RESUMO
AMPK has been reported to facilitate hypoxic pulmonary vasoconstriction but, paradoxically, its deficiency precipitates pulmonary hypertension. Here we show that AMPK-α1/α2 deficiency in smooth muscles promotes persistent pulmonary hypertension of the new-born. Accordingly, dual AMPK-α1/α2 deletion in smooth muscles causes premature death of mice after birth, associated with increased muscularisation and remodeling throughout the pulmonary arterial tree, reduced alveolar numbers and alveolar membrane thickening, but with no oedema. Spectral Doppler ultrasound indicates pulmonary hypertension and attenuated hypoxic pulmonary vasoconstriction. Age-dependent right ventricular pressure elevation, dilation and reduced cardiac output was also evident. KV1.5 potassium currents of pulmonary arterial myocytes were markedly smaller under normoxia, which is known to facilitate pulmonary hypertension. Mitochondrial fragmentation and reactive oxygen species accumulation was also evident. Importantly, there was no evidence of systemic vasculopathy or hypertension in these mice. Moreover, hypoxic pulmonary vasoconstriction was attenuated by AMPK-α1 or AMPK-α2 deletion without triggering pulmonary hypertension.
Assuntos
Hipertensão Pulmonar , Proteínas Quinases Ativadas por AMP , Animais , Hipóxia , Camundongos , Mortalidade Prematura , Músculo Liso , Miócitos de Músculo Liso , Artéria Pulmonar , VasoconstriçãoRESUMO
BACKGROUND AND PURPOSE: Over past decades, targeted therapies and immunotherapy have improved survival and reduced the morbidity of patients with BRAF-mutated melanoma. However, drug resistance and relapse hinder overall success. Therefore, there is an urgent need for novel compounds with therapeutic efficacy against BRAF-melanoma. This prompted us to investigate the antiproliferative profile of a tachykinin-peptide from the Octopus kaurna, Octpep-1 in melanoma. EXPERIMENTAL APPROACH: We evaluated the cytotoxicity of Octpep-1 by MTT assay. Mechanistic insights on viability and cellular damage caused by Octpep-1 were gained via flow cytometry and bioenergetics. Structural and pharmacological characterization was conducted by molecular modelling, molecular biology, CRISPR/Cas9 technology, high-throughput mRNA and calcium flux analysis. In vivo efficacy was validated in two independent xerograph animal models (mice and zebrafish). KEY RESULTS: Octpep-1 selectively reduced the proliferative capacity of human melanoma BRAFV600E -mutated cells with minimal effects on fibroblasts. In melanoma-treated cells, Octpep-1 increased ROS with unaltered mitochondrial membrane potential and promoted non-mitochondrial and mitochondrial respiration with inefficient ATP coupling. Molecular modelling revealed that the cytotoxicity of Octpep-1 depends upon the α-helix and polyproline conformation in the C-terminal region of the peptide. A truncated form of the C-terminal end of Octpep-1 displayed enhanced potency and efficacy against melanoma. Octpep-1 reduced the progression of tumours in xenograft melanoma mice and zebrafish. CONCLUSION AND IMPLICATIONS: We unravel the intrinsic anti-tumoural properties of a tachykinin peptide. This peptide mediates the selective cytotoxicity in BRAF-mutated melanoma in vitro and prevents tumour progression in vivo, providing a foundation for a therapy against melanoma.
Assuntos
Antineoplásicos , Melanoma , Trifosfato de Adenosina , Animais , Antineoplásicos/farmacologia , Cálcio , Linhagem Celular Tumoral , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologia , Camundongos , Mutação , Octopodiformes/química , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/uso terapêutico , RNA Mensageiro , Espécies Reativas de Oxigênio , Taquicininas/genética , Taquicininas/uso terapêutico , Peixe-Zebra/genéticaRESUMO
Melanoma is the main cause of skin cancer deaths, with special emphasis in those cases carrying BRAF mutations that trigger the mitogen-activated protein kinases (MAPK) signaling and unrestrained cell proliferation in the absence of mitogens. Current therapies targeting MAPK are hindered by drug resistance and relapse that rely on metabolic rewiring and Akt activation. To identify new drug candidates against melanoma, we investigated the molecular mechanism of action of the Octopus Kaurna-derived peptide, Octpep-1, in human BRAF(V600E) melanoma cells using proteomics and RNAseq coupled with metabolic analysis. Fluorescence microscopy verified that Octpep-1 tagged with fluorescein enters MM96L and NFF cells and distributes preferentially in the perinuclear area of MM96L cells. Proteomics and RNAseq revealed that Octpep-1 targets PI3K/AKT/mTOR signaling in MM96L cells. In addition, Octpep-1 combined with rapamycin (mTORC1 inhibitor) or LY3214996 (ERK1/2 inhibitor) augmented the cytotoxicity against BRAF(V600E) melanoma cells in comparison with the inhibitors or Octpep-1 alone. Octpep-1-treated MM96L cells displayed reduced glycolysis and mitochondrial respiration when combined with LY3214996. Altogether these data support Octpep-1 as an optimal candidate in combination therapies for melanoma BRAF(V600E) mutations.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Melanoma/tratamento farmacológico , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Sirolimo/farmacologia , Neoplasias Cutâneas/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Melanoma/enzimologia , Melanoma/genética , Melanoma/patologia , Transdução de Sinais , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologiaRESUMO
BACKGROUND AND PURPOSE: The NO/cGMP pathway represents a major physiological signalling controlling tone in pulmonary arteries (PA), and drugs activating this pathway are used to treat pulmonary arterial hypertension. Kv channels expressed in PA smooth muscle cells (PASMCs) are key determinants of vascular tone. We aimed to analyse the contribution of Kv 1.5 and Kv 7 channels in the electrophysiological and vasodilating effects evoked by NO donors and the GC stimulator riociguat in PA. EXPERIMENTAL APPROACH: Kv currents were recorded in isolated rat PASMCs using the patch-clamp technique. Vascular reactivity was assessed in a wire myograph. KEY RESULTS: The NO donors diethylamine NONOate diethylammonium (DEA-NO) and sodium nitroprusside hyperpolarized the membrane potential and induced a bimodal effect on Kv currents (augmenting the current between -40 and -10 mV and decreasing it at more depolarized potentials). The hyperpolarization and the enhancement of the current were suppressed by Kv 7 channel inhibitors and by the GC inhibitor ODQ but preserved when Kv 1.5 channels were inhibited. Additionally, DEA-NO enhanced Kv 7.5 currents in COS7 cells expressing the KCNQ5 gene. Riociguat increased Kv currents at all potentials ≥-40 mV and induced membrane hyperpolarization. Both effects were prevented by Kv 7 inhibition. Likewise, PA relaxation induced by NO donors and riociguat was attenuated by Kv 7 inhibitors. CONCLUSIONS AND IMPLICATIONS: NO donors and riociguat enhance Kv 7 currents, leading to PASMC hyperpolarization. This mechanism contributes to NO/cGMP-induced PA vasodilation. Our study identifies Kv 7 channels as a novel mechanism of action of vasodilator drugs used in the treatment of pulmonary arterial hypertension.
Assuntos
GMP Cíclico/fisiologia , Canais de Potássio KCNQ/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Óxido Nítrico/fisiologia , Artéria Pulmonar/fisiologia , Animais , Células COS , Chlorocebus aethiops , Hidrazinas/farmacologia , Canal de Potássio Kv1.5/fisiologia , Masculino , Miócitos de Músculo Liso/fisiologia , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Artéria Pulmonar/citologia , Ratos Wistar , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
Hypoxic pulmonary vasoconstriction (HPV), which aids ventilation-perfusion matching in the lungs, is triggered by mechanisms intrinsic to pulmonary arterial smooth muscles. The unique sensitivity of these muscles to hypoxia is conferred by mitochondrial cytochrome c oxidase subunit 4 isoform 2, the inhibition of which has been proposed to trigger HPV through increased generation of mitochondrial reactive oxygen species. Contrary to this model, we have shown that the LKB1-AMPK-α1 signaling pathway is critical to HPV. Spectral Doppler ultrasound revealed that deletion of the AMPK-α1 catalytic subunit blocked HPV in mice during mild (8% O2) and severe (5% O2) hypoxia, whereas AMPK-α2 deletion attenuated HPV only during severe hypoxia. By contrast, neither of these genetic manipulations affected serotonin-induced reductions in pulmonary vascular flow. HPV was also attenuated by reduced expression of LKB1, a kinase that activates AMPK during energy stress, but not after deletion of CaMKK2, a kinase that activates AMPK in response to increases in cytoplasmic Ca2+ Fluorescence imaging of acutely isolated pulmonary arterial myocytes revealed that AMPK-α1 or AMPK-α2 deletion did not affect mitochondrial membrane potential during normoxia or hypoxia. However, deletion of AMPK-α1, but not of AMPK-α2, blocked hypoxia from inhibiting KV1.5, the classical "oxygen-sensing" K+ channel in pulmonary arterial myocytes. We conclude that LKB1-AMPK-α1 signaling pathways downstream of mitochondria are critical for the induction of HPV, in a manner also supported by AMPK-α2 during severe hypoxia.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Hipóxia , Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Artéria Pulmonar/fisiologia , Transdução de Sinais , Vasoconstrição/fisiologia , Proteínas Quinases Ativadas por AMP/genética , Animais , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/fisiologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Proteínas Serina-Treonina Quinases/genética , Artéria Pulmonar/citologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Regulation of breathing is critical to our capacity to accommodate deficits in oxygen availability and demand during, for example, sleep and ascent to altitude. It is generally accepted that a fall in arterial oxygen increases afferent discharge from the carotid bodies to the brainstem and thus delivers increased ventilatory drive, which restores oxygen supply and protects against hypoventilation and apnoea. However, the precise molecular mechanisms involved remain unclear. We recently identified as critical to this process the AMP-activated protein kinase (AMPK), which is key to the cell-autonomous regulation of metabolic homoeostasis. This observation is significant for many reasons, not least because recent studies suggest that the gene for the AMPK-α1 catalytic subunit has been subjected to natural selection in high-altitude populations. It would appear, therefore, that evolutionary pressures have led to AMPK being utilized to regulate oxygen delivery and thus energy supply to the body in the short, medium and longer term. Contrary to current consensus, however, our findings suggest that AMPK regulates ventilation at the level of the caudal brainstem, even when afferent input responses from the carotid body are normal. We therefore hypothesize that AMPK integrates local hypoxic stress at defined loci within the brainstem respiratory network with an index of peripheral hypoxic status, namely afferent chemosensory inputs. Allied to this, AMPK is critical to the control of hypoxic pulmonary vasoconstriction and thus ventilation-perfusion matching at the lungs and may also determine oxygen supply to the foetus by, for example, modulating utero-placental blood flow.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Oxigênio/metabolismo , Respiração , HumanosRESUMO
The fetal cardiovascular responses to acute hypoxia include a redistribution of the cardiac output toward the heart and the brain at the expense of other organs, such as the intestine. We hypothesized that hypoxia exerts a direct effect on the mesenteric artery (MA) that may contribute to this response. Using wire myography, we investigated the response to hypoxia (Po2 ~2.5 kPa for 20 min) of isolated MAs from 15- to 21-day chicken embryos (E15, E19, E21), and 1- to 45-day-old chickens (P1, P3, P14, P45). Agonist-induced pretone or an intact endothelium were not required to obtain a consistent and reproducible response to hypoxia, which showed a pattern of initial rapid phasic contraction followed by a sustained tonic contraction. Phasic contraction was reduced by elimination of extracellular Ca2+ or by presence of the neurotoxin tetrodotoxin, the α1-adrenoceptor antagonist prazosin, or inhibitors of L-type voltage-gated Ca2+ channels (nifedipine), mitochondrial electron transport chain (rotenone and antimycin A), and NADPH oxidase (VAS2870). The Rho-kinase inhibitor Y27632 impaired both phasic and tonic contraction and, when combined with elimination of extracellular Ca2+, hypoxia-induced contraction was virtually abolished. Hypoxic MA contraction was absent at E15 but present from E19 and increased toward the first days posthatching. It then decreased during the first weeks of life and P45 MAs were unable to sustain hypoxia-induced contraction over time. In conclusion, the results of the present study demonstrate that hypoxic vasoconstriction is an intrinsic feature of chicken MA vascular smooth muscle cells during late embryogenesis and the perinatal period.
Assuntos
Hipóxia/fisiopatologia , Artérias Mesentéricas/embriologia , Artérias Mesentéricas/fisiopatologia , Músculo Liso Vascular/embriologia , Músculo Liso Vascular/fisiopatologia , Vasoconstrição , Animais , Embrião de Galinha , Desenvolvimento Embrionário , Contração MuscularRESUMO
PPARß/δ activation protects against endothelial dysfunction in diabetic models. Elevated glucose is known to impair cAMP-induced relaxation and Kv channel function in coronary arteries (CA). Herein, we aimed to analyse the possible protective effects of the PPARß/δ agonist GW0742 on the hyperglycaemic-induced impairment of cAMP-induced relaxation and Kv channel function in rat CA. As compared with low glucose (LG), incubation under high glucose (HG) conditions attenuated the relaxation induced by the adenylate cyclase activator forskolin in CA and this was prevented by GW0742. The protective effect of GW0742 was supressed by a PPARß/δ antagonist. In myocytes isolated from CA under LG, forskolin enhanced Kv currents and induced hyperpolarization. In contrast, when CA were incubated with HG, Kv currents were diminished and the electrophysiological effects of forskolin were abolished. These deleterious effects were prevented by GW0742. The protective effects of GW0742 on forskolin-induced relaxation and Kv channel function were confirmed in CA from type-1 diabetic rats. In addition, the differences in the relaxation induced by forskolin in CA incubated under LG, HG or HG + GW0742 were abolished by the Kv7 channel inhibitor XE991. Accordingly, GW0742 prevented the down-regulation of Kv7 channels induced by HG. Finally, the preventive effect of GW0742 on oxidative stress and cAMP-induced relaxation were overcome by the pyruvate dehydrogenase kinase 4 (PDK4) inhibitor dichloroacetate (DCA). Our results reveal that the PPARß/δ agonist GW0742 prevents the impairment of the cAMP-mediated relaxation in CA under HG. This protective effect was associated with induction of PDK4, attenuation of oxidative stress and preservation of Kv7 channel function.
Assuntos
Vasos Coronários/metabolismo , AMP Cíclico/metabolismo , Hiperglicemia/metabolismo , Canal de Potássio KCNQ1/metabolismo , PPAR delta/metabolismo , PPAR beta/metabolismo , Animais , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/fisiopatologia , Diabetes Mellitus Experimental , Humanos , Hiperglicemia/genética , Canal de Potássio KCNQ1/genética , Masculino , PPAR delta/genética , PPAR beta/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Tiazóis/administração & dosagem , Vasodilatação/efeitos dos fármacosRESUMO
KEY POINTS: Progression of hypoxic pulmonary hypertension is thought to be due, in part, to suppression of voltage-gated potassium channels (Kv ) in pulmonary arterial smooth muscle by hypoxia, although the precise molecular mechanisms have been unclear. AMP-activated protein kinase (AMPK) has been proposed to couple inhibition of mitochondrial metabolism by hypoxia to acute hypoxic pulmonary vasoconstriction and progression of pulmonary hypertension. Inhibition of complex I of the mitochondrial electron transport chain activated AMPK and inhibited Kv 1.5 channels in pulmonary arterial myocytes. AMPK activation by 5-aminoimidazole-4-carboxamide riboside, A769662 or C13 attenuated Kv 1.5 currents in pulmonary arterial myocytes, and this effect was non-additive with respect to Kv 1.5 inhibition by hypoxia and mitochondrial poisons. Recombinant AMPK phosphorylated recombinant human Kv 1.5 channels in cell-free assays, and inhibited K(+) currents when introduced into HEK 293 cells stably expressing Kv 1.5. These results suggest that AMPK is the primary mediator of reductions in Kv 1.5 channels following inhibition of mitochondrial oxidative phosphorylation during hypoxia and by mitochondrial poisons. ABSTRACT: Progression of hypoxic pulmonary hypertension is thought to be due, in part, to suppression of voltage-gated potassium channels (Kv ) in pulmonary arterial smooth muscle cells that is mediated by the inhibition of mitochondrial oxidative phosphorylation. We sought to determine the role in this process of the AMP-activated protein kinase (AMPK), which is intimately coupled to mitochondrial function due to its activation by LKB1-dependent phosphorylation in response to increases in the cellular AMP:ATP and/or ADP:ATP ratios. Inhibition of complex I of the mitochondrial electron transport chain using phenformin activated AMPK and inhibited Kv currents in pulmonary arterial myocytes, consistent with previously reported effects of mitochondrial inhibitors. Myocyte Kv currents were also markedly inhibited upon AMPK activation by A769662, 5-aminoimidazole-4-carboxamide riboside and C13 and by intracellular dialysis from a patch-pipette of activated (thiophosphorylated) recombinant AMPK heterotrimers (α2ß2γ1 or α1ß1γ1). Hypoxia and inhibitors of mitochondrial oxidative phosphorylation reduced AMPK-sensitive K(+) currents, which were also blocked by the selective Kv 1.5 channel inhibitor diphenyl phosphine oxide-1 but unaffected by the presence of the BKCa channel blocker paxilline. Moreover, recombinant human Kv 1.5 channels were phosphorylated by AMPK in cell-free assays, and K(+) currents carried by Kv 1.5 stably expressed in HEK 293 cells were inhibited by intracellular dialysis of AMPK heterotrimers and by A769662, the effects of which were blocked by compound C. We conclude that AMPK mediates Kv channel inhibition by hypoxia in pulmonary arterial myocytes, at least in part, through phosphorylation of Kv 1.5 and/or an associated protein.
Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Hipóxia/fisiopatologia , Canal de Potássio Kv1.5/fisiologia , Mitocôndrias/metabolismo , Células Musculares/fisiologia , Animais , Células HEK293 , Humanos , Masculino , Fosforilação Oxidativa , Artéria Pulmonar/citologia , Ratos Sprague-DawleyRESUMO
Perhaps the defining characteristic of pulmonary arteries is the process of hypoxic pulmonary vasoconstriction (HPV) which, under physiological conditions, supports ventilation-perfusion matching in the lung by diverting blood flow away from oxygen deprived areas of the lung to oxygen rich regions. However, when alveolar hypoxia is more widespread, either at altitude or with disease (e.g., cystic fibrosis), HPV may lead to hypoxic pulmonary hypertension. HPV is driven by the intrinsic response to hypoxia of pulmonary arterial smooth muscle and endothelial cells, which are acutely sensitive to relatively small changes in pO2 and have evolved to monitor oxygen supply and thus address ventilation-perfusion mismatch. There is now a consensus that the inhibition by hypoxia of mitochondrial oxidative phosphorylation represents a key step towards the induction of HPV, but the precise nature of the signalling pathway(s) engaged thereafter remains open to debate. We will consider the role of the AMP-activated protein kinase (AMPK) and liver kinase B1 (LKB1), an upstream kinase through which AMPK is intimately coupled to changes in oxygen supply via mitochondrial metabolism. A growing body of evidence, from our laboratory and others, suggests that modulation of the LKB1-AMPK signalling pathway underpins both hypoxic pulmonary vasoconstriction and the development of pulmonary hypertension.
Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Hipertensão Pulmonar/etiologia , Proteínas Serina-Treonina Quinases/fisiologia , Artéria Pulmonar/fisiologia , Transdução de Sinais/fisiologia , Vasoconstrição , Quinases Proteína-Quinases Ativadas por AMP , Animais , Hipóxia Celular , Humanos , Mitocôndrias/fisiologiaRESUMO
KEY POINTS: Adult animals that have been perinatally exposed to oxygen-rich atmospheres (hyperoxia), recalling those used for oxygen therapy in infants, exhibit a loss of hypoxic pulmonary vasoconstriction, whereas vasoconstriction elicited by depolarizing agents is maintained. Loss of pulmonary hypoxic vasoconstriction is not linked to alterations in oxygen-sensitive K(+) currents in pulmonary artery smooth muscle cells. Loss of hypoxic vasoconstriction is associated with early postnatal oxidative damage and corrected by an antioxidant diet. Perinatal hyperoxia damages carotid body chemoreceptor cell function and the antioxidant diet does not reverse it. The hypoxia-elicited increase in erythropoietin plasma levels is not affected by perinatal hyperoxia. The potential clinical significance of the findings in clinical situations such as pneumonia, chronic obstructive pulmonary disease or general anaesthesia is considered. ABSTRACT: Adult mammalians possess three cell systems that are activated by acute bodily hypoxia: pulmonary artery smooth muscle cells (PASMC), carotid body chemoreceptor cells (CBCC) and erythropoietin (EPO)-producing cells. In rats, chronic perinatal hyperoxia causes permanent carotid body (CB) atrophy and functional alterations of surviving CBCC. There are no studies on PASMC or EPO-producing cells. Our aim is to define possible long-lasting functional changes in PASMC or EPO-producing cells (measured as EPO plasma levels) and, further, to analyse CBCC functional alterations. We used 3- to 4-month-old rats born and reared in a normal atmosphere or exposed to perinatal hyperoxia (55-60% O2 for the last 5-6 days of pregnancy and 4 weeks after birth). Perinatal hyperoxia causes an almost complete loss of hypoxic pulmonary vasoconstriction (HPV), which was correlated with lung oxidative status in early postnatal life and prevented by antioxidant supplementation in the diet. O2 -sensitivity of K(+) currents in the PASMC of hyperoxic animals is normal, indicating that their inhibition is not sufficient to trigger HPV. Perinatal hyperoxia also abrogated responses elicited by hypoxia on catecholamine and cAMP metabolism in the CB. An increase in EPO plasma levels elicited by hypoxia was identical in hyperoxic and control animals, implying a normal functioning of EPO-producing cells. The loss of HPV observed in adult rats and caused by perinatal hyperoxia, comparable to oxygen therapy in premature infants, might represent a previously unrecognized complication of such a medical intervention capable of aggravating medical conditions such as regional pneumonias, atelectases or general anaesthesia in adult life.
Assuntos
Hiperóxia/fisiopatologia , Hipóxia/fisiopatologia , Artéria Pulmonar/fisiopatologia , Animais , Antioxidantes/uso terapêutico , Corpo Carotídeo/fisiopatologia , Eritropoetina/sangue , Feminino , Hiperóxia/tratamento farmacológico , Gravidez , Ratos Wistar , VasoconstriçãoRESUMO
Quercetin is a dietary flavonoid which exerts vasodilator, antiplatelet and antiproliferative effects and reduces blood pressure, oxidative status and end-organ damage in humans and animal models of systemic hypertension. We hypothesized that oral quercetin treatment might be protective in a rat model of pulmonary arterial hypertension. Three weeks after injection of monocrotaline, quercetin (10 mg/kg/d per os) or vehicle was administered for 10 days to adult Wistar rats. Quercetin significantly reduced mortality. In surviving animals, quercetin decreased pulmonary arterial pressure, right ventricular hypertrophy and muscularization of small pulmonary arteries. Classic biomarkers of pulmonary arterial hypertension such as the downregulated expression of lung BMPR2, Kv1.5, Kv2.1, upregulated survivin, endothelial dysfunction and hyperresponsiveness to 5-HT were unaffected by quercetin. Quercetin significantly restored the decrease in Kv currents, the upregulation of 5-HT2A receptors and reduced the Akt and S6 phosphorylation. In vitro, quercetin induced pulmonary artery vasodilator effects, inhibited pulmonary artery smooth muscle cell proliferation and induced apoptosis. In conclusion, quercetin is partially protective in this rat model of PAH. It delayed mortality by lowering PAP, RVH and vascular remodeling. Quercetin exerted effective vasodilator effects in isolated PA, inhibited cell proliferation and induced apoptosis in PASMCs. These effects were associated with decreased 5-HT2A receptor expression and Akt and S6 phosphorylation and partially restored Kv currents. Therefore, quercetin could be useful in the treatment of PAH.
Assuntos
Hipertensão Pulmonar/tratamento farmacológico , Quercetina/uso terapêutico , Animais , Perfilação da Expressão Gênica , Hipertensão Pulmonar/genética , Hipertrofia Ventricular Direita/fisiopatologia , Masculino , Potenciais da Membrana , Ratos , Ratos Wistar , Análise de SobrevidaRESUMO
Insulin resistance and diabetes are current clinical concerns due to their increasing prevalence in western societies and in developing countries. Cardiovascular alterations, affecting both macro- and microcirculation, are among the major causes of illness and premature death within patients with insulin resistance or diabetes. However, the detrimental effects of insulin resistance and diabetes in the lungs are less clinically apparent, or at least masked by the progression of these metabolic diseases on other target organs. Epidemiological and experimental data suggest a link between pulmonary arterial hypertension and diabetes. Thereby, hemodynamic derangements in uncontrolled diabetes or insulin resistance are predisposing factors leading to early pulmonary alterations that in association with a second hit might accelerate the onset of pulmonary vascular disease and pulmonary hypertension. The present article reviewed the current knowledge about the effects of insulin resistance and diabetes in a territory which has received little attention until recently: the pulmonary circulation.
Assuntos
Diabetes Mellitus Tipo 1/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Hemodinâmica , Resistência à Insulina , Artéria Pulmonar/fisiopatologia , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/epidemiologia , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Hipertensão Pulmonar/epidemiologia , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/fisiopatologia , Canais de Potássio/genética , Canais de Potássio/metabolismo , Artéria Pulmonar/metabolismo , Testes de Função Respiratória , Resistência Vascular/fisiologiaRESUMO
AIMS: A variety of vessels, such as resistance pulmonary arteries (PA) and fetoplacental arteries and the ductus arteriosus (DA) are specialized in sensing and responding to changes in oxygen tension. Despite opposite stimuli, normoxic DA contraction and hypoxic fetoplacental and PA vasoconstriction share some mechanistic features. Activation of neutral sphingomyelinase (nSMase) and subsequent ceramide production has been involved in hypoxic pulmonary vasoconstriction (HPV). Herein we aimed to study the possible role of nSMase-derived ceramide as a common factor in the acute oxygen-sensing function of specialized vascular tissues. RESULTS: The nSMase inhibitor GW4869 and an anticeramide antibody reduced the hypoxic vasoconstriction in chicken PA and chorioallantoic arteries (CA) and the normoxic contraction of chicken DA. Incubation with interference RNA targeted to SMPD3 also inhibited HPV. Moreover, ceramide and reactive oxygen species production were increased by hypoxia in PA and by normoxia in DA. Either bacterial sphingomyelinase or ceramide mimicked the contractile responses of hypoxia in PA and CA and those of normoxia in the DA. Furthermore, ceramide inhibited voltage-gated potassium currents present in smooth muscle cells from PA and DA. Finally, the role of nSMase in acute oxygen sensing was also observed in human PA and DA. INNOVATION: These data provide evidence for the proposal that nSMase-derived ceramide is a critical player in acute oxygen-sensing in specialized vascular tissues. CONCLUSION: Our results indicate that an increase in ceramide generation is involved in the vasoconstrictor responses induced by two opposite stimuli, such as hypoxia (in PA and CA) and normoxia (in DA).
Assuntos
Vasos Sanguíneos/metabolismo , Ceramidas/metabolismo , Oxigênio/metabolismo , Compostos de Anilina/farmacologia , Animais , Compostos de Benzilideno/farmacologia , Vasos Sanguíneos/efeitos dos fármacos , Ceramidas/farmacologia , Embrião de Galinha , Canal Arterial/metabolismo , Expressão Gênica , Humanos , Hipóxia/metabolismo , Artéria Pulmonar/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Canais de Potássio Shaw/metabolismo , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Vasoconstrição/fisiologiaRESUMO
OBJECTIVES: Acute lung injury and acute respiratory distress syndrome are characterized by increased pulmonary artery pressure and ventilation-perfusion mismatch. We analyzed the changes in the pulmonary vascular function in a model of ventilator-induced acute lung injury. DESIGN: Controlled in vivo laboratory study. SETTING: Animal research laboratory. SUBJECTS: Anesthetized male Sprague-Dawley rats. INTERVENTIONS: Rats were ventilated for 120 minutes using low tidal volume ventilation (control group, tidal volume 9 mL/kg, positive end-expiratory pressure 5 cm H2O, n = 15), high tidal volume ventilation (high tidal volume group, tidal volume 25 mL/kg, zero positive end-expiratory pressure, n = 14), or high tidal volume ventilation plus the poly-(adenosine diphosphate-ribose) polymerase inhibitor 3-aminobenzamide (10 mg/kg IP, high tidal volume group + 3-aminobenzamide group, n = 7). Vascular rings from small pulmonary arteries were mounted in a myograph for isometric tension recording. Lung messenger RNA and protein expression were analyzed by reverse transcriptase-polymerase chain reaction and Western blot, respectively. MEASUREMENTS AND MAIN RESULTS: High tidal volume ventilation impaired phenylephrine- and acetylcholine-induced responses in pulmonary arteries in vitro, which were accompanied by induction of inducible nitric oxide synthase messenger RNA and protein. These effects, as well as hypoxemia and hypotension, were prevented by 3-aminobenzamide. Hypoxic pulmonary vasoconstriction and responses to exogenous sphingomyelinase were increased, whereas the responses to serotonin, Kv current density, and inhibition of Kv currents by hypoxia were unaffected by high tidal volume. CONCLUSIONS: High tidal volume ventilation-induced pulmonary vascular dysfunction was characterized by reduced alpha-adrenergic-induced vasoconstriction, reduced endothelium-dependent vasodilatation, and enhanced hypoxic pulmonary vasoconstriction.
Assuntos
Respiração com Pressão Positiva/efeitos adversos , Artéria Pulmonar/fisiopatologia , Volume de Ventilação Pulmonar/fisiologia , Lesão Pulmonar Induzida por Ventilação Mecânica/fisiopatologia , Animais , Benzamidas/farmacologia , Endotélio Vascular/fisiopatologia , Inibidores Enzimáticos/farmacologia , Hipotensão/fisiopatologia , Hipotensão/prevenção & controle , Hipóxia/fisiopatologia , Hipóxia/prevenção & controle , Masculino , Miografia , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases , Respiração com Pressão Positiva/métodos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologiaRESUMO
Although type 1 and type 2 diabetes are strongly associated with systemic cardiovascular morbidity, the relationship with pulmonary vascular disease had been almost disregarded until recent epidemiological data revealed that diabetes might be a risk factor for pulmonary hypertension. Recent experimental studies suggest that diabetes induces changes in lung function insufficient to elevate pulmonary pressure. The aim of this study was to assess the effects of diabetes on the sensitivity to other risk factors for pulmonary hypertension. We therefore analysed the effects of the combination of diabetes with exposure to moderate hypoxia on classical markers of pulmonary hypertension. Control (saline-treated) and diabetic (70 mg kg(-1) streptozotocin-treated) male Wistar-Kyoto rats were followed for 4 weeks and exposed to normoxia or moderate normobaric hypoxia (14%) for another 2 weeks. Hypoxia, but not diabetes, strongly reduced voltage-gated potassium currents, whereas diabetes, but not hypoxia, induced pulmonary artery endothelial dysfunction. Both factors independently induced pulmonary vascular remodelling and downregulated the lung bone morphogenetic protein receptor type 2. However, diabetes, but not hypoxia, induced pulmonary infiltration of macrophages, which was markedly increased when both factors were combined. Diabetes plus hypoxia induced a modest increase in diastolic and mean pulmonary artery pressure and right ventricular weight, while each of the two factors alone had no significant effect. The pattern of changes in markers of pulmonary hypertension was different for moderate hypoxia and diabetes, with no synergic effect except for macrophage recruitment, and the combination of both factors was required to induce a moderate elevation in pulmonary arterial pressure.
Assuntos
Diabetes Mellitus Tipo 1/complicações , Hipertensão Pulmonar/etiologia , Hipóxia/complicações , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Diabetes Mellitus Experimental/complicações , Regulação para Baixo , Hipertrofia Ventricular Direita/etiologia , Pulmão/patologia , Pulmão/fisiopatologia , Macrófagos/imunologia , Masculino , Artéria Pulmonar/patologia , Artéria Pulmonar/fisiopatologia , Ratos , Ratos Endogâmicos WKY , Doenças Vasculares/etiologiaRESUMO
The molecular mechanisms underlying hypoxic pulmonary vasoconstriction (HPV) are not yet properly understood. Mitochondrial electron transport chain (ETC) and NADPH oxidase have been proposed as possible oxygen sensors, with derived reactive oxygen species (ROS) playing key roles in coupling the sensor(s) to the contractile machinery. We have recently reported that activation of neutral sphingomyelinase (nSMase) and protein kinase C ζ (PKCζ) participate in the signalling cascade of HPV. Herein, we studied the significance of nSMase in controlling ROS production rate in rat pulmonary artery (PA) smooth muscle cells and thereby HPV in rat PA. ROS production (analyzed by dichlorofluorescein and dihydroethidium fluorescence) was increased by hypoxia in endothelium-denuded PA segments and their inhibition prevented hypoxia-induced voltage-gated potassium channel (K(V) ) inhibition and pulmonary vasoconstriction. Consistently, H(2) O(2) , or its analogue t-BHP, decreased K(V) currents and induced a contractile response, mimicking the effects of hypoxia. Inhibitors of mitochondrial ETC (rotenone) and NADPH oxidase (apocynin) prevented hypoxia-induced ROS production, K(V) channel inhibition and vasoconstriction. Hypoxia induced p47(phox) phosphorylation and its interaction with caveolin-1. Inhibition of nSMase (GW4869) or PKCζ prevented p47(phox) phosphorylation and ROS production. The increase in ceramide induced by hypoxia (analyzed by immunocytochemistry) was inhibited by rotenone. Exogenous ceramide increased ROS production in a PKCζ sensitive manner. We propose an integrated signalling pathway for HPV which includes nSMase-PKCζ-NADPH oxidase as a necessary step required for ROS production and vasoconstriction.
Assuntos
Hipóxia/metabolismo , Hipóxia/fisiopatologia , NADPH Oxidases/metabolismo , Artéria Pulmonar/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Vasoconstrição/fisiologia , Doença Aguda , Animais , Ceramidas/antagonistas & inibidores , Ceramidas/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Transporte de Elétrons/fisiologia , Hipóxia/enzimologia , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Fosforilação/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Proteína Quinase C/metabolismo , Artéria Pulmonar/enzimologia , Ratos , Ratos Wistar , Rotenona/farmacologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Desacopladores/farmacologia , Vasoconstrição/efeitos dos fármacosRESUMO
BACKGROUND: Insulin resistance and obesity are strongly associated with systemic cardiovascular diseases. Recent reports have also suggested a link between insulin resistance with pulmonary arterial hypertension. The aim of this study was to analyze pulmonary vascular function in the insulin resistant obese Zucker rat. METHODS: Large and small pulmonary arteries from obese Zucker rat and their lean counterparts were mounted for isometric tension recording. mRNA and protein expression was measured by RT-PCR or Western blot, respectively. KV currents were recorded in isolated pulmonary artery smooth muscle cells using the patch clamp technique. RESULTS: Right ventricular wall thickness was similar in obese and lean Zucker rats. Lung BMPR2, KV1.5 and 5-HT2A receptor mRNA and protein expression and KV current density were also similar in the two rat strains. In conductance and resistance pulmonary arteries, the similar relaxant responses to acetylcholine and nitroprusside and unchanged lung eNOS expression revealed a preserved endothelial function. However, in resistance (but not in conductance) pulmonary arteries from obese rats a reduced response to several vasoconstrictor agents (hypoxia, phenylephrine and 5-HT) was observed. The hyporesponsiveness to vasoconstrictors was reversed by L-NAME and prevented by the iNOS inhibitor 1400W. CONCLUSIONS: In contrast to rat models of type 1 diabetes or other mice models of insulin resistance, the obese Zucker rats did not show any of the characteristic features of pulmonary hypertension but rather a reduced vasoconstrictor response which could be prevented by inhibition of iNOS.
Assuntos
Hemodinâmica , Hipertensão Pulmonar/etiologia , Resistência à Insulina , Músculo Liso Vascular/fisiopatologia , Obesidade/complicações , Artéria Pulmonar/fisiopatologia , Animais , Western Blotting , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Hipertensão Pulmonar Primária Familiar , Hemodinâmica/efeitos dos fármacos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/fisiopatologia , Hipertrofia Ventricular Direita/etiologia , Hipertrofia Ventricular Direita/fisiopatologia , Resistência à Insulina/genética , Canal de Potássio Kv1.5/genética , Canal de Potássio Kv1.5/metabolismo , Masculino , Potenciais da Membrana , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Obesidade/genética , Obesidade/metabolismo , Obesidade/fisiopatologia , Técnicas de Patch-Clamp , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Zucker , Receptor 5-HT2A de Serotonina/genética , Receptor 5-HT2A de Serotonina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vasoconstritores/farmacologia , Vasodilatadores/farmacologiaRESUMO
Recent epidemiological data suggest that diabetes is a risk factor for pulmonary arterial hypertension. The aim of the present study was to analyze the link between type 1 diabetes and pulmonary arterial dysfunction in rats. Male Sprague-Dawley rats were randomly divided into a control group (saline) and a diabetic group (70 mg/kg streptozotocin). After 6 weeks, diabetic animals showed a down-regulation of the lung bone morphogenetic protein receptor type 2, up-regulation of 5-hydroxytryptamine (5-HT) 2A receptors and cyclooxygenase-2 (COX-2) proteins as measured by Western blot analysis, and increased contractile responses to 5-HT in isolated intrapulmonary arteries. The hyper-responsiveness to 5-HT was endothelium-independent and unaffected by inhibition of nitric-oxide synthase but prevented by indomethacin, the selective COX-2 inhibitor N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide (NS-398), superoxide dismutase, and the NADPH oxidase inhibitor apocynin or chronic treatment with insulin. However, diabetic rats at 6 weeks did not develop elevated right ventricular pressure or pulmonary artery muscularization, whereas a longer exposure (4 months) to diabetes induced a modest, but significant, increase in right ventricular systolic pressure. In conclusion, type 1 diabetes mellitus in rats induces a number of changes in lung protein expression and pulmonary vascular reactivity characteristic of clinical and experimental pulmonary arterial hypertension but insufficient to elevate pulmonary pressure. Our results further strengthen the link between diabetes and pulmonary arterial hypertension.
Assuntos
Ciclo-Oxigenase 2/biossíntese , Diabetes Mellitus Tipo 1/metabolismo , Endotélio Vascular/metabolismo , Estresse Oxidativo/fisiologia , Artéria Pulmonar/metabolismo , Serotonina/metabolismo , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Diabetes Mellitus Tipo 1/enzimologia , Endotélio Vascular/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/fisiologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Artéria Pulmonar/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Serotonina/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologiaRESUMO
Neutral sphingomyelinase (nSMase)-derived ceramide has been proposed as a mediator of hypoxic pulmonary vasoconstriction (HPV), a specific response of the pulmonary circulation. Voltage-gated K(+) (K(v)) channels are modulated by numerous vasoactive factors, including hypoxia, and their inhibition has been involved in HPV. Herein, we have analyzed the effects of ceramide on K(v) currents and contractility in rat pulmonary arteries (PA) and in mesenteric arteries (MA). The ceramide analog C6-ceramide inhibited K(v) currents in PA smooth muscle cells (PASMC). Similar effects were obtained after the addition of bacterial sphingomyelinase (SMase), indicating a role for endogenous ceramide in K(v) channel regulation. K(v) current was reduced by stromatoxin and diphenylphosphine oxide-1 (DPO-1), selective inhibitors of K(v)2.1 and K(v)1.5 channels, respectively. The inhibitory effect of ceramide was still present in the presence of stromatoxin or DPO-1, suggesting that this sphingolipid inhibited both components of the native K(v) current. Accordingly, ceramide inhibited K(v)1.5 and K(v)2.1 channels expressed in Ltk(-) cells. Ceramide-induced effects were reduced in human embryonic kidney 293 cells expressing K(v)1.5 channels but not the regulatory subunit K(v)ß2.1. The nSMase inhibitor GW4869 reduced the thromboxane-endoperoxide receptor agonist U46619-induced, but not endothelin-1-induced pulmonary vasoconstriction that was partly restored after addition of exogenous ceramide. The PKC-ζ pseudosubstrate inhibitor (PKCζ-PI) inhibited the K(v) inhibitory and contractile effects of ceramide. In MA ceramide had no effect on K(v) currents and GW4869 did not affect U46619-induced contraction. The effects of SMase were also observed in human PA. These results suggest that ceramide represents a crucial signaling mediator in the pulmonary vasculature.