Nitric oxide regulates transforming growth factor-beta signaling in endothelial cells.
ABSTRACT Many forms of vascular disease are characterized by increased transforming growth factor (TGF)-beta1 expression and endothelial dysfunction. Smad proteins are a key step in TGF-beta-initiated signal transduction. We hypothesized that NO may regulate endothelial TGF-beta-dependent gene expression. We show that NO inhibits TGF-beta/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-1alpha suppressed NO/cGMP inhibition of TGF-beta-induced gene expression. Inversely, overexpression of PKG-1alpha catalytic subunit blocked TGF-beta-induced gene transactivation. Furthermore NO delayed and reduced phosphorylated Smad2/3 nuclear translocation, an effect mediated by PKG-1, whereas NG-nitro-L-arginine methyl ester augmented Smad phosphorylation and gene expression in response to TGF-beta. Aortas from endothelial NO synthase-deficient mice showed enhanced basal TGF-beta1 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-beta 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-beta/Smad2 signaling by directing the proteasomal degradation of activated Smad.
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ABSTRACT: Endothelial dysfunction and enhanced transforming growth factor-β (TGF-β)/Smad3 signalling are common features of progressive renal fibrosis. This study investigated a potential link between these mechanisms. In unilateral ureteric obstruction (UUO) we observed an acute (6 hr) down-regulation of nitric oxide synthase 3 (NOS3/eNOS) levels and increased phosphorylation of the linker region of Smad3 at T179 and S208 in Smad3/JNK complexes. These events preceded Smad3 C-terminal domain phosphorylation and the induction of myofibroblast proliferation at 48 hrs. Mice deficient in NOS3 showed enhanced myofibroblast proliferation and collagen accumulation compared to wild type mice in a 7 day UUO model. This was associated with enhanced phosphorylation of Smad3 T179 and S208 by 92% and 88%, respectively, whereas Smad3-C-terminal phosphorylation was not affected. Resolvin D1 (RvD1) can suppress renal fibrosis in the UUO model, and further analysis herein showed that RvD1 protected against endothelial dysfunction and suppressed Smad3/JNK complex formation with a consequent reduction in phosphorylation of Smad3 T179 and S208 by 78% and 65%, respectively, while Smad3 C-terminal phosphorylation was unaltered. In vitro, conditioned media from mouse microvascular endothelial cells (MMEC) treated with a general inhibitor of nitric oxide synthase (L-NAME) augmented the proliferation and collagen production of renal fibroblasts (NRK49F cells) compared to control MMEC media and this was associated with increased phosphorylation of JNK and Smad3 T179 and S208, whereas Smad3-C-terminal domain phosphorylation was unaffected. The addition of RvD1 to L-NAME treated MMEC abrogated these effects of the conditioned media on renal fibroblasts. Finally, Smad3 T179/V and S208/A mutations significantly inhibit TGF-β1 induced up-regulation collagen I promoter. In conclusion, these data suggest that endothelial dysfunction can exacerbate renal interstitial fibrosis through increased fibroblast proliferation and collagen production via enhanced Smad3 linker phosphorylation.PLoS ONE 01/2013; 8(12):e84063. · 3.53 Impact Factor
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ABSTRACT: Although calcimimetics were developed to block parathyroid hormone synthesis, some reports suggest that they may also reduce blood pressure by unknown mechanisms. Calcimimetic-induced changes in the synthesis of endothelial vasoactive factors could be involved. Wistar rats were treated with the calcimimetic R-568, and systolic blood pressure (SBP) was registered with a tail-cuff sphygmomanometer, the content of endothelial nitric oxide synthase (eNOS) and endothelin-converting enzyme (ECE-1) in tissue was evaluated by immunohistochemistry and western blot, circulating levels of endothelin-1 (ET-1) were measured by ELISA. R-568 reduced SBP and circulating levels of ET-1, without changes in eNOS expression. In contrast, R-568 increased the lung and vascular content of ECE-1. In order to analyze the mechanisms involved, we studied the effect of R-568 on human endothelial cells. R-568 did not modify neither eNOS protein content nor pre-pro-ET-1 mRNA expression, but increased ECE-1 protein content, and decreased ET-1 synthesis and ECE-1 activity. The inhibition of ECE-1 activity was very strong, similar to the classic ECE inhibitor phosphoramidon, the addition of exogenous zinc restored enzymatic activity. Moreover, the amount of zinc in immunoprecipitated ECE from R-568 treated cells was 3-fold less than in control cells. In conclusion, R-568 inhibits ECE by expelling zinc from the enzyme, with the subsequent decrease in enzymatic activity and reducing circulating levels of ET-1, which may be responsible for the lower SBP observed in R-568-treated rats. This descent would be partially compensated by the increased synthesis of the ECE-1 itself, and by other homeostatic mechanisms that regulate SBP.Pharmacological Research 08/2013; · 4.35 Impact Factor
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ABSTRACT: Cardiovascular lesions, including coronary artery stenosis, are frequently associated and can cause sudden death in patients with genetic defects of glycosaminoglycan (GAG) metabolism. Early diagnosis of coronary artery lesions is difficult, although potentially lifesaving. Histopathological similarities between atherosclerotic changes in adults and in patients with genetic GAG metabolism defects have been known. Atherosclerosis is the result of a complex process involving metabolism of GAGs and proteoglycans preceded by endothelial dysfunction as a key event. Decreased nitric oxide (NO) bioavailability is considered the hallmark of endothelial dysfunction. Reduced NO synthase (NOS) has been reported in atherosclerotic arteries. Impairment in reactive hyperemia-digital peripheral arterial tonometry (RH-PAT) with EndoPAT has been validated to correlate coronary microvascular function in patients with atherosclerosis. RH-PAT is thought to reflect endothelial NO production. Immunohistological staining of endothelial NOS was performed in the stenotic lesions in the coronary artery of a 3-year-old patient with Mucopolysaccharidosis-I, showing decreased activities. This prompted a study to measure endothelial function in patients with GAG metabolism defects for early diagnosis of endothelial dysfunction in the coronary arteries as an early sign of coronary artery changes. Evaluation by RH-PAT in 30 patients with variable genetic defects in GAG metabolism revealed significantly decreased Reactive Hyperemia Indexes compared with 12 controls. Evaluation of endothelial function with RH-PAT in patients with GAG metabolism defects may detect coronary artery lesions that can be underdiagnosed by the other measures such as coronary angiography. Use of this method may prove vital in the management of patients with GAG metabolism defects.Journal of Inherited Metabolic Disease 07/2013; · 4.07 Impact Factor