Angiotensin II activates the Smad pathway in vascular smooth muscle cells by a transforming growth factor-β-independent mechanism

Background - Angiotensin II (Ang II) participates in vascular fibrosis. Transforming growth factor-beta (TGF-beta) is considered the most important fibrotic factor, and Smad proteins are essential components of the TGF-beta signaling system. Our aim was to investigate whether Ang II activates the Smad pathway in vascular cells and its potential role in fibrosis, evaluating connective tissue growth factor ( CTGF) and extracellular matrix (ECM) proteins. Methods and Results - Systemic infusion of Ang II into Wistar rats increased aortic Smad2, phosphorylated-Smad2, and Smad4 expression, associated with CTGF upregulation. In growth-arrested vascular smooth muscle cells, Ang II treatment for 20 minutes caused Smad2 phosphorylation, nuclear translocation of phosphorylated-Smad2 and Smad4, and increased Smad DNA-binding activity. Ang II also caused Smad overexpression and Smad-dependent gene transcription. The AT(1) antagonist losartan diminished Ang II - induced Smad activation. The blockade of endogenous TGF-beta did not modify the activation of Smad caused by Ang II. The p38 mitogen-activated protein kinase ( MAPK) inhibitor SB203580 diminished Ang II - induced Smad2 phosphorylation. These data show that Ang II activates the Smad pathway via AT1 receptors and MAPK activation independently of TGF-beta. Transient transfection with Smad7, which interferes with receptor-mediated activation of Smad2, diminished Ang II - induced CTGF promoter activation, gene and protein expression, and fibronectin and type-1 procollagen overexpression, showing that Smad activation is involved in Ang II - induced fibrosis. Conclusions - Our results show that Ang II activates the Smad signaling system in vascular cells in vivo and in vitro. Smad proteins are involved in Ang II - induced CTGF and ECM overexpression independently of TGF-beta. This novel finding suggests that Smad activation could be involved in the profibrogenic effects of Ang II in vascular diseases.