The Mechanism Of Treponema Pallidum Membrane Protein Tp47 Stimulates Vascular Cells To Mediate THP-1 Cells Migration,Adhesion And Angiogenesis

Background and ObjectiveSyphilis is a chronic sexually transmitted diseases caused by Treponema pallidum(T.pallidum).The pathologic features of syphilis are characterized by vascular involvement with endarteritis,periarteritis and angiogenesis.Vascular reactivity play a crucial role in inflammation.Vascular endothelial cells and smooth muscle cells,as two main type cells of vascular wall,play a crucial role in this process.Although T.pallidum has structural similarities to classical Gram-negatiae bacteria,such as having outer and inner membranes and a periplasmic space,it lacks lipopolysaccharide,a potent pro-inflammatory glycolipid,and does not produce any known toxic proteins.Therefore,most of the symptoms and tissue damage related to syphilis are due to activation of the host inflammatory and immune responses.Little is known about how T.pallidum causes the protean manifestations of syphilis.Tp47,as a rich membrane protein of T.pallidum,has a strong immunogenicity,which plays an important role in the immune pathological damage caused by T.pallidum.However,the functions of Tp47 in vascular inflammation and angiogenesis caused by T.pallidum infection are still unclear.In this study,in order to clarify whether Tp47 paly an impotant role and related mechanisms in endarteritis,periarteritis and angiogenesis caused by T.pallidum.We analyze the effect of Tp47 on human umbilical vein endothelial cells(HUVECs)and human dermal vascular smooth muscle cells(HDVSMCs).Frist,we detected the expression of cytokines stimulated by Tp47 in HUVECs and invesgated whether Tp47 promoted the migration and adhesion of THP-1 cells to Tp47-stimulated HUVECs.Second,whether Tp47 have an effect on promoting angiogenesis was analyzed.The role of matrix metalloproteinase and related signaling pathways in this process were invesgated.Third,we detected the expression of F-actin,chemotaxis and adhesion factors in HDVSMCs stimulated by Tp47,and analysed the effct of Tp47 on prompting the migration and adhesion of THP-1cells to HDVSMCs.In addition,the related signal pathway was invesgated in this process.This study focus on the effect of Tp47 on vascular wall cells will helpful for investigating the immunopathogenesis of T.pallidum.Methods1.When HUVECs were stimulated by 50?g/mL Tp47,the protein expression of inflammatory factors were detected by Human Inflammation Antibody Array.The changed expression of cytokines were further detected by RT-PCR.The chemotaxis and adhesion assays were used to analyze the migration and adhesion of THP-1 cells to Tp47-treated HUVECs.2.When HUVECs were stimulated by Tp47,the metalloproteinases(MMPs)were detected by RayBio Matrix Metalloproteinase Antibody Array.The expression of MMPs and tissue inhibitors of matrix metalloproteinases(TIMPs)were further detected by RT-PCR and ELISA.the proliferation of HUVECs were detected by CCK-8 assay.The migration ability was detected by transwell and scratch assay.The angiogenesis induced by Tp47 was assessed by endothelial tube formation assay in vitro and zebrafish assay in vivo.The mRNA expression of MMPs and TIMPs were detected by RT-PCR.The protein expression of MMPs and TIMPs were detected by ELISA.The activation of signaling pathways were detected by western blotting.3.When HDVSMCs were stimulated by Tp47,the RT-PCR was used to detect the mRNA expression of monocyte chemoattractant protein-1(MCP-1)and intercellular adhesion molecule-1(ICAM-1).ELISA was used to detect the protein expression of MCP-1.Western blotting and flow cytometry were used to detect the expression of ICAM-1,The chemotaxis and adhesion assays were used to analyze the migration and adhesion of THP-1 cells to Tp47-treated HDVSMCs.Western blotting was used to detect the activation of signaling pathways in this process.The change of F-actin detected by immunofluorescence.At the same time,the expression of MCP-1 and ICAM-1 were detected when HDVSMCs were stimulated by T.pallidum.The migration and adhesion of THP-1 cells to T.pallidum-treated HDVSMCs were also analysed.Results?.When HUVECs were stimulated by50 ?g/mL Tp47 for 24 hours,compared to control group,Tp47 significantly promoted granulocyte-macrophage colony-stimulating factor(GM-CSF),ICAM-1,IL-3,IL-6,IL6sR,IL-8,interferon-inducible protein 10(IP-10)and MCP-1 expression(P<0.05),there is no significant differences among the EOTAXIN,EOTAXIN-2,GCSF,IFN-?,I-309,IL-1?,IL-1b,IL-2,IL-4,IL-7,IL-10,IL-11,IL-12p40,IL-12p70,IL-13,IL-15,IL-16,IL-17,MCP-2,M-CSF,MIG,MIP-1?,MIP-1?,MIP-1?,RANTES,TGF-?1,TNF-?,TNF-?,STNFRI,STNF RII,PDGF-BB and TIMP-2.To further demonstrate our finding mentioned above,the mRNA expression of GM-CSF,ICAM-1,IL-6,IL-8,IP-10 and MCP-1 were detected by RT-PCR.The results showed that Tp47 significantly increased the mRNAs expression of GM-CSF,ICAM-1,IL-6,IL-8,IP-10 and MCP-1 in a dose-dependent manner.Tp47 could promote THP-1 cells migrate to HUVECs,which could be significantly suppressed by the anti-MCP-1 or anti-IL-8 neutralizing antibody,respectively.Meanwhile,Tp47 could promote THP-1 cells adhere to HUVECs in a dose-dependent manner,which could be significantly suppressed by the anti-ICAM-1 neutralizing antibody.2.Frist,the Matrix Metalloproteinase Antibody Array found that the protein expression of MMP-1 and MMP-10 of HUVEC were significantly increased by Tp47.the protein expression of TIMP-1 and TIMP-2 have no significant difference between control and Tp47 group.Following,the HUVECs were stimulated with different dose of Tp47.Tp47 markedly increased MMP-1 and MMP-10 mRNA expression in a dose-dependent manner.Meanwhile,the total and activity protein of MMP-1 and MMP-10 were significantly increased by Tp47.the mRNA and protein expression of TlMP-1 and TIMP-2 have no significant difference between groups.Second,Tp47 significantly prompted proliferation,migration and tube formation ability in vitro of HUVECs.In addition,it was observed that Tp47 significantly promoted intestinal angiogenesis in zebrafish.After HUVECs were transfected with siRNAMMP-1 or siRNAMMP-10,the total and activity protein of MMP-1 or MMP-10 induced by Tp47 were significantly inhibited,and the proliferation,migration,and tube formation ability in vitro enhanced by Tp47 were significantly weakened.Finally,Tp47 could activate the AKT/mTOR/S6 signaling pathway,which could be significantly inhibited by AKT inhibitor(LY294002)and mTOR inhibitor(rapamycin).The LY294002 and rapamycin could inhibit t the total and activity protein of MMP-1 and MMP-10 promoted by Tp47,but there is no effect on mRNA and protein expression of TIMP-1 and TIMP-2.At the same time,the proliferation,migration,and tube formation in vitro of HUVECs stimulated by Tp47 were significantly inhibited by LY294002 and rapamycin.3.Tp47 and T.pallidum increased MCP-1 and ICAM-1 mRNA and protein expression levels in a dose-and time-dependent manner.Meanwhile,Tp47 and T.pallidum could promote the migration and adhesion of THP-1 cells to HDVSMCs.The migration and adhesion of THP-1 cells to HDVSMCs were significantly suppressed by the anti-MCP-1 or anti-ICAM-1 neutralizing antibody,respectively.Further studies revealed that treatment of HDVSMCs with Tp 47 activated AKT,p38 MAPK and NF-?B signaling pathway,the inhibitor of AKT(LY294002),p38 MAPK(SB203580)and NF-?B(BAY1 1-7082)suppressedMCP-1 and ICAM-1 expression induced by Tp47.They also prevented F-actin reorganization and redistribution in HDVSMCs.In addition,the migration and adhesion of THP-1 cells to Tp47-treated HDVSMCs were significantly decreased by pretreatment of LY294002,SB203580 and BAY11-7082.Conclusions1.Tp47 prompt the mRNA and protein expression of GM-CSF,ICAM-1,IL-6,IL-8,IP-10 and MCP-1 in HUVECs.Meanwhile,Tp47 promotes the migration and adherence of THP-1 cells to HUVECs by inducing MCP-1,IL-8 and ICAM-1 expression.2.Tp47 prompt the expression of MMP-1 and MMP-10 through the AKT/mTOR/S6 signaling pathway,which disrupt the MMP/TIMP balance and prompt angiogenesis.3.Tp47 promotes the migration and adherence of THP-1 cells to HDVSMCs by inducing MCP-1 and ICAM-1 expression,which is mediated through activation of the AKT,p3 8 MAPK and NF-?B pathways.In summary,our research find that Tp47 stimulates vascular cells to mediate THP-1 cells migration,adhesion and angiogenesis.These findings will provide new insight to investigate the pathogenesis of T.pallidum and scientific basis for further exploring the molecular pathogenesis of Tp47.