RESUMO
Arterial stiffness plays a causal role in development of systolic hypertension. 20-hydroxyeicosatetraeonic acid (20-HETE), a cytochrome P450 (CYP450)-derived arachidonic acid metabolite, is known to be elevated in resistance arteries in hypertensive animal models and loosely associated with obesity in humans. However, the role of 20-HETE in the regulation of large artery remodeling in metabolic syndrome has not been investigated. We hypothesized that elevated 20-HETE in metabolic syndrome increases matrix metalloproteinase 12 (MMP12) activation leading to increased degradation of elastin, increased large artery stiffness and increased systolic blood pressure. 20-HETE production was increased ~7 fold in large, conduit arteries of metabolic syndrome (JCR:LA-cp, JCR) vs. normal Sprague-Dawley (SD) rats. This correlated with increased elastin degradation (~7 fold) and decreased arterial compliance (~75% JCR vs. SD). 20-HETE antagonists blocked elastin degradation in JCR rats concomitant with blocking MMP12 activation. 20-HETE antagonists normalized, and MMP12 inhibition (pharmacological and MMP12-shRNA-Lnv) significantly improved (~50% vs. untreated JCR) large artery compliance in JCR rats. 20-HETE antagonists also decreased systolic (182⯱â¯3â¯mmHg JCR, 145⯱â¯3â¯mmHg JCRâ¯+â¯20-HETE antagonists) but not diastolic blood pressure in JCR rats. Whereas diastolic pressure was fully angiotensin II (Ang II)-dependent, systolic pressure was only partially Ang II-dependent, and large artery stiffness was Ang II-independent. Thus, 20-HETE-dependent regulation of systolic blood pressure may be a unique feature of metabolic syndrome related to high 20-HETE production in large, conduit arteries, which results in increased large artery stiffness and systolic blood pressure. These findings may have implications for management of systolic hypertension in patients with metabolic syndrome.
Assuntos
Pressão Sanguínea , Ácidos Hidroxieicosatetraenoicos/metabolismo , Hipertensão/enzimologia , Hipertensão/fisiopatologia , Metaloproteinase 12 da Matriz/metabolismo , Síndrome Metabólica/enzimologia , Síndrome Metabólica/fisiopatologia , Rigidez Vascular , Animais , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Complacência (Medida de Distensibilidade) , Citocromo P-450 CYP4A/metabolismo , Família 4 do Citocromo P450/metabolismo , Diástole/efeitos dos fármacos , Elastina/metabolismo , Ativação Enzimática/efeitos dos fármacos , Hipertensão/complicações , Losartan/farmacologia , Masculino , Síndrome Metabólica/complicações , Proteólise/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Ratos Sprague-Dawley , Rigidez Vascular/efeitos dos fármacosRESUMO
As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.
Assuntos
Benchmarking , Ensaio de Proficiência Laboratorial/estatística & dados numéricos , Lipídeos/sangue , Humanos , Cooperação Internacional , Metabolismo dos Lipídeos/fisiologia , Lipídeos/normas , Variações Dependentes do Observador , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Coronary collateral growth (CCG) is impaired in metabolic syndrome (MetS). microRNA-145 (miR-145-Adv) delivery to our rat model of MetS (JCR) completely restored and neutrophil depletion significantly improved CCG. We determined whether low endogenous levels of miR-145 in MetS allowed for elevated production of 20-hydroxyeicosatetraenoic acid (20-HETE), which, in turn, resulted in excessive neutrophil accumulation and endothelial dysfunction leading to impaired CCG. Rats underwent 0-9 days of repetitive ischemia (RI). RI-induced cardiac CYP4F (neutrophil-specific 20-HETE synthase) expression and 20-HETE levels were increased (4-fold) in JCR vs. normal rats. miR-145-Adv and 20-HETE antagonists abolished and neutrophil depletion (blocking antibodies) reduced (~60%) RI-induced increases in CYP4F expression and 20-HETE production in JCR rats. Impaired CCG in JCR rats (collateral-dependent blood flow using microspheres) was completely restored by 20-HETE antagonists [collateral-dependent zone (CZ)/normal zone (NZ) flow ratio was 0.76 ± 0.07 in JCR + 20-SOLA, 0.84 ± 0.05 in JCR + 20-HEDGE vs. 0.11 ± 0.02 in JCR vs. 0.84 ± 0.03 in normal rats]. In JCR rats, elevated 20-HETE was associated with excessive expression of endothelial adhesion molecules and neutrophil infiltration, which were reversed by miR-145-Adv. Endothelium-dependent vasodilation of coronary arteries, endothelial nitric oxide synthase (eNOS) Ser1179 phosphorylation, eNOS-dependent NO·- production and endothelial cell survival were compromised in JCR rats. These parameters of endothelial dysfunction were completely reversed by 20-HETE antagonism or miR-145-Adv delivery, whereas neutrophil depletion resulted in partial reversal (~70%). We conclude that low miR-145 in MetS allows for increased 20-HETE, mainly from neutrophils, which compromises endothelial cell survival and function leading to impaired CCG. 20-HETE antagonists could provide viable therapy for restoration of CCG in MetS.NEW & NOTEWORTHY Elevated 20-hydroxyeicosatetraenoic acid (20-HETE) impairs coronary collateral growth (CCG) in metabolic syndrome by eliciting endothelial dysfunction and apoptosis via excessive neutrophil infiltration. 20-HETE antagonists completely restore coronary collateral growth in metabolic syndrome. microRNA-145 (miR-145) is an upstream regulator of 20-HETE production in metabolic syndrome; low expression of miR-145 in metabolic syndrome promotes elevated production of 20-HETE.
Assuntos
Circulação Colateral/efeitos dos fármacos , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/crescimento & desenvolvimento , Endotélio Vascular/patologia , Ácidos Hidroxieicosatetraenoicos/metabolismo , Síndrome Metabólica/patologia , Animais , Anticorpos Bloqueadores/farmacologia , Arteríolas/efeitos dos fármacos , Capilares/efeitos dos fármacos , Moléculas de Adesão Celular/biossíntese , Vasos Coronários/patologia , Endotélio Vascular/metabolismo , Ácidos Hidroxieicosatetraenoicos/antagonistas & inibidores , Masculino , Síndrome Metabólica/metabolismo , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Neutrófilos/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Angiogenesis is an important adaptation for recovery from peripheral ischemia. Here, we determined whether 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to ischemia-induced angiogenesis and assessed its underlying molecular and cellular mechanisms using a mouse hindlimb-ischemia angiogenesis model. Hindlimb blood flow was measured by Laser Doppler Perfusion Imaging and microvessel density was determined by CD31 and tomato lectin staining. We found that systemic and local administration of a 20-HETE synthesis inhibitor, DDMS, or a 20-HETE antagonist, 6,15-20-HEDGE significantly reduced blood flow recovery and microvessel formation in response to ischemia. 20-HETE production, measured by LC/MS/MS, was markedly increased in ischemic muscles (91±11 vs. 8±2pg/mg in controls), which was associated with prominent upregulation of the 20-HETE synthase, CYP4A12. Immunofluorescence co-localized increased CYP4A12 expression in response to ischemia to CD31-positive EC in the ischemic hindlimb microvessels. We further showed that ischemia increased HIF-1α, VEGF, and VEGFR2 expression in gracilis muscles and that these increases were negated by DDMS and 6,15-20-HEDGE. Lastly, we showed that ERK1/2 of MAPK is a component of 20-HETE regulated ischemic angiogenesis. Taken together, these data indicate that 20-HETE is a critical contributor of ischemia-induced angiogenesis in vivo.
Assuntos
Ácidos Hidroxieicosatetraenoicos/metabolismo , Isquemia/metabolismo , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Transdução de Sinais , Indutores da Angiogênese/farmacologia , Inibidores da Angiogênese/farmacologia , Animais , Velocidade do Fluxo Sanguíneo , Hipóxia Celular , Células Cultivadas , Família 4 do Citocromo P450/metabolismo , Células Endoteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Membro Posterior , Humanos , Ácidos Hidroxieicosatetraenoicos/antagonistas & inibidores , Ácidos Hidroxieicosatetraenoicos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/tratamento farmacológico , Isquemia/fisiopatologia , Camundongos Endogâmicos BALB C , Neovascularização Fisiológica/efeitos dos fármacos , Fluxo Sanguíneo Regional , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Circulating endothelial progenitor cells (EPC) contribute to postnatal neovascularization. We identified the cytochrome P450 4A/F-20-hydroxyeicosatetraenoic acid (CYP4A/F-20-HETE) system as a novel regulator of EPC functions associated with angiogenesis in vitro. Here, we explored cellular mechanisms by which 20-HETE regulates EPC angiogenic functions and assessed its contribution to EPC-mediated angiogenesis in vivo. Results showed that both hypoxia and vascular endothelial growth factor (VEGF) induce CYP4A11 gene and protein expression (the predominant 20-HETE synthases in human EPC), and this is accompanied by an increase in 20-HETE production by ~1.4- and 1.8-fold, respectively, compared with the control levels. Additional studies demonstrated that 20-HETE and VEGF have a synergistic effect on EPC proliferation, whereas 20-HETE antagonist 20-HEDGE or VEGF-neutralizing antibody negated 20-HETE- or VEGF-induced proliferation, respectively. These findings are consistent with the presence of a positive feedback regulation on EPC proliferation between the 20-HETE and the VEGF pathways. Furthermore, we found that 20-HETE induced EPC adhesion to fibronectin and endothelial cell monolayer by 40 ± 5.6 and 67 ± 10%, respectively, which was accompanied by a rapid induction of very late antigen-4 and chemokine receptor type 4 mRNA and protein expression. Basal and 20-HETE-stimulated increases in adhesion were negated by the inhibition of the CYP4A-20-HETE system. Lastly, EPC increased angiogenesis in vivo by 3.6 ± 0.2-fold using the Matrigel plug angiogenesis assay, and these increases were markedly reduced by the local inhibition of 20-HETE system. These results strengthened the notion that 20-HETE regulates the angiogenic functions of EPC in vitro and EPC-mediated angiogenesis in vivo.
Assuntos
Endotélio Vascular/fisiologia , Ácidos Hidroxieicosatetraenoicos/metabolismo , Neovascularização Fisiológica , Células-Tronco/fisiologia , Animais , Anticorpos Neutralizantes/farmacologia , Adesão Celular , Hipóxia Celular , Proliferação de Células , Citocromo P-450 CYP4A , Sistema Enzimático do Citocromo P-450/metabolismo , Endotélio Vascular/citologia , Retroalimentação Fisiológica , Feminino , Fibronectinas/fisiologia , Humanos , Ácidos Hidroxieicosatetraenoicos/antagonistas & inibidores , Integrina alfa4beta1/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microvasos/fisiologia , Receptores CXCR4/metabolismo , Células-Tronco/citologia , Fator A de Crescimento do Endotélio Vascular/imunologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
PURPOSE: Cyclooxygenase (COX)-, lipoxygenase (LOX)-, and cytochrome P450 monooxygenase (CYP)-derived eicosanoids have been implicated in ocular surface inflammation and neovascularization. These eicosanoids are subjected to regulation by enzymes, such as heme oxygenases (HOs) and ferritin. METHODS: Quantitative polymerase chain reaction and lipidomics based on liquid chromatography-tandem mass spectrometry were performed on pterygia from patients undergoing surgical pterygium excision. Control tissues consisted of donor corneas. In addition, lipidomics based on liquid chromatography-tandem mass spectrometry was performed on tears collected from patients before the surgery. RESULTS: Messenger RNA (mRNA) expression of HO-2, the constitutive HO isoform, was upregulated by 40% in pterygia compared with control tissue, whereas the mRNA level of the inducible form, HO-1, was downregulated by more than 50%. Levels of CYP4B1 mRNA showed an approximate 2-fold increase in pterygia compared with control. Lipidomic analysis of tissues indicated a moderate elevation in Prostaglandin E2 and thromboxane B2 levels in pterygia compared with control. Among the LOX-derived metabolites, the antiinflammatory-hydroxyeicosatetraenoic acid (15-HETE) levels were significantly reduced in pterygia (79.3 ± 48.11 pg/mg protein) compared with control (586.2 ± 213.5 pg/mg protein), whereas the proinflammatory LOX- and CYP4B1-derived 12-HETE levels were 10-fold higher in pterygia (2768 ± 832.3 pg/mg protein) compared with control (231.4 ± 87.35 pg/mg protein). Prostaglandin E2 and HETEs were also present in tears from patients with pterygium but were not detected in tears from healthy volunteers. The mRNA expression levels of both light and heavy chain ferritin were 60% and 30% lower, respectively, in pterygia compared with control. CONCLUSIONS: We believe that a dysfunctional HO-ferritin system leads to increased levels of proinflammatory mediators, thus contributing to the inflammation characteristic of pterygia.
Assuntos
Ferritinas/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase-1/genética , Pterígio/genética , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Adulto , Hidrocarboneto de Aril Hidroxilases/genética , Cromatografia Líquida de Alta Pressão , Dinoprostona/metabolismo , Feminino , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Isoenzimas , Masculino , Pessoa de Meia-Idade , Pterígio/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Lágrimas/metabolismo , Tromboxano B2/metabolismoRESUMO
BACKGROUND: Circulating endothelial progenitor cells (EPCs) are recruited from the blood system to sites of ischemia and endothelial damage, where they contribute to the repair and development of blood vessels. Since numerous eicosanoids including leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) have been shown to exert potent pro-inflammatory activities, we examined their levels in chronic diabetic patients with severe cardiac ischemia in conjunction with the level and function of EPCs. RESULTS: Lipidomic analysis revealed a diabetes-specific increase (p<0.05) in inflammatory and angiogenic eicosanoids including the 5-lipoxygenase-derived LTB (4.11±1.17 vs. 0.96±0.27 ng/ml), the lipoxygenase/CYP-derived 12-HETE (117.08±35.05 vs. 24.34±10.03 ng/ml), 12-HETrE (17.56±4.43 vs. 4.15±2.07 ng/ml), and the CYP-derived 20-HETE (0.32±0.04 vs. 0.06±0.05 ng/ml) the level of which correlated with BMI (p=0.0027). In contrast, levels of the CYP-derived EETs were not significantly (p=0.36) different between these two groups. EPC levels and their colony-forming units were lower (p<0.05) with a reduced viability in diabetic patients compared with non-diabetics. EPC function (colony-forming units (CFUs) and MTT assay) also negatively correlated with the circulating levels of HgA1C. CONCLUSION: This study demonstrates a close association between elevated levels of highly pro-inflammatory eicosonoids, diabetes and EPC dysfunction in patients with cardiac ischemia, indicating that chronic inflammation impact negatively on EPC function and angiogenic capacity in diabetes.
Assuntos
Diabetes Mellitus/sangue , Eicosanoides/sangue , Células Endoteliais/metabolismo , Isquemia Miocárdica/sangue , Células-Tronco/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/sangue , Antígeno AC133 , Idoso , Antígenos CD/sangue , Índice de Massa Corporal , Sobrevivência Celular , Cromatografia Líquida , Diabetes Mellitus/fisiopatologia , Feminino , Citometria de Fluxo , Glicoproteínas/sangue , Humanos , Ácidos Hidroxieicosatetraenoicos/sangue , Leucotrieno B4/sangue , Lipídeos/sangue , Masculino , Pessoa de Meia-Idade , Isquemia Miocárdica/fisiopatologia , Peptídeos/sangue , Espectrometria de Massas em Tandem , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/sangueRESUMO
Arachidonate 5-lipoxygenase (ALOX5) expression and activity has been implicated in tumor pathogenesis, yet its role in papillary thyroid carcinoma (PTC) has not been characterized. ALOX5 protein and mRNA were upregulated in PTC compared to matched, normal thyroid tissue, and ALOX5 expression correlated with invasive tumor histopathology. Evidence suggests that PTC invasion is mediated through the induction of matrix metalloproteinases (MMPs) that can degrade and remodel the extracellular matrix (ECM). A correlation between MMP-9 and ALOX5 protein expression was established by immunohistochemical analysis of PTC and normal thyroid tissues using a tissue array. Transfection of ALOX5 into a PTC cell line (BCPAP) increased MMP-9 secretion and cell invasion across an ECM barrier. The ALOX5 product, 5(S)-hydroxyeicosatetraenoic acid also increased MMP-9 protein expression by BCPAP in a dose-dependent manner. Inhibitors of MMP-9 and ALOX5 reversed ALOX5-enhanced invasion. Here we describe a new role for ALOX5 as a mediator of invasion via MMP-9 induction; this ALOX5/MMP9 pathway represents a new avenue in the search for functional biomarkers and/or potential therapeutic targets for aggressive PTC.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Ácidos Hidroxieicosatetraenoicos/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Invasividade Neoplásica , Neoplasias da Glândula Tireoide/metabolismo , Adulto , Idoso , Araquidonato 5-Lipoxigenase/genética , Biomarcadores Tumorais , Carcinoma , Carcinoma Papilar , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Matriz Extracelular/metabolismo , Feminino , Humanos , Ácidos Hidroxieicosatetraenoicos/farmacologia , Inibidores de Lipoxigenase/farmacologia , Masculino , Inibidores de Metaloproteinases de Matriz , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/patologia , Adulto JovemRESUMO
The aim of this study is to test whether the Cyp2c44 epoxygenase-dependent metabolism of arachidonic acid prevents the hypertensive effect of a high K (HK) intake by inhibiting the epithelial sodium channel (ENaC) activity. A HK intake elevated Cyp2c44 mRNA expression and 11,12-epoxyeicosatrienoic acid levels in the cortical collecting duct in Cyp2c44(+/+) mice (wild-type [wt]). However, an HK intake failed to increase 11,12-epoxyeicosatrienoic acid formation in the cortical collecting ducts of Cyp2c44(-/-) mice. Moreover, increasing K intake enhanced arachidonic acid-induced inhibition of ENaC in the wt but not in Cyp2c44(-/-) mice. In contrast, 11,12-epoxyeicosatrienoic acid, a Cyp2c44 metabolite, inhibited ENaC in the wt and Cyp2c44(-/-) mice. The notion that Cyp2c44 is the epoxygenase responsible for mediating the inhibitory effects of arachidonic acid on ENaC is further suggested by the observation that inhibiting Cyp-epoxygenase increased the whole-cell Na currents in principal cells of wt but not in Cyp2c44(-/-) mice. Feeding mice with an HK diet raised the systemic blood pressures of Cyp2c44(-/-) mice but was without an effect on wt mice. Moreover, application of amiloride abolished the HK-induced hypertension in Cyp2c44(-/-) mice. The HK-induced hypertension of Cyp2c44(-/-) mice was accompanied by decreasing 24-hour urinary Na excretion and increasing the plasma Na concentration, and the effects were absent in wt mice. In contrast, disruption of the Cyp2c44 gene did not alter K excretion. We conclude that Cyp2c44 epoxygenase mediates the inhibitory effect of arachidonic acid on ENaC and that Cyp2c44 functions as an HK-inducible antihypertensive enzyme responsible for inhibiting ENaC activity and Na absorption in the aldosterone-sensitive distal nephron.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Potássio na Dieta/farmacologia , Sódio/metabolismo , Absorção/fisiologia , Animais , Ácido Araquidônico/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Sistema Enzimático do Citocromo P-450/deficiência , Sistema Enzimático do Citocromo P-450/genética , Família 2 do Citocromo P450 , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Canais Epiteliais de Sódio/metabolismo , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Hipertensão/prevenção & controle , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp , Potássio/farmacologiaRESUMO
Increased vascular synthesis of 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) is associated with increased vascular contraction, endothelial dysfunction, and endothelial activation; all are believed to account for 20-HETE prohypertensive properties. We demonstrated previously that the 20-HETE-dependent inhibition of NO production is mediated through inhibitor of κB kinase (IKK), suggesting a cross-talk between 20-HETE-mediated endothelial dysfunction and activation. In this study, we examined the temporal relationship among blood pressure, endothelial dysfunction, and endothelial activation and the role of IKK in the rat model of androgen-driven 20-HETE-mediated hypertension. In Sprague-Dawley rats treated with 5α-dihydrotestosterone, renal vascular 20-HETE levels increased by day 2 of treatment from 17.7±2.4 to 57.7±9.7 ng/mg, whereas blood pressure elevation reached significance by day 3 (132.7±1.7 versus 117.2±0.8 mm Hg). In renal interlobar arteries, when compared with vehicle, 5α-dihydrotestosterone treatment increased the sensitivity to phenylephrine-induced vasoconstriction by 3.5-fold, decreased acetylcholine-induced vasorelaxation, and increased nuclear factor κB activity, all of which were attenuated by treatment with the 20-HETE antagonist, 20 hydroxyeicosa-6(Z),15(Z)-dienoic acid, (20-6,15-HEDE). Cotreatment with parthenolide, an IKK inhibitor, attenuated the androgen-dependent 20-HETE-mediated elevation in blood pressure (from 133.7±3.1 to 109.8±3.0 mm Hg). In addition, parthenolide treatment negated 20-HETE-mediated inhibition of the relaxing response to acetylcholine and 20-HETE-mediated increase in vascular nuclear factor κB activity. These findings suggest that inhibition of IKK attenuates the androgen-dependent 20-HETE-mediated increase in blood pressure by inhibiting both 20-HETE-dependent endothelial activation and dysfunction.
Assuntos
Androgênios/farmacologia , Di-Hidrotestosterona/farmacologia , Endotélio Vascular/fisiopatologia , Ácidos Hidroxieicosatetraenoicos/metabolismo , Hipertensão/induzido quimicamente , Hipertensão/fisiopatologia , Quinase I-kappa B/metabolismo , Androgênios/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Di-Hidrotestosterona/metabolismo , Endotélio Vascular/efeitos dos fármacos , Hipertensão/metabolismo , Quinase I-kappa B/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Artéria Renal/efeitos dos fármacos , Artéria Renal/fisiopatologia , Sesquiterpenos/farmacologia , Vasoconstrição/efeitos dos fármacosRESUMO
High dietary potassium stimulates the renal expression of cytochrome P450 (CYP) epoxygenase 2C23, which metabolizes arachidonic acid (AA). Because the AA metabolite 11,12-epoxyeicosatrienoic acid (11,12-EET) can inhibit the epithelial sodium channel (ENaC) in the cortical collecting duct, we tested whether dietary potassium modulates ENaC function. High dietary potassium increased 11,12-EET in the isolated cortical collecting duct, an effect mimicked by inhibiting the angiotensin II type I receptor with valsartan. In patch-clamp experiments, a high potassium intake or treatment with valsartan enhanced AA-induced inhibition of ENaC, an effect mediated by a CYP-epoxygenase-dependent pathway. Moreover, high dietary potassium and valsartan each augmented the inhibitory effect of 11,12-EET on ENaC. Liquid chromatography/mass spectrometry showed that the rate of EET conversion to dihydroxyeicosatrienoic acids (DHET) was lower in renal tissue obtained from rats on a high-potassium diet than from those on a control diet, but this was not a result of altered expression of soluble epoxide hydrolase (sEH). Instead, suppression of sEH activity seemed to be responsible for the 11,12-EET-mediated enhanced inhibition of ENaC in animals on a high-potassium diet. Patch-clamp experiments demonstrated that 11,12-DHET was a weak inhibitor of ENaC compared with 11,12-EET, whereas 8,9- and 14,15-DHET were not. Furthermore, inhibition of sEH enhanced the 11,12-EET-induced inhibition of ENaC similar to high dietary potassium. In conclusion, high dietary potassium enhances the inhibitory effect of AA and 11,12-EET on ENaC by increasing CYP epoxygenase activity and decreasing sEH activity, respectively.
Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido Araquidônico/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Canais Epiteliais de Sódio/metabolismo , Túbulos Renais Coletores/metabolismo , Potássio/administração & dosagem , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animais , Citocromo P-450 CYP2J2 , Epóxido Hidrolases/metabolismo , Técnicas In Vitro , Masculino , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/metabolismoRESUMO
Endothelial dysfunction and activation occur in the vasculature and are believed to contribute to the pathogenesis of cardiovascular diseases. We have shown that 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE), a cytochrome P450 4A-derived eicosanoid that promotes vasoconstriction in the microcirculation, uncouples endothelial nitric-oxide synthase (eNOS) and reduces nitric oxide (NO) levels via the dissociation of the 90-kDa heat shock protein (HSP90) from eNOS. It also causes endothelial activation by stimulating nuclear factor-kappaB (NF-kappaB) and increasing levels of pro-inflammatory cytokines. In this study, we examined signaling mechanisms that may link 20-HETE-induced endothelial dysfunction and activation. Under conditions in which 20-HETE inhibited NO production, it also stimulated inhibitor of NF-kappaB (IkappaB) phosphorylation. Both effects were prevented by inhibition of tyrosine kinases and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK). It is noteworthy that inhibitor of IkappaB kinase (IKK) activity negated the 20-HETE-mediated inhibition of NO production. Immunoprecipitation experiments revealed that treatment of ionophore-stimulated cells with 20-HETE brings about a decrease in HSP90-eNOS association and an increase in HSP90-IKKbeta association, suggesting that the activation by 20-HETE of NF-kappaB is linked to its action on eNOS. Furthermore, addition of inhibitors of tyrosine kinase MAPK and IKK restored the 20-HETE-mediated impairment of acetylcholine-induced relaxation in rat renal interlobar arteries. The results indicate that 20-HETE mediates eNOS uncoupling and endothelial dysfunction via the activation of tyrosine kinase, MAPK, and IKK, and these effects are linked to 20-HETE-mediated endothelial activation.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Ácidos Hidroxieicosatetraenoicos/farmacologia , Quinase I-kappa B/metabolismo , Microcirculação/efeitos dos fármacos , Óxido Nítrico Sintase/metabolismo , Vasoconstrição/efeitos dos fármacos , Animais , Aorta/citologia , Bovinos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Células Endoteliais/metabolismo , Endotélio Vascular/enzimologia , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/farmacologia , Ácidos Hidroxieicosatetraenoicos/fisiologia , Técnicas In Vitro , Masculino , Óxido Nítrico/metabolismo , Ratos , Ratos Sprague-Dawley , Artéria Renal/efeitos dos fármacos , Artéria Renal/enzimologia , Artéria Renal/metabolismoRESUMO
Heme oxygenase (HO) and cytochrome P450 (P450)-derived epoxyeicosatrienoic acids (EETs) participate in vascular protection, and recent studies suggest these two systems are functionally linked. We examined the consequences of HO deficiency on P450-derived EETs with regard to body weight, adiposity, insulin resistance, blood pressure, and vascular function in HO-2-null mice. The HO-2-null mice were obese, displayed insulin resistance, and had high blood pressure. HO-2 deficiency was associated with decreases in cyp2c expression, EET levels, HO-1 expression, and HO activity and with an increase in superoxide production and an impairment in the relaxing response to acetylcholine. In addition, HO-2-null mice exhibited increases in serum levels of tumor necrosis factor (TNF)-alpha and macrophage chemoattractant protein (MCP)-1 and a decrease in serum adiponectin levels. Treatment of HO-2-null mice with a dual-activity EET agonist/soluble epoxide hydrolase inhibitor increased renal and vascular EET levels and HO-1 expression, lowered blood pressure, prevented body weight gain, increased insulin sensitivity, reduced subcutaneous and visceral fat, and decreased serum TNF-alpha and MCP-1, while increasing adiponectin and restoring the relaxing responses to acetylcholine. The decrease in cyp2c expression and EETs levels in HO-2-null mice underscores the importance of the HO system in the regulation of epoxygenase levels and suggests that protection against obesity-induced cardiovascular complications requires interplay between these two systems. A deficiency in one of these protective systems may contribute to the adverse manifestations associated with the clinical progression of the metabolic syndrome.
Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Heme Oxigenase (Desciclizante)/fisiologia , Síndrome Metabólica/enzimologia , Síndrome Metabólica/metabolismo , Ácido 8,11,14-Eicosatrienoico/agonistas , Ácido 8,11,14-Eicosatrienoico/metabolismo , Adiponectina/biossíntese , Adiponectina/sangue , Tecido Adiposo/metabolismo , Animais , Aorta/enzimologia , Aorta/metabolismo , Glicemia/metabolismo , Pressão Sanguínea/fisiologia , Western Blotting , Peso Corporal/fisiologia , Quimiocina CCL2/biossíntese , Quimiocina CCL2/sangue , Sistema Enzimático do Citocromo P-450/biossíntese , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase-1/biossíntese , Córtex Renal/enzimologia , Córtex Renal/metabolismo , Proteínas de Membrana/biossíntese , Síndrome Metabólica/fisiopatologia , Síndrome Metabólica/prevenção & controle , Camundongos , Camundongos Knockout , Fenótipo , Superóxidos/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/sangue , Vasodilatação/fisiologiaRESUMO
We have previously reported that adenoviral-mediated delivery of cytochrome P-450 (CYP) 4A2, which catalyzes the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE), results in endothelial dysfunction and hypertension in Sprague-Dawley (SD) rats (Wang JS, Singh H, Zhang F, Ishizuka T, Deng H, Kemp R, Wolin MS, Hintze TH, Abraham NG, Nasjletti A, Laniado-Schwartzman M. Circ Res 98: 962-969, 2006). In this study, we targeted the vascular endothelium by using a lentivirus construct expressing CYP4A2 under the control of the endothelium-specific promoter VE-cadherin (VECAD-4A2) and examined the effect of long-term CYP4A2 overexpression on blood pressure and kidney function in SD rats. A bolus injection of VECAD-4A2 increased blood pressure (P < 0.001) by 26, 36, and 30 mmHg 10, 20, and 30 days postinjection, respectively. Arteries from VECAD-4A2-transduced rats produced increased levels of 20-HETE (P < 0.01), expressed lower levels of endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (p-eNOS) (P < 0.05), generated higher levels of superoxide anion, and displayed decreased relaxing responsiveness to acetylcholine (P < 0.05). Proteinuria increased by twofold in VECAD-4A2-transduced rats compared with controls. Treatment of VECAD-4A2-transduced rats with HET0016, an inhibitor of 20-HETE biosynthesis, not only attenuated the increase in blood pressure (P < 0.05) but also improved vascular function (acetylcholine-induced relaxations) and reduced plasma creatinine and proteinuria. HET0016 treatment decreased oxidative stress and increased the phosphorylated state of key proteins that regulate endothelial function, including eNOS, AKT, and AMPK. Collectively, these findings demonstrate that augmentation of vascular endothelial 20-HETE levels results in hypertension, endothelial dysfunction, and renal injury, which is offset by HET0016 through a reduction in vascular 20-HETE coupled with a lessening of oxidative stress and the amplification of pAKT, pAMPK, and p-eNOS levels leading to normalization of endothelial responses.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Endotélio Vascular/metabolismo , Ácidos Hidroxieicosatetraenoicos/biossíntese , Hipertensão/metabolismo , Proteinúria/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Acetilcolina , Animais , Antígenos CD/genética , Pressão Sanguínea , Caderinas/genética , Linhagem Celular , Sistema Enzimático do Citocromo P-450/genética , DNA Complementar , Marcação de Genes , Técnicas de Transferência de Genes , Humanos , Lentivirus , Óxido Nítrico Sintase Tipo III/metabolismo , Oxirredução , Fosforilação , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Superóxidos/metabolismo , VasodilataçãoRESUMO
Atherosclerosis is now recognized as an inflammatory disease involving the vascular wall. Recent results indicate that acute inflammation does not simply passively resolve as previously assumed but is actively terminated by a homeostatic process that is governed by specific lipid-derived mediators initiated by lipoxygenases. Experiments with animals and humans support a proinflammatory role for the 5-lipoxygenase system. In contrast, results from animal experiments show a range of responses with the 12/15-lipoxygenase pathways in atherosclerosis. To date, the only two clinical epidemiology human studies both support an antiatherogenic role for 12/15-lipoxygenase downstream actions. We tested the hypothesis that atherosclerosis results from a failure in the resolution of local inflammation by analyzing apolipoprotein E-deficient mice with 1) global leukocyte 12/15-lipoxygenase deficiency, 2) normal enzyme expression, or 3) macrophage-specific 12/15-lipoxygenase overexpression. Results from these indicate that 12/15-lipoxygenase expression protects mice against atherosclerosis via its role in the local biosynthesis of lipid mediators, including lipoxin A(4), resolvin D1, and protectin D1. These mediators exert potent agonist actions on macrophages and vascular endothelial cells that can control the magnitude of the local inflammatory response. Taken together, these findings suggest that a failure of local endogenous resolution mechanisms may underlie the unremitting inflammation that fuels atherosclerosis.
Assuntos
Aterosclerose/etiologia , Ácidos Docosa-Hexaenoicos/metabolismo , Lipoxinas/biossíntese , Vasculite/complicações , Animais , Apolipoproteínas E/genética , Araquidonato 12-Lipoxigenase/genética , Araquidonato 12-Lipoxigenase/fisiologia , Araquidonato 15-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/fisiologia , Aterosclerose/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Interleucinas/biossíntese , Lipoxinas/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Vasculite/metabolismoRESUMO
PURPOSE: Heme oxygenase (HO) is considered a fundamental endogenous immunomodulatory, cytoprotective, and anti-inflammatory system. This protective function is primarily ascribed to the inducible HO-1. The authors examined the effect of HO-1 induction on corneal inflammation and wound healing in mice undergoing epithelial injury. METHODS: C57BL6 mice were treated with SnCl(2) the day before epithelial injury and once daily thereafter. The corneal epithelium was removed with the use of a corneal rust ring remover in anesthetized mice. Reepithelialization was measured by fluorescein staining. The inflammatory response was examined by histology and was quantified by the myeloperoxidase assay. Inflammatory lipid mediators were detected and quantified by LC/MS/MS-based lipidomic analysis. HO-1 expression was assessed by real-time PCR, and HO activity was determined by measuring HO-dependent carbon monoxide production. RESULTS: Epithelial injury caused a time-dependent transient increase in HO-1 expression and HO activity that was significantly amplified by treatment with SnCl(2), resulting in a twofold to threefold increase in mRNA levels and a similar increase in corneal HO activity. Induction of HO-1 was associated with a significant acceleration of wound healing when compared with a vehicle-treated group and with attenuation of the inflammatory response, evidenced by a significant decrease in the number of infiltrating cells and by a significant reduction in the expression and production of proinflammatory lipid mediators and cytokines. CONCLUSIONS: Increased expression of HO-1 provides a mechanism that modulates inflammation and promotes wound closure; pharmacologic amplification of this system may constitute a novel strategy to treat corneal inflammation while accelerating wound repair after injury.
Assuntos
Epitélio Corneano/fisiologia , Heme Oxigenase-1/biossíntese , Ceratite/prevenção & controle , Cicatrização/fisiologia , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Quimiocina CXCL2/metabolismo , Cromatografia Líquida , Indução Enzimática/efeitos dos fármacos , Epitélio Corneano/lesões , Heme Oxigenase-1/genética , Interleucina-1alfa/metabolismo , Ceratite/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Peroxidase/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em Tandem , Fatores de Tempo , Compostos de Estanho/farmacologiaRESUMO
Newly described lipoxygenase (LOX)-generated lipid mediators, that is, resolvins and protectins as well as lipoxins, are both anti-inflammatory and proresolving. We aimed to determine whether these lipid mediators are present in bone marrow and whether their lipidomic profiles are altered following ovariectomy or dietary supplementation with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) ethyl esters. Female rats were ovariectomised or sham-operated. Shams and one ovariectomised group received a diet devoid of omega-3 long-chain polyunsaturated fatty acids. The remaining ovariectomised rats received either 0.5 g EPA or DHA ethyl ester/kg body weight/day for 4 months. Bone marrow was analyzed using both GC to determine fatty acid composition and mediator lipidomics by LC/MS/MS profiling for the presence of LOX-pathway lipid mediators derived from arachidonic acid (AA), EPA, and DHA. LOX-derived products including lipoxins, resolvin D1, resolvin E1, and protectin D1 were identified in bone marrow by the presence of diagnostic ions in their corresponding MS-MS spectra. The proportion of AA relative to DHA and of AA-derived relative to DHA-derived mediators in bone marrow was higher in ovariectomised compared to sham-operated rats. DHA or EPA ethyl ester supplementation increased the percentage of DHA and EPA in bone marrow and increased the proportion of LOX mediators biosynthesized from DHA or EPA, respectively. Given the potent bioactivities of the lipoxins, resolvins, and protectins, the presence and changes in profile postovariectomy and with EPA and DHA ethyl ester supplementation may be of interest in bone marrow function and as a potential source of these mediators in vivo.
Assuntos
Ácidos Graxos Ômega-3/metabolismo , Inflamação/patologia , Lipídeos/fisiologia , Ovariectomia , Animais , Medula Óssea/metabolismo , Medula Óssea/patologia , Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Eicosapentaenoico/administração & dosagem , Ácido Eicosapentaenoico/análogos & derivados , Ácido Eicosapentaenoico/metabolismo , Ácidos Graxos Ômega-3/administração & dosagem , Feminino , Inflamação/metabolismo , Metabolismo dos Lipídeos/fisiologia , Lipídeos/análise , Lipoxinas , Lipoxigenase/metabolismo , Espectrometria de Massas , RatosRESUMO
Resolvin D1 (RvD1) and protectin D1 (Neuroprotectin D1, PD1/NPD1) are newly identified anti-inflammatory lipid mediators biosynthesized from docosahexaenoic acid (DHA). In this report, the spectra-structure correlations and fragmentation mechanisms were studied using electrospray low-energy collision-induced dissociation tandem mass spectrometry (MS/MS) for biogenic RvD1 and PD1, as well as mono-hydroxy-DHA and related hydroperoxy-DHA. The loss of H2O and CO2 in the spectra indicates the number of functional group(s). Chain-cut ions are the signature of the positions and numbers of functional groups and double bonds. The observed chain-cut ion is equivalent to a hypothetical homolytic-segment (cc, cm, mc, or mm) with addition or extraction of up to 2 protons (H). The alpha-cleavage ions are equivalent to: [cc + H], with H from the hydroxyl through a beta-ene or gamma-ene rearrangement; [cm - 2H], with 2H from hydroxyls of PD1 through a gamma-ene rearrangement, or 1H from the hydroxyl and the other H from the alpha-carbon of mono-HDHA through an alpha-H-beta-ene rearrangement; [mc - H], with H from hydroxyl through a beta-ene or gamma-ene rearrangement, or from the alpha-carbon through an alpha-H-beta-ene rearrangement; or [mm] through charge-direct fragmentations. The beta-ene or gamma-ene facilitates the H shift to gamma position and alpha-cleavage. Deuterium labeling confirmed the assignment of MS/MS ions and the fragmentation mechanisms. Based on the MS/MS spectra and fragmentation mechanisms, we identified RvD1, PD1, and mono-hydroxy-DHA products in human neutrophils and blood, trout head-kidney, and stroke-injury murine brain-tissue.
Assuntos
Ácidos Docosa-Hexaenoicos/química , Mediadores da Inflamação/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Cromatografia Líquida de Alta Pressão , Ácidos Docosa-Hexaenoicos/análise , Mediadores da Inflamação/química , Espectrometria de Massas por Ionização por Electrospray/instrumentaçãoRESUMO
Resolvin E1 (RvE1, 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid) is a novel anti-inflammatory lipid mediator recently found in humans, mice, and fish in vivo. To identify endogenous RvE1 and other eicosapentaenoic acid (EPA)-derived lipid mediators using electrospray low-energy collision-induced dissociation tandem mass spectrometry (MS/MS), the MS/MS product ion spectra of these compounds were correlated with their structures, and the MS/MS fragmentation mechanisms were studied. Deuterium labeling confirmed the proposed correlations and the fragmentation mechanisms. beta-cleavage was observed for RvE1, and beta and gamma cleavages were seen for leukotriene B5; however, alpha-cleavage was more common. The positions and numbers of hydroxyls and double bonds of these lipid mediators can be deduced from the MS/MS spectra. The MS/MS fragmentation generating chain-cut ions involved beta-ene, gamma-ene, or alpha-H-beta-ene rearrangement, depending on the specific structure. The m/z value of a detected chain-cut ion from RvE1 or from an EPA-derived product is equal to the corresponding hypothetical homolytic segment (cc, cm, mc, or mm) with the addition or extraction of up to two hydrogen atoms (H) from hydroxyls or an alpha-carbon; namely, the m/z value of an alpha-cleavage-generated ion is equal to [cc+H], [cm-2H], [mc-H], or [mm]. Wideband activation increased the signal intensities of chain-cut ions, and therefore was better for trace analysis of RvE1 in biological samples. RvE1, LTB5, PGE3, and other EPA-derived lipid mediators were found in trout brain or head-kidney via this approach on the basis of MS/MS spectra and fragmentation mechanisms. Negative ion electrospray low-collision-energy MS/MS spectra provide adequate data to elucidate and identify the structures of RvE1 and other EPA-derived lipid mediators at levels below a few picomoles in trout samples.