Epoxyeicosatrienoic Acids Research Articles

Quantitative metabolic analysis of plasma extracellular vesicles for the diagnosis of severe acute pancreatitis

BackgroundSevere acute pancreatitis (SAP) is the most common gastrointestinal disease and is associated with unpredictable seizures and high mortality rates. Despite improvements in the treatment of acute pancreatitis, the timely and accurate diagnosis of SAP remains highly challenging. Previous research has shown that extracellular vesicles (EVs) in the plasma have significant potential for the diagnosis of SAP since the pancreas can release EVs that carry pathological information into the peripheral blood in the very early stages of the disease. However, we know very little about the metabolites of EVs that might play a role in the diagnosis of SAP.MethodsHere, we performed quantitative metabolomic analyses to investigate the metabolite profiles of EVs isolated from SAP plasma. We also determined the metabolic differences of EVs when compared between healthy controls, patients with SAP, and those with mild acute pancreatitis (MAP).ResultsA total of 313 metabolites were detected, mainly including organic acids, amino acids, fatty acids, and bile acids. The results showed that the metabolic composition of EVs derived from SAP and MAP was significantly different from those derived from healthy controls and identified specific differences between EVs derived from patients with SAP and MAP. On this basis, we identified four biomarkers from plasma EVs for SAP detection, including eicosatrienoic acid (C20:3), thiamine triphosphate, 2-Acetylfuran, and cis-Citral. The area under the curve (AUC) was greater than 0.95 for both discovery (n = 30) and validation (n = 70) sets.ConclusionsOur data indicate that metabolic profiling analysis of plasma EVs and the screening of potential biomarkers are of significant potential for improving the early diagnosis and severity differentiation of acute pancreatitis.Graphical abstract

Antinociception role of 14,15-epoxyeicosatrienoic acid in a central post-stroke pain model in rats mediated by anti-inflammation and anti-apoptosis effect

Central post stroke pain (CPSP) is an intractable neuropathic pain syndrome that occurs after the acute focal lesion of the central nervous system (CNS) due to a cerebrovascular cause. Epoxyeicosatrienoic acids (EETs) exert many pharmacological effects in vivo and in vitro, such as anti-apoptosis, anti-inflammatory, and anti-oxidative stress. Neuroinflammation and apoptosis are the potential pathophysiological mechanisms of neuropathic pain. This study aimed to investigate whether 14,15-EET has an antinociception effect on CPSP rats through its anti-inflammation and anti-apoptosis mechanisms. Rats were treated with type IV collagenase (CPSP group) or saline (Sham group) via injection with a Hamilton syringe into the ventral posterior lateral nucleus (VPL) according to the stereotaxic coordinates. We first tested the mechanical withdrawal threshold, as well as neuroinflammation- and apoptosis-related protein expressions in the per-lesion site of CPSP and Sham rats. Sprague-Dawley rats were randomly divided into five groups, as follows: vehicle; EET at 0.025, 0.05, and 0.1 μg; and EET (0.1 μg) + EEZE (3.25 ng). EET or and vehicle were administered into VPL nuclei three consecutive days after hemorrhagic stroke. Immunostaining, ELISA, and Western blot were performed to evaluate neuroinflammation and apoptosis. Hemorrhagic stroke induced mechanical allodynia, glial activation, neuroinflammation, and apoptosis-related protein upregulation. However, early treatment with 14,15-EET inhibited glial cell activation, decreased proinflammatory cytokines and apoptosis-related protein, and alleviated the pain behavior of CPSP rats. Our results provided strong evidence that antinociception produced by 14,15-EET is partly mediated by the inhibition of neuroinflammation and apoptosis.

Association between FADS Gene Expression and Polyunsaturated Fatty Acids in Breast Milk.

Polyunsaturated fatty acid (PUFA) in breast milk provides physiological benefits for offspring and is closely related to endogenous biosynthesis in lactating women. Few studies have addressed the association between fatty acid desaturase (FADS) gene expression patterns and fatty acids in breast milk. This research aimed to explore the differences in PUFA levels among breast milk groups with different levels of FADS gene expression and provide a scientific basis for precision nutrition strategies. A total of 50 healthy women 42–45 days postpartum were included in this study. A basic information questionnaire and breast milk samples were collected. Eight types of PUFA were detected, and RNA was extracted from breast milk. The transcription level of the FADS gene was detected using real-time quantitative PCR. Significant differences in the content of gamma-linolenic acid and eicosatrienoic acid (C20:3n6) were found in breast milk among FADS1 gene transcription groups (p = 0.009, p = 0.042, respectively). No significant differences in PUFA were found among the FADS2 and FADS3 gene expression groups. The results demonstrated that n-6 PUFA was associated with the mRNA expression levels of the FADS1 gene. They are of great significance in developing new methods and diets to optimize infant feeding using breast milk.

Epoxyeicosatrienoic Acid Analog and 20-HETE Antagonist Combination Prevent Hypertension Development in Spontaneously Hypertensive Rats.

Numerous studies indicate a significant role for cytochrome P-450-dependent arachidonic acid metabolites in blood pressure regulation, vascular tone, and control of renal function. Epoxyeicosatrienoic acids (EETs) exhibit a spectrum of beneficial effects, such as vasodilatory activity and anti-inflammatory, anti-fibrotic, and anti-apoptotic properties. 20-Hydroxyeicosatetraenoic acid (20-HETE) is a potent vasoconstrictor that inhibits sodium reabsorption in the kidney. In the present study, the efficiency of EET-A (a stable analog of 14,15-EET) alone and combined with AAA, a novel receptor antagonist of 20-HETE, was tested in spontaneously hypertensive rats (SHR). Adult SHR (16 weeks old) were treated with two doses of EET-A (10 or 40 mg/kg/day). In the following experiments, we also tested selected substances in the prevention of hypertension development in young SHR (6 weeks old). Young rats were treated with EET-A or the combination of EET-A and AAA (both at 10 mg/kg/day). The substances were administered in drinking water for 4 weeks. Blood pressure was measured by telemetry. Once-a-week observation in metabolic cages was performed; urine, blood, and tissue samples were collected for further analysis. The combined treatment with AAA + EET-A exhibited antihypertensive efficiency in young SHR, which remained normotensive until the end of the observation in comparison to a control group (systolic blood pressure, 134 ± 2 versus 156 ± 5 mmHg, respectively; p < 0.05). Moreover the combined treatment also increased the nitric oxide metabolite excretion. Considering the beneficial impact of the combined treatment with EET-A and AAA in young rats and our previous positive results in adult SHR, we suggest that it is a promising therapeutic strategy not only for the treatment but also for the prevention of hypertension.

Anthracycline derivatives inhibit cardiac CYP2J2

Anthracycline chemotherapeutics are highly effective, but their clinical usefulness is hampered by adverse side effects such as cardiotoxicity. Cytochrome P450 2J2 (CYP2J2) is a cytochrome P450 epoxygenase in human cardiomyocytes that converts arachidonic acid (AA) to cardioprotective epoxyeicosatrienoic acid (EET) regioisomers. Herein, we performed biochemical studies to understand the interaction of anthracycline derivatives (daunorubicin, doxorubicin, epirubicin, idarubicin, 5-iminodaunorubicin, zorubicin, valrubicin, and aclarubicin) with CYP2J2. We utilized fluorescence polarization (FP) to assess whether anthracyclines bind to CYP2J2. We found that aclarubicin bound the strongest to CYP2J2 despite it having large bulky groups. We determined that ebastine competitively inhibits anthracycline binding, suggesting that ebastine and anthracyclines may share the same binding site. Molecular dynamics and ensemble docking revealed electrostatic interactions between the anthracyclines and CYP2J2, contributing to binding stability. In particular, the glycosamine groups in anthracyclines are stabilized by binding to glutamate and aspartate residues in CYP2J2 forming salt bridge interactions. Furthermore, we used iterative ensemble docking schemes to gauge anthracycline influence on EET regioisomer production and anthracycline inhibition on AA metabolism. This was followed by experimental validation of CYP2J2-mediated metabolism of anthracycline derivatives using liquid chromatography tandem mass spectrometry fragmentation analysis and inhibition of CYP2J2-mediated AA metabolism by these derivatives. Taken together, we use both experimental and theoretical methodologies to unveil the interactions of anthracycline derivatives with CYP2J2. These studies will help identify alternative mechanisms of how anthracycline cardiotoxicity may be mediated through the inhibition of cardiac P450, which will aid in the design of new anthracycline derivatives with lower toxicity.

Epoxyeicosatrienoic acids and soluble epoxide hydrolase in physiology and diseases of the central nervous system.

Epoxyeicosatrienoic acids (EETs) are fatty acid signaling molecules synthesized by cytochrome P450 epoxygenases from arachidonic acid. The biological activity of EETs is terminated when being metabolized by soluble epoxide hydrolase (sEH), a process that serves as a key regulator of tissue EETs levels. EETs act through several signaling pathways to mediate various beneficial effects, including anti-inflammation, anti-apoptosis, and anti-oxidation with relieve of endoplasmic reticulum stress, thereby sEH has become a potential therapeutic target in cardiovascular disease and cancer therapy. Enzymes for EET biosynthesis and metabolism are both widely detected in both neuron and glial cells in the central nervous system (CNS). Recent studies discovered that astrocyte-derived EETs not only mediate neurovascular coupling and neuronal excitability by maintaining glutamate homeostasis but also glia-dependent neuroprotection. Genetic ablation as well as pharmacologic inhibition of sEH has greatly helped to elucidate the physiologic actions of EETs, and maintaining or elevating brain EETs level has been demonstrated beneficial effects in CNS disease models. Here, we review the literature regarding the studies on the bioactivity of EETs and their metabolic enzyme sEH with special attention paid to their action mechanisms in the CNS, including their modulation of neuronal activity, attenuation of neuroinflammation, regulation of cerebral blood flow, and improvement of neuronal and glial cells survival. We further reviewed the recent advance on the potential application of sEH inhibition for treating cerebrovascular disease, epilepsy, and pain disorder.

Hyperhomocysteinemia dysregulates plasma levels of polyunsaturated fatty acids-derived eicosanoids.

Hyperhomocysteinemia (HHcy) contributes to the incidence of many cardiovascular diseases (CVD). Our group have previously established crucial roles of eicosanoids and homocysteine in the incidence of vascular injury in diabetic retinopathy and renal injury. Using cystathionine-β-synthase heterozygous mice (cβs+/-) as a model of HHcy, the current study was designed to determine the impact of homocysteine on circulating levels of lipid mediators derived from polyunsaturated fatty acids (PUFA). Plasma samples were isolated from wild-type (WT) and cβs+/- mice for the assessment of eicosanoids levels using LC/MS. Plasma 12/15-lipoxygenase (12/15-LOX) activity significantly decreased in cβs+/- vs. WT control mice. LOX-derived metabolites from both omega-3 and omega-6 PUFA were also reduced in cβs+/- mice compared to WT control (P < 0.05). Contrary to LOX metabolites, cytochrome P450 (CYP) metabolites from omega-3 and omega-6 PUFA were significantly elevated in cβs+/- mice compared to WT control. Epoxyeicosatrienoic acids (EETs) are epoxides derived from arachidonic acid (AA) metabolism by CYP with anti-inflammatory properties and are known to limit vascular injury, however their physiological role is limited by their rapid degradation by soluble epoxide hydrolase (sEH) to their corresponding diols (DiHETrEs). In cβs+/- mice, a significant decrease in the plasma EETs bioavailability was obvious as evident by the decrease in EETs/ DiHETrEs ratio relative to WT control mice. Cyclooxygenase (COX) metabolites were also significantly decreased in cβs+/- vs. WT control mice. These data suggest that HHcy impacts eicosanoids metabolism through decreasing LOX and COX metabolic activities while increasing CYP metabolic activity. The increase in AA metabolism by CYP was also associated with increase in sEH activity and decrease in EETs bioavailability. Dysregulation of eicosanoids metabolism could be a contributing factor to the incidence and progression of HHcy-induced CVD.

Orally active epoxyeicosatrienoic acid analogs in hypertension and renal injury.

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites synthesized by cytochrome P450 epoxygenases. Biological activities for EETs include vasodilation, decreasing inflammation, opposing apoptosis, and inhibiting renal sodium reabsorption. These actions are beneficial in lowering blood pressure and slowing kidney disease progression. Furthermore, evidence in human and experimental animal studies have found that decreased EET levels contribute to hypertension and kidney diseases. Consequently, EET mimics/analogs have been developed as a potential therapeutic for hypertension and acute and chronic kidney diseases. Their development has resulted in EET analogs that are orally active with favorable pharmacological profiles. Analogs for 8,9-EET, 11,12-EET, and 14,15-EET have been tested in several hypertension and kidney disease animal models. More recently, kidney targeted EET analogs have been synthesized and tested against drug-induced nephrotoxicity. Experimental evidence has demonstrated compelling therapeutic potential for EET analogs to oppose cardiovascular and kidney diseases. These EET analogs lower blood pressure, decrease kidney inflammation, improve vascular endothelial function, and decrease kidney fibrosis and apoptosis. Overall, these preclinical studies support the likelihood that EET analogs will advance to clinical trials for hypertension and associated comorbidities or acute and chronic kidney diseases.

Metabolomic changes in polyunsaturated fatty acids and eicosanoids as diagnostic biomarkers in Mycobacterium avium ssp. paratuberculosis (MAP)-inoculated Holstein–Friesian heifers

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative organism of Johne’s disease, a chronic granulomatous enteritis of ruminants. We have previously used naturally MAP-infected heifer calves to document metabolomic changes occurring in MAP infections. Herein, we used experimentally MAP-inoculated heifer calves to identify biomarkers for MAP infections. At 2-weeks of age, 20 Holstein–Friesian (HF) calves were experimentally inoculated with MAP. These calves, along with 20 control calves, were sampled biweekly up to 13-months of age and then monthly up to 19-months of age. Sera were assessed using flow infusion electrospray high-resolution mass spectrometry (FIE-HRMS) on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer for high throughput, sensitive, non-targeted metabolite fingerprinting. Partial least squares-discriminate analysis (PLS-DA) and hierarchical cluster analysis (HCA) discriminated between MAP-inoculated and control heifer calves. Out of 34 identified metabolites, six fatty acyls were able to differentiate between experimental groups throughout the study, including 8, 11, 14-eicosatrienoic acid and cis-8, 11, 14, 17-eicosatetraenoic acid which were also detected in our previous study and so further suggested their value as biomarkers for MAP infection. Pathway analysis highlighted the role of the alpha-linoleic acid and linoleic acid metabolism. Within these pathways, two broad types of response, with a rapid increase in some saturated fatty acids and some n-3 polyunsaturated fatty acids (PUFAs) and later n-6 PUFAs, became predominant. This could indicate an initial anti-inflammatory colonisation phase, followed by an inflammatory phase. This study demonstrates the validity of the metabolomic approach in studying MAP infections. Nevertheless, further work is required to define further key events, particularly at a cell-specific level.

Analysis of the gut microbiome to validate a mouse model of pellagra.

Pellagra is caused by an abnormal intake and/or use of niacin, but its phenotypes are diverse. The phenotypes of pellagra can also be atypical, such as nausea. We previously reported a mouse model of pellagra-related nausea. However, the mechanism of this model is unclear. In this study, we found that the gut microbiota, which is thought to be a source of niacin, played an important role in the development of pellagra-related nausea in germ-free mice. We also investigated the gut microbiome. We compared urinary niacin metabolite levels and the dermal response between mice fed a normal diet and those fed a low-niacin diet to investigate the putative trigger of pellagra. Epoxyeicosatrienoic and hydroxyeicosatetraenoic acid levels were higher in mice fed a low-niacin diet compared with those fed a normal diet. Furthermore, histological studies indicated a dermatological response to the low-niacin diet. Interestingly, higher levels of oxidised fatty acids in response to the germ-free state were also observed. These findings indicate successful establishment of our newly established mouse model of pellagra via the gut microbiota. We believe that this model could enable the discovery of the putative cause of pellagra and phenotypes of pellagra that have not been recognised yet.

Interaction of aging with lipoxygenase deficiency initiates hypersplenism, cardiac dysfunction, and profound leukocyte directed non-resolving inflammation.

In the process of physiological cardiac repair, splenic leukocyte-activated lipoxygenases (LOXs) are essential for the biosynthesis of specialized pro-resolving lipid mediators as a segment of an active process of acute inflammation in splenocardiac manner. In contrast, young 12/15LOX-/- mice use a compensatory mechanism that amplifies epoxyeicosatrienoic acid mediators after myocardial infarction, improving cardiac repair, function, and survival. Next, we tested whether deletion of 12/15LOX impacted the genesis of chronic inflammation in progressive aging. To test the risk factor of aging, we used the inter-organ hypothesis and assessed heart and spleen leukocyte population along with the number of inflammation markers in age-related 12/15LOX-/- aging mice (2months, 6months, 13months) and compared with C57BL/6J (WT; wild type) as controls (2months). The 12/15LOX-/- aging mice showed an age-related increase in spleen mass (hypersplenism) and decreased marginal zone area. Results suggest increased interstitial fibrosis in the heart marked with the inflammatory mediator (PGD2) level in 12/15LOX-/- aging mice than WT controls. From a cellular perspective, the quantitative measurement of immune cells indicates that heart and spleen leukocytes (CD11b+ and F4/80+ population) were reduced in 12/15LOX-/- aging mice than WT controls. At the molecular level, analyses of cytokines in the heart and spleen suggest amplified IFN-γ, with reduced COX-1, COX-2, and ALOX5 expression in the absence of 12/15LOX-derived mediators in the spleen. Thus, aging of 12/15LOX-/- mice increased spleen mass and altered spleen and heart structure with activation of multiple molecular and cellular pathways contributing to age-related integrative and inter-organ inflammation.

ROS-responsive nanoparticle-mediated delivery of CYP2J2 gene for therapeutic angiogenesis in severe hindlimb ischemia.

With critical limb ischemia (CLI) being a multi-factorial disease, it is becoming evident that gene therapy with a multiple bio-functional growth factor could achieve better therapeutic outcomes. Cytochrome P450 epoxygenase-2J2 (CYP2J2) and its catalytic products epoxyeicosatrienoic acids (EETs) exhibit pleiotropic biological activities, including pro-angiogenic, anti-inflammatory and cardiovascular protective effects, which are considerably beneficial for reversing ischemia and restoring local blood flow in CLI. Here, we designed a nanoparticle-based pcDNA3.1-CYP2J2 plasmid DNA (pDNA) delivery system (nanoparticle/pDNA complex) composed of a novel three-arm star block copolymer (3S-PLGA-po-PEG), which was achieved by conjugating three-armed PLGA to PEG via the peroxalate ester bond. Considering the multiple bio-functions of CYP2J2-EETs and the sensitivity of the peroxalate ester bond to H2O2, this nanoparticle-based gene delivery system is expected to exhibit excellent pro-angiogenic effects while improving the high oxidative stress and inflammatory micro-environment in ischemic hindlimb. Our study reports the first application of CYP2J2 in the field of therapeutic angiogenesis for CLI treatment and our findings demonstrated good biocompatibility, stability and sustained release properties of the CYP2J2 nano-delivery system. In addition, this nanoparticle-based gene delivery system showed high transfection efficiency and efficient VEGF expression in vitro and in vivo. Intramuscular injection of nanoparticle/pDNA complexes into mice with hindlimb ischemia resulted in significant rapid blood flow recovery and improved muscle repair compared to mice treated with naked pDNA. In summary, 3S-PLGA-po-PEG/CYP2J2-pDNA complexes have tremendous potential and provide a practical strategy for the treatment of limb ischemia. Moreover, 3S-PLGA-po-PEG nanoparticles might be useful as a potential non-viral carrier for other gene delivery applications.

2-(Piperidin-4-yl)acetamides as Potent Inhibitors of Soluble Epoxide Hydrolase with Anti-Inflammatory Activity.

The pharmacological inhibition of soluble epoxide hydrolase (sEH) has been suggested as a potential therapy for the treatment of pain and inflammatory diseases through the stabilization of endogenous epoxyeicosatrienoic acids. Numerous potent sEH inhibitors (sEHI) have been developed, however many contain highly lipophilic substituents limiting their availability. Recently, a new series of benzohomoadamantane-based ureas endowed with potent inhibitory activity for the human and murine sEH was reported. However, their very low microsomal stability prevented further development. Herein, a new series of benzohomoadamantane-based amides were synthetized, fully characterized, and evaluated as sEHI. Most of these amides were endowed with excellent inhibitory potencies. A selected compound displayed anti-inflammatory effects with higher effectiveness than the reference sEHI, TPPU.

The soluble epoxide hydrolase inhibitor GSK2256294 decreases the proportion of adipose pro-inflammatory T cells

Adipose tissue contains a complex immune environment and is a central contributor to heightened systemic inflammation in obese persons. Epoxyeicosatrienoic acids (EETs) are lipid signaling molecules that decrease inflammation in obese animals, but their effect on inflammation in humans is unknown. The enzyme soluble epoxide hydrolase (sEH) hydrolyzes EETs to less active diols, and we hypothesized that pharmacologic sEH inhibition would decrease adipose inflammation in obese individuals. We treated obese prediabetic adults with the sEH inhibitor GSK2256294 versus placebo in a crossover design, collected subcutaneous abdominal adipose tissue via lipoaspiration and characterized the tissue T cell profile. Treatment with GSK2256294 decreased the percentage of pro-inflammatory T cells producing interferon-gamma (IFNγ), but not interleukin (IL)-17A, and decreased the amount of secreted tumor necrosis factor-alpha (TNFα). Understanding the contribution of the EET/sEH pathway to inflammation in obesity could lead to new strategies to modulate adipose and systemic inflammation.

Mesquite pod (Prosopis juliflora) meal on meat quality of pasture-finishing lambs.

This study was carried out to validate the inclusion of up to 750g/kg of mesquite pod (Prosopis juliflora) meal in the diet and evaluate the effects on carcass characteristics and meat quality for lambs finished in pasture. Forty male, non-castrated, crossbred Santa Inês lambs, with an initial body weight (24.2 ± 3.1kg), and approximately 120days old were used. The animals were kept in a total area of 4ha, divided in four paddocks of 0.62ha each (10 animals/paddocks), on pastures of Massai (Panicum maximum cv. Massai) with drinkers and feeders during the finishing phase. Dietary treatments based on mesquite pod meal inclusion levels (g/kg of dry matter): CON, without mesquite pod meal; MPM25, 250g/kg of mesquite pod meal; MPM50, 500g/kg of mesquite pod meal; and MPM75, 750g/kg of mesquite pod meal. No treatment effect were detected (P > 0.05) for carcass measures, carcass characteristics, chemical composition of longissimus thoracis muscle, tissue composition, and lipid oxidation. Lamb meat color values, such as lightness (L*) and yellowness (b*), were not affected (P > 0.05) by mesquite pod meal inclusion on the diets, whereas for redness (a*), HUE-angle, and chroma were influenced (P < 0.05). Palmitic acid had a quadratic effect, while oleic acid, eicosatrienoic acid, saturated fatty acids, monounsaturated fatty acids, and PUFA:SFA had a linear course (P < 0.05). In conclusion, the mesquite pod meal can be used as an energy feed source up to 750g/kg of dry matter in the diet, without changing the carcass characteristics and meat quality of lambs finished in pasture.

Dual use of a local Protosiphon isolate BENHA2020 for biodiesel production and antioxidant activity of lipid-free biomass: A novel biorefinery approach for biomass valorization

The present study aimed to isolate and evaluate a local Protosiphon sp. for biodiesel production and antioxidant activity of its residual lipid-free biomass. Phylogenetic analysis confirmed that the isolate is Protosiphon botryoides. In the control medium using 250 mg L−1 NaNO3 as a nitrogen source, cells showed 37.7 dw% lipid content at stationary phase. At different nitrogen levels, increasing of nitrogen concentration resulted in obvious increase in growth, showing the maximum biomass productivity of 0.133 g L−1 d−1 at middle exponential phase using 750 mg L−1 NaNO3, with simultaneous reduction in lipid content, reaching its lowest recorded value of 27.9 dw%. Due to enhancement of lipid content of N-deficient culture (125 mg L−1 NaNO3), it showed the highest lipid productivity of 41.3 mg L−1 d−1. Fatty acid analysis confirmed the dominance of linoleic acid, eicosatrienoic acid, oleic acid, and palmitic acid. Biodiesel characteristics complied with the international standards and showed better properties than some other reported biodiesel promising microalgae. In addition, lipid-free biomass showed high radical scavenging activity of 55.3%, which confirms its suitability as additional nutritional and pharmaceutical material for enhanced biomass valorization.

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.

Low-dose angiotensin II supplementation restores flow-induced dilation mechanisms in cerebral arteries of Sprague-Dawley rats on a high salt diet.

Salt-induced suppression of angiotensin II contributes to impaired endothelium-dependent vascular reactivity. The present study investigated the effect of chronic low-dose angiotensin II (ANG II) supplementation on the mechanisms of flow-induced dilation (FID) and oxidative stress at the cellular and molecular level in middle cerebral arteries (MCA) of male Sprague-Dawley rats fed high salt diet. Rats (10 weeks old) were randomly assigned to a low salt diet group (0.4% NaCl in rat chow); high salt diet group (7 days 4% NaCl in rat chow) or HS+ANG II group [7 days high salt diet with 3 days ANG II administration via osmotic minipumps (100 ng/kg per min on days 4-7)]. FID was determined in absence/presence of the NOS inhibitor L-NAME, the non-selective cyclooxygenase (COX-1,2) inhibitor indomethacin, a selective inhibitor of CYP450 epoxygenase activity (MS-PPOH) and the superoxide dismutase mimetic TEMPOL. Gene expression of antioxidative enzymes, and of genes and proteins involved in FID mechanisms were determined by RT-qPCR and western blot. Vascular nitric oxide and superoxide/reactive oxygen species levels were assessed by direct fluorescence. Serum systemic oxidative stress parameters were measured by spectrophotometry. Chronic low-dose ANG II supplementation in high salt fed rats restored FID of MCAs, which was nitric oxide, prostanoid and epoxyeicosatrienoic acid dependent. ANG II changed the protein/gene expression of COXs, HIF-1α and VEGF and significantly increased GPx4 and EC-SOD antioxidative enzyme expression, decreased systemic oxidative stress, decreased superoxide/ROS levels and increased nitric oxide bioavailability in the vascular wall. Physiological levels of circulating ANG II are crucial to maintain the HIF-1α dependent mechanisms of FID and vascular oxidative balance without affecting mean arterial pressure.

New Alkoxy- Analogues of Epoxyeicosatrienoic Acids Attenuate Cisplatin Nephrotoxicity In Vitro via Reduction of Mitochondrial Dysfunction, Oxidative Stress, Mitogen-Activated Protein Kinase Signaling, and Caspase Activation.

The usage of cisplatin, a highly potent chemotherapeutic, is limited by its severe nephrotoxicity. Arachidonic acid (ARA)-derived epoxyeicosatrienoic acids (EETs) and soluble epoxide hydrolase (sEH) inhibitors were shown to ameliorate this dose-limiting side effect, but both approaches have some pharmacological limitations. Analogues of EETs are an alternative avenue with unique benefits, but the current series of analogues face concerns regarding their structure and mimetic functionality. Hence, in this study, regioisomeric mixtures of four new ARA alkyl ethers were synthesized, characterized, and assessed as EET analogues against the concentration- and time-dependent toxicities of cisplatin in porcine proximal tubular epithelial cells. All four ether groups displayed bioisostere activity, ranging from marginal for methoxy- (1), good for n-propoxy- (4), and excellent for ethoxy- (2) and i-propoxy- (3). Compounds 2 and 3 displayed cytoprotective effects comparable to that of an EET regioisomeric mixture (5) against high, acute cisplatin exposures but were more potent against low to moderate, chronic exposures. Compounds 2 and 3 (and 5) acted through stabilization of the mitochondrial transmembrane potential and attenuation of reactive oxygen species, leading to reduced phosphorylation of mitogen-activated protein kinases p38 and JNK and decreased activation of caspase-9 and caspase-3. This study demonstrates that alkoxy- groups are potent and more metabolically stable bioisostere alternatives to the epoxide within EETs that enable sEH-independent activity. It also illustrates the potential of ether-based mimics of EETs and other epoxy fatty acids as promising nephroprotective agents to tackle the clinically relevant side effect of cisplatin without compromising its antineoplastic function.

Differential contribution of renal cytochrome P450 enzymes to kidney endothelial dysfunction and vascular oxidative stress in obesity

Arachidonic acid (AA)-derived cytochrome P450 (CYP) derivatives, epoxyeicosatrienoic acids (EETs) and 20-hidroxyeicosatetranoic acid (20-HETE), play a key role in kidney tubular and vascular functions and blood pressure. Altered metabolism of CYP epoxygenases and CYP hydroxylases has differentially been involved in the pathogenesis of metabolic disease-associated vascular complications, although the mechanisms responsible for the vascular injury are unclear. The present study aimed to assess whether obesity-induced changes in CYP enzymes may contribute to oxidative stress and endothelial dysfunction in kidney preglomerular arteries. Endothelial function and reactive oxygen species (ROS) production were assessed in interlobar arteries of obese Zucker rats (OZR) and their lean counterparts lean Zucker rats (LZR) and the effects of CYP2C and CYP4A inhibitors sulfaphenazole and HET0016, respectively, were examined on the endothelium-dependent relaxations and O2− and H2O2 levels of preglomerular arteries. Non-nitric oxide (NO) non-prostanoid endothelium-derived hyperpolarization (EDH)-type responses were preserved but resistant to the CYP epoxygenase blocker sulfaphenazole in OZR in contrast to those in LZR. Sulfaphenazole did not further inhibit reduced arterial H2O2 levels, and CYP2C11/CYP2C23 enzymes were downregulated in intrarenal arteries from OZR. Renal EDH-mediated relaxations were preserved in obese rats by the enhanced activity and expression of endothelial calcium-activated potassium channels (KCa). CYP4A blockade restored impaired NO-mediated dilatation and inhibited augmented O2− production in kidney arteries from OZR. The current data demonstrate that both decreased endothelial CYP2C11/ CYP2C23-derived vasodilator H2O2 and augmented CYP4A-derived 20-HETE contribute to endothelial dysfunction and vascular oxidative stress in obesity. CYP4A inhibitors ameliorate arterial oxidative stress and restore endothelial function which suggests its therapeutic potential for the vascular complications of obesity-associated kidney injury.