Jun-Kun Zhan1, Yan-Jiao Wang1, Yi Wang1, Sha Wang1, Pan Tan1, Wu Huang1, You-Shuo Liu2. 1. Department of Geriatrics, Second Xiangya Hospital, Institute of Aging and Geriatrics, Central South University, Changsha, Hunan, P.R. China. 2. Department of Geriatrics, Second Xiangya Hospital, Institute of Aging and Geriatrics, Central South University, Changsha, Hunan, P.R. China. Electronic address: Liuyoushuo@yeah.net.
Abstract
BACKGROUND: Vascular calcification is a major risk factor for cardiovascular diseases. Osteoblastic differentiation of vascular smooth muscle cells (VSMCs) is a key step in vascular calcification, but the molecular mechanisms driving the differentiation remain elusive. In this study, the involvement of mammalian target of rapamycin (mTOR) signalling in osteoblastic differentiation of VSMCs is investigated. METHODS: Calcification of VSMCs was induced in vitro using β-glycerophosphate (β-GP). Real-time polymerase chain reaction was used to measure messenger RNA (mRNA) expression, and Western blot was used to detect protein expression. Inhibition of mTOR expression was established by small interfering RNA (siRNA) and mTOR inhibitors. RESULTS: The model for osteoblastic differentiation of VSMCs was established in vitro by treating mouse VSMCs with 10 mM β-GP for 3-15 days. Overexpression of mTOR was observed in differentiated VSMCs. Downregulation of mTOR by siRNA or rapamycin significantly inhibited osteoblastic differentiation of VSMCs and decreased the expression and phosphorylation of mTOR and P70 ribosomal S6 kinase in a time- and concentration-dependent manner. Furthermore, adiponectin inhibited the mRNA and protein expression of mTOR in β-GP-treated VSMCs in a time- and concentration-dependent manner. CONCLUSIONS: mTOR signalling plays a crucial role in the osteoblastic differentiation of VSMCs. Rapamycin and adiponectin might inhibit vascular calcification through regulation of the mTOR pathway.
BACKGROUND:Vascular calcification is a major risk factor for cardiovascular diseases. Osteoblastic differentiation of vascular smooth muscle cells (VSMCs) is a key step in vascular calcification, but the molecular mechanisms driving the differentiation remain elusive. In this study, the involvement of mammalian target of rapamycin (mTOR) signalling in osteoblastic differentiation of VSMCs is investigated. METHODS: Calcification of VSMCs was induced in vitro using β-glycerophosphate (β-GP). Real-time polymerase chain reaction was used to measure messenger RNA (mRNA) expression, and Western blot was used to detect protein expression. Inhibition of mTOR expression was established by small interfering RNA (siRNA) and mTOR inhibitors. RESULTS: The model for osteoblastic differentiation of VSMCs was established in vitro by treating mouseVSMCs with 10 mM β-GP for 3-15 days. Overexpression of mTOR was observed in differentiated VSMCs. Downregulation of mTOR by siRNA or rapamycin significantly inhibited osteoblastic differentiation of VSMCs and decreased the expression and phosphorylation of mTOR and P70 ribosomal S6 kinase in a time- and concentration-dependent manner. Furthermore, adiponectin inhibited the mRNA and protein expression of mTOR in β-GP-treated VSMCs in a time- and concentration-dependent manner. CONCLUSIONS:mTOR signalling plays a crucial role in the osteoblastic differentiation of VSMCs. Rapamycin and adiponectin might inhibit vascular calcification through regulation of the mTOR pathway.