[OxLDL/β2GPI/anti-β2GPI complex promotes calcification and inflammatory cytokine expression in A7r5 cells]

Objective To evaluate the effect of oxidized low-density lipoprotein/β2-glycoprotein I/anti-β2 glycoprotein I antibody (oxLDL/β2GPI/anti-β2GPI) complex on the calcification and inflammatory cytokine expression in A7r5 rat vascular smooth muscle cells, and to find out the role of Toll-like receptor 4 (TLR4) signal in this process. Methods The A7r5 cells were intervened with oxLDL, oxLDL/β2GPI complex, oxLDL/anti-β2GPI complex, β2GPI/anti-β2GPI complex, and oxLDL/β2GPI/anti-β2GPI complex for different time, with or without TLR4 inhibitor (TAK-242). Alizarin red staining was used to observe the calcification. Real-time quantitative PCR was applied to detect the total mRNA levels of actin-associated protein SM22α (trangelin) and Runt-related transcription factor 2 (RUNX2). The protein levels of SM22α and RUNX2 were measured by Western blotting. Tumor necrosis factor α (TNF-α) was detected by ELISA. Different concentrations of TNF-α were adopted to stimulate A7r5 cells, and the above methods were operated to examine the changes of SM22 and RUNX2. Results When A7r5 cells were incubated by oxLDL/β2GPI/anti-β2GPI complex, more calcified nodules were observed, the expression of SM22α was reduced and RUNX2 expression was enhanced at both mRNA and protein levels. And the complex increased the expression of inflammation cytokine TNF-α. TLR4 inhibitor TAK-242 reversed the phenomenon. Different concentrations of TNF-α could reduce mRNA and protein expression of SM22α while raise RUNX2 expression. Conclusion oxLDL/β2GPI/anti-β2GPI complex can increase the expression of cytokine TNF-α, and thus quicken calcification procedure in A7r5 cells, along with the change of the traditional contraction phenotype into the osteogenic phenotype. TLR4 receptor participates in this process.