RESUMO
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 (IC50) 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.
Assuntos
Inibidores Enzimáticos/farmacologia , Simulação de Acoplamento Molecular , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Pirróis/farmacologia , Quinoxalinas/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Pirróis/síntese química , Pirróis/química , Quinoxalinas/síntese química , Quinoxalinas/química , Sais/síntese química , Sais/química , Sais/farmacologia , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Regulação para Baixo , Doenças Metabólicas/etiologia , Proteínas Serina-Treonina Quinases/genética , Animais , Feminino , Gluconeogênese , Inflamação/etiologia , Inflamação/genética , Resistência à Insulina , Lipólise , Masculino , Doenças Metabólicas/genética , Camundongos , Camundongos Endogâmicos BALB CRESUMO
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-Iß 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-3ß-dependent activation of the transcription factor, cAMP response element-binding protein, which is responsible for PKG-Iß 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-Iß 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-Iß pathway activation.
Assuntos
Angiotensina II/efeitos adversos , Cardiomegalia/enzimologia , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Hipertensão/enzimologia , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas p21(ras)/deficiência , Angiotensina II/farmacologia , Animais , Cardiomegalia/induzido quimicamente , Cardiomegalia/genética , Cardiomegalia/prevenção & controle , Proteína Quinase Dependente de GMP Cíclico Tipo I/genética , Embrião de Mamíferos/enzimologia , Embrião de Mamíferos/patologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Fibroblastos/enzimologia , Fibroblastos/patologia , Deleção de Genes , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Hipertensão/induzido quimicamente , Hipertensão/patologia , Camundongos , Camundongos KnockoutRESUMO
Patients with Stage 5 chronic kidney disease who are on hemodialysis (HD) remain in a chronic inflammatory state, characterized by the accumulation of uremic toxins that induce endothelial damage and cardiovascular disease (CVD). Our aim was to examine microvesicles (MVs), monocyte subpopulations, and angiopoietins (Ang) to identify prognostic markers in HD patients with or without diabetes mellitus (DM). A total of 160 prevalent HD patients from 10 centers across Spain were obtained from the Biobank of the Nephrology Renal Network (Madrid, Spain): 80 patients with DM and 80 patients without DM who were matched for clinical and demographic criteria. MVs from plasma and several monocyte subpopulations (CD142+/CD16+, CD14+/CD162+) were analyzed by flow cytometry, and the plasma concentrations of Ang1 and Ang2 were quantified by ELISA. Data on CVD were gathered over the 5.5 yr after these samples were obtained. MV level, monocyte subpopulations (CD14+/CD162+ and CD142+/CD16+), and Ang2-to-Ang1 ratios increased in HD patients with DM compared with non-DM patients. Moreover, MV level above the median (264 MVs/µl) was associated independently with greater mortality. MVs, monocyte subpopulations, and Ang2-to-Ang1 ratio can be used as predictors for CVD. In addition, MV level has a potential predictive value in the prevention of CVD in HD patients. These parameters undergo more extensive changes in patients with DM.
Assuntos
Angiopoietina-1/sangue , Angiopoietina-2/sangue , Micropartículas Derivadas de Células/metabolismo , Nefropatias Diabéticas/sangue , Nefropatias Diabéticas/terapia , Células Endoteliais/metabolismo , Diálise Renal , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/terapia , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Micropartículas Derivadas de Células/patologia , Nefropatias Diabéticas/diagnóstico , Nefropatias Diabéticas/mortalidade , Progressão da Doença , Células Endoteliais/patologia , Feminino , Humanos , Mediadores da Inflamação/sangue , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Valor Preditivo dos Testes , Prevalência , Diálise Renal/efeitos adversos , Diálise Renal/mortalidade , Insuficiência Renal Crônica/diagnóstico , Insuficiência Renal Crônica/mortalidade , Espanha/epidemiologia , Fatores de Tempo , Resultado do TratamentoRESUMO
Within the past decade tremendous efforts have been made to understand the mechanism behind aquaporin-2 (AQP2) water channel trafficking and recycling, to open a path toward effective diabetes insipidus therapeutics. A recent study has shown that integrin-linked kinase (ILK) conditional-knockdown mice developed polyuria along with decreased AQP2 expression. To understand whether ILK also regulates AQP2 trafficking in kidney tubular cells, we performed in vitro analysis using LLCPK1 cells stably expressing rat AQP2 (LLC-AQP2 cells). Upon treatment of LLC-AQP2 cells with ILK inhibitor cpd22 and ILK-siRNA, we observed increased accumulation of AQP2 in the perinuclear region, without any significant increase in the rate of endocytosis. This perinuclear accumulation did not occur in cells expressing a serine-256-aspartic acid mutation that retains AQP2 in the plasma membrane. We then examined clathrin-mediated endocytosis after ILK inhibition using rhodamine-conjugated transferrin. Despite no differences in overall transferrin endocytosis, the endocytosed transferrin also accumulated in the perinuclear region where it colocalized with AQP2. These accumulated vesicles also contained the recycling endosome marker Rab11. In parallel, the usual vasopressin-induced AQP2 membrane accumulation was prevented after ILK inhibition; however, ILK inhibition did not measurably affect AQP2 phosphorylation at serine-256 or its dephosphorylation at serine-261. Instead, we found that inhibition of ILK increased F-actin polymerization. When F-actin was depolymerized with latrunculin, the perinuclear located AQP2 dispersed. We conclude that ILK is important in orchestrating dynamic cytoskeletal architecture during recycling of AQP2, which is necessary for its subsequent entry into the exocytotic pathway.
Assuntos
Aquaporina 2/metabolismo , Citoesqueleto/metabolismo , Exocitose/fisiologia , Rim/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Linhagem Celular , Citoesqueleto/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Exocitose/efeitos dos fármacos , Rim/efeitos dos fármacos , Masculino , Camundongos , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genéticaRESUMO
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.
RESUMO
The coordination compound of the antihypertensive ligand irbesartan (irb) with copper(II) (CuIrb) was synthesized and characterized by FTIR, FT-Raman, UV-visible, reflectance and EPR spectroscopies. Experimental evidence allowed the implementation of structural and vibrational studies by theoretical calculations made in the light of the density functional theory (DFT). This compound was designed to induce structural modifications on the ligand. No antioxidant effects were displayed by both compounds, though CuIrb behaved as a weak 1,1-diphenyl-2-picrylhydrazyl radical (DPPH(·)) scavenger (IC50 = 425 µM). The measurements of the contractile capacity on human mesangial cell lines showed that CuIrb improved the antihypertensive effects of the parent medication. In vitro cell growth inhibition against prostate cancer cell lines (LNCaP and DU 145) was measured for CuIrb, irbesartan and copper(II). These cell lines have been selected since the angiotensin II type 1 (AT1) receptor (that was blocked by the angiotensin receptor blockers, ARB) has been identified in them. The complex exerted anticancer behavior (at 100 µM) improving the activity of the ligand. Flow cytometry determinations were used to determine late apoptotic mechanisms of cell death. Experimental and DFT characterization of an irbesartan copper(II) complex has been performed. The complex exhibits low scavenging activity against DPPH(·) and significant growth inhibition of LNCaP and DU 145 prostate cancer cell lines. Flow cytometry determinations were used to determine late apoptotic mechanisms of cell death. This compound improved the antihypertensive effect of irbesartan. This effect was observed earlier for the mononuclear Cu-candesartan complex, but not in structurally modified sartans forming dinuclear or octanuclear Cu-sartan compounds.
Assuntos
Compostos de Bifenilo/química , Cobre/química , Compostos Organometálicos/química , Compostos Organometálicos/farmacologia , Tetrazóis/química , Anti-Hipertensivos/química , Anti-Hipertensivos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Irbesartana , Modelos Moleculares , Conformação Molecular , Estresse Oxidativo/efeitos dos fármacos , Teoria Quântica , Relação Estrutura-AtividadeRESUMO
KEY POINTS: Patients with chronic kidney disease have a higher risk of developing cardiovascular diseases than the general population. Their vascular endothelium is dysfunctional, among other things, because it is permanently exposed to uraemic toxins, several of which have poor clearance by conventional dialysis. Recent studies have demonstrated the important role of integrin-linked kinase (ILK) in the maintenance of endothelial integrity and in this study we investigate the involvement of ILK in the mechanism underlying vascular endothelial damage that occurs in uraemia. For the first time, we demonstrate the implication of ILK in the protection against endothelial cell damage (inhibition of proliferation, toxicity, oxidative stress and programed cell death) induced by uraemic serum from chronic kidney disease patients and uraemic toxins. This molecular mechanism may have clinical relevance because it highlights the importance of maintaining high levels of ILK activity to help preserve endothelial integrity, at least in early stages of chronic kidney disease. ABSTRACT: Patients with chronic kidney disease (CKD) have a higher risk of developing cardiovascular diseases. Their vascular endothelium is dysfunctional, among other things, because it is permanently exposed to uraemic toxins, several of which, mostly protein-bound compounds such as indoxyl sulfate (IS) and p-cresyl sulphate, having poor clearance by conventional dialysis, induce endothelial toxicity. However, the molecular mechanism by which uraemic toxins regulate early stages of endothelial dysfunction remains unclear. Recent studies have demonstrated the important role of integrin-linked kinase (ILK) in the maintenance of endothelial integrity. In this study, we investigate the involvement of ILK in the mechanism underlying vascular endothelial damage that occurs in uraemia. First, we show that incubation of EA.hy926 cells with human uraemic serum from CKD patients upregulates ILK activity. This ILK activation also occurs when the cells are exposed to IS (25-100 µg ml(-1)), p-cresol (10-100 µg ml(-1)) or both combined, compared to human serum control. Next, we observed that high doses of both toxins together induce a slight decrease in cell proliferation and increase apoptosis and reactive oxygen species production. Interestingly, these toxic effects displayed a strong increase when the ILK protein is knocked down by small interfering RNA, even at low doses of uraemic toxins. Abrogation of AKT has demonstrated the ILK/AKT signalling pathway involved in these processes. This study has demonstrated the implication of ILK in the protection against endothelial cell damage induced by uraemic toxins, a molecular mechanism that could play a protective role in the early stages of endothelial dysfunction observed in uraemic patients.
Assuntos
Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Uremia/metabolismo , Cresóis/toxicidade , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Indicã/toxicidade , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
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.
Assuntos
Aquaporina 2/fisiologia , Água Corporal/metabolismo , Proteínas da Matriz Extracelular/fisiologia , Capacidade de Concentração Renal/fisiologia , Túbulos Renais Coletores/metabolismo , Poliúria/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Aquaporina 2/biossíntese , Aquaporina 2/genética , Aquaporina 3/biossíntese , Aquaporina 3/genética , Arginina Vasopressina/sangue , Transporte Biológico Ativo , Membrana Celular/química , Polaridade Celular , Células Cultivadas , Colágeno Tipo I/farmacologia , Desamino Arginina Vasopressina/farmacologia , Diabetes Insípido Nefrogênico/metabolismo , Modelos Animais de Doenças , Túbulos Renais Coletores/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Concentração Osmolar , Pressão Osmótica/fisiologia , Fosforilação , Poliúria/genética , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Receptores de Vasopressinas/biossíntese , Receptores de Vasopressinas/genéticaRESUMO
TNF-like weak inducer of apoptosis (TWEAK) is an inflammatory cytokine that activates the FGF-inducible 14 receptor. Both TWEAK and the FGF-inducible 14 receptor are constitutively expressed in the kidney. TWEAK has been shown to modulate several biological responses, such as inflammation, proliferation, differentiation, and apoptosis, that contribute to kidney injury. However, the role of TWEAK in fibrosis and TWEAK-activated intracellular signaling pathways remain poorly understood. We tested the hypothesis that TWEAK can be a potent inducer of renal fibrosis by increasing transforming growth factor (TGF)-ß1 expression (a well-known switch in the fibrosis process) through PKG-I downregulation. We showed that in human mesangial cells, TWEAK increased TGF-ß1 expression and activity, leading to higher levels of the extracellular matrix protein fibronectin and decreased PKG-I expression and activity via the Ras pathway. PKG-I activation with 8-bromo-cGMP, Ras inactivation with dominant negative Ras, or Ras pathway inhibition with the ERK1/2 inhibitor PD-98059 resulted in the prevention of TWEAK-induced TGF-ß1 upregulation. In vivo, exogenous administration of TWEAK to wild-type mice downregulated kidney PKG-I and increased kidney TGF-ß1 expression. These effects were blunted in H-Ras knockout mice. Together, these data demonstrate, for the first time, the key role of PKG-I in TGF-ß1 induction by TWEAK in kidney cells.
Assuntos
Injúria Renal Aguda/metabolismo , Apoptose/fisiologia , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fatores de Necrose Tumoral/metabolismo , Animais , Células Cultivadas , Citocina TWEAK , Modelos Animais de Doenças , Progressão da Doença , Fibrose/metabolismo , Flavonoides/farmacologia , Genes ras/genética , Rim/metabolismo , Células Mesangiais/metabolismo , Camundongos , Camundongos Knockout , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Despite the presence of vitamin D receptor (VDR) in endothelial cells, the effect of vitamin D on endothelial function is unknown. An unbalanced production of vasoactive endothelial factors such as nitric oxide (NO) or endothelin-1 (ET-1) results in endothelial dysfunction, which can alter the normal cardiovascular function. Present experiments were devoted to assess the effect of active vitamin D (calcitriol) on the synthesis of endothelial vasoactive factors. The results were that, in cells, calcitriol increased ET-1 and NO productions, which were measured by ELISA and fluorimetric assay, respectively. Calcitriol also increased endothelin-converting enzyme-1 (ECE-1) and endothelial-nitric oxide synthase (eNOS) activities, their mRNA (qPCR), their protein expressions (Western-blot), and their promoter activities (transfection assays). Calcitriol did not change prepro-ET-1 mRNA. The effect was specific to VDR activation because when VDR was silenced by siRNA, the observed effects disappeared. Mechanisms involved in each upregulation differed. ECE-1 upregulation depended on AP-1 activation, whereas eNOS upregulation depended directly on VDR activation. To evaluate the in vivo consequences of acute calcitriol treatment, normal Wistar rats were treated with a single ip injection of 400 ng/kg calcitriol and euthanized 24 h later. Results confirmed those observed in cells, that production and expression of both factors were increased by calcitriol. Besides, calcitriol-treated rats showed a slight rise in mean blood pressure, which decreased when pretreated with FR-901533, an ECE-1 antagonist. We conclude that calcitriol increases the synthesis of both ET-1 and NO in endothelial cells. However, the ET-1 upregulation seems to be biologically more relevant, as animals acutely treated with calcitriol show slight increases in blood pressure.
Assuntos
Calcitriol/farmacologia , Células Endoteliais/efeitos dos fármacos , Endotelinas/metabolismo , Óxido Nítrico/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/genética , Células Cultivadas , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Enzimas Conversoras de Endotelina , Humanos , Masculino , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Ratos , Ratos Wistar , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
ILK (integrin-linked kinase) is an intracellular serine/threonine kinase involved in cell-matrix interactions. ILK dysregulation has been described in chronic renal disease and modulates podocyte function and fibrosis, whereas data about its role in inflammation are scarce. AngII (angiotensin II) is a pro-inflammatory cytokine that promotes renal inflammation. AngII blockers are renoprotective and down-regulate ILK in experimental kidney disease, but the involvement of ILK in the actions of AngII in the kidney has not been addressed. Therefore we have investigated whether ILK signalling modulates the kidney response to systemic AngII infusion in wild-type and ILK-conditional knockout mice. In wild-type mice, AngII induced an inflammatory response, characterized by infiltration of monocytes/macrophages and lymphocytes, and up-regulation of pro-inflammatory factors (chemokines, adhesion molecules and cytokines). AngII activated several intracellular signalling mechanisms, such as the NF-κB (nuclear factor κB) transcription factor, Akt and production of ROS (reactive oxygen species). All these responses were prevented in AngII-infused ILK-deficient mice. In vitro studies characterized further the mechanisms regulating the inflammatory response modulated by ILK. In cultured tubular epithelial cells ILK blockade, by siRNA, inhibited AngII-induced NF-κB subunit p65 phosphorylation and its nuclear translocation. Moreover, ILK gene silencing prevented NF-κB-related pro-inflammatory gene up-regulation. The results of the present study demonstrate that ILK plays a key role in the regulation of renal inflammation by modulating the canonical NF-κB pathway, and suggest a potential therapeutic target for inflammatory renal diseases.
Assuntos
Angiotensina II/farmacologia , Nefrite/enzimologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Células Cultivadas , Feminino , Inativação Gênica , Humanos , Mediadores da Inflamação/metabolismo , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Camundongos Knockout , NF-kappa B/metabolismo , Nefrite/induzido quimicamente , Nefrite/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/fisiologia , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Regulação para Cima/efeitos dos fármacosRESUMO
RATIONALE: Atherosclerotic lesions develop in regions of disturbed flow, whereas laminar flow protects from atherogenesis; however, the mechanisms involved are not completely elucidated. Integrins are mechanosensors of shear stress in endothelial cells, and integrin-linked kinase (ILK) is important for blood vessel integrity and cardiovascular development. OBJECTIVES: To explore the role of ILK in vascular function by studying conditionally ILK-deficient (cKO) mice and human atherosclerotic arteries. RESULTS: ILK expression was detected in the endothelial cell layer of nonatherosclerotic vessels but was absent from the endothelium of atherosclerotic arteries. Live ultrasound imaging revealed that acetylcholine-mediated vasodilatation was impaired in cKO mice. These mice exhibited lowered agonist-induced nitric oxide synthase (NOS) activity and decreased cyclic guanosine monophosphate and nitrite production. ILK deletion caused endothelial NOS (eNOS) uncoupling, reflected in reduced tetrahydrobiopterin (BH4) levels, increased BH2 levels, decreased dihydrofolate reductase expression, and increased eNOS-dependent generation of superoxide accompanied by extensive vascular protein nitration. ILK reexpression prevented eNOS uncoupling in cKO cells, whereas superoxide formation was unaffected by ILK depletion in eNOS-KO cells, indicating eNOS as a primary source of superoxide anion. eNOS and ILK coimmunoprecipitated in aortic lysates from control animals, and eNOS-ILK-shock protein 90 interaction was detected in human normal mammary arteries but was absent from human atherosclerotic carotid arteries. eNOS-ILK interaction in endothelial cells was prevented by geldanamycin, suggesting heat shock protein 90 as a binding partner. CONCLUSIONS: Our results identify ILK as a regulatory partner of eNOS in vivo that prevents eNOS uncoupling, and suggest ILK as a therapeutic target for prevention of endothelial dysfunction related to shear stress-induced vascular diseases.
Assuntos
Artérias/fisiopatologia , Aterosclerose/fisiopatologia , Endotélio Vascular/fisiopatologia , Óxido Nítrico Sintase Tipo III/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Sistema Vasomotor/fisiologia , Idoso , Idoso de 80 Anos ou mais , Animais , Bovinos , Células Cultivadas , GMP Cíclico/fisiologia , Modelos Animais de Doenças , Regulação para Baixo/fisiologia , Feminino , Proteínas de Choque Térmico HSP90/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Óxido Nítrico/fisiologia , Transdução de Sinais/fisiologiaRESUMO
A new Cu(II) complex with the antihypertensive drug telmisartan, [Cu8Tlm16]·24H2O (CuTlm), was synthesized and characterized by elemental analysis and electronic, FTIR, Raman and electron paramagnetic resonance spectroscopy. The crystal structure (at 120 K) was solved by X-ray diffraction methods. The octanuclear complex is a hydrate of but otherwise isostructural to the previously reported [Cu8Tlm16] complex. [Cu8Tlm16]·24H2O crystallizes in the tetragonal P4/ncc space group with a = b = 47.335(1), c = 30.894(3) Å, Z = 4 molecules per unit cell giving a macrocyclic ring with a double helical structure. The Cu(II) ions are in a distorted bipyramidal environment with a somewhat twisted square basis, cis-coordinated at their core N2O2 basis to two carboxylate oxygen and two terminal benzimidazole nitrogen atoms. Cu8Tlm16 has a toroidal-like shape with a hydrophobic nanometer hole, and their crystal packing defines nanochannels that extend along the crystal c-axis. Several biological activities of the complex and the parent ligand were examined in vitro. The antioxidant measurements indicate that the complex behaves as a superoxide dismutase mimic with improved superoxide scavenger power as compared with native sartan. The capacity of telmisartan and its copper complex to expand human mesangial cells (previously contracted by angiotensin II treatment) is similar to each other. The antihypertensive effect of the compounds is attributed to the strongest binding affinity to angiotensin II type 1 receptor and not to the antioxidant effects. The cytotoxic activity of the complex and that of its components was determined against lung cancer cell line A549 and three prostate cancer cell lines (LNCaP, PC-3, and DU 145). The complex displays some inhibitory effect on the A549 line and a high viability decrease on the LNCaP (androgen-sensitive) line. From flow cytometric analysis, an apoptotic mechanism was established for the latter cell line. Telmisartan and CuTlm show antibacterial and antifungal activities in various strains, and CuTlm displays improved activity against the Staphylococcus aureus strain as compared with unbounded copper(II).
Assuntos
Antibacterianos/síntese química , Anti-Hipertensivos/síntese química , Antineoplásicos/síntese química , Antioxidantes/síntese química , Benzimidazóis/química , Benzoatos/química , Cobre/química , Antibacterianos/química , Antibacterianos/farmacologia , Anti-Hipertensivos/química , Anti-Hipertensivos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Bactérias/efeitos dos fármacos , Linhagem Celular , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Nanoestruturas , TelmisartanRESUMO
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.
Assuntos
Compostos de Anilina/farmacologia , Ácido Aspártico Endopeptidases/metabolismo , Cálcio/agonistas , Metaloendopeptidases/metabolismo , Animais , Ácido Aspártico Endopeptidases/análise , Pressão Sanguínea/efeitos dos fármacos , Linhagem Celular , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotelina-1/sangue , Endotelina-1/metabolismo , Enzimas Conversoras de Endotelina , Humanos , Masculino , Metaloendopeptidases/análise , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/análise , Óxido Nítrico Sintase Tipo III/metabolismo , Fenetilaminas , Propilaminas , Ratos , Ratos WistarRESUMO
Integrin-linked kinase (ILK) is an intracellular effector of cell-matrix interactions and regulates many cellular processes, including growth, proliferation, survival, differentiation, migration, invasion and angiogenesis. The present work analyzes the role of ILK in wound healing in adult animals using a conditional knock-out of the ILK gene generated with the tamoxifen-inducible Cre-lox system (CRE-LOX mice). Results show that ILK deficiency leads to retarded wound closure in skin. Intracellular mechanisms involved in this process were analyzed in cultured mouse embryonic fibroblast (MEF) isolated from CRE-LOX mice and revealed that wounding promotes rapid activation of phosphatidylinositol 3-kinase (PI3K) and ILK. Knockdown of ILK resulted in a retarded wound closure due to a decrease in cellular proliferation and loss of HGF protein expression during the healing process, in vitro and in vivo. Alterations in cell proliferation and wound closure in ILK-deficient MEF or mice could be rescued by exogenous administration of human HGF. These data demonstrate, for the first time, that the activation of PI3K and ILK after skin wounding are critical for HGF-dependent tissue repair and wound healing.
Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Cicatrização/fisiologia , Animais , Proliferação de Células , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Fator de Crescimento de Hepatócito/genética , Humanos , Masculino , Camundongos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Cicatrização/genéticaRESUMO
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.
Assuntos
Grafite , Lipólise , Camundongos , Ratos , Animais , Glicerol , Aumento de Peso , Obesidade/tratamento farmacológico , Obesidade/etiologia , Obesidade/metabolismo , Camundongos Transgênicos , Hipertrofia/complicações , IntegrinasRESUMO
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 (AT1 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.
Assuntos
Anti-Hipertensivos , Hipertensão , Hipertrofia Ventricular Esquerda , Zinco , Animais , Ratos , Anti-Hipertensivos/química , Anti-Hipertensivos/farmacologia , Compostos de Bifenilo/farmacologia , Pressão Sanguínea , Hipertensão/complicações , Hipertensão/tratamento farmacológico , Hipertrofia Ventricular Esquerda/tratamento farmacológico , Metaloproteinase 2 da Matriz , Miócitos Cardíacos , Ratos Endogâmicos SHR , Tetrazóis/farmacologia , Tetrazóis/uso terapêutico , Zinco/farmacologiaRESUMO
Renal fibrosis is the final outcome of many clinical conditions that lead to chronic renal failure, characterized by a progressive substitution of cellular elements by extracellular-matrix proteins, in particular collagen type I. The aim of this study was to identify the mechanisms responsible for human mesangial cell survival, conditioned by changes in extracellular-matrix composition. Our results indicate that collagen I induces apoptosis in cells but only after inactivation of the pro-survival factor NFκB by either the super-repressor IκBα or the PDTC inhibitor. Collagen I activates a death pathway, through ILK/GSK-3ß-dependent Bim expression. Moreover, collagen I significantly increases NFκB-dependent transcription, IκBα degradation and p65/NFκB translocation to the nucleus; it activates ß1 integrin and this is accompanied by increased activity of ILK which leads to AKT activation. Knockdown of ILK or AKT with small interfering RNA suppresses the increase in NFκB activity. NFκB mediates cell survival through the antiapoptotic protein Bcl-xL. Our data suggest that human mesangial cells exposed to abnormal collagen I are protected against apoptosis by a complex mechanism involving integrin ß1/ILK/AKT-dependent NFκB activation with consequent Bcl-xL overexpression, that opposes a simultaneously activated ILK/GSK-3ß-dependent Bim expression and this dual mechanism may play a role in the progression of glomerular dysfunction.
Assuntos
Apoptose , Matriz Extracelular/metabolismo , Células Mesangiais/citologia , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Sobrevivência Celular , Colágeno Tipo I/metabolismo , Matriz Extracelular/química , Humanos , Integrina beta1/metabolismo , Células Mesangiais/metabolismo , NF-kappa B/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Insuficiência RenalRESUMO
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 sGCß1 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 ß1 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 sGCß1 upregulation was attributable to sGCß1 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 sGCß1 3' untranslated region. HuR silencing reversed the effects of H(2)O(2) on sGCß1 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 sGCß1 mRNA stabilization.