RESUMO
Myocardial perfusion and coronary vascular resistance are regulated by signaling metabolites released from the local myocardium that act either directly on the VSMC or indirectly via stimulation of the endothelium. A prominent mechanism of vasodilation is EDH of the arteriolar smooth muscle, with EETs and H(2)O(2) playing important roles in EDH in the coronary microcirculation. In some cases, EETs and H(2)O(2) are released as transferable hyperpolarizing factors (EDHFs) that act directly on the VSMCs. By contrast, EETs and H(2)O(2) can also promote endothelial KCa activity secondary to the amplification of extracellular Ca(2+) influx and Ca(2+) mobilization from intracellular stores, respectively. The resulting endothelial hyperpolarization may subsequently conduct to the media via myoendothelial gap junctions or potentially lead to the release of a chemically distinct factor(s). Furthermore, in human isolated coronary arterioles dilator signaling involving EETs and H(2)O(2) may be integrated, being either complimentary or inhibitory depending on the stimulus. With an emphasis on the human coronary microcirculation, this review addresses the diverse and integrated mechanisms by which EETs and H(2)O(2) regulate vessel tone and also examines the hypothesis that myoendothelial microdomain signaling facilitates EDH activity in the human heart.
Assuntos
Vasos Coronários/metabolismo , Eicosanoides/metabolismo , Endotélio Vascular/metabolismo , Junções Comunicantes/metabolismo , Peróxido de Hidrogênio/metabolismo , Músculo Liso Vascular/metabolismo , Miocárdio/metabolismo , Vasodilatação/fisiologia , Animais , Sinalização do Cálcio/fisiologia , HumanosRESUMO
The protective actions of prostacyclin (PGI(2) ) are mediated by cyclic AMP (cAMP) which is reduced by type 4 phosphodiesterases (PDE4) which hydrolyze cAMP. Superoxide (O2(-)) from NADPH oxidase (Nox) is associated with impaired PGI(2) bioactivity. The objective of this study, therefore, was to study the relationship between Nox and PDE4 expression in human umbilical vein endothelial cells (HUVECs). HUVECs were incubated with the thromboxane A(2) analog, U46619, 8-isoprostane F(2α) (8IP), or tumor necrosing factor alpha (TNFα) [±iloprost (a PGI(2) analog)] and the expression of PDE4A, B, C, and D and splice variants thereof assessed using Western blotting and qPCR and mRNA silencing of Nox4 and Nox5. Effects on cell replication and angiogenesis were also studied. U46619, 8IP, and TNFα increased the expression of Nox 4 and Nox 5 and all PDE4 isoforms as well as cell replication and tubule formation (index of angiogenesis), effects inhibited by mRNA silencing of Nox4 (but not Nox5) and iloprost and rolipram. These data demonstrate that upregulation of Nox4 leads to an upregulation of PDE4A, B, and D and increased hydrolysis of cAMP which in turn augments cell replication and angiogenesis. This mechanism may be central to vasculopathies associated with endothelial dysfunction since the PGI(2)-cAMP signaling axis plays a key role in mediating functions that include hemostasis and angiogenesis.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Células Endoteliais da Veia Umbilical Humana/enzimologia , Isoenzimas/metabolismo , NADPH Oxidases/metabolismo , Regulação para Cima , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Processamento Alternativo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Dinoprosta/análogos & derivados , Dinoprosta/farmacologia , Inibidores Enzimáticos/metabolismo , Inativação Gênica , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Iloprosta/farmacologia , Isoenzimas/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , NADPH Oxidase 4 , NADPH Oxidase 5 , NADPH Oxidases/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Vasoconstritores/farmacologia , Vasodilatadores/farmacologiaRESUMO
Despite the exploration of a large number of disparate drugs in animal models and clinical trials, no pharmacological intervention, with the exception of aggressive lipid lowering therapy has reduced late vein graft failure in man. The importance of devising more effective strategies is exemplified by the enormous economic consequences of vein graft failure. Worldwide, there are currently more than 1,000,000 coronary artery bypass graft surgery (CABG) operations a year, the same number of patients undergoing infrainguinal bypass for vascular diseases of the lower limb. The pathophysiology of vein graft failure is complex, involving disparate factors that include adhesion of platelets and leukocytes, rheological forces, metalloproteinase expression, proliferation and migration of vascular smooth muscle cells, neointima formation, oxidative stress, hypoxia and neural re-organisation. Although this diverse etiology may seem to preclude any single drug type as being effective in mediating vein graft failure: one factor that is involved in every facet of vein graft pathobiology is endothelin-1 (ET-1). As such a single drug type (ET(A) antagonist) may prove to be the magic bullet in this scenario. Thus, in this review, we will consider the etiology of vein graft disease in relation to ET-1 and will then present an argument (with evidence) that specific ET(A) receptor antagonists constitute a potentially effective means of preventing vein graft failure.
Assuntos
Antagonistas do Receptor de Endotelina A , Endotelina-1/fisiologia , Oclusão de Enxerto Vascular/tratamento farmacológico , Rejeição de Enxerto/tratamento farmacológico , Veias/efeitos dos fármacos , Endotelina-1/farmacologia , Endotelina-1/uso terapêutico , Oclusão de Enxerto Vascular/metabolismo , Oclusão de Enxerto Vascular/prevenção & controle , Rejeição de Enxerto/metabolismo , Rejeição de Enxerto/prevenção & controle , Humanos , Terapia de Alvo Molecular , Neointima/fisiopatologia , Receptor de Endotelina A/fisiologia , Veias/metabolismo , Veias/fisiopatologiaRESUMO
OBJECTIVES: To explore the possible role of of 8-isoprostane F(2α) (8-IPF(2α) ) in the aetiology of erectile dysfunction (ED), as the over-production of superoxide (O(2)(-)) derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase results in the formation of 8-IPF(2α) in vascular tissue, which has similar properties to thromboxane A(2) (TXA(2) ). TXA(2) is vasoconstrictor and up-regulates the expression of NADPH oxidase and phosphodiesterase type 5 (PDE5). MATERIALS AND METHODS: Cavernosal vascular smooth muscle cells (CVSMCs) were incubated with 8-IPF(2α) or the TXA(2) analogue, U46619, ±sildenafil, iloprost (a stable prostacyclin [PGI(2) ] analogue) or the nitric oxide (NO) donor NONOate for 16 h. The formation of O(2)(-) was then measured, PDE5 expression assessed using Western blotting and PGI(2) and 8-IPF(2α) formation measured using enzyme-linked immunoassays. RESULTS: 8-IPF(2α) promoted the formation of O(2)(-) , an effect inhibited by apocynin (an NADPH oxidase inhibitor) and up-regulated the expression of PDE5. Under identical incubation conditions, 8-IPF(2α) induced an increase in the formation of 8-IPF(2α) but reduced the formation of PGI(2) . All, these effects were reversed by sildenafil, iloprost, NONOate and picotamide. CONCLUSIONS: These data show that O(2) (-) derived from NADPH oxidase influences the relative balance of PGI(2) and 8-IPF(2α) in CVSMCs, which in turn alters the degree of PDE5 expression. This is a novel pathogenic mechanism underlying ED and a novel mechanism of action of sildenafil.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Dinoprosta/análogos & derivados , Impotência Vasculogênica/etiologia , Miócitos de Músculo Liso/metabolismo , Pênis/metabolismo , Inibidores da Fosfodiesterase 5/farmacologia , Animais , Western Blotting , Dinoprosta/metabolismo , Dinoprosta/farmacologia , Ensaio de Imunoadsorção Enzimática , Iloprosta/farmacologia , Masculino , Músculo Liso Vascular/citologia , NADP/metabolismo , Óxido Nítrico , Estresse Oxidativo/efeitos dos fármacos , Pênis/efeitos dos fármacos , Ácidos Ftálicos/farmacologia , Piperazinas/farmacologia , Purinas/farmacologia , Coelhos , Citrato de Sildenafila , Sulfonas/farmacologia , Superóxidos/metabolismo , Regulação para CimaRESUMO
OBJECTIVE: To test the possibility that folic acid (FA) may be a means of treating erectile dysfunction (ED) in diabetes mellitus (DM), by studying the effect of FA administration to DM rabbits on cavernosal function and intrapenile oxidative stress. MATERIALS AND METHODS: To investigate the effect of administering FA to DM rabbits on erectile function and oxidative stress the formation of superoxide (O(2)(-)), 8-isoprostane F(2 alpha) (8-IPF(2 alpha)) and prostacyclin (as 6-keto-PGF(1 alpha)) were assessed, as well as carbachol- and electrical field stimulated (EFS) relaxation and p47(phox) content (active component of NADPH oxidase complex). Non-ketotic DM was induced in New Zealand rabbits with alloxan and FA administered orally daily for 1 month. Rabbits were killed, penises excised and segments prepared. These were mounted in an organ bath and relaxation elicited with carbachol or EFS. O(2)(-) release was measured spectrophotometrically, p47(phox) expression by Western blotting and 8-IPF(2 alpha) and 6-keto-PGF(1 alpha) formation by enzyme-linked immunosorbant assay. Blood was collected for measurement of homocysteine, red blood cell (RBC) folate and glucose. RESULTS: In cavernosal tissue from DM rabbits, carbachol-and EFS-induced relaxation was significantly impaired compared with the untreated controls. O(2)(-) release, p47(phox) expression and 8-IPF(2 alpha) formation were all enhanced and 6-keto-PGF(1 alpha) formation reduced compared with the controls. All these effects were reversed by FA. Plasma total homocysteine was reduced and RBC folate elevated. CONCLUSIONS: The administration of FA may constitute a strategy for reducing ED in patients with DM.
Assuntos
Antioxidantes/farmacologia , Disfunção Erétil/tratamento farmacológico , Ácido Fólico/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Ereção Peniana/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Antioxidantes/administração & dosagem , Western Blotting , Diabetes Mellitus Experimental/complicações , Avaliação Pré-Clínica de Medicamentos , Disfunção Erétil/fisiopatologia , Ácido Fólico/administração & dosagem , Masculino , Estresse Oxidativo/fisiologia , Ereção Peniana/fisiologia , Coelhos , Resultado do Tratamento , Vasodilatadores/administração & dosagemRESUMO
OBJECTIVE To study the effect of the H(2)S-donating derivative of sildenafil (ACS6) compared to sildenafil citrate and sodium hydrosulphide (NaHS) on relaxation, superoxide formation and NADPH oxidase and type 5 phosphodiesterase (PDE5) expression in isolated rabbit cavernosal tissue and smooth muscle cells (CSMCs), and in vivo on indices of oxidative stress induced with buthionine sulphoximine (BSO). MATERIALS AND METHODS Relaxation was studied in an organ bath in response to carbachol and after incubation with interleukin-1beta for 12 h. CSMCs were incubated with tumour-necrosis factor-alpha or the thromboxane A(2) (TXA(2)) analogue, U46619, with or with no sildenafil citrate, ACS6 or NaHS for 16 h. Superoxide formation and the expression of p47(phox) (an active subunit of the NADPH oxidase complex) and PDE5 protein was then assessed using Western blotting. Rats were also treated with BSO (with or with no sildenafil citrate or ACS6) for 7 days; cavernosal cGMP, cAMP, glutathionine and plasma TXA(2) and 8-isoprostane F(2alpha) was measured by enzyme-linked immunosorbent assay. RESULTS ACS6 and sildenafil citrate relaxed cavernosal smooth muscle equipotently; NaHS alone had little effect at up to 100 microm. The formation of superoxide and expression of p47(phox) and PDE5 was reduced by ACS6, sildenafil citrate and NaHS (order of potency: ACS6 > sildenafil citrate > NaHS). The effects of ACS6 were blocked by inhibitors of protein kinase A (PKA) and PKG. In rats treated with BSO, both ASC6 and sildenafil citrate reduced the increased plasma levels of TXA(2) and 8-isoprostane F(2alpha) but increased cGMP, cAMP and glutathionine levels in corpus cavernosum. CONCLUSIONS By virtue of a dual action on PKA and PKG activation, ACS6 not only promotes erection, acutely, but might also have a long-term beneficial effect through inhibition of oxidative stress and downregulation of PDE5.
Assuntos
Disfunção Erétil/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Ereção Peniana/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Piperazinas/farmacologia , Sulfonas/farmacologia , Animais , Western Blotting , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Regulação para Baixo , Masculino , Relaxamento Muscular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Inibidores de Fosfodiesterase/química , Inibidores de Fosfodiesterase/uso terapêutico , Piperazinas/química , Piperazinas/uso terapêutico , Purinas , Coelhos , Ratos , Citrato de Sildenafila , Sulfonas/química , Sulfonas/uso terapêuticoRESUMO
The activity of NADPH oxidase (NOX) is blocked by nitric oxide (NO). Hydrogen sulfide (H(2)S) is also produced by blood vessels. It is reasonable to suggest that H(2)S may have similar actions to NO on NOX. In order to test this hypothesis, the effect of sodium hydrosulfide (NaHS) on O(2)(-) formation, the expression of NOX-1 (a catalytic subunit of NOX) and Rac(1) activity (essential for full NOX activity) in isolated vascular smooth muscle cells (hVSMCs) was investigated. hVSMCs were incubated with the thromboxane A(2) analogue U46619 +/- NaHS for 1 or 16 h, and O(2)(-) formation, NOX-1 expression and Rac(1) activity were assessed. The possible interaction between H(2)S and NO was also studied by using an NO synthase inhibitor, L-NAME, and an NO donor, DETA-NONOate. The role of K(ATP) channels was studied by using glibenclamide. NaHS inhibited O(2)(-) formation following incubation of 1 h (IC(50), 30 nM) and 16 h (IC(50), 20 nM), blocked NOX-1 expression and inhibited Rac(1) activity. These inhibitory effects of NaHS were mediated by the cAMP-protein-kinase-A axis. Exogenous H(2)S prevents NOX-driven intravascular oxidative stress through an a priori inhibition of Rac(1) and downregulation of NOX-1 protein expression, an effect mediated by activation of the adenylylcyclase-cAMP-protein-kinase-G system by H(2)S.
Assuntos
Sulfeto de Hidrogênio/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , NADPH Oxidases/metabolismo , Sulfetos/farmacologia , Superóxidos/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Adenilil Ciclases/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Glibureto/farmacologia , Humanos , Canais KATP/antagonistas & inibidores , Canais KATP/metabolismo , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , NADPH Oxidase 1 , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Compostos Nitrosos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
There is evidence that plasma homocysteine augments angiopathy in patients with diabetes mellitus. Although lowering homocysteine with folic acid improves endothelial function, the precise mechanisms underlying this effect are unknown. To study this area further, the effect of administration of folic acid to diabetic rabbits on intraaortic oxidative stress was studied by assessing the formation of superoxide (O(2)(-)), 8-isoprostane F(2alpha) (8-IPF(2alpha)), and prostacyclin (as 6-keto-PGF(1alpha)) as well as acetylcholine-stimulated relaxation and gp47(phox) content. Nonketotic diabetes mellitus was induced in New Zealand rabbits with alloxan, and low- and high-dose folic acid was administered daily for 1 month. Rabbits were killed, aortae were excised, and rings were prepared. Rings were mounted in an organ bath, and relaxation was elicited with acetylcholine. The O(2)(-) release was measured spectrophotometrically; the gp47(phox) expression, by Western blotting; and the 8-IPF(2alpha) and 6-keto-PGF(1alpha) formation, by enzyme-linked immunosorbent assay. Blood was collected for measurement of homocysteine, red blood cell folate, and glucose. In aortae from the diabetic rabbits, acetylcholine-induced relaxation was significantly impaired compared with that in untreated controls. The O(2)(-) release, p47(phox) expression, and 8-IPF(2alpha) formation were all enhanced and 6-keto-PGF(1alpha) formation was reduced compared with controls. All these effects were reversed by both low- and high-dose folic acid. Plasma total homocysteine was reduced by high-dose, but not low-dose, folic acid. Red blood cell folate was elevated in both groups. The improvement of endothelial function in patients receiving folic acid may be due to inhibition of nicotinamide adenine nucleotide phosphate oxidase (NADPH) oxidase expression and therefore conservation of nitric oxide and prostacyclin bioavailability, 2 vasculoprotective factors.
Assuntos
Aorta/metabolismo , Diabetes Mellitus Experimental/metabolismo , Ácido Fólico/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Aloxano , Animais , Peso Corporal , Dinoprosta/análogos & derivados , Dinoprosta/biossíntese , Epoprostenol/biossíntese , Ácido Fólico/uso terapêutico , Homocisteína/sangue , Masculino , NADPH Oxidases/fisiologia , Coelhos , Superóxidos/metabolismoRESUMO
AIM: To study the effects of homocysteine and copper on type 5 phosphodiesterase (PDE5) expression in cavernosal vascular smooth muscle cells (CVSMCs) and to investigate superoxide (O(2)(.-)) derived from nicotinamide adenine dinucleotide phosphate oxidase as homocysteine and copper generate O(2)(.-), and O(2)(.-) upregulates PDE5 expression. METHODS: CVSMCs derived from rabbit penis were incubated with homocysteine or copper chloride with or without superoxide dismutase (SOD), catalase, sildenafil citrate, or apocynin (nicotinamide adenine dinucleotide phosphate inhibitor) for 16 h. The expression of PDE5 and of glyceraldehyde-3-phosphate dehydrogenase (internal standard) was assessed using Western blot analysis. In parallel, O(2)(.-) was measured spectrophotometrically. RESULTS: CuCl(2) alone (up to 10 micromol/L) and homocysteine alone (up to 100 micromol/L) had no effect on O(2)(.-) formation in CVSMCs compared to controls. In combination, however, homocysteine and CuCl(2) markedly increased O(2)(.-) formation, an effect blocked by SOD, catalase, apocynin, and sildenafil (1 micromol/L) when co-incubated over the same time course. PDE5 expression was also significantly increased in CVSMCs incubated with homocysteine and CuCl(2), compared to controls. This effect was also negated by 16-h co-incubation with SOD, catalase, apocynin and sildenafil. CONCLUSION: This represents a novel pathogenic mechanism underlying ED, and indicates that the therapeutic actions of prolonged sildenafil use are mediated in part through inhibition of this pathway.
Assuntos
Cobre/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/biossíntese , Homocisteína/farmacologia , Miócitos de Músculo Liso/enzimologia , Pênis/enzimologia , Animais , Western Blotting , Quelantes/farmacologia , Interpretação Estatística de Dados , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Masculino , Miócitos de Músculo Liso/efeitos dos fármacos , NADPH Oxidases/antagonistas & inibidores , Penicilamina/farmacologia , Pênis/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Piperazinas/farmacologia , Purinas/farmacologia , Coelhos , Espécies Reativas de Oxigênio/metabolismo , Citrato de Sildenafila , Sulfonas/farmacologiaRESUMO
Autologous saphenous vein is used as a conduit to bypass atherosclerotic lesions in both the coronary artery (coronary artery bypass graft surgery [CABG]) and in femoral arteries (infrainguinal bypass graft surgery [IIBS]). Despite the undoubted success and benefits of the procedures, graft failure occurs in 50% of cases within 10 years after surgery. A principal cause of late vein graft failure is intimal and medial hyperplasia and superimposed atherogenesis. Apart from lipid lowering therapy, no intervention has hitherto proved clinically effective in preventing late vein graft failure which clearly constitutes a major clinical and economic problem that needs to be urgently resolved. However, we have studied the effect of external synthetic stents and sheaths in pig models of vein into artery interposition grafting and found them to have a profound effect on vein graft remodelling and thickening. In this review, therefore, we will summarise the mechanisms underlying vein graft failure and how these stents influence these processes and the possible mechanisms involved as well as the application of these devices in preventing vein graft failure clinically.
Assuntos
Bioprótese , Prótese Vascular , Rejeição de Enxerto/fisiopatologia , Sobrevivência de Enxerto/fisiologia , Veia Safena/fisiopatologia , Veia Safena/transplante , Stents , Animais , Biologia/métodos , Rejeição de Enxerto/prevenção & controle , Veia Safena/patologia , SuínosRESUMO
Although ceruloplasmin (CP), a copper containing metalloenzyme, possesses antioxidant properties (e.g. ferroxidase activity), elevated circulating CP is associated with cardiovascular disease (CVD). This ambivalence is possibly due to the capacity of CP, via its coppers, to promote vasculopathic effects that include lipid oxidation, negation of nitric oxide bioactivity and endothelial cell apoptosis. In turn, these effects that are mediated by increased formation of reactive oxygen species (ROS), such as superoxide and hydrogen peroxide. There is also evidence that risk factors for CVD (in particular, diabetes mellitus and hyperhomocysteinaemia) may augment the vasculopathic impact of CP. In turn, it appears that ROS disrupt copper binding to CP, thereby impairing its normal protective function while liberating copper which in turn may promote oxidative pathology. The objective of this review, therefore, is to consider the epidemiology and pathophysiology of CP in relation to CVD, with particular emphasis on the relationship between CP and oxidative stress.
Assuntos
Ceruloplasmina/metabolismo , Cobre/metabolismo , Estresse Oxidativo/fisiologia , Doenças Vasculares/etiologia , Doenças Vasculares/metabolismo , Animais , HumanosRESUMO
Central to the aetiology of Acute Respiratory Distress Syndrome (ARDS) is superoxide, the principal source of which is nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase). To test whether superoxide may influence NADPH oxidase expression directly, the effect of incubation of superoxide with porcine pulmonary arterial endothelial cells on the expression of gp91(phox) (a catalytic subunit of NADPH oxidase) and superoxide formation was investigated. Since iloprost has been purported to be potentially effective in treating ARDS, the effect of iloprost on superoxide-mediated effects was also studied. Pulmonary artery endothelial cells were incubated with xanthine/xanthine oxidase which generates superoxide, or tumour necrosis factor alpha (TNFalpha) or thromboxane A(2) analogue, U46619 (+/- superoxide dismutase [SOD] or catalase or iloprost) for 16 h. Cells were then washed and superoxide formation assessed spectrophometrically and gp91(phox) expression using Western blotting. The role of NADPH oxidase was also studied in the above settings using apocynin, an NADPH oxidase inhibitor. Superoxide, TNFalpha and U46619 elicited an increase in the formation of superoxide and induced gp91(phox) expression in pulmonary artery endothelial cells following a 16 h incubation an effect blocked by the continual presence of SOD and apocynin but not catalase. Apocynin completely inhibited superoxide formation induced with xanthine/xanthine oxidase after the 16 h incubation. Rotenone and allopurinol were without effect. Iloprost inhibited the formation of superoxide and gp91(phox) expression. These data demonstrate that superoxide upregulates gp91(phox) expression in pulmonary artery endothelial cells and thus augments superoxide formation, an effect blocked by iloprost. This constitutes a novel mechanism by which vascular superoxide creates a self-perpetuating cascade that may be of importance to the etiology of ARDS and other vasculopathies.
Assuntos
Células Endoteliais/metabolismo , Glicoproteínas de Membrana/biossíntese , NADPH Oxidases/biossíntese , Superóxidos/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Animais , Western Blotting , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Iloprosta/farmacologia , Masculino , Modelos Biológicos , Artéria Pulmonar/citologia , Superóxido Dismutase/farmacologia , Superóxidos/farmacologia , Suínos , Fator de Necrose Tumoral alfa/farmacologia , Vasoconstritores/farmacologia , Vasodilatadores/farmacologia , Xantina/metabolismo , Xantina Oxidase/metabolismoRESUMO
Although hyperhomocysteinaemia is a risk factor for cardiovascular disease, the mechanisms underlying this association have not been elucidated. It has been demonstrated, however, that copper augments the inhibitory effect of homocysteine on nitric oxide (NO)-mediated relaxation of the rat aorta through increased superoxide formation, which reacts with NO thereby reducing the bioavailability of NO. Since it follows that the administration of a copper chelator may blunt the pathogenic impact of hyperhomocysteinaemia, in vivo, the effect of penicillamine administration on NO-dependent relaxation and superoxide formation in the aortae of hyperhomocysteinaemic rabbits was studied. New Zealand White rabbits were fed a methionine-rich (20 g/kg chow) diet for 1 month+/-penicillamine administered orally (10 mg/kg/day) and aortic relaxation elicited with acetylcholine and superoxide measured. The role of NADPH oxidase was also studied using a range of inhibitors and western analysis of gp47(phox) (a catalytic subunit of NADPH oxidase). The methionine-rich diet markedly increased plasma total homocysteine levels. In hyperhomocysteinaemic rabbits there was a marked reduction of acetylcholine-stimulated relaxation and an increase in superoxide formation that were both inhibited with superoxide dismutase and apocynin, an NADPH oxidase inhibitor. Gp47(phox) expression was also increased in aortae from methionine fed rabbits. Penicillamine administration significantly reduced plasma total copper in methionine-fed rabbits compared to controls. Impaired acetylcholine-stimulated relaxation, increased superoxide formation and increased gp47(phox) expression in aortae from methionine-fed rabbits was reversed by penicillamine administration. These data indicate that hyperhomocysteinaemia augments the formation of arterial superoxide through an increase in NADPH oxidase expression/activity which in turn reduces NO bioavailability. Since these effects were reversed by penicillamine, these data consolidate the hypothesis that copper plays a role in mediating homocysteine-induced vasculopathy.
Assuntos
Aorta/efeitos dos fármacos , Hiper-Homocisteinemia/fisiopatologia , Penicilamina/farmacologia , Superóxidos/metabolismo , Vasodilatação/efeitos dos fármacos , Acetofenonas/farmacologia , Acetilcolina/farmacologia , Administração Oral , Animais , Aorta/metabolismo , Aorta/fisiopatologia , Western Blotting , GMP Cíclico/metabolismo , Suplementos Nutricionais , Relação Dose-Resposta a Droga , Endotélio Vascular/fisiopatologia , Inibidores Enzimáticos/farmacologia , Homocisteína/sangue , Hiper-Homocisteinemia/sangue , Técnicas In Vitro , Metionina/administração & dosagem , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Oniocompostos/farmacologia , Penicilamina/administração & dosagem , Coelhos , Fatores de Tempo , Vasodilatadores/farmacologiaRESUMO
The increased expression and activity of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex has emerged as a major common factor in the etiology of all forms of cardiovascular diseases since the upregulation of intravascular NADPH oxidase results in the formation of superoxide (O(2)(-)), which in turn promotes vasculopathy. An ever-increasing number of drugs commonly used in cardiovascular medicine have been shown to influence NADPH oxidase expression and activity. These include nitric oxide donors, nitroaspirin, eicosanoids, phosphodiesterase inhibitors, corticosteroids, antioxidants, and specific inhibitors. The objective of this review is to discuss these drugs in relation to the mechanisms underlying their effects on NADPH oxidase activity and the expression and therapeutic implications of these effects.
Assuntos
Fármacos Cardiovasculares/farmacologia , Doenças Cardiovasculares/tratamento farmacológico , NADPH Oxidases/metabolismo , Doenças Cardiovasculares/fisiopatologia , Eicosanoides/fisiologia , Disfunção Erétil/tratamento farmacológico , Disfunção Erétil/fisiopatologia , Glucocorticoides/uso terapêutico , Humanos , Masculino , Inibidores de Fosfodiesterase/uso terapêutico , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Purinas , Citrato de Sildenafila , Sulfonas , Regulação para Cima/fisiologiaRESUMO
BACKGROUND: Although nonsteroidal antiinflammatory drugs (NSAIDs) are ineffective in treating acute respiratory distress syndrome (ARDS), inhalational NO has proved to be useful. NO-donating NSAIDs may therefore be more effective in treating ARDS than NSAIDs alone. Because oxidant stress is central to the pathophysiology of ARDS, the effect of nitroaspirins (NCX 4016, NCX 4040, and NCX 4050) compared with morpholinosydnonimine (SIN-1; an NO donor) and aspirin (ASA) on superoxide (O2*-) formation and gp91phox (an active catalytic subunit of NADPH oxidase) expression in pig pulmonary artery vascular smooth muscle cells (PAVSMCs) and endothelial cells (PAECs) was investigated. METHODS AND RESULTS: Cultured PAVSMCs and PAECs were incubated with lipopolysaccharide (LPS), tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1alpha (with or without NO-ASA, SIN-1, or ASA) for 16 hours, and O2*- release was measured by use of the reduction of ferricytochrome c. The expression of gp91(phox) was assessed by use of Western blotting. LPS, TNF-alpha, and IL-1alpha all stimulated the formation of O2*- and expression of gp91(phox) in both PAVSMCs and PAECs, an effect inhibited by NADPH oxidase inhibitors, diphenyleneiodonium, and apocynin. SIN-1, NCX 4016, and NCX 4050 but not ASA alone inhibited the formation of O2*- and expression of gp91(phox). CONCLUSIONS: LPS and cytokines promote the formation of O2*- in PAVSMCs and PAECs through an augmentation of NADPH oxidase activity, which in turn is prevented by NO. Thus, NO may play a protective role in preventing excess O2*- formation, but its negation by O2*- may augment the progress of ARDS. The inhibitory effect of nitroaspirins suggests that they may be therapeutically useful in treating ARDS through the suppression of NADPH oxidase upregulation and O2*- formation.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Aspirina/análogos & derivados , Aspirina/farmacologia , Glicoproteínas/biossíntese , Molsidomina/análogos & derivados , Molsidomina/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , NADPH Oxidases/biossíntese , Nitrocompostos/farmacologia , Artéria Pulmonar/citologia , Superóxidos/metabolismo , Acetofenonas/farmacologia , Animais , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Indução Enzimática/efeitos dos fármacos , Epoprostenol/biossíntese , Glicoproteínas/genética , Guanilato Ciclase/antagonistas & inibidores , Interleucina-1/farmacologia , Lipopolissacarídeos/toxicidade , Masculino , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , NADPH Oxidases/genética , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Oniocompostos/farmacologia , Síndrome do Desconforto Respiratório/metabolismo , Sus scrofa , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
1. Prednisolone, a potent anti-inflammatory drug, has proved ineffective in treating acute respiratory distress syndrome (ARDS). ARDS is associated with superoxide (O(2)(*-)) generation, which negates nitric oxide (NO). NO also downregulates NADPH oxidase and inhibits O(2)(*-) formation. A possible reason for the lack of effect of prednisolone may due to an inhibition of eNOS expression. In order to test this proposal, the effect of prednisolone on O(2)(*-) formation and the expression of gp91(phox) (catalytic subunit of NADPH oxidase) and eNOS in pig pulmonary artery (PA) segments and PA endothelial cells (PAECs) and PA vascular smooth muscle cells (PAVSMCs) was investigated. 2. PA segments and cells were incubated with prednisolone and tumour necrosis factor-alpha (TNF-alpha) for 16 h. O(2)(*-) formation was measured spectrophometrically and gp91(phox) and eNOS expression by Western blotting. The role of the NO-cGMP axis was studied using morpholinosydnonimine hydrochloride, the diethylamine/NO complex (DETA-NONOate), the guanylyl cyclase inhibitor, 1H-{1,2,4}oxadiazolo{4,3-a}quinoxalin-1-one (ODQ) and the stable cGMP analogues, 8-bromo cGMP and 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP). NO release was studied using a fluorescence assay and O(2)(*-)-NO interactions with a nitrite/nitrate assay. 3. Prednisolone elicited significant increase in O(2)(*-) formation in intact PA segments and PAECs, but not PAVSMCs, in a concentration-dependent manner. In endothelium-denuded segments, prednisolone slightly enhanced O(2)(*-) release. TNF-alpha further increased prednisolone-enhanced O(2)(*-) formation in intact PA segments and PAECs. NADPH oxidase inhibitor, apocynin, inhibited O(2)(*-) formation. Increased O(2)(*-) release and gp91(phox) expression in PAECs elicited by prednisolone was blocked by SIN-1 (3-morpholinosydnonimine hydrochloride), DETA-NONOate, 8-pCPT-cGMP and 8-bromo cGMP. The effects of SIN-1 on gp91(phox) expression were reversed by ODQ. Finally, eNOS protein expression was significantly reduced by prednisolone. 4. Prednisolone increases O(2)(*-) in porcine PAECs through a downregulation of endogenous eNOS expression. Since the NO-cGMP axis inhibits gp91(phox) expression, the resultant decrease in endogenous NO formation then augments NADPH oxidase activity, which in turn results in increased O(2)(*-) formation. Since O(2)(*-) promotes inflammation, this mechanism may explain why prednisolone is ineffective in treating ARDS. Therapeutically, the coadministration of an NO donor may render prednisolone more effective in treating ARDS.
Assuntos
Células Endoteliais/metabolismo , NADPH Oxidases/biossíntese , Óxido Nítrico Sintase/biossíntese , Prednisolona/farmacologia , Artéria Pulmonar/metabolismo , Superóxidos/metabolismo , Animais , Western Blotting , Células Endoteliais/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Masculino , Óxido Nítrico Sintase Tipo III , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacos , SuínosRESUMO
Acute respiratory distress syndrome (ARDS) is associated with increased superoxide (O(2)(*-)) formation in the pulmonary vasculature and negation of the bioavailability of nitric oxide (NO). Since NO inhibits NADPH oxidase expression through a cyclic GMP-mediated mechanism, sildenafil, a type V phosphodiesterase inhibitor, may be therapeutically effective in ARDS through an augmentation of NO-mediated inhibition of NADPH oxidase. Therefore, the effect of sildenafil citrate and NO-donating sildenafil (NCX 911) on O(2)(*-) formation and gp91(phox) (active catalytic subunit of NADPH oxidase) expression was investigated in cultured porcine pulmonary artery endothelial cells (PAECs). PAECs were incubated with 10 nM TXA(2) analogue, 9,11-dideoxy-9alpha,11alpha-methanoepoxy-prostaglandin F(2alpha) (U46619) (+/-sildenafil or NCX 911), for 16 h and O(2)(*-) formation measured spectrophometrically and gp91(phox) using Western blotting. The role of the NO-cGMP axis was studied using morpholinosydnonimine hydrochloride (SIN-1), the diethylamine/NO complex (DETA-NONOate), the guanylyl cyclase inhibitor, 1H-{1,2,4}oxadiazolo{4,3-a}quinoxalin-1-one (ODQ), and the protein kinase G inhibitor, 8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-cGMPS). NO release was studied using a fluorescence assay and O(2)(*-)-NO interactions by measuring nitrites. After a 16-h incubation with 10 nM U46619, both NCX 911 and sildenafil elicited a concentration-dependent inhibition of O(2)(*-) formation and gp91(phox) expression, NCX 911 being more potent (IC(50); 0.26 nM) than sildenafil citrate (IC(50); 1.85 nM). These inhibitory effects were reversed by 1 microM ODQ and 10 microM Rp-8-Br-cGMPS. NCX 911 stimulated the formation of cGMP in PAECs and generated NO in a cell-free system to a greater degree than sildenafil citrate. The inhibitory effect of sildenafil was augmented by 1 muM SIN-1 and blocked partially by the eNOS inhibitor 10 microM N(5)-(1-iminoethyl)-ornithine (L-NIO). Acutely, sildenafil and NCX 911 also inhibited O(2)(*-) formation, again blocked by 1 microM ODQ. NCX 911 reacted with O(2)(*-) generated by xanthine oxidase, an effect that was inhibited by superoxide dismutase (500 U ml(-1)). Since O(2)(*-) formation plays contributory role in ARDS, both sildenafil citrate and NCX 911 may be indicated for treating ARDS through suppression of NADPH oxidase expression and therefore of O(2)(*-) formation and preservation of NO bioavailability.
Assuntos
Glicoproteínas de Membrana/antagonistas & inibidores , NADPH Oxidases/antagonistas & inibidores , Piperazinas/farmacologia , Superóxidos/antagonistas & inibidores , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Animais , Células Cultivadas , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , GMP Cíclico/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Guanilato Ciclase/antagonistas & inibidores , Masculino , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Ornitina/análogos & derivados , Ornitina/farmacologia , Oxidiazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Artéria Pulmonar , Purinas , Quinoxalinas/farmacologia , Citrato de Sildenafila , Sulfonas , Superóxidos/metabolismo , Suínos , Tionucleotídeos/farmacologiaRESUMO
Despite the exploration of a large number of disparate drugs in animal models and clinical trials, no pharmacological intervention, with the exception of aggressive lipid lowering therapy has reduced late vein graft failure in man. The importance of devising more effective strategies is exemplified by the considerable economic consequences of vein graft failure. Worldwide, there are currently more than 1,000,000 coronary artery bypass graft surgery (CABG) operations a year, the same number of patients undergoing infrainguinal bypass (IIBS) for vascular diseases of the lower limb. The pathophysiology of vein graft failure is complex, involving disparate factors that include adhesion of platelets and leukocytes, rheological forces, metalloproteinase expression, proliferation and migration of vascular smooth muscle cells, neointima formation, oxidative stress, hypoxia and neural re-organisation. Although this diverse aetiology may seem to preclude any single drug type as being effective in preventing vein graft failure, one factor that is involved in every facet of vein graft pathobiology is endothelin-1 (ET-1). Thus, in this review, we will consider the diverse aetiology of vein graft disease in relation to ET-1 and will then present an argument (with evidence) that ET-1(A) (ET(A)) receptor antagonists constitute a potentially effective means of preventing vein graft failure.
Assuntos
Antagonistas do Receptor de Endotelina A , Endotelina-1/metabolismo , Oclusão de Enxerto Vascular/prevenção & controle , Músculo Liso Vascular/efeitos dos fármacos , Animais , Oclusão de Enxerto Vascular/metabolismo , Oclusão de Enxerto Vascular/fisiopatologia , Hemodinâmica/efeitos dos fármacos , Humanos , Músculo Liso Vascular/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Veias/efeitos dos fármacos , Veias/metabolismoRESUMO
Hypercholesterolaemia promotes erectile dysfunction through increased superoxide formation and negation of nitric oxide (NO) bioactivity in cavernosal tissue. The source of superoxide has not been clearly defined, however. Sildenafil (Viagra), the standard therapy for erectile dysfunction, may also be rendered more effective by the presence of an NO donor. One drug that intrinsically fulfils this criterion is sildenafil nitrate (NCX 911), an NO donating derivative of sildenafil. The objective of this study, therefore, was to determine the source of superoxide and its effect on erectile function in corpus cavernosum from hypercholesterolaemic rabbits and to determine whether NCX 911 confers an improvement over sildenafil citrate in this model. Hypercholesterolaemia elicited an increase in superoxide formation by rabbit cavernosal tissue and a reduction of carbachol-stimulated relaxation both of which were reversed by diphenylene iodonium chloride and apocynin (NADPH oxidase inhibitors). In response to sodium nitroprusside, hypercholesterolaemia also caused an attenuation of cavernosal relaxation which was not reversed with NADPH oxidase inhibitors. Both sildenafil citrate and NCX 911 significantly reversed impaired carbachol-stimulated relaxation and inhibited superoxide formation by cavernosal tissue from hypercholesterolaemic rabbits, NCX 911 being more potent. NCX 911 also augmented cavernosal cGMP levels, an effect blocked by the guanylyl cyclase inhibitor, 1H-{1,2,4}oxadiazolo {4,3-a}quinoxalin-1-one (ODQ). These data demonstrate that hypercholesterolaemia promotes erectile dysfunction through an augmentation of superoxide derived from NADPH oxidase in cavernosal tissue. It also indicates that NO donating sildenafil may be therapeutically more beneficial than conventional sildenafil in treating erectile dysfunction with an oxidative stress-related aetiology.
Assuntos
Hipercolesterolemia/fisiopatologia , Relaxamento Muscular/efeitos dos fármacos , Pênis/efeitos dos fármacos , Piperazinas/farmacologia , Superóxidos/metabolismo , Acetofenonas/farmacologia , Alopurinol/farmacologia , Animais , Carbacol/farmacologia , GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Masculino , NADPH Oxidases/antagonistas & inibidores , NG-Nitroarginina Metil Éster/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Nitroprussiato/farmacologia , Oniocompostos/farmacologia , Oxidiazóis/farmacologia , Pênis/metabolismo , Pênis/fisiopatologia , Inibidores de Fosfodiesterase/farmacologia , Purinas , Quinoxalinas/farmacologia , Coelhos , Rotenona/farmacologia , Citrato de Sildenafila , Sulfonas , Desacopladores/farmacologia , Xantina Oxidase/antagonistas & inibidoresRESUMO
Since the precise role of sarcoplasmic reticular Ca2+ in mediating vascular smooth muscle cells (VSMC) proliferation is unknown, the effect of pre-incubation with thapsigargin on extracellular signal regulated kinase (ERK) activation, the translocation of activated of ERK 1/2 to the nucleus, cyclin D1 expression, the onset of S phase and cytosolic Ca2+ levels were studied. Human saphenous vein VSMCs (hVSMC) were incubated with 10 nM thapsigargin for 24 h followed by stimulation with fetal calf serum and the activation of ERK1/2 and cyclin D1 assessed by western blotting, the intracellular distribution of ERK1/2 using indirect immunofluorescence, the onset of S-phase with the incorporation of bromodeoxyuridine and sarcoplasmic reticular Ca2+ status using FURA-2. Thapsigargin had a marginal effect on ERK1/2 activation only at 5 min and 10 min after stimulation with fetal calf serum. In contrast, the rapid translocation of ERK1/2 to the nucleus was completely blocked by thapsigargin. S phase was delayed by 8 h by thapsigargin which co-incided with the recovery of cytosolic Ca2+ levels and cyclin D1 expression. It is concluded that the inhibitory effect of thapsigargin (depletion of Ca2+ pools) on hVSMC replication is mediated through the inhibition of translocation of activated ERK1/2 to the nucleus and not to the phosphorylation of ERK, per se, which in turn prevents cyclin D1 expression and thus progression of the cell cycle.