骨成型蛋白4通过Kv1.5调控平滑肌细胞增殖迁移促进冠脉介入术后再狭窄的机制

Neointimal hyperplasia is the major cause of restenosis after percutaneous coronary intervention, which is associated with the proliferation and migration of vascular smooth muscle cells (VSMCs). It is reported that bone morphogenetic protein-4 (BMP-4) influencen blood vessel behavior. Our previous study showed balloon injury significantly stimulated arterial neointimal formation in SD rat, compared with sham carotids, BMP-4 expression and serum activity were markedly upregulated, BMP-4 inhibitor prevented neointimal formation; BMP-4 promoted proliferation and migration of VSMCs, BMP-4 inhibitor Noggin inhibited VSMCs proliferation and migration which induced by BMP-4. Therefore, We put forward the hypothethsis : BMP-4 promoted neointimal hyperplasia through increasing proliferation and migration of VSMCs. we will use a series of molecularbiological and electrophysiological techniques to carry out the following research: ①The effect of BMP-4 on neointimal hyperplasia after balloon injury; ②The role of BMP-4 which have been shown to regulate cell cycle and MMPs expression, in proliferation and migration of VSMCs; ③ The role of inhibition of BMP-4 protein expression in neointimal hyperplasia after injury. This project will provided the new drug target and theoretical basis for prevention and treatment restenosis after percutaneous coronary intervention.

经皮冠状动脉介入（PCI）术后管腔再狭窄发生率高。血管内膜增生是其重要的病理基础。血管平滑肌细胞（VSMCs）增殖迁移可引起内膜增生。骨成型蛋白4（BMP-4）参与调控血管功能。前期研究表明大鼠颈动脉损伤后出现内膜增生，成熟型和非成熟型BMP-4表达增高，血浆BMP-4活性升高，拮抗BMP-4活性抑制血管损伤后内膜增生；BMP-4促进VSMCs增殖迁移，BMP-4抑制剂拮抗由BMP-4表达升高引起的VSMCs增殖迁移。因此我们提出研究课题：研究BMP-4通过调控VSMCs增殖迁移促进血管损伤后内膜增生的机制。本课题将应用电生理学和分子生物学等手段阐明①BMP-4表达变化在血管损伤后内膜增生中的生物学意义；②BMP-4促进VSMCs增殖迁移的分子机制（BMP-4-Kv1.5-CaMKⅡδ）；③拮抗BMP-4对损伤后血管内膜增生的防治作用。本课题为PCI术后再狭窄的防治提供新靶点和理论依据。

血管平滑肌细胞异常增殖迁移及内皮细胞修复滞后是经皮冠状动脉介入术（PCI）后再狭窄的病理基础。结果显示，血管损伤后出现内膜增生，伴有平滑肌细胞表型转化和内皮修复滞后，损伤血管处及血清中BMP-4表达显著升高。病理检测表明给予BMP-4抑制剂DMH1后，BMP-4表达降低，抑制血管平滑肌细胞转型、胶原分泌、活性氧生成以及促进内皮迁移修复，抑制内膜增生进展。体外培养HA-VSMCs细胞，结果表明BMP-4诱导细胞表型转化，促进细胞迁移；BMP-4通过诱导MCT-4表达增加培养基乳酸浓度，诱导培养基酸化；乳酸诱导细胞表型转化，促进细胞迁移；应用MCT-4 siRNA靶向抑制MCT-4表达后，显著抑制BMP-4诱导的细胞表型转化及迁移；BMP-4通过激活smad-4诱导MCT-4表达，增加培养基中乳酸浓度；BMP-4促进HA-VSMCs细胞葡萄糖摄取，LDHA表达；BMP-4促进NOX-4表达，增加细胞内钙离子浓度。体外培养HUVEC细胞，结果表明BMP-4促进细胞迁移，增加细胞内ROS生成及FoXO-3表达；ROS清除剂可以逆转BMP-4对细胞迁移的抑制作用；应用FoXO-3 siRNA靶向抑制FoXO-3表达，显著抑制BMP-4诱导的ROS生成并可以逆转BMP-4对细胞迁移的抑制作用；BMP-4诱导细胞SOD-1、Keap-1、Nrf-2表达，细胞转染FoXO-3 siRNA后，此作用被抑制。结论：1）血管损伤时BMP-4表达升高，抑制体内BMP-4表达，抑制平滑肌细胞转型，促进内皮细胞迁移修复，抑制内膜增生；2）BMP-4通过smad-4增加MCT-4表达，促进乳酸释放，诱导细胞外微环境酸化，促进平滑肌转型和迁移；3）BMP-4通过FoXO-3/Keap-1/Nrf-2途径调控氧化应激水平，抑制内皮细胞迁移修复。该项目为PCI术后血管再狭窄提供新的治疗思路及治疗靶点。