Fibulin-2 is essential for angiotensin II-induced myocardial fibrosis mediated by transforming growth factor (TGF)-β

Fibrosis is an ominous pathological process in failing myocardium, but its pathogenesis is poorly understood. We recently reported that loss of an extracellular matrix (ECM) protein, fibulin-2, protected against ventricular dysfunction after myocardial infarction (MI) in association with absence of activation of transforming growth factor (TGF)-beta signaling and suppressed upregulation of ECM protein expression during myocardial remodeling. Here we investigated the role of fibulin-2 in the development of myocardial hypertrophy and fibrosis induced by continuous pressor-dosage of angiotensin II (Ang II) infusion. Both wild type (WT) and fibulin-2 null (Fbln2K0) mice developed comparable hypertension and myocardial hypertrophy by Ang II infusion. However, myocardial fibrosis with significant upregulation of collagen type I and III mRNA was only seen in WT but not in Fbln2K0 mice. Transforming growth factor (TGF)-beta(1) mRNA and its downstream signal, Smad2, were significantly upregulated in WT by Ang II, whereas there were no Ang II-induced changes in Flbn2KO, suggesting fibulin-2 is necessary for Ang II-induced TGF-beta signaling that induces myocardial fibrosis. To test whether fibulin-2 is sufficient for Ang II-induced TGF-beta upregulation, isolated Flbn2KO cardiac fibroblasts were treated with Ang II after transfecting with fibulin-2 expression vector or pretreating with recombinant fibulin-2 protein. Ang II-induced TGF-beta signaling in Fbln2KO cells was partially rescued by exogenous fibulin-2, suggesting that fibulin-2 is required and probably sufficient for Ang II-induced TGF-beta activation. Smad2 phosphorylation was induced just by adding recombinant fibulin-2 to KO cells, suggesting that extracellular interaction between fibulin-2 and latent TGF-beta triggered initial TGF-beta activation. Our study indicates that Ang II cannot induce TGF-beta activation without fibulin-2 and that fibulin-2 has an essential role in Ang II-induced TGF-beta signaling and subsequent myocardial fibrosis. Fibulin-2 can be considered as a critical regulator of TGF-beta that induces myocardial fibrosis.