The Role Of OxLDL/β2GPI/Anti-β2GPI Complex On Promoting Calcification And Inflammatory Cytokines Expression In A7r5 Cells

Objective: Vascular calcification is a middle-to-late course of cardiovascular disease and is the result of ectopic deposition of calcium and phosphorus crystals.Vascular calcification involves the expression of multiple bone / osteogenic proteins and is closely related to metabolic diseases such as atherosclerosis(AS).Oxidized low-density lipoprotein(ox LDL)is the key lipid for atheromatous plaques.Its excessive accumulation in the body causes cells to develop into foam cells,which is the initial stage of AS.It has been found that patients with anti-phospholipid syndrome(APS)generally have the same clinical features of atherosclerosis.Abundant β2GPI antigens and anti-β2GPI antibodies in such patients can combine with ox LDL to form complex ox LDL/β2GPI/anti-β2GPI complexes,having an adverse effect on the disease.Vascular smooth muscle cells are located in the medial wall of blood vessels.In the development of AS,smooth muscle cells can not only express high levels of inflammatory factors,but also undergo phenotypic changes while possessing the functions of other cells: such as transformation to macrophages phagocytosing lipids;transition to osteoblasts,promoting calcification.The previous results of research confirmed that the ox LDL/β2GPI/ anti-β2GPI complex mediates macrophage-like transformation of A7r5 and affects lipid transport through the TLR4-JNK1/2 signaling molecule,but there is no specific mechanism in calcification.Therefore,this study want to further investigate whether the ox LDL/β2GPI/anti-β2GPI complex can mediate bone / osteoblast-like transformation in A7r5 rats vascular smooth muscle cells through inflammation molecules,and whether the TLR4 pathway plays a relevant role in the process.Methods:(1)The A7r5 cells were stimulated by ox LDL,ox LDL/β2GPI,ox LDL/anti-β2GPI,β2GPI/anti-β2GPI,ox LDL/β2GPI/anti-β2GPI,TAK-242+ ox LDL/β2GPI/anti-β2GPI or 2.6 mmol/L Pi.Alizarin red S staining was used to detect mineralization in the cells.(2)The A7r5 cells were incubated with ox LDL,ox LDL/β2GPI,ox LDL/anti-β2GPI,β2GPI/anti-β2GPI,or ox LDL/β2GPI/anti-β2GPI.Real-time quantitative PCR(RT-q PCR)was used to analysis m RNA levels of SM22α and Runx2 in different stimulation groups.Western blot was applied to detect protein levels of SM22α and Runx2.A7r5 cells were pretreated with TAK-242 to test whether TLR4 involved in this process.(3)The expression of ALP and TNF-α in the cells induced by ox LDL,ox LDL/β2GPI,ox LDL/anti-β2GPI,β2GPI/anti-β2GPI,ox LDL/β2GPI/ anti-β2GPI were measured by ELISA kits.(4)TNF-α with different concentration was used to stimulate A7r5 cells,SM22α and Runx2 m RNA and protein levels were detected via RT-q PCR and Western blot.Results(1)The mineralized nodules in the cells were significantly increased after induced with ox LDL(50 μg/m L)/β2GPI(100 μg/m L)/anti-β2GPI(100 μg/m L)complex,while pretreating with TAK-242,the mineralized nodules can be partially declined(p<0.01).(2)The complex of ox LDL/β2GPI/anti-β2GPI can decrease the m RNA and protein levels of SM22α of cells,while increasing the expression of Runx2 and ALP,compared with the blank(p<0.05).TLR4 inhibitor TAK-242 can partially reverse the expression of SM22α and Runx2 in the cells.(3)After treatment with ox LDL/β2GPI/anti-β2GPI complex,the protein level of TNF-α in the cells was significantly increased(p<0.05).SM22α could go down compared with blank,while Runx2 could go up after different concentrations of TNF-α stimulation on cells.Conclusions:(1)ox LDL/β2GPI/anti-β2GPI complex can reduce the surface contraction marker SM22α level in A7r5 cells and increase the expression of bone/osteoblast marker Runx2 and ALP,which can promote the cell calcification.TLR4 inhibitor can reserve this process,suggesting that TLR4 may play an important role in A7r5 cell calcification inducing by ox LDL/β2GPI/anti-β2GPI.(2)Treatment of ox LDL/β2GPI/anti-β2GPI complex on A7r5 cells,the expression of TNF-α in cells can be increased.The stimulation of TNF-α on cells can induce calcification of cells,suggesting that ox LDL/β2GPI/anti-β2GPI complex may promote calcification of cells through inflammatory molecules.It is suggested that reducing the expression of inflammatory molecules may effectively slow down the process of calcification.