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Therapeutic hypothermia mitigates the sepsis-increased permeability in EA. hy926 cells by preserving Rap1 expression

Background: To determine the effect and potential mechanisms of therapeutic hypothermia (TH) on the permeability of septic cells. Main methods: Human EA. hy926 cells were transfected with, or without, control or Rap1-specic siRNA and treated with 2 µg/ml of lipopolysaccharide (LPS), followed by cultured in normal temperature (NT) or a temporary therapeutic hypothermia (TH) for 10 h. The cellular permeability of each group of cells was determined by transwell permeability assay and the relative levels of ras-proximate-1 (Rap1), RhoA (a small GTP enzyme of the Rho family), Ve-cadherin expression and myosin light chain (MLC) phosphorylation were quantied by western blot and immunouorescent assays. Results: Compared with the control group, LPS stimulation increased cellular permeability, which was enhanced by Rap1 silencing in EA. hy926 cells under a NT condition, but signicantly mitigated by TH. Furthermore, LPS up-regulated RhoA expression and MLC phosphorylation, but reduced Rap1 and Ve-cadherin expression, which were also enhanced by Rap1 silencing, but signicantly mitigated by TH. Immunouorescent analyses indicated that LPS signicantly increased phosphorylated MLC, but decreased Ve-cadherin expression, which were deteriorated by Rap1 silencing, but signicantly mitigated by TH in EA. hy926 cells. Conclusions: TH signicantly mitigated the sepsis-increased permeability of EA. hy926 cells by enhancing the Rap1 expression to attenuate the RhoA/MLC signaling.


Background
Sepsis is a life-threatening condition and characterized by aberrant host responses to infection, leading to multiple organ dysfunction [1]. There are 1.5 million cases of sepsis in the United States yearly, accounting for about 30% of hospital deaths [2] and patients with septic shock have a mortality rate up to 50% [3]. The economic impact of sepsis on the United States is estimated for more than $20 billion yearly [4]. Although extensive studies have led to a great approach there is no effective therapies to control septic shock in the clinic [5]. During the pathogenesis of septic shock, infectious and in ammatory factors destroy endothelial barriers, damage vascular endothelial cells and activate the coagulation system, together with immunosuppression and immunoregulation, contributing to multiple organ dysfunction [6]. Our previous study has shown that temporary therapeutic hypothermia (TH) can regulate immune response, protect epithelial cells and reduce vascular endothelial injury, suggesting that TH may be a potential therapeutic strategy for septic shock [7]. However, whether and how TH regulate the sepsisinduced endothelial cell permeability have not been clari ed.
Sepsis can result in severe endothelial dysfunction [8,9], leading to increased permeability and tissue edema, which contribute to organ failure and mortality [10]. Cyclic adenosine monophosphate (cAMP) is an important intracellular signaling molecule that regulates the endothelial barrier function [11]. The cAMP can promote ras-proximate-1 (Rap1) and Ve-cadherin expression to enhance endothelial barrier function [12]. The Rap1 can stabilize epithelial cell-cell connections [13]. Furthermore, Rap1 can inhibit the RhoA, a small GTP enzyme of the Rho family, and Rho-associated coiled coil-containing protein kinase (ROCK) signaling to preserve epithelial barrier function [14,15]. Actually, ROCK can induce actin contraction, inhibit myosin light chain (MLC) phosphatase, prevent MLC dephosphorylation to increase MLC phosphorylation and vascular endothelial permeability [16,17]. Accordingly, increased Rap1 activity can inhibit the RhoA/ROCK/MLC signaling to preserve endothelial barrier function and decrease endothelial permeability [15,[18][19][20][21] We hypothesize that TH may mitigate the sepsis-increased cellular permeability by enhancing Rap1 activity to inhibit the RhoA /MLC signaling. To address the hypothesis, we employed human Ea. hy926 and Rap1 silenced Ea. hy926 cells to test the impact of TH on the LPS-induced cellular permeability and LPS-modulated Rap1, Ve-cadherin, RhoA expression and MLC phosphorylation. Our ndings indicated that TH enhanced Rap1 and Ve-cadherin to mitigate the LPS-increased RhoA expression, MLC phosphorylation and cellular permeability in Ea. hy926 cells.

TH mitigates the LPS-increased cell permeability
First, we tested whether TH could modulate the LPS-increased cell permeability in EA. Hy926 cells using transwell chamber assay. As shown in Fig. 1, in comparison with that in the control cells, LPS stimulation signi cantly increased cell permeability (** P < 0.01), which was signi cantly mitigated by TH (# P < 0.05) although the permeability in the TH group remained signi cantly higher than that in the control (* P < 0.05). Furthermore, transfection with control siRNA did not alter the permeability in EA. Hy926 cells while Rap1 silencing signi cantly elevated the LPS-increased cellular permeability (# P < 0.05) and it signi cantly reduced the effect of TH (& P < 0.05). Hence, TH mitigated the LPS-increased cellular permeability in EA. Hy926 cells in a Rap1-dependent manner.
TH signi cantly enhances the Rap1 expression to attenuate the LPS-enhanced RhoA/MLC signaling in EA. Hy926 cells.
The Rap1 can modulate the RhoA/MLC signaling and Ve-cadherin expression [14,15]. Accordingly, we characterized the relative levels of Ve-cadherin and RhoA expression and MLC phosphorylation in the different groups of EA. Hy926 cells by western blot. As shown in Fig. 2, in comparison with that in the control group, LPS stimulation signi cantly decreased Rap1 and Ve-cadherin expression, but increased RhoA expression and MLC phosphorylation in the NT group of EA. Hy926 cells (** P < 0.01 for all), which were further enhanced in the Rap1 silenced EA. Hy926 cells (# P < 0.05 or ## P < 0.01). In contrast, TH signi cantly enhanced the Rap1 and Ve-cadherin expression, relative to that in the NT condition (# P < 0.05 or ## P < 0.01), which were decreased in the Rap1 silenced EA. Hy926 cells (& P < 0.05 or && P < 0.01). Furthermore, TH also decreased the RhoA expression and MLC phosphorylation in EA. Hy926 cells, relative to that in the NT condition (# P < 0.05), while the therapeutic effect were weakened by Rap1speci c siRNA1 and Rap1-speci c siRNA2 silence respectively .
Immuno uorescent analyses indicated that compared with that in the control, LPS stimulation signi cantly decreased Ve-cadherin expression (** P < 0.01), but increased phosphorylated MLC signals even in the Rap1-silenced EA. Hy926 cells under a NT condition (** P < 0.01) (Fig. 3). In contrast, TH signi cantly mitigated the effects of LPS on Ve-cadherin expression and phosphorylated MLC signaling in EA. Hy926 cells (# P < 0.05), and the effect of TH on Ve-cadherin expression was signi cantly reduced in the Rap1 siRNA2-silenced EA. Hy926 cells (& P < 0.05). Thus, TH signi cantly enhanced the Rap1 expression to attenuate the LPS-enhanced RhoA/MLC signaling in EA. Hy926 cells.

Discussion
Sepsis can increase cell permeability, and lead to capillary leakage syndrome (CLS), which causes severe hypoproteinemia, hypovolemia, tissue hypoperfusion, edema, shock and multiple organ dysfunction syndrome [22][23][24]. Our previous studies have shown that TH can improve the permeability of septic cells [7]. In this study, we found that TH signi cantly mitigated the LPS-increased permeability in EA. Hy926 cells. Given that cellular permeability is crucial for the pathogenesis of CLS the decreased permeability by TH suggests that TH may be valuable for control of septic shock, like VEGF (vascular endothelial growth factor) antagonist [25], nitric oxide inducer [26], inhibition of MLC phosphorylation [27], protection of cell connection [28].
The Rap1/Rho/MLC signaling is a critical regulator of cell permeability. We found that compared with the control group, LPS stimulation signi cantly decreased Rap1 and Ve-cadherin expression, but increased RhoA expression and MLC phosphorylation in EA. hy926 cells, which were enhanced by Rap1 silencing. In contrast, TH signi cantly mitigated the effects of LPS by preserving Rap1 and Ve-cadherin expression and reducing the LPS-stimulated RhoA expression and MLC phosphorylation in EA. hy926 cells. Such inhibitory effects of TH were attenuated by Rap1 silencing. Such novel data demonstrated that TH mitigated the LPS-increased permeability by preserving Rap1 expression in EA. hy926 cells.
The available data indicated that Rap1 were crucial for cell-matrix adhesion and cell-cell adhesion. Rap1 inhibits the RhoA activity, which can activate ROCK to promote non-muscle myosin II activation and actin contraction, and stress ber formation to enhance local adhesion [16]. In addition, activated ROCK can promote MLC phosphorylation to increase permeability [18]. Therefore, Rap1 can reduce vascular permeability under both resting and stress conditions and dynamically regulate the barrier function of endothelial cells [29]. During the process of sepsis, Rap1 inhibits RhoA and Rac activities to preserve endothelial cell permeability [30]. Actually, inhibition of Rap1 can increase vascular permeability to deteriorate ARDS (acute respiratory distress syndrome) [31] while enhancement of Rap1 activity can accelerate the recovery of LPS-induced lung injury and vascular endothelial cell function [32].
Previous studies have shown that many factors, such as histamine, bradykinin, platelet activating factor and thrombin, increase vascular permeability by regulating Ve-cadherin expression in endothelial cells [16,17,33,34]. We found that LPS stimulation increased the MLC phosphorylation in EA. hy926 cells, which explained why LPS increased cell permeability [35], consistent with previous observations [36,37]. However, TH mitigated the LPS-decreased Ve-cadherin expression such data indicated that TH preserved Rap1 expression to inhibit the LPS-increased RhoA expression and MLC phosphorylation and LPSdecreased Ve-cadherin expression in EA. hy926 cells [14,20,21,38]. Thus, the Rap1/RhoA/MLC signaling may be valuable targets for intervention of LPS-induced high endothelial cell permeability. We are interested in further investigating the therapeutic effects of TH in vivo and the potential mechanisms underlying the action of TH during the process of septic shock.

Conclusion
Our data indicated that TH signi cantly mitigated the LPS-increased cell permeability in EA. hy926 cells by preserving Rap1 and Ve-cadherin expression to reduce the LPS-increased RhoA expression and MLC phosphorylation. Therefore, TH may be valuable for control of septic shock by mitigating cell permeability.

Methods
Human EA. Hy926 cells were from Zhong Qiao Xin Zhou Biotechnology(ZQ0079) and cultured in 10% FBS DMEM at 37℃ in 5% CO2. The cells (5 × 104 cells/well) were cultured in 6-well plates for 24 h and treated with, or without, lipopolysaccharide (2 µg/ml) at 37℃ for 4 h. The cells were further cultured at 32℃ (therapeutic hypothermia, TH) for 4 h, followed by cultured at 37℃ for 2 h. The control cells were cultured at 37℃ (normal temperature, NT) for 10 h.

Transwell analysis of cell permeability
The impact of TH on LPS-increased cell permeability in EA. Hy926 cells was determined as described previously [39]. Brie y, EA. Hy926 cells (5 × 104 cells/well) were cultured in 24-well transwell plates up to formation of a monolayer and stimulated in triplicate with LPS (2 µg/ml) for NT or TH culture.

Western blot
The relative levels of Rap1, RhoA, Ve-cadherin, MLC expression and MLC phosphorylation in individual groups of cells were quanti ed by Western blot. Brie y, the different groups of cells were harvested and lyzed in lysis buffer, followed by centrifuged. After quanti cation of protein concentrations, the cell lysates (50 µg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and transferred onto polyvinylidene di uoride (PVDF) membranes. After being blocked, the membranes were probed with primary antibodies including anti-Rap1 (Abcam, ab181858

Statistical analysis
Data are expressed as the mean ± SD. The difference among groups was analyzed ANOVA and post hoc least signi cant test and the difference between groups was determined by Student's T test using SPSS software window 17. Statistical signi cance was de ned when a P-value of < 0.05. Availability of data and materials

Abbreviations
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests. No potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Funding
No.
Authors' contributions SY and DW made conception and design of the study and completed the experiment. SY and DW analyzed and interpreted the data. SY drafted the manuscript. All authors read and approved the nal manuscript.   TH mitigates the LPS-modulated Ve-cadherin expression and MLC phosphorylation in EA. Hy926 cells.

Tables
Following transfected with control or Rap1-speci c siRNA and treated with, or without, LPS, the different groups of EA. Hy926 cells were cultured in NT or TH for 10 h and stained with uorescent anti-Vecadherin or anti-phosphorylated MLC, followed by photoimaged under a uorescent microscope. Data are representative images (magni cation x 200) or expressed as the mean ± SD of each group of cells from three separate experiments. *P < 0.05, **P < 0.01 vs. the NT control group; #P < 0.05, ##P < 0.01 vs. the NT LPS group; &P < 0.05, &&P < 0.01 vs. the TH LPS group.