钙化是动脉粥样硬化斑块破裂的主要危险因素之一。在动脉粥样硬化发展进程中，钙化位点处骨形态发生蛋白2(bone morphogenetic protein 2，BMP2)基因的高表达预示该基因在血管钙化过程中扮演重要角色。替尼泊苷是一种DNA拓扑异构酶II抑制剂，被广泛运用于肿瘤治疗中。本课题组前期研究结果表明替尼泊苷激活巨噬细胞三磷酸腺苷结合盒转运体A1蛋白表达，从而促进巨噬细胞胆固醇外流，预示DNA拓扑异构酶II抑制剂具有抑制动脉粥样硬化潜能。本项目以载脂蛋白E敲除(apoE-/-)小鼠为实验模型，将8周龄的apoE KO小鼠随机分为三组：第一组小鼠连续喂食高脂食物(21%脂肪，0.5%胆固醇)16周(对照组)；第二组小鼠喂食添加了替尼泊苷的高脂食物16周(预防组)；第三组小鼠喂食高脂食物12周后，使用添加了替尼泊苷的高脂食物继续喂食4周(治疗组)。实验结束后处死小鼠并评估小鼠动脉粥样硬化以及动脉粥样硬化伴随的血管钙化相关指标。在体外，我们使用人主动脉平滑肌细胞研究替尼泊苷影响血管钙化的分子机制。. 实验结果发现替尼泊苷能够抑制动脉粥样硬化的发生发展，动脉粥样硬化伴随的血管钙化也被替尼泊苷显著抑制。机制上，替尼泊苷以p53依赖的方式抑制BMP2/(p-Smad1/5/8)/RUNX2信号通路，从而抑制血管钙化的发生。进一步研究表明BMP2为miR-203-3p的靶基因，p53通过激活miR-203-3p抑制BMP2信号通路从而发挥抑制血管钙化作用。. 通过本项目的完成，我们明确了DNA拓扑异构酶II抑制剂具有抑制动脉粥样硬化以及内膜钙化的作用，为血管钙化防治提供新的理论依据。