| Objective:Atherosclerosis(AS)is a chronic inflammatory disease that occurs exclusively in the middle and large arteries.The dysfunction of cells caused by stimulation of lipid components such as oxidized low density lipoprotein(oxLDL)is considered to be a key factor in the pathological process of AS.Studies have shown that autoimmune diseases,such as antiphospholipid syndrome(APS),are association with AS.β2 glycoprotein I(β2GPI),the autoantigen of APS,can enhance the pathogenic effect of oxLDL by forming a complex with it.In addition,anti-β2GPI antibodies have also been proved to binding to oxLDL and enhance oxLDL-mediated cell damage.Toll-like receptor 4(TLR4)is the main pattern recognition receptor(PPAR)that mediates APS inflammation.In addition,it also plays an important role in the pathological process of AS.Activation of TLR4 promotes the expression of monocyte chemotactic protein 1(MCP-1)in endothelial cells and smooth muscle cells.The cytokine accelerates the inflammatory process of AS by promoting the adherence of monocytes to the vascular endothelium.TLR4 promotes the expression of matrix metalloproteinase 9(MMP-9)in smooth muscle cells(SMC),which accelerates the migration of SMC into intima by degrading the extracellular matrix.Then,the SMC phagocytize oxLDL and other lipid components,and converts to foam cells that participate in the formation of AS plaques.Although the role of β2GPI,anti-β2GPI antibody,and oxLDL/β2GPI complex in AS has been widely reported,the involvement of oxidized low-density lipoprotein/β2 glycoprotein I/anti-β2 glycoprotein I antibody complex(oxLDL/β2GPI/β2GPI-Ab)in AS the pathology remains to be further explored.Our previous study found that oxLDL/β2GPI/β2GPI-Ab complexes promoted the foam cell formation and the secretion of cytokines,such as interleukin-1β(IL-1β),MCP-1 and other proinflammatory cytokines in macrophages in a TLR4-dependent manner This study was aimed to observe the effects of oxLDL/β2GPI/β2GPI-Ab complex on the chemotaxis,migration,lipid accumulation and other functions of rat aortic smooth muscle cell line A7r5 and the role of TLR4 signaling pathway.Methods:(1)A7r5 cells were treated with serum-free media,oxLDL,oxLDL/β2GPI complex,β2GPI-Ab,β2GPI/β2GPI-Ab complex,and oxLDL/β2GPI/β2GPI-Ab complex in each experimental group,Wound-Healing Assay was used to evaluate the migration ability of A7r5 cells.quantitative analysis of cell migration distance was evaluated by Image J 1.44 software.(2)The mRNA levels of MCP-1,MMP-9,ACAT1 after media,oxLDL,oxLDL/β2GPI complex,β2GPI-Ab,β2GPI/β2GPI-Ab complex,oxLDL/β2GPI/β2GPI-Ab complex treatments were measured by Real-time quantitative PCR(RT-qPCR).The protein levels of MCP-1,MMP-9 after media,oxLDL,oxLDL/β2GPI complex,β2GPI-Ab,β2GPI/β2GPI-Ab complex,oxLDL/β2GPI/β2GPI-Ab complex treatments were measured by Enzyme-linked immunosorbent assay(ELISA)(3)To explore the role of TLR4 in the above process,A7r5 cells were pretreated with TAK-242,then stimulated by media,oxLDL,oxLDL/β2GPI complex,β2GPI-Ab,β2GPI/β2GPI-Ab complex,oxLDL/β2GPI/β2GPI-Ab complex.the mRNA level of ACAT1,MCP-1,and MMP-9 was detected by RT-qPCR.The protein level of MCP-1 and MMP-9 in the supernatant was detected by ELISA.(4)A7r5 cells pretreated with or without TAK-242 were stimulated by oxLDL or oxLDL,oxLDL/β2GPI/β2GPI-Ab complex.Then the content of total cholesterol(TC)and free cholesterol(FC)in them were measured by corresponding commercial kits.Then the level of intracellular cholesterol ester(CE)was calculated by TC and FC.Results(1)After the stimulation of oxLDL/β2GPI/β2GPI-Ab complex,the migration of A7r5 cells was most obvious(68.33±3.12 μm),and the cells on both sides were basically attached.The difference from the media group was statistically significant(p<0.05).After TAK-242 pretreatment,the migration distance was significantly shorter compared with oxLDL/β2GPI/β2GPI-Ab complex group that did not pretreat with TAK-242(p<0.05).(2)After the stimulation of oxLDL/β2GPI/β2GPI-Ab complex,the mRNA and protein expression levels of MCP-1 and MMP-9 were increased in comparison to media group(p<0.05).After TAK-242 pretreatment,the mRNA and protein expression levels of MCP-1 and MMP-9 were significantly decreased in comparation to the corresponding group that did not pretreat with TAK-242(p<0.05).(3)After treatment with oxLDL/β2GPI/β2GPI-Ab complex,the TC,FC,and CE contents of A7r5 were significantly higher than those of media(p<0.05),and the mRNA level of AC AT1 was also increased(p<0.05)..After pretreatment with TAK-242,the expression of TC and FC was significantly inhibited compared to the group without TAK-242(p<0.05).Conclusions:(1)oxLDL/β2GPI/β2GPI-Ab complex increase the expression of MMP-9,an A7r5 migration-related molecule,and promote A7r5 migration,suggesting that oxLDL/β2GPI/β2GPI-Ab complexes play an important role in AS-related SMC migration.The blockage of TLR4 significantly inhibited this trend,suggesting that TLR4 is an important signaling molecule in this process.(2)oxLDL/β2GPI/β2GPI-Ab complexes increase the expression of chemokine MCP-1,suggesting that oxLDL/β2GPI/β2GPI-Ab complexes play an important role in AS-associated SMC chemotaxis.The blockage of TLR4 significantly inhibited this trend,suggesting that TLR4 is an important signaling molecule in this process.(3)oxLDL/β2GPI/β2GPI-Ab complex can promote intracellular cholesterol accumulation and AC AT1 expression in A7r5.This phenomenon was significantly inhibited by the pretreatment with TAK-242,suggesting that oxLDL/β2GPI/β2GPI-Ab complexes can effectively promote the lipid accumulation a in A7r5,and TLR4 is an important signaling molecule in this process. |
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