| Objective:Atherosclerosis(AS)is a kind of vascular disease characterized by chronic inflammation involving multiple cells.Recently,the autoimmune mechanism of AS has gain increasing attention.Anti-?2 glycoprotein I(anti-?2GPI)is the main autoantibody in patients with antiphospholipid syndrome(APS).Anti-?2GPI could bind to ?2GPI and oxidized low-density lipoprotein(oxLDL),forming oxLDL/?2GPI/anti-?2GPI antibody complex.This complex has been proven to be a proatherogenic autoimmune complex.Previous studies in our group have confirmed that oxLDL/?2GPI/anti-?2GPI antibody complex could enhance the foam cell differentiation and tissue factor(TF)secretion of rat peritoneal macrophage,playing a crucial role in pathological mechanism of AS.Vascular smooth muscle cell(VSMC)is one of the main cell components involved in AS plaque formation.The abnormal proliferation,migration,foaming and apoptosis of VSMC are key points in the development of AS.Therefore,the aim of this paper is to explore the effects of oxLDL/?2GPI/anti-?2GPI antibody complex on proatherogenic functional activation of VSMC and the underlying signaling molecule regulation mechanismsMethods:(1)A7r5 and HCASMC cells were divided into 6 groups:blank control group,oxLDL group,oxLDL/?2GPI group,oxLDL/anti-?2GPI antibody group,oxLDL/?2GPI/anti-?2GPI antibody complex group and ?2GPI/anti-?2GPI antibody group.After a certain period of stimulation,oil red O staining was used to observe the formation of foam cells;Transwell assay was used to detect cell migration;Western blot was used to detect the expression of PCNA,cleaved-caspase-3,Bcl-2,and Bax;CCK-8 and EdU were used to detect the cell proliferation activity;Annexin V-FITC/PI flow cytometry was used to detect apoptosis rate(2)A7r5 and HCASMC cells was stimulated with oxLDL/?2GPI/anti-?2GPI antibody complex for different time.The expression of proliferation and apoptosis-related proteins was measured by Western blot;CCK-8 and EdU were used to assess cell proliferation;apoptosis rate was detected Annexin V-FITC/PI flow cytometry.The time related NF-?B and MAPKs signal molecules activation during stimulation of oxLDL/?2GPI/anti-?2GPI antibody complex were examined by Western blot(3)A7r5 cells were incubated with 6 different stimuli.The expression of TLR4,TLR2,NF-?B,and MAPKs were observed by Western blot or RT-PCR.In order to confirm the participation of TLR4 and TLR2 in the signaling pathway activation of VSMC during the stimulation of oxLDL/?2GPI/anti-?2GPI antibody complex,after pretreatment of A7r5 cells with TLR4 inhibitor(TAK-242)or TLR2 inhibitor(anti-TLR2 antibody),the activation of NF-?B,and MAPKs were detected by Western blot(4)A7r5 cells were pretreated with TAK-242,NF-?B inhibitor(PDTC)or MAPKs inhibitor(SB203580 and U0126),respectively.Then cells were detected by oil red O staining or Transwell following stimulation of oxLDL/?2GPI/anti-?2GPI antibody complex;A7r5 cells were pretreated with TAK-242,anti-TLR2 antibody,PDTC,SB203580 and U0126,respectively.After stimulation of oxLDL/?2GPI/anti-?2GPI antibody complex,proliferation and apoptosis related proteins expression were observed by Western blot;cell viability was determined by EdU assay;cell apoptosis was observed by mitochondrial membrane potential fluorescence staining.(5)A7r5 cells were pretreated with PDTC,U0126 or SB203580.After stimulation of oxLDL/?2GPI/anti-?2GPI antibody complex,the activation of NF-?B,ERK1/2 and p38 MAPK was detected by Western blot(6)ROS and SOD alterations were measured in A7r5 cells after different time-stimulation of oxLDL/?2GPI/anti-?2GPI antibody complex;then ROS and SOD changes were measured after pretreatment of NADPH oxidase inhibitors(Apocynin and DPI)(7)A7r5 cells were pretreated with Apocynin and DPI before stimulation of oxLDL/?2GPI/anti-?2GPI antibody complex for a certain period of time Proliferation and apoptosis-related proteins expression were observed by Western blot;cell proliferation was detected by CCK-8 and EdU;apoptosis rate was examined by Annexin V-FITC/PI flow cytometry(8)The production of ROS induced by oxLDL/?2GPI/anti-?2GPI antibody in A7r5 cells was detected after pretreatment of TAK-242 and anti-TLR2 antibody.Then A7r5 cells were pretreated with Apocynin and DPI,followed by stimulation of oxLDL/?2GPI/anti-?2GPI antibody complex for a certain period of time,the activation of NF-?B and MAPKs were detected by Western blot.Results:(1)The oxLDL/?2GPI/anti-?2GPI antibody complex had a significant promoting effect on foam cell formation,migration,proliferation and apoptosis of VSMCs,which was statistically significant compared with various control groups(p<0.05)(2)The biphasic effect of oxLDL/?2GPI/anti-?2GPI antibody complex on the proliferation and apoptosis of VSMC was closely related to the stimulation time:when the stimulation time was short(HCASMC 6 h or A7r5 cells 24 h),induced proliferation was obvious;while longer time(HCASMC 24 h or A7r5 cells 48 h),apoptosis was increased.At the same time,during the activation of A7r5 cells,the activation of intracellular signaling molecules had a similar time correlation:when the stimulation time is short(24 h),NF-?B and ERK1/2 are activated;when the stimulation time was longer(48 h),p38 MAPK activation was up-regulated(3)The oxLDL/?2GPI/anti-?2GPI antibody complex significantly promoted the expression of TLR4 and TLR2(p<0.05).When compared with control group,the activation of NF-?B,ERK1/2 and p38 MAPK were all also significantly enhanced in oxLDL/?2GPI/anti-?2GPI antibody complex group(p<0.05).TAK-242 could significantly inhibit the activation of NF-?B induced by the oxLDL/?2GPI/anti-?2GPI antibody complex,and anti-TLR2 antibody could significantly block the activation of p3 8 MAPK.(4)TAK-242,PDTC and U0126 could partly inhibit the foam cell formation,migration and proliferation of VSMC induced by the oxLDL/?2GPI/anti-?2GPI antibody complex and promote induced apoptosis;anti-TLR2 antibody and SB203580 could partly inhibit the apoptosis of VSMC induced by oxLDL/?2GPI/anti-?2GPI antibody complex and promote the induced proliferation(5)Both PDTC and U0126 could promote the activation of p38 MAPK induced by oxLDL/?2GPI/anti-?2GPI antibody complex;however,SB203580 could simultaneously up-regulate oxLDL/?2GPI/anti-?2GPI antibody complex-induced NF-?B and ERK1/2 activation(6)During the stimulation of oxLDL/?2GPI/anti-?2GPI antibody complex,the production of ROS and SOD were time correlated:when the time was short,the production of ROS increased slightly and the activity of SOD significantly increased;when the time was longer,ROS production was increased significantly and SOD activity decreased dramatically.Apocynin and DPI could also inhibit oxLDL/?2GPI/anti-?2GPI antibody complex-induced ROS production(7)Apocynin and DPI could simultaneously inhibit the proliferation and apoptosis of VSMCs induced by the oxLDL/?2GPI/anti-?2GPI antibody complex.(8)TAK-242 and anti-TLR2 antibody had no significant effect on ROS production induced by oxLDL/?2GPI/anti-?2GPI antibody complex,while Apocynin and DPI could significantly inhibit activation of NF-?B,ERK1/2 and p38 MAPK induced by oxLDL/?2GPI/anti-?2GPI antibody complexConclusions:(1)The oxLDL/?2GPI/anti-?2GPI antibody complex is a proatherogenic autoimmune complex which could promote a variety of functional activations of VSMC,including foam cell differentiation,migration,proliferation and apoptosis,consequently accelerating the development of AS plaque(2)TLR4/NF-?B and ERK 1/2 can positively regulate foam cell formation,migration and proliferation of VSMC induced by oxLDL/?2GPI/anti-?2GPI antibody complex,however,have a negative regulation effect on the apoptosis of VSMCs induced by this complex.TLR2/p38 MAPK has a positive regulation effect on apoptosis of VSMC induced by oxLDL/?2GPI/anti-?2GPI antibody complex,and negatively regulate the proliferation of VSMCs induced by this complex(3)The dual effects on VSMC proliferation and apoptosis induced by oxLDL/?2GPI/anti-?2GPI antibody complex are regulated by a signal molecular network constituted by time-dependent activated NF-?B,ERK1/2 and p38 MAPK.There is a feedback mechanism for competitive activation between NF-?B or ERK 1/2 and p38 MAPK(3)The oxLDL/?2GPI/anti-?2GPI antibody complex participate in the regulation of the signal molecular network composed of NF-?B,ERK1/2 and p38 MAPK by changing the production of ROS mediated by NADPH oxidase,thereby exerting promoting dual effects on proliferation and apoptosis in VSMC after different time of stimulation.TLR4/NF-?B and TLR2/p38 MAPK signaling pathways also act as bypass mechanisms to participate in regulating this signaling molecular network. |
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