RESUMO
BACKGROUND: Epoxyeicosatrienoic acids (EETs) derived from cytochrome P450 (CYP)-dependent metabolism of arachidonic acid are increased in the plasma of women with preeclampsia as compared with normal pregnancy and are significantly higher in fetal than in maternal plasma and erythrocytes. We hypothesized that differences in EET synthesis or metabolism in the feto-placental unit contributed to the observed differences in circulating EETs. METHOD: To evaluate EETs, formation as well as the expression of relevant CYP isoforms and the metabolizing enzyme, soluble epoxide hydrolase (sEH), biopsies of placenta were collected from 19 normal pregnancy and 10 preeclampsia at the time of cesarean section delivery. EETs were extracted from tissue homogenates and analyzed by liquid chromatography coupled with tandem mass spectrometry. RESULTS: Both cis-EETs and trans-EETs were detected in the placenta. Concentration of total EETs was higher in the placenta from preeclampsia compared with normal pregnancy (2.37â±â1.42âng/mg vs. 1.20â±â0.72âng/mg, meanâ±âSD, Pâ<â0.01), especially the 5,6-, 8,9- and 11,12-EETs, measured in a subgroup of tissue samples (normal pregnancyâ=â10, preeclampsiaâ=â5). By immunohistochemistry, sEH, CYP2J2, CYP4A11 were present in placental villi with different pattern distribution, whereas CYP2C8 was not detectable. Neither were CYP2J2, CYP4A11, and CYP2C8 detected in the umbilical cord. Western blot analysis of placenta homogenates showed reduced expression of sEH in preeclampsia as compared with normal pregnancy. CONCLUSION: Increased EETs in the placenta and umbilical cord are associated with the presence of CYP2J2, whereas reduced expression of sEH in preeclampsia may be the key factor of increased EETs in the placenta.
Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Eicosanoides/metabolismo , Epóxido Hidrolases/metabolismo , Compostos de Epóxi/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Ácido 8,11,14-Eicosatrienoico/metabolismo , Adulto , Animais , Estudos de Casos e Controles , Citocromo P-450 CYP2C8/análise , Citocromo P-450 CYP2J2 , Citocromo P-450 CYP4A/análise , Sistema Enzimático do Citocromo P-450/análise , Sistema Enzimático do Citocromo P-450/metabolismo , Eicosanoides/biossíntese , Epóxido Hidrolases/análise , Eritrócitos/metabolismo , Feminino , Feto/metabolismo , Humanos , Imuno-Histoquímica , Placenta/química , Placenta/enzimologia , Pré-Eclâmpsia/enzimologia , Gravidez , Cordão Umbilical/químicaRESUMO
Cyclooxygenase (COX)-2 expression is increased in the kidney of rats made diabetic with streptozotocin and associated with enhanced release of prostaglandins stimulated by arachidonic acid (AA). Treatment of diabetic rats with nitro-L-arginine methyl ester (L-NAME) to inhibit nitric oxide synthase or with tempol to reduce superoxide prevented these changes, suggesting the possibility that peroxynitrite (ONOO) may be the stimulus for the induction of renal COX-2 in diabetes. Consequently, we tested the effects of an ONOO decomposition catalyst, 5,10,15,20-tetrakis(N-methyl-4'-pyridyl)porphyrinato iron(III) (FeTMPyP), which was administered for 3-4 wk after the induction of diabetes. FeTMPyP treatment normalized the twofold increase in the expression of nitrotyrosine, a marker for ONOO formation, in the diabetic rat and prevented the increase in renal COX-2 expression without modifying the two- to threefold increases in renal release of prostaglandins PGE(2) and 6-ketoPGF(1α) in response to AA. FeTMPyP treatment of diabetic rats reduced the elevated creatinine clearance and urinary excretion of TNF-α and transforming growth factor (TGF)-ß, suggesting a renoprotective effect. Double immunostaining of renal sections and immunoprecipitation of COX-2 and nitrotyrosine suggested nitration of COX-2 in diabetic rats. In cultured human umbilical vein endothelial cells (HUVECs) exposed to elevated glucose (450 mg/dl) or ONOO derived from 3-morpholinosydnonimine (SIN-1), expression of COX-2 was increased and was prevented when endothelial cells were treated with FeTMPyP. These results indicate that elevated glucose increases the formation of ONOO, which contributes to the induction of renal COX-2 in the diabetic rat.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Diabetes Mellitus Experimental/metabolismo , Metaloporfirinas/administração & dosagem , Ácido Peroxinitroso/metabolismo , Tirosina/análogos & derivados , 6-Cetoprostaglandina F1 alfa/metabolismo , Animais , Ácido Araquidônico/metabolismo , Células Cultivadas , Creatinina/urina , Inibidores de Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Humanos , Rim/enzimologia , Masculino , Ratos , Ratos Wistar , Fator de Crescimento Transformador beta/urina , Fator de Necrose Tumoral alfa/urina , Tirosina/metabolismoRESUMO
We examined the hypothesis that vascular and renal dysfunction caused by angiotensin II (Ang II) through increased levels of blood pressure, inflammatory cytokines, and oxidative stress in Sprague-Dawley rats can be prevented by lentiviral-mediated delivery of endothelial heme oxygenase (HO)-1. We targeted the vascular endothelium using a lentiviral construct expressing human HO-1 under the control of the endothelium-specific promoter VE-cadherin (VECAD-HO-1) and examined the effect of long-term human HO-1 expression on blood pressure in Ang II-mediated increases in blood pressure and oxidant stress. A bolus injection of VECAD-HO-1 into the renal artery resulted in expression of human HO-1 for up to 6-9 weeks. Sprague-Dawley rats were implanted with Ang II minipumps and treated with lentivirus carrying either the HO-1 or green fluorescent protein. Renal tissue from VECAD-HO-1-transduced rats expresses human HO-1 mRNA and proteins without an effect on endogenous HO-1. Infusion of Ang II increased blood pressure (p < 0.001) but decreased vascular relaxation in response to acetylcholine, endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (peNOS) levels, and renal and plasma levels of adiponectin (p < 0.05); in contrast, plasma tumor necrosis factor-α and monocyte chemoattractant protein-1 levels increased. Ang II-treated animals had higher levels of superoxide anion and inducible nitric oxide synthase and increased urinary protein and plasma creatinine levels. Lentiviral transduction with the VECAD-HO-1 construct attenuated the increase in blood pressure (p < 0.05), improved vascular relaxation, increased plasma adiponectin, and prevented the elevation in urinary protein and plasma creatinine in Ang II-treated rats. Endothelial-specific expression of HO-1 also reduced oxidative stress and levels of inflammatory cytokines resulting in increased expression of the anti-apoptotic proteins phosphorylated AKT, phosphorylated AMP-activated protein kinase, peNOS, and eNOS. Collectively, these findings demonstrate that endothelial-specific increases in HO-1 expression attenuate Ang II hypertension and the associated vascular dysfunction that is associated with increases in adiponectin and peNOS and reductions in oxidative stress and levels of inflammatory cytokines.
Assuntos
Angiotensina II/farmacologia , Endotélio Vascular/metabolismo , Terapia Genética , Heme Oxigenase-1 , Hipertensão/terapia , Lentivirus , Adiponectina/sangue , Adiponectina/metabolismo , Angiotensina II/metabolismo , Animais , Animais Geneticamente Modificados , Biomarcadores/sangue , Pressão Sanguínea/efeitos dos fármacos , Modelos Animais de Doenças , Vetores Genéticos/genética , Células HEK293 , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Hipertensão/enzimologia , Hipertensão/genética , Inflamação/patologia , Rim/efeitos dos fármacos , Rim/metabolismo , Lentivirus/genética , Óxido Nítrico Sintase/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Superóxidos/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatação/genéticaAssuntos
Endotélio Vascular/fisiopatologia , Inflamação/fisiopatologia , NF-kappa B/fisiologia , Obesidade/fisiopatologia , Estresse Oxidativo/fisiologia , Animais , Glicemia/metabolismo , Ciclo-Oxigenase 2/metabolismo , Gorduras na Dieta/efeitos adversos , Modelos Animais de Doenças , Humanos , Camundongos , Obesidade/etiologia , Obesidade/metabolismo , Superóxido Dismutase/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The beneficial effects of coronary angioplasty are limited by the proliferation and migration of vascular smooth muscle cells leading to restenosis. We hypothesized that increased activity of phosphodiesterase (PDE) after angioplasty in response to growth factors such as platelet-derived growth factor (PDGF)-BB and fibroblast growth factor (FGF), leads to reduced cAMP levels, which, in turn, may contribute to vascular smooth muscle cell proliferation. In rats subjected to angioplasty, aortic expression and activity of PDE3/PDE4 were increased within 24 h and associated with reduced phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a substrate for cAMP-dependent protein kinase A (PKA). Inhibition of PDE3 increased VASP phosphorylation in aortic rings from rats subjected to angioplasty, whereas inhibition of PDE4 or stimulation of adenylate cyclase with isoproterenol was without effect; however, combined inhibition of PDE3 and PDE4 produced a synergistic effect on VASP phosphorylation. In cultured vascular smooth muscle cells, exposure to PDGF-BB resulted in increased expression of PDE3, which was prevented by an inhibitor of PI3 kinase but not by inhibitors of the MAP kinase signaling pathway. In contrast, FGF increased the expression of PDE4 in vascular smooth muscle cells but did not influence expression of PDE3. This study shows that angioplasty results in increased expression/activity of PDE, possibly arising from stimulation by PDGF-BB and FGF, and decreased cAMP levels, which may promote restenosis. These results provide a rational explanation for the beneficial effects of PDE inhibitors.
Assuntos
Angioplastia com Balão , Moléculas de Adesão Celular/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/análise , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/análise , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Animais , Aorta Torácica/enzimologia , Becaplermina , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/fisiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/fisiologia , Fatores de Crescimento de Fibroblastos/farmacologia , Masculino , Músculo Liso Vascular/enzimologia , Fosforilação , Fator de Crescimento Derivado de Plaquetas/fisiologia , Proteínas Proto-Oncogênicas c-sis , Ratos , Ratos Sprague-DawleyRESUMO
It is known that cAMP and cGMP are important for vasorelaxation, and cyclic nucleotide phosphodiesterases (PDEs) regulate their levels. Balloon angioplasty (BAL) is associated with reduced cAMP and cGMP levels, and inhibition of PDE-3 reduces restenosis. In this study, we found that BAL increased PDE-3 activity, which affected vasoreactivity of rat aortic rings 24-h post-BAL; these were compared with intact (INT) and ex vivo endothelium-denuded rings (RUB) from sham rats. In BAL and RUB rings, vasorelaxant responses to ACh were abolished. The EC(50) for phenylephrine (PE) was 1.8-fold less in RUB than in INT or BAL, whereas the maximal contractile effect of PE was greater in BAL than in INT or RUB. PDE-3 inhibitors reduced the maximal response to PE by >65% in BAL compared with 10-30% in INT and RUB; the reduction of the maximal response to U-46619 was 37% in BAL compared with 8% in INT with no reduction in RUB. PDE-4 inhibitors reduced PE-induced tone by <30% in an endothelium-dependent manner. Vasorelaxant responses to agonists that utilize cAMP were greatly impaired in BAL and RUB rings, and inhibition of PDE-3 enhanced the vasorelaxant responses in BAL or RUB. Inhibition of PDE-4 increased vasorelaxant responses to isoproterenol (ISO) to a much lesser degree. Thus PDE-3 and PDE-4 inhibitors exhibited differential effects on PE-induced tone and vasorelaxant responses to ISO. Inhibition of PDE-3 also produced a greater increase in cAMP in BAL than INT or RUB rings. These results suggest that increased PDE-3 activity after BAL may promote a vasospastic state and that the reduction in cAMP may, possibly, influence vessel remodeling.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Angioplastia com Balão/efeitos adversos , Aorta/efeitos dos fármacos , AMP Cíclico/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Animais , Aorta/enzimologia , Aorta/lesões , Aorta/fisiopatologia , Ácidos Carboxílicos/farmacologia , Cilostazol , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Ácidos Cicloexanocarboxílicos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiopatologia , Técnicas In Vitro , Masculino , Milrinona/farmacologia , Nitrilas/farmacologia , Fosforilação , Quinolonas/farmacologia , Ratos , Ratos Sprague-Dawley , Tetrazóis/farmacologia , Regulação para Cima , Vasoconstritores/farmacologia , Vasodilatadores/farmacologiaRESUMO
Our initial studies on renal cyclooxygenase (COX)-2 expression and activity addressed the critical role of angiotensin II (Ang II) in increasing tumor necrosis factor alpha (TNF) that eventuated in expression of COX-2 in the medullary thick ascending limb (mTAL) of the nephron. COX-2 supplanted the dominant oxygenase, the cytochrome P450 (CYP) enzyme, omega-hydroxylase, that synthesized 20-hydroxyeicosatetraenoic acid (20-HETE). These findings served as the basis for additional studies on: 1) the role of glucocorticoids in regulating COX-2 expression and activity in the mTAL; and 2) the utilization of the same signaling pathways in response to stimulation of the mTAL calcium receptor (CaR). These studies of mTAL COX-2 expression which are addressed in the first part of this chapter are followed by explorations of the expression of COX-2 in preglomerular microvessels (PGMV) and the relationship of COX-2 to 20-HETE, the principal eicosanoid of PGMV. The third and last component of this chapter explores the signaling events, focusing on COX-2, which are set in motion by diabetes.