Abstract-Many forms of vascular disease are characterized by increased transforming growth factor (TGF)-␤ 1 expression and endothelial dysfunction. Smad proteins are a key step in TGF-␤-initiated signal transduction. We hypothesized that NO may regulate endothelial TGF-␤-dependent gene expression. We show that NO inhibits TGF-␤/Smad-regulated gene transactivation in a cGMP-dependent manner. NO effects were mimicked by a soluble analogue of cGMP. Inhibition of cGMP-dependent protein kinase 1 (PKG-1) or overexpression of dominant-negative PKG-1␣ suppressed NO/cGMP inhibition of TGF-␤-induced gene expression. Inversely, overexpression of PKG-1␣ catalytic subunit blocked TGF-␤-induced gene transactivation. Furthermore NO delayed and reduced phosphorylated Smad2/3 nuclear translocation, an effect mediated by PKG-1, whereas N G -nitro-L-arginine methyl ester augmented Smad phosphorylation and gene expression in response to TGF-␤. Aortas from endothelial NO synthase-deficient mice showed enhanced basal TGF-␤ 1 and collagen type I expression; endothelial cells from these animals showed increased Smad phosphorylation and transcriptional activity. Proteasome inhibitors prevented the inhibitory effect of NO on TGF-␤ signaling. NO reduced the metabolic life of ectopically expressed Smad2 and enhanced its ubiquitination. Taken together, these results suggest that the endothelial NO/cGMP/PKG pathway interferes with TGF-␤/Smad2 signaling by directing the proteasomal degradation of activated Smad. Key Words: nitric oxide Ⅲ endothelial cells Ⅲ vascular remodeling Ⅲ transforming growth factor-␤ T ransforming growth factor (TGF)-␤ plays a major role in the vascular response to injury by controlling both cellular proliferation and extracellular matrix turnover through the Smad-signaling pathway. [1][2][3] Ligand binding leads to phosphorylation and nuclear translocation of receptor-activated Smads (R-Smads), Smad2/3, which modulate the transcription of a large number of genes. Smad7 and Smad6, inhibitory Smads (I-Smad), antagonize TGF-␤ signaling. Smad7/6 expression is induced by TGF-␤ in the endothelium, providing an autoregulatory negative feedback loop on TGF-␤ signaling. 4 The response to TGF-␤ in the cardiovascular system is tightly controlled. Mice deficient in TGF-␤ 1 die in utero because of vascular defects. 5 Smad1-deficient mice fail to establish chorion-allantoic circulation, whereas Smad5-deficient embryos have defects in yolk sac vasculature with enlarged blood vessels. 6,7 Mice deficient in the accessory receptor endoglin exhibit embryonic lethality, with cardiovascular and angiogenesis defects associated with abnormal vascular smooth muscle cell development. 8 Smad6-deficient mice develop aortic ossification and elevated blood pressure. 9 TGF-␤ is highly expressed in injured arteries, and TGF-␤-dependent effects play a role in the pathogenesis of atherosclerosis, coronary artery disease, transplant arteriosclerosis, hypertension, diabetes, myocardial remodeling, and restenosis. 10 -14 Blood vessels overexpressing TG...