Pharmacological manipulation of arachidonic acid-epoxygenase results in divergent effects on renal damage.

Kidney damage is markedly accelerated by high-salt (HS) intake in stroke-prone spontaneously hypertensive rats (SHRSP). Epoxyeicosatrienoic acids (EETs) are epoxygenase products of arachidonic acid which possess vasodepressor, natriuretic, and anti-inflammatory activities. We examined whether up-regulation (clofibrate) or inhibition [N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH)] of epoxygenase would alter systolic blood pressure (SBP) and/or renal pathology in SHRSP on HS intake (1% NaCl drinking solution). Three weeks of treatment with clofibrate induced renal cortical protein expression of CYP2C23 and increased urinary excretion of EETs compared with vehicle-treated SHRSP. SBP and urinary protein excretion (UPE) were significantly lowered with clofibrate treatment. Kidneys from vehicle-treated SHRSP, which were on HS intake for 3 weeks, demonstrated focal lesions of vascular fibrinoid degeneration, which were markedly attenuated with clofibrate treatment. In contrast, 2 weeks of treatment with the selective epoxygenase inhibitor, MS-PPOH, increased UPE without significantly altering neither urinary EET levels nor SBP. Kidneys from vehicle-treated SHRSP, which were on HS intake for 11 days, demonstrated occasional mild damage whereas kidneys from MS-PPOH-treated rats exhibited widespread malignant nephrosclerosis. These results suggest that pharmacological manipulation of epoxygenase results in divergent effects on renal damage and that interventions to increase EET levels may provide therapeutic strategies for treating salt-sensitive hypertension and renal damage.

Effects of 5,14-HEDGE, a 20-HETE mimetic, on lipopolysaccharide-induced changes in MyD88/TAK1/IKKß/IκB-α/NF-κB pathway and circulating miR-150, miR-223, and miR-297 levels in a rat model of septic shock.

OBJECTIVES: We have previously demonstrated that a stable synthetic analog of 20-hydroxyeicosatetraenoic acid (20-HETE), N-(20-hydroxyeicosa-5[Z],14[Z]-dienoyl)glycine (5,14-HEDGE), which mimics the effects of endogenously produced 20-HETE, prevents vascular hyporeactivity, hypotension, tachycardia, inflammation, and mortality in a rodent model of septic shock. The present study was performed to determine whether decreased renal and cardiovascular expression and activity of myeloid differentiation factor 88 (MyD88)/transforming growth factor-activated kinase 1 (TAK1)/inhibitor of κB (IκB) kinase ß (IKKß)/IκB-α/nuclear factor-κB (NF-κB) pathway and reduced circulating microRNA (miR)-150, miR-223, and miR-297 expression levels participate in the protective effect of 5,14-HEDGE against hypotension, tachycardia, and inflammation in response to systemic administration of lipopolysaccharide (LPS). METHODS: Conscious male Wistar rats received saline (4 ml/kg) or LPS (10 mg/kg) at time 0. Blood pressure and heart rate were measured using a tail-cuff device. Separate groups of LPS-treated rats were given 5,14-HEDGE (30 mg/kg) 1 h after injection of saline or LPS. The rats were killed 4 h after LPS challenge and blood, kidney, heart, thoracic aorta, and superior mesenteric artery were collected for measurement of the protein expression. RESULTS: LPS-induced fall in blood pressure and rise in heart rate were associated with increased MyD88 expression and phosphorylation of TAK1 and IκB-α in cytosolic fractions of the tissues. LPS also caused an increase in both unphosphorylated and phosphorylated NF-κB p65 proteins in the cytosolic and nuclear fractions as well as nuclear translocation of NF-κB p65. In addition, serum miR-150, miR-223, and miR-297 expression levels were increased in LPS-treated rats. These effects of LPS were prevented by 5,14-HEDGE. CONCLUSIONS: These results suggest that downregulation of MyD88/TAK1/IKKß/IκB-α/NF-κB pathway as well as decreased circulating miR-150, miR-223, and miR-297 expression levels participate in the protective effect of 5,14-HEDGE against hypotension, tachycardia, and inflammation in the rat model of septic shock.

20-HETE induces remodeling of renal resistance arteries independent of blood pressure elevation in hypertension.

20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P-450 (Cyp)-derived arachidonic acid metabolite that has been shown to increase smooth muscle contractions and proliferation, stimulate endothelial dysfunction and activation, and promote hypertension. We examined if 20-HETE contributes to microvascular remodeling in hypertension. In Sprague-Dawley rats, administration of the 20-HETE biosynthesis inhibitor HET0016 or the 20-HETE antagonist N-20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) prevented 5α-dihydrotestosterone (DHT)-induced increases in blood pressure as well as abrogated DHT-induced increases in the media-to-lumen ratio (M/L), media thickness, and collagen IV deposition in renal interlobar arteries. Reserpine prevented blood pressure elevation in DHT-treated rats but did not affect microvascular remodeling (M/L, media thickness, and collagen deposition); under these conditions, treatment with the 20-HETE antagonist attenuated microvascular remodeling, suggesting that 20-HETE contributes to DHT-induced vascular remodeling independent of blood pressure elevation. In Cyp4a14(-/-) mice, which display androgen-driven and 20-HETE-dependent hypertension, treatment with the 20-HETE antagonist abolished remodeling of renal resistance arteries measured as media thickness (24 ± 1 vs. 15 ± 1 µm) and M/L (0.29 ± 0.03 vs. 0.17 ± 0.01). Moreover, in Cyp4a12 transgenic mice in which the expression of Cyp4a12-20-HETE synthase is driven by a tetracycline-sensitive promoter, treatment with doxycycline resulted in blood pressure elevation (140 ± 4 vs. 92 ± 5 mmHg) and a significant increase in remodeling of renal resistance arteries (media thickness: 23 ± 1 vs. 16 ± 1 µm; M/L: 0.39 ± 0.04 vs. 0.23 ± 0.02); these increases were abrogated by cotreatment with 20-HEDE. This study demonstrated that 20-HETE is a key regulator of microvascular remodeling in hypertension; its effect is independent of blood pressure elevation and androgen levels.

Stable EET urea agonist and soluble epoxide hydrolase inhibitor regulate rat pulmonary arteries through TRPCs.

Epoxyeicosatrienoic acids (EETs), cytochrome P450-derived metabolites of arachidonic acid, have been reported to increase intracellular calcium concentration in aortic vascular smooth muscle cells (SMCs). As EETs are labile, we synthesized a new stable urea EET analog with agonist and soluble epoxide hydrolase (sEH) inhibitor properties. We refer to this analog, 12-(3-hexylureido)dodec-8-enoic acid, as 8-HUDE. Measuring tension of vascular rings, intracellular calcium signaling by confocal laser scanning microscopy and gene expression by reverse-transcription-PCR and western blots, we examined the effects of 8-HUDE on pulmonary vascular tone and calcium signaling in rat pulmonary artery (PA) SMCs (PASMCs). 8-HUDE increased the tension of rat PAs to 145% baseline, whereas it had no effect on the tension of mesenteric arteries (MAs). The 8-HUDE-induced increase in vascular tone was abolished by removal of extracellular Ca(2+) or by pretreatment with either La(3+) or SKF96365, which are inhibitors of canonical transient receptor potential channels (TRPCs). Furthermore, 8-HUDE-evoked increases in [Ca(2+)](i) in PASMCs could be blunted by inhibition of TRPC with SKF96365, removal of extracellular calcium or depletion of intracellular calcium stores with caffeine, cyclopiazonic acid or 2-aminoethoxydiphenyl borate, but not by the voltage-activated calcium channel blocker nifedipine. In addition to immediate effects on calcium signaling, 8-HUDE upregulated the expression of TRPC1 and TRPC6 at both mRNA and protein levels in rat PASMCs, whereas it suppressed the expression of sEH. Our observations suggest that 8-HUDE increases PA vascular tone through increased release of calcium from intracellular stores, enhanced [Ca(2+)](i) influx in PASMCs through store-operated Ca(2+) channels and modulated the expression of TRPC and sEH proteins in a proconstrictive manner.

Inhibition of 20-HETE synthesis and action protects the kidney from ischemia/reperfusion injury.

20-Hydroxyeicosatetraenoic acid (20-HETE) production is increased in ischemic kidney tissue and may contribute to ischemia/reperfusion (I/R) injury by mediating vasoconstriction and inflammation. To test this hypothesis, uninephrectomized male Lewis rats were exposed to warm ischemia following pretreatment with either an inhibitor of 20-HETE synthesis (HET0016), an antagonist (20-hydroxyeicosa-6(Z),15(Z)-dienoic acid), an agonist (20-hydroxyeicosa-5(Z),14(Z)-dienoic acid), or vehicle via the renal artery and the kidneys were examined 2 days after reperfusion. Pretreatment with either the inhibitor or the antagonist attenuated I/R-induced renal dysfunction as shown by improved creatinine clearance and decreased plasma urea levels, compared to controls. The inhibitor and antagonist also markedly reduced tubular lesion scores, inflammatory cell infiltration, and tubular epithelial cell apoptosis. Administering the antagonist accelerated the recovery of medullary perfusion, as well as renal medullary and cortical re-oxygenation, during the early reperfusion phase. In contrast, the agonist did not improve renal injury and reversed the beneficial effect of the inhibitor. Thus, 20-HETE generation and its action mediated kidney injury due to I/R. Whether or not these effects are clinically important will need to be tested in appropriate human studies.

8,9-Epoxyeicosatrienoic acid analog protects pulmonary artery smooth muscle cells from apoptosis via ROCK pathway.

Epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid (AA) catalyzed by cytochrome P450 (CYP), have many essential biologic roles in the cardiovascular system including inhibition of apoptosis in cardiomyocytes. In the present study, we tested the potential of 8,9-EET and derivatives to protect pulmonary artery smooth muscle cells (PASMCs) from starvation induced apoptosis. We found 8,9-epoxy-eicos-11(Z)-enoic acid (8,9-EET analog (214)), but not 8,9-EET, increased cell viability, decreased activation of caspase-3 and caspase-9, and decreased TUNEL-positive cells or nuclear condensation induced by serum deprivation (SD) in PASMCs. These effects were reversed after blocking the Rho-kinase (ROCK) pathway with Y-27632 or HA-1077. Therefore, 8,9-EET analog (214) protects PASMC from serum deprivation-induced apoptosis, mediated at least in part via the ROCK pathway. Serum deprivation of PASMCs resulted in mitochondrial membrane depolarization, decreased expression of Bcl-2 and enhanced expression of Bax, all effects were reversed by 8,9-EET analog (214) in a ROCK dependent manner. Because 8,9-EET and not the 8,9-EET analog (214) protects pulmonary artery endothelial cells (PAECs), these observations suggest the potential to differentially promote apoptosis or survival with 8,9-EET or analogs in pulmonary arteries.

20-HETE-induced nitric oxide production in pulmonary artery endothelial cells is mediated by NADPH oxidase, H2O2, and PI3-kinase/Akt.

We have shown that 20-hydroxyeicosatetraenoic acid (20-HETE) increases both superoxide and nitric oxide (NO) production in bovine pulmonary artery endothelial cells (BPAECs). The current study was designed to determine mechanisms underlying 20-HETE-stimulated NO release, and particularly the role of NADPH oxidase, reactive oxygen species, and PI3-kinase in stimulated NO release. Intracellular hydrogen peroxide (H(2)O(2)) and NO production were detected by dichlorofluorescein or dihydrorhodamine and diaminofluorescein fluorescence, respectively. Activation of endothelial nitric oxide synthase (eNOS) (Ser1179) and Akt (Ser473) was assessed by comparing the ratio of phosphorylated to total protein expression by Western blotting. Addition of 20-HETE to BPAECs caused an increase in superoxide and hydrogen peroxide, but not peroxynitrite. 20-HETE-evoked activation of Akt and eNOS, as well as enhanced NO release, are dependent on H(2)O(2) as opposed to superoxide in that these endpoints are blocked by PEG-catalase and not PEG-superoxide dismutase. Similarly, 20-HETE-stimulated NO production in BPAECs is blocked by NADPH oxidase inhibitors apocynin or gp91 blocking peptide, and by PI3-kinase/Akt blockers wortmannin, LY-294002, or Akt inhibitor, implicating NADPH oxidase, PI3-kinase, and Akt signaling pathways, respectively, in this process. Together, these data suggest the following scheme: 20-HETE stimulates NADPH oxidase-dependent formation of superoxide. Superoxide is rapidly dismutated to hydrogen peroxide, which then mediates activation of PI3-kinase/Akt, phosphorylation of eNOS, and enhanced release of NO from eNOS in response to 20-HETE in BPAECs.

Epoxyeicosatrienoic acid agonist rescues the metabolic syndrome phenotype of HO-2-null mice.

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.

14,15-Epoxyeicosa-5,8,11-trienoic acid (14,15-EET) surrogates containing epoxide bioisosteres: influence upon vascular relaxation and soluble epoxide hydrolase inhibition.

All-cis-14,15-epoxyeicosa-5,8,11-trienoic acid (14,15-EET) is a labile, vasodilatory eicosanoid generated from arachidonic acid by cytochrome P450 epoxygenases. A series of robust, partially saturated analogues containing epoxide bioisosteres were synthesized and evaluated for relaxation of precontracted bovine coronary artery rings and for in vitro inhibition of soluble epoxide hydrolase (sEH). Depending upon the bioisostere and its position along the carbon chain, varying levels of vascular relaxation and/or sEH inhibition were observed. For example, oxamide 16 and N-iPr-amide 20 were comparable (ED(50) 1.7 microM) to 14,15-EET as vasorelaxants but were approximately 10-35 times less potent as sEH inhibitors (IC(50) 59 and 19 microM, respectively); unsubstituted urea 12 showed useful activity in both assays (ED(50) 3.5 microM, IC(50) 16 nM). These data reveal differential structural parameters for the two pharmacophores that could assist the development of potent and specific in vivo drug candidates.

Protective actions of epoxyeicosatrienoic acid: dual targeting of cardiovascular PI3K and KATP channels.

Epoxyeicosatrienoic acid(s) (EETs) have been shown to protect cardiovascular tissue against apoptosis dependent on activation of targets such as ATP-sensitive K+ (KATP) channels (sarcolemmal and mitochondrial), calcium-activated K+ channels, extracellular signal-regulated kinase or phosphoinositide 3-kinase (PI3K). We tested if EETs protect human atrial tissue ex vivo from hypoxia/reoxygenation (H/R) injury, and compared our results with myocardium from two rodent species, rats and mice. EETs reduced myocardial caspase 3 activity in all three species and protected against loss of mitochondrial membrane potential in primary cultures of neonatal rat ventricular myocytes submitted to H/R. In addition, EETs protected mouse pulmonary arteries ex vivo exposed to H/R. Myocardium and pulmonary arteries from genetically engineered mice having elevated plasma levels of EETs (Ephx2-/-) exhibited protection from H/R-induced injury over that of wild type controls, suggesting that endogenously produced EETs may have pro-survival effects. Electrophysiological studies in myocytes demonstrated that EETs can stimulate KATP currents even when PI3K is inhibited. Similarly, activation of PI3K/Akt occurred in the presence of the KATP channel blocker glibenclamide. Based upon loss of protection with EETs in the presence of either wortmannin (a PI3K inhibitor) or glibenclamide, simultaneous activation of at least 2 pathways, PI3K and KATP channels respectively, appears to be required for protection. In conclusion, we demonstrate that exogenous and endogenous EETs have powerful pro-survival effects in cardiovascular tissues including diseased human myocardium, mediated by activation of not only one but at least two pathways, PI3K and KATP channels.

Protective effect of 20-HETE analogues in experimental renal ischemia reperfusion injury.

While it is known that the arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to ischemic injury in the heart and brain, its role in kidney injury is unclear. Here we determined the effects on ischemia-reperfusion injury of the 20-HETE analogues, 20-hydroxyeicosa-5(Z), 14(Z)-dienoic acid (5,14-20-HEDE), and N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14-20-HEDGE), and of the inhibitor of 20-HETE synthesis N-hydroxy-N-(4-butyl-2 methylphenyl) formamidine (HET0016). Using Sprague-Dawley rats we found that while treatment with the inhibitor exacerbated renal injury, infusion of both 5,14-20-HEDE and 5,14-20-HEDGE significantly attenuated injury when compared to vehicle or inhibitor-treated rats. Medullary blood flow, measured by laser-Doppler flowmetry, decreased to half of the baseline one hour after reperfusion in the control rats, but 5,14-20-HEDGE completely prevented this. Treatment of control animals with 5,14-20-HEDGE increased urine output and sodium excretion without altering their mean arterial pressure or glomerular filtration rate. Our results suggest that 20-HETE analogues protect the kidney from ischemia-reperfusion injury by inhibiting renal tubular sodium transport and preventing the post-ischemic fall in medullary blood flow. Analogues of 20-HETE may be useful in the treatment of acute ischemic kidney injury.

20-Hydroxyeicosatetraenoic acid stimulates nuclear factor-kappaB activation and the production of inflammatory cytokines in human endothelial cells.

Endothelial dysfunction is associated with endothelial cell activation, i.e., up-regulation of surface cell adhesion molecules and the release of proinflammatory cytokines. 20-Hydroxyeicosatetraenoic acid (HETE), a major vasoactive eicosanoid in the microcirculation, has been implicated in the regulation of endothelial cell function through its angiogenic and pro-oxidative properties. We examined the effects of 20-HETE on endothelial cell activation in vitro. Cells transduced with adenovirus containing either CYP4A1 or CYP4A2 produced higher levels of 20-HETE, and they demonstrated increased expression levels of the adhesion molecule intercellular adhesion molecule (ICAM) (4-7-fold) and the oxidative stress marker 3-nitrotyrosine (2-3-fold) compared with cells transduced with control adenovirus. Treatment of cells with 20-HETE markedly increased levels of prostaglandin (PG) E(2) and 8-epi-isoprostane PGF(2alpha), commonly used markers of activation and oxidative stress, and most prominently, interleukin-8, a potent neutrophil chemotactic factor whose overproduction by the endothelium is a key feature of vascular injury. 20-HETE at nanomolar concentrations increased inhibitor of nuclear factor-kappaB phosphorylation by 2 to 5-fold within 5 min, which was followed with increased nuclear translocation of nuclear factor-kappaB (NF-kappaB). Likewise, 20-HETE activated the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway by stimulating phosphorylation of ERK1/2. Inhibition of NF-kappaB activation and inhibition of ERK1/2 phosphorylation inhibited 20-HETE-induced ICAM expression. It seems that 20-HETE triggers NF-kappaB and MAPK/ERK activation and that both signaling pathways participate in the cellular mechanisms by which 20-HETE activates vascular endothelial cells.

Hydrogen peroxide inhibits cytochrome p450 epoxygenases: interaction between two endothelium-derived hyperpolarizing factors.

The cytochrome P450 epoxygenase (CYP)-derived metabolites of arachidonic acid the epoxyeicosatrienoic acids (EETs) and hydrogen peroxide (H2O2) both function as endothelium-derived hyperpolarizing factors (EDHFs) in the human coronary microcirculation. However, the relative importance of and potential interactions between these 2 vasodilators remain unexplored. We identified a novel inhibitory interaction between CYPs and H2O2 in human coronary arterioles, where EDHF-mediated vasodilatory mechanisms are prominent. Bradykinin induced vascular superoxide and H2O2 production in an endothelium-dependent manner and elicited a concentration-dependent dilation that was reduced by catalase but not by 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE), 6-(2-propargyloxyphenyl)hexanoic acid, sulfaphenazole, or iberiotoxin. However, in the presence of catalase, an inhibitory effect of these compounds was unmasked. In a tandem-bioassay preparation, application of bradykinin to endothelium-intact donor vessels elicited dilation of downstream endothelium-denuded detectors that was partially inhibited by donor-applied catalase but not by detector-applied EEZE; however, EEZE significantly inhibited dilation in the presence of catalase. EET production by human recombinant CYP 2C9 and 2J2, 2 major epoxygenase isozymes expressed in human coronary arterioles, was directly inhibited in a concentration-dependent fashion by H2O2 in vitro, as observed by high-performance liquid chromatography (HPLC); however, EETs were not directly sensitive to oxidative modification. H2O2 inhibited dilation to arachidonic acid but not to 11,12-EET. These findings suggest that an inhibitory interaction exists between 2 EDHFs in the human coronary microcirculation. CYP epoxygenases are directly inhibited by H2O2, and this interaction may modulate vascular EET bioavailability.