Heat stress-induced disruption of endothelial barrier function is via PAR1 signaling and suppressed by Xuebijing injection.

Qiulin Xu,Jingxian Liu,Xiaohua Guo,Zhenglian Wang,Yanan Liu,Qiaobing Huang,Gengbiao Zhou,Lei Su,Cheng Huang

doi:10.1371/journal.pone.0118057

Abstract

Increased vascular permeability leading to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) is central to the pathogenesis of heatstroke. Protease-activated receptor 1 (PAR1), the receptor for thrombin, plays a key role in disruption of endothelial barrier function in response to extracellular stimuli. However, the role of PAR1 in heat stress-induced endothelial hyper-permeability is unknown. In this study, we measured PAR1 protein expression in heat-stressed human umbilical venous endothelial cells (HUVECs), investigated the influences of PAR1 on endothelial permeability, F-actin rearrangement, and moesin phosphorylation by inhibiting PAR1 with its siRNA, neutralizing antibody (anti-PAR1), specific inhibitor(RWJ56110), and Xuebijing injection (XBJ), a traditional Chinese medicine used for sepsis treatment, and evaluated the role of PAR1 in heatstroke-related ALI/ARDS in mice by suppressing PAR1 with RWJ56110, anti-PAR1and XBJ. We found that heat stress induced PAR1 protein expression 2h after heat stress in endothelial cells, caused the release of endothelial matrix metalloprotease 1, an activator of PAR1, after 60 or 120 min of heat stimulation, as well as promoted endothelial hyper-permeability and F-actin rearrangement, which were inhibited by suppressing PAR1 with RWJ56110, anti-PAR1 and siRNA. PAR1 mediated moesin phosphorylation, which caused F-actin rearrangement and disruption of endothelial barrier function. To corroborate findings from in vitro experiments, we found that RWJ56110 and the anti-PAR1 significantly decreased lung edema, pulmonary microvascular permeability, protein exudation, and leukocytes infiltrations in heatstroke mice. Additionally, XBJ was found to suppress PAR1-moesin signal pathway and confer protective effects on maintaining endothelial barrier function both in vitro and in vivo heat-stressed model, similar to those observed above with the inhibition of PAR1. These results suggest that PAR1 is a potential therapeutic target in heatstroke.

Highlights

Despite several decades of researches in pharmacologic therapy, heatstroke remains a major clinical problem with high morbidity and mortality and has a high incidence of multiple organ dysfunction syndromes (MODS)
We found that heat stress induced Protease-activated receptor 1 (PAR1) protein expression 2h after heat stress in endothelial cells, caused the release of endothelial matrix metalloprotease 1, an activator of PAR1, after 60 or 120 min of heat stimulation, as well as promoted endothelial hyper-permeability and F-actin rearrangement, which were inhibited by suppressing PAR1 with RWJ56110, anti-PAR1 and siRNA
Increased vascular permeability leading to pulmonary edema is one of critical components in the pathogenesis of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) [15,16]

Summary

Introduction

Despite several decades of researches in pharmacologic therapy, heatstroke remains a major clinical problem with high morbidity and mortality and has a high incidence of multiple organ dysfunction syndromes (MODS). Diffused endothelium injury and disruption of endothelial barrier function leading to vascular endothelial hyper-permeability are central to the pathogenesis of ALI/ARDS. It is important to clarify the mechanisms of endothelial hyper-permeability induced by heat stress, which will provide novel insights in pharmacologic treatment for heatstroke. Studies reported that PAR1 activated by matrix metalloprotease 1 (MMP-1) and thrombin regulates endothelial barrier function in some situations[2,4,5]. It remains unclear whether PAR1 is involved in heast stress-induced endothelial hyper-permeability

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