Connective tissue growth factor expression after angiotensin II exposure is dependent on transforming growth factor-ß signaling via the canonical Smad-dependent pathway in hypertensive induced myocardial fibrosis.

ABSTRACT

INTRODUCTION: Transforming growth factor-ß (TGF-ß) and connective tissue growth factor (CTGF) are often described as the initial pro-fibrotic mediators upregulated early in fibrosis models dependent on angiotensin II (Ang-II). In the present study, we explore the mechanistic link between TGF-ß and CTGF expression by using a novel TGF-ß trap. MATERIALS AND METHODS: NIH/3T3 fibroblasts were subjected to TGF-ß with or without TGF-ß trap or 1D11 antibody, CTGF or CTGF plus TGF-ß for six or 24 hours, and then used for quantitative real-time polymerase chain reaction (qRT-PCR) or immunocytochemistry. Male C57BL/6 mice were infused with Ang-II and randomly assigned TGF-ß trap for six or 24 hours. Hearts were harvested for histological analyses, qRT-PCR and western blotting. RESULTS: Exogenous TGF-ß-induced fibroblasts resulted in significant upregulation of CTGF, TGF-ß and type I collagen transcript levels in vitro. Additionally, TGF-ß promoted the differentiation of fibroblasts into α-SMA+ myofibroblasts. CTGF expression was reduced by the addition of TGF-ß trap or neutralizing antibody, confirming that its expression is dependent on TGF-ß signaling. In contrast, exogenous CTGF did not appear to have an effect on fibroblast production of pro-fibrotic transcripts or fibroblast differentiation. Ang-II infusion in vivo led to a significant increase in TGF-ß and CTGF mRNA expression at six and 24 hours with corresponding changes in Smad2 phosphorylation (pSmad2), indicative of increased TGF-ß signaling. Ang-II animals that received the TGF-ß trap demonstrated reduced CTGF mRNA levels and pSmad2 at six hours, suggesting that early CTGF expression is dependent on TGF-ß signaling. CONCLUSIONS: We demonstrated that CTGF expression is dependent on TGF-ß signaling both in vitro and in vivo in a model of myocardial fibrosis. This also suggests that early myocardial CTGF mRNA expression (six hours) after Ang-II exposure is likely dependent on latent TGF-ß activation via the canonical Smad-dependent pathway in resident cardiac cells.