Abstract 2950: Fibulin-2 is Essential for Angiotensin II-Induced Transforming Growth Factor-β-Mediated Extracellular Matrix Remodeling in Isolated Mouse Cardiac Fibroblasts
Introduction Angiotensin II (Ang II) is a potent neurohormone inducing cardiac remodeling. Transforming growth factor (TGF)-β serves as a principal downstream mediator of Ang II. Recently we found that cardiac fibulin-2 is up-regulated by Ang II and that the absence of fibulin-2 attenuates Ang II-induced myocardial remodeling in association with TGF-β inactivation in vivo. Here we tested the hypothesis that fibulin-2 is essential for Ang II-induced TGF-β-activation in isolated mouse cardiac fibroblasts.
Materials and methods Fibroblasts were isolated from either wild type (WT) or fibulin-2 knockout (KO) mouse hearts. WT and KO cells were treated with Ang II (10−7 M/L for 24 hours) with/without TGF-β neutralizing antibody (10 ng/ml). KO cells were treated with Ang II with/without recombinant fibulin-2 (0.5 μg/ml or 2.5 μg/ml). For extracellular matrix (ECM) synthesis, mRNA levels of collagen type I, type III, fibulin-2, TGF-β1, and MMP-2 were assessed by real-time RT-PCR. TGF-β1 protein in the conditioned medium was measured by ELISA. Activation levels of Smad2 and ERK1/2 were assessed by Western analysis.
Results For all the above ECM proteins except fibulin-2, baseline mRNA expression levels were comparable between WT and KO cells. With Ang II treatment, the increase in TGF-β1 mRNA, TGF-β1 protein in the medium, and pSmad2/Smad2 ratio from the baseline was significantly higher in WT than in KO, so was the increase of other ECM proteins. In WT, Ang II-induced expression of these ECM proteins was partially but significantly reduced by TGF-β neutralizing antibody, whereas in KO the Ang II-induced expression of these ECM proteins was not affected by TGF-β antibody. Ang II-induced TGF-β-independent up-regulation of ECM markers was comparable between WT and KO. In KO cells, recombinant fibulin-2 dose-dependently enhanced Ang II-induced TGF-β signaling assessed by Smad2 and ERK1/2 phosphorylation.
Conclusion Our study demonstrates that Ang II enhances ECM synthesis via TGF-β-dependent and TGF-β-independent fashion and that fibulin-2 is essential for TGF-β-dependent signaling pathways and ECM synthesis in response to Ang II. Our findings suggest that fibulin-2 is a novel and essential extracellular enhancer of TGF-β in Ang II-induced ECM remodeling.
This research has received full or partial funding support from the American Heart Association, Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).