Effect Of Bone Morphogentic Protein-7 On Vascular Smooth Muscle Cells Calcification Induced By High Phosphorus

Objective This study is aimed to investigate the effect of BMP-7 on vascular smooth muscle cells (VSMCs) calcification induced by high phosphorus in vitro by determining the calcium depocition and expression of Pit-1 and Cbfα-1.Methods The explants derived from thoracic aorta were used for primary culture of vascular smooth muscle cells and identification of cells was carried on by morphological identification and direct immunohistochemical staining ofα-SMA, Passage 3 to 8 of VSMCs were used for experiments. Briefly, VSMCs were placed in various culture media, including normal phosphorus medium (Pi 1.3 mmol/L), high phosphorus medium (Pi 2.6 mmol/L ) , high phosphorus plus rhBMP-7 medium ( Pi2.6mmol/L + rhBMP-7 100ng/ml ) , normal phosphorus plus rhBMP-7 medium (Pi1.3mmol/L+ rhBMP-7 100ng/ml),calcium deposition was quantified and visualized by Alizarin stain method at day 6. Pit-1 mRNA levels was determined by RT-PCR, and Cbfα-1 expression was semi-quantified by RT-PCR and Western Blot at day 3. All of the experiments were repeated for 3 times at least.Results1. Under inverted phase contrast microscope we found VSMCs were characterized by the ribbon spindle shape and showed"hill and valley"morphological appearance. Direct immunohistochemical staining ofα-SMA showed positive staining in the cultured VSMCs, and there were more than 95% VSMCs in the cell cultures.2. Compared with normal phosphorus group, the calcium deposition was significantly increased in high phosphorus group (P<0.01),and BMP-7 reduced the increasing calcium deposition induced by high phosphorus(P<0.01), as were also shown in Alizarin stain.3. Compared with normal phosphorus group, the Pit-1 mRNA expression was significantly increased in high phosphorus group (P<0.01), and BMP-7 reduced the increasing Pit-1 mRNA expression (P <0.01).4. As were shown in RT-PCR and Western Blot, Compared with normal phosphorus group, the Cbfα-1 expression was significantly increased in high phosphorus group(P<0.01), and BMP-7 reduced the increasing Cbfα-1 mRNA(P <0.01) and protein (P=0.02) expression.Conclusion1. Phosphorus induces VSMCs calcification in vitro.2. BMP-7 inhibits the transformation of vascular smooth muscle cells into osteoblast-like cells induced by high phosphorus in vitro.3. BMP-7 inhibits the calcium deposition of vascular smooth muscle cells induced by high phosphorus in vitro.