Introduction: As the primary cells of the heart valve responsible for valve formation and tissue homeostasis, valvular interstitial cells (VICs) are a diverse, dynamic, and highly plastic population with quiescent, activated, and osteoblastic phenotypes.1 Using ω-functionalized alkanethiol self-assembled monolayers (SAMs) on gold [CH3 (hydrophobic), OH (hydrophilic), COO- (negative at physiological pH), and NH3+ (positive at physiological pH)], we have identified several novel in vitro mechanisms for activated/healthy and osteoblastic differentiation of VICs in response to surface chemistry. Most importantly, previous studies have shown that NH3+ surfaces results in rapid and robust calcium nodule formation while COO-surfaces prevent calcification. While osteoblastic media resulted in distinct morphological changes little calcified nodule formation was exhibited. In the current study we have identified possible mechanisms of calcification through differential integrin expression and binding to surfaces.
Materials and Methods: SAMs Fabrication & Characterization: SAMs were created as previously described2 and characterized by x-ray photoelectron spectroscopy (XPS), sessile drop goniometry, optical ellipsometry, and atomic force microscopy (AFM). Cell Studies: Primary VICs were obtained from porcine aortic heart valves via collagenase digestion3 with endothelial cells removal using CD31-coated magnetic dnyabeads. Samples were seeded at 21,500 cells/cm2 and maintained in 10% fetal bovine serum supplemented growth media with 10mM β-glycerophosphate, 10-6M ascorbic acid, & 10-7M dexamethasone supplemented osteoblastic controls.4 Genetic analysis (qPCR) of α-smooth muscle actin (αSMA) and bone gamma-carboxyglutamic acid-containing protein (BGLAP/osteocalcin) was conducted using TaqMan probes with glyceraldehydes 3-phophatase dehyderogenase (GAPDH) as an endogenous control. Calcium deposition was stained with 10% alizarin red solution.4Integrin expression was assessed using α/β integrin-mediated cell adhesion array (Millipore, ECM535) or immunofluorescent imagining and flowcytometry.
Results and Discussion: Characterization: XPS, contact angle measurements (CH3-105.9°±0.9, OH-14.9°±1.5, COO--31.8°±2.9, & NH3+-46.6°±4.9), film thickness, and AFM confirm uniform SAM formation and correspond with published values.2 Cellular Studies: While COO- environments show no signs of calcification, the increased expression of RGD binding integrin subunits (αv, α5) may play a role in inhibiting calcium deposition in this cell type.2,5 While both NH3+ and osteoblastic environments exhibit high β1 integrin expression as an indicator of differentiation,2,5 rapid calcified nodule formation and increased osteocalcin expression over osteoblastic media suggest alternative mechanisms of osteoblastic differentiation. Furthermore, increased expression of RGD binding (α5) within osteoblastic environments may explain the lack of early calcification exhibited.
Conclusions: Distinct phenotypic change in VICs due to variations in material chemistry are an important factor in disease progression. In well-defined environments, the expression of specific integris has been shown to correlate with differentiation potential. 2,5 Through identification of variations in integrin binding subunits for osteoblastic-like disease in differing models of progression as well as non-calcifying in vitro environments we can now begin to identify possible mechanisms for disease progression. These results provide alternative models for in vitroVIC expansion as well as directing disease development of VICs without the addition of exogenous signaling factors.
References: 1) Liu AC, Am J Path. 2007, 171:1407-1416. 2) Keselowsky BG, JBMR, 2003, 66A:247-259. 3) Johnson CM, J Mol Cell Card, 1987, 19:1185-1193. 4) Cloyd KL, PLOSone, 2012, 7:e48154. 5) Keselowsky BG, PNAS, 2005, 102(17):5953-5957.
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