RESUMEN
Arachidonic acid (AA)-derived cytochrome P450 (CYP) derivatives, epoxyeicosatrienoic acids (EETs) and 20-hidroxyeicosatetranoic acid (20-HETE), play a key role in kidney tubular and vascular functions and blood pressure. Altered metabolism of CYP epoxygenases and CYP hydroxylases has differentially been involved in the pathogenesis of metabolic disease-associated vascular complications, although the mechanisms responsible for the vascular injury are unclear. The present study aimed to assess whether obesity-induced changes in CYP enzymes may contribute to oxidative stress and endothelial dysfunction in kidney preglomerular arteries. Endothelial function and reactive oxygen species (ROS) production were assessed in interlobar arteries of obese Zucker rats (OZR) and their lean counterparts lean Zucker rats (LZR) and the effects of CYP2C and CYP4A inhibitors sulfaphenazole and HET0016, respectively, were examined on the endothelium-dependent relaxations and O2- and H2O2 levels of preglomerular arteries. Non-nitric oxide (NO) non-prostanoid endothelium-derived hyperpolarization (EDH)-type responses were preserved but resistant to the CYP epoxygenase blocker sulfaphenazole in OZR in contrast to those in LZR. Sulfaphenazole did not further inhibit reduced arterial H2O2 levels, and CYP2C11/CYP2C23 enzymes were downregulated in intrarenal arteries from OZR. Renal EDH-mediated relaxations were preserved in obese rats by the enhanced activity and expression of endothelial calcium-activated potassium channels (KCa). CYP4A blockade restored impaired NO-mediated dilatation and inhibited augmented O2- production in kidney arteries from OZR. The current data demonstrate that both decreased endothelial CYP2C11/ CYP2C23-derived vasodilator H2O2 and augmented CYP4A-derived 20-HETE contribute to endothelial dysfunction and vascular oxidative stress in obesity. CYP4A inhibitors ameliorate arterial oxidative stress and restore endothelial function which suggests its therapeutic potential for the vascular complications of obesity-associated kidney injury.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Endotelio Vascular/metabolismo , Riñón/metabolismo , Obesidad/metabolismo , Estrés Oxidativo , Arteria Renal/metabolismo , Amidinas/farmacología , Animales , Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromo P-450 CYP2J2/metabolismo , Citocromo P-450 CYP4A/metabolismo , Familia 2 del Citocromo P450/metabolismo , Peróxido de Hidrógeno/metabolismo , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Ácidos Hidroxieicosatetraenoicos/metabolismo , Riñón/irrigación sanguínea , Masculino , Obesidad/fisiopatología , Ratas Zucker , Especies Reactivas de Oxígeno/metabolismo , Arteria Renal/efectos de los fármacos , Arteria Renal/fisiopatología , Esteroide 16-alfa-Hidroxilasa/metabolismo , Sulfafenazol/farmacología , Vasodilatación/efectos de los fármacosRESUMEN
ABSTRACT: Arachidonic acid-derived lipid mediators play crucial roles in the development and progression of cardiovascular diseases. Eicosanoid metabolites generated by lipoxygenases and cytochrome P450 enzymes produce several classes of molecules, including the epoxyeicosatrienoic acid (EET) and hydroxyeicosatetraenoic acids (HETE) family of bioactive lipids. In general, the cardioprotective effects of EETs have been documented across a number of cardiac diseases. In contrast, members of the HETE family have been shown to contribute to the pathogenesis of ischemic cardiac disease, maladaptive cardiac hypertrophy, and heart failure. The net effect of 12(S)- and 20-HETE depends upon the relative amounts generated, ratio of HETEs:EETs produced, timing of synthesis, as well as cellular and subcellular mechanisms activated by each respective metabolite. HETEs are synthesized by and affect multiple cell types within the myocardium. Moreover, cytochrome P450-derived and lipoxygenase- derived metabolites have been shown to directly influence cardiac myocyte growth and the regulation of cardiac fibroblasts. The mechanistic data uncovered thus far have employed the use of enzyme inhibitors, HETE antagonists, and the genetic manipulation of lipid-producing enzymes and their respective receptors, all of which influence a complex network of outcomes that complicate data interpretation. This review will summarize and integrate recent findings on the role of 12(S)-/20-HETE in cardiac diseases.
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Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Enfermedades Cardiovasculares/fisiopatología , Ácidos Hidroxieicosatetraenoicos/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/antagonistas & inhibidores , Animales , Animales Modificados Genéticamente , Cardiomegalia/fisiopatología , Sistema Enzimático del Citocromo P-450/metabolismo , Insuficiencia Cardíaca/fisiopatología , Humanos , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Isquemia Miocárdica/fisiopatología , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Diabetic cardiomyopathy (DCM) is a well-established complication of type 1 and type 2 diabetes associated with a high rate of morbidity and mortality. DCM is diagnosed at advanced and irreversible stages. Therefore, it is of utmost need to identify novel mechanistic pathways involved at early stages to prevent or reverse the development of DCM. In vivo experiments were performed on type 1 diabetic rats (T1DM). Functional and structural studies of the heart were executed and correlated with mechanistic assessments exploring the role of cytochromes P450 metabolites, the 20-hydroxyeicosatetraenoic acids (20-HETEs) and epoxyeicosatrienoic acids (EETs), and their crosstalk with other homeostatic signaling molecules. Our data displays that hyperglycemia results in CYP4A upregulation and CYP2C11 downregulation in the left ventricles (LV) of T1DM rats, paralleled by a differential alteration in their metabolites 20-HETEs (increased) and EETs (decreased). These changes are concomitant with reductions in cardiac outputs, LV hypertrophy, fibrosis, and increased activation of cardiac fetal and hypertrophic genes. Besides, pro-fibrotic cytokine TGF-ß overexpression and NADPH (Nox4) dependent-ROS overproduction are also correlated with the observed cardiac functional and structural modifications. Of interest, these observations are attenuated when T1DM rats are treated with 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA), which blocks EETs metabolism, or N-hydroxy-N'-(4-butyl-2-methylphenol)Formamidine (HET0016), which inhibits 20-HETEs formation. Taken together, our findings confer pioneering evidence about a potential interplay between CYP450-derived metabolites and Nox4/TGF-ß axis leading to DCM. Pharmacologic interventions targeting the inhibition of 20-HETEs synthesis or the activation of EETs synthesis may offer novel therapeutic approaches to treat DCM.
Asunto(s)
Ácido Araquidónico/metabolismo , Cardiomiopatías/etiología , Sistema Enzimático del Citocromo P-450/fisiología , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Tipo 1/complicaciones , Ácidos Hidroxieicosatetraenoicos/fisiología , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animales , Cardiomiopatías/tratamiento farmacológico , Cardiomiopatías/metabolismo , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Masculino , NADPH Oxidasa 4/fisiología , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , EstreptozocinaRESUMEN
OBJECTIVES: The global morbidity and mortality related to hypertension and associated disorders increases continuously and novel therapeutic strategies are still in high demand. Increasing evidence suggests the important role in blood pressure regulation of cytochrome P-450-dependent metabolites of arachidonic acid. Epoxyeicosatrienoic acids (EETs) induce vasodilation and natriuresis, and have renoprotective and anti-inflammatory properties. 20-HETE is an arachidonic acid metabolite with both prohypertensive and antihypertensive activities. To explore the pathophysiological role of arachidonic acid metabolites in more detail, we examined the antihypertensive efficiency of EET-A, a stable analog of 14,15-EET, and of AAA, a novel antagonist of the 20-HETE receptors. METHODS: Male spontaneously hypertensive rats (SHR) were treated for 5 weeks with EET-A, AAA or the combination; age-matched untreated SHR and normotensive Wistar-Kyoto rats served as controls. EET-A and AAA were administered in drinking water at 10âmg/kg/day each. SBP was measured by telemetry and urine, blood, and tissue samples were collected for relevant analyses. RESULTS: EET-A or AAA given alone had no significant effect on SHR blood pressure. In contrast, combined treatment with AAA and EET-A was significantly antihypertensive, causing a decrease in SBP from 180â±â3 to 160â±â5âmmHg (Pâ<â0.05). Additionally, the combined treatment attenuated cardiac hypertrophy, decreased kidney ANG II level, increased natriuresis, and increased the excretion of nitric oxide metabolites. CONCLUSION: Considering the beneficial impact of the combined treatment with EET-A and AAA on SHR blood pressure and cardiovascular and renal function, we suggest that the treatment is a promising therapeutic strategy for human hypertension.
Asunto(s)
Antihipertensivos/farmacología , Presión Sanguínea/efectos de los fármacos , Eicosanoides/farmacología , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Hipertensión/metabolismo , Animales , Masculino , Ratas , Ratas Endogámicas SHRRESUMEN
PURPOSE: Recent studies have shown that 20-hydroxyeicosatetraenoic acid (20-HETE) is a key molecule in sustaining androgen-mediated prostate cancer cell survival. Thus, the aim of this study was to determine whether 20-HETE can affect the metastatic potential of androgen-insensitive prostate cancer cells, and the implication of the newly described 20-HETE receptor, GPR75, in mediating these effects. METHODS: The expression of GPR75, protein phosphorylation, actin polymerization and protein distribution were assessed by western blot and/or fluorescence microscopy. Additionally, in vitro assays including epithelial-mesenchymal transition (EMT), metalloproteinase-2 (MMP-2) activity, scratch wound healing, transwell invasion and soft agar colony formation were used to evaluate the effects of 20-HETE agonists/antagonists or GPR75 gene silencing on the aggressive features of PC-3 cells. RESULTS: 20-HETE (0.1â¯nM) promoted the acquisition of a mesenchymal phenotype by increasing EMT, the release of MMP-2, cell migration and invasion, actin stress fiber formation and anchorage-independent growth. Also, 20-HETE augmented the expression of HIC-5, the phosphorylation of EGFR, NF-κB, AKT and p-38 and the intracellular redistribution of p-AKT and PKCα. These effects were impaired by GPR75 antagonism and/or silencing. Accordingly, the inhibition of 20-HETE formation with N-hydroxy-N'-(4-n-butyl-2-methylphenyl) formamidine (HET0016) elicited the opposite effects. CONCLUSIONS: The present results show for the first time the involvement of the 20-HETE-GPR75 receptor in the activation of intracellular signaling known to be stimulated in cell malignant transformations leading to the differentiation of PC-3 cells towards a more aggressive phenotype. Targeting the 20-HETE/GPR75 pathway is a promising and novel approach to interfere with prostate tumor cell malignant progression.
Asunto(s)
Ácidos Hidroxieicosatetraenoicos/metabolismo , Neoplasias de la Próstata/patología , Receptores Acoplados a Proteínas G/metabolismo , Amidinas/farmacología , Andrógenos/metabolismo , Movimiento Celular/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Ácidos Hidroxieicosatetraenoicos/agonistas , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas con Dominio LIM/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Células PC-3 , Neoplasias de la Próstata/tratamiento farmacológico , ARN Interferente Pequeño/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/genética , Transducción de Señal/efectos de los fármacosRESUMEN
The arachidonic acid pathway metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to ischemia/reperfusion brain injury. Inhibition of 20-HETE formation can protect the developing brain from global ischemia. Here, we examined whether treatment with the 20-HETE synthesis inhibitor N-hydroxy-N-4-butyl-2-methylphenylformamidine (HET0016) can protect the immature brain from traumatic brain injury (TBI). Male rats at postnatal day 9-10 underwent controlled cortical impact followed by intraperitoneal injection with vehicle or HET0016 (1 mg/kg, 5 min and 3 h post-injury). HET0016 decreased the lesion volume by over 50% at 3 days of recovery, and this effect persisted at 30 days as the brain matured. HET0016 decreased peri-lesion gene expression of proinflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin-1ß [IL-1ß]) at 1 day and increased reparative cytokine (IL-4, IL-10) expression at 3 days. It also partially preserved microglial ramified processes, consistent with less activation. HET0016 decreased contralateral hindlimb foot faults and improved outcome on the novel object recognition memory task 30 days after TBI. In cultured BV2 microglia, HET0016 attenuated the lipopolysaccharide-evoked increase in release of TNF-α. Our data show that HET0016 improves acute and long-term histologic and functional outcomes, in association with an attenuated neuroinflammatory response after contusion of an immature rat brain.
Asunto(s)
Amidinas/farmacología , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Daño por Reperfusión/patología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Lesiones Traumáticas del Encéfalo/inducido químicamente , Citocinas/metabolismo , Modelos Animales de Enfermedad , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Masculino , Ratas Sprague-Dawley , Daño por Reperfusión/inducido químicamenteRESUMEN
We hypothesized that vascular actions of 20-hydroxyeicosatetraenoic acid (20-HETE), the product of cytochrome P450 (CYP450)-dependent ω-hydroxylase, potentiate prohypertensive actions of angiotensin II (ANG II) in Cyp1a1-Ren-2 transgenic rats, a model of ANG II-dependent malignant hypertension. Therefore, we evaluated the antihypertensive effectiveness of 20-HETE receptor antagonist (AAA) in this model. Malignant hypertension was induced in Cyp1a1-Ren-2 transgenic rats by activation of the renin gene using indole-3-carbinol (I3C), a natural xenobiotic. Treatment with AAA was started either simultaneously with induction of hypertension or 10 days later, during established hypertension. Systolic blood pressure (SBP) was monitored by radiotelemetry, indices of renal and cardiac injury, and kidney ANG II levels were determined. In I3C-induced hypertensive rats, early AAA treatment reduced SBP elevation (to 161 ± 3 compared with 199 ± 3 mmHg in untreated I3C-induced rats), reduced albuminuria, glomerulosclerosis index, and cardiac hypertrophy (P<0.05 in all cases). Untreated I3C-induced rats showed augmented kidney ANG II (405 ± 14 compared with 52 ± 3 fmol/g in non-induced rats, P<0.05) which was markedly lowered by AAA treatment (72 ± 6 fmol/g). Remarkably, in TGR with established hypertension, AAA also decreased SBP (from 187 ± 4 to 158 ± 4 mmHg, P<0.05) and exhibited organoprotective effects in addition to marked suppression of kidney ANG II levels. In conclusion, 20-HETE antagonist attenuated the development and largely reversed the established ANG II-dependent malignant hypertension, likely via suppression of intrarenal ANG II levels. This suggests that intrarenal ANG II activation by 20-HETE is important in the pathophysiology of this hypertension form.
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Antihipertensivos/farmacología , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Hipertensión Maligna/tratamiento farmacológico , Riñón/efectos de los fármacos , Amidas/farmacología , Angiotensina II/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Animales , Citocromo P-450 CYP1A1/genética , Ácidos Hidroxieicosatetraenoicos/metabolismo , Hipertensión Maligna/inducido químicamente , Hipertensión Maligna/metabolismo , Indoles/toxicidad , Riñón/metabolismo , Masculino , Ratas TransgénicasAsunto(s)
Presión Sanguínea/fisiología , Endotelio Vascular/fisiopatología , Tasa de Filtración Glomerular/fisiología , Ácidos Hidroxieicosatetraenoicos/metabolismo , Hipertensión/metabolismo , Animales , Inhibidores del Citocromo P-450 CYP3A/uso terapéutico , Humanos , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Hipertensión/tratamiento farmacológico , Hipertensión/fisiopatología , Vasodilatación/fisiologíaRESUMEN
20-Hydroxyeicosatetraenoicacid (20-HETE) is an important mediator that regulates vascular tone and blood pressure (BP). Although various experimental animal hypertension models demonstrated that 20-HETE contributes to increased vascular resistance and BP, these effects have not been studied in Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME)-induced hypertension model. In this study, we investigated the effects of 20-HETE on the vascular responsiveness and BP in an L-NAME-induced hypertension. Wistar Albino rats were used in this study. Hypertension was induced by the addition of L-NAME to drinking water for 5 weeks. The study was performed in three stages: first, BP changes were monitored in real time in the presence of 20-HETE enzymatic inhibitor, N-hydroxy-N´-(4-butly-2-methylphenyl)-formamidine (HET-0016) for 1 h. Second, vascular responses of the conduit and resistance arteries were investigated in the presence or absence of HET-0016 in the organ bath. Third, BP was monitored weekly in some hypertensive animals treated with HET-0016 and vascular responses were investigated at the end of the experiment. We demonstrated an increase in 20-HETE levels in the resistance arteries of hypertensive animals. 20-HETE inhibition by HET-0016 significantly decreased BP in L-NAME-induced hypertension model. In addition, HET-0016 treatment caused significant improvement in vascular dilator and constrictor responses in the conduit and resistance arteries. This study demonstrates an important role of 20-HETE in increasing BP and altering vascular responsiveness in L-NAME-induced hypertension model, which suggests a possible involvement of 20-HETE in essential hypertension development in humans.
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Amidinas/farmacología , Presión Sanguínea/efectos de los fármacos , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Ácidos Hidroxieicosatetraenoicos/metabolismo , Hipertensión/fisiopatología , Resistencia Vascular/efectos de los fármacos , Animales , Inhibidores Enzimáticos/farmacología , Hipertensión/inducido químicamente , Masculino , NG-Nitroarginina Metil Éster , Ratas , Ratas Wistar , Técnicas de Cultivo de Tejidos , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacosRESUMEN
Little is currently known of the role(s) of the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE) in hypertensive pregnancies. We hypothesized that specific inhibition of 20-HETE would attenuate increases in blood pressure in the reduced uterine perfusion pressure (RUPP) rat model of preeclampsia. Specific 20-HETE synthesis inhibitor HET0016 (1mg/kg) was administered daily to RUPP rats from gestational days 14-18. Blood pressure (BP) increased in RUPP rats and was decreased with HET0016 administration. BP was unchanged in NP+HET0016 rats. Fetal death greatly increased in RUPP rats and was reduced in RUPP+HET0016 rats. 20-HETE levels increased modestly in RUPP rats compared to NP and was reduced in both NP+HET0016 and RUPP+HET0016 rats. Furthermore, circulating levels of HETEs, EET, and DHETE were significantly altered between groups. HET0016 shifted CYP metabolism toward EETs, as indicated by a decrease in plasma 20-HETE:EETs in RUPP+HET0016 rats compared to RUPP. In conclusion, 20-HETE inhibition in RUPP rats reduces BP and fetal death, and is associated with an increase in EET/20-HETE ratio.
Asunto(s)
Presión Sanguínea/efectos de los fármacos , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Preeclampsia/fisiopatología , Amidinas/farmacología , Animales , Sistema Enzimático del Citocromo P-450/metabolismo , Modelos Animales de Enfermedad , Femenino , Preeclampsia/enzimología , Embarazo , RatasRESUMEN
20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) has been linked to pro-hypertensive and anti-hypertensive actions through its ability to promote vasoconstriction and inhibit Na transport in the ascending limb of the loop of Henle, respectively. In this study, we assessed the effects of 20-HETE blockade on blood pressure, renal hemodynamics, and urinary sodium excretion in Cyp4a14(-/-) male mice, which display androgen-driven 20-HETE-dependent hypertension. Administration of 2,5,8,11,14,17-hexaoxanonadecan-19-yl 20-hydroxyicosa-6(Z),15(Z)-dienoate (20-SOLA), a water-soluble 20-HETE antagonist, in the drinking water normalized the blood pressure of male Cyp4a14(-/-) hypertensive mice (±124 vs. ±153 mmHg) while having no effect on age-matched normotensive wild-type (WT) male mice. Hypertension in Cyp4a14(-/-) male mice was accompanied by decreased renal perfusion and reduced glomerular filtration rates, which were corrected by treatment with 20-SOLA. Interestingly, Cyp4a14(-/-) male mice treated with 20-SOLA displayed increased urinary sodium excretion that was paralleled by the reduction of blood pressure suggestive of an antinatriuretic activity of endogenous 20-HETE in the hypertensive mice. This interpretation is in line with the observation that the natriuretic response to acute isotonic saline loading in hypertensive Cyp4a14(-/-) male mice was significantly impaired relative to that in WT mice; this impairment was corrected by 20-SOLA treatment. Hence, endogenous 20-HETE appears to promote sodium conservation in hypertensive Cyp4a14(-/-) male mice, presumably, as a result of associated changes in renal hemodynamics and/or direct stimulatory action on tubular sodium reabsorption.
Asunto(s)
Presión Sanguínea/efectos de los fármacos , Familia 4 del Citocromo P450/genética , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Natriuresis/efectos de los fármacos , Animales , Antihipertensivos/farmacología , Femenino , Tasa de Filtración Glomerular/genética , Túbulos Renales/metabolismo , Masculino , Ratones , Ratones Noqueados , Polietilenglicoles , Circulación Renal/genética , Siloxanos , Sodio/orinaRESUMEN
Recent data demonstrated the role of CYP1B1 in cardiovascular disease. It was, therefore, necessary to examine whether the inhibition of CYP1B1 and hence inhibiting the formation of its metabolites, using 2,4,3',5'-tetramethoxystilbene (TMS), would have a cardioprotective effect against angiotensin II (Ang II)-induced cardiac hypertrophy. For this purpose, male Sprague Dawley rats were treated with Ang II with or without TMS (300 µg/kg every third day i.p.). Thereafter, cardiac hypertrophy and the formation of mid-chain HETEs and arachidonic acid were assessed. In vitro, RL-14 cells were treated with Ang II (10 µM) in the presence and absence of TMS (0.5 µM). Then, reactive oxygen species, mitogen-activated protein kinase phosphorylation levels, and nuclear factor-kappa B-binding activity were determined. Our results demonstrated that TMS protects against Ang II-induced cardiac hypertrophy as indicated by the improvement in cardiac functions shown by the echocardiography as well as by reversing the increase in heart weight to tibial length ratio caused by Ang II. In addition, the cardioprotective effect of TMS was associated with a significant decrease in cardiac mid-chain HETEs levels. Mechanistically, TMS inhibited reactive oxygen species formation, the phosphorylation of ERK1/2, p38 mitogen-activated protein kinase, and the binding of p65 NF-κB.
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Angiotensina II/toxicidad , Cardiomegalia/metabolismo , Cardiomegalia/prevención & control , Citocromo P-450 CYP1B1/antagonistas & inhibidores , Citocromo P-450 CYP1B1/metabolismo , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Ácidos Hidroxieicosatetraenoicos/metabolismo , Animales , Cardiomegalia/inducido químicamente , Cardiotónicos/farmacología , Cardiotónicos/uso terapéutico , Células Cultivadas , Humanos , Masculino , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Estilbenos/farmacología , Estilbenos/uso terapéutico , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
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.
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Circulación Colateral/efectos de los fármacos , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/crecimiento & desarrollo , Endotelio Vascular/patología , Ácidos Hidroxieicosatetraenoicos/metabolismo , Síndrome Metabólico/patología , Animales , Anticuerpos Bloqueadores/farmacología , Arteriolas/efectos de los fármacos , Capilares/efectos de los fármacos , Moléculas de Adhesión Celular/biosíntesis , Vasos Coronarios/patología , Endotelio Vascular/metabolismo , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Masculino , Síndrome Metabólico/metabolismo , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patología , Neutrófilos/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
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.
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Ácidos Hidroxieicosatetraenoicos/metabolismo , Isquemia/metabolismo , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica , Transducción de Señal , Inductores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/farmacología , Animales , Velocidad del Flujo Sanguíneo , Hipoxia de la Célula , Células Cultivadas , Familia 4 del Citocromo P450/metabolismo , Células Endoteliales/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Miembro Posterior , Humanos , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Ácidos Hidroxieicosatetraenoicos/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Isquemia/tratamiento farmacológico , Isquemia/fisiopatología , Ratones Endogámicos BALB C , Neovascularización Fisiológica/efectos de los fármacos , Flujo Sanguíneo Regional , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
UNLABELLED: Background/Aims . High salt (HS) intake may elevate blood pressure (BP), also in animals without genetic salt sensitivity. The development of salt-dependent hypertension could be mediated by endogenous vasoactive agents; here we examined the role of vasodilator epoxyeicosatrienoic acids (EETs) and vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE). METHODS: In conscious Wistar rats on HS diet systolic BP (SBP) was examined after chronic elevation of EETs using 4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (c-AUCB), a blocker of soluble epoxide hydrolase, or after inhibition of 20-HETE with 1-aminobenzotriazole (ABT). Thereafter, in acute experiments the responses of renal artery blood flow (Transonic probe) and renal regional perfusion (laser-Doppler) to intrarenal acetylcholine (ACh) or norepinephrine were determined. RESULTS: HS diet increased urinary 20-HETE excretion. The SBP increase was not reduced by c-AUCB but prevented by ABT until day 5 of HS exposure. Renal vasomotor responses to ACh or norepinephrine were similar on standard and HS diet. ABT but not c-AUCB abolished the responses to ACh. Conclusions . 20-HETE seems to mediate the early-phase HS diet-induced BP increase while EETs are not engaged in the process. Since HS exposure did not alter renal vasodilator responses to Ach, endothelial dysfunction is not a critical factor in the mechanism of salt-induced blood pressure elevation.
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Presión Sanguínea/efectos de los fármacos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Circulación Renal/efectos de los fármacos , Sodio en la Dieta/efectos adversos , Acetilcolina/metabolismo , Animales , Presión Arterial , Epóxido Hidrolasas/sangre , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Masculino , Óxido Nítrico/fisiología , Norepinefrina/metabolismo , Concentración Osmolar , Ratas , Ratas Wistar , Triazoles/farmacologíaRESUMEN
Vasoconstrictive and vasodilatory eicosanoids generated after cardiac arrest (CA) may contribute to cerebral vasomotor disturbances and neurodegeneration. We evaluated the balance of vasodilator/vasoconstrictor eicosanoids produced by cytochrome P450 (CYP) metabolism, and determined their role on cortical perfusion, functional outcome, and neurodegeneration after pediatric asphyxial CA. Cardiac arrest of 9 and 12 minutes was induced in 16- to 18-day-old rats. At 5 and 120 minutes after CA, we quantified the concentration of CYP eicosanoids in the cortex and subcortical areas. In separate rats, we inhibited 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis after CA and assessed cortical cerebral blood flow (CBF), neurologic deficit score, neurodegeneration, and edema. After 9 minutes of CA, vasodilator eicosanoids markedly increased versus sham. Conversely, after 12 minutes of CA, vasoconstrictor eicosanoid 20-HETE increased versus sham, without compensatory increases in vasodilator eicosanoids. Inhibition of 20-HETE synthesis after 12 minutes of CA decreased cortical 20-HETE levels, increased CBF, reduced neurologic deficits at 3 hours, and reduced neurodegeneration and edema at 48 hours versus vehicle-treated rats. In conclusion, cerebral vasoconstrictor eicosanoids increased after a pediatric CA of 12 minutes. Inhibition of 20-HETE synthesis improved cortical perfusion and short-term neurologic outcome. These results suggest that alterations in CYP eicosanoids have a role in cerebral hypoperfusion and neurodegeneration after CA and may represent important therapeutic targets.
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Amidinas/uso terapéutico , Asfixia Neonatal/tratamiento farmacológico , Edema Encefálico/tratamiento farmacológico , Corteza Cerebral/irrigación sanguínea , Circulación Cerebrovascular/efectos de los fármacos , Paro Cardíaco/tratamiento farmacológico , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Fármacos Neuroprotectores/uso terapéutico , Animales , Asfixia Neonatal/complicaciones , Asfixia Neonatal/fisiopatología , Agua Corporal/metabolismo , Química Encefálica/efectos de los fármacos , Edema Encefálico/etiología , Corteza Cerebral/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/metabolismo , Eicosanoides/metabolismo , Paro Cardíaco/complicaciones , Paro Cardíaco/fisiopatología , Masculino , Enfermedades Neurodegenerativas/prevención & control , Ratas , Ratas Sprague-Dawley , Resultado del TratamientoRESUMEN
The severity of perinatal hypoxia-ischemia and the delay in initiating therapeutic hypothermia limit the efficacy of hypothermia. After hypoxia-ischemia in neonatal piglets, the arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) has been found to contribute to oxidative stress at 3 h of reoxygenation and to eventual neurodegeneration. We tested whether early administration of a 20-HETE synthesis inhibitor after reoxygenation augments neuroprotection with 3-hour delayed hypothermia. In two hypothermic groups, whole body cooling from 38.5 to 34°C was initiated 3 h after hypoxia-ischemia. Rewarming occurred from 20 to 24 h; then anesthesia was discontinued. One hypothermic group received a 20-HETE inhibitor at 5 min after reoxygenation. A sham-operated group and another hypoxia-ischemia group remained normothermic. At 10 days of recovery, resuscitated piglets with delayed hypothermia alone had significantly greater viable neuronal density in the putamen, caudate nucleus, sensorimotor cortex, CA3 hippocampus, and thalamus than did piglets with normothermic recovery, but the values remained less than those in the sham-operated group. In piglets administered the 20-HETE inhibitor before hypothermia, the density of viable neurons in the putamen, cortex and thalamus was significantly greater than in the group with hypothermia alone. Cytochrome P450 4A, which can synthesize 20-HETE, was expressed in piglet neurons in these regions. We conclude that early treatment with a 20-HETE inhibitor enhances the therapeutic benefit of delayed hypothermia in protecting neurons in brain regions known to be particularly vulnerable to hypoxia-ischemia in term newborns.
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Amidinas/farmacología , Citocromo P-450 CYP4A/metabolismo , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Hipotermia Inducida/métodos , Hipoxia-Isquemia Encefálica/terapia , Fármacos Neuroprotectores/farmacología , Amidinas/administración & dosificación , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Ácidos Hidroxieicosatetraenoicos/biosíntesis , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Fármacos Neuroprotectores/administración & dosificación , PorcinosRESUMEN
We recently demonstrated that 12/15-lipoxygenase (LOX) derived metabolites, hydroxyeicosatetraenoic acids (HETEs), contribute to diabetic retinopathy (DR) via NADPH oxidase (NOX) and disruption of the balance in retinal levels of the vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). Here, we test whether PEDF ameliorates retinal vascular injury induced by HETEs and the underlying mechanisms. Furthermore, we pursue the causal relationship between LOX-NOX system and regulation of PEDF expression during DR. For these purposes, we used an experimental eye model in which normal mice were injected intravitreally with 12-HETE with/without PEDF. Thereafter, fluorescein angiography (FA) was used to evaluate the vascular leakage, followed by optical coherence tomography (OCT) to assess the presence of angiogenesis. FA and OCT reported an increased vascular leakage and pre-retinal neovascularization, respectively, in response to 12-HETE that were not observed in the PEDF-treated group. Moreover, PEDF significantly attenuated the increased levels of vascular cell and intercellular adhesion molecules, VCAM-1 and ICAM-1, elicited by 12-HETE injection. Accordingly, the direct relationship between HETEs and PEDF has been explored through in-vitro studies using Müller cells (rMCs) and human retinal endothelial cells (HRECs). The results showed that 12- and 15-HETEs triggered the secretion of TNF-α and IL-6, as well as activation of NFκB in rMCs and significantly increased permeability and reduced zonula occludens protein-1 (ZO-1) immunoreactivity in HRECs. All these effects were prevented in PEDF-treated cells. Furthermore, interest in PEDF regulation during DR has been expanded to include NOX system. Retinal PEDF was significantly restored in diabetic mice treated with NOX inhibitor, apocynin, or lacking NOX2 up to 80% of the control level. Collectively, our findings suggest that interfering with LOX-NOX signaling opens up a new direction for treating DR by restoring endogenous PEDF that carries out multilevel vascular protective functions.
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Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/antagonistas & inhibidores , Retinopatía Diabética/tratamiento farmacológico , Proteínas del Ojo/farmacología , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Factores de Crecimiento Nervioso/farmacología , Retina/efectos de los fármacos , Neovascularización Retiniana/tratamiento farmacológico , Serpinas/farmacología , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacología , Acetofenonas/farmacología , Animales , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/metabolismo , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Ependimogliales/efectos de los fármacos , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Regulación de la Expresión Génica , Humanos , Ácidos Hidroxieicosatetraenoicos/farmacología , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Inyecciones Intravítreas , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , NADPH Oxidasa 2 , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Retina/metabolismo , Retina/patología , Neovascularización Retiniana/genética , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/patología , Transducción de Señal , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismo , Proteína de la Zonula Occludens-1/genéticaRESUMEN
20-HETE is a potent inducer of endothelial ACE in vitro and administration of lisinopril or losartan attenuates blood pressure in models of 20-HETE-dependent hypertension. The present study was undertaken to further define the relationship between 20-HETE and the renin-angiotensin system in hypertension using an angiotensinogen-deficient mouse (Agt+/-). Treatment of male AGT+/- with 5α-dihydrotestosterone (DHT) increased systolic BP from 102±2 to 125±3mmHg; in comparison, the same treatment raised BP in wild type (WT) from 110±2 to 138±2mmHg. DHT increased vascular 20-HETE levels in AGT+/- and WT from 1.5±0.7 and 2.1±0.6 to 13.0±2.0 and 15.8±4.0ng/mg, respectively. Concurrent treatment with the 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) prevented the increases in BP in both AGT+/- and WT mice. Administration of 20-HEDE at the peak of the DHT-induced BP increase (12 days) reduced BP to basal levels after 48h. Interestingly, basal levels of renal microvascular EETs were higher in AGT+/- compared to WT (55.2±9.7 vs 20.0±4.1ng/mg) and treatment of AGT+/- with DHT decreased the levels of EETs (28.4±5.1ng/mg). DHT-mediated changes in vascular EET level were not observed in WT mice. Vascular Cyp4a12 and ACE protein levels were increased in both AGT+/- and WT by 30-40% and decreased with concomitant administration of 20-HEDE. Lisinopril was as effective as 20-HEDE in preventing DHT-mediated increases in BP in both AGT+/- and WT mice. This study substantiates our previous findings that the RAS plays an important role in 20-HETE-mediated hypertension. It also proposes a novel interaction between 20-HETE and EETs.
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Andrógenos/efectos adversos , Angiotensinas/deficiencia , Ácidos Hidroxieicosatetraenoicos/metabolismo , Ácidos Hidroxieicosatetraenoicos/farmacología , Hipertensión , Andrógenos/farmacología , Animales , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Ácidos Hidroxieicosatetraenoicos/genética , Hipertensión/inducido químicamente , Hipertensión/tratamiento farmacológico , Hipertensión/genética , Hipertensión/metabolismo , Masculino , Ratones , Ratones MutantesRESUMEN
In the kidney, 20-hydroxyeicosatetraenoic acid (20-HETE) is a primary cytochrome P450 4 (Cyp4)-derived eicosanoid that enhances vasoconstriction of renal vessels and induces hypertension, renal tubular cell hypertrophy, and podocyte apoptosis. Hypertension and podocyte injury contribute to diabetic nephropathy and are strong predictors of disease progression. In this study, we defined the mechanisms whereby 20-HETE affects the progression of diabetic nephropathy. We used Cyp4a14KO male mice that exhibit androgen-sensitive hypertension due to increased Cyp4a12-mediated 20-HETE production. We show that, upon induction of diabetes type 1 via streptozotocin injection, Cyp4a14KO male mice developed worse renal disease than streptozotocin-treated wild-type mice, characterized by increased albuminuria, mesangial expansion, glomerular matrix deposition, and thickness of the glomerular basement membranes. Castration blunted androgen-mediated Cyp4a12 synthesis and 20-HETE production, normalized BP, and ameliorated renal damage in diabetic Cyp4a14KO mice. Notably, treatment with a 20-HETE antagonist or agents that normalized BP without affecting Cyp4a12 expression and 20-HETE biosynthesis also ameliorated diabetes-mediated renal damage and albuminuria in Cyp4a14KO male mice. Taken together, these results suggest that hypertension is the major contributor to 20-HETE-driven diabetes-mediated kidney injury.