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

目的：层流剪切流（LF）改善血管内皮细胞（ECs）的屏障功能；而扰动流（DF）则破坏了屏障。我们之前的研究表明，LF刺激导致ECs中半胱氨酸蛋白酶m-calpain的激活，从而影响RhoA的活性。我们假设m-calpain通过RhoA信号参与剪切模式依赖的EC屏障维持。方法与结果：小鼠主动脉弓下侧以DF为主的内膜中m-calpain表达水平高于邻近区域。钙蛋白酶抑制剂（calpain inhibitor， ALLM）诱导的跨内皮白蛋白通透性升高在下弓明显；此外，Rho激酶（ROCK）抑制剂（Y-27632）可以消除这种升高。同样，短干扰RNA （siRNA）诱导的m-calpain沉默导致主动脉弓RhoA活性增加和高通透性，同时伴随着ROCK抑制剂对下游效应物LIM激酶2 （LIMK2）的敏感磷酸化，内皮细胞中的应激纤维积累和内皮间间隙增加。人脐静脉内皮细胞（HUVECs）暴露于LF可降低RhoA活性；相反，DF促进了活动。sirna诱导的m-calpain沉默进一步加速了df诱导的RhoA过激活、LIMK2磷酸化和细胞骨架重排，导致细胞屏障功能障碍。结论：我们的研究结果显示下弓中m-calpain的表达水平相对较高。m-calpain活性可拮抗df诱导的EC中RhoA/ROCK/LIMK2信号的过度激活和随后的细胞骨架重排，从而改善EC屏障。

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.