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

Xu Q, Liu J, Wang Z, Guo X, Zhou G, Liu Y, Huang Q, Su L - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of ICU, General Hospital of Guangzhou Military Command, Key Laboratory of Tropical Zone Trauma Care and Tissue Repair of PLA, Guangzhou, China; Postdoctoral Workstation, Huabo Bio-pharmaceutical Research Institute, Guangzhou, China.

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.

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PAR1 mediated endothelial hyper-permeability and F-actin rearrangement induced by heat stress.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. (a) 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) TER was determined similar to Fig. 1A (n = 4, *P < 0.05, vs. PBS group, $ vs. control siRNA group, # vs. IgG group, P < 0.05). (c) Following above heat stress, the cells were recovered at 37°C for 2h, and nuclei (blue) and F-actin (red) were stained as Fig. 1B noted, scale bar: 20μm.
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pone.0118057.g003: PAR1 mediated endothelial hyper-permeability and F-actin rearrangement induced by heat stress.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. (a) 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) TER was determined similar to Fig. 1A (n = 4, *P < 0.05, vs. PBS group, $ vs. control siRNA group, # vs. IgG group, P < 0.05). (c) Following above heat stress, the cells were recovered at 37°C for 2h, and nuclei (blue) and F-actin (red) were stained as Fig. 1B noted, scale bar: 20μm.

Mentions: To examine whether PAR1 was involved in heat stress-induced elevation of endothelial permeability, we decreased PAR1 protein expressions in endothelial cells using siRNA transfection (Fig. 3A), or inhibited PAR1 activation with its specific inhibitor, RWJ56110, and its neutralizing antibody, ATAP2 (anti-PAR1). We found RWJ56110, anti-PAR1 and PAR1 siRNA attenuated heat stress-induced elevation of endothelial permeability, suggesting disruption of endothelial barrier function induced by heat stress is partially dependent on PAR1 (Fig. 3B, S1A).


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

Xu Q, Liu J, Wang Z, Guo X, Zhou G, Liu Y, Huang Q, Su L - PLoS ONE (2015)

PAR1 mediated endothelial hyper-permeability and F-actin rearrangement induced by heat stress.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. (a) 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) TER was determined similar to Fig. 1A (n = 4, *P < 0.05, vs. PBS group, $ vs. control siRNA group, # vs. IgG group, P < 0.05). (c) Following above heat stress, the cells were recovered at 37°C for 2h, and nuclei (blue) and F-actin (red) were stained as Fig. 1B noted, scale bar: 20μm.
© Copyright Policy
Related In: Results  -  Collection

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pone.0118057.g003: PAR1 mediated endothelial hyper-permeability and F-actin rearrangement induced by heat stress.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. (a) 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) TER was determined similar to Fig. 1A (n = 4, *P < 0.05, vs. PBS group, $ vs. control siRNA group, # vs. IgG group, P < 0.05). (c) Following above heat stress, the cells were recovered at 37°C for 2h, and nuclei (blue) and F-actin (red) were stained as Fig. 1B noted, scale bar: 20μm.
Mentions: To examine whether PAR1 was involved in heat stress-induced elevation of endothelial permeability, we decreased PAR1 protein expressions in endothelial cells using siRNA transfection (Fig. 3A), or inhibited PAR1 activation with its specific inhibitor, RWJ56110, and its neutralizing antibody, ATAP2 (anti-PAR1). We found RWJ56110, anti-PAR1 and PAR1 siRNA attenuated heat stress-induced elevation of endothelial permeability, suggesting disruption of endothelial barrier function induced by heat stress is partially dependent on PAR1 (Fig. 3B, S1A).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of ICU, General Hospital of Guangzhou Military Command, Key Laboratory of Tropical Zone Trauma Care and Tissue Repair of PLA, Guangzhou, China; Postdoctoral Workstation, Huabo Bio-pharmaceutical Research Institute, Guangzhou, China.

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.

Show MeSH
Related in: MedlinePlus