BMP ‐9 regulates the osteoblastic differentiation and calcification of vascular smooth muscle cells through an ALK 1 mediated pathway

The process of vascular calcification shares many similarities with that of physiological skeletal mineralization, and involves the deposition of hydroxyapatite crystals in arteries. However, the cellular mechanisms responsible have yet to be fully explained. Bone morphogenetic protein ( BMP ‐9) has been shown to exert direct effects on both bone development and vascular function. In the present study, we have investigated the role of BMP ‐9 in vascular smooth muscle cell ( VSMC ) calcification. Vessel calcification in chronic kidney disease ( CKD ) begins pre‐dialysis, with factors specific to the dialysis milieu triggering accelerated calcification. Intriguingly, BMP ‐9 was markedly elevated in serum from CKD children on dialysis. Furthermore, in vitro studies revealed that BMP ‐9 treatment causes a significant increase in VSMC calcium content, alkaline phosphatase ( ALP ) activity and m RNA expression of osteogenic markers. BMP ‐9‐induced calcium deposition was significantly reduced following treatment with the ALP inhibitor 2,5‐Dimethoxy‐ N ‐(quinolin‐3‐yl) benzenesulfonamide confirming the mediatory role of ALP in this process. The inhibition of ALK 1 signalling using a soluble chimeric protein significantly reduced calcium deposition and ALP activity, confirming that BMP ‐9 is a physiological ALK 1 ligand. Signal transduction studies revealed that BMP ‐9 induced Smad2, Smad3 and Smad1/5/8 phosphorylation. As these Smad proteins directly bind to Smad4 to activate target genes, si RNA studies were subsequently undertaken to examine the functional role of Smad4 in VSMC calcification. Smad4‐si RNA transfection induced a significant reduction in ALP activity and calcium deposition. These novel data demonstrate that BMP ‐9 induces VSMC osteogenic differentiation and calcification via ALK 1, Smad and ALP dependent mechanisms. This may identify new potential therapeutic strategies for clinical intervention.