RESUMO
Fibrotic processes in the liver of non-alcoholic steatohepatitis (NASH) patients cause microcirculatory dysfunction in the organ which increases blood vessel resistance and causes portal hypertension. Assessing blood vessel function in the liver is challenging, necessitating the development of novel methods in normal and fibrotic tissue that allow for drug screening and translation toward pre-clinical settings. Cultures of precision cut liver slices (PCLS) from normal and fibrotic rat livers were used for blood vessel function analysis. Live recording of vessel diameter was used to assess the response to endothelin-1, serotonin and soluble guanylate cyclase (sGC) activation. A cascade of contraction and relaxation events in response to serotonin, endothelin-1, Ketanserin and sGC activity could be established using vessel diameter analysis of rat PCLS. Both the sGC activator BI 703704 and the sGC stimulator Riociguat prevented serotonin-induced contraction in PCLS from naive rats. By contrast, PCLS cultures from the rat CCl4 NASH model were only responsive to the sGC activator, thus establishing that the sGC enzyme is rendered non-responsive to nitric oxide under oxidative stress found in fibrotic livers. The role of the sGC pathway for vessel relaxation of fibrotic liver tissue was identified in our model. The obtained data shows that the inhibitory capacities on vessel contraction of sGC compounds can be translated to published preclinical data. Altogether, this novel ex vivo PCLS method allows for the differentiation of drug candidates and the translation of therapeutic approaches towards the clinical use.
Assuntos
Cirrose Hepática/fisiopatologia , Fígado/irrigação sanguínea , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Guanilil Ciclase Solúvel/fisiologia , Vasoconstrição , Trifosfato de Adenosina/metabolismo , Animais , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/fisiologia , Tetracloreto de Carbono , Endotelina-1/farmacologia , Ketanserina/farmacologia , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Estresse Oxidativo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Ratos Sprague-Dawley , Ratos Wistar , Serotonina/farmacologia , Transdução de Sinais , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacosRESUMO
Cigarette smoking induces vascular endothelial dysfunction characterized by impaired nitric oxide (NO) bioavailability. There are two types of soluble guanylate cyclase (sGC), which is a cellular target of NO: NO-sensitive reduced form (the heme moiety with a ferrous iron) and NO-insensitive oxidized (the heme moiety with a ferric iron)/heme-free form. This study investigated the influence of cigarette smoking on NO-sensitive and NO-insensitive sGC-mediated vascular tone regulation in organ chamber experiments with isolated rat and human arteries. The rats were subcutaneously administered phosphate-buffered saline (PBS), nicotine-free cigarette smoke extract (N(-)-CSE) or nicotine-containing cigarette smoke extract (N(+)-CSE) for 4 weeks. Plasma thiobarbituric acid reactive substance (TBARS) levels were higher in the N(+)-CSE group than those in the N(-)-CSE group, and TBARS levels for these groups were higher than those for the PBS group. In the aorta and the pulmonary artery in rats administered N(-)-CSE or N(+)-CSE, acetylcholine-induced relaxation was significantly impaired compared with that in rats administered PBS; there was no significant difference in the relaxation between the N(-)-CSE and N(+)-CSE groups. However, sodium nitroprusside (NO-sensitive sGC stimulant)- and BAY 60-2770 (NO-insensitive sGC stimulant)-induced relaxations were not different among the three groups, regardless of the vessel type. In addition, in the human gastroepiploic artery, the relaxant responses to these sGC-targeting drugs were identical between nonsmokers and smokers. These findings suggest that NO-sensitive and NO-insensitive sGC-mediated vascular tone regulation functions normally even in blood vessels damaged by cigarette smoking.
Assuntos
Fumar Cigarros/fisiopatologia , Artéria Gastroepiploica/fisiopatologia , Óxido Nítrico/fisiologia , Artéria Pulmonar/fisiopatologia , Guanilil Ciclase Solúvel/fisiologia , Vasodilatação/efeitos dos fármacos , Acetilcolina/farmacologia , Adulto , Animais , Aorta/efeitos dos fármacos , Feminino , Artéria Gastroepiploica/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Artéria Pulmonar/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/sangue , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Vasodilatação/fisiologia , Adulto JovemRESUMO
H2S causes vasorelaxation however there is considerable heterogeneity in the reported pharmacological mechanism of this effect. This study examines the contribution of endogenously released H2S in the regulation of vascular tone and the mechanism of H2S-induced vasorelaxation in small resistance-like arteries. Mesenteric arteries from C57 and eNOS-/- mice were mounted in myographs to record isometric force. Vasorelaxation responses to NaHS were examined in the presence of various inhibitors of vasorelaxation pathways. Expression and activity of the H2S-producing enzyme, cystathionine-γ-lyase (CSE), were also examined. CSE was expressed in vascular smooth muscle and perivascular adipose cells from mouse mesenteric artery. The substrate for CSE, L-cysteine, caused a modest vasorelaxation (35%) in arteries from C57 mice and poor vasorelaxation (10%) in arteries from eNOS-/- mice that was sensitive to the CSE inhibitor DL-propargylglycine. The fast H2S donor, NaHS, elicited a full and biphasic vasorelaxation response in mesenteric arteries (EC50 (1) 8.7 µM, EC50 (2) 0.6 mM), which was significantly inhibited in eNOS-/- vessels (P < 0.05), unaffected by endothelial removal, or blockers at any point in the NO via soluble guanylate cyclase and cGMP (NO-sGC-cGMP) vasorelaxation pathway. Vasorelaxation to NaHS was significantly inhibited by blocking K+ channels of the KCa and KV subtypes and the Cl-/HCO3- exchanger (P < 0.05). Further experiments showed that NaHS can significantly inhibit voltage-gated Ca2+ channel function (P < 0.05). The vasorelaxant effect of H2S in small resistance-like arteries is complex, involving eNOS, K+ channels, Cl-/HCO3- exchanger, and voltage-gated Ca2+ channels. CSE is present in the smooth muscle and periadventitial adipose tissue of these resistance-like vessels and can be activated to cause modest vasorelaxation under these in vitro conditions.
Assuntos
Sulfeto de Hidrogênio , Artérias Mesentéricas/fisiologia , Vasodilatação , Animais , Canais de Cálcio/fisiologia , Cloretos/fisiologia , GMP Cíclico/fisiologia , Cistationina gama-Liase/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/farmacologia , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase Tipo III/genética , Canais de Potássio/fisiologia , Guanilil Ciclase Solúvel/fisiologia , Vasodilatação/efeitos dos fármacosRESUMO
Background Nitric oxide (NO) has been heavily implicated in migraine. Nitroglycerin is a prototypic NO-donor, and triggers migraine in humans. However, nitroglycerin also induces oxidative/nitrosative stress and is a source of peroxynitrite - factors previously linked with migraine etiology. Soluble guanylyl cyclase (sGC) is the high affinity NO receptor in the body, and the aim of this study was to identify the precise role of sGC in acute and chronic migraine. Methods We developed a novel brain-bioavailable sGC stimulator (VL-102), and tested its hyperalgesic properties in mice. We also determined the effect of VL-102 on c-fos and calcitonin gene related peptide (CGRP) immunoreactivity within the trigeminovascular complex. In addition, we also tested the known sGC inhibitor, ODQ, within the chronic nitroglycerin migraine model. Results VL-102-evoked acute and chronic mechanical cephalic and hind-paw allodynia in a dose-dependent manner, which was blocked by the migraine medications sumatriptan, propranolol, and topiramate. In addition, VL-102 also increased c-fos and CGRP expressing cells within the trigeminovascular complex. Importantly, ODQ completely inhibited acute and chronic hyperalgesia induced by nitroglycerin. ODQ also blocked hyperalgesia already established by chronic nitroglycerin, implicating this pathway in migraine chronicity. Conclusions These results indicate that nitroglycerin causes migraine-related pain through stimulation of the sGC pathway, and that super-activation of this receptor may be an important component for the maintenance of chronic migraine. This work opens the possibility for negative sGC modulators as novel migraine therapies.
Assuntos
Hiperalgesia/induzido quimicamente , Hiperalgesia/enzimologia , Transtornos de Enxaqueca/enzimologia , Guanilil Ciclase Solúvel/fisiologia , Antagonistas Adrenérgicos beta/administração & dosagem , Antagonistas Adrenérgicos beta/uso terapêutico , Regulação Alostérica , Animais , Anticonvulsivantes/administração & dosagem , Anticonvulsivantes/uso terapêutico , Peptídeo Relacionado com Gene de Calcitonina/biossíntese , Modelos Animais de Doenças , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/uso terapêutico , Feminino , Hiperalgesia/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transtornos de Enxaqueca/induzido quimicamente , Transtornos de Enxaqueca/tratamento farmacológico , Transtornos de Enxaqueca/etiologia , Terapia de Alvo Molecular , Óxido Nítrico/efeitos adversos , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Nitroglicerina/farmacologia , Oxidiazóis/administração & dosagem , Oxidiazóis/metabolismo , Oxidiazóis/uso terapêutico , Propranolol/administração & dosagem , Propranolol/uso terapêutico , Proteínas Proto-Oncogênicas c-fos/biossíntese , Quinoxalinas/administração & dosagem , Quinoxalinas/metabolismo , Quinoxalinas/uso terapêutico , Agonistas do Receptor 5-HT1 de Serotonina/administração & dosagem , Agonistas do Receptor 5-HT1 de Serotonina/uso terapêutico , Guanilil Ciclase Solúvel/metabolismo , Sumatriptana/administração & dosagem , Sumatriptana/uso terapêutico , Topiramato/administração & dosagem , Topiramato/uso terapêuticoRESUMO
Biomarkers are increasingly being investigated in the treatment of pulmonary vascular disease. In particular, the signaling pathways targeted by therapies for pulmonary arterial hypertension provide biomarkers that potentially can be used to guide therapy and to assess clinical response as an alternative to invasive procedures such as right-sided cardiac catheterization. Moreover, the growing use of combination therapy for both the initial and subsequent treatment of pulmonary arterial hypertension highlights the need for biomarkers in this treatment approach. Currently approved therapies for pulmonary arterial hypertension target 3 major signaling pathways: the nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate pathway, the endothelin pathway, and the prostacyclin pathway. Although the main biomarker used in practice and evaluated in clinical trials is N-terminal pro-brain natriuretic peptide, other putative biomarkers include the endogenous nitric oxide (NO) synthase inhibitor asymmetric dimethylarginine, NO metabolites including S-nitrosothiols and nitrite, exhaled NO, endothelins, cyclic guanosine monophosphate, cyclic adenosine monophosphate, and atrial natriuretic peptide. This review describes accessible biomarkers, related to the actual molecules targeted by current therapies, for measuring and predicting response to the individual pulmonary arterial hypertension treatment classes as well as combination therapy.
Assuntos
Hipertensão Pulmonar/metabolismo , Óxido Nítrico/fisiologia , Biomarcadores/metabolismo , GMP Cíclico/fisiologia , Humanos , Hipertensão Pulmonar/fisiopatologia , Hipertensão Pulmonar/terapia , Peptídeos Natriuréticos/metabolismo , Avaliação de Resultados em Cuidados de Saúde , Transdução de Sinais , Guanilil Ciclase Solúvel/fisiologiaRESUMO
Head and neck squamous cell carcinoma (HNSCC) is an aggressive and often fatal disease. Cisplatin is the most common chemotherapeutic drug in the treatment of HNSCC, but intrinsic and acquired resistance are frequent, and severe side effects occur at high doses. The second messenger cyclic GMP (cGMP) is produced by soluble guanylate cyclase (sGC). We previously reported that activation of the cGMP signaling cascade caused apoptosis in HNSCC cells, while others found that this pathway enhances cisplatin efficacy in some cell types. Here we found that sGC stimulators reduced HNSCC cell viability synergistically with cisplatin, and enhanced apoptosis by cisplatin. Moreover, the sGC stimulators effectively reduced viability in cells with acquired cisplatin resistance, and were synergistic with cisplatin. The sGC stimulator BAY 41-2272 reduced expression of the survival proteins EGFR and ß-catenin, and increased pro-apoptotic Bax, suggesting a potential mechanism for the anti-tumorigenic effects of these drugs. The sGC stimulator Riociguat is FDA-approved to treat pulmonary hypertension, and others are being studied for therapeutic use in several diseases. These drugs could provide valuable addition or alternative to cisplatin in the treatment of HNSCC.