AngII (angiotensin II) is a potent neurohormone responsible for cardiac hypertrophy, in which TGF (transforming growth factor)-β serves as a principal downstream mediator. We recently found that ablation of fibulin-2 in mice attenuated TGF-β signalling, protected mice against progressive ventricular dysfunction, and significantly reduced the mortality after experimental MI (myocardial infarction). In the present study, we investigated the role of fibulin-2 in AngII-induced TGF-β signalling and subsequent cardiac hypertrophy. We performed chronic subcutaneous infusion of AngII in fibulin-2 null (Fbln2−/− ), heterozygous (Fbln2+/− ) and WT (wild-type) mice by a mini-osmotic pump. After 4 weeks of subpressor dosage of AngII infusion (0.2 μg/kg of body weight per min), WT mice developed significant hypertrophy, whereas the Fbln2−/− showed no response. In WT, AngII treatment significantly up-regulated mRNAs for fibulin-2, ANP (atrial natriuretic peptide), TGF-β1, Col I (collagen type I), Col III (collagen type III), MMP (matrix metalloproteinase)-2 and MMP-9, and increased the phosphorylation of TGF-β-downstream signalling markers, Smad2, TAK1 (TGF-β-activated kinase 1) and p38 MAPK (mitogen-activated protein kinase), which were all unchanged in AngII-treated Fbln2−/− mice. The Fbln2+/− mice consistently displayed AngII-induced effects intermediate between WT and Fbln2−/− . Pressor dosage of AngII (2 mg/kg of body weight per min) induced significant fibrosis in WT but not in Fbln2−/− mice with comparable hypertension and hypertrophy in both groups. Isolated CFs (cardiac fibroblasts) were treated with AngII, in which direct AngII effects and TGF-β-mediated autocrine effects was observed in WT. The latter effects were totally abolished in Fbln2−/− cells, suggesting that fibulin-2 is essential for AngII-induced TGF-β activation. In conclusion our data indicate that fibulin-2 is essential for AngII-induced TGF-β-mediated cardiac hypertrophy via enhanced TGF-β activation and suggest that fibulin-2 is a potential therapeutic target to inhibit AngII-induced cardiac remodelling.

Remodeling of the cardiac extracellular matrix (ECM) is an integral part of wound healing and ventricular adaptation after myocardial infarction (MI), but the underlying mechanisms remain incompletely understood. Fibulin-2 is an ECM protein upregulated during cardiac development and skin wound healing, yet mice lacking fibulin-2 do not display any identifiable phenotypic abnormalities. To investigate the effects of fibulin-2 deficiency on ECM remodeling after MI, we induced experimental MI by permanent coronary artery ligation in both fibulin-2 null and wild-type mice. Fibulin-2 expression was up-regulated at the infarct border zone of the wild-type mice. Acute myocardial tissue responses after MI, including inflammatory cell infiltration and ECM protein synthesis and deposition in the infarct border zone, were markedly attenuated in the fibulin-2 null mice. However, the fibulin-2 null mice had significantly better survival rate after MI compared to the wild-type mice as a result of less frequent cardiac rupture and preserved left ventricular function. Up-regulation of TGF-β signaling and ECM remodeling after MI were attenuated in both ischemic and non-ischemic myocardium of the fibulin-2 null mice compared to the wild type counterparts. Increase in TGF-β signaling in response to angiotensin II was also lessened in cardiac fibroblasts isolated from the fibulin-2 null mice. The studies provide the first evidence that absence of fibulin-2 results in decreased up-regulation of TGF-β signaling after MI and protects against ventricular dysfunction, suggesting that fibulin-2 may be a potential therapeutic target for attenuating the progression of ventricular remodeling.

Background: Angiotensin-II (Ang-II) is a potent neurohormone responsible for progression of cardiac remodeling in which TGF-β serves as a principal downstream mediator. In our previous study, genetic deletion of fibulin-2 attenuated progression of ventricular dysfunction after experimental myocardial infarction (MI). Because Ang-II plays a central role in post-MI ventricular remodeling, we tested the hypothesis that fibulin-2 modulates Ang-II-induced cardiac remodeling. Methods: Subpressor dosage of Ang-II (0.2 μg/kg/min) was infused over 4 weeks by mini-osmotic-pump in age matched wild-type (WT), heterozygous, and fibulin-2 null (Fbln2 −/− ) adult male mice. Sham mice received normal saline. Results: There was no blood pressure change throughout Ang-II treatment. WT developed significant left ventricular (LV) hypertrophy by Ang-II, whereas Ang-II-treated Fbln2 −/− mice showed no noticeable hypertrophy compared with sham: LV/body weight ratio (WT 4.83±0.18 vs. Fbln2 −/− 4.01± 0.12 mg/g, p < 0.05) and LV posterior wall thickness by echocardiogram (WT 0.76± 0.03 vs. Fbln2 −/− 0.71± 0.02 mm, p < 0.05). Atrial natriuretic peptide (ANP) mRNA expression was significantly increased in Ang-II-treated WT compared with sham, but not in Ang-II-treated Flbn2 −/− . Ang-II also induced significant up-regulation in fibulin-2, Collagen I, Collagen III, and MMP-2 mRNA level in WT, but not in Fbln2 −/− . Both TGF-β1 mRNA and protein expression were significantly up-regulated in Ang-II-treated WT, but were unchanged in Ang-II-treated Fbln2 −/− compared with sham. Activation of TGF-β downstream signaling proteins, phosphorylated forms of Smad2, TGF-β-activated kinase 1 (TAK1), and p38MAPK, were all significantly increased in Ang-II-treated WT, as opposed to no increase in Ang-II-treated Fbln2 −/− compared with sham. Heterozygous mice showed intermediate increase in LV hypertrophy, matrix protein synthesis, and activation of TGF-β downstream signaling pathways between WT and Fbln2 −/− . Conclusions: Our data suggest that fibulin-2 enhances Ang-II-induced myocardial hypertrophy via up-regulation of TGF-β and its downstream signaling pathways in dose-dependent fashion and that fibulin-2 is required for Ang-II-induced TGF-β activation. This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).