RESUMO
Platelet-activating factor (PAF) is a potent mediator of inflammation that plays a crucial role in atherosclerosis. The purpose of this study was to evaluate the effect of a dietary supplement containing mainly plant extracts on PAF actions and metabolism in healthy volunteers. A double-blind, placebo-controlled, 8 weeks' duration study was performed. Healthy volunteers were randomly allocated into the supplement or the placebo group and fifty-eight of them completed the study. The supplement contained plant extracts (Aloe gel, grape juice, Polygonum cuspidatum) and vitamins. The activities of PAF metabolic enzymes: the two isoforms of acetyl-CoA:lyso-PAF acetyltransferase, cytidine 5'-diphospho-choline:1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (PAF-cholinephosphotransferase) and platelet-activating factor-acetylhydrolase (PAF-AH) in leucocytes and lipoprotein associated phospholipase-A2 in plasma were measured along with several markers of endothelial function. Platelet aggregation against PAF, ADP and thrombin receptor activating peptide was measured in human platelet-rich plasma by light transmission aggregometry. No difference was observed on soluble vascular cell adhesion molecule-1, sP-selectin and IL-6 levels at the beginning or during the study period between the two groups. Concerning PAF metabolism enzymes' activity, no difference was observed at baseline between the groups. PAF-AH activity was only increased in the supplement group at 4 and 8 weeks compared with baseline levels. In addition, supplement consumption led to lower platelet sensitivity against PAF and ADP compared with baseline levels. However, a trial effect was only observed when platelets were stimulated by PAF. In conclusion, supplementation with plant extracts and vitamins ameliorates platelet aggregation primarily against PAF and secondarily against ADP and affects PAF catabolism by enhancing PAF-acetylhydrolase activity in healthy subjects.
Assuntos
Suplementos Nutricionais , Extratos Vegetais/farmacologia , Fator de Ativação de Plaquetas/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Difosfato de Adenosina/metabolismo , Adulto , Biomarcadores/sangue , Plaquetas/metabolismo , Método Duplo-Cego , Feminino , Voluntários Saudáveis , Humanos , Leucócitos/metabolismo , MasculinoRESUMO
The evaluation was implemented concerning impact of anticoagulants used during venous blood sampling, on aggregation of thrombocytes and acetylsalicylic acid effect in vitro in 111 patients with suspicion to chronic myelo-proliferative tumors and 16 healthy volunteers. The vacutainers (Becton Dickinson) with 3.2% citrate, with heparin (Becton Dickinson) and S-Monovette system (Sarstedt AG & Co) with recombinant herudin were applied. The analysis of aggregation was implemented using the technique of impedance in whole blood before and after preliminary incubation of blood samples with acetylsalicylic acid effect in 0.1 mM concentration. The induction was implemented by ATP in final concentration of 5 mkM. It is demonstrated that ATP-induced amplitude of aggregation under application of heparin and hirudin is significantly higher in comparison with its level in samples of citrate blood. At that, acetylsalicylic acid effect paradoxically increases amplitude of aggregation in samples with heparin but not in samples with citrate or hirudin. The application of hirudin permits evaluating impact of acetylsalicylic acid effect both at aggregation and disaggregation component of thrombocyte functions under erythrocytosis and thrombocytosis and can be recommended as a preferable approach in testing individual sensitivity of patients to acetylsalicylic acid effect.
Assuntos
Anticoagulantes/farmacologia , Aspirina/farmacologia , Plaquetas/efeitos dos fármacos , Transtornos Mieloproliferativos/sangue , Trifosfato de Adenosina/química , Adulto , Idoso , Plaquetas/patologia , Citratos/farmacologia , Impedância Elétrica , Feminino , Voluntários Saudáveis , Heparina/farmacologia , Hirudinas/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Fator de Ativação de Plaquetas/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacosRESUMO
It was the objective of this study to determine whether the intrinsic platelet response to adenosine diphosphate (ADP) before thienopyridine exposure contributes to residual platelet reactivity to ADP despite high level P2Y12 blockade by prasugrel (60 mg loading dose [LD]), 10 mg daily maintenance dose [MD]) or high-dose clopidogrel (600 mg LD, 150 mg daily MD). High residual platelet function during clopidogrel therapy is associated with poor clinical outcomes. It remains unknown whether the relationship between platelet reactivity prior to treatment with clopidogrel (300 mg LD, 75 mg daily MD) and residual on-treatment platelet reactivity is maintained after more potent P2Y12 inhibition. PRINCIPLE-TIMI 44 was a randomised, double-blind, two-phase crossover study of prasugrel compared with high-dose clopidogrel in 201 patients undergoing cardiac catheterisation for planned percutaneous coronary intervention. ADP-stimulated platelet-monocyte aggregates, platelet surface P-selectin and platelet aggregation were measured pre-treatment, during LD (6 h and 18-24 h) and MD (15 d). Correlations of pre-treatment to on-treatment values were determined by Spearman rank order. Prasugrel resulted in greater platelet inhibition than high-dose clopidogrel for each measure. However, for both drugs, pre-treatment reactivity to ADP predicted 6 h, 18-24 h and 15 day reactivity to ADP (correlations 0.24-0.62 for platelet-monocyte aggregates and P-selectin). In conclusion, a patient's intrinsic platelet response to ADP before exposure to thienopyridines contributes to residual platelet reactivity to ADP despite high level P2Y12 blockade with high-dose clopidogrel or even higher level P2Y12 blockade with prasugrel. Patients who are hyper-responsive to ADP pre-treatment are more likely to be hyper-responsive to ADP on-treatment, which may be relevant to therapeutic strategies.
Assuntos
Piperazinas/administração & dosagem , Inibidores da Agregação Plaquetária/administração & dosagem , Antagonistas do Receptor Purinérgico P2/administração & dosagem , Tiofenos/administração & dosagem , Ticlopidina/análogos & derivados , Difosfato de Adenosina/farmacologia , Idoso , Clopidogrel , Estudos Cross-Over , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fator de Ativação de Plaquetas/efeitos dos fármacos , Fator de Ativação de Plaquetas/fisiologia , Cloridrato de Prasugrel , Receptores Purinérgicos P2Y12/efeitos dos fármacos , Ticlopidina/administração & dosagemRESUMO
Platelet-activating factor (PAF) is a potent inflammatory mediator, which seems to play a role in the pathogenesis of several AIDS manifestations such as AIDS dementia complex, Kaposi's sarcoma, and HIV-related nephropathy. PAF antagonists have been studied in these conditions with promising results. In order to examine the possible interactions between PAF and antiretroviral therapy, we studied the effect of nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors against PAF biological activities and its basic biosynthetic enzymes dithiothreitol-insensitive PAF-cholinephosphotransferase (PAF-CPT) and lyso-PAF-acetyltransferase (Lyso-PAF-AT), as well as its main degradative enzyme PAF-acetylhydrolase, of human mesangial cell line (HMC). We also studied the effect of several backbones and highly active antiretroviral therapy (HAART) regimens against PAF activity. Among the drugs tested, several inhibited PAF-induced platelet aggregation in a concentration-depended manner, with tenofovir, efavirenz, and ritonavir exhibiting the higher inhibitory effect. In addition, when these drugs were combined in backbones and HAART regimens based on American antiretroviral therapy proposals, they also synergistically exhibited an inhibitory effect against PAF-induced platelet aggregation. Several of these drugs have also inhibited in vitro microsomal PAF-CPT activity, and concentrations of lopinavir-r or tenofovir-DF (similar to their IC(50) against PAF-induced platelet aggregation) exhibited the same effect against PAF-CPT and Lyso-PAF-AT when added in the cell medium of cultured HMC. In addition, in naïve patients treated with one of the most potent anti-PAF HAART regimens (efavirenz/emtricitabine/tenofovir-DF) for a period of 1 month, a significant reduction of the specific activity of PAF-CPT of washed human leukocytes of these patients was also observed, compared with its levels before the HAART treatment. These promising results need to be further studied and confirmed by additional in vivo tests in order to optimize HAART efficacy.
Assuntos
Antirretrovirais/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Infecções por HIV/tratamento farmacológico , Fator de Ativação de Plaquetas/efeitos dos fármacos , 1-Alquil-2-acetilglicerofosfocolina Esterase/efeitos dos fármacos , Acetiltransferases/efeitos dos fármacos , Células Cultivadas , Diacilglicerol Colinofosfotransferase/efeitos dos fármacos , Humanos , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/metabolismo , Fator de Ativação de Plaquetas/metabolismo , Agregação Plaquetária/efeitos dos fármacosRESUMO
Experiments in cultured endothelial cell monolayers demonstrate that increased intracellular cAMP strongly inhibits the acute permeability responses by both protein kinase A (PKA)-dependent and -independent pathways. The contribution of the PKA-independent pathways to the anti-inflammatory mechanisms of cAMP in intact mammalian microvessels has not been systematically investigated. We evaluated the role of the cAMP-dependent activation of the exchange protein activated by cAMP (Epac), a guanine nucleotide exchange factor for the small GTPase Rap1, in rat venular microvessels exposed to the platelet-activating factor (PAF). The cAMP analog 8-pCPT-2'-O-methyl-cAMP (O-Me-cAMP), which stimulates the Epac/Rap1 pathway but has no effect on PKA, significantly attenuated the PAF increase in microvessel permeability as measured by hydraulic conductivity (Lp). We also demonstrated that PAF induced a rearrangement of vascular endothelial (VE)-cadherin seen as numerous lateral spikes and frequent short breaks in the otherwise continuous peripheral immunofluorescent label. Pretreatment with O-Me-cAMP completely prevented the PAF-induced rearrangement of VE-cadherin. We conclude that the action of the Epac/Rap1 pathway to stabilize cell-cell adhesion is a significant component of the activity of cAMP to attenuate an acute increase in vascular permeability. Our results indicate that increased permeability in intact microvessels by acute inflammatory agents such as PAF is the result of the decreased effectiveness of the Epac/Rap1 pathway modulation of cell-cell adhesion.
Assuntos
Permeabilidade Capilar/fisiologia , Fator de Ativação de Plaquetas/fisiologia , Transdução de Sinais/fisiologia , Circulação Esplâncnica/fisiologia , Proteínas rap1 de Ligação ao GTP/fisiologia , Agonistas Adrenérgicos beta/farmacologia , Animais , Caderinas/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Colforsina/farmacologia , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Indicadores e Reagentes , Isoproterenol/farmacologia , Masculino , Microscopia Confocal , Inibidores de Fosfodiesterase/farmacologia , Fator de Ativação de Plaquetas/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Rolipram/farmacologia , Transdução de Sinais/efeitos dos fármacos , Circulação Esplâncnica/efeitos dos fármacos , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
The authors have carried out a comparative analysis of the interaction between two types of suture material with blood components, as well as studied the effect of heparin-mediated modification on the sorption and contact-activating processes in the zone of the anastomosis. The blood-compatible properties of the latter was assesses in vitro. It was determined that by minute 120 of the contact with blood, the largest amount of protein is had been absorbed by the anastomoses performed using the Prolene thread - 112 microg/cm2. Heparin-mediated modification made it possible to dramatically decrease the amount of the absorbed proteins. On the anastomoses performed with TiNi, additional treatment with heparin lead to an inconsiderable decrease in the protein amount. When identifying the absorbed proteins, we revealed dependence on the type of the suture material and modification with heparin. After a 60-minute contact with blood in the area of the anastomosis made with TiNi, absorbed were: albumin, immunoglobulins A, G, and transferrin. When using the Prolene thread, fibrinogen was noted to join. Additional heparinization exerted a favourable effect on the sorption processes in the area of the anastomosis wherein predominantly albumin and immunoglobulins A and G are predominantly absorbed. The parameters of the peak values and the rate of blood platelet aggregation were minimal in the area of the anastomoses done with TiNi with an additional treatment with heparin.
Assuntos
Suturas , Resistência à Tração , Adsorção , Anastomose Cirúrgica , Fibrinolíticos/farmacologia , Heparina/farmacologia , Humanos , Fator de Ativação de Plaquetas/efeitos dos fármacosRESUMO
Platelet activating factor (PAF; beta-acetyl-gamma-O-hexadecyl-l-alpha-phosphatidylcholine) triggers a rapid pro-inflammatory gene expression program in primary cultures of human neural (HN) cells. Two genes and gene products consistently induced after PAF treatment are the cytosoluble prostaglandin synthase cycloooxygenase-2 (COX-2) and the pro-apoptotic tumor necrosis factor alpha (TNFalpha). Both of these mediators are associated with the activation of inflammatory signaling, neural cell dysfunction, apoptosis and brain cell death, and both have been found to be up-regulated after brain injury in vivo. In this study we investigated the effects of the non-halogenated synthetic glucocorticoid budesonide epimer R (BUDeR), the novel PAF antagonist LAU-0901, and the electron spin trap and free radical scavenger phenyl butyl nitrone (PBN), upon early COX-2 and TNFalpha gene activation and prostaglandin E(2) (PGE(2)) release in PAF-stressed primary HN cells. The data indicate that these three biochemically unrelated classes of inflammatory repressors act synergistically in modulating PAF-induced up-regulation of COX-2, TNFalpha, and PGE(2) by quenching oxidative stress or inflammatory signaling, resulting in increased HN cell survival. These, or analogous classes of compounds, may be useful in the design of more effective combinatorial pharmacotherapeutic strategies in the treatment of complex neuro-inflammatory disorders.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Encefalite/metabolismo , Neurônios/metabolismo , Fator de Ativação de Plaquetas/metabolismo , Estresse Fisiológico/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Anti-Inflamatórios/farmacologia , Budesonida/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Óxidos N-Cíclicos/farmacologia , Ciclo-Oxigenase 2/efeitos dos fármacos , Di-Hidropiridinas/farmacologia , Dinoprostona/metabolismo , Sinergismo Farmacológico , Encefalite/tratamento farmacológico , Encefalite/fisiopatologia , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Humanos , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Fator de Ativação de Plaquetas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Estresse Fisiológico/fisiopatologia , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologia , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Fish oils contain several active compounds that modify cell activity and influence various functions of the body. Shark liver oils are rich in alkylglycerols and squalene, but contain relatively low amounts of n-3 polyunsaturated fatty acids. Alkylglycerols may control immune response possibly throw modification of platelet activating factor (PAF) and diacylglycerol (DAG) production. Squalene enhances antigen presentation and induction of inflammatory response. Moreover, alkylglycerols and squalene have antitumour activity, that is possibly based on different mechanisms, ie., induction of apoptosis of neoplastic cells, suppression of signal transduction, inhibition of angiogenesis and promoting of transmembrane transport of cytotoxic agents. Shark liver oil has been found to be useful in treatment of conditions resulted from inadequate immune response, and in adjunctive treatment of several types of cancer.
Assuntos
Óleos de Peixe/farmacologia , Óleos de Peixe/uso terapêutico , Esqualeno/farmacologia , Esqualeno/uso terapêutico , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Diglicerídeos/biossíntese , Humanos , Inflamação/imunologia , Neoplasias/tratamento farmacológico , Neovascularização Patológica/prevenção & controle , Fator de Ativação de Plaquetas/efeitos dos fármacos , Tubarões , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND AND AIM: The consumption of olive oil has been associated with lower incidence of cardiovascular disease in the Mediterranean countries. This may be due in part to the action of platelet-activating factor (PAF) antagonists which we have previously demonstrated to be present in olive oil. In order to assess the in vivo effects of olive oil lipids and PAF in the development of atherosclerosis, the effects of diet supplementation with olive oil (OO), olive oil polar lipid extract (OOPLE) and olive oil neutral lipid extract (OONLE) were studied in rabbits fed a cholesterol-enriched diet. METHODS AND RESULTS: Rabbits were fed for 45 days with atherogenic diet (Group A) supplemented with OO (Group B), OOPLE (Group C) or OONLE (Group D). Lipoprotein profiles, plasma in vitro oxidation, blood PAF levels, PAF-induced platelet aggregation and PAF-acetylhydrolase (PAF-AH) activity, were measured on day 0 and 45. Atherosclerotic lesions formed in the aortic wall and wall elasticity were assessed on day 45. Changes in lipid profile were in accordance with previous studies. Blood PAF levels were higher in group A and decreased in group D on day 45. In rabbits fed an atherogenic diet (Group A) blood PAF and PAF-AH increased, atherosclerotic lesions formed and the elasticity of vessel walls declined. In animals fed olive oil (Group B) or OOPLE (Group C) blood PAF-AH increased, platelet aggregation was attenuated, less oxidation occurred in plasma, lesion thickness was reduced and vessel walls retained elasticity. Most of these beneficial changes were not seen in animals fed OONLE (Group D) although blood PAF and plasma oxidation were lower. CONCLUSIONS: The antiatherogenic effects of OO result from OOPLE. The beneficial effect of these factors is linked to PAF metabolism and proaggregant activity.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Aterosclerose/prevenção & controle , Lipoproteínas/sangue , Óleos de Plantas/farmacologia , Fator de Ativação de Plaquetas , Agregação Plaquetária/efeitos dos fármacos , Animais , Aorta/patologia , Aorta/ultraestrutura , Aterosclerose/patologia , Dieta Aterogênica , Modelos Animais de Doenças , Hiperlipidemias/complicações , Hiperlipidemias/dietoterapia , Hiperlipidemias/metabolismo , Masculino , Azeite de Oliva , Oxirredução , Óleos de Plantas/química , Fator de Ativação de Plaquetas/antagonistas & inibidores , Fator de Ativação de Plaquetas/efeitos dos fármacos , Fator de Ativação de Plaquetas/metabolismo , CoelhosRESUMO
Lung fibrosis is a common side effect of the chemotherapeutic agent, bleomycin. Current evidence suggests that reactive oxygen species may play a key role in the development of lung fibrosis. The present study examined the effect of mesna on bleomycin-induced lung fibrosis in rats. Animals were divided into three groups: (1) saline control group; (2) Bleomycin group in which rats were injected with bleomycin (15 mg/kg, i.p.) three times a week for four weeks; (3) Bleomycin and mesna group, in which mesna was given to rats (180 mg/kg/day, i.p.) a week prior to bleomycin and daily during bleomycin injections for 4 weeks until the end of the treatment. Bleomycin treatment resulted in a pronounced fall in the average body weight of animals. Bleomycin-induced pulmonary injury and lung fibrosis was indicated by increased lung hydroxyproline content, and elevated nitric oxide synthase, myeoloperoxidase, platelet activating factor, and tumor necrosis factor-alpha in lung tissues. On the other hand, bleomycin induced a reduction in reduced glutathione concentration and angiotensin converting enzyme activity in lung tissues. Moreover, bleomycin-induced severe histological changes in lung tissues revealed as lymphocytes and neutrophils infiltration, increased collagen deposition and fibrosis. Co-administration of bleomycin and mesna reduced bleomycin-induced weight loss and attenuated lung injury as evaluated by the significant reduction in hydroxyproline content, nitric oxide synthase activity, and concentrations of myeoloperoxidase, platelet activating factor, and tumor necrosis factor-alpha in lung tissues. Furthermore, mesna ameliorated bleomycin-induced reduction in reduced glutathione concentration and angiotensin activity in lung tissues. Finally, histological evidence supported the ability of mesna to attenuate bleomycin-induced lung fibrosis and consolidation. Thus, the findings of the present study provide evidence that mesna may serve as a novel target for potential therapeutic treatment of lung fibrosis.
Assuntos
Bleomicina/efeitos adversos , Mesna/efeitos adversos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/tratamento farmacológico , Animais , Bleomicina/administração & dosagem , Bleomicina/antagonistas & inibidores , Esquema de Medicação , Ensaios de Seleção de Medicamentos Antitumorais , Glutationa/antagonistas & inibidores , Glutationa/efeitos dos fármacos , Glutationa/metabolismo , Hidroxiprolina/efeitos adversos , Hidroxiprolina/química , Hidroxiprolina/metabolismo , Injeções Intraperitoneais , Pulmão/química , Pulmão/efeitos dos fármacos , Pulmão/ultraestrutura , Masculino , Mesna/administração & dosagem , Mesna/farmacocinética , Óxido Nítrico Sintase/química , Óxido Nítrico Sintase/metabolismo , Peptidil Dipeptidase A/efeitos dos fármacos , Peptidil Dipeptidase A/metabolismo , Peroxidase/efeitos dos fármacos , Peroxidase/metabolismo , Fator de Ativação de Plaquetas/efeitos dos fármacos , Fator de Ativação de Plaquetas/metabolismo , Fibrose Pulmonar/fisiopatologia , Ratos , Ratos Wistar , Fatores de Tempo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Aumento de Peso/efeitos dos fármacos , Redução de Peso/efeitos dos fármacosRESUMO
BACKGROUND: Tea extracts have antiallergic and anti-inflammatory actions in rats and mice. However the mechanism through which tea polyphenols act in vivo are still incompletely understood. We found inhibitory effects of black tea extracts on an fMLP-induced aggregating response in a rabbit platelet-polymorphonuclear leukocyte (PMN) system. METHOD: To elucidate whether 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) production in PMNs and/or PAF-stimulated platelet activation were inhibited, the effects of tea polyphenols were investigated on the enzyme activity of acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine acetyltransferase (EC 2.3.1.67), PAF biosynthesis in A23187-activated rabbit PMNs, and rabbit platelet aggregation. By comparing the inhibitory effects of 31 galloyl esters and gallic acid, the structure-inhibitory activity relationship was characterized. RESULTS: Theaflavin and its galloyl esters and pentagalloyl glucose were found to be potent inhibitors of the acetyltransferase (IC(50) = 28-58 microM) and the PAF biosynthesis as well as (-)-epicatechin-3-O-gallate (IC(50) = 72 +/- 13 microM) and (-)-epigallocatechin-3-O-gallate (IC(50) = 46 +/- 6 microM). On the other hand, flavan-3-ols without galloyl group at C-3 and gallic acid did not show significant enzyme inhibition. In addition, theaflavin and its galloyl esters (IC(50) = 32-77 microM) and geranyl gallate, farnesyl gallate and geranylgeranyl gallate (IC(50) = 6.4-7.6 microM) were found to be potent inhibitors of PAF- and TPA-induced rabbit platelet aggregation but not A23187-induced aggregation. CONCLUSIONS: Theaflavin and its galloyl esters in black tea extract, and isoprenyl gallates were potent inhibitors of PAF synthesis and platelet aggregation and these activities may be relevant to the claimed therapeutic effects of tea extracts.
Assuntos
Acetiltransferases/biossíntese , Acetiltransferases/efeitos dos fármacos , Flavonoides/farmacologia , Fenóis/farmacologia , Fator de Ativação de Plaquetas/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Chá/química , Animais , Biflavonoides/farmacologia , Catequina/farmacologia , Quelantes/farmacologia , Modelos Animais , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Extratos Vegetais/farmacologia , Polifenóis , Coelhos , Rheum/química , Fatores de TempoRESUMO
Vascular endothelial growth factor (VEGF-A) is an inducer of endothelial cell (EC) proliferation, migration, and synthesis of inflammatory agents such as platelet-activating factor (PAF). Recently, neuropilin-1 (NRP-1) has been described as a coreceptor of KDR which potentiates VEGF-A activity. However, the role of NRP-1 in numerous VEGF-A activities remains unclear. To assess the contribution of NRP-1 to VEGF-A mediated EC proliferation, migration, and PAF synthesis, we used porcine aortic EC (PAEC) recombinantly expressing Flt-1, NRP-1, KDR or KDR and NRP-1. Cells were stimulated with VEGF-A, which binds to Flt-1, KDR and NRP-1, and VEGF-C, which binds to KDR only. VEGF-A was 12.4-fold more potent than VEGF-C in inducing KDR phosphorylation in PAEC-KDR. VEGF-A and VEGF-C showed similar potency to mediate PAEC-KDR proliferation, migration, and PAF synthesis. On PAEC-KDR/NRP-1, VEGF-A was 28.6-fold more potent than VEGF-C in inducing KDR phosphorylation and PAEC-KDR/NRP-1 proliferation (1.3-fold), migration (1.7-fold), and PAF synthesis (4.6-fold). These results suggest that cooperative binding of VEGF-A to KDR and NRP-1 enhances KDR phosphorylation and its biological activities. Similar results were obtained with bovine aortic EC that endogenously express both KDR and NRP-1 receptors. In contrast, stimulation of PAEC-Flt-1 and PAEC-NRP-1 with VEGF-A or VEGF-C did not induce proliferation, migration, or PAF synthesis. In conclusion, the presence of NRP-1 on EC preferentially increases KDR activation by VEGF-A as well as KDR-mediated biological activities, and may elicit novel intracellular events. On the other hand, VEGF-A and VEGF-C have equipotent biological activities on EC in absence of NRP-1.
Assuntos
Fatores de Crescimento Endotelial/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Neuropilina-1/fisiologia , Fator de Ativação de Plaquetas/biossíntese , Animais , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/efeitos dos fármacos , Proteínas da Matriz Extracelular/efeitos dos fármacos , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Neuropilina-1/efeitos dos fármacos , Neuropilina-1/genética , Neuropilina-1/metabolismo , Fosforilação/efeitos dos fármacos , Fator de Ativação de Plaquetas/efeitos dos fármacos , Ligação Proteica/fisiologia , Suínos , Transfecção , Fator A de Crescimento do Endotélio Vascular , Fator C de Crescimento do Endotélio VascularRESUMO
Despite the intensive clinical use of 1-deamino-8-D-arginine vasopressin (desmopressin; DDAVP) for 20 years, its mechanism of action is still not completely explained. It has been proposed that DDAVP stimulates release of a 'second messenger' which in turn stimulates release of von Willebrand factor (vWF) from endothelial cells. Platelet-activating factor (PAF) and interleukin (IL)-6 were individually proposed to be mediators for haemostatic action. The aim of this study was to investigate cellular-based PAF levels in patients with haemophilia A (HA) and von Willebrand disease (vWD) before and after DDAVP treatment and also to look for any probable relationship between the haemostatic response of DDAVP and cellular PAF activities. In total, 20 patients (11 HA and nine vWD) were enrolled in the study. DDAVP was given subcutaneously as a single dose (0.3 microg kg(-1)). Ten patients responded to DDAVP and were defined as the 'able group' (four mild HA, six type 1 vWD). The remaining 10 patients did not respond to DDAVP and were defined as the 'unable group' (seven severe HA, three type 3 vWD). Released (extracellular) and intracellular (intraleucocyte) PAF levels under the stimulation of specific agents (A23187 and Zymosan) were measured by high-performance liquid chromatography and radioimmunoassay. Extracellular and intracellular PAF activities were not detected without stimulation in healthy children whereas significantly higher PAF levels were found in the patients (extracellular: 37.5 +/- 34.4 ng per 10(7) cells; intracellular: 24.8 +/- 23.5 ng per 10(7) cells; P=0.0001). Intracellular PAF levels obtained from in vitro unstimulated cells were significantly higher in DDAVP-responsive (able) patients in comparison to DDAVP-unresponsive (unable) patients (52.1 +/- 18.5 vs. 28.9 +/- 8.0 ng per 10(7)cells). After in vitro stimulation by A23187, intracellular PAF activities were significantly higher in patients than in controls (209.3 +/- 26.1 vs. 172 +/- 18.1 ng per 10(7) cells). Intracellular PAF levels obtained from in vitro stimulated cells by A23187 were also significantly higher in the 'able' patients in comparison to the 'unable' patients (277 +/- 43.5 vs. 225 +/- 30 ng per 10(7)cells). In conclusion, cellular PAF activities are significantly higher in patients with HA and vWD. We also suggest that PAF, especially intracellular PAF mediates intracellular signalling and may be one of the important mediators for the haemostatic response of DDAVP.
Assuntos
Desamino Arginina Vasopressina/administração & dosagem , Hemofilia A/sangue , Hemostáticos/administração & dosagem , Leucócitos/metabolismo , Fator de Ativação de Plaquetas/metabolismo , Doenças de von Willebrand/sangue , Adolescente , Adulto , Calcimicina/farmacologia , Criança , Pré-Escolar , Desamino Arginina Vasopressina/uso terapêutico , Feminino , Hemofilia A/tratamento farmacológico , Humanos , Ionóforos/farmacologia , Masculino , Fator de Ativação de Plaquetas/efeitos dos fármacos , Resultado do Tratamento , Doenças de von Willebrand/tratamento farmacológicoRESUMO
In the present study we examined the effect of recombinant human erythropoietin (rhEPO) on intestinal malondialdehyde (MDA) as an index of lipid peroxidation, related to iron-catalysed free radical reaction and platelet-activating factor (PAF) synthesis in the experimental model of necrotizing enterocolitis (NEC). Three groups, each consisting of eight 1-day-old Wistar albino rat pups, were studied; Group 1, hypoxia-reoxygenation; Group 2, hypoxia-reoxygenation and rhEPO pretreatment; Group 3, control. rhEPO was given 750 U/kg/week by intraperitoneal injection three times a week for 2 weeks. On day 15th of life, hypoxia was induced by placing rat pups in a 100% CO2 chamber for 5 min. After hypoxia, the rat pups were reoxygenated for 10 min with 100% oxygen and returned to their mothers. All pups were killed at 4h following hypoxia-reoxygenation. The abdomen was opened and representative samples of injured areas were taken for histopathologic examination. MDA and PAF levels were determined in the intestine. Significantly increased intestinal MDA content was found in Group 1 rat pups compared to Group 2 and Group 3 pups (p < 0.001 and p < 0.001, respectively). However, PAF concentrations were highly elevated in the intestine of Group 1 and Group 2 pups (p>0.05) when compared to the intestine of Group 3 pups (p < 0.001 and p < 0.001, respectively). Histopathologic findings did not differ between Groups 1 and 2. The present study demonstrates that oxygen-derived free radicals and PAF are involved in the pathophysiological mechanism of the development of NEC. This study also shows that administration of rhEPO significantly decreases lipid peroxidation; however, PAF generation was not inhibited in hypoxia-induced bowel necrosis.
Assuntos
Enterocolite Necrosante/tratamento farmacológico , Enterocolite Necrosante/fisiopatologia , Eritropoetina/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Malondialdeído/metabolismo , Fator de Ativação de Plaquetas/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Feminino , Injeções Intraperitoneais , Masculino , Fator de Ativação de Plaquetas/metabolismo , Probabilidade , Ratos , Ratos Wistar , Proteínas Recombinantes , Valores de Referência , Sensibilidade e Especificidade , Estatísticas não ParamétricasRESUMO
BACKGROUND: Leukotrienes have been implicated in the selective infiltration of eosinophils into the bronchial mucosa in asthma. OBJECTIVE: We studied whether eosinophil transmigration through cultured human umbilical vein endothelial cells (HUVECs) can be blocked by a specific cysteinyl LT1-receptor-antagonist. METHODS: Unstimulated and stimulated eosinophils from patients with asthma and normal controls were subjected to confluent human umbilical vein endothelial cell (HUVEC) monolayers separating the upper and lower chamber of Transwell culture plates. Unstimulated eosinophils or cells pre-incubated in the presence of the eosinophil activating cytokines GM-CSF or IL-13 were placed in the upper chambers while PAF, a potent chemoattractant factor for eosinophils, was added to the lower chamber. Migration of eosinophils was quantified by a beta-glucuronidase assay. RESULTS: The assumption that eosinophils express CysLT1 (cysteinyl-leukotriene 1)-receptors was based on our demonstration of mRNA-expression for the CysLT-1-receptor by polymerase chain reaction on purified eosinophils. The chemotactic response to PAF was significantly reduced when eosinophils were pre-incubated with montelukast for 15 min. When eosinophils were pre-incubated with GM-CSF and/or IL-13, the migratory response to PAF was also significantly reduced by montelukast. CONCLUSION: From these data we conclude that the specific cysteinyl LT1-receptor antagonist montelukast can inhibit PAF-induced eosinophil transmigration through cultured HUVEC monolayers.
Assuntos
Acetatos/antagonistas & inibidores , Antiasmáticos/antagonistas & inibidores , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Eosinófilos/efeitos dos fármacos , Eosinófilos/fisiologia , Antagonistas de Leucotrienos/farmacologia , Proteínas de Membrana , Quinolinas/antagonistas & inibidores , Veias Umbilicais/citologia , Veias Umbilicais/efeitos dos fármacos , Adulto , Asma/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ciclopropanos , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucina-13/farmacologia , Masculino , Fator de Ativação de Plaquetas/efeitos dos fármacos , Fator de Ativação de Plaquetas/fisiologia , RNA Mensageiro/genética , Receptores de Leucotrienos/genética , Sensibilidade e Especificidade , SulfetosRESUMO
Platelet-activating factor (PAF) is an early product of the inflammatory environment, influencing development and resolution of inflammation. Its production is greater in neutrophils and macrophages, which predominantly synthesize 1-alkyl sn-2 acetyl glycerophosphocholine (GPC) than in nongranulocytes (B cells and endothelial cells), which lack a respiratory burst and synthesize 1-acyl sn-2 acetyl GPC as their major PAF species. This study investigated whether the respiratory burst was responsible for the quantitative and qualitative differences in sn-2 acetyl GPC species generation by neutrophils and macrophages versus those cells lacking the NADPH oxidase complex. The myeloid cell line PLB-985 (capable of differentiation into neutrophils) was used to test this hypothesis, since these cells had previously been generated with a non-functional respiratory burst (X-CGD PLB-985). Differentiated PLB-985 cells underwent a large respiratory burst in response to PMA (phorbol ester), and smaller respiratory bursts in response to A23187 (calcium ionophore), and the bacterial polypeptide fMLP (receptor mediated activation). Concurrently, treated cells were assessed for production of 1-hexadecyl and 1-palmitoyl sn-2 acetyl GPC species by gas chromatography/mass spectrometry. Neither cell type generated these lipid species in response to PMA, but both cell types generated equal levels of sn-2 acetyl GPC in response to A23187, with five times more 1-hexadecyl than 1-palmitoyl species. Upon fMLP activation, X-CGD PLB-985 cells produced significantly less 1-hexadecyl and 1-palmitoyl sn-2 acetyl GPC in comparison to the wild-type PLB-985 cells. These findings suggest phagocytic oxidant production by NADPH oxidase is not essential for sn-2 acetyl GPC generation, but appears important for optimal production of PAF in response to some stimuli.
Assuntos
Glicerilfosforilcolina/análogos & derivados , N-Formilmetionina Leucil-Fenilalanina/análogos & derivados , NADPH Oxidases/metabolismo , Fator de Ativação de Plaquetas/análogos & derivados , Fator de Ativação de Plaquetas/metabolismo , Calcimicina , Cálcio/metabolismo , Linhagem Celular , Cromatografia Gasosa-Espectrometria de Massas , Glicerilfosforilcolina/análise , Glicerilfosforilcolina/metabolismo , Humanos , Ionóforos , Leucemia Mieloide , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Fosfatidilcolinas , Fator de Ativação de Plaquetas/análise , Fator de Ativação de Plaquetas/efeitos dos fármacos , Receptores de Formil Peptídeo , Receptores Imunológicos/efeitos dos fármacos , Receptores Imunológicos/metabolismo , Receptores de Peptídeos/efeitos dos fármacos , Receptores de Peptídeos/metabolismo , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Stimulation of rat peritoneal macrophages by thapsigargin (46.1 nM) increased levels of tumor necrosis factor-alpha and prostaglandin E2 in the conditioned medium. Platelet-activating factor (PAF) was not detected in the conditioned medium, but the level of cell-associated PAF was increased transiently by thapsigargin. The PAF receptor antagonists such as E6123 ((S)-(+)-6-(2-chlorophenyl)-3-cyclopro-panecarbonyl-8,11-dim ethyl-2,3,4,5-tetrahydro-8 H-pyrido[4',3':4,5]thieno [3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine), L-652,73 1 (2,5-bis(3,4,5-trimethoxyphenyl) tetrahydrofuran) and CV-6209 (2-[N-acetyl-N-(2-methoxy-3-octadecyl-carbamoyloxy propoxycarbonyl)aminomethyl]-1-ethylpyridinium chloride) inhibited thapsigargin-induced production of tumor necrosis factor-alpha. The cyclooxygenase inhibitor indomethacin inhibited prostaglandin E2 production, and further enhanced thapsigargin-induced tumor necrosis factor-alpha production in parallel with further increase in cell-associated PAF production. The enhancement of tumor necrosis factor-alpha production induced by thapsigargin plus indomethacin was also inhibited by E6123, L-652,731 and CV-6209. However, exogenously added PAF up to 100 nM did not stimulate production of tumor necrosis factor-alpha. The level of tumor necrosis factor-alpha mRNA was increased by thapsigargin, but was lowered by the PAF receptor antagonist E6123, suggesting that the inhibition of tumor necrosis factor-alpha production by the PAF receptor antagonist is induced at the level of mRNA for tumor necrosis factor-alpha. These findings suggested that concurrently produced cell-associated PAF in thapsigargin-stimulated macrophages up-regulates production of tumor necrosis factor-alpha by acting as an intracellular signaling molecule and the PAF receptor antagonists might penetrate into the cells and antagonize the action of intracellular PAF.
Assuntos
Macrófagos Peritoneais/metabolismo , Fator de Ativação de Plaquetas/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Animais , Azepinas/farmacologia , Células Cultivadas , Dinoprostona/biossíntese , Inibidores Enzimáticos/farmacologia , Furanos/farmacologia , Imidazóis/farmacologia , Indometacina/farmacologia , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/efeitos dos fármacos , Masculino , Compostos Organofosforados/farmacologia , Fator de Ativação de Plaquetas/efeitos dos fármacos , Fator de Ativação de Plaquetas/farmacologia , Compostos de Piridínio/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Tapsigargina/farmacologia , Triazóis/farmacologia , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genéticaRESUMO
We have recently shown that modified natural pulmonary surfactant Curosurf inhibits the synthesis of type II phospholipase A2 (sPLA2-II) by cultured guinea-pig alveolar macrophages (AM). The goal of the present study was to identify the surfactant components and the mechanisms involved in this process. We show that protein-free artificial surfactant (AS) mimicked the inhibitory effect of Curosurf, suggesting that phospholipid components of surfactant play a role in the inhibition of sPLA2-II expression. Among surfactant phospholipids, dioleylphosphatidylglycerol (DOPG) was the most effective in inhibiting the synthesis of sPLA2-II. By contrast, the concentrations of platelet-activating factor (PAF)-acetylhydrolase and lysophospholipase activities remained unchanged, indicating that inhibition of sPLA2-II synthesis was caused by a specific effect of surfactant. The effect of DOPG on sPLA2-II synthesis was concentration-dependent and was accompanied by a rapid and time-dependent uptake of DOPG by AM whereas dipalmitoylphosphatidylcholine (DPPC) was only marginally taken up. Curosurf, AS, and DOPG inhibited tumor necrosis factor-alpha (TNF-alpha) secretion, a key step in the induction of sPLA2-II synthesis by AM, in contrast to DPPC which had only a marginal effect. We conclude that phospholipid components, especially DOPG, play a major role in the inhibition of sPLA2-II synthesis by surfactant and that this effect can be explained, at least in part, by an impairment of TNF-alpha secretion.
Assuntos
Produtos Biológicos , Macrófagos Alveolares/efeitos dos fármacos , Fosfatidilgliceróis/farmacologia , Fosfolipases A/antagonistas & inibidores , Fosfolipídeos , 1,2-Dipalmitoilfosfatidilcolina/farmacologia , 1-Alquil-2-acetilglicerofosfocolina Esterase , Animais , Células Cultivadas , Expressão Gênica , Fosfolipases A2 do Grupo II , Cobaias , Lisofosfolipase/efeitos dos fármacos , Lisofosfolipase/metabolismo , Macrófagos Alveolares/citologia , Macrófagos Alveolares/enzimologia , Masculino , Fosfolipases A/efeitos dos fármacos , Fosfolipases A/genética , Fosfolipases A/metabolismo , Fosfolipases A2 , Fator de Ativação de Plaquetas/efeitos dos fármacos , Fator de Ativação de Plaquetas/metabolismo , Surfactantes Pulmonares/farmacologia , RNA Mensageiro/metabolismo , Espectrometria de Fluorescência , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismoAssuntos
Complexo AIDS Demência/metabolismo , Macrófagos/metabolismo , Microglia/metabolismo , Fator de Ativação de Plaquetas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Complexo AIDS Demência/tratamento farmacológico , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , HIV-1/metabolismo , Humanos , Macrófagos/efeitos dos fármacos , Microglia/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fator de Ativação de Plaquetas/efeitos dos fármacos , Receptores de AMPA/efeitos dos fármacos , Receptores de AMPA/metabolismoRESUMO
Platelet-activating factor (PAF) has been shown, in the rat model of pleural inflammation, to induce the generation of an intermediate proteic factor able to cause eosinophil proliferation in vitro. This study was undertaken to investigate the effect of the anti-mitotic compound doxorubicin on PAF-induced eosinophilia in rats, in order to evaluate the contribution of local cell proliferation to this phenomenon. The late eosinophil infiltration caused by another chemoattractant leukotriene B4 was used for comparison. We observed that local treatment with doxorubicin (20 and 40 microg/cavity), given 6 h after PAF (1 microg/cavity), suppressed the eosinophil accumulation within 24 h, whilst only the higher dose was effective when the drug was given 12 h post-PAF. An effect on chemotaxis was ruled out, since local doxorubicin (40 microg/cavity) failed to modify the eosinophil migration noted 24 h after leukotriene B4 (0.5 microg/cavity) and the neutrophil/eosinophil infiltration noted at 6 h after PAF injection. Transfer of the pleural fluids collected 6 h after PAF from donors to recipient rats caused significant eosinophil accumulation in the recipient rats, an effect which was inhibited by the co-administration of doxorubicin (40 microg/cavity). No inhibitory effect was noted when the drug was given 6 h after the pleural fluids were transferred. We also found no change in the number of blood or bone marrow eosinophils after PAF stimulation. We conclude that doxorubicin selectively impaired the late eosinophil accumulation triggered by PAF in the pleural cavity of rats, clearly indicating that local cell proliferation seems to contribute to the development of this inflammatory response.