caa a22 4500 605521360 CHVBK 20210128100739.0 cr unu---uuuuu 210128e20150401xx s 000 0 eng 10.1007/s00223-015-9962-z doi (NATIONALLICENCE)springer-10.1007/s00223-015-9962-z Rat Aortic Smooth Muscle Cells Cultured on Hydroxyapatite Differentiate into Osteoblast-Like Cells via BMP-2-SMAD-5 Pathway [Elektronische Daten] [Pranjal Nahar-Gohad, Neeraj Gohad, Chen-Chih Tsai, Rajendra Bordia, Naren Vyavahare] Vascular calcification 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 calcification 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 first characterized vascular origin of Vsmcs by studying the expression of smooth muscle cell markers: myosin heavy chain and smooth muscle actin along with SM22α 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 inupregulation 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 confirming their role in osteogenesis of Vsmcs. Springer Science+Business Media New York, 2015 Calcium-sensing receptor (CaSR) nationallicence Hydroxyapatite (HA) nationallicence Bone morphogenetic protein 2 (BMP-2) nationallicence Vascular smooth muscle cells (Vsmcs) nationallicence Osteogenic markers nationallicence BMP-2 inhibitor LDN nationallicence SMAD-5: Sma- and Mad-related protein 5 nationallicence VSMCs : Vascular smooth muscle cells nationallicence GAPDH : Glyceraldehyde 3-phosphate dehydrogenase nationallicence SMA : Alpha smooth muscle actin nationallicence MHC : Myosin heavy chain nationallicence BMP-2 : Bone morphogenetic protein 2 nationallicence SMAD-5 : Sma- and Mad-related protein 5 nationallicence Col2a1 : Collagen type II alpha 1 nationallicence RUNX2 : Runt-related transcription factor 2 nationallicence ALP : Alkaline phosphatase nationallicence DAPI : 4′,6-Diamidino-2-phenylindole, dihydrochloride nationallicence HA : Hydroxyapatite nationallicence MGP : Matrix-gla protein nationallicence Nahar-Gohad Pranjal Department of Bioengineering, Clemson University, 501 Rhodes Research Center, 29634, Clemson, SC, USA aut Gohad Neeraj Department of Biological Sciences, Clemson University, 132 Long Hall, 29634, Clemson, SC, USA aut Tsai Chen-Chih Department of Material Sciences, Clemson University, 161 Sirrine Hall, 29634, Clemson, SC, USA aut Bordia Rajendra Department of Material Sciences, Clemson University, 161 Sirrine Hall, 29634, Clemson, SC, USA aut Vyavahare Naren Department of Bioengineering, Clemson University, 501 Rhodes Research Center, 29634, Clemson, SC, USA aut Calcified Tissue International Springer US; http://www.springer-ny.com 96/4(2015-04-01), 359-369 0171-967X 96:4<359 2015 96 223 https://doi.org/10.1007/s00223-015-9962-z text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00223-015-9962-z text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Nahar-Gohad Pranjal Department of Bioengineering, Clemson University, 501 Rhodes Research Center, 29634, Clemson, SC, USA aut NATIONALLICENCE 700 1- Gohad Neeraj Department of Biological Sciences, Clemson University, 132 Long Hall, 29634, Clemson, SC, USA aut NATIONALLICENCE 700 1- Tsai Chen-Chih Department of Material Sciences, Clemson University, 161 Sirrine Hall, 29634, Clemson, SC, USA aut NATIONALLICENCE 700 1- Bordia Rajendra Department of Material Sciences, Clemson University, 161 Sirrine Hall, 29634, Clemson, SC, USA aut NATIONALLICENCE 700 1- Vyavahare Naren Department of Bioengineering, Clemson University, 501 Rhodes Research Center, 29634, Clemson, SC, USA aut NATIONALLICENCE 773 0- Calcified Tissue International Springer US; http://www.springer-ny.com 96/4(2015-04-01), 359-369 0171-967X 96:4<359 2015 96 223