DGLA from the Microalga Lobosphaera Incsa P127 Modulates Inflammatory Response, Inhibits iNOS Expression and Alleviates NO Secretion in RAW264.7 Murine Macrophages.

Microalgae have been considered as a renewable source of nutritional, cosmetic and pharmaceutical compounds. The ability to produce health-beneficial long-chain polyunsaturated fatty acids (LC-PUFA) is of high interest. LC-PUFA and their metabolic lipid mediators, modulate key inflammatory pathways in numerous models. In particular, the metabolism of arachidonic acid under inflammatory challenge influences the immune reactivity of macrophages. However, less is known about another omega-6 LC-PUFA, dihomo-γ-linolenic acid (DGLA), which exhibits potent anti-inflammatory activities, which contrast with its delta-5 desaturase product, arachidonic acid (ARA). In this work, we examined whether administrating DGLA would modulate the inflammatory response in the RAW264.7 murine macrophage cell line. DGLA was applied for 24 h in the forms of carboxylic (free) acid, ethyl ester, and ethyl esters obtained from the DGLA-accumulating delta-5 desaturase mutant strain P127 of the green microalga Lobosphaera incisa. DGLA induced a dose-dependent increase in the RAW264.7 cells' basal secretion of the prostaglandin PGE1. Upon bacterial lipopolysaccharide (LPS) stimuli, the enhanced production of pro-inflammatory cytokines, tumor necrosis factor alpha (TNFα) and interleukin 1ß (IL-1ß), was affected little by DGLA, while interleukin 6 (IL-6), nitric oxide, and total reactive oxygen species (ROS) decreased significantly. DGLA administered at 100 µM in all forms attenuated the LPS-induced expression of the key inflammatory genes in a concerted manner, in particular iNOS, IL-6, and LxR, in the form of free acid. PGE1 was the major prostaglandin detected in DGLA-supplemented culture supernatants, whose production prevailed over ARA-derived PGE2 and PGD2, which were less affected by LPS-stimulation compared with the vehicle control. An overall pattern of change indicated DGLA's induced alleviation of the inflammatory state. Finally, our results indicate that microalgae-derived, DGLA-enriched ethyl esters (30%) exhibited similar activities to DGLA ethyl esters, strengthening the potential of this microalga as a potent source of this rare anti-inflammatory fatty acid.

Oral administration of whole dihomo-γ-linolenic acid-producing yeast suppresses allergic contact dermatitis in mice.

Dihomo-γ-linolenic acid (DGLA, C20: 3n-6) is known to have an anti-inflammatory activity, but its range of effects was not well studied because of its limited natural sources. We addressed these issues by constructing an yeast Saccharomyces cerevisiae strain having a complete metabolic pathway for DGLA synthesis by introducing two desaturase and one elongase genes to convert endogenous oleic acid to DGLA. Taking advantage of well-known safety of S. cerevisiae, we previously investigated the efficacy of heat-killed whole DGLA-producing yeast cells on irritant contact dermatitis, and showed that oral intake of this yeast significantly suppressed inflammatory reactions, whereas no such suppression was observed by the intake of 25 times the amount of purified DGLA. Since this method is considered to be a simple and efficient way to suppress inflammation, we examined its effectiveness against allergic contact dermatitis (ACD) in this study and showed that this method was also effective against ACD.

A synthetic epoxyeicosatrienoic acid analogue prevents the initiation of ischemic acute kidney injury.

AIM: Imbalances in cytochrome P450 (CYP)-dependent eicosanoid formation may play a central role in ischemic acute kidney injury (AKI). We reported previously that inhibition of 20-hydroxyeicosatetraenoic acid (20-HETE) action ameliorated ischemia/reperfusion (I/R)-induced AKI in rats. Now we tested the hypothesis that enhancement of epoxyeicosatrienoic acid (EET) actions may counteract the detrimental effects of 20-HETE and prevent the initiation of AKI. METHODS: Male Lewis rats underwent right nephrectomy and ischemia was induced by 45 min clamping of the left renal pedicle followed by up to 48 h of reperfusion. Circulating CYP-eicosanoid profiles were compared in patients who underwent cardiac surgery with (n = 21) and without (n = 38) developing postoperative AKI. RESULTS: Ischemia induced an about eightfold increase of renal 20-HETE levels, whereas free EETs were not accumulated. To compensate for this imbalance, a synthetic 14,15-EET analogue was administered by intrarenal infusion before ischemia. The EET analogue improved renal reoxygenation as monitored by in vivo parametric MRI during the initial 2 h reperfusion phase. The EET analogue improved PI3K- as well as mTORC2-dependent rephosphorylation of Akt, induced inactivation of GSK-3ß, reduced the development of tubular apoptosis and attenuated inflammatory cell infiltration. The EET analogue also significantly alleviated the I/R-induced drop in creatinine clearance. Patients developing postoperative AKI featured increased preoperative 20-HETE and 8,9-EET levels. CONCLUSIONS: Pharmacological interventions targeting the CYP-eicosanoid pathway could offer promising new options for AKI prevention. Individual differences in CYP-eicosanoid formation may contribute to the risk of developing AKI in clinical settings.

A liposome-based formulation containing equol, dihomo-γ-linolenic acid and propionyl-l-carnitine to prevent and treat hair loss: A prospective investigation.

Hair loss is a common aesthetic disorder that can be triggered by genetic, inflammatory, hormonal, and environmental factors acting on hair follicles and their life cycle. There are several types of hair loss that differ in causes, symptoms, and spatial and temporal progression. Androgenic alopecia, a common form of hair loss, is the consequence of a decreased microcirculation of the scalp as well as the toxic action of elevated dihydrotestosterone levels on the hair bulbs. In the present study, the lotions TRINOV Lozione Anticaduta Uomo and TRINOV Lozione Anticaduta Donna, containing dihomo-γ-linolenic acid (DGLA), S-equol, and propionyl-l-carnitine, were tested on 30 men and 30 women (mean age of men was 46.6 ± 6.4 years; mean age of women was 49.5 ± 9.0) with signs of androgenic alopecia, respectively. DGLA is a precursor of the prostaglandin PGE1, which acts by improving microcirculation; S-equol inhibits 5α-reductases, thus preventing the transformation of testosterone into dihydrotestosterone; and propionyl-l-carnitine promotes lipid metabolism, stimulating energy production. These three molecules are loaded into liposomes for their effective transdermal delivery. Daily topical applications of the lotions resulted in a hair count that significantly increased for women and marginally increased for men after 6 months of treatment. Furthermore, significant increase in anagen hair and a significant decrease in telogen hair were observed starting from 3 months in male and 1 month in female patients. Thus, the formulations under investigation were effective in attenuating androgenic alopecia-related hair loss in men and women.

Dihomo-γ-linolenic acid inhibits xenograft tumor growth in mice bearing shRNA-transfected HCA-7 cells targeting delta-5-desaturase.

BACKGROUND: We previously demonstrated that knockdown of delta-5-desaturase via siRNA transfection together with dihomo-γ-linolenic acid supplementation inhibited colon cancer cell growth and migration, by promoting the production of the anti-cancer byproduct 8-hydroxyoctanoic acid from Cyclooxygenase-2-catalyzed dihomo-γ-linolenic acid peroxidation. Here, we extend our study to investigate the effects of delta-5-desaturase-knockdown and the resulting intensified dihomo-γ-linolenic acid peroxidation in xenograft tumor mice model. METHODS: Four-week old nude mice bearing the human colon cancer cell HCA-7/C29 vs. its delta-5-desaturase knockdown analog (via shRNA transfection) were subject to 4-week treatments of: vehicle control, dihomo-γ-linolenic acid supplementation, 5-Fluorouracil, and combination of dihomo-γ-linolenic acid and 5-Fluorouracil. Tumor growth was monitored during the treatment. At the endpoint, the mice were euthanized and the tumor tissues were collected for further mechanism analysis. RESULTS: Delta-5-desaturase knockdown (shRNA) together with dihomo-γ-linolenic acid supplementation increased 8-hydroxyoctanoic acid production to a threshold level in xenograft tumors, which consequently induced p53-dependent apoptosis and reduced tumors significantly. The promoted 8-hydroxyoctanoic acid formation was also found to suppress the tumors' metastatic potential via regulating MMP-2 and E-cadherin expressions. In addition, our in vivo data showed that delta-5-desaturase knockdown along with dihomo-γ-linolenic acid supplementation resulted in anti-tumor effects comparable to those of 5-Fluorouracil. CONCLUSIONS: We have demonstrated that our paradigm-shifting strategy of knocking down delta-5-desaturase and taking advantage of overexpressed Cyclooxygenase-2 in tumor cells can be used for colon cancer suppression. Our research outcome will lead us to develop a better and safer anti-cancer therapy for patients.

Serum n-6 polyunsaturated fatty acids and risk of death: the Kuopio Ischaemic Heart Disease Risk Factor Study.

Background: The cardioprotective properties of linoleic acid (LA), a major n-6 (ω-6) polyunsaturated fatty acid (PUFA), have been recognized, but less is known about its associations with other causes of death. Relatively little is also known about how the minor n-6 PUFAs-γ-linolenic acid (GLA), dihomo-γ-linolenic acid (DGLA), and arachidonic acid (AA)-relate to mortality risk. Objective: We investigated the associations of serum n-6 PUFAs, an objective biomarker of exposure, with risk of death in middle-aged and older men and whether disease history modifies the associations. Design: We included 2480 men from the prospective Kuopio Ischaemic Heart Disease Risk Factor Study, aged 42-60 y at baseline in 1984-1989. The stratified analyses by baseline disease status included 1019 men with a history of cardiovascular disease (CVD), cancer, or diabetes and 1461 men without a history of disease. Results: During the mean follow-up of 22.4 y, 1143 deaths due to disease occurred. Of these, 575 were CVD deaths, 317 were cancer deaths, and 251 were other-cause deaths. A higher serum LA concentration was associated with a lower risk of death from any cause (multivariable-adjusted HR for the highest compared with the lowest quintile: 0.57; 95% CI: 0.46, 0.71; P-trend < 0.001) and with deaths due to CVD (extreme-quintile HR: 0.54; 95% CI: 0.40, 0.74; P-trend < 0.001) and non-CVD or noncancer causes (HR: 0.48; 95% CI: 0.30, 0.76; P-trend = 0.001). Serum AA had similar, although weaker, inverse associations. Serum GLA and DGLA were not associated with risk of death, and none of the fatty acids were associated with cancer mortality. The results were generally similar among those with or without a history of major chronic disease (P-interaction > 0.13). Conclusions: Our findings showed an inverse association of a higher biomarker of LA intake with total and CVD mortality and little concern for risk, thus supporting the current dietary recommendations to increase LA intake for CVD prevention. The finding of an inverse association of serum AA with the risk of death needs replication in other populations.

Soluble Epoxide Hydrolase Inhibitor and 14,15-Epoxyeicosatrienoic Acid-Facilitated Long-Term Potentiation through cAMP and CaMKII in the Hippocampus.

Epoxyeicosatrienoic acids (EETs) are derived from arachidonic acid and metabolized by soluble epoxide hydrolase (sEH). The role of EETs in synaptic function in the central nervous system is still largely unknown. We found that pharmacological inhibition of sEH to stabilize endogenous EETs and exogenous 14,15-EET significantly increased the field excitatory postsynaptic potential (fEPSP) response in the CA1 area of the hippocampus, while additionally enhancing high-frequency stimulation- (HFS-) induced long-term potentiation (LTP) and forskolin- (FSK-) induced LTP. sEH inhibitor (sEHI) N-[1-(oxopropyl)-4-piperidinyl]-N'-[4-(trifluoromethoxy) phenyl)-urea (TPPU) and exogenous 14,15-EET increased HFS-LTP, which could be blocked by an N-methyl-D-aspartate (NMDA) receptor subunit NR2B antagonist. TPPU- or 14,15-EET-facilitated FSK-mediated LTP can be potentiated by an A1 adenosine receptor antagonist and a phosphodiesterase inhibitor, but is prevented by a cAMP-dependent protein kinase (PKA) inhibitor. sEHI and 14,15-EET upregulated the activation of extracellular signal-regulated kinases (ERKs) and Ca2+/calmodulin- (CaM-) dependent protein kinase II (CaMKII). Phosphorylation of synaptic receptors NR2B and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR1 was increased by TPPU and 14,15-EET administration. These results indicated that EETs increased NMDAR- and FSK-mediated synaptic potentiation via the AC-cAMP-PKA signaling cascade and upregulated the ERKs and CaMKII, resulting in increased phosphorylation of NR2B and GluR1 in the hippocampus.

Orally Active Epoxyeicosatrienoic Acid Analogs.

Biologically active epoxyeicosatrienoic acid (EET) regioisomers are synthesized from arachidonic acid by cytochrome P450 epoxygenases of endothelial, myocardial, and renal tubular cells. EETs relax vascular smooth muscle and decrease inflammatory cell adhesion and cytokine release. Renal EETs promote sodium excretion and vasodilation to decrease hypertension. Cardiac EETs reduce infarct size after ischemia-reperfusion injury and decrease fibrosis and inflammation in heart failure. In diabetes, EETs improve insulin sensitivity, increase glucose tolerance, and reduce the renal injury. These actions of EETs emphasize their therapeutic potential. To minimize metabolic inactivation, 14,15-EET agonist analogs with stable epoxide bioisosteres and carboxyl surrogates were developed. In preclinical rat models, a subset of agonist analogs, termed EET-A, EET-B, and EET-C22, are orally active with good pharmacokinetic properties. These orally active EET agonists lower blood pressure and reduce cardiac and renal injury in spontaneous and angiotensin hypertension. Other beneficial cardiovascular actions include improved endothelial function and cardiac antiremodeling actions. In rats, EET analogs effectively combat acute and chronic kidney disease including drug- and radiation-induced kidney damage, hypertension and cardiorenal syndrome kidney damage, and metabolic syndrome and diabetes nephropathy. The compelling preclinical efficacy supports the prospect of advancing EET analogs to human clinical trials for kidney and cardiovascular diseases.

Epoxyeicosanoids prevent intervertebral disc degeneration in vitro and in vivo.

Intervertebral disc (IVD) degeneration is considered a common cause of low back pain. In the degenerating IVD, the production of pro-inflammatory cytokines, including IL-1 and TNF-α, progressively increases, contributing to the degenerative process. Epoxyeicosatrienoic acids (EETs), synthesized from arachidonic acid by cytochrome P450 enzymes, act as autocrine and paracrine effectors in regulating inflammation, cardiovascular functions, and angiogenesis. EETs were shown to be especially potent promoters of tissue regeneration. Considering their anti-inflammatory and anti-catabolic potential, we investigated whether EETs can influence IVD degeneration. We found that 14,15-EET protected rat nucleus pulposus (NP) cells against death induced by treatment with H2O2and TNF-α in vitro. At the molecular level, 14,15-EET significantly inhibited the NF-κB pathway, which plays essential roles in the degeneration and survival of NP cells. As a result, 14,15-EET efficiently prevented the matrix remodeling response of NP cells to TNF-α. Using a needle-punctured rat tail model, the influence of 14,15-EET on IVD degeneration in vivo was evaluated using radiographs, magnetic resonance images (MRI), and histological analysis. We observed that 14,15-EET prevented IVD degeneration. Our findings demonstrated that 14,15-EET can enhance the survival of NP cells and inhibit IVD degeneration. The EET pathway may be a novel therapeutic target against IVD degeneration.

Effect of tailored dietary guidance for pregnant women on nutritional status: A double-cohort study.

This double cohort study aimed to evaluate the effect of tailored dietary guidance for pregnant women on dietary intake, nutritional status, and infant birth weight. Healthy pregnant women were recruited at an antenatal clinic during two phases over 2 years. The historical controls were analyzed a year prior to the intervention group. In both groups, data were collected at 19-26 gestational weeks (baseline) and at 34-37 gestational weeks (outcome measurement). The intervention included the following: (a) assessments of maternal dietary nutritional intake using the brief self-administered diet history questionnaire, (b) individual feedback based on the assessments of maternal nutritional status, (c) tailored guidance for a healthy diet, (d) original cooking recipes, and (e) goal sharing. Mann-Whitney U test was used to compare the outcome data between the groups. Of the 378 eligible women, 309 women had follow-up questionnaire data. Blood samples were obtained from 202 women. Despite a lack of improvement in reported dietary intake, plasma eicosapentaenoic acid (p = .002), docosahexaenoic acid (p < .001), arachidonic acid (p < .001), and dihomo-gamma-linolenic acid (p < .001) concentrations as well as maternal weight gain (p = .019) were significantly higher in the intervention group. However, serum folate (p = .031) concentration was significantly lower in the intervention group, and there were no significant differences between the groups in 25-hydroxy vitamin D levels, blood count, average birth weight, and rate of low birth weight infants. Assessment-based tailored guidance individualized to maternal dietary intake might partially contribute to improved nutrition in pregnant women.

Epoxygenated Fatty Acids Inhibit Retinal Vascular Inflammation.

The objective of the present study was to assess the effect of elevating epoxygenated fatty acids on retinal vascular inflammation. To stimulate inflammation we utilized TNFα, a potent pro-inflammatory mediator that is elevated in the serum and vitreous of diabetic patients. In TNFα-stimulated primary human retinal microvascular endothelial cells, total levels of epoxyeicosatrienoic acids (EETs), but not epoxydocosapentaenoic acids (EDPs), were significantly decreased. Exogenous addition of 11,12-EET or 19,20-EDP when combined with 12-(3-adamantane-1-yl-ureido)-dodecanoic acid (AUDA), an inhibitor of epoxide hydrolysis, inhibited VCAM-1 and ICAM-1 expression and protein levels; conversely the diol product of 19,20-EDP hydrolysis, 19,20-DHDP, induced VCAM1 and ICAM1 expression. 11,12-EET and 19,20-EDP also inhibited leukocyte adherence to human retinal microvascular endothelial cell monolayers and leukostasis in an acute mouse model of retinal inflammation. Our results indicate that this inhibition may be mediated through an indirect effect on NFκB activation. This is the first study demonstrating a direct comparison of EET and EDP on vascular inflammatory endpoints, and we have confirmed a comparable efficacy from each isomer, suggesting a similar mechanism of action. Taken together, these data establish that epoxygenated fatty acid elevation will inhibit early pathology related to TNFα-induced inflammation in retinal vascular diseases.

CYP2J2 and its metabolites (epoxyeicosatrienoic acids) attenuate cardiac hypertrophy by activating AMPKα2 and enhancing nuclear translocation of Akt1.

Cytochrome P450 epoyxgenase 2J2 and epoxyeicosatrienoic acids (EETs) are known to protect against cardiac hypertrophy and heart failure, which involve the activation of 5'-AMP-activated protein kinase (AMPK) and Akt. Although the functional roles of AMPK and Akt are well established, the significance of cross talk between them in the development of cardiac hypertrophy and antihypertrophy of CYP2J2 and EETs remains unclear. We investigated whether CYP2J2 and its metabolites EETs protected against cardiac hypertrophy by activating AMPKα2 and Akt1. Moreover, we tested whether EETs enhanced cross talk between AMPKα2 and phosphorylated Akt1 (p-Akt1), and stimulated nuclear translocation of p-Akt1, to exert their antihypertrophic effects. AMPKα2(-/-) mice that overexpressed CYP2J2 in heart were treated with Ang II for 2 weeks. Interestingly, overexpression of CYP2J2 suppressed cardiac hypertrophy and increased levels of atrial natriuretic peptide (ANP) in the heart tissue and plasma of wild-type mice but not AMPKα2(-/-) mice. The CYP2J2 metabolites, 11,12-EET, activated AMPKα2 to induce nuclear translocation of p-Akt1 selectively, which increased the production of ANP and therefore inhibited the development of cardiac hypertrophy. Furthermore, by co-immunoprecipitation analysis, we found that AMPKα2ß2γ1 and p-Akt1 interact through the direct binding of the AMPKγ1 subunit to the Akt1 protein kinase domain. This interaction was enhanced by 11,12-EET. Our studies reveal a novel mechanism in which CYP2J2 and EETs enhanced Akt1 nuclear translocation through interaction with AMPKα2ß2γ1 and protect against cardiac hypertrophy and suggest that overexpression of CYP2J2 might have clinical potential to suppress cardiac hypertrophy and heart failure.

Knockdown of delta-5-desaturase promotes the anti-cancer activity of dihomo-γ-linolenic acid and enhances the efficacy of chemotherapy in colon cancer cells expressing COX-2.

Cyclooxygenase (COX), commonly overexpressed in cancer cells, is a major lipid peroxidizing enzyme that metabolizes polyunsaturated fatty acids (ω-3s and ω-6s). The COX-catalyzed free radical peroxidation of arachidonic acid (ω-6) can produce deleterious metabolites (e.g. 2-series prostaglandins) that are implicated in cancer development. Thus, COX inhibition has been intensively investigated as a complementary therapeutic strategy for cancer. However, our previous study has demonstrated that a free radical-derived byproduct (8-hydroxyoctanoic acid) formed from COX-catalyzed peroxidation of dihomo-γ-linolenic acid (DGLA, the precursor of arachidonic acid) can inhibit colon cancer cell growth. We thus hypothesize that the commonly overexpressed COX in cancer (~90% of colon cancer patients) can be taken advantage to suppress cell growth by knocking down delta-5-desaturase (D5D, a key enzyme that converts DGLA to arachidonic acid). In addition, D5D knockdown along with DGLA supplement may enhance the efficacy of chemotherapeutic drugs. After knocking down D5D in HCA-7 colony 29 cells and HT-29 cells (human colon cancer cell lines with high and low COX levels, respectively), the antitumor activity of DGLA was significantly enhanced along with the formation of a threshold range (~0.5-1.0µM) of 8-hydroxyoctanoic acid. In contrast, DGLA treatment did not inhibit cell growth when D5D was not knocked down and only limited amount of 8-hydroxyoctanoic acid was formed. D5D knockdown along with DGLA treatment also enhanced the cytotoxicities of various chemotherapeutic drugs, including 5-fluorouracil, regorafenib, and irinotecan, potentially through the activation of pro-apoptotic proteins, e.g. p53 and caspase 9. For the first time, we have demonstrated that the overexpressed COX in cancer cells can be utilized in suppressing cancer cell growth. This finding may provide a new option besides COX inhibition to optimize cancer therapy. The outcome of this translational research will guide us to develop a novel ω-6-based diet-care strategy in combination with current chemotherapy for colon cancer prevention and treatment.

Relationship between polyunsaturated fatty acid levels in maternal diets and human milk in the first month post-partum.

BACKGROUND: The present study evaluated the relationship between dietary fatty acid (FA) intakes and human milk FA levels. METHODS: Healthy lactating women (n = 514) from Northern China participated in the study. Dietary intake was assessed with a 24-h dietary recall questionnaire and evaluated using golden key maternal nutrition software (Wincome, Shanghai, China) and China Food Composition 2009. Human milk FA composition was determined by gas chromatography. RESULTS: The maternal daily median intakes of linoleic acid (LA), α-linolenic acid (ALA) and arachidonic acid (AA) were 19.93 g, 3.08 g and 16.33 mg, respectively. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) intakes were below the recommended levels. FA levels in 100 g of human milk were 0.363 g LA, 0.038 g γ-linolenic acid (GLA), 0.052 g dihomo γ-linolenic acid (DGLA), 0.144 g ALA, 0.079 g AA, 0.007 g EPA, 0.018 g docosatetraenoic acid (DTA) and 0.048 g DHA. Multiple linear regression analysis revealed that human milk DGLA levels were negatively correlated with dietary LA intake (ß = -0.223, P = 0.030), and human milk GLA and DTA levels were negatively correlated with dietary ALA intake (ß = -2.189, P = 0.031; ß = -2.252, P = 0.027) after adjusting for possible confounding factors. CONCLUSIONS: The results of the present study suggest the presence of competitive interactions between n-3 fatty acids (ALA) and n-6 fatty acids (GLA and DTA).

EETs Elicit Direct Increases in Pulmonary Arterial Pressure in Mice.

OBJECTIVE: The biological role of epoxyeicosatrienoic acids (EETs) in the regulation of pulmonary circulation is currently under debate. We hypothesized that EETs initiate increases in right ventricular systolic pressure (RVSP) via perhaps, pulmonary vasoconstriction. METHODS: Mice were anesthetized with isoflurane. Three catheters, inserted into the left jugular vein, the left carotid artery, and the right jugular vein, were used for infusing EETs, monitoring blood pressure (BP), and RVSP respectively. BP and RVSP were continuously recorded at basal conditions, in response to administration of 4 regioisomeric EETs (5,6-EET; 8,9-EET; 11,12-EET, and 14,15-EET; 1, 2, 5 and 10 ng/g body weight (BW) for each EET), and during exposure of mice to hypoxia. RESULTS: All 4 EETs initiated dose-dependent increases in RVSP, though reduced BP. 11,12-EET elicited the greatest increment in RVSP among all EET isoforms. To clarify the direct elevation of RVSP in a systemic BP-independent manner, equivalent amounts of 14,15-EET were injected over 1 and 2 minutes respectively. One-minute injection of 14,15-EET elicited significantly faster and greater increases in RVSP than the 2-minute injection, whereas their BP changes were comparable. Additionally, direct injection of low doses of 14,15-EET (0.1, 0.2, 0.5, and 1 ng/g BW) into the right ventricle caused significant increases in RVSP without effects on BP, confirming that systemic vasodilation-induced increases in venous return are not the main cause for the increased RVSP. Acute exposure of mice to hypoxia significantly elevated RVSP, as well as 14,15-EET-induced increases in RVSP. CONCLUSIONS: EETs directly elevate RVSP, a response that may play an important role in the development of hypoxia-induced pulmonary hypertension (PH).

Epoxides Derived from Dietary Dihomo-Gamma-Linolenic Acid Induce Germ Cell Death in C. elegans.

Dietary fats are not created equally, slight differences in structure lead to crucial differences in function. Muticellular organisms use polyunsaturated fatty acid as substrates to produce potent signaling molecules crucial for many physiological processes, including reproduction. Here we explored the mechanism responsible for germ cell loss induced by dietary supplementation of dihomo-gamma-linolenic acid (DGLA, 20:3n-6) in the roundworm Caenorhabditis elegans. In this study we found that C. elegans CYP-33E2 activity produces a range of epoxy and hydroxy metabolites from dietary DGLA. Knockdown of cyp-33E2 suppressed the DGLA-induced sterility phenotype. Additionally, direct exposure of two specific DGLA-derived epoxy products, 8,9- and 14,15-epoxyeicosadienoic acids, produced germ cell abnormalities in the C. elegans gonad. We propose that sterility is mediated by the production of toxic DGLA-derived epoxides that trigger germ cell destruction. These studies are the first to establish a biological activity for a CYP-produced metabolite of DGLA.

Orally active epoxyeicosatrienoic acid analog does not exhibit antihypertensive and reno- or cardioprotective actions in two-kidney, one-clip Goldblatt hypertensive rats.

This study examined the effects of a novel orally active 14,15-epoxyeicosatrienoic acid analog (EET-A) on blood pressure (BP) and myocardial infarct size (IS) in two-kidney, one-clip (2K1C) Goldblatt hypertensive rats during sustained phase of hypertension. Between days 31 and 35 after clip placement the rats were treated with EET-A and BP was monitored by radiotelemetry; sham-operated normotensive rats were used as controls. Tissue concentrations of epoxyeicosatrienoic acids served as a marker of production of epoxygenase metabolites. The rats were subjected to acute myocardial ischemia/reperfusion (I/R) injury and IS was determined. We found that EET-A treatment did not lower BP in 2K1C rats and did not alter availability of biologically active epoxygenase metabolites in 2K1C or in sham-operated rats. The myocardial IS was significantly smaller in untreated 2K1C rats as compared with normotensive controls and EET-A reduced it in controls but not in 2K1C rats. Our findings suggest that during the phase of sustained hypertension 2K1C Goldblatt hypertensive rats exhibit increased cardiac tolerance to I/R injury as compared with normotensive controls, and that in this animal model of human renovascular hypertension short-term treatment with EET-A does not induce any antihypertensive and cardioprotective actions.

Epoxyeicosatrienoic acids regulate macrophage polarization and prevent LPS-induced cardiac dysfunction.

Macrophages, owning tremendous phenotypic plasticity and diverse functions, were becoming the target cells in various inflammatory, metabolic and immune diseases. Cytochrome P450 epoxygenase 2J2 (CYP2J2) metabolizes arachidonic acid to form epoxyeicosatrienoic acids (EETs), which possess various beneficial effects on cardiovascular system. In the present study, we evaluated the effects of EETs treatment on macrophage polarization and recombinant adeno-associated virus (rAAV)-mediated CYP2J2 expression on lipopolysaccharide (LPS)-induced cardiac dysfunction, and sought to investigate the underlying mechanisms. In vitro studies showed that EETs (1µmol/L) significantly inhibited LPS-induced M1 macrophage polarization and diminished the proinflammatory cytokines at transcriptional and post-transcriptional level; meanwhile it preserved M2 macrophage related molecules expression and upregulated anti-inflammatory cytokine IL-10. Furthermore, EETs down-regulated NF-κB activation and up-regulated peroxisome proliferator-activated receptors (PPARα/γ) and heme oxygenase 1 (HO-1) expression, which play important roles in regulating M1 and M2 polarization. In addition, LPS treatment in mice induced cardiac dysfunction, heart tissue damage and infiltration of M1 macrophages, as well as the increase of inflammatory cytokines in serum and heart tissue, but rAAV-mediated CYP2J2 expression increased EETs generation in heart and significantly attenuated the LPS-induced harmful effects, which mechanisms were similar as the in vitro study. Taken together, the results indicate that CYP2J2/EETs regulates macrophage polarization by attenuating NF-κB signaling pathway via PPARα/γ and HO-1 activation and its potential use in treatment of inflammatory diseases.

Novel orally active epoxyeicosatrienoic acid (EET) analogs attenuate cisplatin nephrotoxicity.

Nephrotoxicity severely limits the use of the anticancer drug cisplatin. Oxidative stress, inflammation, and endoplasmic reticulum (ER) stress contribute to cisplatin-induced nephrotoxicity. We developed novel orally active epoxyeicosatrienoic acid (EET) analogs and investigated their prophylactic effect in cisplatin-induced nephrotoxicity in rats. Cisplatin-induced nephrotoxicity was manifested by increases in blood urea nitrogen, plasma creatinine, urinary N-acetyl-ß-(d)-glucosaminidase activity, kidney injury molecule 1, and histopathology. EET analogs (10 mg/kg/d) attenuated cisplatin-induced nephrotoxicity by reducing these renal injury markers by 40-80% along with a 50-70% reduction in renal tubular cast formation. This attenuated renal injury is associated with reduced oxidative stress, inflammation, and ER stress evident from reduction in related biomarkers and in the renal expression of genes involved in these pathways. Moreover, we demonstrated that the attenuated nephrotoxicity correlated with decreased apoptosis that is associated with 50-90% reduction in Bcl-2 protein family mediated proapoptotic signaling, reduced renal caspase-12 expression, and a 50% reduction in renal caspase-3 activity. We further demonstrated in vitro that the protective activity of EET analogs does not compromise the anticancer effects of cisplatin. Collectively, our data provide evidence that EET analogs attenuate cisplatin-induced nephrotoxicity by reducing oxidative stress, inflammation, ER stress, and apoptosis without affecting the chemotherapeutic effects of cisplatin.

Relationship between bradykinin-induced relaxation and endogenous epoxyeicosanoid synthesis in human bronchi.

Epoxyeicosanoids (EETs) are produced by cytochrome P-450 epoxygenase; however, it is not yet known what triggers their endogenous production in epithelial cells. The relaxing effects of bradykinin are known to be related to endogenous production of epithelial-derived hyperpolarizing factors (EpDHF). Because of their effects on membrane potential, EETs have been reported to be EpDHF candidates (Benoit C, Renaudon B, Salvail D, Rousseau E. Am J Physiol Lung Cell Mol Physiol 280: L965-L973, 2001.). Thus, we hypothesized that bradykinin (BK) may stimulate endogenous EET production in human bronchi. To test this hypothesis, the relaxing and hyperpolarizing effects of BK and 14,15-EET were quantified on human bronchi, as well as the effects of various enzymatic inhibitors on these actions. One micromolar BK or 1 µM 14,15-EET induced a 45% relaxation on the tension induced by 30 nM U-46619 [a thromboxane-prostanoid (TP)-receptor agonist]. These BK-relaxing effects were reduced by 42% upon addition of 10 nM iberiotoxin [a large-conductance Ca(2+)-sensitive K(+) (BK(Ca)) channel blocker], by 27% following addition of 3 µM 14,15-epoxyeicosa-5(Z)-enoic acid (an EET antagonist), and by 32% with 3 µM N-methanesulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH, an epoxygenase inhibitor). Hence, BK and 14,15-EET display net hyperpolarizing effects on airway smooth muscle cells that are related to the activation of BK(Ca) channels and ultimately yielding to relaxation. Data also indicate that 3 µM MS-PPOH reduced the hyperpolarizing effects of BK by 43%. Together, the present data support the current hypothesis suggesting a direct relationship between BK and the production of EET regioisomers. Because of its potent anti-inflammatory and relaxing properties, epoxyeicosanoid signaling may represent a promising target in asthma and chronic obstructive pulmonary disease.