EET Formation Research Articles

Involvement of peptidylarginine deiminase 4 in eosinophil extracellular trap formation and contribution to citrullinated histone signal in thrombi

BackgroundExtracellular traps formed by neutrophils (NETs) and eosinophils (EETs) have been described in coronary thrombi, contributing to thrombus stability. A key mechanism during NET formation is histone modification by the enzyme PAD4. Citrullinated histones, the product of PAD4 activity, are often attributed to neutrophils. Eosinophils also express high levels of PAD4. ObjectivesWe aimed to explore the contribution of PAD4 to EET formation. MethodsWe performed immunohistological analyses on thrombi, including a large, intact, and eosinophil-containing thrombus retrieved from the right coronary artery using an aspiration catheter and stroke thrombi from thrombectomy retrieval. We studied eosinophils for their capability to form PAD4-dependent EETs in response to strong ET-inducing agonists as well as activated platelets and bacteria. ResultsHistopathology and immunofluorescence microscopy identified a coronary thrombus rich in platelets and neutrophils, with distinct areas containing von Willebrand factor and citrullinated histone H3 (H3Cit). Eosinophils were also identified in leukocyte-rich areas. The majority of the H3Cit+ signal colocalized with myeloperoxidase, but some colocalized with eosinophil peroxidase, indicating EETs. Eosinophils isolated from healthy volunteers produced H3Cit+ EETs, indicating an involvement of PAD4 activity. The selective PAD4 inhibitor GSK484 blocked this process, supporting PAD4 dependence of H3Cit+ EET release. Citrullinated histones were also present in EETs produced in response to live Staphylococci. However, limited evidence for EETs was found in mouse models of venous thrombosis or infective endocarditis. ConclusionAs in NETosis, PAD4 can catalyze the formation of EETs. Inhibition of PAD4 decreases EET formation, supporting the future utility of PAD4 inhibitors as possible antithrombotic agents.

Aspergillus Enhances Eosinophil and Neutrophil Extracellular DNA Trap Formation in Chronic Rhinosinusitis.

Chronic rhinosinusitis (CRS) is characterized by inflammatory cell infiltration in the sinonasal mucosa. Eosinophil and neutrophil extracellular traps (EETs and NETs, respectively) are prominently found in CRS. This study aimed to investigate the effect of airborne fungi, Alternaria alternata and Aspergillus fumigatus, on EET and NET formation. Nasal epithelial cells, eosinophils, and neutrophils were isolated from eosinophilic CRS (ECRS), non-ECRS (NECRS), and healthy control. We determined eosinophil and neutrophil transepithelial migration after fungal treatment. We then determined the release of EETs and NETs by fungi using Sytox Green staining and determined the role of reactive oxygen species (ROS) using ROS inhibitors. We identified more abundant EETs and NETs in ECRS than in NECRS. A. alternata and A. fumigatus enhanced eosinophil and neutrophil transepithelial migration. A. fumigatus strongly induced EET and NET formation in CRS and, simultaneously, suppressed fungal metabolic activity. EET formation in CRS is associated with nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase and NET formation with NADPH-oxidase and mitochondrial ROS. A. fumigatus, but not A. alternata, induced EET and NET formation, and peripheral blood eosinophils and neutrophils exhibited different immune responses against A. fumigatus following the inflammatory status of the host. Aspergillus-fumigatus-induced EET and NET formation plays a crucial role in CRS pathogenesis.

Association between CYP4A11 and EPHX2 genetic polymorphisms and chronic kidney disease progression in hypertensive patients

BackgroundThere is evidence indicating that some metabolites of arachidonic acid produced by cytochromes P450 (CYP) and epoxide hydroxylase (EPHX2), such as hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (EETs) or dihydroxyeicosatrienoic acids (DHETEs), play an important role in blood pressure regulation and they could contribute to the development of hypertension (HT) and kidney damage. Therefore, the main aim of the study was to evaluate whether the genetic polymorphisms of CYP2C8, CYP2C9, CYP2J2, CYP4F2, CYP4F11 and EPHX2, responsible for the formation of HETEs, EETs and DHETEs, are related to the progression of impaired renal function in a group of patients with hypertension. Methods151 HT patients from a hospital nephrology service were included in the study. Additionally, a group of 87 normotensive subjects were involved in the study as control group. For HT patients, a general biochemistry analysis, estimated glomerular filtration rate and genotyping for different CYPs and EPHX2 variant alleles were performed. ResultsCYP4A11 rs3890011, rs9332982 and EPHX2 rs41507953 polymorphisms, according to the dominant model, presented a high risk of impaired kidney function, with odds ratios (OR) of 2.07 (1.00–4.32; P=0.049), 3.02 (1.11–8.23; P=0.030) and 3.59 (1.37–9.41; P=0.009), respectively, and the EPHX2 rs1042032 polymorphism a greater risk according to the recessive model (OR=6.23; 95% CI=1.50–25.95; P=0.007). However, no significant differences in allele frequencies between HT patients and in normotensive subjects for any of the SNP analyzed. In addition, the patients with diagnosis of dyslipidemia (n=90) presented higher frequencies of EPHX2 K55R (rs41507953) and *35A>G (rs1042032) variants than patients without dyslipidemia, 4% vs. 14% (P=0.005) and 16 vs. 27% (P=0.02), respectively. ConclusionsIn this study has been found higher odds of impaired renal function progression associated with rs3890011 and rs9332982 (CYP4A11) and rs41507953 and rs1042032 (EPHX2) polymorphisms, which may serve as biomarkers for improve clinical interventions aimed at avoiding or delaying, in chronic kidney disease patients, progress to end-stage kidney disease needing dialysis or kidney transplant.

Rottlerin renders a selective and highly potent CYP2C8 inhibition to impede EET formation for implication in cancer therapy

CYP2C8 is a crucial CYP isoform responsible for the metabolism of xenobiotics and endogenous molecules. CYP2C8 converts arachidonic acid to epoxyeicosatrienoic acids (EETs) that cause cancer progression. Rottlerin possess significant anticancer actions. However, information on its CYP inhibitory action is lacking in the literature and therefore, we aimed to explore the same using in silico, in vitro, and in vivo approaches. Rottlerin showed highly potent and selective CYP2C8 inhibition (IC50 < 0.1 μM) compared to negligible inhibition (IC50 > 10 μM) for seven other experimental CYPs in human liver microsomes (HLM) (in vitro) using USFDA recommended index reactions. Mechanistic studies reveal that rottlerin could reversibly (mixed-type) block CYP2C8. Molecular docking (in silico) results indicate a strong interaction could occur between rottlerin and the active site of human CYP2C8. Rottlerin boosted the plasma exposure of repaglinide and paclitaxel (CYP2C8 substrates) by delaying their metabolism using the rat model (in vivo). Multiple-dose treatment of rottlerin with CYP2C8 substrates lowered the CYP2C8 protein expression and up-regulated & down-regulated the mRNA for CYP2C12 & CYP2C11 (rat homologs), respectively, in rat liver tissue. Rottlerin substantially hindered the EET formation in HLM. Overall results of rottlerin on CYP2C8 inhibition and EET formation insinuate further exploration for cancer therapy.

VEGF-A: A Novel Mechanistic Link Between CYP2C-Derived EETs and Nox4 in Diabetic Kidney Disease.

Diabetes is associated with an alteration in cytochrome P450 2C11 (CYP2C11)-derived epoxyeicosatrienoic acid (EET) bioavailability. Decreased CYP2C11-derived EET bioavailability mediates hyperglycemia-induced glomerular injury. Decreased CYP2C11-derived EET bioavailability is associated with increased reactive oxygen species production, NADPH oxidase activity, and Nox4 expression in type 1 diabetes. Decreased CYP2C11-derived EET formation mediates hyperglycemia-induced glomerular injury through the activation of the vascular endothelial growth factor A (VEGF-A) signaling pathway. Inhibiting VEGF signaling using anti-VEGF or SU5416 attenuates type 1 diabetes-induced glomerular injury by decreasing NADPH oxidase activity and NOX4 expression.

Effects of multi-kinase inhibitors on the activity of cytochrome P450 2J2

1. Cytochrome P450 2J2 (CYP2J2) shows high expression in extrahepatic tissues, including the heart and kidney and in tumours. Inhibition of CYP2J2 has attracted attention for cancer treatment because it metabolises arachidonic acid (AA) to epoxyeicosatrienoic acid (EET), which inhibits apoptosis and promotes tumour growth. Multi-kinase inhibitor (MKI) is a molecular-targeted drug with antitumor activities. This study aimed to clarify the inhibitory effects of MKIs on CYP2J2 activity. We also investigated whether MKIs affected CYP2J2-catalysed EET formation from AA. 2. Twenty MKIs showed different inhibitory potencies against astemizole O-demethylation in CYP2J2. In particular, apatinib, motesanib, and vatalanib strongly inhibited astemizole O-demethylation. These three MKIs exhibited competitive inhibition with inhibition constant (K i) values of 9.3, 15.4, and 65.0 nM, respectively. Apatinib, motesanib, and vatalanib also inhibited CYP2J2-catalysed 14,15-EET formation from AA. 3. In simulations of docking to CYP2J2, the U energy values of apatinib, motesanib, and vatalanib were low, and measured −84.5, −69.9, and −52.3 kcal/mol, respectively. 4. In conclusion, apatinib, motesanib, and vatalanib strongly inhibited CYP2J2 activity, suggesting that the effects of a given CYP2J2 substrate may be altered upon the administration of these MKIs.

Monolayer autoxidation of arachidonic acid to epoxyeicosatrienoic acids as a model of their potential formation in cell membranes

In light of the importance of epoxyeicosatrienoic acids (EETs) in mammalian pathophysiology, a nonenzymatic route that might form these monoepoxides in cells is of significant interest. In the late 1970s, a simple system of arranging linoleic acid molecules on a monolayer on silica was devised and shown to yield monoepoxides as the main autoxidation products. Here, we investigated this system with arachidonic acid and characterized the primary products. By the early stages of autoxidation (∼10% conversion of arachidonic acid), the major products detected by LC-MS and HPLC-UV were the 14,15-, 11,12-, and 8,9-EETs, with the 5,6-EET mainly represented as the 5-δ-lactone-6-hydroxyeicosatrienoate as established by 1H-NMR. The EETs were mainly the cis epoxides as expected, with minor trans configuration EETs among the products. 1H-NMR analysis in four deuterated solvents helped clarify the epoxide configurations. EET formation in monolayers involves intermolecular reaction with a fatty acid peroxyl radical, producing the EET and leaving an incipient and more reactive alkoxyl radical, which in turn gives rise to epoxy-hydro(pero)xides and other polar products. The monolayer alignment of fatty acid molecules resembles the arrangements of fatty acids in cell membranes and, under conditions of lipid peroxidation, this intermolecular mechanism might contribute to EET formation in biological membranes.

Eosinophil extracellular trap formation is closely associated with disease severity in chronic rhinosinusitis regardless of nasal polyp status

Chronic rhinosinusitis (CRS) is a heterogeneous inflammatory airway disease involving non-eosinophilic and eosinophilic phenotypes, which translate to various endotypes. Activated eosinophils and neutrophils are known to generate extracellular traps consisting of DNA and cytotoxic granule proteins. We sought to investigate the presence of eosinophil and neutrophil extracellular traps (EETs and NETs, respectively) in human CRS tissues and to clarify the associations with their clinical features. Nasal polyp (NP) or ethmoid tissue slides of 43 subjects from endoscopic sinus surgery for CRS were analysed. Quantitative analysis of EETs and NETs was performed by confocal microscopy using immunofluorescent staining. For correlation study, the presence of NPs, number of infiltrating tissue eosinophils, preoperative Lund–Mackay scores, and other comorbidities were analysed. EET formation was observed to varying degrees in all CRS groups and was correlated with the number of tissue eosinophils (r = 0.83, p < 0.001) regardless of the presence of NPs. Patients with more EETs demonstrated higher Lund–Mackay scores (r = 0.51, p = 0.009), blood eosinophilia (r = 0.80, p < 0.001), and decreased olfactory function (r = −0.65, p < 0.001). No correlation between the extent of EET formation and the presence of atopy or asthma was apparent. However, none of the CRS groups containing neutrophils formed NETs in this study. Eosinophilic CRS indicates the presence of EETs. Formation of EETs could have a role in clinical decision-making and prediction of treatment outcome of CRS, regardless of NP status.

Regulation of CYP2J2 and EET Levels in Cardiac Disease and Diabetes.

Cytochrome P450 2J2 (CYP2J2) is a known arachidonic acid (AA) epoxygenase that mediates the formation of four bioactive regioisomers of cis-epoxyeicosatrienoic acids (EETs). Although its expression in the liver is low, CYP2J2 is mainly observed in extrahepatic tissues, including the small intestine, pancreas, lung, and heart. Changes in CYP2J2 levels or activity by xenobiotics, disease states, or polymorphisms are proposed to lead to various organ dysfunctions. Several studies have investigated the regulation of CYP2J2 and EET formation in various cell lines and have demonstrated that such regulation is tissue-dependent. In addition, studies linking CYP2J2 polymorphisms to the risk of developing cardiovascular disease (CVD) yielded contradictory results. This review will focus on the mechanisms of regulation of CYP2J2 by inducers, inhibitors, and oxidative stress modeling certain disease states in various cell lines and tissues. The implication of CYP2J2 expression, polymorphisms, activity and, as a result, EET levels in the pathophysiology of diabetes and CVD will also be discussed.

Enzymatic and free radical formation of cis- and trans- epoxyeicosatrienoic acids in vitro and in vivo

Epoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid (AA) oxidation that have important cardioprotective and signaling properties. AA is an ω-6 polyunsaturated fatty acid (PUFA) that is prone to autoxidation. Although hydroperoxides and isoprostanes are major autoxidation products of AA, EETs are also formed from the largely overlooked peroxyl radical addition mechanism. While autoxidation yields both cis- and trans-EETs, cytochrome P450 (CYP) epoxygenases have been shown to exclusively catalyze the formation of all regioisomer cis-EETs, on each of the double bonds. In plasma and red blood cell (RBC) membranes, cis- and trans-EETs have been observed, and both have multiple physiological functions. We developed a sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay that separates cis- and trans- isomers of EETs and applied it to determine the relative distribution of cis- vs. trans-EETs in reaction mixtures of AA subjected to free radical oxidation in benzene and liposomes in vitro. We also determined the in vivo distribution of EETs in several tissues, including human and mouse heart, and RBC membranes. We then measured EET levels in heart and RBC of young mice compared to old. Formation of EETs in free radical reactions of AA in benzene and in liposomes exhibited time- and AA concentration-dependent increase and trans-EET levels were higher than cis-EETs under both conditions. In contrast, cis-EET levels were overall higher in biological samples. In general, trans-EETs increased with mouse age more than cis-EETs. We propose a mechanism for the non-enzymatic formation of cis- and trans-EETs involving addition of the peroxyl radical to one of AA's double bonds followed by bond rotation and intramolecular homolytic substitution (SHi). Enzymatic formation of cis-EETs by cytochrome P450 most likely occurs via a one-step concerted mechanism that does not allow bond rotation. The ability to accurately measure circulating EETs resulting from autoxidation or enzymatic reactions in plasma and RBC membranes will allow for future studies investigating how these important signaling lipids correlate with heart disease outcomes.

Recombinant human deoxyribonuclease therapy improves airway resistance and reduces DNA extracellular traps in a murine acute asthma model

ABSTRACTPurpose: Asthma is a highly prevalent chronic inflammatory lung disease characterized by airway hyperresponsiveness to allergens, airway edema, and increased mucus secretion. Such mucus can be liquefied by recombinant human deoxyribonuclease (rhDNase), in which efficacy of rhDNase has been well documented in patients with cystic fibrosis, but little studied in asthma. In the present study, we investigated whether rhDNase intranasal administration improved inflammation and pulmonary function in an experimental model of asthma. Methods: Mice were sensitized by two subcutaneous injections of ovalbumin (OVA), on days 0 and 7, followed by three intranasal challenges with OVA on days 14, 15, and 16. A control group, replacing OVA by DPBS, was included. On days 15 and 16, after 2 hours of OVA challenge, mice received 1 mg/mL of intranasal rhDNase. Results: We showed that rhDNase decreased significantly the airway resistance and reduced EETs formation and globet cells hyperplasia. Conclusions: Our results suggest that extracellular DNA in mucus play a role in lower airways obstruction in OVA asthma protocol and that the treatment with rhDNase improved lung function and DNA extracellular traps, with no direct cellular anti-inflammatory effects.

Molecular mechanisms and cell signaling of 20-hydroxyeicosatetraenoic acid in vascular pathophysiology.

Cytochrome P450s enzymes catalyze the metabolism of arachidonic acid to epoxyeicosatrienoic acids (EETs), dihydroxyeicosatetraenoic acid and hydroxyeicosatetraeonic acid (HETEs). 20-HETE is a vasoconstrictor that depolarizes vascular smooth muscle cells by blocking K+ channels. EETs serve as endothelial derived hyperpolarizing factors. Inhibition of the formation of 20-HETE impairs the myogenic response and autoregulation of renal and cerebral blood flow. Changes in the formation of EETs and 20-HETE have been reported in hypertension and drugs that target these pathways alter blood pressure in animal models. Sequence variants in CYP4A11 and CYP4F2 that produce 20-HETE, UDP-glucuronosyl transferase involved in the biotransformation of 20-HETE and soluble epoxide hydrolase that inactivates EETs are associated with hypertension in human studies. 20-HETE contributes to the regulation of vascular hypertrophy, restenosis, angiogenesis and inflammation. It also promotes endothelial dysfunction and contributes to cerebral vasospasm and ischemia-reperfusion injury in the brain, kidney and heart. This review will focus on the role of 20-HETE in vascular dysfunction, inflammation, ischemic and hemorrhagic stroke and cardiac and renal ischemia reperfusion injury.

IMUNITAS ALAMIAH: EOSINOPHIL EXTRACELLULAR TRAPS (EET)

Eosinophils play a role in the defense against viruses, bacteria, fungi and parasites, as well as be involved in the pathogenesis of allergic and autoimmune diseases. The role of eosinophils in the defense against bacterial run by enzymes found in the granules such as MBP, ECP, EPO, and EDN, and spending EET. EET formation is determined by the presence of free radicals and intracellular calcium. The antimicrobial function of EET played by Histon.Keywords: eosinophil , Eosinophils extracellular traps

Effects of Angiotensin II Receptor Blockers on Metabolism of Arachidonic Acid &lt;i&gt;via&lt;/i&gt; CYP2C8

Arachidonic acid (AA) is metabolized to epoxyeicosatrienoic acids (EETs) via cytochrome enzymes such as CYP 2C9, 2C8 and 2J2. EETs play a role in cardioprotection and regulation of blood pressure. Recently, adverse reactions such as sudden heart attack and fatal myocardial infarction were reported among patients taking angiotensin II receptor blockers (ARBs). As some ARBs have affinity for these CYP enzymes, metabolic inhibition of AA by ARBs is a possible cause for the increase in cardiovascular events. In this study, we quantitatively investigated the inhibitory effects of ARBs on the formation of EETs and further metabolites, dihydroxyeicosatrienoic acids (DHETs), from AA via CYP2C8. In incubations with recombinant CYP2C8 in vitro, the inhibitory effects were compared by measuring EETs and DHETs by HPLC-MS/MS. Inhibition of AA metabolism by ARBs was detected in a concentration-dependent manner with IC50 values of losartan (42.7 µM), telmisartan (49.5 µM), irbesartan (55.6 µM), olmesartan (66.2 µM), candesartan (108 µM), and valsartan (279 µM). Losartan, telmisartan and irbesartan, which reportedly accumulate in the liver and kidneys, have stronger inhibitory effects than other ARBs. The lower concentration of EETs leads to less protective action on the cardiovascular system and a higher incidence of adverse effects such as sudden heart attack and myocardial infarction in patients taking ARBs.

Deletion of the Murine Cytochrome P450 Cyp2j Locus by Fused BAC-Mediated Recombination Identifies a Role for Cyp2j in the Pulmonary Vascular Response to Hypoxia

Epoxyeicosatrienoic acids (EETs) confer vasoactive and cardioprotective functions. Genetic analysis of the contributions of these short-lived mediators to pathophysiology has been confounded to date by the allelic expansion in rodents of the portion of the genome syntenic to human CYP2J2, a gene encoding one of the principle cytochrome P450 epoxygenases responsible for the formation of EETs in humans. Mice have eight potentially functional genes that could direct the synthesis of epoxygenases with properties similar to those of CYP2J2. As an initial step towards understanding the role of the murine Cyp2j locus, we have created mice bearing a 626-kb deletion spanning the entire region syntenic to CYP2J2, using a combination of homologous and site-directed recombination strategies. A mouse strain in which the locus deletion was complemented by transgenic delivery of BAC sequences encoding human CYP2J2 was also created. Systemic and pulmonary hemodynamic measurements did not differ in wild-type, null, and complemented mice at baseline. However, hypoxic pulmonary vasoconstriction (HPV) during left mainstem bronchus occlusion was impaired and associated with reduced systemic oxygenation in null mice, but not in null mice bearing the human transgene. Administration of an epoxygenase inhibitor to wild-type mice also impaired HPV. These findings demonstrate that Cyp2j gene products regulate the pulmonary vascular response to hypoxia.

20-HETE and EETs in Diabetic Nephropathy: A Novel Mechanistic Pathway

Diabetic nephropathy (DN), a major complication of diabetes, is characterized by hypertrophy, extracellular matrix accumulation, fibrosis and proteinuria leading to loss of renal function. Hypertrophy is a major factor inducing proximal tubular epithelial cells injury. However, the mechanisms leading to tubular injury is not well defined. In our study, we show that exposure of rats proximal tubular epithelial cells to high glucose (HG) resulted in increased extracellular matrix accumulation and hypertrophy. HG treatment increased ROS production and was associated with alteration in CYPs 4A and 2C11 expression concomitant with alteration in 20-HETE and EETs formation. HG-induced tubular injury were blocked by HET0016, an inhibitor of CYPs 4A. In contrast, inhibition of EETs promoted the effects of HG on cultured proximal tubular cells. Our results also show that alteration in CYPs 4A and 2C expression and 20HETE and EETs formation regulates the activation of the mTOR/p70S6Kinase pathway, known to play a major role in the development of DN. In conclusion, we show that hyperglycemia in diabetes has a significant effect on the expression of Arachidonic Acid (AA)-metabolizing CYPs, manifested by increased AA metabolism, and might thus alter kidney function through alteration of type and amount of AA metabolites.

Role of CYP1A2 polymorphisms in breast cancer risk in women

Cytochrome P4501A2 (CYP1A2) is a key enzyme in the etiology of breast cancer (BC). It is involved in breast carcinogen activation [aromatic (AAs) and heterocyclic amines (HAs), polycyclic aromatic hydrocarbons (PAHs)], in the production of beneficial oestrogen [2-hydroxyestrone (2-OHE1)] and in converting arachidonic acid (AAc) to epoxyeicosatrienoic acids (EETs), which have anti-inflammatory properties. Within a hospital-based case-control study, the effect of functional CYP1A2 variants [-3860G/A (rs2069514), -2467T/delT (rs3569413), -163C/A (rs762551)] and their interactions with environmental factors in BC risk was investigated. The study population included 125 BC cases and 43 non-cancer controls. Genotyping was performed in RT-PCR using Taqman assays. The gene-environment interaction was appraised using a case-only study design. We found that the -3860A variant, independently from environmental factors, as well as by interacting with fried foods (p=0.025) and indoor exposure to pollutants (p=0.050), reduced the risk of BC (p=0.025), whereas its interaction with coffee (p=0.045) increased the BC risk. This is the first study indicating that the -3860A variant, by decreasing CYP1A2 activity, modifies BC risk by interacting with environmental factors, thereby supporting the hypothesis that reduced CYP1A2 activity contributes to BC risk in different ways, for example, it may be protective by reducing the activation of pro-carcinogens such as AAs, HAs and PAHs, but would increase risk by reducing the beneficial formation of 2-OHE1 and EETs.

Abstract 348: Urinary CYP Eicosanoid Excretion Correlates with Glomerular Filtration in African-Americans with Chronic Kidney Disease

Previous studies have indicated that cytochrome P450 (CYP) metabolites of arachidonic acid, ie, 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) play an important role in the regulation of renal tubular function and vascular tone. More recent studies suggest that variants in the CYP4A11 and CYP4F2 genes are linked to the development of hypertension in a variety of human population studies. However, little is known about the role of 20-HETE or EETs in the pathogenesis of hypertension or diabetic induced renal disease because CYP eicosanoids have not been measured in patients with CKD. The present study profiled HETEs and the dihydroxy metabolites of EETs (DHETs) levels, from spot urines at the time of their clinic visit using LC/MS/MS in 106 African-American patients from the University of Mississippi (UMC) Chronic Kidney Disease (CKD) Clinic with various etiologies of renal disease. Informed consent was obtained for this UMMC IRB approved protocol. Significant positive correlations were found between urinary 5,6- DHETE, 8,9-DHETE, 11,12-DIHETE, 14,15-DHETE, 20-HETE and 19-, 15- and 8-HETE levels and estimated GFR (eGFR) as derived from the MDRD. The magnitude of the influence of urinary eicosanoid levels on eGFR was relatively profound since the slopes of these relationships indicated that there is a 5-10% decrement eGFR associated with each ng/ml fall in urinary eicosanoid levels. These results suggest that a decline in the renal formation of 20-HETE and/or EETs may contribute to the pathogenesis of CKD. At very least, urinary CYP eicosanoids may serve as a useful biomarker for progressive disease.

Rhythmic expression of vasoactive compounds in the rat brain and cerebral vasculature

In mammals, circadian variation is evident in hormone levels, arterial pressure, heart rate and cerebral blood flow (CBF). In humans there is an increased stroke incidence in the morning. CYP2C11 and CYP4X1 are cytochrome P450 epoxygenases expressed in rat brain which convert arachidonic acid (AA) to vasodilatory epoxyeicosatrienoic acids (EETs). We have shown that these genes are expressed in a circadian or biphasic circadian manner in rat brain and vasculature. Here we investigate rhythms in cultured astrocytes and cerebral microvascular endothelial cells over 48 hours using RT‐PCR. Using Western blotting, we measured CYP2C11 and CYP4X1 protein levels over 24 hours and CYP450 enzyme activity by measuring EETs and 20‐HETE formation following addition of the substrate AA. We show Cyp2c11, Cyp4x1 and Cyp4a2 mRNA expression is rhythmic with a 12 hour period although the phase differs between genes. This pattern was also apparent at the protein level. Preliminary P450 activity data suggest rhythmic formation of EETs and 20‐HETE with 12 and 24 hour periods. Our data indicate CYP450 enzymes have rhythmic mRNA and protein expression and activity levels, suggesting their involvement in regulation of rhythmic physiological phenomena, such as CBF. Our findings will lead to a better understanding of the mechanisms underlying increased morning stroke incidence in humans.

Vitamin C activation of the biosynthesis of epoxyeicosatrienoic acids

The cardiovascular effects of vitamin C (VitC) could be mediated by epoxyeicosatrienoic acids (EETs). We aimed to study the mechanism of VitC-dependent microsomal formation of cis- and trans-EETs and the regulation of EET levels in rat isolated perfused kidneys and in vivo. VitC biphasically stimulated rat kidney microsomal cis- and trans-EET formation in a ratio of 1:2, involving the participation of lipid hydroperoxides (LOOHs), Fe2+, and cytochrome P450 (CYP). Levels of LOOHs correlated with microsomal EET production. LOOH stimulation of CYP isoforms resulted in preferred trans-over cis-EET formation from arachidonic acid and was associated with the cleavage of LOOHs, which indicated a CYP peroxygenase activity. EETs contributed to VitC-induced vasodilator responses in rat isolated perfused kidneys. VitC (1 mg/ml) given in the drinking water for 9 days doubled rat urinary EET excretion, increased plasma levels of EETs, mostly trans-EETs, by 40%, and reduced plasma levels of 20-hydroxyeicosatetraenoic acid. Depletion of VitC in brain cortex and kidney tissues by more than 20- and 50-fold, respectively, in gulonolactone oxidase-knockout mice was associated with mild increases in tissue EETs. These data suggest that LOOHs are a determinant factor for EET formation in vivo in which VitC exerts a key regulatory effect. VitC-activated CYP peroxygenase activity may represent a CYP interaction with lipoxygenases and cyclooxygenases to mediate the cardiovascular effects of VitC via formation of EETs.