Therapeutic Hypothermia Mitigates the Sepsis-Increased Permeability in EA. hy926 Cells by Preserving Rap1 Expression

To determine the effect and potential mechanisms of therapeutic hypothermia (TH) on the permeability of septic cells. Human EA. hy926 cells were transfected with, or without, control or ras-proximate-1 (Rap1)-specific siRNA and treated with 2 μg/mL of lipopolysaccharide (LPS). The cells were cultured in normal temperature (NT) or a temporary TH for 10 hours. The cellular permeability of each group of cells was determined by transwell permeability assay. The relative levels of Rap1, RhoA (a small GTP enzyme of the Rho family), VE-cadherin expression, and myosin light chain (MLC) phosphorylation were quantified by Western blot and immunofluorescent assays. Compared with the control group, LPS stimulation increased cellular permeability in EA. hy926 cells under an NT condition, but significantly mitigated by TH. The effect of TH decreased after Rap1 silencing. Furthermore, LPS upregulated RhoA expression and MLC phosphorylation, but reduced Rap1 and VE-cadherin expression, which were also enhanced by Rap1 silencing, but significantly mitigated by TH. Immunofluorescent analyses indicated that LPS significantly increased phosphorylated MLC, but decreased VE-cadherin expression, which were further deteriorated by Rap1 silencing, but significantly mitigated by TH in EA. hy926 cells. TH significantly mitigated the sepsis-increased permeability of EA. hy926 cells by enhancing the Rap1 expression to attenuate the RhoA/MLC signaling.