高糖状态下血管壁微环境中内皮细胞源性外泌体调控动脉中膜钙化的机制研究

Arterial medial calcification is a common disease in patients with diabetes, leading to a significant increased occurrence of cardiovascular events. The transdifferentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells is the key process of arterial medial calcification. It is known that vascular wall microenvironment consists of endothelial cells and VSMCs. Previous studies have focused more on the effects of various factors on the transdifferentiation of VSMCs, whereas few studies have investigated the role of endothelial cells on the transdifferentiation of VSMCs. Our previous studies found that high glucose stimulates endothelial cells to secrete miR-151-3p enriched exosomes, which are uptaken by VSMCs and then promote VSMCs transdifferentiation into osteoblast-like cells. The present project aims at studying the mechanism by which endothelial cells regulate the transition of VSMCs into osteoblast-like cells through secreting exosomes under high glucose stimulation and the role of endothelial cells-derived miR-151-3p in arterial medial calcification by using mouse model of diabetes, endothelial cells-specific miR-151-3p knockout mice and clinical samples. The present project will strengthen the theory of vascular wall microenvironment, illustrate the mechanism of the communication between endothelial cells and VSMCs, and shed new light on the pathogenesis, prevention and treatment of arterial medial calcification.

动脉中膜钙化常见于糖尿病患者，导致心血管事件显著增高。血管平滑肌细胞（VSMCs）向成骨样细胞转分化是其细胞学基础。内皮细胞和VSMCs构成血管壁微环境。既往研究多关注各种因子对VSMCs转分化的影响，而对内皮细胞调控VSMCs的转分化研究则很少。我们的前期研究发现：高糖刺激内皮细胞分泌富含miR-151-3p的外泌体，被VSMCs摄取后，可促进VSMCs向成骨样细胞转分化。本项目研究高糖刺激内皮细胞分泌外泌体，并调控VSMCs向成骨样细胞转分化的分子机制；拟在糖尿病性动脉中膜钙化小鼠、内皮细胞特异性敲除miR-151-3p小鼠以及临床标本中证实内皮细胞源性miR-151-3p调控动脉中膜钙化。本项目的实施将阐明高糖调节内皮细胞与VSMCs之间的对话机制，夯实血管壁微环境的理论基础，进而揭示动脉中膜钙化新的发病机制，为动脉中膜钙化的防治提供新的调控靶点。

动脉中膜钙化常见于慢性肾脏病和2型糖尿病，与心血管事件密切相关的。但是目前动脉中膜钙化的发病机制尚不清楚。在项目的资助下，一方面，我们发现高磷处理的内皮细胞（ECs）上清中分离的外泌体（ECs-Exos）能够被VSMC和动脉中膜摄取，促进血管平滑肌细胞（VSMC）钙化和小鼠动脉中膜钙化。进一步，我们通过测序分析发现miR-670-3p在高磷组ECs-Exos中的表达明显增高，并且下调ECs-Exos中miR-670-3p能够阻断ECs-Exos的促进VSMC钙化作用。进一步研究发现，ECs特异性miR-670-3p敲除小鼠能够部分阻断尿毒症小鼠的动脉中膜钙化，而ECs特异性miR-670-3p转基因小鼠促进小鼠的动脉中膜钙化，以上研究提示ECs源性miR-670-3p调控动脉中膜钙化。机制研究表明，IGF-1是miR-670-3p的下游靶点，并参与调节VSMC钙化。另一方面，我们发现糖尿病状态下糖基化终末期产物刺激ECs分泌富集miR-126-5p的外泌体，这些外泌体可以被动脉中膜VSMC摄取，发挥促进VSMC钙化和动脉中膜钙化作用。此外，我们还发现长链非编码RNA H19通过结合miR-140-5p，上调Satb2表达，从而调控血管平滑肌细胞向成骨样细胞分化及矿化。而脂肪因子omentin-1 可以通过激活 AMPK/Akt 信号通路调控血管平滑肌细胞向成骨样细胞分化，并可以在小鼠体内抑制动脉钙化的形成。通过项目的实施，我们发现高糖和高磷状态下内皮细胞通过旁分泌特定外泌体调控动脉中膜钙化的作用机制，揭示了动脉微环境内细胞间的对话模式，丰富了对脂肪-血管轴的认识，为认识动脉钙化发病机制开辟新的思路，为动脉钙化的防治提供新的治疗靶点。