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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.

Glomerular diseases are characterized by an abnormal synthesis of extracellular matrix proteins, such as collagen type I. Evidence that growth on collagen type I downregulates soluble guanylyl cyclase (sGC) expression and the responsiveness of human mesangial cells to nitric oxide (NO) by activating specific integrin signals involving integrin-linked kinase (ILK) is presented. Human mesangial cells were grown on collagen type I or IV for 24 to 72 h. Compared with collagen IV, growth on collagen I reduced the protein expression and NO-stimulated enzyme activity of sGC. This downregulation was effected at the level of transcription, because steady-state sGC mRNA expression was reduced on collagen I, but inhibition of transcription with Actinomycin D revealed no differences in transcript stability between the two culture conditions. Collagen I also reduced the capacity of cells to relax in response to NO after H2O2 challenge and inhibited NO-induced phosphorylation of vasodilator-activated phosphoprotein, a target of cyclic guanine monophosphate-dependent protein kinase. Examination of the surface expression of integrins, the receptors for extracellular matrix components, revealed that alpha1 and alpha2 integrin subunits were more abundant on cells that were grown on collagen IV and that surface expression of beta1 integrin did not vary with collagen type. However, growth on collagen I induced beta1 integrin to adopt its active conformation, and this activation of beta1 integrin was accompanied by increased activity of its downstream effector ILK. Dominant-negative suppression of ILK signaling relieved the suppression of sGC expression and NO-induced vasodilator-activated phosphoprotein phosphorylation induced by collagen I.

The mechanisms involved in the progression of chronic renal disease (CRD) have not been completely clarified. A role for hyperfiltration and increased intraglomerular pressure was proposed about twenty years ago, and experimental and clinical evidence supports, at least partially, this hypothesis. Moreover, a lot of experimental data point to the importance of different autacoids, including prostanoids, endothelial vasoactive factors, reactive oxygen species, cytokines and growth factors, in the genesis of the changes that characterize CRD. However, alternative mechanisms of progression may be involved such as the presence of abnormal extracellular matrix (ECM) proteins in kidney structures. By modifying the phenotype of renal cells, they may constitute a key factor in disease progression. Experimental studies support this hypothesis. Human mesangial cells cultured on collagen I (COLI) synthesize increased amounts of TGFss1 compared with cells cultured on collagen IV (COLIV). As a consequence of this increased TGFBeta1 synthesis, they also produce more collagens and fibronectin. Human umbilical vein endothelial cells cultured on COLI show a different pattern of endothelial vasoactive factor synthesis, compared with those on COLIV: they synthesize more endothelin-1 and less nitric oxide. Integrins and integrin-linked kinase (ILK) play a significant role in the genesis of these changes. Although an extrapolation of these data to human diseases can not be performed, they point to alternative mechanisms of chronic renal damage progression or renal dysfunction in kidney diseases. They also point to potential therapeutic targets such as integrins and ILK.

Objective: Since mesangial and endothelial cells interact in the kidney, the present experiments were designed to analyze the ability of human mesangial cells (HMC) to modulate endothelin-1 (ET-1) synthesis by human umbilical vein endothelial cells (HuVEC)., Methods and Results: The supernatants of HuVEC/HMC contained significantly lower amounts of ET-1 than those of HuVEC alone. This effect was not due to a decreased prepro-ET-1 mRNA expression and was only partially the consequence of HMC-dependent ET-1 degradation. Therefore, we tested the influence of the coculture on endothelin-converting enzyme-1 (ECE-1), and found a significant reduction of its mRNA and protein levels as well as a decreased activity in HuVEC/HMC as compared to HuVEC alone. Using a pharmacological blockade approach (sulotrobam, BN52021, losartan or catalase), losartan was shown to completely abolish down-regulation of ECE-1 observed in HuVEC/HMC. Angiotensin II (AII) induced a dose and time-dependent inhibition of ECE-1 expression in HuVEC., Conclusions: These results support the importance of cross-talk among different cell types in the regulation of vascular or renal function. ET-1, and particularly ECE-1, might constitute a target in this regulation. In addition, locally synthesized AII could be one of the mediators involved in the down-regulation of ECE-1., (Copyright 2005 S. Karger AG, Basel.)

Background: Vascular diseases are characterized by the presence of structural changes and the progressive loss of endothelial function. Although the biochemical basis of these structural changes have started to be outlined, it seems that accumulation of normal extracellular matrix proteins as well as the appearance of interstitial collagens, mainly collagen type I, characterize this process. On the other hand, a role for endothelial vasoactive factors has been proposed in the genesis of endothelial dysfunction, and it is generally accepted that changes in extracellular matrix composition may modify cell behavior., Methods: Experiments were designed to test the influence of the supporting matrix on endothelin-1 (ET-1) synthesis by endothelial cells. Northern blot experiments were performed to analyze the prepro-endothelin-1 (prepro-ET-1) mRNA expression. ET-1 production was measured by ELISA., Results: Cells grown on collagen type I (Col I) showed an increase of prepro-ET-1 mRNA level when compared with cells cultured on collagen type IV (Col IV). According to these results, the release of ET-1 to culture medium was also higher in Col I-grown cells than in those cultured on Col IV. Treatment of cells with a peptide that interferes with Col I integrins (D6Y), or with protein tyrosine kinase inhibitors such as genistein and herbimycin, completely abolished the effect of Col I. Moreover, experiments with antibodies against integrins suggest that these cell surface receptors could be involved in the modulation of ET-1 system by extracellular matrix., Conclusions: These results suggest that the presence of an abnormal extracellular matrix could stimulate endothelin synthesis by human endothelial cells, through integrin activation.

In the last years, reactive oxygen species (ROS) have been proposed as mediators of proliferative/hypertrophic responses to angiotensin II (Ang II), both in vivo and in vitro. However, the hypothesis that the Ang II-dependent cell contraction could be mediated by ROS, particularly H2O2, has not been tested. Present experiments were devoted to test this hypothesis and to analyze the possible mechanisms involved. Catalase (CAT) prevented the increased myosin light chain phosphorylation and the decreased planar cell surface area (PCSA) induced by 1 microM Ang II in cultured rat vascular smooth muscle cells (VSMC). This preventive effect of CAT was also detected when 1 microM platelet-activating factor (PAF) was used as a contractile agonist instead of Ang II. Similar results were found when using horseradish peroxidase as an H2O2 scavenger or cultured rat mesangial cells. In vascular smooth muscle cells, CAT modified neither the binding of labeled Ang II nor the Ang II-induced inositol 1,4,5-trisphosphate (IP3) synthesis. However, it completely abolished the Ang II-dependent calcium peak, in a dose-dependent fashion. CAT-loaded cells (increased intracellular CAT concentration over 3-fold) did not show either a decreased PCSA or an increased intracellular calcium concentration after Ang II treatment. Ang II stimulated the H2O2 synthesis by cultured cells, and the presence of CAT in the extracellular compartment significantly diminished the Ang II-dependent increased intracellular H2O2 concentration. The physiological importance of these findings was tested in rat thoracic aortic rings: CAT prevented the contraction elicited by Ang II. In summary, present experiments point to H2O2 as a critical intracellular metabolite in the regulation of cell contraction.