Roles Of Phosphorylation Of N-WASP In Promoting Chlamydia Pneumoniae-Infected Vascular Endothelial Cell Migration And Angiogenesis

Objective:Using the model of vascular endothelial cell(VEC)infected with Chlamydia pneumoniae(C.pn)in vitro,to observe the effects of C.pn infection on angiogenesis and VEC migration,to explore the roles of phosphorylation of N-WASP in VEC migration and angiogenesis induced by C.pn infection.Methods:1.C.pn AR-39 was propagated in HEp-2 cells in vitro.Indirect immunofluorescence was used to determine inclusion-forming unit(IFU)in the infected HEp-2 cells.The successful infection of VECs with C.pn was confirmed by a murine monoclonal antibody against C.pn under fluorescence microscope.2.Capillary tube formation assay was performed to observe the effects of C.pn infection on angiogenesis.3.Wound-healing assay and Transwell assay were performed to observe the effects of C.pn infection on VEC migration.4.The subcellular location of myofilament and N-WASP in C.pn-infected VECs was determined by immunofluorescence with FITC-phalloidin and a polyclonal rabbit anti-humanN-WASP antibody under confocal laser scanning microscopy.5.The total RNA was respectively extracted from VECs which were infected with C.pn for 0 h,2 h,4 h,6 h,8 h,10 h,12 h,14 h,16 h,18 h,20 hand 24 h.Reverse transcription-polymerase chain reaction(RT-PCR)was used to detect the mRNA expressions ofN-WASP in C.pn-infected VECs at the indicated time points.6.The total protein was respectively extracted from VECs which were infected with C.pn for 0 h,2 h,4 h,6 h,8 h,10 h,12 h,14 h,16 h,18 h,20 h and 24 h.Western blot was used to detect the expressions of total N-WASP and phosphorylated N-WASP(Y256)in C.pn-infected VECs at the indicated time points.7.CCK-8 assay was performed to determine the effects of the N-WASP specific inhibitor Wiskostain at differet concentrations on VEC viability.8.After the activity of N-WASP was inhibited by Wiskostain,Western blot was used to analyze the changes in the expressions of total N-WASP and phosphorylated N-WASP in VECs in each group.Wound-healing assay and Transwel assay was performed to observe the changes in VEC migration ability in each group.Capillary tube formation assay was conducted to determine the ability of VECs in each group to form micro lumen.Results:1.At 60 h postinfection,the clusters of bright apple-green botryoidal inclusions were obseved in HEp-2 cells under the fluorescence microscope,indicating the successful infection of HEp-2 cells with C.pn.The IFU of C.pn was 5.93×108/ml.2.The clusters of typical bright apple-green botryoidal inclusions were obseved in VECs 60 h after the infection with 5×105 IFU of C.pn suspension,suggesting the successful establishment of the mode of VECs infected with C.pn.3.Capillary tube formation assay showed that the ability of VECs infected with C.pn for 24 h to form micro lumen was significantly increased compared with the normal control group(P<0.05).4.The results from wound-healing assay revealed that the areas of the recovery of VECs infected with C.pn for 24 h were remarkably larger than that in the control VECs(P<0.05).The results from Transwel assay showed that the number of C.pn infected-VECs migrated through the membrane was more than that of the control VECs(P<0.05).5.The actin polymerization assay showed that in the normal control group,myofilament exhibited the green fluorescence with the diffuse distribution in cytoplasm and without specific polymerization;N-WASP glowed with a red fluorescence with the uniform distribution in membrane and cytoplasm,but more in membrane.After C.pn infection,actin polymerization was significantly enhanced at the cellular " leading edge" and myofilament was arranged in bundles.Co-localization of N-WASP and myofilament was observed in C.pn infected-VECs.6.RT-PCR results showed that there was no significant difference in the mRNA expressions of N-WASP in the VECs between the different time points after C.pn infection(P>0.05).7.Western blot analysis showed that the expression of phosphorylated N-WASP(Y256)in VECs was up-regulated 6 h after C.pn infection.With the increase in the infection time,the phosphorylation level of N-WASP was increased gradually to reach a peak at 10 to 12 h postinfection and then began to decrease at 14 h postinfection until 24 h after infection.No significant changes in the expressions of total N-WASP were detected during the infection.8.The results from CCK-8 assay showed that the N-WASP specific inhibitor Wiskostain at the concentrations of less than 5 ?M did not reduce the viability of VECs compared with the normal control group,whereas Wiskostain at the concentrations of more than 10 ?M resulted in a reduction of viability compared with normal control group(P<0.05).Moreover,with the increase in Wiskostain concentrations,a more significant reduction in the vitality of VECs was obseved.9.The immunofluorescence assay showed that C.pn infection rate in VECs with or without the pretreatment with Wiskostain(5 ?M)had no obvious difference(P>0.05),indicating that C.pn infection is not affected by Wiskostain at the indicated concentrations and during the experiments.10.After VEC was pretreated with Wiskostain(5 ?M),the results from Western blot showed that the expression of phosphorylated N-WASP(Y256)in VECs was significantly downregulated.In C.pn infected-VECs pretreated with Wiskostain(5?M),the expression level of phosphorylated N-WASP(Y256)was lower than that in C.pn infected-VECs(P<0.05).The expression level of phosphorylated N-WASP(Y256)in VECs pretreated with Wiskostain(5?M)also lower than that in C.pn infected-VECs(P<0.05).No obvious differences in the expressioins of total N-WASP were detected between VECs in each group.11.After VEC was pretreated with Wiskostain(5 ?M),the results from capillary tube formation assay showed that the ability of VECs infected with C.pn to form microlumen was significantly lower than that C.pn infected-VECs(P<0.05).In addition,the migration ability of C.pn infected-VECs pretreated with Wiskostain(5?M)was significantly lower than that in C.pn infected-VECs(P<0.05).Transwell assay showed that the number of C.pn infected-VECs pretreated with Wiskostain(5 ?M)that had migrated through the membrane was less than that of C.pn infected-VECs(P<0.05).Conclusions:C.pn infection could promote VEC migration and angiogenesis significantly possibly by inducing the phosphorylation of N-WASP(phospho Y256).