title: BMP-9 regulates the osteoblastic differentiation and calcification of vascular smooth muscle cells through an ALK1 mediated pathway.
abstract: 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 mRNA 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 ALK1 signalling using a soluble chimeric protein significantly reduced calcium deposition and ALP activity, confirming that BMP-9 is a physiological ALK1 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, siRNA studies were subsequently undertaken to examine the functional role of Smad4 in VSMC calcification. Smad4-siRNA 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 ALK1, Smad and ALP dependent mechanisms. This may identify new potential therapeutic strategies for clinical intervention.
authors.affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Midlothian, UK.
authors.forename: Dongxing
authors.initials: D
authors.lastname: Zhu
authors.forename: Neil Charles Wallace
authors.initials: NC
authors.lastname: Mackenzie
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Shanahan
authors.forename: Rukshana C
authors.initials: RC
authors.lastname: Shroff
authors.forename: Colin
authors.initials: C
authors.lastname: Farquharson
authors.forename: Vicky Elizabeth
authors.initials: VE
authors.lastname: MacRae
mesh_terms: D030201:Activin Receptors, Type I / Q000378:metabolism*; D030301:Activin Receptors, Type II; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002113:Calcification, Physiologic / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D003956:Dialysis; D055427:Growth Differentiation Factor 2 / Q000097:blood / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008810:Mice, Inbred C57BL; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects; D010710:Phosphates / Q000494:pharmacology; D051436:Renal Insufficiency, Chronic / Q000097:blood; D015398:Signal Transduction / Q000187:drug effects; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D055427:Growth Differentiation Factor 2; D010710:Phosphates; D030201:Activin Receptors, Type I; D030301:Activin Receptors, Type II; C498931:Acvrl1 protein, mouse; D000469:Alkaline Phosphatase
keywords: ALK1; BMP-9; vascular calcification; vascular smooth muscle cells