RESUMO
BACKGROUND AND PURPOSE: Oxidative stress plays a key role in the vascular and metabolic abnormalities associated with obesity. Herein, we assessed whether obesity can increase coronary vasoconstriction induced by hydrogen peroxide (H2 O2 ) and the signalling pathways involving COX-2 and superoxide (O2.- ) generation. EXPERIMENTAL APPROACH: Contractile responses to H2 O2 and O2.- generation were measured in coronary arteries from genetically obese Zucker rats (OZR) and compared to lean Zucker rats (LZR). KEY RESULTS: Both basal and H2 O2 -stimulated O2.- production were enhanced in coronary arteries from OZR, but H2 O2 -induced vasoconstriction was unchanged. The selective COX-2 inhibitor NS398 significantly reduced H2 O2 -induced contractions in endothelium-denuded arteries from LZR and OZR, but only in endothelium-intact arteries from LZR. PGI2 (IP) receptor antagonism modestly reduced the vasoconstrictor action of H2 O2 while antagonism of the PGE2 receptor 4 (EP4 ) enhanced H2 O2 contractions in arteries from OZR but not LZR. Basal release of COX-2-derived PGE2 was higher in coronary arteries from OZR where the selective agonist of EP4 receptors TCS 2519 evoked potent relaxations. COX-2 was up-regulated after acute exposure to H2 O2 in coronary endothelium and vascular smooth muscle (VSM) and inhibition of COX-2 markedly reduced H2 O2 -elicited O2.- generation in coronary arteries and myocardium. Expression of Nox subunits in VSM and NADPH-stimulated O2.- generation was enhanced and contributed to H2 O2 vasoconstriction in arteries from obese rats. CONCLUSION AND IMPLICATIONS: COX-2 contributes to cardiac oxidative stress and to the endothelium-independent O2.- -mediated coronary vasoconstriction induced by H2 O2 in obesity, which is offset by the release of COX-2-derived endothelial PGE2 acting on EP4 vasodilator receptors.
Assuntos
Vasos Coronários/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Peróxido de Hidrogênio/farmacologia , Obesidade/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Animais , Vasos Coronários/metabolismo , Masculino , Obesidade/enzimologia , Ratos , Ratos ZuckerRESUMO
Mercury (Hg) has many harmful vascular effects by increasing oxidative stress, inflammation and vascular/endothelial dysfunction, all of which may contribute to cerebrovascular diseases development. We aimed to explore the effects of chronic low-mercury concentration on vascular function in cerebral arteries and the mechanisms involved. Basilar arteries from control (vehicle-saline solution, im) and mercury chloride (HgCl2)-treated rats for 30 days (first dose 4.6µg/kg, subsequent dose 0.07µg/kg/day, im, to cover daily loss) were used. Vascular reactivity, protein expression, nitric oxide (NO) levels and superoxide anion (O2(-)) production were analyzed. HgCl2 exposure increased serotonin contraction and reduced the endothelium-dependent vasodilatation to bradykinin. After NO synthase inhibition, serotonin responses were enhanced more in control than in mercury-treated rats while bradykinin-induced relaxation was abolished. NO levels were greater in control than Hg-treated rats. Tiron and indomethacin reduced vasoconstriction and increased the bradykinin-induced relaxation only in HgCl2-treated rats. Vascular O2(-) production was greater in mercury-treated when compared to control rats. Protein expressions of endothelial NO synthase, copper/zinc (Cu/Zn), Manganese (Mn) and extracellular-superoxide dismutases were similar in cerebral arteries from both groups. Results suggest that Hg treatment increases cerebrovascular reactivity by reducing endothelial negative modulation and NO bioavailability; this effect seems to be dependent on increased reactive oxygen species and prostanoids generation. These findings show, for the first time, that brain vasculature are also affected by chronic mercury exposure and offer further evidence that even at small concentration, HgCl2 is hazardous and might be an environmental risk factor accounting for cerebral vasospasm development.
Assuntos
Transtornos Cerebrovasculares/induzido quimicamente , Transtornos Cerebrovasculares/patologia , Células Endoteliais/efeitos dos fármacos , Cloreto de Mercúrio/farmacologia , Vasoconstrição/efeitos dos fármacos , Análise de Variância , Animais , Artéria Basilar/citologia , Peso Corporal/efeitos dos fármacos , Bradicinina/farmacologia , Relação Dose-Resposta a Droga , Ingestão de Líquidos/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Hidrazinas/farmacologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Ratos , Ratos Wistar , Serotonina/farmacologia , Superóxidos/metabolismo , Vasodilatadores/farmacologiaRESUMO
Chronic exposure to low lead concentration produces hypertension; however, the underlying mechanisms remain unclear. We analyzed the role of oxidative stress, cyclooxygenase-2-dependent pathways and MAPK in the vascular alterations induced by chronic lead exposure. Aortas from lead-treated Wistar rats (1st dose: 10 µg/100g; subsequent doses: 0.125µg/100g, intramuscular, 30days) and cultured aortic vascular smooth muscle cells (VSMCs) from Sprague Dawley rats stimulated with lead (20µg/dL) were used. Lead blood levels of treated rats attained 21.7±2.38µg/dL. Lead exposure increased systolic blood pressure and aortic ring contractile response to phenylephrine, reduced acetylcholine-induced relaxation and did not affect sodium nitroprusside relaxation. Endothelium removal and L-NAME left-shifted the response to phenylephrine more in untreated than in lead-treated rats. Apocynin and indomethacin decreased more the response to phenylephrine in treated than in untreated rats. Aortic protein expression of gp91(phox), Cu/Zn-SOD, Mn-SOD and COX-2 increased after lead exposure. In cultured VSMCs lead 1) increased superoxide anion production, NADPH oxidase activity and gene and/or protein levels of NOX-1, NOX-4, Mn-SOD, EC-SOD and COX-2 and 2) activated ERK1/2 and p38 MAPK. Both antioxidants and COX-2 inhibitors normalized superoxide anion production, NADPH oxidase activity and mRNA levels of NOX-1, NOX-4 and COX-2. Blockade of the ERK1/2 and p38 signaling pathways abolished lead-induced NOX-1, NOX-4 and COX-2 expression. Results show that lead activation of the MAPK signaling pathways activates inflammatory proteins such as NADPH oxidase and COX-2, suggesting a reciprocal interplay and contribution to vascular dysfunction as an underlying mechanisms for lead-induced hypertension.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Chumbo/toxicidade , Sistema de Sinalização das MAP Quinases/fisiologia , Músculo Liso Vascular/metabolismo , Estresse Oxidativo/fisiologia , Vasoconstrição/fisiologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Chumbo/administração & dosagem , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Vasoconstrição/efeitos dos fármacosRESUMO
AIMS: Connective tissue growth factor (CTGF/CCN2) is a developmental gene upregulated in pathological conditions, including cardiovascular diseases, whose product is a matricellular protein that can be degraded to biologically active fragments. Among them, the C-terminal module IV [CCN2(IV)] regulates many cellular functions, but there are no data about redox process. Therefore, we investigated whether CCN2(IV) through redox signaling regulates vascular responses. RESULTS: CCN2(IV) increased superoxide anion (O2(â¢-)) production in murine aorta (ex vivo and in vivo) and in cultured vascular smooth muscle cells (VSMCs). In isolated murine aorta, CCN2(IV), via O2(â¢-), increased phenylephrine-induced vascular contraction. CCN2(IV) in vivo regulated several redox-related processes in mice aorta, including increased nonphagocytic NAD(P)H oxidases (Nox)1 activity, protein nitrosylation, endothelial dysfunction, and activation of the nuclear factor-κB (NF-κB) pathway and its related proinflammatory factors. The role of Nox1 in CCN2(IV)-mediated vascular responses in vivo was investigated by gene silencing. The administration of a Nox1 morpholino diminished aortic O2(â¢-) production, endothelial dysfunction, NF-κB activation, and overexpression of proinflammatory genes in CCN2(IV)-injected mice. The link CCN2(IV)/Nox1/NF-κB/inflammation was confirmed in cultured VSMCs. Epidermal growth factor receptor (EGFR) is a known CCN2 receptor. In VSMCs, CCN2(IV) activates EGFR signaling. Moreover, EGFR kinase inhibition blocked vascular responses in CCN2(IV)-injected mice. INNOVATION AND CONCLUSION: CCN2(IV) is a novel prooxidant factor that in VSMCs induces O2(â¢-) production via EGFR/Nox1 activation. Our in vivo data demonstrate that CCN2(IV) through EGFR/Nox1 signaling pathway induces endothelial dysfunction and activation of the NF-κB inflammatory pathway. Therefore, CCN2(IV) could be considered a potential therapeutic target for redox-related cardiovascular diseases.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Receptores ErbB/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , NADH NADPH Oxirredutases/metabolismo , NF-kappa B/metabolismo , Animais , Imuno-Histoquímica , Masculino , Camundongos , Microscopia Confocal , NADPH Oxidase 1RESUMO
Lysyl oxidase (LOX) is an extracellular matrix-modifying enzyme that seems to play a critical role in vascular remodelling. However, the lack of viable LOX-deficient animal models has been an obstacle to deep in LOX biology. In this study we have developed a transgenic mouse model that over-expresses LOX in vascular smooth muscle cells (VSMC) to clarify whether LOX could regulate VSMC phenotype and vascular remodelling. The SM22α proximal promoter drove the expression of a transgene containing the human LOX cDNA. Two stable transgenic lines, phenotypically indistinguishable, were generated by conventional methods (TgLOX). Transgene expression followed the expected SMC-specific pattern. In TgLOX mice, real-time PCR and immunohistochemistry evidenced a strong expression of LOX in the media from aorta and carotid arteries, coincident with a higher proportion of mature collagen. VSMC isolated from TgLOX mice expressed high levels of LOX pro-enzyme, which was properly secreted and processed into mature and bioactive LOX. Interestingly, cell proliferation was significantly reduced in cells from TgLOX mice. Transgenic VSMC also exhibited low levels of Myh10 (marker of SMC phenotypic switching), PCNA (marker of cell proliferation) and MCP-1, and a weak activation of Akt and ERK1/2 in response to mitogenic stimuli. Accordingly, neointimal thickening induced by carotid artery ligation was attenuated in TgLOX mice that also displayed a reduction in PCNA and MCP-1 immunostaining. Our results give evidence that LOX plays a critical role in vascular remodelling. We have developed a new animal model to study the role of LOX in vascular biology.
Assuntos
Proteína-Lisina 6-Oxidase/metabolismo , Remodelação Vascular/genética , Animais , Artérias Carótidas/patologia , Movimento Celular , Proliferação de Células , Quimiocina CCL2/metabolismo , Colágeno/química , Colágeno/metabolismo , DNA Complementar/metabolismo , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Cadeias Pesadas de Miosina/metabolismo , Miosina não Muscular Tipo IIB/metabolismo , Fenótipo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Regiões Promotoras Genéticas , Transfecção , Transgenes , CicatrizaçãoRESUMO
C5-unsubstituted-C6-aryl-1,4-dihydropyridines were prepared by a CAN-catalyzed multicomponent reaction from chalcones, ß-dicarbonyl compounds, and ammonium acetate. These compounds were able to block Ca(2+) entry after a depolarizing stimulus and showed an improved Cav1.3/Cav1.2 selectivity in comparison with nifedipine. Furthermore, they were able to protect neuroblastoma cells against Ca(2+) overload and oxidative stress models. Their selectivity ratio makes them highly interesting for the treatment of neurological disorders where Ca(2+) dyshomeostasis and high levels of oxidative stress have been demonstrated. Furthermore, their low potency toward the cardiovascular channel subtype makes them safer by reducing their probable side effects, in comparison to classical 1,4-dihydropyridines. Some compounds afforded good protective profile in a postincubation model that simulates the real clinical situation of ictus patients, offering a therapeutic window of opportunity of great interest for patient recovery after a brain ischemic episode. Good activities were also found in acute ischemia/reperfusion models of oxygen and glucose deprivation.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Canais de Cálcio Tipo L/efeitos dos fármacos , Di-Hidropiridinas/síntese química , Fármacos Neuroprotetores/síntese química , Traumatismo por Reperfusão/prevenção & controle , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Linhagem Celular Tumoral , Di-Hidropiridinas/farmacologia , Hipocampo/irrigação sanguínea , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo , Ratos , Ratos Endogâmicos WKYRESUMO
OBJECTIVE: Leptin acts as a cardiac profibrotic factor. However, the mechanisms underlying this effect are unclear. Therefore, we sought to elucidate the mediators involved in this process and the potential role of leptin in cardiac fibrosis associated with obesity. METHODS: Male Wistar rats were fed either a high-fat diet (HFD; 33.5% fat), or a standard diet (3.5% fat) for 6 weeks. RESULTS: HFD animals show cardiac hypertrophy, fibrosis and an increase in O2- production as evaluated by dihydroethidium. Echocardiographic parameters of cardiac structure and systolic function were similar in both groups. Cardiac levels of leptin, collagen I, galectin-3 and transforming growth factor ß (TGF-ß) were higher in HFD than in controls. In cardiac myofibroblasts, leptin (10-100âng/ml) increased O2-, collagen I, galectin-3, TGF-ß and connective tissue growth factor production (CTGF). These effects were prevented by the presence of either melatonin (10âmmol/l) or the inhibitor of mTOR, rapamycin (10âmmol/l). Blockage of galectin-3 activity by N-acetyllactosamine (LacNac 10âmmol/l) reduced both collagen I and O2(*-) production induced by leptin. The p70S6 kinase activation/phosphorylation, the downstream mediator of mTOR, induced by leptin was not modified by melatonin. Leptin reduced the metalloproteinase (MMP) 2 activity and the presence of melatonin, rapamycin or LacNac were unable to prevent it. CONCLUSION: The data suggest that leptin locally produced in the heart could participate in the fibrosis observed in HFD by affecting collagen turnover. Collagen synthesis induced by leptin seems to be mediated by the production of galectin-3, TGF-ß and CTGF through oxidative stress increased by activation of mTOR pathway.
Assuntos
Cardiomiopatias/metabolismo , Fibrose/metabolismo , Galectina 3/metabolismo , Leptina/fisiologia , Obesidade/metabolismo , Estresse Oxidativo , Serina-Treonina Quinases TOR/metabolismo , Animais , Células Cultivadas , Gorduras na Dieta/administração & dosagem , Masculino , Fosforilação , Ratos , Ratos Wistar , Superóxidos/metabolismoRESUMO
Erectile dysfunction frequently coexists with coronary artery disease and has been proposed as a potential marker for silent coronary artery disease in type 2 diabetes. In the present study, we comparatively assessed the structural and functional changes of both penile arteries (PAs) and coronary arteries (CAs) from a prediabetic animal model. PAs and CAs from 17- to 18-wk-old obese Zucker rats (OZRs) and from their control counterparts [lean Zucker rats (LZRs)] were mounted in microvascular myographs to evaluate vascular function, and stained arteries were subjected to morphometric analysis. Endothelial nitric oxide (NO) synthase (eNOS) protein expression was also assessed. The internal diameter was reduced and the wall-to-lumen ratio was increased in PAs from OZRs, but structure was preserved in CAs. ACh-elicited relaxations were severely impaired in PAs but not in CAs from OZRs, although eNOS expression was unaltered. Contractions to norepinephrine and 5-HT were significantly enhanced in both PAs and CAs, respectively, from OZRs. Blockade of NOS abolished endothelium-dependent relaxations in PAs and CAs and potentiated norepinephrine and 5-HT contractions in arteries from LZRs but not from OZRs. The vasodilator response to the phosphodiesterase 5 inhibitor sildenafil was reduced in both PAs and CAs from OZRs. Pretreatment with SOD reduced the enhanced vasoconstriction in both PAs and CAs from OZRs but did not restore ACh-induced relaxations in PAs. In conclusion, the present results demonstrate vascular inward remodeling in PAs and a differential impairment of endothelial relaxant responses in PAs and CAs from insulin-resistant OZRs. Enhanced superoxide production and reduced basal NO activity seem to underlie the augmented vasoconstriction in both PAs and CAs. The severity of the structural and functional abnormalities in PAs might anticipate the vascular dysfunction of the more preserved coronary vascular bed.
Assuntos
Vasos Coronários/patologia , Disfunção Erétil/patologia , Disfunção Erétil/fisiopatologia , Pênis/irrigação sanguínea , Estado Pré-Diabético/patologia , Estado Pré-Diabético/fisiopatologia , Acetilcolina/farmacologia , Animais , Artérias/patologia , Artérias/fisiologia , Colesterol/sangue , Vasos Coronários/fisiopatologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Inibidores Enzimáticos/farmacologia , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/metabolismo , Nitroarginina/farmacologia , Obesidade/patologia , Obesidade/fisiopatologia , Piperazinas/farmacologia , Purinas/farmacologia , Ratos , Ratos Zucker , Citrato de Sildenafila , Sulfonas/farmacologia , Superóxidos/metabolismo , Triglicerídeos/sangue , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologia , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Vasodilatadores/farmacologiaRESUMO
This study investigated the mechanisms underlying the response to hydrogen peroxide (H(2)O(2)) in mesenteric resistance arteries from spontaneously hypertensive rats (SHRs) and normotensive Wistar Kyoto (WKY) rats. Arteries were mounted in microvascular myographs for isometric tension recording and for simultaneous measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)), superoxide anion (O(2)(.)) production was evaluated by dihydroethidium fluorescence and confocal microscopy, and thromboxane A(2) (TXA(2)) production was evaluated by enzyme immunoassay. H(2)O(2) (1-100 microM) induced biphasic responses characterized by a transient endothelium-dependent contraction followed by relaxation. Simultaneous measurements of tension and Ca(2+) showed a greater effect of H(2)O(2) in arteries from hypertensive than normotensive rats. The cyclooxygenase (cox) inhibitor, indomethacin [1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1-H-indole-3-acetic acid] (1 microM); the COX-1 inhibitor, SC-58560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl pyrazole] (1 microM); the thromboxane (TXA(2)) synthase inhibitor, furegrelate [5-(3-pyridinylmethyl)-2-benzofurancarboxylic acid, sodium salt] (10 microM); and the TXA(2)/prostaglandin H(2) receptor antagonist, SQ 29,548 ([1S-[1.alpha.,2.alpha.(Z),3.alpha.,4.alpha.]]-7-[3-[[2-[(phenylamino) carbonyl] hydrazino] methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid)) (1 microM) abolished H(2)O(2) contraction in arteries from WKY rats but only reduced it in SHRs. The O(2)(.) scavenger, tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt) (1 mM), and the NADPH oxidase inhibitor, apocynin (4'-hydroxy-3'-methoxyacetophenone) (0.3 mM), decreased H(2)O(2) contraction in arteries from SHRs but not in WKY rats. H(2)O(2) induced TXA(2) and O(2)(.) production that was greater in SHRs than in WKY rats. The TXA(2) analog, U46619 [9,11-di-deoxy-11 alpha,9 alpha-epoxymethano prostaglandin F(2 alpha) (0.1 nM-1 microM)], also increased O(2)(.) production in SHR vessels. H(2)O(2)-induced TXA(2) production was decreased by SC-58560. H(2)O(2)-induced O(2)(.) production was decreased by tiron, apocynin, and SQ 29,548. In conclusion, the enhanced H(2)O(2) contraction in resistance arteries from SHRs seems to be mediated by increased TXA(2) release from COX-1 followed by elevations in vascular smooth muscle [Ca(2+)](i) levels and O(2)(.) production. This reveals a new mechanism of oxidative stress-induced vascular damage in hypertension.