Influences of heat stress on PAR1 protein expression and releases of endothelial MMP-1.

<p><b>(a)</b> Mono-layer HUVECs were cultured at 37°C or subjected to heat stress at 41°C for 2h, and time courses of PAR1 protein expression post heat stress were determined by western blot (n = 4, *P < 0.05, **P < 0.01, vs. 37°C). <b>(b)</b> Mono-layer HUVECs were subjected to heat stress at different temperatures for 2h, followed by recovery at 37°C for 4h, and PAR1 protein expressions were determined by western blot. Representative images of western blot and quantitative analysis of PAR1 protein normalized to β-actin were shown (n = 4, *vs. 37°C, ^#vs. 39°C, ^&vs. 41°C, P < 0.05). (c) Mono-layer HUVECs were subjected to heat stress (HS) at 41°C for 60 min or 120 min respectively, MMP-1 in medium were determined by western blot. Representative images of western blot and quantitative analysis of PAR1 protein normalized to β-actin were shown (n = 4, *vs. control, ^#vs. PBS in corresponding HS group, P < 0.05).</p

Heat Stress-Induced Disruption of Endothelial Barrier Function Is via PAR1 Signaling and Suppressed by Xuebijing Injection

<div><p>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.</p></div

Influences of heat stress on Trans-endothelial resistance (TER) and F-actin rearrangement.

<p><b>(a)</b> Mono-layer HUVECs were subjected to heat stress at 37°C, 39°C, 41°C, or 43°C for 2h, respectively, and TER was examined with an EVOM2 meter and a STX2 electrode at 37°C. Values are presented as a percentage relative to that prior to heat stress (n = 4, *P < 0.05, vs. 37°C, #P < 0.05, vs. 39°C). <b>(b)</b> Mono-layer HUVECs were subjected to heat stress at 37°C, 39°C, 41°C, or 43°C for 2h respectively, followed by recovery at 37°C for 2h, and then stained with DAPI (nuclei, blue) and rhodamine-phalloidin (F-actin, red). Representative F-actin, nuclei, and merged images were shown (scale bar: 20μm).</p

Heat stress increased phosphorylation levels for of moesin via PAR1 signal.

<p>Representative images of western blot and quantitative analysis of phosphorylation levels of moesin normalized to total moesin were shown (n = 4). <b>(a)</b> Mono-layer HUVECs were subjected to heat stress at 41°C for 2h, and time courses of moesin phosphorylation post heat stress were determined by western blot (*P < 0.05, **P < 0.01, vs. 37°C). <b>(b)</b> Mono-layer HUVECs were subjected to heat stress at different temperature for 2h, followed by recovery at 37°C for 2h, and phosphorylation levels of moesin was determined by western blot (*vs. 37°C, ^#vs. 39°C, ^&vs. 41°C, P < 0.05). <b>(c)</b> Mono-layer HUVECs were incubated with RWJ56110 (RWJ; 5μM), control IgG (IgG), anti-PAR1, PAR1 siRNA, or control siRNA, followed by heat stress at 39°C for 2h, and moesin phosphorylation was determined by western blot (*P < 0.05, vs. PBS at 37°C, ^#P < 0.01, vs. corresponding control at 41°C).</p

Influences of XBJ on PAR1 protein expression and phosphorylation levels of moesin.

<p>Mono-layer HUVECs were treated with PBS or XBJ at different concentrations for 30min, followed by heat stress for 2h at 41°C and recovery at 37°C for 2h. PAR1 protein <b>(a)</b> and phosphorylated moesin <b>(b)</b> were determined by western blot. Representative images of western blot and quantitative analysis of normalized protein levels were shown (n = 4, *P < 0.05, vs. 37°C, ^#P < 0.05, vs. PBS at 41°C).</p

PAR1 mediated endothelial hyper-permeability and F-actin rearrangement induced by heat stress.

<p>Mono-layer HUVECs were treated with RWJ56110 (RWJ; 5μM), PAR1 neutralizing antibody (anti-PAR1), control IgG (IgG), PAR1 siRNA, or negative control siRNA, followed by heat stress at 41°C for 2h. Mono-layer HUVECs were transfected with control siRNA or PAR1 siRNA respectively, or treated with RWJ, anti-PAR1 and IgG, followed by heat stress at 41°C or cultured at 37°C for 2h, PAR1 protein expressions were determined by western blot. <b>(a)</b> Representative images of western blot and quantitative analysis of PAR1 protein normalized to β-actin were shown (n = 4, * vs. PBS group, P < 0.05). <b>(b)</b> TER was determined similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118057#pone.0118057.g001" target="_blank">Fig. 1A</a> (n = 4, *P < 0.05, vs. PBS group, $ vs. control siRNA group, # vs. IgG group, P < 0.05). <b>(c)</b> Following above heat stress, the cells were recovered at 37°C for 2h, and nuclei (blue) and F-actin (red) were stained as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118057#pone.0118057.g001" target="_blank">Fig. 1B</a> noted, scale bar: 20μm.</p

XBJ and suppression of PAR1 diminished ALI associated with heatstroke (HS).

<p>Mice were treated with XBJ (4ml/kg, intraperitoneally), RWJ56110 (RWJ; 25μg/kg, intraperitoneally), and PAR1 neutralizing antibody (anti-PAR1; 2mg/kg, subcutaneously) for 30min, followed by heat insult until Tc reached 42.7°C, and then recovered at room temperature for 2h. <b>(a)</b> Lung wet weight (LWW) to lung dry weight (LDW) ratio, <b>(b)</b> Envans blue leakage, <b>(c)</b> BAL proteins, and <b>(d)</b> BAL leukocytes numbers were determined (n = 6, *vs. sham, ^#vs. HS, P < 0.05). <b>(e)</b> Representative images of lung stained with H&E (scale bar: 10μm).</p