Calcium phosphate crystals promotes vascular calcification through BMP-2/Smad signaling

Abstract

Objective To investigate the role of BMP-2/Smad signaling pathway in the osteogenic differentiation of human aortic smooth muscle cells (HASMCs) caused by hyperphosphatemia-induced calcium phosphate (CaP) crystals. Methods High-phosphate medium was incubated at 37℃ for 3 days. CaP crystals and supernatant were isolated by ultracentrifugation. Scanning electron microscope and energy dispersive X-ray spectroscopy were performed for analysis of physicochemical characteristics of CaP crystals. HASMCs were cultured in vitro, and divided into high-phosphate, control, crystals and supernatant groups. Calcification was visualized by Alizarin red staining. Calcium loads in cells were quantified by o-cresolphthalein complexone method. Protein expression of bone morphogenetic protein-2 (BMP-2), Runt-related transcription factor 2 (RUNX2), osteopontin (OPN), phospho-Smad1/5/9 (p-Smad1/5/9) were quantified by Western blotting. After knockdowns of BMP-2 and Smad1 with small hairpin RNA (shRNA) interfering respectively in HASMCs, protein expressions were measured by Western blotting. Results High-phosphate medium induced the formation of CaP crystals. Compared with the cells in control group, CaP crystals significantly induced HASMCs calcification, increased calcium loads and up-regulated the levels of BMP-2, RUNX2 and OPN proteins (all P<0.05). After the addition of CaP crystals into HASMCs, the level of p-Smad 1/5/9 protein peaked at 30 min (P<0.05). After BMP-2 was knocked down in HASMCs, the expression of p-Smad1 caused by CaP crystals was blocked completely, and the expressions of RUNX2 and OPN caused by CaP crystals were reduced significantly (all P<0.05). After Smad1 was knocked down in HASMCs, the expressions of RUNX2 and OPN caused by CaP crystals were decreased significantly (all P<0.05). Conclusions Hyperphosphatemia-induced CaP crystals promoted osteogenic differentiation of HASMCs through the BMP-2/Smad signaling pathway. Key words: Renal insufficiency, chronic; Calcinosis; Calcium phosphates; Myocytes, smooth muscle