TLR4/NF-κB/Ceramide signaling contributes to Ox-LDL-induced calcification of human vascular smooth muscle cells

Vascular calcification is a major feature of advanced atherosclerosis and highly associated with cardiovascular diseases. Oxidized low density lipoprotein (Ox-LDL) has been recognized as a critical risk factor for atherosclerosis and osteogenic differentiation of vascular smooth muscle cells (VSMCs). Previous studies have demonstrated that toll like receptor 4 (TLR4) is highly expressed in atherosclerotic lesions and participates in the progression of atherosclerosis. However, the role of TLR4 in vascular calcification remains unknown. In this study, we investigated whether TLR4 modulates vascular calcification induced by Ox-LDL. TLR4 expression was up-regulated in cultured human VSMCs treated with Ox-LDL. Knockdown of TLR4 by small interfering RNA (siRNA) significantly reduced Ox-LDL-induced calcification, detected by alizarin red staining and calcium content assay. TLR4 siRNA also decreased the mRNA expression of bone-related proteins including Msx2, osterix, BMP2 and KLF4, but increased the expression of VSMC contractile proteins including SMA and SM22α in VSMCs. In addition, Ox-LDL stimulated nuclear translocation of nuclear factor kappa B (NK-κB) p65. These effects of Ox-LDL on VSMCs were reversed by TLR4 siRNA. Furthermore, NK-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), attenuated Ox-LDL-induced VSMC calcification, which was rescued by C2-ceramide treatment. In conclusion, these findings suggest that TLR4 regulates VSMC calcification induced by Ox-LDL through activation of NK-κB, highlighting the critical role of TLR4/NK-κB signaling in vascular calcification.