P450 Metabolites Of Arachidonic Acid Research Articles

Effects of Erchen Decoction on Oxidative Stress-Related Cytochrome P450 Metabolites of Arachidonic Acid in Dyslipidemic Mice with Phlegm-Dampness Retention Syndrome: A Randomized, Controlled Trial on the Correspondence between Prescription and Syndrome.

Phlegm-dampness retention (PDR) syndrome is one of the main syndromes of dyslipidemia. This study investigated the effects of Erchen decoction (ECD) on concentrations of two oxidative stress-related cytochrome P450 (CYP450) metabolites of arachidonic acid—14,15-dihydroxyeicosatrienoic acid (14,15-DHET) and 20-hydroxyeicosatetraenoic acid (20-HETE)—in mice with dyslipidemia and phlegm-dampness retention (PDR) syndrome (n = 5 C57BL/6J mice and n = 30 apolipoprotein E knockout mice). Murine models of the disease and syndrome were established using multifactor stimulation. Then, all mice were assigned to normal, model, low- (L-), medium- (M-), or high- (H-) dose ECD groups or to a control or an unmatched prescription-syndrome (unmatched P-S) group; five mice were included in each group. Dose formulations were administered by oral gavage for 30 days to animals in the corresponding groups. We detected and analyzed hematoxylin and eosin (HE) staining characteristics of the mouse aorta and serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), peroxynitrite (ONOO−), 14,15-DHET, and 20-HETE concentrations in each group. TC and LDL-C concentrations significantly decreased in the M-ECD versus control group (P < 0.05); however, the TC and LDL-C concentrations were not significantly different in the unmatched P-S versus model group (P > 0.05). After treatment in the P-S correspondence groups (L-ECD, M-ECD, and H-ECD groups), the concentration of ONOO− decreased to different degrees in each group. Among these groups, the concentration of ONOO− significantly decreased in the L-ECD, M-ECD, and H-ECD groups versus the model group (P < 0.05). However, the concentration of ONOO− was not significantly different in the unmatched P-S versus the model group (P > 0.05). From the perspective of aortic HE staining, the P-S group experienced an improved endothelium structure after treatment. 14,15-DHET concentrations significantly increased in the normal, M-ECD, and H-ECD groups versus the model group; in the H-ECD versus the L-ECD group; and in the H-ECD versus the control group (all P < 0.05) to various extents after different doses of the prescription. 20-HETE concentrations pronouncedly decreased in the M-ECD versus normal group; in the M- and H-ECD groups versus the model group; in the M-ECD versus the L-ECD group; in the M-ECD versus the control group; and in the M-ECD versus the unmatched P-S (P < 0.05). However, the concentrations of 14,15-DHET and 20-HETE in the model group were not significantly different from those of the unmatched P-S (P > 0.05). In this study, ECD reversed blood lipid indexes and ameliorated oxidative stress-related metabolites, elevating serum 14,15-DHET and lowering serum 20-HETE in mice with dyslipidemia and PDR syndrome via CYP450 pathways of arachidonic acid metabolism. The efficacy of ECD relies on correspondence between the prescription and the syndrome. These findings scientifically validate the treatment according to traditional Chinese medicine syndrome differentiation. ECD can strengthen the protective effect on the vascular endothelium by driving out pathogenic factors and strengthening healthy resistance. Its efficacy may be related to the adjustment of the polarization state of macrophages.

Pharmacological regulation of cytochrome P450 metabolites of arachidonic acid attenuates cardiac injury in diabetic rats

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-β 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.

AUDA as a modulator of lipid profile and endothelial function in diet induced hypercholesterolemia in rats

Background: Epoxyeicosatrienoic acids (EETs) are cytochrome P450 metabolites of arachidonic acid produced by the vascular endothelium and act as important regulators of vascular tone. Objectives: to study the effect of soluble epoxide hydrolase inhibition AUDA on endothelial function in hypercholesterolemic rats. Methods: Hypercholesterolemia was induced by using Matos diet for 8 weeks and it significantly increased TC, TG, and LDL-C and decreased HDL-C. AUDA, was used with a dose of 0.35 mg/ kg for 10 weeks via gastric gavage every other day. Results: AUDA treated group has significantly lower total-C, TG, LDL-C and higher HDL-C as compared to untreated group. AUDA also improved vascular reactivity and vasodilatory response to different doses of Acetyl choline. Histopathological examination of aortic rings showed reduced development of atherosclerotic changes in the vascular wall with no effect on e-NOS expression in vascular endothelial cells. Conclusions: We can conclude that AUDA treatment has improved lipid profile and protect against development of atherosclerotic histopathological changes.

Alleviation of Mechanical Allodynia by 14,15-Epoxyeicosatrienoic Acid in a Central Poststroke Pain Model: Possible Role of Allopregnanolone and δ-Subunit-Containing Gamma-Aminobutyric Acid A Receptors

Central poststroke pain (CPSP) is a neuropathic pain syndrome arising after a lesion of the central nervous system owing to cerebrovascular insult. Impaired daily activities and reduced quality of life in people suffering from CPSP justify the need for improved treatment. The detailed mechanism of CPSP is not well understood, but central disinhibition has been suggested. Recent reports indicated that epoxyeicosatrienoic acids (EETs), the cytochrome P450 metabolites of arachidonic acid, promoted neuronal survival after stroke, displayed antinociception in peripheral inflammatory pain, and reduced neuronal excitability in seizure model. Here, we tested the hypothesis that 14,15-EET may attenuate CPSP by suppressing thalamic disinhibition through neurosteroids−δ-subunit-containing gamma-aminobutyric acid A receptors (δGABAAR) signaling. In this study, we used a rat model of thalamic hemorrhagic stroke to induce CPSP. Pain behavioral tests revealed that CPSP rats exhibited mechanical allodynia, starting at day 7 postlesion and lasting at least 4 weeks. Analysis of the perithalamic lesion tissue from the brain of CPSP rats demonstrated a decrease of 14,15-EET content, steroidogenic acute regulatory protein expression, and allopregnanolone (AP) production. This was accompanied by reduced δGABAAR expression in the medial thalamus at 4 weeks postlesion. Intrathalamic injection of exogenous 14,15-EET into the ventral posterior lateral nucleus attenuated mechanical allodynia, induced a marked increase in the abundance of the steroidogenic acute regulatory protein and AP along the lesion site and a concomitant increase in δGABAAR expression in the medial thalamus under CPSP condition. However, this antinociceptive effect could be eliminated by the 5α-reductase inhibitor finasteride or dutasteride or GABAAR antagonist bicuculline. Moreover, compared with the current first-line drug gabapentin for central neuropathic pain, an early treatment of EET showed greater efficacy in the secondary prevention of CPSP. Taken together, this study provided a proof of concept that EETs may have anti-CPSP effect by reserving normal thalamic inhibition through AP-δGABAAR signaling. PerspectiveAgents targeting EETs may serve as potential therapeutic options for stroke, the use of which at the initial period could not only block further nerve damage but also prevent the occurrence of CPSP.

An improved Ultra-High Performance Liquid chromatography-tandem mass spectrometry method for simultaneous quantitation of cytochrome P450 metabolites of arachidonic acid in human plasma

This study aims to develop a straightforward, sensitive UHPLC–MS/MS method to quantify 15 eicosanoids derived from arachidonic acid in human plasma. Tert-Butyl methyl ether was used on the liquid–liquid extraction method and significantly reduced the expense and time. The method showed excellent linearity for all analytes, with regression coefficients higher than 0.99 over a wide range of concentrations from 0.01 ng mL−1 to 100 ng mL−1. The recovery rates were over 65.00%, and the matrix effects ranged from 8.42% to 40.00%. The limits of detection ranged from 6 pg mL−1 to 10 pg mL−1, and all of the limits of quantification were 20 − 33 pg mL−1. For the broad concentration range, the RE% for accuracy and precision were less than ± 15%. Moreover, trans-4-{4-[3-(4-Trifluoromethoxyphenyl)-ureido] cyclohexyloxy} benzoic acid (t-TUCB) pretreatment extended the window of detection for as much as 30 days. Eicosanoid signaling is altered in various neurological diseases, including pain, Alzheimer’s disease and major depressive disorder. Therefore, this rapid, robust quantitative profiling of 15 eicosanoids in plasma could provide a distinct eicosanoid fingerprint for precision medicine in these patients.

Drug-Disease Interaction: Effect of Inflammation and Nonsteroidal Anti-Inflammatory Drugs on Cytochrome P450 Metabolites of Arachidonic Acid

Inflammatory conditions increase cardiovascular (CV) risk. Some nonsteroidal anti-inflammatory drugs (NSAIDs) that are used to treat pain and inflammation are also associated with CV complications. Inflammation, but not NSAIDs, disrupts the balance of vasodilator and vasoconstrictor components of the renin-angiotensin system within the heart. Herein, we report the effect of both inflammation and NSAIDs (rofecoxib, celecoxib, and meloxicam) on the physiologically active cytochrome P450 metabolites of arachidonic acid (ArA) in the rat with adjuvant arthritis. After oral administration of 7 daily therapeutically equivalent doses of NSAIDs or vehicle, the anti-inflammatory response, as well as the ArA metabolites and drug concentrations in plasma, heart and kidneys were assessed. Inflammation in the form of adjuvant arthritis caused a significant tissue-dependent imbalance of ArA metabolites by elevating the ratio of cardiotoxic 20-hydroxyeicosatetraenoic acid over cardioprotective epoxyeicosatrienoic acids in the heart, and reducing the ratio in the kidney. The observed imbalance was augmented by cardiotoxic rofecoxib but not by other examined NSAIDs with known milder cardiotoxicity. The cardio-renal toxicity of NSAIDs with known severe CV side effects may be due to altered cytochrome P450–mediated ArA acid metabolism. The ArA metabolism profile may be a marker of NSAIDs safety and toxicity.

Effect of Cytochrome P450 Metabolites of Arachidonic Acid in Nephrology.

Thirty-five years ago, a third pathway for the metabolism of arachidonic acid by cytochrome P450 enzymes emerged. Subsequent work revealed that 20-hydroxyeicosatetraenoic and epoxyeicosatrienoic acids formed by these pathways have essential roles in the regulation of renal tubular and vascular function. Sequence variants in the genes that produce 20-hydroxyeicosatetraenoic acid are associated with hypertension in humans, whereas the evidence supporting a role for variants in the genes that alter levels of epoxyeicosatrienoic acids is less convincing. Studies in animal models suggest that changes in the production of cytochrome P450 eicosanoids alter BP. However, the mechanisms involved remain controversial, especially for 20-hydroxyeicosatetraenoic acid, which has both vasoconstrictive and natriuretic actions. Epoxyeicosatrienoic acids are vasodilators with anti-inflammatory properties that oppose the development of hypertension and CKD; 20-hydroxyeicosatetraenoic acid levels are elevated after renal ischemia and may protect against injury. Levels of this eicosanoid are also elevated in polycystic kidney disease and may contribute to cyst formation. Our review summarizes the emerging evidence that cytochrome P450 eicosanoids have a role in the pathogenesis of hypertension, polycystic kidney disease, AKI, and CKD.

TPPU protects tau from H2O2-induced hyperphosphorylation in HEK293/tau cells by regulating PI3K/AKT/GSK-3β pathway.

Neurofibrillary pathology of abnormally hyperphosphorylated tau is a hallmark of Alzheimer's disease (AD) and other tauopathies. Phosphatidylinositol 3-kinase (PI3K)/Akt/glycogen synthase kinase-3 beta (GSK-3β) signaling pathway is pivotal for tau phosphorylation. Inhibition of soluble epoxide hydrolase (sEH) metabolism has been shown to effectively increase the accumulation of epoxyeicosatrienoic acids (EETs), which are cytochrome P450 metabolites of arachidonic acid and have been demonstrated to have neuroprotective effects. However, little is known about the role of sEH in tau phosphorylation. The present study investigated the role of a sEH inhibitor, 1-(1-propanoylpiperidin-4-yl)-3-[4-(trifluoromethoxy)phenyl] urea (TPPU), on H2O2-induced tau phosphorylation and the underlying signaling pathway in human embryonic kidney 293 (HEK293)/Tau cells. We found that the cell viability was increased after TPPU treatment compared to control in oxidative stress. Western blotting and immunofluorescence results showed that the levels of phosphorylated tau at Thr231 and Ser396 sites were increased in H2O2-treated cells but dropped to normal levels after TPPU administration. H2O2 induced an obvious decreased phosphorylation of GSK-3β at Ser9, an inactive form of GSK-3β, while there were no changes of phosphorylation of GSK-3β at Tyr216. TPPU pretreatment maintained GSK-3β Ser 9 phosphorylation. Moreover, Western blotting results showed that TPPU upregulated the expression of p-Akt. The protective effects of TPPU were found to be inhibited by wortmannin (WT, a specific PI3K inhibitor). In conclusion, these results suggested that the protective effect of TPPU on H2O2-induced oxidative stress is associated with PI3K/Akt/GSK-3β pathway.

Soluble Epoxide Hydrolase Inhibition and Epoxyeicosatrienoic Acid Treatment Improve Vascularization of Engineered Skin Substitutes.

Autologous engineered skin substitutes comprised of keratinocytes, fibroblasts, and biopolymers can serve as an adjunctive treatment for excised burns. However, engineered skin lacks a vascular plexus at the time of grafting, leading to slower vascularization and reduced rates of engraftment compared with autograft. Hypothetically, vascularization of engineered skin grafts can be improved by treatment with proangiogenic agents at the time of grafting. Epoxyeicosatrienoic acids (EETs) are cytochrome P450 metabolites of arachidonic acid that are inactivated by soluble epoxide hydrolase (sEH). EETs have multiple biological activities and have been shown to promote angiogenesis. Inhibitors of sEH (sEHIs) represent attractive therapeutic agents because they increase endogenous EET levels. We investigated sEHI administration, alone or combined with EET treatment, for improved vascularization of engineered skin after grafting to mice. Engineered skin substitutes, prepared using primary human fibroblasts and keratinocytes, were grafted to full-thickness surgical wounds in immunodeficient mice. Mice were treated with the sEHI 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), which was administered in drinking water throughout the study period, with or without topical EET treatment, and were compared with vehicle-treated controls. Vascularization was quantified by image analysis of CD31-positive areas in tissue sections. At 2 weeks after grafting, significantly increased vascularization was observed in the TPPU and TPPU + EET groups compared with controls, with no evidence of toxicity. The results suggest that sEH inhibition can increase vascularization of engineered skin grafts after transplantation, which may contribute to enhanced engraftment and improved treatment of full-thickness wounds.

20-HETE Activates the Transcription of Angiotensin-Converting Enzyme via Nuclear Factor-κB Translocation and Promoter Binding.

Increased vascular 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 arachidonic acid metabolite, promotes vascular dysfunction, injury, and hypertension that is dependent, in part, on the renin angiotensin system (RAS). We have shown that, in human microvascular endothelial cells, 20-HETE increases angiotensin-converting enzyme (ACE) mRNA, protein, and ACE activity via an epidermal growth factor receptor (EGFR)/tyrosine kinase/mitogen-activated protein kinase (MAPK)/inhibitor of κB kinase (IKK)β-mediated signaling pathway. In this work, we show that, similar to epidermal growth factor (EGF), 20-HETE (10 nM) activates EGFR by stimulating tyrosine phosphorylation; however, unlike 20-HETE, EGF does not induce ACE expression, and pretreatment with a neutralizing antibody against EGF does not prevent the 20-HETE-mediated ACE induction. Inhibition of nuclear factor κB (NF-κB) activation prevented the 4.58-fold (±0.78; P < 0.05) 20-HETE-mediated induction of ACE. The 20-HETE increased NF-κB-binding activity in nuclear extracts and the activity of both the somatic and germinal ACE promoters by 4.37-fold (±0.18; P < 0.05) and 2.53-fold (± 0.24; P < 0.05), respectively. The 20-HETE-stimulated ACE promoter activity was abrogated by the 20-HETE antagonist 20-hydroxy-6,15-eicosadienoic acid and by inhibitors of EGFR, MAPK, IKKβ, and NF-κB activation. Sequence analysis demonstrated the presence of two and one putative NF-κB binding sites on the human somatic and germinal ACE promoters, respectively. Chromatin immunoprecipitation assay indicated that 20-HETE stimulates the translocation and subsequent binding of NF-κB to each of the putative binding sites (S1, 3.43 ± 0.3-fold enrichment versus vehicle; S2, 3.72 ± 0.68-fold enrichment versus vehicle; S3, 3.20 ± 0.18-fold enrichment versus vehicle; P < 0.05). This is the first study to identify NF-κB as a transcriptional factor for ACE and to implicate a distinct EGFR/MAPK/IKK/NF-κB signaling cascade underlying 20-HETE-mediated transcriptional activation of ACE mRNA and stimulation of ACE activity.

Analysis of cytochrome P450 metabolites of arachidonic acid by stable isotope probe labeling coupled with ultra high-performance liquid chromatography/mass spectrometry

Cytochrome P450 metabolites of arachidonic acid (AA) belong to eicosanoids and are potent lipid mediators of inflammation. It is well-known that eicosanoids play an important role in numerous pathophysiological processes. Therefore, quantitative analysis of cytochrome P450 metabolites of AA, including hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatreinoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs) can provide crucial information to uncover underlying mechanisms of cytochrome P450 metabolites of AA related diseases. Herein, we developed a highly sensitive method to identify and quantify HETEs, EETs, and DHETs in lipid extracts of biological samples based on stable isotope probe labeling coupled with ultra high-performance liquid chromatography/mass spectrometry. To this end, a pair of stable isotope probes, 2-dimethylaminoethylamine (DMED) and d4-2-dimethylaminoethylamine (d4-DMED), were utilized to facilely label eicosanoids. The heavy labeled eicosanoid standards were prepared and used as internal standards for quantification to minimize the matrix and ion suppression effects in mass spectrometry analysis. In addition, the detection sensitivities of DMED labeled eicosanoids improved by 3–104 folds in standard solution and 5–138 folds in serum matrix compared with unlabeled analytes. Moreover, a good separation of eicosanoids isomers was achieved upon DMED labeling. The established method provided substantial sensitivity (limit of quantification at sub-picogram), high specificity, and broad linear dynamics range (3 orders of magnitude). We further quantified cytochrome P450 metabolites of AA in rat liver, heart, brain tissues and human serum using the developed method. The results showed that 19 eicosanoids could be distinctly detected and the contents of 11-, 15-, 16-, 20-HETE, 5,6-EET, and 14,15-EET in type 2 diabetes mellitus patients and 5-, 11-, 12-, 15-, 16-, 20-HETE, 8,9-EET, and 5,6-DHET in myeloid leukemia patients had significant changes, demonstrating that these eicosanoids may have important roles on the pathogenesis of type 2 diabetes mellitus and myeloid leukemia.

Cytochrome P450 eicosanoids in hypertension and renal disease.

Purpose of reviewCytochrome (CYP) P450 metabolites of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) contribute to the regulation of renal tubular and vascular function. This review highlights the results of the recent genetic studies in humans and rodent models, indicating that these eicosanoids participate in the control of blood pressure (BP), chronic kidney disease (CKD), renal ischemia–reperfusion injury (IRI) and polycystic kidney disease (PKD).Recent findingsEndogenous 20-HETE has been reported to play an essential role in the myogenic and tubuloglomerular feedback responses in the afferent arteriole, and a deficiency of 20-HETE contributes to the development of hypertension and renal injury in Dahl S rats. Mutations in CYP4A11 and CYP4F2 have been linked to elevated BP in humans. EETs have been shown to regulate epithelial sodium channel in the collecting duct, lower BP and have renoprotective properties. 20-HETE also opposes the development of CKD and IRI, and may play a role in PKD.SummaryThese studies indicate that CYP P450 metabolites of arachidonic acid play an important role in the control of BP, CKD, AKI and PKD. Drugs targeting these pathways could be useful in the treatment of IRI and CKD.

Effect of compound danshen preparation on the plasma and heart concentrations of cytochrome P-450 metabolites of arachidonic acid in rats with myocardial ischemia and reperfusion

Effect of compound danshen preparation on the plasma and heart concentrations of cytochrome P-450 metabolites of arachidonic acid in rats with myocardial ischemia and reperfusion

Abstract 024: 20-HETE Activates the Transcription of Endothelial Angiotensin Converting Enzyme (ACE) via NF-kB Translocation and Promoter Binding

Increased vascular 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450-arachidonic acid metabolite, promotes vascular dysfunction, injury and hypertension that is dependent, in part, on the renin angiotensin system (RAS). We showed that in cultured human microvascular endothelial cells (HMVEC), 20-HETE (5 nM) increases angiotensin converting enzyme (ACE) mRNA, protein, and cellular and extracellular ACE activity via EGFR-tyrosine kinase-MAPK-IKKβ- signaling pathway. Here we show that treatment of HMVEC with EGF (100 ng/ml) alone or with 20-HETE (10 nM) did not induce or further increase ACE mRNA levels. Pretreatment of cells with a neutralizing antibody against EGF did not prevent the 20-HETE- or 20-HETE+EGF-mediated ACE induction, indicating that the induction of ACE by 20-HETE is EGF-independent. The NF-kB inhibitor JSH-23 prevented the 4.58-fold (±0.78; p&lt;0.05) 20-HETE (10nM)-mediated induction of ACE. EMSA of 20-HETE-treated cells showed increased NF-kB binding activity in nuclear extracts. In cells transfected with luciferase promoter-reporter constructs for the somatic or germinal ACE promoter regions, 20-HETE (10 nM) increased promoter activity by 4.37-fold (±0.18; p&lt;0.05) and 2.53-fold (± 0.24; p&lt;0.05), respectively. 20-HETE-stimulated ACE promoter activity was abrogated by the 20-HETE antagonist 20-HEDE and by inhibitors of EGFR, MAPK, IKKβ and NF-kB activation. Sequence analysis demonstrated the presence of two (Chr 17: S1: 61554061-61554075 , S2: 61554157-61554171) and one (Chr 17: S3: 61562250-61562264) putative NF-kB binding sites on the human somatic and germinal ACE promoters, respectively. CHIP assay indicated that 20-HETE stimulates the translocation and subsequent binding of NF-kB to each of the putative binding sites (S1: 3.43±0.3, S2: 3.72±0.68, S3: 3.20±0.18-fold enrichment vs vehicle; p&lt;0.05). In vivo, increased vascular 20-HETE levels are associated with increased phospho-IKK and ACE levels that are prevented by treatment with 20-HETE antagonists. This is the first study to identify NF-kB as a transcriptional factor for ACE and to implicate a distinct EGFR/MAPK/IKK/NF-kB signaling cascade underlying 20-HETE-mediated transcriptional activation of ACE mRNA and stimulation of ACE activity.

Increases in Levels of Epoxyeicosatrienoic and Dihydroxyeicosatrienoic Acids (EETs and DHETs) in Liver and Heart in Vivo by 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) and in Hepatic EET:DHET Ratios by Cotreatment with TCDD and the Soluble Epoxide Hydrolase Inhibitor AUDA

The environmental toxin and carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) binds and activates the transcription factor aryl hydrocarbon receptor (AHR), inducing CYP1 family cytochrome P450 enzymes. CYP1A2 and its avian ortholog CYP1A5 are highly active arachidonic acid epoxygenases. Epoxygenases metabolize arachidonic acid to four regioisomeric epoxyeicosatrienoic acids (EETs) and selected monohydroxyeicosatetraenoic acids (HETEs). EETs can be further metabolized by epoxide hydrolases to dihydroxyeicosatrienoic acids (DHETs). As P450-arachidonic acid metabolites affect vasoregulation, responses to ischemia, inflammation, and metabolic disorders, identification of their production in vivo is needed to understand their contribution to biologic effects of TCDD and other AHR activators. Here we report use of an acetonitrile-based extraction procedure that markedly increased the yield of arachidonic acid products by lipidomic analysis over a standard solid-phase extraction protocol. We show that TCDD increased all four EETs (5,6-, 8,9-, 11,12-, and 14,15-), their corresponding DHETs, and 18- and 20-HETE in liver in vivo and increased 5,6-EET, the four DHETs, and 18-HETE in heart, in a chick embryo model. As the chick embryo heart lacks arachidonic acid-metabolizing activity, the latter findings suggest that arachidonic acid metabolites may travel from their site of production to a distal organ, i.e., heart. To determine if the TCDD-arachidonic acid-metabolite profile could be altered pharmacologically, chick embryos were treated with TCDD and the soluble epoxide hydrolase inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA). Cotreatment with AUDA increased hepatic EET-to-DHET ratios, indicating that the in vivo profile of P450-arachidonic acid metabolites can be modified for potential therapeutic intervention.

Abstract 159: Inhibition of Ace Does Not Prevent 20-hete-mediated Vascular Injury in Cyp4a12tg Mice

20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450-arachidonic acid metabolite in the microcirculation, increases smooth muscle contraction and proliferation, stimulates endothelial dysfunction and activation and promotes hypertension that is dependent, in part, on the renin angiotensin system (RAS). We showed that in cultured human microvascular endothelial cells, 20-HETE increases angiotensin converting enzyme (ACE) mRNA by 3.1-fold (± 0.16; p&lt;0.05), ACE protein by 4.2-fold (±0.99; p&lt;0.05) and cellular and extracellular ACE activity by 2.2-fold (±0.09; p&lt;0.05) and 1.96-fold (±0.16; p&lt;0.05), respectively. The contribution of ACE to vascular 20-HETE-mediated microvascular remodeling in hypertension was assessed using the Cyp4a12 transgenic mouse in which the expression of the Cyp4a12-20-HETE synthase is driven by a tetracyclin (doxycycline, DOX)-sensitive promoter. Treatment of Cyp4a12tg mice with DOX increased blood pressure (133±6 vs. 107±3 mmHg; p&lt;0.05) and remodeling of renal resistance arteries (M/L, 0.15±0.01 vs. 0.07±0.01; p&lt;0.05); these increases were abrogated by co-treatment with the 20-HETE antagonist, 20-HEDGE. Renal ACE protein expression was significantly higher in DOX-treated (1.6 ± 0.17 fold; p&lt;0.05) as compared to untreated Cyp4a12tg mice; co-treatment with 20-HEDGE prevented the increase in ACE expression. Lisinopril treatment of Cyp4a12tg mice concurrently receiving DOX prevented the blood pressure increase (133±6 vs. 104±2 mmHg; p&lt;0.05) but did not interfere with renal microvascular 20-HETE production (1.05±0.18 vs 1.09±0.30 ng/mg). Co-treatment with lisinopril and DOX attenuated but did not prevent remodeling of renal microvessels (M/L, 0.07±0.0 vs. 0.15±0.01 vs. 0.11±0.01; p&lt;0.05 in water, DOX and DOX+Lisinopril, respectively). In contrast, co-treatment with 20-HEDGE prevented DOX-induced remodeling of resistance arteries of Cyp4a12tg mice. This study demonstrates that 20-HETE is a key determinant of microvascular remodeling in DOX-induced Cyp4a12tg hypertensive mice. 20-HETE’s mechanism only partially relies on activation of the RAS. Thus, the 20-HETE-driven microvascular remodeling does not fully depend on the upregulation of ACE and elevation of blood pressure.

UHPLC–MS/MS analysis of arachidonic acid and 10 of its major cytochrome P450 metabolites as free acids in rat livers: Effects of hepatic ischemia

The cytochrome P450 metabolites of arachidonic acid (AA) are mostly present in tissues, such as the liver, as bound to phospholipids, with only a small fraction available as free acids. The purpose of this study was to develop and validate a UHPLC-MS/MS method for quantitation of free liver concentrations of AA and four epoxygenated (5,6-, 8,9-, 11,12-, and 14,15-EET), four dihydroxylated (5,6-, 8,9-, 11,12-, and 14,15-DHET), and two ω/(ω-1) hydroxylated (19- and 20-HETEs) metabolites of AA in rat livers using deuterated internal standards. The analytes were rapidly and efficiently (79-92%) recovered from 100mg of fresh liver into methanol. After evaporation, the reconstituted samples were injected either undiluted (for the simultaneous analysis of the metabolites) into a gradient or diluted (for AA analysis) into an isocratic UHPLC system with run times of 5 and 2 min, respectively. Mass spectrometry was conducted using multiple reaction monitoring in negative mode. The method was linear (r(2)≥ 0.98) in the concentration ranges tested for metabolites (0.19-120 ng/g liver) and AA (7.8-500 μg/g liver). The lower limit of quantitation of the assay was between 0.57 and 5.6 pg injected on column for different AA metabolites. The assay was validated (n=5) based on acceptable intra- and inter-run accuracy and precision values. Additionally, matrix effect was minimal for most analytes. Freeze-thaw of samples drastically increased the free liver concentrations of analytes, presumably due to their release from the membrane storage sites. Therefore, fresh liver samples should be used for analysis. However, the methanolic extracts may be stored at -80°C for at least two weeks without any compromise. The method was successfully used in the measurement of all the analytes in the rats subjected to 60 min of hepatic ischemia (n=6) or sham operation (n=6). Ischemia resulted in significantly higher free concentrations of AA and most of its studied metabolites. The method is precise, accurate, and sensitive for measurement of free liver concentrations of AA and its P450 metabolites in the rat liver.

Vascular Effects of Urocortins 2 and 3 in Healthy Volunteers

BackgroundUrocortin 2 and urocortin 3 are endogenous peptides with an emerging role in cardiovascular pathophysiology. We assessed their pharmacodynamic profile and examined the role of the endothelium in mediating their vasomotor effects in vivo in man.Methods and ResultsEighteen healthy male volunteers (23±4 years) were recruited into a series of double‐blind, randomized crossover studies using bilateral forearm venous occlusion plethysmography during intra‐arterial urocortin 2 (3.6 to 120 pmol/min), urocortin 3 (1.2 to 36 nmol/min), and substance P (2 to 8 pmol/min) in the presence or absence of inhibitors of cyclooxygenase (aspirin), cytochrome P450 metabolites of arachidonic acid (fluconazole), and nitric oxide synthase (L‐NMMA). Urocortins 2 and 3 evoked arterial vasodilatation (P<0.0001) without tachyphylaxis but with a slow onset and offset of action. Inhibition of nitric oxide synthase with L‐NMMA reduced vasodilatation to substance P and urocortin 2 (P≤0.001 for both) but had little effect on urocortin 3 (P>0.05). Neither aspirin nor fluconazole affected vasodilatation induced by any of the infusions (P>0.05 for all). In the presence of all 3 inhibitors, urocortin 2– and urocortin 3–induced vasodilatation was attenuated (P<0.001 for all) to a greater extent than with L‐NMMA alone (P≤0.005).ConclusionsUrocortins 2 and 3 cause potent and prolonged arterial vasodilatation without tachyphylaxis. These vasomotor responses are at least partly mediated by endothelial nitric oxide and cytochrome P450 metabolites of arachidonic acid. The role of urocortins 2 and 3 remains to be explored in the setting of human heart failure, but they have the potential to have major therapeutic benefits.Clinical Trial Registrationhttp://www.clinicaltrials.gov//. Unique identifier: NCT01096706 and NCT01296607.

Maternal and Fetal Epoxyeicosatrienoic Acids in Normotensive and Preeclamptic Pregnancies

Epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) are cytochrome P450 metabolites of arachidonic acid posited to act in the circulatory adaptation to pregnancy and the development of preeclampsia. Red blood cells (RBCs) may function as major contributors of cis- and trans-EETs. We performed paired analyses of EETs, dihydroxyeicosatrienoic acids (DHETs), and 20-HETE in RBCs, plasma, and urine from preeclamptic and normotensive pregnant and nonpregnant women. Blood from fetal and maternal circulation was collected. EETs, DHETs, and 20-HETE were analyzed by gas chromatography and liquid chromatography mass spectrometry. Vascular function and inflammation indices were analyzed. Plasma EET is higher in normotensive (median, range; 9.9, 6.3-25.2ng/mL n = 29) and preeclamptic (10.9, 6.0-48.0ng/mL, n = 19) women than in nonpregnant controls (7.3, 3.7-10.2ng/mL, n = 19) and correlate with RBC EETs, C-reactive protein, and arterial stiffness. Renal production of EETs, measured as urinary DHETs, was reduced in preeclamptic (4.5, 1.6-24.5ng/mg creatinine) compared to normotensive (11.4, 1.6-44.5ng/mg creatinine) pregnancies. EETs are 3- to 5-fold greater in fetoplacental than in maternal circulation (RBCs 36.6, 13.1-69.4 vs. 12.5, 6.4-12.0ng/10(9) cells; plasma 31.6, 8.5-192.6 vs. 12.0, 6.8-48.0ng/mL). Both cis- and trans-EETs are present in fetal RBCs. RBCs contribute to elevated levels of EETs in the fetoplacental circulation. EETs may modulate systemic and fetoplacental hemodynamics in normal and preeclamptic pregnancies. Decreased renal EET generation may be associated with the development of maternal renal dysfunction and hypertension in preeclampsia.

Cytochrome P450 metabolites of arachidonic acid are elevated in stroke patients compared with healthy controls

CYP450AAM [arachidonic acid metabolites of the CYP450 (cytochrome P450) enzyme system] have a range of biological functions. CYP450AAM are involved in the pathogenesis of hypertension, renal function and vascular function, yet their role in stroke has not been clarified. We aimed at determining the levels of circulating CYP450 metabolites in patients with acute ischaemic stroke (<96h) compared with healthy age- and gender-matched controls. This was a retrospective case-controlled study of 44 acute ischaemic stroke patients and 44 matched controls. A subset of acute ischaemic stroke patients was available for follow-up. Acute ischaemic stroke patients had elevated plasma CYP450AAM, including 20-HETE (20-hydroxyeicosatetraenoic acid) (1921±170 compared with 1108±170pmol/l, P<0.001), EETs (epoxyeicosatrienoic acids) (77.88±3.34 compared with 35.35±3.34 nmol/l, P<0.0001) and DiHETEs (dihydroxyeicosatetraenoic acids) (92.87±4.61 compared with 68.17±4.61 nmol/l, P<0.0001), as well as increased plasma F2-isoprostane levels (3754±538 compared with 1947±538pmol/l, P<0.02), the latter a marker of oxidative stress, compared with controls. In a subset analysis of the stroke patients, plasma 20-HETE, EETs and F2-isoprostanes were attenuated 30days after the stroke. Baseline 20-HETE levels were also associated with lesion size and functional indices within the stroke patients. The present study highlights the elevation in CYP450AAM and oxidative stress in acute ischaemic stroke patients. Further investigation of the effect this has on long-term clinical outcome or whether this can be modified by treatment is warranted.