Search

Abstract: While arginine-glycine-aspartic acid-based peptidomimetics have been employed for the treatment of cardiovascular disorders and cancer, their use in other contexts remains to be explored. Arginine-glycine-aspartic acid-serine induces Transforming growth factor-β1 transcription in human mesangial cells, but the molecular mechanisms involved have not been studied extensively. We explored whether this effect could be due to Activator protein-1 activation and studied the potential pathways involved. Addition of arginine-glycine-aspartic acid-serine promoted Activator protein-1 binding to its cognate sequence within the Transforming growth factor-β1 promoter as well as c-jun and c-fos protein abundance. Moreover, this effect was suppressed by curcumin, a c-Jun N terminal kinase inhibitor, and was absent when the Activator protein-1 cis-regulatory element was deleted. Activator protein-1 binding was dependent on the activity of integrin linked kinase, as transfection with a dominant negative mutant suppressed both Activator protein-1 binding and c-jun and c-fos protein increment. Integrin linked kinase was, in turn, dependent on Phosphoinositol-3 kinase activity. Arginine-glycine-aspartic acid-serine stimulated Phosphoinositol-3 kinase activity, and Transforming growth factor-β1 promoter activation was abrogated by the use of Phosphoinositol-3 kinase specific inhibitors. In summary, we propose that arginine-glycine-aspartic acid-serine activates Integrin linked kinase via the Phosphoinositol-3 kinase pathway and this leads to activation of c-jun and c-fos and increased Activator protein-1 binding and Transforming growth factor-β1 promoter activity. These data may contribute to understand the molecular mechanisms involved in the cellular actions of arginine-glycine-aspartic acid-related peptides and enhance their relevance as these products evolve into clinical therapeutic use. [Copyright &y& Elsevier]

Background: Traditional biomarkers of chronic kidney disease (CKD) detect the disease in its late stages and hardly predict associated vascular damage. Integrin-linked kinase (ILK) is a scaffolding protein and a serine/threonine protein kinase that plays multiple roles in several pathophysiological processes during renal damage. However, the involvement of ILK as a biomarker of CKD and its associated vascular problems remains to be fully elucidated., Methods: CKD was induced by an adenine-rich diet for 6 weeks in mice. We used an inducible ILK knockdown mice (cKD-ILK) model to decrease ILK expression. ILK content in mice's peripheral blood mononuclear cells (PBMCs) was determined and correlated with renal function parameters and with the expression of ILK and fibrosis and inflammation markers in renal and aortic tissues. Also, the expression of five miRNAs that target ILK was analyzed in whole blood of mice., Results: The adenine diet increased ILK expression in PBMCs, renal cortex, and aortas, and creatinine and urea nitrogen concentrations in the plasma of WT mice, while these increases were not observed in cKD-ILK mice. Furthermore, ILK content in PBMCs directly correlated with renal function parameters and with the expression of renal and vascular ILK and fibrosis and inflammation markers. Finally, the expression of the five miRNAs increased in the whole blood of adenine-fed mice, although only four correlated with plasma urea nitrogen, and of those, three were downregulated in cKD-ILK mice., Conclusions: ILK, in circulating mononuclear cells, could be a potential biomarker of CKD and CKD-associated renal and vascular damage., (© 2024. The Author(s).)

Background: Diagnosis of nontuberculous mycobacteria (NTM) infections remains a challenge. In this study, we describe the evaluation of an immunological NTM-interferon (IFN)-γ release assay (IGRA) that we developed using glycopeptidolipids (GPLs) as NTM-specific antigens., Methods: We tested the NTM-IGRA in 99 samples from pediatric patients. Seventy-five were patients with lymphadenitis: 25 were NTM confirmed, 45 were of unknown etiology but compatible with mycobacterial infection and 5 had lymphadenitis caused by an etiologic agent other than NTM. The remaining 24 samples were from control individuals without lymphadenitis (latently infected with M. tuberculosis , uninfected controls and active tuberculosis patients). Peripheral blood mononuclear cells were stimulated overnight with GPLs. Detection of IFN-γ producing cells was evaluated by enzyme-linked immunospot assay., Results: NTM culture-confirmed lymphadenitis patient samples had a significantly higher response to GPLs than the patients with lymphadenitis of unknown etiology but compatible with mycobacterial infection ( P < 0.001) and lymphadenitis not caused by NTM ( P < 0.01). We analyzed the response against GPLs in samples from unknown etiology lymphadenitis but compatible with mycobacterial infection cases according to the tuberculin skin test (TST) response, and although not statistically significant, those with a TST ≥5 mm had a higher response to GPLs when compared with the TST <5 mm group., Conclusions: Stimulation with GPLs yielded promising results in detecting NTM infection in pediatric patients with lymphadenitis. Our results indicate that the test could be useful to guide the diagnosis of pediatric lymphadenitis. This new NTM-IGRA could improve the clinical handling of NTM-infected patients and avoid unnecessary misdiagnosis and treatments., Competing Interests: R.V.H., I.L., J.B.T. and J.D. are registered as inventors on a patent (WO 2019/234296 A1) filed by Institut d’Investigació Germans Trias i Pujol, CIBER, and The Ohio State University Innovation Foundation, disclosing the use of GPLs for NTM infection diagnosis. Such patent has been licensed by Genome Identification Diagnostics (GenID) GmbH. R.V.H. is currently employed by GenID but not at the moment this study was performed. I.L. is funded by the Miguel Servet programme, Instituto de Salud Carlos III (Spain). A.S.A. received funding from Fundació la Marató TV3 exp 202134-30-31 Projecte Escoles Sentinella (Departament de Salut i d’Educació de la Generalitat de Catalunya). F.B.A. has participated on Pfizer, Merck Sharp and Dohme (MSD) and Glaxo Smith Kline (GSK)’s Advisory Boards, has received honoraria for presentations from Pfizer and MSD and his institution receives payments from MSD and GSK. J.R.S. has received consulting fees, payment honoraria, payment for expert testimony and support attending meetings and/or travels from ViiV, Gilead and MSD. The other authors have no conflicts of interest to disclose., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

White adipose tissue (WAT) hypertrophy is caused by the excessive storage of triglycerides (TGs) and is associated with obesity. We previously demonstrated that extracellular matrix mediator integrin beta1 (INTB1) and its downstream effector integrin linked kinase (ILK) are implicated in obesity establishment. We also considered in our previous works that ILK upregulation is a therapeutical strategy to reduce WAT hypertrophy. Carbon based nanomaterials (CNMs) have interesting potential to modify cell differentiation but have been never studied to change the properties of adipocytes., Methods: GMC is a new graphene-based CNM that was tested for biocompatibility and functionality in cultured adipocytes. MTT, TG content, lipolysis quantification, and transcriptional changes were determined. Specific INTB1 blocking antibody and ILK depletion with specific siRNA were used to study the intracellular signalling. We complemented the study using subcutaneous WAT (scWAT) explants from transgenic ILK knockdown mice (cKD-ILK). GMC was topically administrated in the dorsal area of high fat diet-induced obese rats (HFD) for 5 consecutive days. The scWAT weights and some intracellular markers were analyzed after the treatment., Results: graphene presence was characterized in GMC. It was non-toxic and effective in reducing TG content in vitro in a dose-dependent manner. GMC rapidly phosphorylated INTB1 and increased the expression and activity of hormone sensitive lipase (HSL), the lipolysis subproduct glycerol, and the expression of glycerol and fatty acid transporters. GMC also reduced the expression of adipogenesis markers. Pro-inflammatory cytokines were unaffected. ILK was overexpressed, and INTB1 or ILK blockade avoided functional GMC effects. Topical administration of GMC in HFD rats overexpressed ILK in scWAT, and their weight gains were reduced, while systemic (renal, hepatic) toxicity parameters were unaffected., Conclusions: GMC is safe and effective in reducing hypertrophied scWAT weight when topically applied and it can be considered of interest in anti-obesogenic strategies. GMC increases lipolysis and reduces adipogenesis inside adipocytes by mechanisms that imply the activation of INTB1, the overexpression of ILK, and changes in the expression and activity of several markers related to fat metabolism.

Hypertension is the most common cause of left ventricular hypertrophy, contributing to heart failure progression. Candesartan (Cand) is an angiotensin receptor antagonist widely used for hypertension treatment. Structural modifications were previously performed by our group using Zinc (ZnCand) as a strategy for improving its pharmacological properties. The measurements showed that ZnCand exerts a stronger interaction with the angiotensin II receptor, type 1 (AT 1 receptor), reducing oxidative stress and intracellular calcium flux, a mechanism implied in cell contraction. These results were accompanied by the reduction of the contractile capacity of mesangial cells. In vivo experiments showed that the complex causes a significant decrease in systolic blood pressure after 8 weeks of treatment in spontaneously hypertensive rats (SHR). The reduction of heart hypertrophy was evidenced by echocardiography, the histologic cross-sectional area of cardiomyocytes, collagen content, the B-type natriuretic peptide (BNP) marker and connective tissue growth factor (CTGF) and the matrix metalloproteinase 2 (MMP-2) expression. Besides, the complex restored the redox status. In this study, we demonstrated that the complexation with Zn(II) improves the antihypertensive and cardiac effects of the parental drug., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Cardiovascular disease is an important cause of death in patients with chronic kidney disease (CKD). Protein-bound uremic toxins, such as p-cresyl and indoxyl sulfate (IS), are poorly removed during hemodialysis, leading to vascular endothelial dysfunction and leukocyte extravasation. These processes can be related to dynamic adhesion structures called podosomes. Several studies have indicated the role of integrin-linked kinase (ILK) in the accumulation of integrin-associated proteins in podosomes. Here, we investigated the involvement of ILK and podosome formation in the adhesion and extravasation of monocytes under p-cresol (pc) and IS exposure. Incubation of THP-1 human monocyte cells with these toxins upregulated ILK kinase activity. Together, both toxins increased cell adhesion, podosome formation, extracellular matrix degradation, and migration of THP-1 cells, whereas ILK depletion with specific small interfering RNAs suppressed these processes. Interestingly, F-actin colocalized with cortactin in podosome cores, while ILK was colocalized in podosome rings under toxin stimulation. Podosome Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) and AKT protein depletion demonstrated that monocyte adhesion depends on podosome formation and that the ILK/AKT signaling pathway is involved in these processes. Ex vivo experiments showed that both toxins induced adhesion and podosome formation in leukocytes from wild-type mice, whereas these effects were not observed in leukocytes of conditional ILK-knockdown animals. In summary, under pc and IS stimulation, monocytes increase podosome formation and transmigratory capacity through an ILK/AKT signaling pathway-dependent mechanism, which could lead to vascular injury. Therefore, ILK could be a potential therapeutic target for the treatment of vascular damage associated with CKD., (© 2022. The Author(s).)

Protein tyrosine phosphatase 1B (PTP1B) is a very promising target for the treatment of metabolic disorders such as type II diabetes mellitus. Although it was validated as a promising target for this disease more than 30 years ago, as yet there is no drug in advanced clinical trials, and its biochemical mechanism and functions are still being studied. In the present study, based on our experience generating PTP1B inhibitors, we have developed and implemented a scaffold-hopping approach to vary the pyrrole ring of the pyrrolo[1,2-a]quinoxaline core, supported by extensive computational techniques aimed to explain the molecular interaction with PTP1B. Using a combination of docking, molecular dynamics and end-point free-energy calculations, we have rationally designed a hypothesis for new PTP1B inhibitors, supporting their recognition mechanism at a molecular level. After the design phase, we were able to easily synthesize proposed candidates and their evaluation against PTP1B was found to be in good concordance with our predictions. Moreover, the best candidates exhibited glucose uptake increments in cellulo model, thus confirming their utility for PTP1B inhibition and validating this approach for inhibitors design and molecules thus obtained., (© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

Protein tyrosine phosphatase (PTP1B) is an interesting therapeutical target for diabetes, obesity, heart disease and cancer. As such, inhibition of PTP1B using orally administered drugs is still being pursued by academia and pharmaceutical companies. The failure of catalytic-site inhibitors led to the focus in this field being switched to allosteric inhibitors. To date, the non-competitive inhibitors that have reached clinical trials target the site formed by the α3/α6/α7 tunnel or the site found in a disordered C-terminal non-catalytic segment. Herein, pyrrolo[1,2-a]quinoxal-5-inium salts and 4,5-dihydropyrrolo[1,2-a]quinoxalines are synthesized from pyrrolo[1,2-a]quinoxalines by alkylation and reduction, respectively. These compounds showed no toxicity in HepG2 cells and exhibited inhibitory activity against PTP1B, with inhibition percentages of between 37% and 53% at 1 μM and activities (IC 50 ) of between 0.25 and 1.90 μM. The inhibitory activity against T-cell protein tyrosine phosphatase (TC-TPT) was also assayed, with 4,5-dihydropyrrolo[1,2-a]quinoxalines being found to be slightly more active and selective. Compounds from the two series behave as insulin mimetics since they exhibit enhancement of glucose uptake in C2C12 cells. Computational docking studies provide information about the putative binding mode for both series and the preference for the α3/α6/α7 allosteric tunnel., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

PTP1B dephosphorylates insulin receptor and substrates to modulate glucose metabolism. This enzyme is a validated therapeutic target for type 2 diabetes, but no current drug candidates have completed clinical trials. Pyrrolo[1,2-a]quinoxalines substituted at positions C1-C4 and/or C7-C8 were found to be nontoxic to cells and good inhibitors in the low- to sub-micromolar range, with the 4-benzyl derivative being the most potent inhibitor (0.24 μm). Some analogues bearing chlorine atoms at C7 and/or C8 kept potency and showed good selectivity compared to TCPTP (selectivity index >40). The most potent inhibitors behaved as insulin mimetics by increasing glucose uptake. The 4-benzyl derivative inhibited insulin receptor substrate 1 and AKT phosphorylation. Molecular docking and molecular dynamics simulations supported a putative binding mode for these compounds to the allosteric α3/α6/α7 pocket, but inconsistent results in enzyme inhibition kinetics were obtained due to the high tendency of these inhibitors to form stable aggregates. Computational calculations supported the druggability of inhibitors., (© 2020 Wiley-VCH GmbH.)

Intravesical Mycobacterium bovis Bacillus Calmette-Guérin (BCG) immunotherapy remains the gold-standard treatment for non-muscle-invasive bladder cancer patients, even though half of the patients develop adverse events to this therapy. On exploring BCG-alternative therapies, Mycolicibacterium brumae , a nontuberculous mycobacterium, has shown outstanding anti-tumor and immunomodulatory capabilities. As no infections due to M. brumae in humans, animals, or plants have been described, the safety and/or toxicity of this mycobacterium have not been previously addressed. In the present study, an analysis was made of M. brumae - and BCG-intravenously-infected severe combined immunodeficient (SCID) mice, M. brumae -intravesically-treated BALB/c mice, and intrahemacoelic-infected- Galleria mellonella larvae. Organs from infected mice and the hemolymph from larvae were processed to count bacterial burden. Blood samples from mice were also taken, and a wide range of hematological and biochemical parameters were analyzed. Finally, histopathological alterations in mouse tissues were evaluated. Our results demonstrate the safety and non-toxic profile of M. brumae . Differences were observed in the biochemical, hematological and histopathological analysis between M. brumae and BCG-infected mice, as well as survival curves rates and colony forming units (CFU) counts in both animal models. M. brumae constitutes a safe therapeutic biological agent, overcoming the safety and toxicity disadvantages presented by BCG in both mice and G. mellonella animal models.

Background/aims: Diabetes type 2, metabolic syndrome or non-alcoholic fatty liver disease are insulin resistance-related metabolic disorders, which lack a better prognosis before their full establishment. We studied the importance of the intracellular scaffold protein integrin linked kinaes (ILK) as a key modulator in the initial pathogenesis and the early progression of those insulin resistance- related disorders., Methods: Adult mice with a global transgenic downregulation of ILK expression (cKD-ILK) and littermates without that depletion (CT) were fed with either standard (STD) or high fat (HFD) diets during 2 and 6 weeks. Weights, blood glucose and other systemic biochemical parameters were determined in animals under fasting conditions and after glucose or pyruvate intraperitoneal injections to test their tolerance. In RNA or proteins extracted from insulin-sensitive tissues, we determined by reverse transcription-quantitative PCR and western blot the expression of ILK, metabolites transporters and other metabolism and inflammatory markers. Glucose uptake capacity was studied in freshly isolated tissues., Results: HFD feeding was able to early and progressively increase glycaemia, insulinemia, circulating glycerol, body weight gain, liver-mediated gluconeogenesis along this time lapse, but cKD-ILK have all these systemic misbalances exacerbated compared to CT in the same HFD time lapse. Interestingly, the tisular expression of ILK in HFD-fed CT was dramatically downregulated in white adipose tissue (WAT), skeletal muscle and liver at the same extent of the original ILK downregulation of cKD-ILK. We previously published that basal STD-fed cKD-ILK compared to basal STD-CT have different expression of glucose transporters GLUT4 in WAT and skeletal muscle. In the same STD-fed cKD-ILK, we observed here the increased expressions of hepatic GLUT2 and WAT pro-inflammatory cytokines TNF-α and MCP-1. The administration of HFD exacerbated the expression changes in cKD-ILK of these and other markers related to the imbalanced metabolism observed, such as WAT lipolysis (HSL), hepatic gluconeogenesis (PCK-1) and glycerol transport (AQP9)., Conclusion: ILK expression may be taken as a predictive determinant of metabolic disorders establishment, because its downregulation seems to correlate with the early imbalance of glucose and glycerol transport and the subsequent loss of systemic homeostasis of these metabolites., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Kidney fibrosis is one of the main pathological findings of progressive chronic kidney disease (CKD) although the pathogenesis of renal scar formation remains incompletely explained. Integrin-linked kinase (ILK), a major scaffold protein between the extracellular matrix (ECM) and intracellular signaling pathways, is involved in several pathophysiological processes during renal damage. However, ILK contribution in the CKD progress remains to be fully elucidated. In the present work, we studied 1) the renal functional and structural consequences of CKD genesis and progression when ILK is depleted and 2) the potential of ILK depletion as a therapeutic approach to delay CKD progression. We induced an experimental CKD model, based on an adenine-supplemented diet on adult wild-type (WT) and ILK-depleted mice, with a tubulointerstitial damage profile resembling that is observed in human CKD. The adenine diet induced in WT mice a progressive increase in plasma creatinine and urea concentrations. In the renal cortex it was also observed tubular damage, interstitial fibrosis and progressive increased ECM components, pro-inflammatory and chemo-attractant cytokines, EMT markers and TGF-β1 expressions. These observations were highly correlated to a simultaneous increase of ILK expression and activity. In adenine-fed transgenic ILK-depleted mice, all these changes were prevented. Additionally, we evaluated the potential role of ILK depletion to be applied after the disease induction, as an effective approach to interventions in human CKD subjects. In this scenario, two weeks after the establishment of adenine-induced CKD, ILK was abrogated in WT mice and stabilized renal damage, avoiding CKD progression. We propose ILK to be a potential target to delay renal disease progression., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Patients with chronic kidney disease present with an accumulation of uraemic toxins, which have been identified as pathogenic agents associated with cardiovascular mortality, which is very high is this patient group. A phenomenon common to the progressive renal dysfunction and associated vascular damage, is the abnormal accumulation of extracellular matrix (ECM) proteins in the renal or vascular structures., Objective: To determine the contribution of uraemia or the uraemic toxins to the production of cytokinins and ECM in aortas of uraemic animals or human aortic smooth muscle cells (HASMCs)., Materials and Methods: Mice were used with uraemia induced by a diet rich in adenine (0.2%) for 2, 4 or 6 weeks. Kidney function was evaluated by means of urine volume, plasma levels of creatinine, urea, fractional excretion of sodium, and vascular damage using histology, as well as protein expression using RT-qPCR. The HASMCs were incubated in vitro with uraemic toxins: p-cresol 10-100 (μg/ml) and indoxyl-sulphate25-100 (μg/ml) alone or simultaneously. The protein expression was evaluated using Western blot and confocal microscopy., Results: The administration of adenine produced progressive kidney damage in the mice, thickening of the aortic wall, and increasing the expression of TGF-β1 and ECM proteins. The toxins at high doses and combined also induced the expression of TGF-β1 and ECM proteins by the HASMCs., Conclusions: The uraemia produced by an adenine rich diet or high doses of uraemic toxins induced the abnormal deposit of ECM proteins in the vascular wall or its production by HASMCs. The understanding of the mechanisms that underlie this pathophysiological process may be useful in the prevention of cardiovascular damage associated with the progress of chronic kidney disease, a disease, at the moment that is irreversible and occasional silent until its diagnosis in advanced stages., (Copyright © 2018 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.)

A series of new azolopyrimidine-peptide hybrids and indolomethylideneimidazolones were obtained and evaluated as calpain inhibitors. The hybrid compounds were inactive, whereas some members of the initial azolomethylideneimidazolone series showed interesting calpain inhibitory activity. By using 4b as a hit compound, a new series of analogs were synthesized by an efficient synthetic procedure based on a multicomponent reaction followed by an unprecedented reaction at the methylene position of the molecule. The best inhibitor found for calpain I (IC 50  = 20 nM) was about 20 times more potent than the hit compound. Studies on 4b showed that its inhibition is consistent with an uncompetitive inhibition mode. This compound did not exhibit cellular toxicity at any of the doses tested (0.1-10 μM) and further studies indicated that it was capable of blockading chemical ischemia induction of apoptosis by preventing sodium azide-dependent calpain activation in intact human kidney tubular epithelial cells. The results of molecular modeling studies rationalized the inhibitory activity found for this series and account, from a structural point of view, for the most active compound identified (4j)., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Ras proteins regulate cell survival, growth, differentiation, blood pressure, and fibrosis in some organs. We have demonstrated that H- ras gene deletion produces mice hypotension via a soluble guanylate cyclase-protein kinase G (PKG)-dependent mechanism. In this study, we analyzed the consequences of H- ras deletion on cardiac remodeling induced by continuous angiotensin II (AngII) infusion and the molecular mechanisms implied. Left ventricular posterior wall thickness and mass and cardiomyocyte cross-sectional area were similar between AngII-treated H-Ras knockout (H -ras -/- ) and control wild-type (H -ras +/+ ) mice, as were extracellular matrix protein expression. Increased cardiac PKG-Iβ protein expression in H -ras -/- mice suggests the involvement of this protein in heart protection. Ex vivo experiments on cardiac explants could support this mechanism, as PKG blockade blunted protection against AngII-induced cardiac hypertrophy and fibrosis markers in H -ras -/- mice. Genetic modulation studies in cardiomyocytes and cardiac and embryonic fibroblasts revealed that the lack of H-Ras down-regulates the B-RAF/MEK/ERK pathway, which induces the glycogen synthase kinase-3β-dependent activation of the transcription factor, cAMP response element-binding protein, which is responsible for PKG-Iβ overexpression in H -ras -/- mouse embryonic fibroblasts. This study demonstrates that H- ras deletion protects against AngII-induced cardiac remodeling, possibly via a mechanism in which PKG-Iβ overexpression could play a partial role, and points to H-Ras and/or downstream proteins as potential therapeutic targets in cardiovascular disease.-Martín-Sánchez, P., Luengo, A., Griera, M., Orea, M. J., López-Olañeta, M., Chiloeches, A., Lara-Pezzi, E., de Frutos, S., Rodríguez-Puyol, M., Calleros, L., Rodríguez-Puyol, D. H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Iβ pathway activation.

Two processes are associated with progressive loss of renal function: 1) decreased aquaporin-2 (AQP2) expression and urinary concentrating capacity (Nephrogenic Diabetes Insipidus, NDI); and 2) changes in extracellular matrix (ECM) composition, e.g. increased collagen I (Col I) deposition, characteristic of tubule-interstitial fibrosis. AQP2 expression is regulated by both the ECM-to-intracellular scaffold protein integrin-linked kinase (ILK) by NFATc/AP1 and other transcription factors. In the present work, we used in vivo and in vitro approaches to examine ILK participation in NFATc3/AP-1-mediated increases in AQP2 gene expression. Both NFATc3 knock-out mice and ILK conditional-knockdown mice (cKD-ILK) display symptoms of NDI (polyuria and reduced AQP2 expression). NFATc3 is upregulated in the renal medulla tubular cells of cKD-ILK mice but with reduced nuclear localization. Inner medullary collecting duct mIMCD3 cells were subjected to ILK depletion and transfected with reporter plasmids. Pharmacological activators or inhibitors determined the effect of ILK activity on NFATc/AP-1-dependent increases in transcription of AQP2. Finally, mIMCD3 cultured on Col I showed reduced activity of the ILK/GSK3β/NFATc/AQP2 axis, suggesting this pathway is a potential target for therapeutic treatment of NDI., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The development of insulin resistance is characterized by the impairment of glucose uptake mediated by glucose transporter 4 (GLUT4). Extracellular matrix changes are induced when the metabolic dysregulation is sustained. The present work was devoted to analyze the possible link between the extracellular-to-intracellular mediator integrin-linked kinase (ILK) and the peripheral tissue modification that leads to glucose homeostasis impairment. Mice with general depletion of ILK in adulthood (cKD-ILK) maintained in a chow diet exhibited increased glycemia and insulinemia concurrently with a reduction of the expression and membrane presence of GLUT4 in the insulin-sensitive peripheral tissues compared with their wild-type littermates (WT). Tolerance tests and insulin sensitivity indexes confirmed the insulin resistance in cKD-ILK, suggesting a similar stage to prediabetes in humans. Under randomly fed conditions, no differences between cKD-ILK and WT were observed in the expression of insulin receptor (IR-B) and its substrate IRS-1 expressions. The IR-B isoform phosphorylated at tyrosines 1150/1151 was increased, but the AKT phosphorylation in serine 473 was reduced in cKD-ILK tissues. Similarly, ILK-blocked myotubes reduced their GLUT4 promoter activity and GLUT4 expression levels. On the other hand, the glucose uptake capacity in response to exogenous insulin was impaired when ILK was blocked in vivo and in vitro , although IR/IRS/AKT phosphorylation states were increased but not different between groups. We conclude that ILK depletion modifies the transcription of GLUT4, which results in reduced peripheral insulin sensitivity and glucose uptake, suggesting ILK as a molecular target and a prognostic biomarker of insulin resistance., (© 2017 Society for Endocrinology.)

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and produces cGMP, which activates cGMP-dependent protein kinases (PKG) and is hydrolyzed by specific phosphodiesterases (PDE). The vasodilatory and cytoprotective capacity of cGMP-axis activation results in a therapeutic strategy for several pathologies. Integrin-linked kinase (ILK), a major scaffold protein between the extracellular matrix and intracellular signaling pathways, may modulate the expression and functionality of the cGMP-axis-related proteins. We introduce ILK as a novel modulator in renal homeostasis as well as a potential target for cisplatin (CIS)-induced acute kidney injury (AKI) improvement. We used an adult mice model of depletion of ILK (cKD-ILK), which showed basal increase of sGC and PKG expressions and activities in renal cortex when compared with wildtype (WT) littermates. Twenty-four h activation of sGC activation with NO enhanced the filtration rate in cKD-ILK. During AKI, cKD-ILK maintained the cGMP-axis upregulation with consequent filtration rates enhancement and ameliorated CIS-dependent tubular epithelial-to-mesenchymal transition and inflammation and markers. To emphasize the role of cGMP-axis upregulation due to ILK depletion, we modulated the cGMP axis under AKI in vivo and in renal cultured cells. A suboptimal dose of the PDE inhibitor ZAP enhanced the beneficial effects of the ILK depletion in AKI mice. On the other hand, CIS increased contractility-related events in cultured glomerular mesangial cells and necrosis rates in cultured tubular cells; ILK depletion protected the cells while sGC blockade with ODQ fully recovered the damage.

Hyperglycemia is involved in the diabetic complication of different organs and can elevate serum osmolarity. Here, we tested whether hyperosmolarity promoted by high glucose levels induces cellular senescence in renal cells. We treated Wistar rats with streptozotocin to induce diabetes or with consecutive daily injections of mannitol to increase serum osmolarity and analyzed p53 and p16 genes in renal cortex by immunohistochemistry. Both diabetic and mannitol treated rats showed a significant increase in serum osmolarity, without significant signs of renal dysfunction, but associated with increased staining for p53 and p16 in the renal cortex. An increase in p53 and p16 expression was also found in renal cortex slices and glomeruli isolated from healthy rats, which were later treated with 30 mM glucose or mannitol. Intracellular mechanisms involved were analyzed in cultured human glomerular mesangial cells treated with 30 mM glucose or mannitol. After treatments, cells showed increased p53, p21 and p16 expression and elevated senescence-associated β-galactosidase activity. Senescence was prevented when myo-inositol was added before treatment. High glucose or mannitol induced constitutive activation of Ras and ERK pathways which, in turn, were activated by oxidative stress. In summary, hyperosmolarity induced renal senescence, particularly in glomerular mesangial cells, increasing oxidative stress, which constitutively activated Ras-ERK 1/2 pathway. Cellular senescence could contribute to the organ dysfunction associated with diabetes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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., (© FASEB.)

Podosomes are integrin-based adhesions fundamental for stabilisation of the leading lamellae in migrating dendritic cells (DCs) and for extracellular matrix (ECM) degradation. We have previously shown that soluble factors and chemokines such as SDF 1-a trigger podosome initiation whereas integrin ligands promote podosome maturation and stability in DCs. The exact intracellular signalling pathways that regulate the sequential organisation of podosomal components in response to extracellular cues remain largely undetermined. The Wiskott Aldrich Syndrome Protein (WASP) mediates actin polymerisation and the initial recruitment of integrins and associated proteins in a circular configuration surrounding the core of filamentous actin (F-actin) during podosome initiation. We have now identified integrin linked kinase (ILK) surrounding the podosomal actin core. We report that DC polarisation in response to chemokines and the assembly of actin cores during podosome initiation require PI3K-dependent clustering of the Wiskott Aldrich Syndrome Protein (WASP) in puncta independently of ILK. ILK is essential for the clustering of integrins and associated proteins leading to podosome maturation and stability that are required for degradation of the subjacent extracellular matrix and the invasive motility of DCs across connective tissue barriers. We conclude that WASP regulates DCs polarisation for migration and initiation of actin polymerisation downstream of PI3K in nascent podosomes. Subsequently, ILK mediates the accumulation of integrin-associated proteins during podosome maturation and stability for efficient degradation of the subjacent ECM during the invasive migration of DCs., (Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.)

Aims: Integrin-linked kinase (ILK) regulates proliferation, differentiation, cell adhesion, and motility in many cell types and has been related to cancer progression, fibrosis, and vascular diseases. We designed the present study to directly explore the effect of ILK deletion on the regulation of vascular tone through the soluble guanylate cyclase (sGC) /protein kinase G (PKG) pathway in healthy adult mice., Methods and Results: Experiments were carried out using a tamoxifen-inducible CRE-LOX system to conditionally delete the ILK gene in adult mice. Mice lacking ILK expression (cKO) presented increased vascular content and increased activity of sGC and PKG, resulting in a more intense vasodilatory response to a single dose of a nitric oxide (NO) donor [sodium nitroprusside (SNP)] or PKG agonist [8-bromoguanosine 3',5'-cyclic monophosphate sodium salt (8-Br)]. Five minutes after SNP or 8-Br administration the reduction in the systolic arterial pressure was enhanced in cKO mice (SNP WT: -7.4 ± 1.2 mmHG; SNP cKO: -14.0 ± 2.5; 8-Br WT: -2.9 ± 1.5 mmHG; 8-Br cKO: -10.0 ± 3.4 mmHG). ILK deletion restored the vascular response to SNP after chronic oral nitrite administration. In addition, ILK deletion also increased hypotensive SNP effect in angiotensin II-treated animals, suggesting a role for ILK in basal and pathological states., Conclusion: Deletion of ILK in adult animals increased the vascular response to NO. These findings show, for the first time, a requirement for ILK in regulating sGC-PKG expression in vivo.

The circulating levels of heat shock proteins (HSP) are increased in cardiovascular diseases; however, the implication of this for the fibrotic process typical of such diseases remains unclear. HSP70 can interact with the vascular smooth muscle cells (SMC), the major producer of extracellular matrix (ECM) proteins, through the Toll-like receptors 4 (TLR4). The transforming growth factor type-β1 (TGF-β1) is a well known vascular pro-fibrotic cytokine that is regulated in part by AP-1-dependent transcriptional mechanisms. We hypothesized that extracellular HSP70 could interact with SMCs, inducing TGF-β1 synthesis and subsequent changes in the vascular ECM. We demonstrate that extracellular HSP70 binds to human aorta SMC TLR4, which up-regulates the AP-1-dependent transcriptional activity of the TGF-β1 promoter. This is achieved through the mitogen activated protein kinases JNK and ERK, as demonstrated by the use of specific blockers and the knockdown of TLR4 with specific small interfering RNAs. The TGF-β1 upregulation increase the expression of the ECM proteins type I collagen and fibronectin. This novel observation may elucidate the mechanisms by which HSP70 contributes in the inflammation and fibrosis present in atherosclerosis and other fibrosis-related diseases., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We report new examples of a series of losartan-hydrocaffeic hybrids that bear novel ester, amide and amine linkers. These compounds were made by linking hydrocaffeic acid to the side chain of losartan at the C-5 position of the imidazole ring through different strategies. Experiments performed in cultured cells demonstrate that these new hybrids retain the ability to block the angiotensin II effect and have increased antioxidant ability. Most of them reduced arterial pressure in rats better or as much as losartan., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

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.

The nitric oxide (NO)-soluble guanylate cyclase (sGC) pathway exerts most of its cellular actions through the activation of the cGMP-dependent protein kinase (PKG). Accumulation of extracellular matrix is one of the main structural changes in pathological conditions characterized by a decreased activity of this pathway, such as hypertension, diabetes, or aging, and it is a well-known fact that extracellular matrix proteins modulate cell phenotype through the interaction with membrane receptors such as integrins. The objectives of this study were 1) to evaluate whether extracellular matrix proteins, particularly fibronectin (FN), modulate PKG expression in contractile cells, 2) to analyze the mechanisms involved, and 3) to evaluate the functional consequences. FN increased type I PKG (PKG-I) protein content in human mesangial cells, an effect dependent on the interaction with β(1)-integrin. The FN upregulation of PKG-I protein content was due to increased mRNA expression, determined by augmented transcriptional activity of the PKG-I promoter region. Akt and the transcription factor CCAAT enhancer-binding protein (C/EBP) mediated the genesis of these changes. FN also increased PKG-I in another type of contractile cell, rat vascular smooth muscle cells (RVSMC). Tirofiban, a pharmacological analog of FN, increased PKG-I protein content in RVSMC and rat aortic walls and magnified the hypotensive effect of dibutyryl cGMP in conscious Wistar rats. The present results provide evidence of a mechanism able to increase PKG-I protein content in contractile cells. Elucidation of this novel mechanism provides a rationale for future pharmacotherapy in certain vascular diseases.

Objective: Hydrogen peroxide (H(2)O(2)) 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 H(2)O(2) on sGC expression and function and to explore the molecular mechanism involved., Methods and Results: H(2)O(2) increased sGCβ1 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. sGCα2 expression increased along with β1 subunit, whereas α1 subunit remained unchanged. Vascular relaxation to an NO donor (sodium nitroprusside) was enhanced by H(2)O(2), and it was prevented by ODQ (sGC inhibitor). cGMP production in both freshly isolated vessels and RASMCs exposed to H(2)O(2) was greatly increased after sodium nitroprusside treatment. The H(2)O(2)-dependent sGCβ1 upregulation was attributable to sGCβ1 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 sGCβ1 3' untranslated region. HuR silencing reversed the effects of H(2)O(2) on sGCβ1 levels and cGMP synthesis., Conclusions: Our results identify H(2)O(2) as an endogenous mediator contributing to the regulation of vascular tone and point to a key role of HuR in sGCβ1 mRNA stabilization.

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.

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.

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.

Objectives: Alterations in NO/cGMP signaling have been associated with vascular dysfunction. Here, we tested whether peptides containing arginine-glycine-aspartic acid (RGD) motifs, commonly found on the binding sites of extracellular matrix to integrins, could increase the expression and function of soluble guanylate cyclase (sGC) in human mesangial cell (HMC), and human aortic smooth muscle (HASMC) cells., Methods and Results: Arginine-glycine-aspartic acid-serine (RGDS) promoted an up-regulation in the sGC beta1 subunit steady-state level, both in HMC and HASMC, in a time- and dose-dependent manner. The cellular effects of RGDS-stimulation of sGC expression was an enhanced cellular response to sodium nitroprusside, resulting in elevated cGMP levels and vasodilator-stimulated phosphoprotein (VASP) phosphorylation in both kinds of cells, and an increased NO relaxing effect on cells precontracted with H(2)O(2) or Angiotensin II. Moreover, RGDS was able to restore the sGC levels that had been previously decreased by long term exposure to NO donors. RGDS effects on sGC regulation were due to the specific interaction with alpha(5)beta(1) integrin. To investigate the intracellular mechanisms activated after RGDS cell treatment, pharmacological kinase inhibitors were used. The effect of RGDS on sGC protein content was completely abolished by treating the cells with c-Jun N-terminal kinase (JNK) inhibitors. In addition, c-fos and c-jun were found in the cell nuclei after RGDS treatment, suggesting that the RGDS effect could be mediated by the AP-1 transcription factor., Conclusion: Results provide evidence of a mechanism able to increase the sGC protein content linked to increased activity in contractile cells, not only in basal conditions, but also after the down-regulation of the receptor by its own substrate. Elucidation of this novel mechanism provides a rationale for future pharmacotherapy in certain vascular diseases.

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.