The Experimental Study Of The Effects Of Different Strains Of Porphyromonas Gingivalis With Different FimA Genotypes On The Function Of Human Umbilical Vein Endothelial Cells

Objective:In order to explore the contribution of P.gingivalis in the development of atherosclerosis and the possible role of P.gingivalis underlying the relationship between periodontitis and atherosclerosis,we observed the effects of different strains of P. gingivalis possessing different fimA genotypes on the expression of cell adhesion molecules,and secretion of pro-inflammatory cytokines and chemokines in HUVECs.Methods:P.gingivalis ATCC 33277(typeⅠfimA gene),WCSP 115(typeⅡfimA gene) and W83(typeⅣfimA gene) were cultured anaerobically in standard condition,meanwhile, HUVECs were cultured in vitro and identified.Different fimA genotypes of P.gingivalis were added to confluent HUVECs monolayers and co-cultured for 2,6 or 24h.At different times,cell culture supematants were assessed for IL-1β,IL-6,TNF-α,IL-8 and MCP-1 production using ELISA analysis,while HUVECs were harvested and processed for FCM and CLSM analysis for ICAM-1 and VCAM-1 expression.Results:(1) The expression of ICAM-1 on the surface of HUVECs intensified after P. gingivalis infected.The up-regulation effects caused by P.gingivalis with typeⅡandⅣfimA genes were stronger than P.gingivalis with typeⅠfimA gene.Under our experiment of condition,infection with P.gingivalis stimulated low expression of VCAM-1 by HUVEC, which showed no significant difference among P gingivalis with typeⅠ,ⅡandⅣfimA genes.Expression of ICAM-1 and VCAM-1 on P.gingivalis-infected HUVECs was confirmed by confocal microscopy.Infection of HUVEC with P.gingivalis resulted in more fluorescence staining of ICAM-1 and VCAM-1 compared with that in uninfected HUVEC cultures.The staining was more extensive on the HUVEC infected by P.gingivalis with typeⅡandⅣfimA genes.(2) The secretion of IL-1βand IL-6 increased significantly by HUVECs which were infected by P.gingivalis with typeⅠ,typeⅡandⅣfimA genes.The up-regulation effects caused by P.gingivalis with typeⅡandⅣfimA genes were stronger than that of P. gingivalis with typeⅠfimA gene,which indicated that the difference of fimA genotypes may lead to different ability of P.gingivalis in modulating the secretion of pro-inflammatory cytokines by HUVEC,and then the different ability to induce inflammatory and immunological reaction in endothelial system.(3) Under our experiment of condition,the secretion of TNF-αincreased significantly by HUVEC,infected by P.gingivalis with typeⅡandⅣfimA genes.While the up-regulation effect was not significant in P.gingivalis with typeⅠfimA gene,which indicated that the secretion of TNF-αfrom HUVEC stimulated by P.gingivalis with typeⅠfimA gene was weaker than that of P.gingivalis with typeⅡandⅣfimA genes.(4) The secretion of IL-8 and MCP-1 increased significantly by HUVEC which was infected by P.gingivalis with typeⅡandⅣfimA genes.The up-regulation effects caused by P.gingivalis with typeⅡandⅣfimA genes were stronger than that of P.gingivalis with typeⅠfimA gene,which indicated that the difference of fimA genotypes may lead to different ability of chemokines induction by P.gingivalis-infected HUVEC,and then the different ability to induce inflammatory and immunological reaction in endothelial tissue.Conclusion:There were different effects in the HUVECs infected by different strains of P. gingivalis with different fimA genotypes,which also had different pathogenicity in periodontal tissue.Compared with P.gingivalis of typeⅠfimA gene,P.gingivalis with typeⅡandⅣfimA genes possesse stronger ability to stimulate HUVECs to up-regulate the expression of cell adhesion molecules,and the induction of production of pro-inflammatory cytokines and chemokines.This indicates that the virulence and pathogenicity of P. gingivalis is related with its fimA genotypes,and P.gingivalis with typeⅡandⅣfimA genes are closely correlated with the development of atherosclerosis.