Plastic Surgery Research Council
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Presenter: Sudheer K Ravuri, PhD
Co-Authors: Philips BJ; Kling RE; Interval JD; Kashkoush AI; Meyer EM; Pfeifer ME; Zimmerlin L; Donnenberg VS; Donnenberg AD; Marra KG; Rubin JP
University of Pittsburgh

Introduction: Human adipose derived stem cells (ASC) may have broad applications to plastic and reconstructive surgery. For soft tissue reconstruction, the ability to induce adipogenesis and angiogenesis is vital for long term graft survival. However the gene expression and cellular fate of these cells is governed by molecular signaling. Wnt signaling is well evidenced in inhibiting adipogenesis and influence cell differentiation. We hypothesized that regulation of this signaling cascade in adipose stem cells could enhance adipogenesis.

Purpose: This study aims to antagonize Wnt signaling by lentiviral overexpression of secreted frizzled-related protein1 (sFRP1) in ASCs to assess adipogenesis and angiogenic growth factor secretion.

Methods: Wnt antagonistic studies were carried out in lentiviral sFRP1 transfected and flow cytometry selected GFP reporter positive ASCs. mRNA gene expression of signaling cascade were analyzed for adipogenic marker genes (PPARy, FABP4, CEBPa), Wnt related genes (sFRP1, LRP5, Wnt10b, GSK3, beta-catenin), Vascular genes (VEGF-A) and Growth factors (TGF≤-b, TNF-a, IL-1b). Recombinant sFRP1 protein and Niclosamid ligand were used as positive controls for Wnt inhibition. Adipogenesis was assessed by adipored assay.

Results: sFPR1 positive cells showed improved adipogenesis in adipored assay and upregulated mRNA expression (sFRP1, PPARy, FABP4 and C/EBPa by 32, 20, 35 and 15-folds respectively). Cell proliferation was not affected but showed 5-fold increase in angiogenic marker, VEGF-A mRNA expression under normoxia (21% O2). Overexpressed sFRP1 levels suppressed beta-catenin, GSK3 and LRP5 by nearly 5-folds and TGF≤-b, TNF-a and IL-1b around 2-folds.

Conclusions: sFRP1 upregulation can greatly improve adipogenic genes expression and high accumulation of lipid in ASCs by antagonizing Wnt signaling. In addition, expression of angiogenic gene, VEGF-A was also increased upon sFRP1 over expression. The enhanced effects of adipogenesis and angiogenesis caused by sFRP1 overexpression elucidate a signaling pathway that can potentially be exploited for adipose tissue engineering and fat grafting.

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