m-Calpain antagonizes RhoA overactivation and endothelial barrier dysfunction under disturbed shear conditions.
OBJECTIVE
It has been reported that laminar shear flow (LF) improves barrier functions in vascular endothelial cells (ECs), whereas disturbed flow (DF) impairs the barrier. Our previous study showed that LF stimulus led to the activation of the 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 signalling.


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 a 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 increased RhoA activity and hyperpermeability in the aortic arch, which was accompanied by ROCK inhibitor-sensitive phosphorylation of downstream effecter LIM kinase 2 (LIMK2), stress fibre accumulation in endothelium and enhanced interendothelial gaps. Exposure of human umbilical vein endothelial cells to LF diminished RhoA activity; in contrast, DF facilitated the activity. siRNA-induced m-calpain silencing further accelerated the DF-induced RhoA overactivation, phosphorylation of LIMK2, and cytoskeletal rearrangement, resulting in barrier dysfunction in the cells.


CONCLUSIONS
Our findings revealed relatively high m-calpain expression levels in the inferior arch. The m-calpain activity antagonizes DF-induced overactivation of RhoA/ROCK/LIMK2 signalling and subsequent cytoskeletal rearrangement in ECs, which leads to barrier improvement.
Miyazaki
Takuro
T
Department of Pharmacology, School of Pharmacy, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. taku@pharm.showa-u.ac.jp
Honda
Kazuo
K
Ohata
Hisayuki
H
D000818:Animals; D001775:Blood Circulation; D002154:Calpain / Q000235:genetics / Q000502:physiology*; D002199:Capillary Permeability; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000737:chemistry; D004730:Endothelium, Vascular / Q000502:physiology*; D006801:Humans; D054461:Lim Kinases / Q000378:metabolism; D008297:Male; D051379:Mice; D010766:Phosphorylation; D012333:RNA, Messenger / Q000032:analysis; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors* / Q000502:physiology
D012333:RNA, Messenger; C102895:LIMK2 protein, human; D054461:Lim Kinases; D002154:Calpain; C449000:m-calpain; D020742:rhoA GTP-Binding Protein