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Calcif Tissue Int (2015) 96:359369
DOI 10.1007/s00223-015-9962-z
ORIGINAL RESEARCH
Rat Aortic Smooth Muscle Cells Cultured on Hydroxyapatite Differentiate into Osteoblast-Like Cells via BMP-2SMAD-5 Pathway
Pranjal Nahar-Gohad Neeraj Gohad
Chen-Chih Tsai Rajendra Bordia
Naren Vyavahare
Received: 14 September 2014 / Accepted: 3 February 2015 / Published online: 1 March 2015 Springer Science+Business Media New York 2015
Abstract Vascular calcication is an important pathological condition associated with increased risk of cardiovascular mortality. Hydroxyapatite (HA) found in such deposits is the same polymorph of calcium (Ca) found in bone, indicating calcication may involve mechanisms akin to bone formation. Vascular smooth muscle cells (Vsmcs) have been shown to undergo phenotypic change to osteoblast-like cells. However, the mechanisms underlying this phenotypic change are unclear, and whether the stimulus to become osteogenic is a result of loss of mineralization inhibitors or early mineral deposits is not known. Our aim in this study is to identify mechanisms and signal transduction pathways that cause differentiation of Vsmcs into osteoblast-like cells in the presence of HA. We rst characterized vascular origin of Vsmcs by studying the expression of smooth muscle cell markers: myosin heavy chain and smooth muscle actin along with SM22a at both mRNA and protein levels. Vsmcs grown on HA exhibited
progressive change in cellular morphology at 3-, 7-, and 14-day time points. Culturing of Vsmcs on HA disc resulted in decrease in media Ca levels and increased expression of Ca-sensing receptor (CaSR) on Vsmcs resulting in upregulation of intracellular CaSR signaling leading to increased BMP-2 secretion. BMP-2 pathway mediated differentiation of Vsmcs to osteoblast-like cells shown by expression of osteogenic markers like runt-related transcription factor 2, osteocalcin, and alkaline phosphatase at mRNA and protein levels. Blocking CaSR by NPS-2143 reduced BMP-2 secretion and blocking the BMP-2 pathway by LDN-193189, a BMP inhibitor, modulated expression of osteogenic markers conrming their role in osteogenesis of Vsmcs.
Keywords Calcium-sensing receptor (CaSR)
Hydroxyapatite (HA) Bone morphogenetic protein 2
(BMP-2) Vascular smooth muscle cells (Vsmcs)
Osteogenic markers BMP-2 inhibitor LDN SMAD-5:
Sma- and Mad-related protein 5
AbbreviationsVSMCs Vascular smooth muscle cellsGAPDH Glyceraldehyde 3-phosphate dehydrogenase SMA Alpha smooth muscle actinMHC Myosin heavy chainBMP-2 Bone morphogenetic protein 2SMAD-5 Sma- and Mad-related protein 5Col2a1 Collagen type II alpha 1RUNX2 Runt-related transcription factor 2ALP Alkaline phosphataseDAPI 40,6-Diamidino-2-phenylindole, dihydrochloride
HA Hydroxyapatite...