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

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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.