TLR4 Activation of Small GTPase RhoA Signaling Mediates Endotoxin-Induced Endothelial Barrier Dysfunction

Abstract

Abstract Sepsis is an uncontrolled activation of immune response, leads to multi-organ failure and high rate of mortality. Studies show the role of increase in endothelial activation and barrier dysfunction significantly contribute to the pathophysiology in sepsis. Endothelial cells form a tight barrier between blood and underlying tissue, and thus maintain an anti-inflammatory milieu. Here, we investigated the role of small GTPase, RhoA activation in regulating the increase in human coronary artery endothelial (HCAE) permeability in response to TLR4-receptor agonist, endotoxin lipopolysaccharide (LPS). Endothelial permeability was determined by determining the influx of Evans blue-labeled albumin across endothelial monolayer on 0.4μ Transwell filters. Our data show that LPS induced RhoA activation and resulted in increase in HCAE cell monolayer permeability. Thus, we tested the hypothesis that LPS mediated induction of RhoA activates pro-inflammatory signaling and eliciting endothelial barrier dysfunction. Knockdown of RhoA using siRNA approach abrogated LPS induced increase in endothelial permeability. Rho-associated kinase (ROCK) is an effector of the RhoA. To further determine the molecular mechanism involved downstream of RhoA activation, we pretreated the cell with ROCK inhibitor (Y-27632) and we found that LPS failed to induce endothelial permeability in cells treated with Y-27642. In conclusion, our study shows that LPS induces activation of the RhoA - ROCK signaling pathway, which leads to increase in endothelial permeability and barrier dysfunction. Therefore, ROCK emerges as a potential drug target to treat sepsis induced endothelial permeability and inflammation.