BMP-9 regulates the osteoblastic differentiation and calcification of vascular smooth muscle cells through an ALK1 mediated pathway.

Dongxing Zhu,Colin Farquharson,Neil Charles Wallace Mackenzie,Rukshana C Shroff,Catherine M Shanahan,Vicky Elizabeth Macrae

doi:10.1111/jcmm.12373

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.

Highlights

Arterial medial calcification (AMC), a hallmark of disease in patients with end-stage kidney disease, is highly correlated with elevated serum phosphate levels and cardiovascular mortality [1, 2]
Bone morphogenetic proteins (BMPs)-9 concentration was determined by reading from the standard curve, which was created by Gen5 software (BioTek) using a four parameter logistic (4-PL) curve-fit
BMP-9 was markedly elevated in serum from children on haemodialysis (234% increase compared to pre-dialysis chronic kidney disease (CKD); P < 0.001; Fig. 1)

Summary

Introduction

Arterial medial calcification (AMC), a hallmark of disease in patients with end-stage kidney disease, is highly correlated with elevated serum phosphate levels and cardiovascular mortality [1, 2]. AMC is recognized as an active, tightly regulated process, sharing many similarities with physiological bone formation [3], and involves the deposition of hydroxyapatite crystals in arteries. Vascular smooth muscle cells (VSMCs), the predominant cell type involved in AMC, can undergo transdifferentiation to a chondrocytic, osteoblastic and osteocytic phenotype in a calcified environment [3, 4]. Bone morphogenetic proteins (BMPs) constitute a group of signalling factors that orchestrate embryonic patterning in development and contribute to post-natal tissue remodelling. Over 20 identified BMP ligands are recognized by type I and type II serine-threonine kinase BMP receptors [5]. Ligand binding induces constitutively active BMP type II receptors to transphosphorylate BMP type I receptors, which in turn phosphorylate the intracellular BMP effector proteins, Smad 1/5/8. Signalling through Smad1/5/8 activation mediates the principal effects of BMPs, activation of additional pathways may further refine cellular effects [6]

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Conclusion