From the Department of Laboratory Medicine and Pathobiology (M.L., M.H.W., A.S.R., D.N., N.S., G.H., M.P.B.).
Ted Rogers Centre for Heart Research (M.L., D.N., N.S., G.H., M.P.B.).
Arterioscler Thromb Vasc Biol. 2018 Aug;38(8):1878-1889. doi: 10.1161/ATVBAHA.118.311238.
Objective- Vascular calcification is a common and severe complication in patients with atherosclerosis which is exacerbated by type 2 diabetes mellitus. Our laboratory recently reported that the collagen receptor discoidin domain receptor 1 (DDR1) mediates vascular calcification in atherosclerosis; however, the underlying mechanisms are unknown. During calcification, vascular smooth muscle cells transdifferentiate into osteoblast-like cells, in a process driven by the transcription factor RUNX2 (runt-related transcription factor 2). DDR1 signals via the phosphoinositide 3-kinase/Akt pathway, which is also central to insulin signaling, and upstream of RUNX2, and this led us to investigate whether DDR1 promotes vascular calcification in diabetes mellitus via this pathway. Approach and Results- Ddr1 ; Ldlr (single knock-out) and Ddr1 ; Ldlr (double knock-out) mice were placed on high-fat diet for 12 weeks to induce atherosclerosis and type 2 diabetes mellitus. Von Kossa staining revealed reduced vascular calcification in the aortic arch of double knock-out compared with single knock-out mice. Immunofluorescent staining for RUNX2 was present in calcified plaques of single knock-out but not double knock-out mice. Primary vascular smooth muscle cells obtained from Ddr1 and Ddr1 mice were cultured in calcifying media. DDR1 deletion resulted in reduced calcification, a 74% reduction in p-Akt levels, and an 88% reduction in RUNX2 activity. Subcellular fractionation revealed a 77% reduction in nuclear RUNX2 levels in Ddr1 vascular smooth muscle cells. DDR1 associated with phosphoinositide 3-kinase, and treatment with the inhibitor wortmannin attenuated calcification. Finally, we show that DDR1 is important to maintain the microtubule cytoskeleton which is required for the nuclear localization of RUNX2. Conclusions- These novel findings demonstrate that DDR1 promotes RUNX2 activity and atherosclerotic vascular calcification in diabetes mellitus via phosphoinositide 3-kinase/Akt signaling.
目的-血管钙化是动脉粥样硬化患者的一种常见且严重的并发症，2 型糖尿病会使其恶化。我们实验室最近报道，胶原蛋白受体盘状结构域受体 1（DDR1）介导动脉粥样硬化中的血管钙化；然而，其潜在机制尚不清楚。在钙化过程中，血管平滑肌细胞向成骨样细胞转分化，这一过程由转录因子 RUNX2（runt 相关转录因子 2）驱动。DDR1 通过磷酸肌醇 3-激酶/Akt 途径发出信号，该途径也是胰岛素信号的核心，并且位于 RUNX2 的上游，这导致我们研究 DDR1 是否通过该途径促进糖尿病中的血管钙化。方法和结果-Ddr1;Ldlr（单敲除）和 Ddr1;Ldlr（双敲除）小鼠接受高脂肪饮食 12 周以诱导动脉粥样硬化和 2 型糖尿病。Von Kossa 染色显示，与单敲除小鼠相比，双敲除小鼠主动脉弓的血管钙化减少。RUNX2 的免疫荧光染色存在于单敲除但不存在于双敲除小鼠的钙化斑块中。从 Ddr1 和 Ddr1 小鼠获得的原代血管平滑肌细胞在钙化培养基中培养。DDR1 缺失导致钙化为单敲除的 74%，p-Akt 水平降低 74%，RUNX2 活性降低 88%。亚细胞分级显示 Ddr1 血管平滑肌细胞中的核 RUNX2 水平降低 77%。DDR1 与磷酸肌醇 3-激酶相关，并用抑制剂wortmannin 处理可减弱钙化。最后，我们表明 DDR1 对于维持微管细胞骨架很重要，微管细胞骨架是 RUNX2 核定位所必需的。结论-这些新发现表明，DDR1 通过磷酸肌醇 3-激酶/Akt 信号促进糖尿病中的 RUNX2 活性和动脉粥样硬化性血管钙化。
