血管支架再内皮化细胞来源及其协同作用参与支架段血管修复的力学生物学机制研究

The implantation of vascular stent introduces a mechanical injure to vessle wall, and the subsequent re-endothlialzation process depends on both of the neighboring vascular endothelial cells (VECs) migration and circulating bone marrow-derived endothelial progenitor cells (EPCs) homing and adhesion. However, the synergistic effect and relative molecular mechanism of two cell population contribute to re-endothlialzation repair are unclear. In this project, the optimized flow chamber system and Tie2-Lac (Tie2-GFP) ApoE-/- transgentic mice were applied to simulate deep injure induced by stent implatation and expansion in vitro and in vivo. Also, the fluorescence labeling and β-gel staining were used to recognize VECs and EPCs, respecively, and to examine the time course of re-endothelialization. In addition, the expression levels and activated sequence of critical proteins in PI3K/AKT/eNOS signaling events would be studied to explore the mechanobiologic mechanism of synergistic effect of VECs and EPCs in re-endothlialzation process. We try to find the answers of the following questions: (1) what kind of endothelial cell repopulation mainly contributes to the stented re-endothelialization? Do adjacent VECs migration and circulating EPCs homing show a synergistic effect on the re-endothelialization repair? (2) How does the fluid shear stress induce the interaction between VECs and EPCs under the condition of deep injured blood vessel? (3) What’s mechanism in regulating synergistic effect of two endothelial cell populations? The finding of this project would help us to understand the mechanobiologic mechanism of re-endothelialization repair, and provide the sugestions for the design and improvement of cardiovascular implants.

血管支架植入对血管造成力学损伤，其再内皮化修复主要依赖于血管内皮细胞（VECs）迁移和内皮祖细胞（EPCs）归巢黏附，然而两种细胞参与再内皮化的协同作用及影响机制尚未明确。本项目拟采用优化的流动腔系统以及ApoE-/-转基因小鼠模型，体内外模拟支架扩张对血管的不同损伤程度；采用免疫荧光、β-gel标记等方法，实时观测再内皮化进程；测定PI3K/AKT/eNOS信号轴关键蛋白表达规律，明确EPCs、VECs之间的相互作用及共同参与再内皮化修复的力学生物学机制。从而回答以下科学问题：①参与支架段血管再内皮化修复的细胞主要来源于VECs还是EPCs？两者是否具有协同作用，各自贡献多少？②血流剪切力、血管损伤程度等不同影响因素如何介导VECs和EPCs的相互作用及再内皮化进程？③调控两者协同作用的分子机制？本项目研究结果有助于揭示血管修复的力学生物学机制，对心血管介入材料的设计改进具有重要的指导意义。

心脑血管疾病是当今威胁人类健康最严重的疾病。冠脉血管支架的植入和扩张对血管造成力学损伤，加速支架植入受损内膜的再内皮化是抑制支架内再狭窄、降低晚期支架内血栓形成的关键。血管支架的再内皮化修复主要依赖于受损内膜血管内皮细胞（VECs）的迁移和来源于骨髓的内皮祖细胞（EPCs）的归巢黏附，然而关于两种细胞参与再内皮化修复的协同作用及影响机制尚未明确。本项目探索了血流剪切力变化、支架扩张对血管损伤程度等影响条件下，VECs黏附和迁移协同作用对支架段血管再内皮化修复的贡献程度及其分子机制。我们通过优化设计的新型流动腔和载玻片模型模拟支架扩张对血管壁的不同损伤程度；分别转染绿色和红色荧光蛋白标记黏附和迁移的VECs，观察血管植入损伤模型邻近VECs的迁移和循环VECs的黏附，评价VECs迁移/黏附参与血管支架植入后内皮修复的各自贡献。我们的研究结果表明：（1）支架植入对支架段上游的血流动力学影响较小，其下游产生流动分离区域，出现扰动流，该扰动区域大小依赖于支架植入后损伤尺度和凸起高度。（2）不同程度和尺度损伤模型下，FSS介导的再内皮化细胞主要来源于邻近VECs迁移，循环VECs黏附贡献较少；而随着损伤尺度的增加，黏附贡献增加，呈现尺度依赖性。（3）FAK抑制剂显著抑制VECs迁移及丝状伪足的形成，伴随着PI3K/AKT/Rho GTPase表达显著下调，证实了VECs迁移和黏附协同作用完成内皮修复依赖于FAK-PI3K-AKT-Rho GTPases 信号通路。（4）构建转基因ApoE-/-小鼠动物在体模型，进一步验证体内支架植入后的再内皮化进程。本项目的研究结果有助于从新的角度阐述血管损伤程度对血流动力学及细胞黏附/迁移的影响，揭示血管支架介入治疗后的再内皮化修复机制，给国内外众多从事血管生物力学和心血管植入物表面改性研究的科研工作者以新的启示。