RESUMO
This study investigates the role of sphingosylphosphorylcholine (SPC) in the mechanisms underlying cerebral vasospasm after subarachnoid hemorrhage (SAH). The levels of SPC were measured in cerebrospinal fluid (CSF) of patients with SAH and also in an experimental canine model. CSF samples were collected from 11 patients with SAH, and from dogs that had received an injection of SPC into the cisterna magna to examine SPC kinetics in the CSF. SPC was assayed using solid-phase extraction and triple quadrupole mass spectrometry. The SPC concentrations in SAH patients on days 3, 8, and 14 after the onset of SAH were significantly higher than those in normal CSF. In the canine model, rapid dilution of SPC in CSF was observed. In combination with data from previous studies, these results suggest that SPC is involved in the development of cerebral vasospasm. Rapid dilution of SPC in CSF suggests that SPC is released into CSF at higher concentrations than those measured in the present study.
Assuntos
Fosforilcolina/análogos & derivados , Esfingosina/análogos & derivados , Hemorragia Subaracnóidea/líquido cefalorraquidiano , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Animais , Calibragem , Cromatografia Líquida de Alta Pressão , Cães , Feminino , Humanos , Cinética , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Fosforilcolina/líquido cefalorraquidiano , Fosforilcolina/química , Análise de Regressão , Extração em Fase Sólida , Esfingosina/líquido cefalorraquidiano , Esfingosina/químicaRESUMO
Hypercholesterolemia is a major risk factor involved in abnormal cardiovascular events. Rho-kinase-mediated Ca(2+) sensitization of vascular smooth muscle (VSM) plays a critical role in vasospasm and hypertension. We recently identified sphingosylphosphorylcholine (SPC) and Src family tyrosine kinase (Src-TK) as upstream mediators for the Rho-kinase-mediated Ca(2+) sensitization. Here we report the strong linkage between cholesterol and the Ca(2+) sensitization of VSM mediated by a novel SPC/Src-TK/Rho-kinase pathway in both humans and rabbits. The extent of the sensitization correlated well with the total cholesterol or low-density lipoprotein cholesterol levels in serum. However, an inverse correlation with the serum level of high-density lipoprotein cholesterol was observed, and a correlation with other cardiovascular risk factors was nil. When cholesterol-lowering therapy was given to patients and rabbits with hypercholesterolemia, the SPC-induced contractions diminished. Depletion of VSM cholesterol by beta-cyclodextrin resulted in a loss of membrane caveolin-1, a marker of cholesterol-enriched lipid raft, and inhibited the SPC-induced Ca(2+) sensitization and translocation of Rho-kinase from cytosol to the cell membrane. Vasocontractions induced by membrane depolarization and by an adrenergic agonist were cholesterol-independent. Our data support the previously unreported concept that cholesterol potentiates the Ca(2+) sensitization of VSM mediated by a SPC/Src-TK/Rho-kinase pathway, and are also compatible with a role for cholesterol-enriched membrane microdomain, a lipid raft. This process may play an important role in the development of abnormal vascular contractions in patients with hypercholesterolemia.
Assuntos
Cálcio/fisiologia , Colesterol/fisiologia , Microdomínios da Membrana/metabolismo , Músculo Liso Vascular/fisiologia , Fosforilcolina/análogos & derivados , Proteínas Serina-Treonina Quinases/fisiologia , Esfingosina/análogos & derivados , Animais , Vasos Coronários/patologia , Feminino , Humanos , Hipercolesterolemia/patologia , Hipercolesterolemia/fisiopatologia , Hipercolesterolemia/terapia , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Contração Muscular , Fosforilcolina/metabolismo , Coelhos , Esfingosina/metabolismo , Quinases Associadas a rhoRESUMO
BACKGROUND AND PURPOSE: Rho-kinase (ROK)-mediated Ca2+ sensitization of vascular smooth muscle (VSM) contraction plays a pivotal role in cerebral vasospasm (CV). We previously demonstrated that sphingosylphosphorylcholine (SPC) induces Ca2+ sensitization through sequential activation of the Src family protein tyrosine kinases (Src-PTKs) and ROK in vitro, and that Ca2+ sensitization is inhibited by eicosapentaenoic acid (EPA) through the selective inactivation of Src-PTK. In this study, we examined whether SPC induced CV in vivo, and, if it did, whether EPA would inhibit CV, as induced by SPC or in an in vivo model of subarachnoid hemorrhage (SAH). METHODS: Changes in the diameter of the canine basilar artery were investigated by angiography after administering SPC into the cisterna magna. Then, Y27632, a specific Rho-kinase inhibitor, or EPA was injected intracisternally and the effects of both agents were investigated. In another experiment using a single-hemorrhage model, Y27632 or EPA was injected on day 7 after SAH and the changes in the diameter of the canine basilar artery were investigated. RESULTS: At cerebrospinal fluid concentrations of 100 and 300 micromol/l, SPC induced severe vasoconstriction (maximum vasoconstriction by SPC (100 micromol/l): 61.8 +/- 8.2%), which was markedly reversed by Y27632 (96.3 +/- 4.4%) or EPA (92.6 +/- 12.8%). SAH caused severe vasospasm on day 7 (67.6 +/- 7.8%), which was significantly blocked by Y27632 (95.5 +/- 10.6%) or EPA (90.0 +/- 4.4%). CONCLUSIONS: SPC is a novel mediator of ROK-induced CV in vivo. The inhibition of CV induced by SPC or after SAH by EPA suggests beneficial roles of EPA in the treatment of CV. Our findings are compatible with the notion that the SPC-ROK pathway may be involved in CV.
Assuntos
Artéria Basilar/efeitos dos fármacos , Ácido Eicosapentaenoico/farmacologia , Fosforilcolina/análogos & derivados , Esfingosina/análogos & derivados , Hemorragia Subaracnóidea/tratamento farmacológico , Vasoconstrição/efeitos dos fármacos , Vasodilatadores/farmacologia , Vasoespasmo Intracraniano/prevenção & controle , Quinases Associadas a rho/metabolismo , Animais , Artéria Basilar/diagnóstico por imagem , Artéria Basilar/enzimologia , Benzopiranos/farmacologia , Angiografia Cerebral , Cisterna Magna/irrigação sanguínea , Cisterna Magna/efeitos dos fármacos , Cisterna Magna/enzimologia , Modelos Animais de Doenças , Cães , Ácido Eicosapentaenoico/administração & dosagem , Feminino , Injeções , Masculino , Fosforilcolina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Esfingosina/metabolismo , Hemorragia Subaracnóidea/complicações , Hemorragia Subaracnóidea/diagnóstico por imagem , Hemorragia Subaracnóidea/enzimologia , Fatores de Tempo , Vasodilatadores/administração & dosagem , Vasoespasmo Intracraniano/diagnóstico por imagem , Vasoespasmo Intracraniano/enzimologia , Vasoespasmo Intracraniano/etiologia , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
Human heart failure is preceded by a process called cardiac remodeling, in which heart chambers progressively enlarge and contractile function deteriorates. Programmed cell death (apoptosis) of cardiac muscle cells has been identified as an essential process in the progression to heart failure. The execution of the apoptotic program entails complex interactions between and execution of multiple molecular subprograms. Endothelin (ET)-1, a potent vasoconstrictor peptide, is synthesized and secreted by cardiomyocytes and induces hypertrophy of cardiomyocytes. The cardiovascular benefit of fish oil containing eicosapentaenoic acid (EPA) in humans and experimental animals was reported. Recently, we found that ET-1-induced cardiomyocytic remodeling could be prevented by pretreatment with EPA. The aim of the present study is to investigate whether there would be any alteration in the expression of important apoptosis-related molecules in ET-1-administered hypertrophied cardiomyocytes. We also sought to determine, if there are alterations in apoptotic molecules, what type of role for EPA would then exist. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats and were cultured for 3 days. At Day 4 of culture, the cardiomyocytes were divided into three groups: control, the ET-1 (0.1 nM)-treated group, and the ET-1 group pretreated with EPA (10 microM). Twenty-four hours after the treatment, the gene expressions of three important molecules related to apoptosis (caspase-3, Bax, and Bcl-2) in three experimental groups were evaluated by real-time polymerase chain reaction. The present study could not demonstrate any significant or representative alteration in any of the above three apoptosis-related important markers in either ET-1-induced hypertrophied cardiomyocytes with or without EPA pretreatment. The present study would at least be able to exclude the involvement of some representative molecules related to apoptosis in ET-1-induced hypertrophied cardiomyocytes. In addition, the present study demonstrates that the antihypertrophic effect of EPA to ET-1-administered cardiomyocytes appears not to modulate the apoptosis signaling cascade.
Assuntos
Apoptose/efeitos dos fármacos , Ácido Eicosapentaenoico/farmacologia , Endotelina-1/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Animais , Animais Recém-Nascidos , Caspase 3 , Caspases/metabolismo , Células Cultivadas , Ventrículos do Coração/citologia , Hipertrofia/induzido quimicamente , Hipertrofia/patologia , Miócitos Cardíacos/citologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína X Associada a bcl-2/metabolismoRESUMO
The cardiovascular benefit of fish oil, including eicosapentaenoic acid (EPA), in humans and experimental animals has been reported. The role of endothelin-1 (ET-1) in cardiac hypertrophy is well known. Endothelin-1 stimulates prepro-ET-1 mRNA expression in cardiomyocytes, and the autocrine/paracrine system of ET-1 is important for cardiomyocyte hypertrophy. Although many studies link EPA to cardiac protection, the effect of EPA on cardiac hypertrophy has yet to be clarified. Recently, we demonstrated that ET-1-induced cardiomyocytic change could be prevented by pretreatment with EPA. The present study investigated the changes of different components of the ET system at the mRNA level in ET-1-administered cardiomyocytes, and examined the effect of EPA pretreatment. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats, cultured in Dulbecco's modified Eagle's medium and Ham F12 supplemented with 0.1% fatty acid-free bovine serum albumin for 3 days. At Day 4 of culture, the cardiomyocytes were divided into 3 groups: control group, ET-1-treated (0.1 nM) group, and ET-1-treated group pretreated with EPA (10 microM). Twenty-four hours after treatment, the gene expressions of different components of the endothelin system in three experimental groups were evaluated by real-time polymerase chain reaction. Prepro-ET-1 mRNA expression was 53% upregulated in ET-1-induced hypertrophied cardiomyocytes and suppressed in the EPA-pretreated group. Endothelin-converting enzyme-1 (ECE-1) was also increased in ET-1-administered cardiomyocytes by 42% compared with the control group and was reversed in the EPA-pretreated group. The two receptors of ET system, ET(A) and ET(B), tended to be increased in the ET-1-treated group, but no statistical significance was seen among study groups. Endothelin-1 increased prepro-ET-1 and ECE-1 mRNA expression in hypertrophied-neonatal cardiomyocytes, and this was reversed with EPA pretreatment. Thus, EPA may play a crucial role in the regression of ET-1-induced cardiomyocyte hypertrophy, partly through the suppression of ET-1 and ECE-1 expression.
Assuntos
Ácido Eicosapentaenoico/farmacologia , Endotelina-1/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Animais , Animais Recém-Nascidos , Ácido Aspártico Endopeptidases/metabolismo , Células Cultivadas , Endotelina-1/farmacologia , Enzimas Conversoras de Endotelina , Ventrículos do Coração/citologia , Hipertrofia/induzido quimicamente , Hipertrofia/patologia , Metaloendopeptidases/metabolismo , RNA Mensageiro/metabolismo , RatosRESUMO
Cardiomyocytes release (or metabolize) several diffusible agents (e.g., nitric oxide [NO], endothelin-1 [ET-1], and angiotensin II) that exert direct effects on myocyte function under various pathologic conditions. Although cardiac hypertrophy is a compensatory mechanism in response to different cardiovascular diseases, there can be a pathologic transition in which the myocardium becomes dysfunctional. Recently, NO has been found to be an important regulator of cardiac remodeling. Specifically, NO has been recognized as a potent antihypertrophic and proapoptotic mediator in cultured cardiomyocytes. We demonstrated that ET-1-induced hypertrophic remodeling in neonatal cardiomyocytes was arrested by pretreatment with eicosapentaenoic acid (EPA), a major component of fish oil. In some recent studies, EPA has demonstrated cardioprotective effects by modulating NO. This study investigated the changes in NO synthase (NOS) in ET-1-induced hypertrophied cardiomyocytes and in total levels of nitrates and nitrites. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats and were cultured in D-MEM/Ham F12 supplemented with 0.1% fatty acid-free bovine serum albumin for 3 days. At Day 4 of culture, the cardiomyocytes were divided into three groups: control group, ET-1 (0.1 nM) group, and ET-1 pretreated with EPA (10 microM) group. NOS gene expression was evaluated 24 hrs after treatment using real-time polymerase chain reaction. Endothelial NOS (eNOS) mRNA expression was decreased in the ET-1 group compared with controls and was unchanged by pretreatment with EPA. mRNA expression of inducible NOS (iNOS) was significantly increased in ET-1-treated cardiomyocytes and was suppressed by EPA pretreatment. Neuronal NOS gene expression and total NO level did not exhibit a statistically significant change in any of the groups. There may be some interaction between ET-1, eNOS, and iNOS in ET-1-induced and EPA-regressed hypertrophied cardiomyocytes that suppress iNOS expression without modulating total NO level or eNOS gene expression.
Assuntos
Ácido Eicosapentaenoico/farmacologia , Endotelina-1/farmacologia , Miócitos Cardíacos/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/análise , Animais , Animais Recém-Nascidos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Hipertrofia/induzido quimicamente , Hipertrofia/patologia , Miócitos Cardíacos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Nitritos/análise , Nitritos/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
cis-Polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) are the major fatty acids contained in fish oil, and are known to affect the various physiological properties of cell membranes in humans. The present study investigated the effects of polyunsaturated fatty acids on endothelin-1 (ET-1) production in human umbilical vein endothelial cells (HUVECs) and on insulin activity. After addition of various concentrations of EPA, docosahexaenoic acid, arachidonic acid, or linoleic acid to a culture medium, the concentration of ET-1 was measured using ELISA, and that of ET-1 mRNA was determined by RT-PCR. The results showed that EPA had the strongest inhibitory effect (p<0.05) on both basal ET-1 production and ET-1 mRNA levels. In addition, insulin (1 micromol/l) markedly increased ET-1 production, and EPA also significantly decreased the effect induced by insulin. Pretreatment with Ca2+ chelator EGTA (1 mmol/l), NOS inhibitor L-NAME (300 micromol/l), or calmodulin antagonist W-7 (300 micromol/l) inhibited NO production by EPA (100 micromol/l), but these pretreatments had no effect on ET-1 production by EPA. These findings suggest that EPA reduces basal and insulin-enhanced ET-1 production by inhibiting ET-1 mRNA production. These effects of EPA may contribute to its vasorelaxant and anti-atherosclerotic effects.
Assuntos
Endotelina-1/genética , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Ácidos Graxos Insaturados/farmacologia , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Células Cultivadas , Quelantes/farmacologia , Interações Medicamentosas , Ácido Egtázico/farmacologia , Ácido Eicosapentaenoico , Endotelina-1/metabolismo , Endotélio Vascular/citologia , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , RNA Mensageiro/análise , Sulfonamidas/farmacologia , Veias Umbilicais/citologiaRESUMO
ω-3 fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have beneficial effects on cardiovascular diseases. Cytochrome P-450 (CYP) 2J2 that is expressed in endothelial cells metabolizes arachidonic acids to biologically active epoxyeicosatrienoic acids (EETs) that possess anti-inflammatory and anti-thrombotic effects. We studied the effects of EPA and DHA on the expression of CYP 2J2 mRNA by reverse transcription-polymerase chain reaction in cultured human umbilical vein endothelial cells and found that EPA, but not DHA, increased the expression of CYP 2J2 mRNA in a dose-dependent and a time-dependent manner. EPA-induced CYP 2J2 expression was significantly inhibited by pretreatment with a peroxisome proliferator-activated receptor (PPAR) γ antagonist, GW9662. EPA, but not DHA, caused a significant increase in cellular levels of 11,12-dihydroxyeicosatrienoic acid that is a stable metabolite of 11,12-EET, which was blocked by pretreatment with GW9662. These data demonstrate that EPA increases CYP 2J2 mRNA expression and 11,12-EET production via PPARγ in endothelial cells and indicate a novel protective role of EPA and PPARγ against vascular inflammation.
Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Sistema Enzimático do Citocromo P-450/genética , Ácido Eicosapentaenoico/farmacologia , Células Endoteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , PPAR gama/fisiologia , RNA Mensageiro/metabolismo , Ácido 8,11,14-Eicosatrienoico/metabolismo , Anilidas/farmacologia , Ácidos Araquidônicos , Células Cultivadas , Sistema Enzimático do Citocromo P-450/fisiologia , Ácidos Docosa-Hexaenoicos/farmacologia , Relação Dose-Resposta a Droga , Humanos , PPAR gama/antagonistas & inibidores , Estimulação Química , Fatores de Tempo , Veias Umbilicais/citologiaRESUMO
We investigated the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the stability of Kv1.5 channel protein. The expression and function of Kv1.5 (Kv1.5-FLAG) in transfected African green monkey kidney fibroblast cells as well as rat atrium were estimated by immunoblotting, immunoprecipitation, immunofluorescence and patch-clamp techniques. Both EPA and DHA immediately blocked Kv1.5 channel current in a dose-dependent manner, accompanied by reduction of their phosphorylation. Chronic treatment (for 12 h) with EPA at lower concentrations (0.3-10 muM) increased the level of Kv1.5-FLAG protein as well as Kv1.5 channel current without changes in its gating kinetics, prolonging its half-life; in contrast, both EPA and DHA at higher concentrations (30-100 muM) decreased the expression of Kv1.5-FLAG. EPA at the higher concentrations also decreased mRNA of Kv1.5 and synapse-associated protein 97 expression. EPA at the lower concentrations increased Kv1.5 expression in the endoplasmic reticulum, Golgi apparatus and cell membrane. EPA-induced increase of Kv1.5 channel expression and current was abolished by pretreatment with the protein transport inhibitor brefeldin A or colchicines, and by the Kv1.5 channel blocker 4-aminopyridine. Oral administration of EPA (30 mg/kg) increased the level of endogenous Kv1.5 in rat atria. These results indicate that chronic treatment with EPA at lower concentrations stabilizes Kv1.5 channel protein in the endoplasmic reticulum and Golgi apparatus thereby enhancing the Kv1.5 channel current on the cell membrane.
Assuntos
Ácido Eicosapentaenoico/análogos & derivados , Fibroblastos/efeitos dos fármacos , Canal de Potássio Kv1.5/biossíntese , Ácido Tióctico/análogos & derivados , Potenciais de Ação/efeitos dos fármacos , Animais , Western Blotting , Células COS , Chlorocebus aethiops , Ácidos Docosa-Hexaenoicos/farmacologia , Relação Dose-Resposta a Droga , Ácido Eicosapentaenoico/farmacologia , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Imunoprecipitação , Oligopeptídeos , Técnicas de Patch-Clamp , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Ratos , Ratos Endogâmicos WKY , Proteínas Recombinantes de Fusão/metabolismo , Ácido Tióctico/farmacologia , TransfecçãoRESUMO
OBJECTIVE: To investigate the anti-inflammatory effect of n-3 eicosapentaenoic acid (EPA) compared with n-6 linoleic acid (LA) in an endometriosis rat model. We focused on the relationship between lipid metabolism and inflammatory reactions in endometriosis based on the hypothesis that a lipid intake imbalance is one of the factors responsible for the recent increase of endometriosis. DESIGN: Prospective, randomized experimental study. SETTING: Animal surgery laboratory in a university hospital. ANIMAL(S): Sprague-Dawley rats (female, 6 weeks old). INTERVENTION(S): Rats were fed a diet with EPA (n = 9) or with LA (n = 9) for 2 weeks. Two weeks after feeding, the uterus was autotransplanted to the peritoneum to construct an endometriosis model. Feeding was continued for a total of 6 weeks. Two and 4 weeks after autotransplantation, three rats of each group were killed and evaluated. MAIN OUTCOME MEASURE(S): Endometriotic lesions were morphologically evaluated and their fatty acid composition was examined. Gene expression in these tissues was evaluated by cDNA microarray analysis and quantative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULT(S): In the EPA group, the n-3:n-6 ratio in each tissue significantly increased and the thickening of the interstitium, an active site for inflammation in endometriosis, was significantly suppressed (0.30 +/- 0.09 mm [EPA group] vs. 0.77 +/- 0.23 mm [LA group]). The mRNA of metalloproteinases, interleukin-1beta, interleukin-1r, prostaglandin E synthase (Ptges), and nuclear factor (NF)-kappaB were reduced in the EPA group. CONCLUSION(S): EPA supplementation might be a valid strategy for the treatment of endometriosis.
Assuntos
Suplementos Nutricionais , Ácido Eicosapentaenoico/administração & dosagem , Endometriose/dietoterapia , Administração Oral , Animais , Endometriose/imunologia , Endometriose/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Ácido Linoleico/administração & dosagem , Metabolismo dos Lipídeos/efeitos dos fármacos , Distribuição Aleatória , Ratos , Ratos Sprague-DawleyRESUMO
Accumulating evidence demonstrates that dietary intake of n-3 polyunsaturated fatty acids (PUFAs) is associated with reduced incidence of cardiovascular events. However, the molecular mechanisms by which n-3 PUFAs prevent atherosclerosis are not fully understood. Here, we examined the effect of eicosapentaenoic acid (EPA), a major n-3 PUFA, on the pathogenesis of atherosclerosis in ApoE-deficient mice. Five-week-old ApoE-deficient male mice were fed on western-type diet supplemented with 5% (w/w) EPA (EPA group, n=7) or not (control group, n=5) for 13 weeks. An analysis of the fatty acid composition of liver homogenates revealed a marked increase of the n-3 PUFA content in the EPA group (n-3/n-6 ratio: 0.20+/-0.01 vs. 2.5+/-0.2, p<0.01). En face Sudan IV staining of the aorta and oil red O-staining of the aortic sinus revealed that EPA significantly suppressed the development of atherosclerotic lesions. We also observed anti-atherosclerotic effects of EPA in LDL-receptor-deficient mice. The lesions of the EPA group contained more collagen (19.6+/-2.4% vs. 32.9+/-3.9%, p<0.05) and smooth muscle cells (1.3+/-0.2% vs. 3.6+/-0.8%, p<0.05) and less macrophages (32.7+/-4.1% vs. 14.7+/-2.0%, p<0.05). Pretreatment with EPA attenuated the up-regulation of VCAM-1, ICAM-1 and MCP-1 in HUVECs as well as the expression of MMP-2 and MMP-9 in macrophage-like cells induced by TNF-alpha. The anti-inflammatory effects of EPA were abrogated when the expression of peroxisome proliferator-activated receptor alpha (PPARalpha) was suppressed. EPA may potentially reduce and stabilize atherosclerotic lesions through its anti-inflammatory effects.
Assuntos
Apolipoproteínas E/deficiência , Aterosclerose/dietoterapia , Gorduras Insaturadas na Dieta/farmacologia , Ácido Eicosapentaenoico/farmacologia , Inflamação/dietoterapia , Animais , Aorta/patologia , Aterosclerose/fisiopatologia , Células Cultivadas , Quimiocina CCL2/metabolismo , Gorduras Insaturadas na Dieta/administração & dosagem , Modelos Animais de Doenças , Ácido Eicosapentaenoico/administração & dosagem , Células Endoteliais , Endotélio Vascular , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Masculino , Camundongos , Camundongos Knockout , Veias Umbilicais/citologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
The cardiovascular benefit of fish oil in humans and experimental animals has been reported. Endothelin (ET)-1 is a well-known cardiac hypertrophic factor. However, although many studies link a fish oil extract, eicosapentaenoic acid (EPA), to cardiac protection, the effects of EPA on cardiac hypertrophy and underlying mechanism(s) are unclear. The present study investigated whether EPA prevents ET-1-induced cardiomyocyte hypertrophy; the potential pathways likely to underlie such an effect were also investigated. Cardiomyocytes were isolated from neonatal rat heart, cultured for 3 days, and then treated for 24 h with vehicle only (control), treated with 0.1 nM ET-1 only, or pretreated with 10 microM EPA and then treated with 0.1 nM ET-1. The cells were harvested, and changes in cell surface area, protein synthesis, expression of a cytoskeletal (alpha-actinin) protein, and cell signaling were analyzed. ET-1 induced a 97% increase in cardiomyocyte surface area, a 72% increase in protein synthesis rate, and an increase in expression of alpha-actinin and signaling molecule [transforming growth factor-beta 1 (TGF-beta 1), c-Jun NH2-terminal kinase (JNK), and c-Jun]. Development of these ET-1-induced cellular changes was attenuated by EPA. Moreover, the hypertrophied cardiomyocytes showed a 1.5- and a 1.7-fold increase in mRNA expression of atrial and brain natriuretic peptides, the classical molecular markers of cardiac hypertrophy, respectively; these changes were also suppressed by EPA. Here we show that ET-1 induces cardiomyocyte hypertrophy and expression of hypertrophic markers, possibly mediated by JNK and TGF-beta 1 signaling pathways. These ET-1-induced effects were blocked by EPA, a major fish oil ingredient, suggesting that fish oil may have beneficial protective effects on cardiac hypertrophy.
Assuntos
Cardiomegalia/induzido quimicamente , Cardiomegalia/prevenção & controle , Ácido Eicosapentaenoico/farmacologia , Endotelina-1/antagonistas & inibidores , Endotelina-1/toxicidade , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Miócitos Cardíacos/patologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Fator Natriurético Atrial/fisiologia , Biomarcadores , Western Blotting , Cardiomegalia/patologia , Tamanho Celular , Células Cultivadas , Endotelina-1/biossíntese , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Imuno-Histoquímica , Peptídeo Natriurético Encefálico/fisiologia , Fosforilação , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta1RESUMO
This study investigated acute and chronic effects of eicosapentaenoic acid (EPA) on voltage-gated Na+ current (I(Na)) expressed in cultured human bronchial smooth muscle cells (hBSMCs). The whole-cell voltage clamp technique and quantitative real-time RT-PCR analysis were applied. The alterations in the fatty acid composition of phospholipids after treatment with EPA were also examined. Extracellular application of EPA produced a rapid and concentration-dependent suppression of tetrodotoxin-sensitive I(Na) with the half-maximal inhibitory concentration of 2 microM. After washing out EPA with albumin, I(Na) returned to the control level. Similar inhibitory effects were observed regarding other fatty acids (docosahexaenoic, arachidonic, stearic, and oleic acids), but EPA was the most potent inhibitor. The effect of EPA on I(Na) was not blocked by nordihydroguaiaretic acid and indometacin, and was accompanied by a significant shift of the steady-state inactivation curve to more negative potentials. In cells chronically treated with EPA, the EPA content of the cell lipid fraction (mol%) increased time-dependently, while arachidonic acid (AA) decreased, resulting in an increase of EPA to AA ratio. Then, the level of mRNA (SCN9A) encoding I(Na) decreased significantly. These results provide novel evidence that EPA not only rapidly inhibits I(Na), but also reduces the mRNA levels of the Na+ channel after cellular incorporation of EPA in cultured hBSMCs.
Assuntos
Brônquios/efeitos dos fármacos , Ácido Eicosapentaenoico/farmacologia , Ativação do Canal Iônico , Músculo Liso/efeitos dos fármacos , Canais de Sódio/efeitos dos fármacos , Brônquios/citologia , Brônquios/metabolismo , Células Cultivadas , Humanos , Imuno-Histoquímica , Músculo Liso/citologia , Músculo Liso/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Sódio/genética , Canais de Sódio/metabolismoRESUMO
Intake of fish oil is known to have cardioprotective effects and reduce cardiovascular mortality. However, it is not widely recognized that eicosapentaenoic acid (EPA), one of the n-3 polyunsaturated fatty acids (PUFAs), exerts beneficial effects against myocardial ischemia/reperfusion injury. The purpose of this study is to investigate whether EPA attenuates the severity of myocardial ischemia/reperfusion injury and which cellular mechanism is involved. Rabbits were treated with or without EPA (600 mg/kg/day) for 2 weeks. Infarct size was measured in open-chest rabbits after 30-minute occlusion of the left anterior descending coronary artery (LAD) and after the subsequent 3-hour reperfusion. In several groups, NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase, or charybdotoxin, a blocker of calcium-activated potassium (K(Ca)) channels, was infused intravenously beginning 20 minutes before LAD occlusion and continuing during reperfusion. Infarct size was reduced in the group treated with EPA compared with the control group (7.2 +/- 1.0% vs 24.6 +/- 2.3%; P < 0.01). The occurrence of ventricular arrhythmias in the reperfusion period tended to decrease in the EPA group. Either L-NAME or charybdotoxin partially blunted or completely abolished the infarct size-limiting effect of EPA, respectively. Eicosapentaenoic acid significantly increased the n-3:n-6 ratio of PUFA. Eicosapentaenoic acid reduces myocardial infarct size, mainly via the opening of K(Ca) channel-mediated and partially NO-mediated mechanisms in rabbit hearts.