Mechanisms underlying endothelial barrier functions through mechanotransductions : regulatory role of m-calpain

目的：已有研究表明，层流剪切流（LF）可以改善血管内皮细胞（EC）的屏障功能，而扰动流（DF）则会损害屏障功能。我们之前的研究表明，LF刺激会导致EC中半胱氨酸蛋白酶m-钙蛋白酶的激活，从而影响RhoA活性。我们假设，m-钙蛋白酶参与剪切模式依赖的EC屏障的维护通过RhoA signaling.Methods和结果：m-钙蛋白酶的表达水平在内膜中的下方面的小鼠主动脉弓DF占主导地位的是高于那些在邻近地区。钙蛋白酶抑制剂（ALLM）诱导的跨内皮白蛋白渗透性升高在下弓中显著;此外，Rho激酶（ROCK）抑制剂（Y-27632）消除了这种升高。类似地，短干扰RNA（siRNA）诱导的m-钙蛋白酶沉默导致主动脉弓中RhoA活性增加和高渗透性，这伴随着下游效应物LIM激酶2（LIMK 2）的ROCK底物敏感性磷酸化、内皮中的应力纤维积累和内皮间间隙增强。暴露于LF的人脐静脉内皮细胞（HUVECs）减少RhoA活性，相反，DF促进活性。siRNA诱导的m-calpain沉默进一步加速DF诱导的RhoA过度激活、LIMK 2磷酸化和细胞骨架重排，导致细胞屏障功能障碍。我们的研究结果显示，在下弓中有相对较高的m-钙蛋白酶表达水平。m-钙蛋白酶活性拮抗DF诱导的RhoA/ROCK/LIMK 2信号过度激活和随后的EC细胞骨架重排，其发挥EC屏障改善。

Aims : It has been reported that laminar shear flow (LF) improves barrier functions in vascular endothelial cells (ECs) ; while disturbed flow (DF) impairs the barrier. Our previous study showed that LF stimulus led to the activation of cysteine protease, m-calpain, in ECs, which can influence RhoA activity. We hypothesized that m-calpain participates in the shear pattern-dependent EC barrier maintenance through RhoA signaling.Methods and results : m-calpain expression levels in the intima in the inferior aspect of mouse aortic arch where DF dominates were higher than those in adjacent regions. Elevation in transendothelial albumin permeability, which was induced by administration of calpain inhibitor (ALLM), was prominent in the inferior arch ; moreover, this elevation was abolished by Rho kinase (ROCK) inhibitor (Y-27632). Similarly, short interfering RNA (siRNA)-induced silencing of m-calpain resulted in increasing RhoA activity and hyper-permeability in the aortic arch, which was accompanied by ROCK inhibitor-sensitive phosphorylation of downstream effecter LIM kinase2 (LIMK2), stress fiber accumulation in endothelium and enhanced interendothelial gaps. Exposure of human umbilical vein endothelial cells (HUVECs) to LF diminished RhoA activity ; in contrast, DF facilitated the activity. siRNA-induced m-calpain silencing further accelerated the DF-induced RhoA over-activation, phosphorylation of LIMK2 and cytoskeletal rearrangement, resulting in barrier dysfunction in the cells.Conclusion : Our findings revealed a relatively high m-calpain expression levels in the inferior arch. The m-calpain activity antagonizes DF-induced over-activation of RhoA/ROCK/LIMK2 signaling and subsequent cytoskeletal rearrangement in ECs, which exerts EC barrier improvement.