BACKGROUND Vascular endothelial cell alignment in the direction of flow is an adaptive response that protects against aortic diseases such as atherosclerosis. The RhoGTPases are known to regulate this alignment. We have shown previously that ARHGAP18 in endothelial cells is a negative regulator of RhoC and its expression is essential in flow-mediated alignment. Depletion of ARHGAP18 inhibits alignment and results in the induction of a pro-inflammatory phenotype. In embryogenesis, ARHGAP18 was identified as a downstream effector of the Yes-associated protein, YAP, which regulates cell shape and size. METHODS We have used siRNA technology to deplete either ARHGAP18 or YAP in human endothelial cells. The in vitro studies were performed under athero-protective, laminar flow conditions. The analysis of YAP activity was also investigated, using high performance confocal imaging, in our ARHGAP18 knockout mutant mice. RESULTS We show here that loss of ARHGAP18, although decreasing the expression of YAP results in its nuclear localisation consistent with activation. We further show that depletion of YAP itself results in its activation as defined by an in increase in its nuclear localisation and an increase in the YAP target gene, CyR61. Depletion of YAP, similar to that observed for ARHGAP18 depletion, results in loss of endothelial cell alignment under high shear stress mediated flow and also in the activation of NFkB, as determined by p65 nuclear localisation. In contrast, ARHGAP18 overexpression results in upregulation of YAP, its phosphorylation, and a decrease in the YAP target gene Cyr61, consistent with YAP inactivation. Finally, in ARHGAP18 deleted mice, in regions where there is a loss of endothelial cell alignment, a situation associated with a priming of the cells to a pro-inflammatory phenotype, YAP shows nuclear localisation. CONCLUSION Our results show that YAP is downstream of ARHGAP18 in mature endothelial cells and that this pathway is involved in the athero-protective alignment of endothelial cells under laminar shear stress. ARHGAP18 depletion leads to a disruption of the junctions as seen by loss of VE-Cadherin localisation to these regions and a concomitant localisation of YAP to the nucleus.

中文翻译：

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需要YAP和RhoC调节剂ARHGAP18来调节流量依赖性内皮细胞的排列。

背景技术沿流动方向的血管内皮细胞排列是一种适应性反应，其可预防诸如动脉粥样硬化的主动脉疾病。已知RhoGTP酶调节这种比对。先前我们已经表明，内皮细胞中的ARHGAP18是RhoC的负调节剂，其表达在流介导的比对中至关重要。ARHGAP18的消耗会抑制排列并导致促炎表型的诱导。在胚胎发生过程中，ARHGAP18被确定为是Yes相关蛋白YAP的下游效应子，它调节细胞的形状和大小。方法我们已使用siRNA技术消耗人内皮细胞中的ARHGAP18或YAP。在动脉粥样硬化保护层流条件下进行了体外研究。还对YAP活性进行了分析，在我们的ARHGAP18基因敲除突变小鼠中使用高效共聚焦成像。结果我们在这里显示了ARHGAP18的缺失，尽管降低了YAP的表达会导致其核定位与激活一致。我们进一步显示，YAP本身的耗竭导致其激活，如其核定位的增加和YAP目标基因CyR61的增加所定义。YAP的耗尽与ARHGAP18耗尽所观察到的相似，在高剪切应力介导的血流下会导致内皮细胞排列的丧失，并且还会导致NFkB的活化（如p65核定位所确定）。相反，ARHGAP18过表达导致YAP的上调，其磷酸化和YAP靶基因Cyr61的减少，这与YAP失活相一致。最后，在ARHGAP18删除的小鼠中，在内皮细胞排列丢失的区域（与将细胞启动至促炎表型有关的情况）中，YAP显示核定位。结论我们的结果表明，YAP在成熟的内皮细胞中位于ARHGAP18的下游，并且该途径参与层状剪切应力下内皮细胞的动脉粥样硬化保护排列。从VE-钙黏着蛋白定位到这些区域的损失以及YAP到细胞核的定位中可以看出，ARHGAP18的消耗导致连接的破坏。结论我们的结果表明，YAP在成熟的内皮细胞中位于ARHGAP18的下游，并且该途径参与层状剪切应力下内皮细胞的动脉粥样硬化保护排列。从VE-钙黏着蛋白定位到这些区域的损失以及YAP到细胞核的定位中可以看出，ARHGAP18的消耗导致连接的破坏。结论我们的结果表明，YAP在成熟的内皮细胞中位于ARHGAP18的下游，并且该途径参与层状剪切应力下内皮细胞的动脉粥样硬化保护排列。从VE-钙黏着蛋白定位到这些区域的损失以及YAP到细胞核的定位中可以看出，ARHGAP18的消耗导致连接的破坏。