RESUMO
Zinc is involved in the expression and function of various transcription factors, including the hypoxia-inducible factor-1 (HIF-1). HIF-1 and its target gene endothelin-1 (ET-1) are activated by intermittent hypoxia (IH), one of the main consequences of obstructive sleep apnea (OSA), and both play a key role in the cardiovascular consequences of IH. Because OSA and IH are associated with zinc deficiency, we investigated the effect of zinc deficiency caused by chelation on the HIF-1/ET-1 pathway and its functional consequences in endothelial cells. Primary human microvascular endothelial cells (HMVEC) were incubated with submicromolar doses of the zinc-specific membrane-permeable chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylene diamine (TPEN, 0.5 µM) or ET-1 (0.01 µM) with or without bosentan, a dual ET-1-receptor antagonist. HIF-1α expression was silenced by transfection with specific siRNA. Nuclear HIF-1 content was assessed by immunofluorescence microscopy and Western blot. Migratory capacity of HMVEC was evaluated with a wound-healing scratch assay. Zinc chelation by TPEN exposure induced the translocation of the cytosolic HIF-1α subunit of HIF-1 to the nucleus as well as an HIF-1-mediated ET-1 secretion by HMVEC. Incubation with either TPEN or ET-1 increased endothelial wound-healing capacity. Both HIF-1α silencing or bosentan abolished this effect. Altogether, these results suggest that zinc deficiency upregulates ET-1 signaling through HIF-1 activation and stimulates endothelial cell migration, suggesting an important role of zinc in the vascular consequences of IH and OSA mediated by HIF-1-ET- signaling.
Assuntos
Movimento Celular/efeitos dos fármacos , Quelantes/farmacologia , Células Endoteliais/efeitos dos fármacos , Endotelina-1/metabolismo , Etilenodiaminas/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Zinco/deficiência , Transporte Ativo do Núcleo Celular , Bosentana/farmacologia , Células Cultivadas , Células Endoteliais/metabolismo , Antagonistas dos Receptores de Endotelina/farmacologia , Endotelina-1/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Via Secretória , Transdução de SinaisRESUMO
Purpose: Obstructive sleep apnea recently has been associated with a higher frequency of ischemic optic neuropathies. Intermittent hypoxia (IH) has been proposed as a major component of obstructive sleep apnea cardiovascular consequences. However, there currently are no pathophysiologic data regarding the effect of IH on the ocular vascular system. Thus, we assessed the impact of chronic IH exposure on the morphology and vascular reactivity of the rat ophthalmic artery (OA). Methods: Rats were exposed to 14 days of IH or normoxia (NX). Ophthalmic artery reactivity was studied using wire myography in rats treated or not with tempol (1 mM/day). Expression of endothelin-1 (ET-1) and its receptors, and of the three nitric oxide synthase (NOS) isoform genes was quantified using quantitative polymerase chain reaction (qPCR) in the retina and optic nerve. Structural alterations (optical and electron microscopy) and superoxide anion production were studied in OA sections. Results: Superoxide ion expression in the OA wall was increased by 23% after IH exposure. Ophthalmic artery contractile response to 3.10-8 M ET-1 was increased by 18.6% and nitric oxide-mediated relaxation was significantly delayed in IH compared to NX rats. In the absence of nitric oxide, cytochrome P450 blockade increased relaxation to acetylcholine in IH rats and delayed it in NX rats. Tempol treatment abolished the IH-induced changes in OA reactivity. Conclusions: These results strongly suggest that chronic IH induces oxidative stress in the rat OA, associated with endothelial dysfunction through alterations of nitric oxide and endothelium-derived hyperpolarising factors (EDHF) pathways.
Assuntos
Endotelina-1/metabolismo , Hipóxia/fisiopatologia , Artéria Oftálmica/fisiopatologia , Estresse Oxidativo , Receptor de Endotelina A/metabolismo , Animais , Doença Crônica , Óxidos N-Cíclicos/farmacologia , Hipóxia/metabolismo , Masculino , Músculo Liso Vascular/fisiologia , Miografia , Óxido Nítrico Sintase/genética , Artéria Oftálmica/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Marcadores de Spin , Superóxidos/metabolismoRESUMO
We investigated the effects of intermittent hypoxia (IH), such as that encountered in severe obstructive sleep apnea (OSA) patients, on the development and severity of myocardial ischemia-related ventricular arrhythmias. Rats were exposed to 14 days of IH (30 s at 5%O2 and 30 s at 21%O2, 8 h·day-1) or normoxia (N, similar air-air cycles) and submitted to a 30-min coronary ligature. Arterial blood pressure (BP) and ECG were recorded for power spectral analysis, ECG interval measurement and arrhythmia quantification. Left ventricular monophasic action potential duration (APD) and expression of L-type calcium (LTCC) and transient receptor potential (TRPC) channels were assessed in adjacent epicardial and endocardial sites. Chronic IH enhanced the incidence of ischemic arrhythmias, in particular ventricular fibrillation (66.7% vs. 33.3% in N rats, p < 0.05). IH also increased BP and plasma norepinephine levels along with increased low-frequency (LF), decreased high-frequency (HF) and increased LF/HF ratio of heart rate and BP variability. IH prolonged QTc and Tpeak-to-Tend intervals, increased the ventricular APD gradient and upregulated endocardial but not epicardial LTCC, TRPC1 and TRPC6 (p < 0.05). Chronic IH, is a major risk factor for sudden cardiac death upon myocardial ischemia through sympathoactivation and alterations in ventricular repolarization, transmural APD gradient and endocardial calcium channel expression.
Assuntos
Arritmias Cardíacas/fisiopatologia , Hipóxia/fisiopatologia , Isquemia Miocárdica/fisiopatologia , Apneia Obstrutiva do Sono/fisiopatologia , Função Ventricular/fisiologia , Animais , Células Cultivadas , Doença Crônica , Vasos Coronários/cirurgia , Morte Súbita Cardíaca , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Masculino , Ratos , Ratos Wistar , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo , Canal de Cátion TRPC6/genética , Canal de Cátion TRPC6/metabolismoRESUMO
Performing hypoxia-reoxygenation cycles in cell culture with a cycle duration accurately reflecting what occurs in obstructive sleep apnea (OSA) patients is a difficult but crucial technical challenge. Our goal was to develop a novel device to expose multiple cell culture dishes to intermittent hypoxia (IH) cycles relevant to OSA with limited gas consumption. With gas flows as low as 200 ml/min, our combination of plate holders with gas-permeable cultureware generates rapid normoxia-hypoxia cycles. Cycles alternating 1 min at 20% O2 followed by 1 min at 2% O2 resulted in Po2 values ranging from 124 to 44 mmHg. Extending hypoxic and normoxic phases to 10 min allowed Po2 variations from 120 to 25 mmHg. The volume of culture medium or the presence of cells only modestly affected the Po2 variations. In contrast, the nadir of the hypoxia phase increased when measured at different heights above the membrane. We validated the physiological relevance of this model by showing that hypoxia inducible factor-1α expression was significantly increased by IH exposure in human aortic endothelial cells, murine breast carcinoma (4T1) cells as well as in a blood-brain barrier model (2.5-, 1.5-, and 6-fold increases, respectively). In conclusion, we have established a new device to perform rapid intermittent hypoxia cycles in cell cultures, with minimal gas consumption and the possibility to expose several culture dishes simultaneously. This device will allow functional studies of the consequences of IH and deciphering of the molecular biology of IH at the cellular level using oxygen cycles that are clinically relevant to OSA.
Assuntos
Barreira Hematoencefálica/metabolismo , Neoplasias da Mama/metabolismo , Técnicas de Cultura de Células , Células Endoteliais/metabolismo , Hipóxia/metabolismo , Oxigênio/metabolismo , Apneia Obstrutiva do Sono/metabolismo , Animais , Técnicas de Cultura de Células/instrumentação , Hipóxia Celular , Linhagem Celular Tumoral , Meios de Cultura/metabolismo , Desenho de Equipamento , Feminino , Gases , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fatores de Tempo , Hipóxia TumoralRESUMO
Cardiac ischemia is one of the leading causes of death worldwide. It is now well established that natriuretic peptides can attenuate the development of irreversible ischemic injury during myocardial infarction. Lebetin 2 (L2) is a new discovered peptide isolated from Macrovipera lebetina venom with structural similarity to B-type natriuretic peptide (BNP). Our objectives were to define the acute cardioprotective actions of L2 in isolated Langendorff-perfused rat hearts after regional or global ischemia-reperfusion (IR). We studied infarct size, left ventricular contractile recovery, survival protein kinases and mitochondrial permeability transition pore (mPTP) opening in injured myocardium. L2 dosage was determined by preliminary experiments at its ability to induce cyclic guanosine monophosphate (cGMP) release without changing hemodynamic effects in normoxic hearts. L2 was found to be as effective as BNP in reducing infarct size after the induction of either regional or global IR. Both peptides equally improved contractile recovery after regional IR, but only L2 increased coronary flow and reduced severe contractile dysfunction after global ischemia. Cardioprotection afforded by L2 was abolished after isatin or 5-hydroxydecanote pretreatment suggesting the involvement of natriuretic peptide receptors and mitochondrial KATP (mitoKATP) channels in the L2-induced effects. L2 also increased survival protein expression in the reperfused myocardium as evidenced by phosphorylation of signaling pathways PKCε/ERK/GSK3ß and PI3K/Akt/eNOS. IR induced mitochondrial pore opening, but this effect was markedly prevented by L2 treatment. These data show that L2 has strong cardioprotective effect in acute ischemia through stimulation of natriuretic peptide receptors. These beneficial effects are mediated, at least in part, by mitoKATP channel opening and downstream activated survival kinases, thus delaying mPTP opening and improving IR-induced mitochondrial dysfunction.
Assuntos
Cardiotônicos/farmacologia , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Venenos de Víboras/farmacologia , Animais , Técnicas In Vitro , Masculino , Poro de Transição de Permeabilidade Mitocondrial , Infarto do Miocárdio/patologia , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
Obstructive sleep apnea (OSA) is now recognized as an independent and important risk factor for cardiovascular diseases such as hypertension, coronary heart disease, heart failure and stroke. Clinical and experimental data have confirmed that intermittent hypoxia is a major contributor to these deleterious consequences. The repetitive occurrence of hypoxia-reoxygenation sequences generates significant amounts of free radicals, particularly in moderate to severe OSA patients. Moreover, in addition to hypoxia, reactive oxygen species (ROS) are potential inducers of the hypoxia inducible transcription factor-1 (HIF-1) that promotes the transcription of numerous adaptive genes some of which being deleterious for the cardiovascular system, such as the endothelin-1 gene. This review will focus on the involvement of the ROS-HIF-1-endothelin signaling pathway in OSA and intermittent hypoxia and discuss current and potential therapeutic approaches targeting this pathway to treat or prevent cardiovascular disease in moderate to severe OSA patients.