Mercedes Griera

Nitric Oxide Regulates Transforming Growth Factor-β Signaling in Endothelial Cells

Many forms of vascular disease are characterized by increased transforming growth factor (TGF)-&bgr;1 expression and endothelial dysfunction. Smad proteins are a key step in TGF-&bgr;–initiated signal transduction. We hypothesized that NO may regulate endothelial TGF-&bgr;–dependent gene expression. We show that NO inhibits TGF-&bgr;/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&agr; suppressed NO/cGMP inhibition of TGF-&bgr;–induced gene expression. Inversely, overexpression of PKG-1&agr; catalytic subunit blocked TGF-&bgr;–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-&bgr;. Aortas from endothelial NO synthase–deficient mice showed enhanced basal TGF-&bgr;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-&bgr; 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-&bgr;/Smad2 signaling by directing the proteasomal degradation of activated Smad.

Losartan-Antioxidant Hybrids: Novel Molecules for the Prevention of Hypertension-Induced Cardiovascular Damage

We report the first examples of a new series of antioxidant-sartan hybrids (AO-sartans), which were made by adding an antioxidant fragment to the hydroxymethyl side chain of losartan. Experiments performed in cultured cells demonstrate that these new hybrids retain the ability to block the angiotensin II effect with increased antioxidant ability. In hypertensive rats, these compounds show properties that suggest they may be more useful than losartan for controlling hypertension and preventing hypertension-induced cardiovascular damage.

Crosstalk Between Mesangial and Endothelial Cells: Angiotensin II Down-Regulates Endothelin- Converting Enzyme 1

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.

H2O2 Regulation of Vascular Function Through sGC mRNA Stabilization by HuR

Objective—Hydrogen peroxide (H2O2) is an important mediator in the vasculature, but its role in the regulation of soluble guanylate cyclase (sGC) activity and expression is not completely understood. The aim of this study was to test the effect of H2O2 on sGC expression and function and to explore the molecular mechanism involved. Methods and Results—H2O2 increased sGC&bgr;1 protein steady-state levels in rat aorta and aortic smooth muscle cells (RASMCs) in a time- and dose-dependent manner, and this effect was blocked by catalase. sGC&agr;2 expression increased along with &bgr;1 subunit, whereas &agr;1 subunit remained unchanged. Vascular relaxation to an NO donor (sodium nitroprusside) was enhanced by H2O2, and it was prevented by ODQ (sGC inhibitor). cGMP production in both freshly isolated vessels and RASMCs exposed to H2O2 was greatly increased after sodium nitroprusside treatment. The H2O2-dependent sGC&bgr;1 upregulation was attributable to sGC&bgr;1 mRNA stabilization, conditioned by the translocation of the mRNA-binding protein HuR from the nucleus to the cytosol, and the increased mRNA binding of HuR to the sGC&bgr;1 3′ untranslated region. HuR silencing reversed the effects of H2O2 on sGC&bgr;1 levels and cGMP synthesis. Conclusion—Our results identify H2O2 as an endogenous mediator contributing to the regulation of vascular tone and point to a key role of HuR in sGC&bgr;1 mRNA stabilization.

Targeted genomic disruption of h-ras induces hypotension through a NO-cGMP-PKG pathway-dependent mechanism.

The aim of the present experiments was to evaluate the differences in arterial pressure between H-Ras lacking mice and control mice and to analyze the mechanisms involved in the genesis of the differences. H-Ras lacking mice and mouse embryonic fibroblasts from these animals were used. Blood pressure was measured using 3 different methods: direct intraarterial measurement in anesthetized animals, tail-cuff sphygmomanometer, and radiotelemetry. H-Ras lacking mice showed lower blood pressure than control animals. Moreover, the aorta protein content of endothelial nitric oxide synthase, soluble guanylyl cyclase, and cyclic guanosine monophosphate–dependent protein kinase was higher in H-Ras knockout mice than in control animals. The activity of these enzymes was increased, because urinary nitrite excretion, sodium nitroprusside–stimulated vascular cyclic guanosine monophosphate synthesis, and phosphorylated vasoactive-stimulated phosphoprotein in aortic tissue increased in these animals. Furthermore, mouse embryonic fibroblasts from H-Ras lacking mice showed higher cyclic guanosine monophosphate–dependent protein kinase promoter activity than control cells. These results strongly support the upregulation of the nitric oxide-cyclic guanosine monophosphate pathway in H-Ras–deficient mice. Moreover, they suggest that H-Ras pathway could be considered as a therapeutic target for hypertension treatment.

Hydrogen peroxide down-regulates inositol 1,4,5-trisphosphate receptor content through proteasome activation

Hydrogen peroxide (H(2)O(2)) is implicated in the regulation of signaling pathways leading to changes in vascular smooth muscle function. Contractile effects produced by H(2)O(2) are due to the phosphorylation of myosin light chain kinase triggered by increases in intracellular calcium (Ca(2+)) from intracellular stores or influx of extracellular Ca(2+). One mechanism for mobilizing such stores involves the phosphoinositide pathway. Inositol 1,4,5-trisphosphate (IP(3)) mobilizes intracellular Ca(2+) by binding to a family of receptors (IP(3)Rs) on the endoplasmic-sarcoplasmic reticulum that act as ligand-gated Ca(2+) channels. IP(3)Rs can be rapidly ubiquitinated and degraded by the proteasome, causing a decrease in cellular IP(3)R content. In this study we show that IP(3)R(1) and IP(3)R(3) are down-regulated when vascular smooth muscle cells (VSMC) are stimulated by H(2)O(2), through an increase in proteasome activity. Moreover, we demonstrate that the decrease in IP(3)R by H(2)O(2) is accompanied by a reduction in calcium efflux induced by IP(3) in VSMC. Also, we observed that angiotensin II (ANGII) induces a decrease in IP(3)R by activation of NADPH oxidase and that preincubation with H(2)O(2) decreases ANGII-mediated calcium efflux and planar cell surface area in VSMC. The decreased IP(3) receptor content observed in cells was also found in aortic rings, which exhibited a decreased ANGII-dependent contraction after treatment with H(2)O(2). Altogether, these results suggest that H(2)O(2) mediates IP(3)R down-regulation via proteasome activity.

Differential Regulation of Soluble Guanylyl Cyclase Expression and Signaling by Collagens: Involvement of Integrin-Linked Kinase

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.

Integrin-linked kinase regulates tubular aquaporin-2 content and intracellular location: a link between the extracellular matrix and water reabsorption

One of the clinical alterations observed in chronic renal disease (CRD) is the impaired urine concentration, known as diabetes insipidus (DI). Tubulointerstitial fibrosis of the kidney is also a pathological finding observed in CRD and involves composition of extracellular matrix (ECM). However, an association between these two events has not been elucidated. In this study, we showed that the extracellular‐to‐intracellular scaffold protein integrin‐linked kinase (ILK) regulates expression of tubular water channel aquaporin‐2 (AQP2) and its apical membrane presence in the renal tubule. Basally, polyuria and decreased urine osmolality were present in ILK conditional‐knockdown (cKD‐ILK) adult mice compared with nondepleted ILK littermates. No changes were observed in arginine‐vasopressin (AVP) blood levels, renal receptor (V2R), or AQP3 expression. However, tubular AQP2 was decreased in expression and apical membrane presence in cKD‐ILK mice, where the canonical V2R/cAMP axis activation is still functional, but independent of the absence of ILK. Thus, cKD‐ILK constitutes a nephrogenic diabetes insipidus (NDI) model. AQP2 and ILK colocalize in cultured inner medullary collecting duct (mIMCD3) cells. Specific ILK siRNAs and collagen I (Col) decrease ILK and AQP2 levels and AQP2 presence on the membrane of tubular mIMCD3 cells, which impairs the capacity of the cells to transport water under hypotonic stress. The present work points to ILK as a therapeutic target in NDI.—Cano‐Peñalver, J. L., Griera, M., Serrano, I., Rodríguez‐Puyol, D., Dedhar, S., de Frutos, S., Rodríguez‐Puyol, M. Integrin‐linked kinase regulates tubular aquaporin‐2 content and intracellular location: a link between the extracellular matrix and water reabsorption. FASEB J. 28, 3645–3659 (2014). www.fasebj.org

Changes in extracellular matrix composition regulate cyclooxygenase-2 expression in human mesangial cells

Glomerular diseases are characterized by a sustained synthesis and accumulation of abnormal extracellular matrix proteins, such as collagen type I. The extracellular matrix transmits information to cells through interactions with membrane components, which directly activate many intracellular signaling events. Moreover, accumulating evidence suggests that eicosanoids derived from cyclooxygenase (COX)-2 participate in a number of pathological processes in immune-mediated renal diseases, and it is known that protein kinase B (AKT) may act through different transcription factors in the regulation of the COX-2 promoter. The present results show that progressive accumulation of collagen I in the extracellular medium induces a significant increase of COX-2 expression in human mesangial cells, resulting in an enhancement in PGE(2) production. COX-2 overexpression is due to increased COX-2 mRNA levels. The study of the mechanism implicated in COX-2 upregulation by collagen I showed focal adhesion kinase (FAK) activation. Furthermore, we observed that the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway by collagen I and collagen I-induced COX-2 overexpression was abolished by PI3K and AKT inhibitors. Additionally, we showed that the cAMP response element (CRE) transcription factor is implicated. Finally, we studied COX-2 expression in an animal model, N(G)-nitro-l-arginine methyl ester hypertensive rats. In renal tissue and vascular walls, COX-2 and collagen type I content were upregulated. In summary, our results provide evidence that collagen type I increases COX-2 expression via the FAK/PI3K/AKT/cAMP response element binding protein signaling pathway.

Tirofiban increases soluble guanylate cyclase in rat vascular walls: pharmacological and pathophysiological consequences

AIMS Our aim was to evaluate whether tirofiban, which mimics the structure of arginine-glycine-aspartic acid (RGD) peptides, up-regulates soluble guanylate cyclase beta1 subunit (sGC-beta1) expression in vascular smooth muscle cells (VSMCs) and in aorta from rats, and to investigate the pharmacological and pathophysiological consequences of this up-regulation. METHODS AND RESULTS Wistar, Wistar Kyoto, and spontaneously hypertensive rats (SHRs) were used. sGC-beta1 content was assessed by immunoblotting. Arterial pressure was recorded using a tail-cuff sphygmomanometer. Sodium nitroprusside (SNP) and isosorbide dinitrate (IDN) were used as nitric oxide (NO) donors. Tirofiban increased the sGC-beta1 content in VSMCs and in aortic walls from rats after 6 h of treatment. Rats treated with tirofiban experienced a more pronounced decrease in their arterial pressure after acute SNP treatment than vehicle-treated rats. Isolated rat aortic rings incubated with tirofiban showed a higher relaxing response to SNP than control rings as well as an increased sGC-beta1 content and SNP-induced cyclic guanosine monophosphate synthesis. Animals receiving IDN for 1 week showed decreased sGC-beta1 in aortic walls and did not respond to SNP treatment with changes in arterial pressure. Tirofiban restored the decreased sGC-beta1 content in IDN-treated rats and promoted a decreased arterial pressure in response to SNP administration. SHRs showed reduced sGC-beta1 levels, and tirofiban increased these levels and led to a higher response to SNP. CONCLUSION Tirofiban increased the sGC-beta1 content in contractile cells and aortic walls of rats, enhancing the response to SNP and reversing the NO donor tachyphylaxis.