Transforming growth factor-beta (TGF-beta) participates in the pathogenesis of multiple cardiovascular diseases, including hypertension, restenosis, atherosclerosis, cardiac hypertrophy and heart failure. TGF-beta exerts pleiotropic effects on cardiovascular cells, regulating cell growth, fibrosis and inflammation. TGF-beta has long been believed to be the most important extracellular matrix regulator. We review the complex mechanisms involved in TGF-beta-mediated vascular fibrosis that includes the Smad signaling pathway, activation of protein kinases and crosstalk between these pathways. TGF-beta blockade diminishes fibrosis in experimental models, however better antifibrotic targets are needed for an effective therapy in human fibrotic diseases. A good candidate is connective tissue growth factor (CTGF), a downstream mediator of TGF-beta-induced fibrosis. Among the different factors involved in vascular fibrosis, Angiotensin II (AngII) has special interest. AngII can activate the Smad pathway independent of TGF-beta and shares with TGF-beta many intracellular signals implicated in fibrosis. Blockers of AngII have demonstrated beneficial effects on many cardiovascular diseases and are now one of the best options to block TGF-beta fibrotic responses. A better knowledge of the intracellular signals of TGF-beta can provide novel therapeutic approaches for fibrotic diseases.

Angiotensin II (AngII) participates in the pathogenesis of renal diseases, through the regulation of two key processes inflammation and fibrosis. AT₁ and AT₂ are the main receptors of AngII. AT₁ mediates most of the actions of AngII. This receptor regulates the expression of profibrotic factors, such as connective tissue growth factor (CTGF). The Smad signalling pathway and the Rho/Rho kinase system are two novel mechanisms involved in AngII-induced matrix regulation recently described. The role of AT₂ receptors in renal pathophysiological processes is not fully elucidated. Experimental data suggest that AT₂ receptors through activation of nuclear factor-κB participate in renal inflammatory cell recruitment. Studies in animal models of kidney injury have shown that the combined blockade of both AT₁ and AT₂ receptors, as well as the inhibition of the NF-κB pathway are necessary to stop the inflammatory process fully. On the whole, these data highlight the complex signalling systems activated by AngII and suggest novel potential targets to block fibrosis and inflammation in renal diseases.

Background— Angiotensin II (Ang II) participates in vascular fibrosis. Transforming growth factor-β (TGF-β) is considered the most important fibrotic factor, and Smad proteins are essential components of the TGF-β 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-β 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 AT ₁ receptors and MAPK activation independently of TGF-β. 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-β. This novel finding suggests that Smad activation could be involved in the profibrogenic effects of Ang II in vascular diseases.

Significance: The expression of multiple isoforms of a protein kinase in cells raises the question of which substrates are preferentially phosphorylated by each isoform. Endothelial cells (ECs) that line all blood vessels express the protein kinases, Akt1 and Akt2, and we demonstrate here that endothelial nitric oxide synthase (eNOS) is a preferential Akt1 substrate and supraphysiological overexpression of Akt2 is needed to phosphorylate eNOS and promote NO release. In mice, the endothelial loss of Akt1, but not Akt2, retards vessel growth, and quantitative assessment of the phosphoproteomes in ECs isolated from these mice shows that Akt1 uniquely phosphorylates several important proteins regulating vascular function. Thus, despite the presence of both isoforms of Akt in the endothelium, substrate phosphorylation by Akt1 is nonredundant.