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Rat Aortic Smooth Muscle Cells Cultured on Hydroxyapatite Differentiate into Osteoblast-Like Cells via BMP-2–SMAD-5 Pathway

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Abstract

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 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 confirming their role in osteogenesis of Vsmcs.

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Abbreviations

VSMCs:

Vascular smooth muscle cells

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

SMA:

Alpha smooth muscle actin

MHC:

Myosin heavy chain

BMP-2:

Bone morphogenetic protein 2

SMAD-5:

Sma- and Mad-related protein 5

Col2a1:

Collagen type II alpha 1

RUNX2:

Runt-related transcription factor 2

ALP:

Alkaline phosphatase

DAPI:

4′,6-Diamidino-2-phenylindole, dihydrochloride

HA:

Hydroxyapatite

MGP:

Matrix-gla protein

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Acknowledgments

The authors would like to thank Jayesh Betala and Dr. Martine LaBerge at the Department of Bioengineering at Clemson University for the gift of rat aortic smooth muscle cells. This study was partially supported by NIH P20GM103444 grant and the Hunter Endowment to NV.

Conflict of Interest

Pranjal Nahar-Gohad, Neeraj Gohad, Chen-Chih Tsai, Rajendra Bordia, and Naren Vyavahare declare that there is no conflict of interest.

Human and Animal Rights and Informed Consent

No Human Subjects were involved in this study. The Clemson University Institutional Animal Care and Use Committee (IACUC) assures that animal care and use are in compliance with all federal, state and local regulations as well as University policy and assurances. This research study has been carried out in compliance with IACUC regulations.

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Authors and Affiliations

  1. Department of Bioengineering, Clemson University, 501 Rhodes Research Center, Clemson, SC, 29634, USA

    Pranjal Nahar-Gohad & Naren Vyavahare

  2. Department of Biological Sciences, Clemson University, 132 Long Hall, Clemson, SC, 29634, USA

    Neeraj Gohad

  3. Department of Material Sciences, Clemson University, 161 Sirrine Hall, Clemson, SC, 29634, USA

    Chen-Chih Tsai & Rajendra Bordia

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  1. Pranjal Nahar-Gohad
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Correspondence to Naren Vyavahare.

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Nahar-Gohad, P., Gohad, N., Tsai, CC. et al. Rat Aortic Smooth Muscle Cells Cultured on Hydroxyapatite Differentiate into Osteoblast-Like Cells via BMP-2–SMAD-5 Pathway. Calcif Tissue Int 96, 359–369 (2015). https://doi.org/10.1007/s00223-015-9962-z

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