title: TLR4/NF-κB/Ceramide signaling contributes to Ox-LDL-induced calcification of human vascular smooth muscle cells.
abstract: 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.
authors.affiliation: Department of Pathophysiolgy, Zhongshan Medical School, Sun Yat-Sen University, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Song
authors.affiliation: Department of Pathophysiolgy, Zhongshan Medical School, Sun Yat-Sen University, China.
authors.forename: Menglin
authors.initials: M
authors.lastname: Hou
authors.affiliation: Department of Histology and Embryology, Key Laboratory of Tissue Construction and Detection of Guangdong Province, Southern Medical University, China.
authors.forename: Zhenlin
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Cardiology, The First Affiliated Hospital, Sun Yat-Sen University, China.
authors.forename: Chufan
authors.initials: C
authors.lastname: Luo
authors.affiliation: Division of Cardiac Surgery, The First Affiliated Hospital, Sun Yat-Sen University, China; The key Laboratory of Assisted Circulation, Ministry of Health, Sun Yat-Sen University, China.
authors.forename: Jing-Song
authors.initials: JS
authors.lastname: Ou
authors.affiliation: Department of Cardiology, Guangdong General Hospital, Guangdong Academy of Medical Sciences and Guangdong Cardiovascular Institute, China.
authors.forename: Huimin
authors.initials: H
authors.lastname: Yu
authors.affiliation: Department of Histology and Embryology, Key Laboratory of Tissue Construction and Detection of Guangdong Province, Southern Medical University, China. Electronic address: yanjy790@163.com.
authors.forename: Jianyun
authors.initials: J
authors.lastname: Yan
authors.affiliation: Department of Pathophysiolgy, Zhongshan Medical School, Sun Yat-Sen University, China. Electronic address: lulihe@mail.sysu.edu.cn.
authors.forename: Lihe
authors.initials: L
authors.lastname: Lu
mesh_terms: D002114:Calcinosis / Q000139:chemically induced* / Q000378:metabolism / Q000473:pathology*; D002454:Cell Differentiation / Q000187:drug effects; D002518:Ceramides / Q000378:metabolism*; D006801:Humans; D000090062:Kruppel-Like Factor 4; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D016328:NF-kappa B / Q000378:metabolism*; D010012:Osteogenesis / Q000187:drug effects; D051197:Toll-Like Receptor 4 / Q000378:metabolism*
chemical_list: D002518:Ceramides; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D008077:Lipoproteins, LDL; D016328:NF-kappa B; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; C099252:oxidized low density lipoprotein
keywords: Calcification; Nuclear factor kappa B; Oxidized low density lipoprotein; Toll like receptor 4; Vascular smooth muscle cells