292Endothelial cell forward migration in a disturbed wall shear stress environment is promoted by ROCK inhibition
292Endothelial cell forward migration in a disturbed wall shear stress environment is promoted by ROCK inhibition
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292 ROCK 抑制促进内皮细胞在受干扰的壁剪切应力环境中向前迁移
DOI：
10.1093/cvr/cvu087.6
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发表时间：
2014
影响因子：
10.8
通讯作者：
Hsiao S
中科院分区：
文献类型：
--
作者：
Hsiao S
目的：支架置入治疗冠状动脉病变引起的内皮细胞损伤和丢失。EC迁移对损伤动脉的修复是由Rho家族GTPases协调的。它还受到壁剪切应力（WSS）的调节，这是一种由流动的血液施加在血管壁上的机械力，其机制尚不清楚。据推测，支架支撑物可能通过诱导WSS局部紊乱来阻碍受损内皮的修复。方法：制备与血流方向垂直的脊线（100 μm高）的腔室载玻片，模拟体外支架动脉。在脊状或非脊状（对照）室的一侧播种的融合EC单层暴露于流动培养基（Ibidi®系统）中。通过计算流体动力学（CFD）模型和粒子速度测定法确定了流型。使用ImageJ®软件进行活细胞成像和分析，可以定量EC迁移速度和定向持久性（DP）。结果：CFD模拟和活细胞成像结果表明，无脊腔载玻片上的EC暴露在13 dyn/cm2的均匀WSS下，并相对均匀地平行于流动方向迁移（平均速度1.13±0.20 μm/min; DP 0.59±0.14）。相比之下，CFD在脊状滑动上观察到显著的空间差异，在脊状的角落有明显的高于生理水平的峰值（约70 dyn/cm2），在脊状的上游/下游有明显的流动再循环区（-4 dyn/cm2）。用荧光标记聚苯乙烯珠（直径2 μm）的粒子测速法验证了这些特征。延时成像进一步揭示了山脊处EC迁移的中断。具体来说，虽然EC可以在高压脊上迁移，但从高压脊下游到达再环流区的EC的迁移方向不均匀（DP为0.25±0.06），且速度降低（0.78±0.18 μm/min）。ROCK抑制剂（Y27632或HA1077）抑制RhoA/ROCK信号通路，通过显著提高DP（0.41±0.04,p= 0.01）和流速（1.16±0.09 μm/min, p= 0.01）促进EC在再循环区正向迁移。结论：支架支架样脊下游的WSS受到干扰，阻止了EC的向前迁移。RhoA/ROCK信号通路的抑制促进了EC的迁移和这些位点的再种群。我们的数据表明，使用ROCK抑制剂治疗可能会促进支架动脉的再内皮化；这个概念目前正在用猪模型进行测试。
Purpose: Stent deployment to treat coronary artery disease causes damage and loss of endothelial cells (EC). Repair of injured arteries by EC migration is co-ordinated by Rho family GTPases. It is also regulated by wall shear stress (WSS), a mechanical force exerted by flowing blood on the vessel wall, via mechanisms that are poorly understood. It was hypothesised that stent struts may impede repair of injured endothelium by inducing localised disturbances in WSS. Methods: To simulate a stented artery in vitro, chamber slides were fabricated with ridges (100 μm high) positioned perpendicularly to the flow direction. Confluent EC monolayers seeded on one side of either ridged or non-ridged (control) chambers were exposed to flowing culture medium (Ibidi® system). Flow patterns were determined by computational fluid dynamic (CFD) modelling and particle velocimetry. Live cell imaging and analysis using ImageJ® software enabled quantitation of EC migration velocity and directional persistence (DP). Results: CFD modelling and live cell imaging indicated that EC on the non-ridged chamber slide were exposed to a uniform WSS of 13 dyn/cm2 and migrated relatively uniformly in parallel with the flow direction (average velocity 1.13±0.20 μm/min; DP 0.59±0.14). By contrast, significant spatial differences in WSS were observed over the ridged slide in CFD, with significant spikes above physiological levels (> 70 dyn/cm2) at the corners of the ridges and distinctive flow recirculation zone immediately upstream/downstream from the ridge (-4 dyn/cm2). These features were verified by particle velocimetry using fluorescently labelled polystyrene beads (2 μm diameter). Time-lapse imaging further revealed interrupted EC migration at the ridges. Specifically, although EC could migrate over the ridges, those that reached the recirculation zone downstream from the ridge migrated with non-uniform directionality (DP 0.25±0.06) and displayed a reduction in velocity (0.78±0.18 μm/min). Inhibition of the RhoA/ROCK signalling pathway with ROCK inhibitors (Y27632 or HA1077) promoted EC forward migration within the recirculation zone by significantly elevating DP (0.41±0.04, p= 0.01) and velocity (1.16±0.09 μm/min, p= 0.01). Conclusions: Disturbed WSS downstream from stent strut-like ridges prevented the forward migration of EC. Inhibition of the RhoA/ROCK signaling pathway promoted EC migration and re-population of these sites. Our data suggest that treatment using a ROCK inhibitor may promote re-endothelialisation of stented arteries; a concept that is currently been tested using a porcine model.