125 Vitamin D Inhibits TGFβ-Induced Myofibroblastic Activation of Primary Human Cardiac Fibroblasts

Abstract

Heart failure (HF) is a growing burden in both developed and developing countries. The vitamin D receptor (VDR) is expressed throughout the cardiovascular system, and numerous epidemiological studies have demonstrated that circulating vitamin D levels are correlated with improved outcomes in CVD, and HF in particular. The potential mechanism by which vitamin D may exert beneficial effects is poorly understood, but data from animal studies, and in the setting of renal failure, suggest vitamin D may inhibit fibrosis. Given the critical role of myocardial remodeling, and the fibrotic response in particular, in the setting of heart failure, we examined the effect of vitamin D treatment on fibrotic responses in primary human cardiac fibroblasts. To date, there are no published reports examining vitamin D signaling in human cardiac cells. Commercially available human primary cardiac fibroblasts (Sciencell, CA, USA) were treated with transforming growth factor (TGF) β1 to induce fibrocyte activation, at a concentration of 1ng/ml, in the presence or absence of active vitamin D (1, 25(OH)D3) at 1ìmol/L for 24, 48, 72 and 96 hours. Fibrocyte protein expression was assessed by Western blotting, immunocytochemistry and fluorescence activated cell sorting. Functional responses of fibroblasts were analyzed by in vitro collagen gel contraction and remodeling assays. Gel contraction was assayed over 96 hours, and cells within gels were subsequently imaged by multiphoton and confocal microscopy to evaluate collagen remodeling and signaling effects, respectively. Treatment with 1, 25(OH)D3 for 48 hours reduced alpha smooth muscle actin (αSMA) and collagen I protein expression to control levels. Fibroblast morphology was visibly more similar to untreated controls as compared to TGFβ1 treated cells at all timepoints. Vitamin D treatment significantly inhibited gel contraction at both 48 and 96 hour timepoints (p<0.01). Imaging of fibroblasts within contracted gels showed increased collagen remodeling with TGFβ1 treatment, which was abrogated by addition of 1, 25(OH)D3. Such 1, 25(OH)D3 treatment decreased β-catenin protein levels, and confocal microscopy demonstrated a reduction in nuclear β-catenin in 1, 25(OH)D3 treated cells. Our data demonstrate that active vitamin D can inhibit TGFβ1-mediated functional and biochemical fibrotic changes in human primary cardiac fibroblasts. The reduced levels of β-catenin seen with vitamin D treatment suggest vitamin D inhibits β-catenin mediated gene transcription, and implicates involvement of the canonical Wnt pathway as a potential mechanism of action.