Chlamydia Pneumoniae Infection Increases Vascular Endothelial Cell Permeability Through Promoting The Internalization Of VE Cadherin Via The Phosphorylation Of VE Cadherin

Objectives: To investigate the effect of Chlamydia pneumoniae(C.pn)infection on the permeability of vascular endothelial cells(VECs)by using the model of human umbilical vein endothelial cell line EA.hy926 infected with C.pn in vitro and to explore the mechanism of C.pn infection-induced increase in VEC permeability and the roles of phosphorylation and internalization of VE cadherin in the increase in VEC permeability induced by C.pn infection.Methods:1.The interendothelial junction of VEC was measured by immunofluorescence.2.The cumulative fluorescence intensity of fluoresceine isothiocyanate(FITC)-bovine serum albumin(BSA)was measured to detect the effect of C.pn infection on VEC permeability.3.CCK-8 kit was used to detect the toxicity of chloroquine to VECs at the working time and working concentrations.4.The effect of C.pn infection on VE cadherin internalization was detected by immunofluorescence.5.CCK-8 kit was used to detect the toxicity of vascular endothelial growth factor(VEGF)to VECs at the working time and working concentrations.6.Western blot was used to detect the expression of VE cadherin protein in C.pn infected-VECs.7.The VEC proteins from cell membrane and cytoplasm were respectively isolated,and the expression levels of VE cadherin in cell membrane and cytoplasm were detected by Western blot.8.CCK-8 kit was used to detect the toxicity of chlorpromazine to VECs at the working time and working concentrations.9.We employed CPZ to investigate the roles of VE cadherin internalization in C.pn infection-induced increase in VEC permeability by measuring the cumulative fluorescence intensity of FITC-BSA.10.CCK-8 kit was used to detect the toxicity of bradykinin to VEC at the working time and working concentrations.11.After the pretreatment of VECs with a Src kinase specific inhibitor PP2,Western blot was used to detect the expression of VE cadherin and phosphorylated VE cadherin at Y658 in VECs to demonstrate the effect of PP2 on VE cadherin phosphorylation induced by C.pn infection.12.The cumulative fluorescence intensity of FITC-BSA was measured after the pretreatment of VECs with PP2 to explore the role of VE cadherin phosphorylation in C.pn infection-induced increase in VEC permeability.13.The VEC proteins from cell membrane and cytoplasm were respectively isolated,and the expression levels of VE cadherin and phosphorylated VE cadherin in cell membrane and cytoplasm were detected by Western blot.PP2 was employed to reveal the role of VE cadherin phosphorylation in C.pn infection-induced VE cadherin internalization.Results:1.Under the fluorescent microscope,the borders of interendothelial junctions were intact.2.After C.pn infection for 24 h,36 h and 48 h,VEC permeability was increased,and the significantly increased VEC permeability was observed at 24 h postinfection(P < 0.05).3.CCK-8 results showed that after VECs were treated with chloroquine at the concentrations of 20 μmol/l,40 μmol/l,60 μmol/l,and 80 μmol/l for 24 h,no significant VEC cytotoxicity was observed,while chloroquine at the concentration of 100 μmol/l had significant VEC cytotoxicity(P < 0.05).4.Under a confocal laser scanning microscope,VE cadherin was located at interendothelial junctions and the borders of interendothelial junctions were intact.At 24 h postinfection,the expression of VE cadherin was significantly decreased on cell membrane,but was distributed diffusely in the cytoplasm.Significant gaps were observed at the interendothelial junctions.After the treatment of VECs with chloroquine,more VE cadherin was translocated from membrane to cytoplasm,indicating the significant VE cadherin internalization.5.The results from CCK-8 assay showed that VEGF at the working time and working concentration had no significant cytotoxicity(P > 0.05).6.Western blot analysis showed that when compared with normal control group,no significant changes in the expressions of VE cadherin were detected in C.pn infection group,VEGF group,C.pn infection + chloroquine group,VEGF + chloroquine group and chloroquine group(P > 0.05).7.The results from Western blot showed that significant decreases in the expressions of VE cadherin on cell membrane were detected in C.pn infection group and VEGF group.Only little VE cadherin was detected in the cytoplasm in the control VECs,and more VE cadherin was found in the VEC cytoplasm in C.pn infection group and VEGF group.After VECs were treated with the lysosome inhibitor chloroquine to inhibit the degradation of the internalized VE cadherin,no obvious changes in the expressions of VE cadherin on cell membrane were detected in C.pn infection + chloroquine group and C.pn infection group,while significant increases in the expressions of VE cadherin in the cytoplasm were observed in C.pn infection + chloroquine group(P < 0.05).No obvious changes in the expressions of VE cadherin on cell membrane were detected in VEGF + chloroquine group and VEGF group,while significant increases in the expressions of VE cadherin in the cytoplasm were observed in VEGF + chloroquine group(P < 0.05),suggesting that C.pn infection can significantly promote VE cadherin internalization.8.CCK-8 results showed that chlorpromazine at the working time and working concentration had no significant VEC cytotoxicity(P > 0.05).9.FITC-BSA assay showed that after chlorpromazine treatment,C.pn infection-induced increase in VEC permeability was significantly inhibited(P < 0.05).10.The results from CCK-8 assay showed that bradykinin at the working time and working concentration had no significant VEC cytotoxicity(P > 0.05).11.Western blot analysis showed that significant increases in VE cadherin phosphorylation were detected in C.pn infection group and bradykinin group,while PP2 could significantly reduce the expressions of phosphorylated VE cadherin induced by C.pn infection(P < 0.05).12.FITC-BSA assay showed that the increased VEC permeability induced by bradykinin can be impaired by the pretreatment of VECs with PP2,and C.pn infection-induced increase in VEC permeability can also be significantly inhibited by PP2(P < 0.05),indicating that C.pn infection could increase VEC permeability by inducing VE cadherin phosphorylation.13.The results from Western blot showed that significant increases in the expressions of phosphorylated VE cadherin on cell membrane were detected in C.pn infection group and bradykinin group,while PP2 could significant inhibit the increases in VE cadherin phosphorylation on cell membrane induced by C.pn infection(P < 0.05).In the cytoplasm,significant increases in VE cadherin phosphorylation were detected in C.pn infection group and bradykinin group,and VE cadherin phosphorylation at Y658 in C.pn infection group was further increased after the treatment with chlorpromazine(P < 0.05).Conclusion: C.pn infection could simulate VE cadherin phosphorylation at Y658,and then promote VE cadherin internalization,thereby increasing VEC permeability.