219 Rock Inhibits Endothelial Migration in Disturbed flow Condition: A Potential Therapeutic Target in PCI
219 Rock Inhibits Endothelial Migration in Disturbed flow Condition: A Potential Therapeutic Target in PCI
复制标题
219 岩石在血流扰动条件下抑制内皮细胞迁移：PCI 中的潜在治疗靶点
DOI：
10.1136/heartjnl-2014-306118.219
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发表时间：
2014
期刊：
影响因子：
5.7
通讯作者：
Hsiao S
中科院分区：
文献类型：
--
作者：
Hsiao S
用于治疗冠状动脉疾病的支架展开引起内皮细胞（EC）的损伤和损失。壁切应力（WSS）是由流动的血液施加在血管壁上的机械力，已被提出作为EC迁移的主要调节剂，其通过涉及GTP酶的Rho家族的下游信号传导事件。然而，控制支架动脉再内皮化的生物力学机制仍然知之甚少。假设支架支柱可能通过诱导WSS中的局部扰动而阻碍受损内皮的修复。为了模拟植入支架的动脉，制造了一个基于聚二甲基硅氮烷的流动室，其脊（100 µ m高）垂直于流动方向。将汇合的EC单层接种在脊状室的一侧（或作为对照的非脊状室），然后使用Ibidi®泵系统暴露于流动的细胞培养基。用延时成像监测EC的迁移行为，并使用ImageJ®软件进行分析。计算速度和方向持续性（DP;迁移效率的度量;绝对距离与等高线长度的比率）。计算流体动力学（CFD）模型和粒子速度测定法用于确定流动室中的流动模式。CFD模型和活细胞成像表明，无脊室载玻片上的EC暴露于13 dyn/cm 2的均匀WSS，并平行于流动方向相对均匀地迁移（平均速度1.13 ± 0.20 µm/min; DP 0.59 ± 0.14）。相比之下，在CFD中的脊状幻灯片上观察到WSS的显着空间差异，在脊的拐角处出现高于生理水平（>70 dyn/cm 2）的显着峰值，并且紧邻脊的上游/下游存在独特的流动再循环区（-4 dyn/cm 2）。这些特征通过使用荧光标记的聚苯乙烯珠（2 μm直径）的粒子测速法进行验证。延时成像进一步显示中断EC迁移的山脊。具体而言，尽管EC可以在脊上迁移，但到达脊下游再循环区的EC以不均匀的方向迁移（DP 0.25 ± 0.06），并显示速度降低（0.78 ± 0.18 µm/min）。用ROCK抑制剂（Y27632或HA 1077）抑制RhoA/ROCK信号通路，通过显著升高DP（0.41 ± 0.04，p = 0.01）和速度（1.16 ± 0.09 µm/min，p = 0.01）促进EC在再循环区内的向前迁移。抑制RhoA/ROCK信号通路促进EC迁移和这些网站的内皮化。我们的数据表明，使用ROCK抑制剂的治疗可能会促进支架动脉的再内皮化;目前正在使用猪模型测试这一概念。
Stent deployment to treat coronary artery disease causes damage and loss of endothelial cells (EC). Wall shear stress (WSS), a mechanical force exerted by flowing blood on the vessel wall, has been proposed as a major regulator of EC migration through downstream signalling events involving the Rho family of GTPases. However, the biomechanical mechanisms that control re-endothelialisation in stented arteries remain poorly understood. It was hypothesised that stent struts may impede repair of injured endothelium by inducing localised disturbances in WSS.Anin vitroplatform was developed to assess the influence of surface features on EC migration under flow. To simulate a stented artery, a polydimethylsilozane-based flow chamber was fabricated with ridges (100 µm-high) positioned perpendicular to the flow direction. Confluent EC monolayers were seeded on one side of the ridged chamber (or on a non-ridged chamber as control) and were then exposed to flowing cell culture medium using the Ibidi® pump system. The migratory behaviour of EC was monitored with time-lapse imaging and analysed using ImageJ® software. Velocity and directional persistence (DP; a measure of migration efficiency; ratio of absolute distance to contour length) were calculated. Computational fluid dynamic (CFD) modelling and particle velocimetry were performed to determine flow patterns in the flow chamber.CFD modelling and live cell imaging indicated that EC on the non-ridged chamber slide were exposed to a uniform WSS of 13 dyn/cm2and 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 form 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).Disturbed WSS generated downstream from stent strut-like ridges prevented the forward migration of EC. Inhibition of the RhoA/ROCK signalling pathway promoted EC migration and endothelialisation 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.