RESUMO
The major ghrelin forms, acylated ghrelin and des-acylated ghrelin, are novel gastrointestinal hormones. Moreover, emerging evidence indicates that these peptides may have other functions including neuro- and vaso-protection. Here, we investigated whether post-stroke treatment with acylated ghrelin or des-acylated ghrelin could improve functional and histological endpoints of stroke outcome in mice after transient middle cerebral artery occlusion (tMCAo). We found that des-acylated ghrelin (1 mg/kg) improved neurological and functional performance, reduced infarct and swelling, and decreased apoptosis. In addition, it reduced blood-brain barrier (BBB) disruption in vivo and attenuated the hyper-permeability of mouse cerebral microvascular endothelial cells after oxygen glucose deprivation and reoxygenation (OGD + RO). By contrast, acylated ghrelin (1 mg/kg or 5 mg/kg) had no significant effect on these endpoints of stroke outcome. Next we found that des-acylated ghrelin's vasoprotective actions were associated with increased expression of tight junction proteins (occludin and claudin-5), and decreased cell death. Moreover, it attenuated superoxide production, Nox activity and expression of 3-nitrotyrosine. Collectively, these results demonstrate that post-stroke treatment with des-acylated ghrelin, but not acylated ghrelin, protects against ischaemia/reperfusion-induced brain injury and swelling, and BBB disruption, by reducing oxidative and/or nitrosative damage.
Assuntos
Barreira Hematoencefálica/metabolismo , Lesões Encefálicas/metabolismo , Grelina/metabolismo , Substâncias Protetoras/metabolismo , Acidente Vascular Cerebral/complicações , Acilação , Animais , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/etiologia , Células Endoteliais/metabolismo , Grelina/administração & dosagem , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxigênio/metabolismo , Substâncias Protetoras/administração & dosagemRESUMO
Available inotropic pharmacotherapy for acute heart failure (HF) remains largely ineffective at ameliorating marked impairments in contractile function. Nitroxyl (HNO), the redox sibling of NOâ¢, has recently attracted interest as a therapeutic approach for acute HF. We now compare the impact of ischaemia-reperfusion (I-R) injury on acute haemodynamic responsiveness of the HNO donor, Angeli's salt (AS), to that of NO and dobutamine. Dose-response curves to bolus doses of AS, diethylamine NONOate (DEA/NO, both 0.001-µmol) and dobutamine (0.1-100 nmol) were performed in rat isolated hearts, following I-R or normoxic perfusion. An additional 10µmol dose of Angeli's salt was included, to permit roughly equivalent inotropic responses to dobutamine. Changes in cardiac contraction, heart rate and coronary flow (CF) were determined. Although AS and DEA/NO elicited comparable dose-dependent increases in CF in normoxic hearts, only AS vasodilation was preserved after I-R. AS and dobutamine elicited dose-dependent inotropic responses in normoxic hearts and I-R blunted inotropic responses to both. Dobutamine however increased heart rate, which was exacerbated by I-R; this was not evident with AS. Further, AS infusion during reperfusion (1µM), in a separate cohort of rat hearts, improved recovery of cardiac contractility, with lower incidence of I-R-induced ventricular fibrillation. In conclusion, these observations suggest that HNO offers haemodynamic advantages over NO following I-R. Although I-R suppresses inotropy to both agents, residual contractile responses to AS following I-R is likely free of concomitant pro-arrhythmic events. HNO donors may thus offer haemodynamic advantages over existing pharmacotherapy in acute HF.
Assuntos
Cardiotônicos/farmacologia , Coração/efeitos dos fármacos , Nitritos/farmacologia , Óxidos de Nitrogênio , Traumatismo por Reperfusão/fisiopatologia , Animais , Dobutamina/farmacologia , Coração/fisiopatologia , Hemodinâmica , Masculino , Contração Miocárdica , Óxido Nítrico/fisiologia , Doadores de Óxido Nítrico/farmacologia , Ratos Sprague-DawleyRESUMO
Studies have utilised immortalised mouse cerebral endothelial cells (bEnd.3) exposed to oxygen glucose deprivation (OGD) to study blood-brain barrier (BBB) disruption after ischaemia. However, there is a paucity of literature describing the duration of OGD (and reoxygenation [RO]) required to best simulate BBB disruption in vivo. In this study we assessed BBB disruption in bEnd.3 cells after exposure to a range of OGD periods, and also after OGD + RO. Exposure of bEnd.3 monolayers to 4, 6, 16, or 24 hours of OGD resulted in a significant increase in permeability. The hyperpermeability after 16 or 24 hours was associated with decreased expression of tight junction proteins (occludin and claudin-5). Furthermore, there was a decrease in cell viability and increased expression of the pro-apoptotic protein, cleaved caspase-3. Exposure of bEnd.3 monolayers to 1 hour OGD+ 23 hours RO exacerbated hyperpermeability relative to 1 hour OGD, which was associated with decreased expression levels of occludin and ZO-1, but no change in cell viability or caspase-3. 4 hours OGD + 23 hours RO exacerbated hyperpermeability, decreased expression levels of tight junction proteins, decreased cell viability, and increased caspase-3 expression. Thus, bEnd.3 cells exhibit hyperpermeability, a loss of tight junction proteins, and undergo cell death, after exposure to prolonged periods of OGD. Moreover, they exhibit exacerbated hyperpermeability, a loss of tight junction proteins, and increased expression of caspase-3 after OGD + RO. These findings will facilitate the use of this cell line in studies of BBB disruption and for the testing of therapeutics.
Assuntos
Permeabilidade Capilar/fisiologia , Circulação Cerebrovascular/fisiologia , Células Endoteliais/metabolismo , Glucose/deficiência , Microvasos/metabolismo , Oxigênio/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Hipóxia Celular/fisiologia , Linhagem Celular Transformada , Sobrevivência Celular/fisiologia , CamundongosRESUMO
In acute myocardial infarction (AMI), the first line of treatment is to rapidly restore blood flow to the ischemic myocardium to limit infarct size. It is now well established that though clearly beneficial, the positive outcomes of this intervention are limited by injury in response to the reperfusion itself in addition to the prior ischemia. This process is described as reperfusion injury and is considered to contribute to the arrhythmias, microvascular dysfunction and impaired cardiac contractility that is observed even after the restoration of coronary blood flow. Thus an important, currently unmet, therapeutic challenge is to address the outcomes of this reperfusion injury. In this article, we review the evidence that flavonols and flavones may prove useful in preserving cardiac function after ischemia and reperfusion and consider the possible mechanisms, in particular, the inhibition of kinases, by which they may exert protection.
Assuntos
Cardiotônicos/química , Cardiotônicos/uso terapêutico , Flavonas/uso terapêutico , Flavonóis/uso terapêutico , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Proteínas Quinases/metabolismo , Animais , Cálcio/metabolismo , Flavonas/química , Flavonóis/química , Humanos , Traumatismo por Reperfusão Miocárdica/patologia , Proteínas Quinases/química , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Flavonols, including 3',4'-dihydroxyflavonol (DiOHF), reduce myocardial ischemia and reperfusion (I/R) injury but their mechanism remains uncertain. To better understand the mechanism of the cardioprotective actions of flavonols we investigated the effect of DiOHF on cardiac function and the activation of protective and injurious signalling kinases after I/R in rat isolated hearts. METHODS: We assessed the effect of global ischemia (20min) and reperfusion (5-30min) on cardiac function and injury in rat isolated, perfused hearts in the absence or presence of DiOHF (10µM) during reperfusion. Western blotting was used to assess changes in the phosphorylation state of kinases known to be involved in injury or protection. RESULTS: DiOHF improved cardiac contractility and reduced perfusion pressure and cell death in the isolated hearts. Phosphorylation of p38MAPK and CaMKII increased during ischemia with no further increase during reperfusion. Phosphorylation of other kinases increased during reperfusion. Phosphorylation of phospholamban (PLN) peaked at 5min of reperfusion whereas phosphorylation of Akt, Erk, STAT3 and JNK2 was highest after 30min. The presence of DiOHF during reperfusion significantly inhibited the activation of PLN and JNK without affecting phosphorylation of the protective kinases Erk1/2 and STAT3. Experiments in vitro demonstrated that DiOHF inhibited CaMKII by competing with ATP but not Ca2+/calmodulin. CONCLUSIONS: It is proposed that DiOHF confers protection against myocardial reperfusion injury by inhibiting CaMKII and subsequent PLN-induced leak of Ca2+ from the sarcoplasmic reticulum as well as by inhibiting JNK2 activation to reduce apoptosis.
Assuntos
Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Cardiotônicos/uso terapêutico , Flavonóis/uso terapêutico , Preparação de Coração Isolado , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Cardiotônicos/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Flavonóis/farmacologia , Preparação de Coração Isolado/métodos , Masculino , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Early reperfusion of the blocked vessel is critical to restore the blood flow to the ischemic myocardium to salvage myocardial tissue and improve clinical outcome. This reperfusion strategy after a period of ischemia, however, may elicit further myocardial damage named myocardial reperfusion injury. The manifestations of reperfusion injury include arrhythmias, myocardial stunning and micro-vascular dysfunction, in addition to significant cardiomyocyte death. It is suggested that an overproduction of reactive oxygen species, intracellular calcium overload and inflammatory cell infiltration are the most important features of myocardial ischemia-reperfusion injury. OBJECTIVE: In this review, various pharmacological interventions to treat myocardial reperfusion injury including the antioxidant flavonols, hydrogen sulfide, adenosine, opioids, incretin-based therapies and cyclosporin A which targets the mitochondrial permeability transition pore are discussed. CONCLUSION: The processes involved in reperfusion injury might provide targets for improved outcomes after myocardial infarction but thus far that aim has not been met in the clinic.
Assuntos
Cardiotônicos/uso terapêutico , Isquemia Miocárdica/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Cálcio/metabolismo , Cardiotônicos/farmacologia , Ciclosporina/farmacologia , Ciclosporina/uso terapêutico , Humanos , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial , Isquemia Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND AND PURPOSE: The NO redox sibling nitroxyl (HNO) elicits soluble guanylyl cyclase (sGC)-dependent vasodilatation. HNO has high reactivity with thiols, which is attributed with HNO-enhanced left ventricular (LV) function. Here, we tested the hypothesis that the concomitant vasodilatation and inotropic actions induced by a HNO donor, Angeli's salt (sodium trioxodinitrate), were sGC-dependent and sGC-independent respectively. EXPERIMENTAL APPROACH: Haemodynamic responses to Angeli's salt (10 pmol-10 µmol), alone and in the presence of scavengers of HNO (L-cysteine, 4 mM) or of NO [hydroxocobalamin (HXC), 100 µM] or a selective inhibitor of sGC [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 10 µM], a CGRP receptor antagonist (CGRP8-37 , 0.1 µM) or a blocker of voltage-dependent potassium channels [4-aminopyridine (4-AP), 1 mM] were determined in isolated hearts from male rats. KEY RESULTS: Angeli's salt elicited concomitant, dose-dependent increases in coronary flow and LV systolic and diastolic function. Both L-cysteine and ODQ shifted (but did not abolish) the dose-response curve of each of these effects to the right, implying contributions from HNO and sGC in both the vasodilator and inotropic actions. In contrast, neither HXC, CGRP8-37 nor 4-AP affected these actions. CONCLUSIONS AND IMPLICATIONS: Both vasodilator and inotropic actions of the HNO donor Angeli's salt were mediated in part by sGC-dependent mechanisms, representing the first evidence that sGC contributes to the inotropic and lusitropic action of HNO in the intact heart. Thus, HNO acutely enhances LV contraction and relaxation, while concomitantly unloading the heart, potentially beneficial actions in failing hearts.