Cardiovascular System ISSN 2052-4358 Original Open Access Periostin as an early marker for elastin mediated vascular smooth muscle cell calciication Ilanchezhian Shanmugam 1 , Aditi Sinha 1 , Russell A. Norris 2 , Roger Markwald 2 and Naren Vyavahare 1* *Correspondence: narenv@clemson.edu 1 Department of Bioengineering, Clemson University, USA. 2 Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, USA. Abstract Rationale: Medial vascular calciication (MAC) has been shown to be an active, cell-mediated process. Although several factors have been shown to promote vascular calciication including diabetes and kidney disease, a more complete understanding of the molecular mechanisms underlying the pathogenesis of this disease is needed. Objective: To ind out earliest markers in elastin mediated vascular smooth muscle cell calciication. Methods and results: We provide evidence that the extracellular matrix protein, periostin, is strongly upregulated during in vitro and in vivo vascular smooth muscle cell calciication. RT-RCR and Western analyses demonstrate that upon stimulation with elastin peptides, rat aortic smooth muscle cells (RASMCs) exhibit a time-dependent increase in periostin expression, which is synergistically enhanced by the addition of transforming growth factor (TGF-β1). his increase was signiicantly attenuated by administering either lactose, an elastin-laminin receptor (ELR) antagonist, or the TGF-β1 receptor antagonist SB431542. As elastin peptides and TGF-β1 have been shown to contribute to RASMC calciication, the potential role of periostin in promoting RASMC calciication was further evaluated. Inhibition of periostin blocked RASMC calciication, whereas overexpression promoted calciication. his periostin-dependent calciication occurs, in part, through the regulation of osteogenic genes: alkaline phosphatase (ALP) and core binding factor 1 (Runx2/Cbfa-1). hese data were furt her corroborated in a rat model of aortic calciic disease whereby periostin expression was conined to the calciied abdominal aorta. Conclusions: Our results demonstrate that periostin is a potent osteogenic molecule in vascular smooth muscle cells and may play a signiicant role in mediating vascular calciication. Keywords: Arteriosclerosis, medial arterial calciication, smooth muscle cells, osteogenesis, calciic disease, elastocalcinosis © 2013 Vyavahare et al; licensee Herbert Publications Ltd. his is an Open Access article distributed under the terms of Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0). his permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction Vascular calcification is a feature of both advanced atherosclerosis and medial artery calcification also known as Monckeberg’s sclerosis [1]. Medial arterial calcification (MAC) is the mineral deposition in the media of arteries and is typically associated with elastin. It occurs independently of atherosclerotic plaque [2]. MAC is observed with particularly high frequency in disorders like end-stage renal disease and diabetes mellitus and it is known to increase mortality and amputation risks [3-4]. Both diabetes and chronic kidney disease have been recognized as pro-inflammatory diseases and they increase expression of tumor necrosis factor-alpha (TNF-α) in arteries which has pro-osteogenic properties [5]. Inflammation in arteries is known to increase matrix degrading enzymes such as matrix metalloproteinases (MMPs) and cathepsins [6-8], which can break down elastic fibers in the media. In diabetic conditions, elevated levels of elastin derived peptides are found in serum [9-11]. It is known that the smooth muscle cells undergo morphological changes to osteoblast-like cells in MAC [12]. We have shown earlier that MMPs play a significant role in elastin degradation and calcification [13]. Rat aortic smooth muscle cells (RASMCs) have shown to increase osteogenic genes with the exposure of elastin fragments along with transforming growth factor, (TGF-β1) [14]. However, how elastin peptides and TGF-β1 triggers this transformation of RASMCs has not been thoroughly investigated. Understanding the role of molecules that regulate the process of elastin mediated vascular calcification is the focus of our study. Osteoblast-specific factor 2 (OSF2), also referred to as periostin, was first identified using subtractive hybridization techniques on MC3T3-E1 osteoblast-like cells and was thought at that time to be bone-specific [15]. Originally termed osteoblast specific factor-2 (OSF-2); it was renamed periostin due to localized expression in the periosteum and the periodontal ligament [16]. Periostin (postn) expression has been implicated in heart valve morphogenesis [17] and in osteogenesis [18]. Periostin is not detected in adult tissues except under conditions of chronic overload, injury, stress, or pathology. Norris et al., were the first to propose that periostin should be classified as a matricellular protein due to its ability to interact with matrix components to serve a structural role and its ability to interact with cell surface receptors and regulate signaling processes [19]. We wanted to test if periostin is the earliest marker in the osteogenic process in RASMCs. Here we show that RASMCs after exposure to elastin fragments and TGF-β1 express high levels of periostin before calcification and blocking of interactions of RASMCs with elastin and TGF- β1 reduce the levels back to the normal cells. Moreover, inhibition of periostin blocked RASMC