RESUMO
Purpose: We comprehensively evaluated the mutational spectrum of Leber congenital amaurosis (LCA) and investigated the molecular diagnostic rate and genotype-phenotype correlation in a Korean cohort. Methods: This single-center retrospective case series included 50 Korean patients with LCA between June 2015 and March 2019. Molecular analysis was conducted using targeted panel-based next-generation sequencing, including deep intronic and regulatory variants or whole exome sequencing. The molecular diagnosis was made based on the inheritance pattern, zygosity, and pathogenicity. Results: Among the 50 patients, 27 patients (54%) were male, and 11 (22%) showed systemic features. Genetic variants highly likely to be causative were identified in 78% (39/50) of cases and segregated into families. We detected two pathogenic or likely pathogenic variants in a gene linked to a recessive trait without segregation analysis in three cases (6.0%). GUCY2D (20%), NMNAT1 (18%), and CEP290 (16%) were the most frequently mutated genes in Korean LCA. Copy number variations were found in three patients, which accounted for 6% of LCA cases. A possible dual molecular diagnosis (Senior-Løken syndrome along with Leigh syndrome, and Joubert syndrome with transposition of the great arteries) was made in two patients (4%). Three of 50 patients were medically or surgically actionable: one patient for RPE65 gene therapy and two patients with WDR19 Senior-Løken syndrome for early preparation for kidney and liver transplantations. Conclusions: This study demonstrated that approximately 4% of patients may have dual molecular diagnoses, and 6% were surgically or medically actionable in LCA. Therefore, accurate molecular diagnosis and careful interpretation of next-generation sequencing results can be of great help in patients with LCA.
Assuntos
Anormalidades Múltiplas/genética , Cerebelo/anormalidades , Ciliopatias/genética , Variações do Número de Cópias de DNA/genética , Anormalidades do Olho/genética , Doenças Renais Císticas/genética , Amaurose Congênita de Leber/diagnóstico , Amaurose Congênita de Leber/genética , Doença de Leigh/genética , Atrofias Ópticas Hereditárias/genética , Retina/anormalidades , Anormalidades Múltiplas/diagnóstico , Adolescente , Adulto , Antígenos de Neoplasias/sangue , Antígenos de Neoplasias/genética , Proteínas de Ciclo Celular/sangue , Proteínas de Ciclo Celular/genética , Criança , Pré-Escolar , Ciliopatias/diagnóstico , Proteínas do Citoesqueleto/sangue , Proteínas do Citoesqueleto/genética , Anormalidades do Olho/diagnóstico , Feminino , Estudos de Associação Genética , Terapia Genética , Guanilato Ciclase/sangue , Guanilato Ciclase/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Peptídeos e Proteínas de Sinalização Intracelular/genética , Doenças Renais Císticas/diagnóstico , Amaurose Congênita de Leber/diagnóstico por imagem , Amaurose Congênita de Leber/terapia , Doença de Leigh/diagnóstico , Masculino , Mutação , Nicotinamida-Nucleotídeo Adenililtransferase/sangue , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Atrofias Ópticas Hereditárias/diagnóstico , Transplante de Órgãos , Linhagem , Receptores de Superfície Celular/sangue , Receptores de Superfície Celular/genética , República da Coreia , Estudos Retrospectivos , Transposição dos Grandes Vasos/genética , cis-trans-Isomerases/genéticaRESUMO
Insulin resistance (IR) is found in chronic hepatitis C (CHC) more frequently than in other chronic liver diseases.Prospective cross-sectional study to evaluate a wide multitest panel to identify factors related with IR in CHC and their possible interactions.In 76 patients with CHC we performed a series of routine laboratory analysis as well as specifically designed serum biochemical tests [retinol, retinol-binding protein 4 (RBP4), 25-OH vitamin D, Vitamin E, lipopolysaccharide-binding protein (LBP), interleukin-6 (IL-6), and cystatin C]. The single nucleotide polymorphisms rs7041 and rs4588 GC-DBP (group-specific component-Vitamin D-binding protein), rs738409 PNPLA3 (patatin-like phospholipase domain containing 3), and rs12979860 IL28B (interleukin-28 B) genes were determined. Insulin sensitivity was established with the HOMA-IR and IR was diagnosed when HOMA-IRâ>â3. Fibrosis staging was assessed with liver biopsy or transient elastography.After backward logistic regression analysis, independent variables associated with IR were Gc1s/Gc1s DBP phenotype, that results from the homozygous carriage of the rs7041G/rs4588C haplotype (Pâ=â0.033); low retinol/RBP4 ratio, reflecting a greater rate of unbound RBP4 (Pâ=â0.005); older age (Pâ=â0.01); high serum tryglicerides (Pâ=â0.026); and advanced (F3-F4) fibrosis stage. The AUROC provided by the multivariate model was 0.950 (95% CIâ=â0.906-0.993).In addition to previously known ones, the Gc1s/Gc1s phenotype variant of DBP and the unbound fraction of plasma RBP4 may be considered as factors related with the incidence, and possibly the risk, of IR in CHC patients.
Assuntos
DNA/genética , Guanilato Ciclase/genética , Hepatite C Crônica/genética , Resistência à Insulina/genética , Polimorfismo Genético , Proteínas Plasmáticas de Ligação ao Retinol/genética , Biomarcadores/sangue , Biópsia , Estudos Transversais , Técnicas de Imagem por Elasticidade , Feminino , Genótipo , Guanilato Ciclase/sangue , Hepatite C Crônica/sangue , Hepatite C Crônica/diagnóstico , Humanos , Fígado/diagnóstico por imagem , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Técnicas de Amplificação de Ácido Nucleico , Estudos Prospectivos , Proteínas Plasmáticas de Ligação ao Retinol/metabolismo , Fatores de RiscoRESUMO
The nitric oxide (NO)/soluble guanylate cyclase (sGC) system is fundamental to endothelial control of vascular tone, but also plays a major role in the negative modulation of platelet aggregation. The phenomenon of platelet NO resistance, or decreased antiaggregatory response to NO, occurs increasingly with advanced age, as well as in the context of cardiovascular disease states such as heart failure, ischemic heart disease, and aortic valve disease. The central causes of NO resistance are "scavenging" of NO and dysfunction of sGC. In the current review, we discuss the roles of several modulators of NO synthesis and of the NO/sGC cascade on changes in platelet physiology with aging, together with potential therapeutic options to reduce associated thrombotic risk.
Assuntos
Plaquetas/metabolismo , Óxido Nítrico/sangue , Fatores Etários , Animais , Plaquetas/citologia , Guanilato Ciclase/sangue , Humanos , Agregação Plaquetária , Receptores Citoplasmáticos e Nucleares/sangue , Transdução de Sinais , Guanilil Ciclase SolúvelRESUMO
BACKGROUND: Impaired tissue responsiveness to nitric oxide (NO) occurs in many cardiovascular diseases as well as with advanced age and is a correlate of poor outcomes. This phenomenon results from oxidative stress, with NO "scavenging" and dysfunction of soluble guanylate cyclase (sGC). Thioredoxin-interacting protein (TXNIP) is a major intracellular regulator of inflammatory activation and redox stress, but its interactions with NO/sGC are poorly understood. We have now evaluated the relationship between platelet TXNIP expression and function of the NO/sGC axis in subjects of varying age and during therapy with ramipril. METHODS & RESULTS: Young (n=42) and aging (n=49) subjects underwent evaluation of platelet TXNIP content. Aging subjects additionally had measurements of platelet NO responsiveness and routine biochemistry. Platelet TXNIP content was greater (376±33 units) in the aging compared to younger subjects (289±13 units; p<0.05). In the aging subjects there was a significant negative correlation (r=-0.50, p<0.001) between platelet TXNIP content and NO responsiveness. In a separate cohort of 15 subjects two week treatment with ramipril, which reversed platelet NO resistance and potentiated sGC activity, also decreased platelet TXNIP content by 40% (p=0.011). CONCLUSIONS: Platelet TXNIP content increases with aging, varies inversely with responsiveness to NO, and diminishes rapidly following treatment with ramipril. These data suggest that TXNIP-induced oxidative stress may be a critical modulator of tissue resistance to NO, a fundamental basis for cardiovascular disease. Analogously suppression of TXNIP expression can potentially be utilized as an index of restoration of cardiovascular homeostasis.
Assuntos
Envelhecimento/sangue , Plaquetas/metabolismo , Proteínas de Transporte/biossíntese , Isquemia Miocárdica/sangue , Óxido Nítrico/sangue , Ramipril/uso terapêutico , Adulto , Idoso , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Plaquetas/efeitos dos fármacos , Western Blotting , Proteínas de Transporte/sangue , Proteínas de Transporte/efeitos dos fármacos , Feminino , Seguimentos , Guanilato Ciclase/sangue , Guanilato Ciclase/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Isquemia Miocárdica/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Adulto JovemRESUMO
Metabolic syndrome (MetS) is a combination of clinical disorders that together increase the risk for cardiovascular disease and diabetes. SHRSP.Z-Lepr(fa)/IzmDmcr (SHRSP.ZF) rats with MetS show impaired nitric oxide-mediated relaxation in coronary and mesenteric arteries, and angiotensin II receptor type 1 blockers protect against dysfunction and oxidative-nitrosative stress independently of metabolic effects. We hypothesize that superoxide contributes to functional deterioration in SHRSP.ZF rats. To test our hypothesis, we studied effects of treatment with tempol, a membrane-permeable radical scavenger, on impaired vasodilation in SHRSP.ZF rats. Tempol did not alter body weight, high blood pressure, or metabolic abnormalities, but prevented impairment of acetylcholine-induced and nitroprusside-induced vasodilation in the coronary and mesenteric arteries. Furthermore, tempol reduced the levels of serum thiobarbituric acid reactive substance (TBARS) and 3-nitrotyrosine content in mesenteric arteries. Systemic administration of tempol elevated the expression of soluble guanylate cyclase (sGC) above basal levels in mesenteric arteries of SHRSP.ZF rats. However, acute treatment with tempol or ebselen, a peroxynitrite scavenger, did not ameliorate impaired relaxation of isolated mesenteric arteries. No nitration of tyrosine residues in sGC was observed; however, sGC mRNA expression levels in the arteries of SHRSP.ZF rats were lower than those in the arteries of Wistar-Kyoto rats. Levels of Thr(496)- and Ser(1177)-phosphorylated endothelial nitric oxide synthase (eNOS) were lower in arteries of SHRSP.ZF rats, and acetylcholine decreased Thr(496)-phosphorylated eNOS levels. These results indicated that prolonged superoxide production, leading to oxidative-nitrosative stress, was associated with impaired vasodilation in SHRSP.ZF rats with MetS. Down-regulated sGC expression may be linked to dysfunction, while reduced NO bioavailability/eNOS activity and modified sGC activity due to superoxide production were excluded as pivotal mechanisms.
Assuntos
Vasos Coronários/patologia , Síndrome Metabólica/fisiopatologia , Nitrogênio/química , Estresse Oxidativo , Vasodilatação/efeitos dos fármacos , Animais , Azóis/farmacologia , Óxidos N-Cíclicos/farmacologia , Modelos Animais de Doenças , Regulação para Baixo , Sequestradores de Radicais Livres/farmacologia , Guanilato Ciclase/sangue , Coração/fisiopatologia , Isoindóis , Masculino , Óxido Nítrico/química , Óxido Nítrico Sintase Tipo III/metabolismo , Compostos Organosselênicos/farmacologia , Fosforilação , RNA Mensageiro/metabolismo , Ratos , Receptor Tipo 1 de Angiotensina/metabolismo , Marcadores de Spin , Superóxidos/química , Tirosina/análogos & derivados , Tirosina/químicaAssuntos
Plaquetas/enzimologia , GMP Cíclico/sangue , Guanilato Ciclase/sangue , Receptores Citoplasmáticos e Nucleares/sangue , Difosfato de Adenosina/farmacologia , Plaquetas/efeitos dos fármacos , Ativação Enzimática , Ativadores de Enzimas/farmacologia , Humanos , Peptídeos Natriuréticos/sangue , Nitroprussiato/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Receptores Acoplados a Guanilato Ciclase/sangue , Guanilil Ciclase SolúvelRESUMO
We assessed the safety, tolerability and preliminary efficacy of riociguat, a soluble guanylate cyclase stimulator, in patients with pulmonary hypertension associated with interstitial lung disease (PH-ILD). In this open-label, uncontrolled pilot trial, patients received oral riociguat (1.0-2.5 mg three times daily) for 12 weeks (n=22), followed by an ongoing long-term extension (interim analysis at 12 months) in those eligible (n=15). Primary end-points were safety and tolerability. Secondary end-points included haemodynamic changes and 6-min walk distance (6MWD). Overall, 104 adverse events were reported, of which 25 were serious; eight of the latter were considered drug-related. After 12 weeks of therapy, mean cardiac output increased (4.4 ± 1.5 L · min(-1) to 5.5 ± 1.8 L · min(-1)), pulmonary vascular resistance (PVR) decreased (648 ± 207 dyn · s(-1) · cm(-5) to 528 ± 181 dyn · s(-1) · cm(-5)) and mean pulmonary artery pressure (mPAP) remained unchanged compared with baseline. Arterial oxygen saturation decreased but mixed-venous oxygen saturation slightly increased. The 6MWD increased from 325 ± 96 m at baseline to 351 ± 111 m after 12 weeks. Riociguat was well tolerated by most patients and improved cardiac output and PVR, but not mPAP. Further studies are necessary to evaluate the safety and efficacy of riociguat in patients with PH-ILD.
Assuntos
Guanilato Ciclase/metabolismo , Hipertensão Pulmonar/tratamento farmacológico , Doenças Pulmonares Intersticiais/tratamento farmacológico , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Administração Oral , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Guanilato Ciclase/sangue , Hemodinâmica , Humanos , Masculino , Pessoa de Meia-Idade , Oxigênio/metabolismo , Projetos Piloto , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Receptores Citoplasmáticos e Nucleares/metabolismo , Guanilil Ciclase Solúvel , Resultado do TratamentoRESUMO
Activation of peroxisome proliferator-activated receptor (PPAR) isoforms (α, ß/δ, and γ) is known to inhibit platelet aggregation. In the present study, we examined whether PPARs-mediated pathways contribute to the antiplatelet activity of magnolol, a compound purified from Magnolia officinalis. Magnolol (20-60 µM) dose-dependently enhanced the activity and intracellular level of PPAR-ß/γ in platelets. In the presence of selective PPAR-ß antagonist (GSK0660) or PPAR-γ antagonist (GW9662), the inhibition of magnolol on collagen-induced platelet aggregation and intracellular Ca(2+) mobilization was significantly reversed. Moreover, magnolol-mediated up-regulation of NO/cyclic GMP/PKG pathway and Akt phosphorylation leading to increase of eNOS activity were markedly abolished by blocking PPAR-ß/γ activity. Additionally, magnolol significantly inhibited collagen-induced PKCα activation through a PPAR-ß/γ and PKCα interaction manner. The arachidonic acid (AA) or collagen-induced thromboxane B(2) formation and elevation of COX-1 activity caused by AA were also markedly attenuated by magnolol. However, these above effects of magnolol on platelet responses were strongly reduced by simultaneous addition of GSK0660 or GW9662, suggesting that PPAR-ß/γ-mediated processes may account for magnolol-regulated antiplatelet mechanisms. Similarly, administration of PPAR-ß/γ antagonists remarkably abolished the actions of magnolol in preventing platelet plug formation and prolonging bleeding time in mice. Taken together, we demonstrate for the first time that the antiplatelet and anti-thrombotic activities of magnolol are modulated by up-regulation of PPAR-ß/γ-dependent pathways.
Assuntos
Compostos de Bifenilo/farmacologia , Lignanas/farmacologia , PPAR gama/agonistas , PPAR beta/agonistas , Inibidores da Agregação Plaquetária/farmacologia , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Cálcio/metabolismo , GMP Cíclico/biossíntese , Proteínas Quinases Dependentes de GMP Cíclico/sangue , Ciclo-Oxigenase 1/sangue , Fibrinolíticos/farmacologia , Guanilato Ciclase/sangue , Camundongos , Camundongos Endogâmicos ICR , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo III/sangue , PPAR gama/antagonistas & inibidores , PPAR gama/sangue , PPAR beta/antagonistas & inibidores , PPAR beta/sangue , Fosforilação , Agregação Plaquetária/efeitos dos fármacos , Proteína Quinase C-alfa/sangue , Proteínas Proto-Oncogênicas c-akt/sangue , Coelhos , Receptores Citoplasmáticos e Nucleares/sangue , Transdução de Sinais , Guanilil Ciclase Solúvel , Tromboxano B2/sangue , Regulação para CimaRESUMO
Nitric oxide (NO) is recognized as one of the most important cardiovascular signaling molecules, with multiple regulatory effects on myocardial and vascular tissue as well as on other tissues and organ systems. With the growth in understanding of the range and mechanisms of NO effects on the cardiovascular system, it is now possible to consider pharmaceutical interventions that directly target NO or key steps in NO effector pathways. This article reviews aspects of the cardiovascular effects of NO, abnormalities in NO regulation in heart failure, and clinical trials of drugs that target specific aspects of NO signaling pathways.
Assuntos
Insuficiência Cardíaca/sangue , Insuficiência Cardíaca/tratamento farmacológico , Óxido Nítrico/sangue , Transdução de Sinais/efeitos dos fármacos , Disponibilidade Biológica , Sistema Cardiovascular/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Guanilato Ciclase/sangue , Insuficiência Cardíaca/fisiopatologia , Humanos , Óxido Nítrico/fisiologia , Estresse Oxidativo/fisiologia , Inibidores da Fosfodiesterase 5/farmacologia , Piperazinas/farmacologia , Purinas/farmacologia , Transdução de Sinais/fisiologia , Citrato de Sildenafila , Sulfonas/farmacologia , Resultado do Tratamento , Disfunção Ventricular Esquerda/tratamento farmacológicoRESUMO
BACKGROUND: Adiposity greatly increases the risk of atherothrombotic events, a pathological condition where a chronic state of oxidative stress is reported to play a major role. This study aimed to investigate the involvement of (NO)-soluble guanylyl cyclase (sGC) signaling pathway in the platelet dysfunction from high fat-fed (HFF) rats. METHODS: Male Wistar rats were fed for 10 weeks with standard chow (SCD) or high-fat diet (HFD). ADP (10 µM)- and thrombin (100 mU/ml)-induced washed platelet aggregation were evaluated. Measurement of intracellular levels of ROS levels was carried out using flow cytometry. Cyclic GMP levels were evaluated using ELISA kits. RESULTS: High-fat fed rats exhibited significant increases in body weight, epididymal fat, fasting glucose levels and glucose intolerance compared with SCD group. Platelet aggregation induced by ADP (n = 8) and thrombin from HFD rats (n = 8) were significantly greater (P < 0.05) compared with SCD group. Platelet activation with ADP increased by 54% the intraplatelet ROS production in HFD group, as measured by flow cytometry (n = 6). N-acetylcysteine (NAC; 1 mM) and PEG-catalase (1000 U/ml) fully prevented the increased ROS production and platelet hyperaggregability in HFD group. The NO donors sodium nitroprusside (SNP; 10 µM) and SNAP (10 µM), as well as the NO-independent soluble guanylyl cyclase stimulator BAY 41-2272 (10 µM) inhibited the platelet aggregation in HFD group with lower efficacy (P < 0.05) compared with SCD group. The cGMP levels in response to these agents were also markedly lower in HFD group (P < 0.05). The prostacyclin analogue iloprost (1 µM) reduced platelet aggregation in HFD and SCD rats in a similar fashion (n = 4). CONCLUSIONS: Metabolic abnormalities as consequence of HFD cause platelet hyperaggregability involving enhanced intraplatelet ROS production and decreased NO bioavailability that appear to be accompanied by potential defects in the prosthetic haem group of soluble guanylyl cyclase.
Assuntos
Plaquetas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Estresse Oxidativo , Agregação Plaquetária , Espécies Reativas de Oxigênio/sangue , Difosfato de Adenosina , Animais , Antioxidantes/farmacologia , Plaquetas/efeitos dos fármacos , GMP Cíclico/sangue , Ativação Enzimática , Ativadores de Enzimas/farmacologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Intolerância à Glucose/sangue , Intolerância à Glucose/etiologia , Teste de Tolerância a Glucose , Guanilato Ciclase/sangue , Resistência à Insulina , Masculino , Óxido Nítrico/sangue , Doadores de Óxido Nítrico/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Testes de Função Plaquetária , Ratos , Ratos Wistar , Receptores Citoplasmáticos e Nucleares/sangue , Transdução de Sinais , Guanilil Ciclase Solúvel , Trombina , Fatores de Tempo , Aumento de PesoRESUMO
OBJECTIVE: The soluble guanylate cyclase (sGC) activator Cinaciguat (BAY 58-2667) represents a novel class of drugs that selectively activate oxidised sGC. The extent of oxidised sGC depends on the patient's oxidative burden. We here describe two platelet-based assays that allow determining the extent of oxidised sGC and thus provide a basis for an individualised pharmacotherapy. METHODS/RESULTS: Platelets obtained from patients with (n=12) and without (n=12) coronary artery disease (CAD) were examined by flow cytometry (P-selectin expression), and Western blots (vasodilator associated phosphoprotein, VASP-phosphorylation). Results were compared to maximal oxidation of sGC achieved by the oxidising agent ODQ (1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one). Treatment of platelets with Cinaciguat resulted in differential activation of oxidised sGC. Platelet P-selectin expression and VASP-phosphorylation revealed significant differences (p=0.012, p=0.039, respectively) between CAD and non-CAD patients. CONCLUSION: We describe platelet-based assays that allow the determination of patients' oxidative status and thus allow the prediction of pharmacological response to direct sGC activators.
Assuntos
Doença da Artéria Coronariana/metabolismo , Guanilato Ciclase/sangue , Heme/química , Oxigênio/química , Idoso , Benzoatos/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Moléculas de Adesão Celular/metabolismo , Doença da Artéria Coronariana/sangue , Feminino , Humanos , Masculino , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Modelos Biológicos , Estresse Oxidativo , Selectina-P/sangue , Fosfoproteínas/metabolismo , FosforilaçãoRESUMO
In this study the effects of nitric oxide (NO) donors on intracellular free calcium ([Ca(2+)](i)) in human platelets was examined. Inhibition of guanylyl cyclase (GC) with either methylene blue or ODQ slightly inhibited the ability of submaximal concentrations of thrombin to increase [Ca(2+)](i) which suggests that a small portion of the thrombin mediated increase in [Ca(2+)](i) was due to an increase in NO and subsequent increase in cGMP and activation of cGMP dependent protein kinase (cGPK). Thrombin predominantly increases [Ca(2+)](i) by stimulating store-operated Ca(2+) entry (SOCE). The NO donor GEA3162 was previously shown to stimulate SOCE in some cells. In platelets GEA3162 had no effect to increase [Ca(2+)](i) however it inhibited the ability of thrombin to increase [Ca(2+)](i) and this effect was reversed by ODQ. The addition of low concentrations (2.0 - 20 nM) of the NO donor sodium nitroprusside (SNP) slightly potentiated the ability of thrombin to increase [Ca(2+)](i) whereas higher concentrations (>200 nM) of SNP inhibited thrombin induced increases in [Ca(2+)](i). Both of these effects of SNP were reversed by ODQ which implies that they were both mediated by cGPK. Ba(2+) influx was stimulated by low concentrations (2.0 nM) of SNP and inhibited by high concentrations (>200 nM) of SNP and both effects were inhibited by ODQ. Previous studies showed that Ba(2+) influx was blocked by the SOCE inhibitors 2-aminoethoxydipheny borate and diethylstilbestrol. It was concluded that low levels of SNP can stimulate SOCE in platelets and this effect may account for the increased aggregation and secretion previously observed with low concentrations of NO donors. Of the proteins known to be involved in SOCE (e.g. stromal interaction molecule 1 (Stim1), Stim2 and Orai1) only Stim2 has cGPK phosphorylation sites. The possibility that Stim2 phosphorylation regulates SOCE in platelets is discussed.
Assuntos
Plaquetas/efeitos dos fármacos , Cálcio/sangue , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/sangue , Nitroprussiato/farmacologia , Triazóis/farmacologia , Plaquetas/metabolismo , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/metabolismo , Moléculas de Adesão Celular/sangue , GMP Cíclico/sangue , Proteínas Quinases Dependentes de GMP Cíclico/sangue , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/sangue , Humanos , Proteínas de Membrana/sangue , Proteínas de Neoplasias/sangue , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/sangue , Fosforilação , Molécula 1 de Interação Estromal , Molécula 2 de Interação Estromal , Trombina/metabolismo , Fatores de TempoRESUMO
BACKGROUND AND PURPOSE: Oncocalyxone A (OncoA) has a concentration-dependent anti-platelet activity. The present study aimed to further understand the mechanisms related to this effect. EXPERIMENTAL APPROACH: Human platelet aggregation was measured by means of a turbidimetric method. OncoA (32-256 microM) was tested against several platelet-aggregating agents, such as adenosine diphosphate (ADP), collagen, arachidonic acid (AA), ristocetin and thrombin. KEY RESULTS: OncoA completely inhibited platelet aggregation with a calculated mean inhibitory concentration (IC50-microM) of 122 for ADP, 161 for collagen, 159 for AA, 169 for ristocetin and 85 for thrombin. The anti-aggregatory activity of OncoA was not inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). OncoA, at a concentration that caused no significant anti-aggregatory activity, potentiated sodium nitroprusside (SNP) anti-aggregatory activity (18.8+/-2.9%-SNP vs 85.0+/-8.2%-SNP+OncoA). The levels of nitric oxide (NO) or cAMP were not altered by OncoA while cGMP levels were increased more than 10-fold by OncoA in resting or ADP-activated platelets. Flow cytometry revealed that OncoA does not interact with receptors for fibrinogen, collagen or P-selectin. Nevertheless, OncoA decreased the binding of antibodies to GP Ibalpha, a glycoprotein that is related both to von Willebrand factor and to thrombin-induced platelet aggregation. CONCLUSION AND IMPLICATIONS: OncoA showed anti-aggregatory activity in platelets that was associated with increased cGMP levels, not dependent on NO and with blocking GP Ibalpha glycoprotein. This new mechanism has the prospect of leading to new anti-thrombotic drugs.
Assuntos
Antraquinonas/farmacologia , AMP Cíclico/biossíntese , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Adolescente , Adulto , Antraquinonas/isolamento & purificação , Antraquinonas/metabolismo , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , AMP Cíclico/sangue , GMP Cíclico/sangue , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/sangue , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Feminino , Citometria de Fluxo , Guanilato Ciclase/sangue , Guanilato Ciclase/metabolismo , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , Inibidores da Agregação Plaquetária/metabolismo , Ligação Proteica , Tromboxano A2/fisiologiaAssuntos
Plaquetas/enzimologia , Óxido Nítrico Sintase Tipo III/sangue , Óxido Nítrico Sintase Tipo II/sangue , Animais , Plaquetas/metabolismo , GMP Cíclico/sangue , Guanilato Ciclase/sangue , Humanos , Camundongos , Óxido Nítrico/sangue , Óxido Nítrico Sintase Tipo II/deficiência , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Transdução de SinaisRESUMO
OBJECTIVES: Platelets, specialized adhesive cells, play key roles in normal and pathological hemostasis through their ability to rapidly adhere to subendothelial matrix proteins (adhesion) and to other activated platelets (aggregation), functions which are inhibited by nitric oxide (NO). Platelets have been reported to be regulated not only by exogenous endothelium-derived NO, but also by two isoforms of NO synthase, endothelial (eNOS) and inducible (iNOS), endogenously expressed in platelets. however, data concerning expression, regulation and function of eNOS AND iNOS in platelets remain controversial. METHODS AND RESULTS: Using important positive (endothelial cells, stimulated macrophages) and negative (eNOS/iNOS knock-out mouse) controls, as well as human platelets highly purified by a newly developed protocol, we now demonstrate that human and mouse platelets do not contain eNOS/iNOS proteins or mRNA. NOS substrate (L-arginine), NOS inhibitors (L-NAME, L-NMMA), and eNOS/iNOS deficiency did not produce detectable functional effects on human and mouse platelets. von Willebrand factor (VWF)/ristocetin treatment of platelets increased cGMP by NO-independent activation of soluble guanylyl cyclase (sGC) which correlated with Src kinase-dependent phosphorylation of sGC beta(1)-subunit-Tyr(192). CONCLUSIONS: Human and mouse platelets do not express eNOS/iNOS. VWF/ristocetin-mediated activation of the sGC/cGMP signaling pathway may contribute to feedback platelet inhibition.
Assuntos
Plaquetas/enzimologia , Guanilato Ciclase/sangue , Óxido Nítrico Sintase/sangue , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , GMP Cíclico/sangue , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Guanilato Ciclase/química , Humanos , Técnicas In Vitro , Camundongos , Camundongos Knockout , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/sangue , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II/sangue , Óxido Nítrico Sintase Tipo II/deficiência , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo III/sangue , Óxido Nítrico Sintase Tipo III/genética , Fosforilação , RNA Mensageiro/sangue , RNA Mensageiro/genética , Ristocetina/farmacologia , Solubilidade , ômega-N-Metilarginina/farmacologia , Quinases da Família src/sangue , Fator de von Willebrand/farmacologiaRESUMO
Circulating tumor cell (CTC) detection in peripheral blood of colon and other epithelial cancer patients is becoming a scientifically recognised indicator for the presence of primary tumors and/or metastasis. The resulting need to further develop CTC detection-based systems for improved diagnosis, prognosis and assessment of therapy efficacy in tumour patients has prompted the application of different approaches, including expression analysis of tissue-specific and epithelial genes. In this context, lack of specificity of the analysed genes remains a fundamental problem for reliable CTC detection. In this study, we have selected a panel of highly specific epithelial genes: cytokeratin 20 (CK20), cytokeratin 19 (CK19), carcinoembryonic antigen (CEA) and guanylyl cyclase C (GCC), and performed RT-PCR analysis to assess their expression in total blood and in different cell fractions of peripheral blood (PBMC and CD45-negative population) of cancer patients and healthy controls. Our results demonstrate that analysis of a single gene in a CTC-enriched population (CD45(-) peripheral blood cells) of cancer patients allows detection of a CTC molecular signature in at most 63.3% of cases, while analysis of all four genes performed in all three sample types increases the detection of positive patient samples to 87.7%. Healthy controls did not show positivity for any combination of these genes, although positivity was observed for the CEA marker alone, which was detected in 3 (6.6%) out of 45 donors, and only in the CD45(-) fraction. Here, we demonstrate that combined analysis of the genes above, in multiple blood fractions, results in a highly specific and sensitive CTC detection system in patients with metastatic solid tumors. Therefore, we believe that validation on a large scale of this approach, which demonstrates higher specificity in patients compared to controls, could become a relevant CTC screening test in patients with established metastatic disease, and furthermore, may also be useful for evaluating the possible presence of CTCs before the onset of clinically manifested metastatic spreading.
Assuntos
Neoplasias da Mama/genética , Antígeno Carcinoembrionário/genética , Neoplasias do Colo/genética , Guanilato Ciclase/genética , Queratina-19/genética , Queratina-20/genética , Células Neoplásicas Circulantes , Receptores de Peptídeos/genética , Biomarcadores Tumorais/análise , Neoplasias da Mama/sangue , Antígeno Carcinoembrionário/sangue , Neoplasias do Colo/sangue , Guanilato Ciclase/sangue , Humanos , Queratina-19/sangue , Queratina-20/sangue , Receptores de Enterotoxina , Receptores Acoplados a Guanilato Ciclase , Receptores de Peptídeos/sangue , Sensibilidade e EspecificidadeRESUMO
It is unknown whether long-term pharmacological stimulation of soluble guanylate cyclase (sGC), elevating intracellular cGMP levels, has a beneficial effect on hypertension. The purpose of this study is to investigate the effects of BAY41-2272, an orally available sGC stimulator, on cardiovascular remodeling in hypertensive rats. Eight-week-old male Wistar rats with hypertension induced by angiotensin II infused subcutaneously at 250 ng/kg per minute were treated orally with a low ([L] 2 mg/kg per day) or high ([H] 10 mg/kg per day) dose of BAY41-2272 for 14 days. BAY41-2272-H partially suppressed the rise in blood pressure and reduced the heart weight (4.20+/-0.34 versus 3.68+/-0.20 mg/g; P<0.01), whereas BAY41-2272-L had no effect. However, both doses decreased the angiotensin II-induced left ventricular accumulation of collagen in the perivascular area (L, -20%, P<0.05; H, -30%, P<0.01) and myocardial interstitium (L, -21%, P<0.05; H, -38%, P<0.01), reducing the number of activated fibroblasts surrounding coronary arteries (L, -74%; H, -79%; P<0.05). BAY41-2272 downregulated the angiotensin II-induced left ventricular gene expression of type 1 collagen (L, -41%, P<0.05; H, -49%, P<0.01) and transforming growth factor-beta1 (L, -49%, P<0.05; H, -65%, P<0.01). cGMP levels were elevated by BAY41-2272 not only in the left ventricle, but also in cultured cardiac fibroblasts, resulting in reduced thymidine incorporation into the cells. Thus, stimulation of sGC by BAY41-2272 attenuates fibrosis of the left ventricle in rats with angiotensin II-induced hypertension partly in a pressure-independent manner, suggesting an important role for sGC generating cGMP in inhibiting cardiovascular remodeling.
Assuntos
Angiotensina II/administração & dosagem , Sistema Cardiovascular/enzimologia , Guanilato Ciclase/metabolismo , Hipertensão/enzimologia , Hipertensão/fisiopatologia , Angiotensina II/farmacologia , Animais , Cardiomegalia/induzido quimicamente , Cardiomegalia/tratamento farmacológico , Cardiomegalia/fisiopatologia , Sistema Cardiovascular/efeitos dos fármacos , Células Cultivadas , Guanilato Ciclase/sangue , Guanilato Ciclase/fisiologia , Hipertensão/tratamento farmacológico , Masculino , Pirazóis/administração & dosagem , Pirazóis/farmacologia , Piridinas/administração & dosagem , Piridinas/farmacologia , Ratos , Ratos Wistar , SolubilidadeRESUMO
Changes in cerebral cytochrome oxidase (COX) activity, nitric oxide (NO)-cyclic GMP (cGMP) pathway and cholinergic muscarinic receptors (MRs) have been reported in rodents acutely exposed to carbon monoxide (CO). These endpoints measurable in lymphocytes may serve as peripheral markers of CO neurotoxicity. The early and delayed effects of repeated and acute in vivo CO inhalation were investigated on COX activity, cGMP formation and MR binding in rat brain and lymphocytes to assess whether each endpoint was similarly affected both centrally and peripherally. Male Wistar rats either inhaled 500 ppm CO, 6 h/day, 5 days/week, 4 weeks (repeated exposure) or 2,400 ppm, 1 h (single exposure). Neither treatment altered brain or lymphocyte COX activity 1 and 7 days post-treatment. Also ineffective were repeated and acute CO treatments towards (3)H-quinuclidinyl benzilate (QNB) binding to MRs in cerebral cortex, hippocampus, striatum, cerebellum (respective controls, mean+/-S.D.: 171 +/- 45, 245 +/- 53, 263 +/- 14 and 77 +/- 7 fmol/mg protein) and lymphocytes (24 +/- 10 fmol/million cells) at the same time points. In lymphocytes control cGMP levels averaged 1.98 +/- 0.99 pmol/mg protein under basal conditions, and 3.94 +/- 0.55 pmol/mg protein after NO-stimulation. One day after chronic treatment cessation, the CO-treated group displayed about a 50% decrease in both basal and NO-stimulated cGMP values, which persisted up to 7 days after, compared to air-exposed rats. Acutely, CO caused a delayed enhancement (+140%) of NO-induced activation of soluble guanylate cyclase. The finding that the NO-cGMP pathway is a target for the delayed effects of CO in peripheral blood cells is in accordance with our data in brain [Hernández-Viadel, M., Castoldi, A.F., Coccini, T., Manzo, L., Erceg, S., Felipo, V., 2004. In vivo exposure to carbon monoxide causes delayed impairment of activation of soluble guanylate cyclase by nitric oxide in rat brain cortex and cerebellum. Journal of Neurochemistry 89, 1,157-1,165], and supports the use of this peripheral endpoint as a biomarker of CO central effects.
Assuntos
Encéfalo/efeitos dos fármacos , Intoxicação por Monóxido de Carbono/sangue , Monóxido de Carbono/toxicidade , GMP Cíclico/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Linfócitos/enzimologia , Receptores Muscarínicos/metabolismo , Animais , Biomarcadores/sangue , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Guanilato Ciclase/sangue , Exposição por Inalação , Masculino , Neurônios/efeitos dos fármacos , Neurônios/patologia , Ratos , Ratos WistarRESUMO
Staging patients with colorectal cancer defines their prognosis and therapeutic management. Unfortunately, histopathology, the current standard for staging, is relatively insensitive for detecting occult micrometastases and a significant fraction of patients are understaged and, consequently, undertreated. Similarly, current approaches to postoperative surveillance of patients with colorectal cancer detect disease recurrence at a point when interventions have little impact on survival. The detection of rare cells in tissue, for accurately staging patients, and in blood, for detecting disease recurrence, could be facilitated by employing sensitive and specific markers of disease. Guanylyl cyclase C (GCC), the receptor for the diarrheagenic bacterial heat-stable enterotoxin, is expressed selectively by cells derived from intestinal mucosa, including normal intestinal cells and colorectal tumor cells, but not by extragastrointestinal tissues and tumors. The nearly uniform expression of relatively high levels by metastatic colorectal tumors suggests that GCC may be a sensitive and specific molecular marker for metastatic colorectal cancer cells. Employing GCC reverse transcriptase PCR, occult colorectal cancer micrometastases were detected in lymph nodes that escaped detection by histopathology. Moreover, marker expression correlated with the risk of disease recurrence. Similarly, GCC reverse transcriptase PCR revealed the presence of tumor cells in blood of all patients examined with metastatic colorectal cancer and, in some studies, was associated with an increased risk of disease recurrence and mortality. These observations suggest that GCC reverse transcriptase PCR is a sensitive and specific technique for identifying tumor cells in extraintestinal sites and may be useful for staging and postoperative surveillance of patients with colorectal cancer.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Guanilato Ciclase/metabolismo , Receptores de Peptídeos/metabolismo , Animais , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/cirurgia , Regulação Neoplásica da Expressão Gênica , Guanilato Ciclase/análise , Guanilato Ciclase/sangue , Humanos , Metástase Linfática/patologia , Estadiamento de Neoplasias , Receptores de Enterotoxina , Receptores Acoplados a Guanilato Ciclase , Receptores de Peptídeos/análise , Receptores de Peptídeos/sangueRESUMO
The molecular regulation of nitric oxide synthase (NOS) in blood platelets is an uncharacterised area of platelet biology. We investigated the mechanism of collagen-stimulated NO synthesis in platelets. Our aim was to identify the key collagen receptor and downstream signalling mechanisms linking collagen to NOS activation. Collagen and the GpVI-specific platelet activator collagen-related peptide (CRP-XL) stimulated NO synthesis, as evidenced by increased [(3)H]L-citrulline production, and cyclic GMP (cGMP) formation. After platelet activation by collagen and CRP-XL was normalised, we found no differences in NOS activation or cGMP formation in response to these agonists. Blocking the interaction of collagen with integrin alpha(2)beta(1), a second collagen receptor, failed to affect NOS activation by collagen. These data indicate that collagen-induced NO synthesis is linked to GpVI activation. cGMP formation in response to collagen and CRP-XL required increased intracellular Ca(2+), Src family kinases, phosphatidylinositol 3-kinase (PI3-K) and protein kinase C. By comparison, Gp VI-independent cGMP formation induced by thrombin was Src kinase-dependent, but was independent of PI3-K and PKC. Thus the mechanisms of collagen- and CRP-XL-induced NOS activation were identical, but distinct from that of thrombin. Platelet activation in response to collagen leads to secretion of adenosine diphosphate (ADP) and thromboxane A(2) (TxA(2)). Our results demonstrate that collagen-stimulated cGMP synthesis was enhanced significantly by platelet-derived ADP and TxA(2). These results reveal that collagen stimulates platelet NOS activation through a specific Ca(2+)-dependent GpVI receptor signalling cascade, and demonstrate that collagen-induced cGMP accrual requires the release of secondary platelet agonists.