Bone Abstracts

The process of vascular calcification shares many similarities with that of skeletal mineralisation, and involves the deposition of hydroxyapatite crystals in arteries and cardiac muscle. However, the cellular mechanisms responsible have yet to be fully elucidated. 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. Murine VSMCs were cultured in calcifying medium containing Na₂HPO₄/NaH₂PO₄ for 14 days. Calcium deposition was confirmed by alizarin red staining. Calcified VSMCs showed increased Runx2, Bmp2 and PiT-1 mRNA expression (P<0.001), which are recognised osteogenic markers of vascular calcification. BMP-9 mRNA expression was significantly up-regulated by 7 days (1.4-fold; P<0.05) in calcified VSMCs. BMP-9 treatment (50 ng/ml) caused a significant increase in VSMC calcium content (3.4-fold; P<0.05), ALP activity (10.1-fold; P<0.001) and mRNA expression of osteogenic markers (P<0.001). BMP-9-induced calcium deposition was significantly reduced (68%; P<0.001) following treatment with the ALP inhibitor 2,5-dimethoxy-N-(quinolin-3-yl)benzenesulfonamide (3 μM) confirming the mediatory role of ALP in this process. BMP receptor expression, including ALK1, ALK2, BMPR-II, ActR-IIA and ActR-IIB, was detected in mouse VSMCs. The inhibition of ALK1 signalling using a soluble chimeric protein (ALK1-Fc) significantly reduced calcium deposition (85%; P<0.001) and ALP activity (33%; P<0.01), confirming that BMP-9 is a physiological ALK1 ligand. Signal transduction studies revealed that BMP-9 (0.5–50 ng/ml) induced Smad1/5/8 and Smad2 phosphorylation. Therefore, as both of these Smad proteins directly bind to Smad4, 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 (72%; P<0.001) and calcium deposition (59%; P<0.05). These novel data demonstrate that BMP-9 induces VSMC calcification through a Smad signalling mechanism. This may identify new potential therapeutic strategies for clinical intervention.