Selective inhibition of prostaglandin D2 biosynthesis in human mast cells to overcome need for multiple receptor antagonists: Biochemical consequences.

BACKGROUND: The major mast cell prostanoid PGD2 is targeted for therapy of asthma and other diseases, because the biological actions include bronchoconstriction, vasodilation and regulation of immune cells mediated by three different receptors. It is not known if the alternative to selectively inhibit the biosynthesis of PGD2 affects release of other prostanoids in human mast cells. OBJECTIVES: To determine the biochemical consequences of inhibition of the hematopoietic prostaglandin D synthase (hPGDS) PGD2 in human mast cells. METHODS: Four human mast cell models, LAD2, cord blood derived mast cells (CBMC), peripheral blood derived mast cells (PBMC) and human lung mast cells (HLMC), were activated by anti-IgE or ionophore A23187. Prostanoids were measured by UPLC-MS/MS. RESULTS: All mast cells almost exclusively released PGD2 when activated by anti-IgE or A23187. The biosynthesis was in all four cell types entirely initiated by COX-1. When pharmacologic inhibition of hPGDS abolished formation of PGD2 , PGE2 was detected and release of TXA2 increased. Conversely, when the thromboxane synthase was inhibited, levels of PGD2 increased. Adding exogenous PGH2 confirmed predominant conversion to PGD2 under control conditions, and increased levels of TXB2 and PGE2 when hPGDS was inhibited. However, PGE2 was formed by non-enzymatic degradation. CONCLUSIONS: Inhibition of hPGDS effectively blocks mast cell dependent PGD2 formation. The inhibition was associated with redirected use of the intermediate PGH2 and shunting into biosynthesis of TXA2 . However, the levels of TXA2 did not reach those of PGD2 in naïve cells. It remains to determine if this diversion occurs in vivo and has clinical relevance.

Cytochrome P450 1A1 enhances Arginase-1 expression, which reduces LPS-induced mouse peritonitis by targeting JAK1/STAT6.

The polarization of macrophages is critical to inflammation and tissue repair, with unbalanced macrophage polarization associated with critical dysfunctions of the immune system. Cytochrome P450 1A1 (CYP1A1) is a hydroxylase mainly controlled by the inflammation-limiting aryl hydrocarbon receptor (AhR), which plays a critical role in mycoplasma infection, oxidative stress injury, and cancer. Arginase-1 (Arg-1) is a surrogate for polarized alternative macrophages and is important to the production of nitric oxide (NO) by the modulation of arginine. In the present study, we found CYP1A1 to be upregulated in IL-4-stimulated mouse peritoneal macrophages (PMs) and human peripheral blood monocytes. Using CYP1A1-overexpressing RAW264.7 cells (CYP1A1/RAW) we found that CYP1A1 augmented Arg-1 expression by strengthening the activation of the JAK1/STAT6 signaling pathway in macrophages treated with IL-4. 15(S)-HETE, a metabolite of CYP1A1 hydroxylase, was elevated in IL-4-induced CYP1A1/RAW cells. Further, in macrophages, the loss-of-CYP1A1-hydroxylase activity was associated with reduced IL-4-induced Arg-1 expression due to impaired 15(S)-HETE generation. Of importance, CYP1A1 overexpressing macrophages reduced the inflammation associated with LPS-induced peritonitis. Taken together, these findings identified a novel signaling axis, CYP1A1-15(S)-HETE-JAK1-STAT6, that may be a promising target for the proper maintenance of macrophage polarization and may also be a means by which to treat immune-related disease due to macrophage dysfunction.

Effects of PPARs/20-HETE on the renal impairment under diabetic conditions.

Diabetic nephropathy (DN) is one of the most severe complications of diabetes mellitus. The pathomolecular events behind DN remain uncertain. Peroxisome proliferator-activated receptors (PPARs) play essential functions in the development of DN. Meanwhile, 20-hydroxyeicosatetraenoic acid (20-HETE) also plays central roles in the regulation of renal function. However, the relationship between PPARs and 20-HETE is rarely studied in DN. It was revealed in our study that both PPARs expression and CYP4A-20-HETE level were decreased under DN conditions in vivo and in vitro. Supplementation with bezafibrate, a PPAR pan-agonist, improved the damage of kidney in DN mice and in high glucose-induced NRK-52E cells, following the up-regulation of PPARs and the increase of CYP4A-20-HETE. PPARα antagonist (MK886), PPARß antagonist (GSK0660), and PPARγ antagonist (GW9662) reversed the protection of bezafibrate in NRK-52E, and abrogated the up-regulation of CYP4A-20-HETE produced by bezafibrate. Noteworthily, 20-HETE synthetase inhibitor, HET0016, also blocked the bezafibrate-mediated improvement of NRK-52E, and abolished the up-regulation of PPARs expression. Collectively, our data suggest that the concurrent down-regulation and interaction of PPARs and 20-HETE play crucial roles in the pathogenesis process of DN, and we provide a novel evidence that PPARs/20-HETE signaling may be served as a therapeutic target for DN patients.

Capillary pericytes regulate cerebral blood flow in health and disease.

Increases in brain blood flow, evoked by neuronal activity, power neural computation and form the basis of BOLD (blood-oxygen-level-dependent) functional imaging. Whether blood flow is controlled solely by arteriole smooth muscle, or also by capillary pericytes, is controversial. We demonstrate that neuronal activity and the neurotransmitter glutamate evoke the release of messengers that dilate capillaries by actively relaxing pericytes. Dilation is mediated by prostaglandin E2, but requires nitric oxide release to suppress vasoconstricting 20-HETE synthesis. In vivo, when sensory input increases blood flow, capillaries dilate before arterioles and are estimated to produce 84% of the blood flow increase. In pathology, ischaemia evokes capillary constriction by pericytes. We show that this is followed by pericyte death in rigor, which may irreversibly constrict capillaries and damage the blood-brain barrier. Thus, pericytes are major regulators of cerebral blood flow and initiators of functional imaging signals. Prevention of pericyte constriction and death may reduce the long-lasting blood flow decrease that damages neurons after stroke.

Heart failure-induced activation of phospholipase iPLA2γ generates hydroxyeicosatetraenoic acids opening the mitochondrial permeability transition pore.

Congestive heart failure typically arises from cardiac myocyte necrosis/apoptosis, associated with the pathological opening of the mitochondrial permeability transition pore (mPTP). mPTP opening decreases the mitochondrial membrane potential leading to the activation of Ca2+-independent phospholipase A2γ (iPLA2γ) and the production of downstream toxic metabolites. However, the array of enzymatic mediators and the exact chemical mechanisms responsible for modulating myocardial mPTP opening remain unclear. Herein, we demonstrate that human heart failure activates specific myocardial mitochondrial phospholipases that increase Ca2+-dependent production of toxic hydroxyeicosatetraenoic acids (HETEs) and attenuate the activity of phospholipases that promote the synthesis of protective epoxyeicosatrienoic acids (EETs). Mechanistically, HETEs activated the Ca2+-induced opening of the mPTP in failing human myocardium, and the highly selective pharmacological blockade of either iPLA2γ or lipoxygenases attenuated mPTP opening in failing hearts. In contrast, pharmacological inhibition of cytochrome P450 epoxygenases opened the myocardial mPTP in human heart mitochondria. Remarkably, the major mitochondrial phospholipase responsible for Ca2+-activated release of arachidonic acid (AA) in mitochondria from non-failing hearts was calcium-dependent phospholipase A2ζ (cPLA2ζ) identified by sequential column chromatographies and activity-based protein profiling. In contrast, iPLA2γ predominated in failing human myocardium. Stable isotope kinetics revealed that in non-failing human hearts, cPLA2ζ metabolically channels arachidonic acid into EETs, whereas in failing hearts, increased iPLA2γ activity channels AA into toxic HETEs. These results mechanistically identify the sequelae of pathological remodeling of human mitochondrial phospholipases in failing myocardium. This remodeling metabolically channels AA into toxic HETEs promoting mPTP opening, which induces necrosis/apoptosis leading to further progression of heart failure.

Heme-thiolate sulfenylation of human cytochrome P450 4A11 functions as a redox switch for catalytic inhibition.

Cytochrome P450 (P450, CYP) 4A11 is a human fatty acid ω-hydroxylase that catalyzes the oxidation of arachidonic acid to the eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE), which plays important roles in regulating blood pressure regulation. Variants of P450 4A11 have been associated with high blood pressure and resistance to anti-hypertensive drugs, and 20-HETE has both pro- and antihypertensive properties relating to increased vasoconstriction and natriuresis, respectively. These physiological activities are likely influenced by the redox environment, but the mechanisms are unclear. Here, we found that reducing agents (e.g. dithiothreitol and tris(2-carboxyethyl)phosphine) strongly enhanced the catalytic activity of P450 4A11, but not of 10 other human P450s tested. Conversely, added H2O2 attenuated P450 4A11 catalytic activity. Catalytic roles of five of the potentially eight implicated Cys residues of P450 4A11 were eliminated by site-directed mutagenesis. Using an isotope-coded dimedone/iododimedone-labeling strategy and mass spectrometry of peptides, we demonstrated that the heme-thiolate cysteine (Cys-457) is selectively sulfenylated in an H2O2 concentration-dependent manner. This sulfenylation could be reversed by reducing agents, including dithiothreitol and dithionite. Of note, we observed heme ligand cysteine sulfenylation of P450 4A11 ex vivo in kidneys and livers derived from CYP4A11 transgenic mice. We also detected sulfenylation of murine P450 4a12 and 4b1 heme peptides in kidneys. To our knowledge, reversible oxidation of the heme thiolate has not previously been observed in P450s and may have relevance for 20-HETE-mediated functions.

Angiotensin II upregulates CYP4A isoform expression in the rat kidney through angiotensin II type 1 receptor.

Angiotensin II (AngII) stimulates the renal production and release of 20-hydroxyeicosatetraenoic acids (20-HETE), which is a major metabolite of arachidonic acid catalyzed by CYP4A isoforms. However, the effects of AngII on CYP4A isoform expression in the kidney and its mechanism remains unclear. To clarify the regulation of CYP4A isoform expression by AngII, we examined the chronic effects of AngII and AngII type 1 receptor (AT1-R) blockade on CYP4A isoform expression. Sprague-Dawley rats were infused with vehicle or AngII for 1 week, and the AngII-infused rats were also treated with or without the AT1-R blocker, candesartan. AngII increased CYP4A isoform protein expression in the renal cortex (CO) and outer medulla (OM) in a dose-dependent manner, and candesartan inhibited the AngII-increased CYP4A expression in a dose-dependent manner. AngII increased the CYP4A isoform mRNA expression in the CO and OM, and candesartan inhibited AngII-increased CYP4A isoform mRNA expression. These results indicated that AngII chronically increased the CYP4A isoform expression in the rat kidney. The AngII-induced CYP4A isoform expression was mediated by AT1-R.

Discovery of rubiarbonone C as a selective inhibitor of cytochrome P450 4F enzymes.

Cytochrome P450 (CYP) enzymes, particularly CYP4A/4F, are the major ω-hydroxylases of arachidonic acid (AA) that can produce 20-hydroxyeicosatetraenoic acid (20-HETE). Although there are dissimilarities in substrate specificity, tissue distribution, and gene regulation between CYP4A and CYP4F, selective CYP4A or 4F inhibitors are currently unavailable. Therefore, this study was designed to develop CYP4F selective inhibitors using a novel inhibitory assay of 20-HETE formation. The assay was established using pooled human kidney microsomes (HKMs) and human recombinant CYP4 enzymes incubated with 1,2,3,4,5-13C AA (13C5 AA) as a substrate to minimize interference by endogenous AA. The intrinsic clearance (Vmax/Km) values were 9.5 µL/min/mg for HKMs and 0.02, 0.9, and 10.1 µL/min/pmol for CYP4A11, CYP4F2, and CYP4F3B, respectively, which suggests a major role for CYP4F in ω-hydroxylation of AA. To validate the assay, we tested well-known pan-CYP4 inhibitor HET0016 along with 50 compounds derived from natural products. Of the screened compounds, rubiarbonone C showed the most potent inhibitory activity. The 50% inhibitory concentrations of rubiarbonone C against CYP4A11, CYP4F2, and 4F3B were > 50, 4.2, and 4.2 µM, respectively. Moreover, epoxyeicosatrienoic acid formation from 13C5 AA was not inhibited by up to 30 µM rubiarbonone C. Meanwhile, in pooled human liver microsomes, CYP1, 2, and 3 family enzymes involved in drug metabolism were not substantially inhibited by rubiarbonone C. Thus, rubiarbonone C is a selective inhibitor of CYP4F and can be used to discriminate among CYP4 family enzymes and evaluate their roles in physiological and pathophysiological conditions.

Preferential Generation of 15-HETE-PE Induced by IL-13 Regulates Goblet Cell Differentiation in Human Airway Epithelial Cells.

Type 2-associated goblet cell hyperplasia and mucus hypersecretion are well known features of asthma. 15-Lipoxygenase-1 (15LO1) is induced by the type 2 cytokine IL-13 in human airway epithelial cells (HAECs) in vitro and is increased in fresh asthmatic HAECs ex vivo. 15LO1 generates a variety of products, including 15-hydroxyeicosatetraenoic acid (15-HETE), 15-HETE-phosphatidylethanolamine (15-HETE-PE), and 13-hydroxyoctadecadienoic acid (13-HODE). In this study, we investigated the 15LO1 metabolite profile at baseline and after IL-13 treatment, as well as its influence on goblet cell differentiation in HAECs. Primary HAECs obtained from bronchial brushings of asthmatic and healthy subjects were cultured under air-liquid interface culture supplemented with arachidonic acid and linoleic acid (10 µM each) and exposed to IL-13 for 7 days. Short interfering RNA transfection and 15LO1 inhibition were applied to suppress 15LO1 expression and activity. IL-13 stimulation induced expression of 15LO1 and preferentially generated 15-HETE-PE in vitro, both of which persisted after removal of IL-13. 15LO1 inhibition (by short interfering RNA and chemical inhibitor) decreased IL-13-induced forkhead box protein A3 (FOXA3) expression and enhanced FOXA2 expression. These changes were associated with reductions in both mucin 5AC and periostin. Exogenous 15-HETE-PE stimulation (alone) recapitulated IL-13-induced FOXA3, mucin 5AC, and periostin expression. The results of this study confirm the central importance of 15LO1 and its primary product, 15-HETE-PE, for epithelial cell remodeling in HAECs.

Functional characterization of a common CYP4F11 genetic variant and identification of functionally defective CYP4F11 variants in erythromycin metabolism and 20-HETE synthesis.

CYP4F11, together with CYP4F2, plays an important role in the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) from arachidonic acid. We identified 21 variants by whole exome sequencing, including 4 non-synonymous variants in Korean subjects. The proteins of the wild-type CYP4F11 and the four coding variants (C276R, D315N, D374Y, and D446N) were expressed in Escherichia coli DH5α cells and purified to give cytochrome P450-specific carbon monoxide difference spectra. Wild-type CYP4F2 was also expressed and purified to compare its activity with the CYP4F11 wild-type. Wild-type CYP4F11 exhibited the highest maximal clearance for erythromycin N-demethylase activity followed by the variants D374Y, D446N, C276R, and D315N. In particular, the CYP4F11 D315N protein showed about 50% decrease in intrinsic clearance compared to the wild type. The ability of wild-type CYP4F11 and the variants to synthesize 20-HETE from arachidonic acid was similar; the CYP4F11 D315N variant, however, showed only 68% of wild-type activity. Furthermore, the ability of CYP4F2 to synthesize 20-HETE was 1.7-fold greater than that of CYP4F11. Overall, our results suggest that the metabolism of CYP4F11 substrates may be reduced in individuals carrying the CYP4F11 D315N genetic variant and individuals carrying the common D446N CYP4F11 variant likely exhibit comparable 20-HETE synthesis as individuals expressing wild-type CYP4F11.

Upregulation of 20-HETE Synthetic Cytochrome P450 Isoforms by Oxygen-Glucose Deprivation in Cortical Neurons.

20-Hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictor, is a cytochrome P450 (CYP) 4A/4F-derived metabolite of arachidonic acid. Inhibition of 20-HETE synthesis protects brain from ischemic injury. However, that protection is not associated with changes in cerebral blood flow. The present study examined whether CYP4A isoforms are expressed in neurons, whether they produce 20-HETE in neurons, and whether neuronally derived 20-HETE exerts direct neurotoxicity after oxygen-glucose deprivation (OGD). The expression of Cyp4a10 and Cyp4a12a mRNA in cultured mouse cortical neurons increased significantly at 1 and 3 h after exposure to 1 h of OGD. Reoxygenation also markedly augmented the expression of CYP4A protein in neurons and increased 20-HETE levels in the culture medium. Cell viability after OGD increased after treatment with a 20-HETE synthesis inhibitor or an antagonist. That effect was reversed by co-administration of a 20-HETE agonist. These results indicate that neurons express Cyp4a10 and 4a12a, that expression of these isoforms is upregulated by OGD stress, and that neuronally derived 20-HETE directly contributes to neuronal death after reoxygenation.

Arachidonic Acid Metabolite as a Novel Therapeutic Target in Breast Cancer Metastasis.

Metastatic breast cancer (BC) (also referred to as stage IV) spreads beyond the breast to the bones, lungs, liver, or brain and is a major contributor to the deaths of cancer patients. Interestingly, metastasis is a result of stroma-coordinated hallmarks such as invasion and migration of the tumor cells from the primary niche, regrowth of the invading tumor cells in the distant organs, proliferation, vascularization, and immune suppression. Targeted therapies, when used as monotherapies or combination therapies, have shown limited success in decreasing the established metastatic growth and improving survival. Thus, novel therapeutic targets are warranted to improve the metastasis outcomes. We have been actively investigating the cytochrome P450 4 (CYP4) family of enzymes that can biosynthesize 20-hydroxyeicosatetraenoic acid (20-HETE), an important signaling eicosanoid involved in the regulation of vascular tone and angiogenesis. We have shown that 20-HETE can activate several intracellular protein kinases, pro-inflammatory mediators, and chemokines in cancer. This review article is focused on understanding the role of the arachidonic acid metabolic pathway in BC metastasis with an emphasis on 20-HETE as a novel therapeutic target to decrease BC metastasis. We have discussed all the significant investigational mechanisms and put forward studies showing how 20-HETE can promote angiogenesis and metastasis, and how its inhibition could affect the metastatic niches. Potential adjuvant therapies targeting the tumor microenvironment showing anti-tumor properties against BC and its lung metastasis are discussed at the end. This review will highlight the importance of exploring tumor-inherent and stromal-inherent metabolic pathways in the development of novel therapeutics for treating BC metastasis.

Strict Regiospecificity of Human Epithelial 15-Lipoxygenase-2 Delineates Its Transcellular Synthesis Potential.

Lipoxins are an important class of lipid mediators that induce the resolution of inflammation and arise from transcellular exchange of arachidonic acid (AA)-derived lipoxygenase products. Human epithelial 15-lipoxygenase-2 (h15-LOX-2), the major lipoxygenase in macrophages, has exhibited strict regiospecificity, catalyzing only the hydroperoxidation of carbon 15 of AA. To determine the catalytic potential of h15-LOX-2 in transcellular synthesis events, we reacted it with the three lipoxygenase-derived monohydroperoxy-eicosatetraenoic acids (HPETE) in humans: 5-HPETE, 12-HPETE, and 15-HPETE. Only 5-HPETE was a substrate for h15-LOX-2, and the steady-state catalytic efficiency (kcat/Km) of this reaction was 31% of the kcat/Km of AA. The only major product of h15-LOX-2's reaction with 5-HPETE was the proposed lipoxin intermediate, 5,15-dihydroperoxy-eicosatetraenoic acid (5,15-diHPETE). However, h15-LOX-2 did not react further with 5,15-diHPETE to produce lipoxins. This result is consistent with the specificity of h15-LOX-2 despite the increased reactivity of 5,15-diHPETE. Density functional theory calculations determined that the radical, after abstracting the C10 hydrogen atom from 5,15-diHPETE, had an energy 5.4 kJ/mol lower than that of the same radical generated from AA, demonstrating the facility of 5,15-diHPETE to form lipoxins. Interestingly, h15-LOX-2 does react with 5S,6R-diHETE, forming LipoxinA4, indicating the gemdiol does not prohibit h15-LOX-2 reactivity. Taken together, these results demonstrate the strict regiospecificity of h15-LOX-2 that circumscribes its role in transcellular synthesis.

Identification of ACSL4 as a biomarker and contributor of ferroptosis.

Ferroptosis, a recently identified form of non-apoptotic cell death, is involved in several physiological and pathological processes. Although lipid peroxidation plays a central role in triggering ferroptosis, the essential regulator of lipid metabolism in ferroptosis remains poorly defined. Here, we show that acyl-CoA synthetase long-chain family member 4 (ACSL4) is required for ferroptotic cancer cell death. Compared with ferroptosis-sensitive cells (e.g., HepG2 and HL60), the expression of ACSL4 was remarkably downregulated in ferroptosis-resistant cells (e.g., LNCaP and K562). In contrast, the expression of other ACSLs, including ACSL1, ACSL3, ACSL5, and ACSL6, did not correlate with ferroptosis sensitivity. Moreover, knockdown of ACSL4 by specific shRNA inhibited erastin-induced ferroptosis in HepG2 and HL60 cells, whereas overexpression of ACSL4 by gene transfection restored sensitivity of LNCaP and K562 cells to erastin. Mechanically, ACSL4-mediated production of 5-hydroxyeicosatetraenoic acid (5-HETE) contributed to ferroptosis. Pharmacological inhibition of 5-HETE production by zileuton limited ACSL4 overexpression-induced ferroptosis. Collectively, these results indicate that ACSL4 is not only a sensitive monitor of ferroptosis, but also an important contributor of ferroptosis.

The role of 20-HETE in cardiovascular diseases and its risk factors.

Arachidonic acid (AA) is metabolized in mammals by enzymes of the CYP4A and 4F families to 20-hydroxyeicosatetraeonic acid (20-HETE) which plays an important role in the regulation of renal function, vascular tone and arterial pressure. In the vasculature, 20-HETE is a potent vasoconstrictor, the up-regulation of which contributes to inflammation, oxidative stress, endothelial dysfunction and an increase in peripheral vascular resistance in models of obesity, diabetes, ischemia/reperfusion, and vascular oxidative stress. Recent studies have established a role for 20-HETE in normal and pathological angiogenic conditions. We discuss in this review the synthesis of 20-HETE and how it and various autacoids, especially the renin-angiotensin system, interact to promote hypertension, vasoconstriction, and vascular dysfunction. In addition, we examine the molecular mechanisms through which 20-HETE induces these actions and the clinical implication of inhibiting 20-HETE production and activity.

Expression of CYP 4A ω-hydroxylase and formation of 20-hydroxyeicosatetreanoic acid (20-HETE) in cultured rat brain astrocytes.

Astrocytes secrete vasodilator and vasoconstrictor factors via end feet processes, altering blood flow to meet neuronal metabolic demand. Compared to what is known about the ability of astrocytes to release factors that dilate local cerebral vasculature, very little is known regarding the source and identity of astrocyte derived constricting factors. The present study investigated if astrocytes express CYP 4A ω-hydroxylase and metabolize arachidonic acid (AA) to 20-hydroxyeicotetraenoic acid (20-HETE) that regulates KCa channel activity in astrocytes and cerebral arterial myocyte contractility. Here we report that cultured astrocytes express CYP 4A2/3 ω-hydroxylase mRNA and CYP 4A protein and produce 20-HETE and the CYP epoxygenase metabolites epoxyeicosatrienoic acids (EETs) when incubated with AA. The production of 20-HETE and EETs was enhanced following stimulation of metabotropic glutamate receptors (mGluR) on the astrocytes. Exogenous application of 20-HETE attenuated, whereas inhibition of 20-HETE production with HET-0016 increased the open state probabilities (NPo) of 71pS and 161pS KCa single-channel currents recorded from astrocytes. Exposure of isolated cerebral arterial myocytes to conditioned media from cultured astrocytes caused shortening of the length of freshly isolated cerebral arterial myocytes that was not evident following inhibition of astrocyte 20-HETE synthesis and action. These findings suggest that astrocytes not only release vasodilator EETs in response to mGluR stimulation but also synthetize and release the cerebral arterial myocyte constrictor 20-HETE that also functions as an endogenous inhibitor of the activity of two types of KCa channel currents found in astrocytes.

Deficiency in the Formation of 20-Hydroxyeicosatetraenoic Acid Enhances Renal Ischemia-Reperfusion Injury.

Ischemia-reperfusion (IR) injury is the most common cause of AKI. The susceptibility to develop AKI varies widely among patients. However, little is known about the genes involved. 20-Hydroxyeicosatetraenoic acid (20-HETE) has an important role in the regulation of renal tubular and vascular function and has been implicated in IR injury. In this study, we examined whether a deficiency in the renal formation of 20-HETE enhances the susceptibility of Dahl salt-sensitive (SS) rats to ischemic AKI. Transfer of chromosome 5 containing the CYP4A genes responsible for the formation of 20-HETE from the Brown Norway (BN) rat onto the SS genetic background increased renal 20-HETE levels after ischemia and reduced plasma creatinine levels (±SEM) 24 hours after IR from 3.7±0.1 to 2.0±0.2 mg/dl in an SS.5(BN)-consomic strain. Transfer of this chromosome also prevented the secondary decline in medullary blood flow and ischemia that develops 2 hours after IR in the susceptible SS strain. Blockade of the synthesis of 20-HETE with HET0016 reversed the renoprotective effects in SS.5(BN) rats. Similar results were observed in an SS.5(Lew)-congenic strain, in which a smaller region of chromosome 5 containing the CYP4A genes from a Lewis rat was introgressed onto the SS genetic background. These results indicate that 20-HETE has a protective role in renal IR injury by maintaining medullary blood flow and that a genetic deficiency in the formation of 20-HETE increases the susceptibility of SS rats to ischemic AKI.

Inhibition of long-chain acyl-CoA synthetase 4 facilitates production of 5, 11-dihydroxyeicosatetraenoic acid via the cyclooxygenase-2 pathway.

Long chain acyl-CoA synthetases (ACSLs) are a family of enzymes that convert free long chain fatty acids into their acyl-CoA forms. Among ACSL enzymes, ACSL4 prefers arachidonic acid (AA) as a substrate and plays an important role in re-esterification of free AA. We previously reported that the suppression of ACSL4 activity by treatment with an ACSL inhibitor or a small interfering RNA markedly enhanced interleukin-1ß (IL-1ß)-dependent prostaglandin (PG) biosynthesis in rat fibroblastic 3Y1 cells. We show here that in addition to these prostanoids, cytokine-dependent production of 5,11-dihydroxyeicosatetraenoic acid (5,11-diHETE), a cyclooxygenase product of 5-hydroxyeicosatetraenoic acid (5-HETE), was enhanced by the inhibition of ACSL4 activity. Treatment of several types of cells with an ACSL inhibitor, triacsin C, markedly enhanced IL-1ß-dependent production of 5,11-diHETE. siRNA-mediated knockdown of ACSL4 also enhanced IL-1ß-dependent production of 5,11-diHETE from 3Y1 cells. The production of 5,11-diHETE was significantly decreased by a cyclooxygenase (COX)-2 selective inhibitor, NS-398, but not by a 5-lipoxygenase activating protein (FLAP) inhibitor, MK-886. The inhibition of ACSL enzymes significantly facilitated release of not only 5-HETE but also 8-HETE, 9-HETE, 11-HETE, 12-HETE, and 15-HETE, independently of IL-1ß stimulation. In vitro analysis showed that a recombinant COX-2 enzyme more effectively metabolized 5(S)-HETE to 5-11-diHETE compared to COX-1 enzyme. From these results, we proposed the following mechanism of 5,11-diHETE biosynthesis in these cells: 1) inhibition of ACSL4 causes accumulation of free AA; 2) the accumulated AA is nonspecifically converted into various HETEs; and 3) among these HETEs, 5-HETE is metabolized into 5,11-diHETE by cytokine-induced COX-2.

Additive Neuroprotection of a 20-HETE Inhibitor with Delayed Therapeutic Hypothermia after Hypoxia-Ischemia in Neonatal Piglets.

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.

The role of epoxyeicosatrienoic acids in the cardiovascular system.

There is increasing evidence suggesting that epoxyeicosatrienoic acids (EETs) play an important role in cardioprotective mechanisms. These include regulating vascular tone, modulating inflammatory responses, improving cardiomyocyte function and reducing ischaemic damage, resulting in attenuation of animal models of cardiovascular risk factors. This review discusses the current knowledge on the role of EETs in endothelium-dependent control of vascular tone in the healthy and in subjects with cardiovascular risk factors, and considers the pharmacological potential of targeting this pathway.