12-HETE Research Articles

Polyunsaturated Fatty Acid-Derived Lipid Mediators as Potential Biomarkers for Leprosy Among Individuals with Asymptomatic Mycobacterium leprae Infection.

Intra-household contacts (HCs) of leprosy patients are at increased risk of infection by Mycobacterium leprae and about ∼5-10% will develop active disease. A prognostic tool to identify HCs with the greatest risk of progressing to active disease would enhance early leprosy diagnosis and optimize prophylactic intervention. Previous metabolomics studies suggest that host lipid mediators derived from ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) are potential biomarkers for leprosy. In this study, we investigated retrospective sera of leprosy HCs by liquid chromatography-mass spectrometry and enzyme-linked immunoassay to determine whether circulating levels of ω-3 and ω-6 PUFA metabolites were altered in HCs that developed leprosy (HCDL) in comparison to those that did not (HCNDL). Sera were collected from HCs at the time of index case diagnosis and before clinical signs/symptoms of leprosy. Our findings showed that HCDL sera exhibited a distinct metabolic profile in comparison to HCDNL. Specifically, arachidonic acid, leukotriene B4, 11-hydroxyeicosatetraenoic acid, prostaglandin D2, and lipoxin A4 were elevated in HCDL. In contrast, prostaglandin E2 levels were reduced in HCDL. The ω-3 PUFAs, docosahexaenoic acid, eicosapentaenoic acid, and the docosahexaenoic acid-derived resolvin D1 and maresin-1 were also elevated in HCDL individuals compared to HCNDL. Principal component analyses provided further evidence that lipid mediators could serve as an early biomarker for progression to active leprosy. A logistic model identified resolvin D1 and D2, and prostaglandin D2 as having the greatest potential for early detection of HCs that will manifest leprosy.

Paradoxical Induction of ALOX15/15B by Cortisol in Human Amnion Fibroblasts: Implications for Inflammatory Responses of the Fetal Membranes at Parturition

Inflammation of the fetal membranes is an indispensable event of parturition, with increasing prostaglandin E2 (PGE2) synthesis as one of the ultimate products that prime labor onset. In addition to PGE2, the fetal membranes also boast a large capacity for cortisol regeneration. It is intriguing how increased PGE2 synthesis is achieved in the presence of increasing amounts of classical anti-inflammatory glucocorticoids in the fetal membranes at parturition. 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) synthesized by lipoxygenase 15/15B (ALOX15/15B) has been shown to enhance inflammation-induced PGE2 synthesis in amnion fibroblasts. Here, we examined whether glucocorticoids could induce ALOX15/15B expression and 15(S)-HETE production to promote PGE2 synthesis in amnion fibroblasts at parturition. We found that cortisol and 15(S)-HETE abundance increased parallelly in the amnion at parturition. Cortisol induced ALOX15/15B expression and 15(S)-HETE production paradoxically in amnion fibroblasts. Mechanism study revealed that this paradoxical induction was mediated by p300-mediated histone acetylation and interaction of glucocorticoid receptor with transcription factors CREB and STAT3. Conclusively, cortisol regenerated in the fetal membranes can paradoxically induce ALOX15/15B expression and 15(S)-HETE production in human amnion fibroblasts, which may further assist in the induction of PGE2 synthesis in the inflammatory responses of the fetal membranes for parturition.

CES1 Releases Oxylipins from Oxidized Triacylglycerol (oxTAG) and Regulates Macrophage oxTAG/TAG Accumulation and PGE2/IL-1β Production.

Triacylglycerols (TAGs) are storage forms of fat, primarily found in cytoplasmic lipid droplets in cells. TAGs are broken down to their component free fatty acids by lipolytic enzymes when fuel reserves are required. However, polyunsaturated fatty acid (PUFA)-containing TAGs are susceptible to nonenzymatic oxidation reactions, leading to the formation of oxylipins that are esterified to the glycerol backbone (termed oxTAGs). Human carboxylesterase 1 (CES1) is a member of the serine hydrolase superfamily and defined by its ability to catalyze the hydrolysis of carboxyl ester bonds in both toxicants and lipids. CES1 is a bona fide TAG hydrolase, but it is unclear which specific fatty acids are preferentially released during lipolysis. To better understand the biochemical function of CES1 in immune cells, such as macrophages, its substrate selectivity when it encounters oxidized PUFAs in TAG lipid droplets requires study. We sought to identify those esterified oxidized fatty acids liberated from oxTAGs by CES1 because their release can activate signaling pathways that enforce the development of lipid-driven inflammation. Gaining this knowledge will help fill data gaps that exist between CES1 and the lipid-sensing nuclear receptors, PPARγ and LXRα, which are important drivers of lipid metabolism and inflammation in macrophages. Oxidized forms of triarachidonoylglycerol (oxTAG20:4) or trilinoleoylglycerol (oxTAG18:2), which contain physiologically relevant levels of oxidized PUFAs (<5 mol %), were incubated with recombinant CES1 to release oxylipins and nonoxidized arachidonic acid (AA) or linoleic acid (LA). CES1 hydrolyzed each oxTAG, yielding regioisomers of hydroxyeicosatetraenoic acids (5-, 11-, 12-, and 15-HETE) and hydroxyoctadecadienoic acids (9- and 13-HODE). Furthermore, human THP-1 macrophages with deficient CES1 levels exhibited a differential response to extracellular stimuli (oxTAGs, lipopolysaccharide, and 15-HETE) as compared to those with normal CES1 levels, including enhanced oxTAG/TAG lipid accumulation and altered cytokine and prostaglandin E2 profiles. This study suggests that CES1 can metabolize oxTAG lipids to release oxylipins and PUFAs, and it further specifies the substrate selectivity of CES1 in the metabolism of bioactive lipid mediators. We suggest that the accumulation of oxTAGs/TAGs within lipid droplets that arise due to CES1 deficiency enforces an inflammatory phenotype in macrophages.

Posttranslational Modification of Human Cytochrome CYP4F11 by 4-Hydroxynonenal Impairs ω-Hydroxylation in Malaria Pigment Hemozoin-Fed Monocytes: The Role in Malaria Immunosuppression.

Malaria is a frequent parasitic infection becomes life threatening due to the disequilibrated immune responses of the host. Avid phagocytosis of malarial pigment hemozoin (HZ) and HZ-containing Plasmodium parasites incapacitates monocyte functions by bioactive lipoperoxidation products 4-hydroxynonenal (4-HNE) and hydroxyeicosatetraenoic acids (HETEs). CYP4F conjugation with 4-HNE is hypothesised to inhibit ω-hydroxylation of 15-HETE, leading to sustained monocyte dysfunction caused by 15-HETE accumulation. A combined immunochemical and mass-spectrometric approach identified 4-HNE-conjugated CYP4F11 in primary human HZ-laden and 4-HNE-treated monocytes. Six distinct 4-HNE-modified amino acid residues were revealed, of which C260 and H261 are localized in the substrate recognition site of CYP4F11. Functional consequences of enzyme modification were investigated on purified human CYP4F11. Palmitic acid, arachidonic acid, 12-HETE, and 15-HETE bound to unconjugated CYP4F11 with apparent dissociation constants of 52, 98, 38, and 73 µM, respectively, while in vitro conjugation with 4-HNE completely blocked substrate binding and enzymatic activity of CYP4F11. Gas chromatographic product profiles confirmed that unmodified CYP4F11 catalysed the ω-hydroxylation while 4-HNE-conjugated CYP4F11 did not. The 15-HETE dose dependently recapitulated the inhibition of the oxidative burst and dendritic cell differentiation by HZ. The inhibition of CYP4F11 by 4-HNE with consequent accumulation of 15-HETE is supposed to be a crucial step in immune suppression in monocytes and immune imbalance in malaria.

Development of UPLC-MS/MS methods for quantitation of glutathione, cysteine, 14,15-epoxyeicosatrienoic acid, and 15-hydroxyeicosatetraenoic acid and application in paraquat poisoning cells

Glutathione (GSH) and cysteine are important antioxidants in maintaining the oxidation-reduction balance. 14,15-epoxyeicosatrienoic acid (14,15-EET) and 15-hydroxyeicosatetraenoic acid (15-HETE) are biological active compounds metabolized from arachidonic acid. To dynamically monitor GSH, cysteine, 14,15-EET, and 15-HETE, an ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method was developed. The derivative GSH, cysteine, and underivatized 14,15-EET, 15-HETE were determined by electrospray ionization source with multiple reaction monitoring. The developed method was well validated and applied to monitor the extracellular concentration of GSH, cysteine, 14,15-EET, and 15-HETE in paraquat (PQ) poisoning LO2 cells. It was shown that the derivation of GSH and cysteine is complete and specific. There was a good linear relationship among GSH, cysteine, 14,15-EET, and 15-HETE. The relative standard deviations of precision and accuracy were all within 15%. After PQ poisoning, the extracellular concentrations of GSH and cysteine were increased at 72 h. 14,15-EET and 15-HETE started to increase at 24 h, earlier than GSH and cysteine. Irreversible pathological alterations appeared at 48 h. In conclusion, a fast and specific UPLC-MS/MS method for the determination of GSH, cysteine, 14,15-EET, and 15-HETE has been developed and validated. PQ induced the increase of 14,15-EET and 15-HETE earlier than that of GSH and cysteine in LO2 cells culture medium.

Association of Arachidonic Acid-Derived Lipid Mediators With Disease Severity in Patients With Relapsing and Progressive Multiple Sclerosis.

Excessive activation of certain lipid mediator (LM) pathways plays a role in the complex pathogenesis of multiple sclerosis (MS). However, the relationship between bioactive LMs and different aspects of CNS-related pathophysiologic processes remains largely unknown. Therefore, in this study, we assessed the association of bioactive LMs belonging to the ω-3/ω-6 lipid classes with clinical and biochemical (serum neurofilament light [sNfL] and serum glial fibrillary acidic protein [sGFAP]) parameters and MRI-based brain volumes in patients with MS (PwMS) and healthy controls (HCs). A targeted high-performance liquid chromatography-tandem mass spectrometry approach was used on plasma samples of PwMS and HCs of the Project Y cohort, a cross-sectional population-based cohort that contains PwMS all born in 1966 in the Netherlands and age-matched HCs. LMs were compared between PwMS and HCs and were correlated with levels of sNfL, sGFAP, disability (Expanded Disability Status Scale [EDSS]), and brain volumes. Finally, significant correlates were included in a backward multivariate regression model to identify which LMs best related to disability. The study sample consisted of 170 patients with relapsing remitting MS (RRMS), 115 patients with progressive MS (PMS), and 125 HCs. LM profiles of patients with PMS significantly differed from those of patients with RRMS and HCs, particularly patients with PMS showed elevated levels of several arachidonic acid (AA) derivatives. In particular, 15-hydroxyeicosatetraenoic acid (HETE) (r = 0.24, p < 0.001) correlated (average r = 0.2, p < 0.05) with clinical and biochemical parameters such as EDSS and sNfL. In addition, higher 15-HETE levels were related to lower total brain (r = -0.24, p = 0.04) and deep gray matter volumes (r = -0.27, p = 0.02) in patients with PMS and higher lesion volume (r = 0.15, p = 0.03) in all PwMS. In PwMS of the same birth year, we show that ω-3 and ω-6 LMs are associated with disability, biochemical parameters (sNfL, GFAP), and MRI measures. Furthermore, our findings indicate that, particularly, in patients with PMS, elevated levels of specific products of the AA pathway, such as 15-HETE, associate with neurodegenerative processes. Our findings highlight the potential relevance of ω-6 LMs in the pathogenesis of MS.

Involvement of p38 MAPK/cPLA2 and arachidonic acid metabolic pathway in Shengmai injection-induced pseudo-allergic reactions.

Adverse reactions to traditional Chinese medicine injections involve pseudo-allergic reactions (PARs). However, in clinical practice, "immediate allergic reactions" and PARs in response to these injections are not often differentiated. This study aimed to clarify the type of reactions produced by Shengmai injections (SMI) and elucidate the possible mechanism. A mouse model was used to evaluate vascular permeability. Metabolomic and arachidonic acid metabolite (AAM) analyses were performed using UPLC-MS/MS, and the p38 MAPK/cPLA2 pathway was detected by western blotting. The first exposure to intravenous SMI rapidly and dose-dependently induced edema and exudative reactions in the ears and lungs. These reactions were not IgE-dependent and were likely to be PARs. Metabolomic analysis showed that endogenous substances were perturbed in SMI-treated mice, in which the arachidonic acid (AA) metabolic pathway was the most affected. SMI substantially increased the levels of AAMs in lung, including prostaglandins (PGs), leukotrienes (LTs), and hydroxy-eicosatetraenoic acids (HETEs). The p38 MAPK/cPLA2 signaling pathway was activated after a single SMI dose. Inhibitors of cyclooxygenase-2 and 5-lipoxygenase enzymes reduced exudation and inflammation in the ears and lungs of mice. Production of inflammatory factors that increase vascular permeability may result in SMI-induced PARs, and p38 MAPK/cPLA2 signaling pathway and downstream AA metabolic pathway are involved in the reactions.

Upregulation of Beta 1 and Arachidonic Acid Metabolizing Enzymes in the Mouse Hearts and Kidneys after Sub Chronic Administration of Rofecoxib.

An imbalance in the levels of arachidonic acid (ARA) metabolites in cardiovascular disorders and drug-induced cardiotoxicity have been previously described. This study aimed to investigate the influence of cyclooxygenase-2 (COX-2) selective inhibitors on the gene expression of ARA-metabolizing genes and beta1 gene in the hearts and kidneys of experimental mice. Thirty-five balb/c mice were divided into five groups with seven mice per group. The groups were then given two distinct types of COX-2 selective inhibitors, rofecoxib and celecoxib, in two different doses equivalent to those used in human treatment for 30 days. The mRNA expression of beta1, ace2, and ARA-metabolizing genes, coxs, lipoxygenases (aloxs), and cytochrome p450 (cyp450s) in mice heart and kidneys were assessed. Genes were analyzed using real-time polymerase chain reaction analysis. In addition, rofecoxib-induced histological alterations were examined. It was found that only the high dose of rofecoxib (5 mg/kg) caused toxicological alterations, a finding that was indicated by a significant increase (P < 0.05) in the relative weight of the mouse hearts and increase in the ventricle wall thickness as observed through pathohistological examination. This increase was associated with a significant increase in the mRNA expression level of the beta1 receptor in both the heart and kidneys of the mice (53- and 12-fold, respectively). The expression of both cox1 and 2 genes was increased 4-fold in the kidneys. In addition, the expression of the alox12 gene increased significantly (by 67-fold in the heart and by 21-fold in the kidney), while alox15 gene expression was upregulated in the heart by 8-fold and 5-fold in the kidney. The genes responsible for synthesizing 20- Hydroxyeicosatetraenoic acid (cyp4a12 and cyp1a1) were significantly upregulated (P < 0.05) in the hearts of high-dose rofecoxib-treated mice by 7- and 17 -fold, respectively. In addition, the expression of epoxyeicosatrienoic acid-synthesizing genes, cyp2c29 and cyp2j5, was increased significantly (P < 0.05) in the hearts of high-dose rofecoxib-treated mice by 4- and 16-fold, respectively. Rofecoxib caused upregulation of the mRNA expression of the beta 1 gene in association with increased expression of ARA-metabolizing genes in mouse hearts and kidneys. These findings may help us understand the molecular cardiotoxic mechanism of rofecoxib.

Distinct eicosanoid patterns in severe recalcitrant nasal polyposis.

Although altered eicosanoid levels are related to disease severity in chronic rhinosinusitis with nasal polyps (CRSwNP), identifying patients prone to recurrent nasal polyps (NPs) is still difficult. We investigated levels of nasally secreted eicosanoids before and after NP surgery in patients with or without NP recurrence (NPR) and explored potential endotypes based on pre-surgical eicosanoid levels. Levels of leukotriene (LT) E4 , LTB4 , prostaglandin (PG) D2 , PGE2 and 15(S) hydroxyeicosatetraenoic acid (15[S]-HETE) were measured in nasal secretions with specific immunoassays at pre-surgery (n=38) and 6 and 12 months post-surgery (n=35), with NPR identified endoscopically. Pre- and post-surgical levels were compared between patients with and without NPR. Eicosanoid patterns among patients were explored with cluster analysis and evaluated with clinical parameters. Patients with recurrent NPs had pronounced pre-surgical levels of nasal 15(S)-HETE, PGD2 and LTE4 . From pre-surgery to 12 months post-surgery, NPR was associated with significant decreases of 15(S)-HETE and PGD2 relative to non-recurrence, whereas levels of LTE4 decreased at 6 months but increased again at 12 months. Clustering revealed three potential endotypes. Clusters 1 and 3 featured high and low eicosanoid levels, respectively. Cluster 2 had higher levels of LTE4 and PGD2 , lower levels of PGE2 and LTB4 , and more cases of recurrent NPs and previous NP surgeries. Elevated nasal LTE4 12 months post-surgery in NP recurrent subjects suggests that postoperative LTE4 measurements may indicate rapid NP regrowth. A distinct nasal eicosanoid profile may be used for the identification of the most severe recalcitrant patients in need of targeted immunomodulatory therapies.

Lethal and Sub-lethal Implications of Sodium Chloride Exposure for Adult Unionid Mussel Species: Eurynia dilatata and Lasmigona costata.

The elevated use of salt as a de-icing agent on roads in Canada is causing an increase in the chloride concentration of freshwater ecosystems. Freshwater Unionid mussels are a group of organisms that are sensitive to increases in chloride levels. Unionids have greater diversity in North America than anywhere else on Earth, but they are also one of the most imperiled groups of organisms. This underscores the importance of understanding the effect that increasing salt exposure has on these threatened species. There are more data on the acute toxicity of chloride to Unionids than on chronic toxicity. This study investigated the effect of chronic sodium chloride exposure on the survival and filtering activity of two Unionid species (Eurynia dilatata, and Lasmigona costata) and assessed the effect on the metabolome in L. costata hemolymph. The concentration causing mortality after 28days of exposure was similar for E. dilatata (1893mg Cl-/L) and L. costata (1903mg Cl-/L). Significant changes in the metabolome of the L. costata hemolymph were observed for mussels exposed to non-lethal concentrations. For example, several phosphatidylethanolamines, several hydroxyeicosatetraenoic acids, pyropheophorbide-a, and alpha-linolenic acid were significantly upregulated in the hemolymph of mussels exposed to 1000mg Cl-/L for 28days. While no mortality occurred in the treatment, elevated metabolites in the hemolymph are an indicator of stress.

17-(R/S)-hydroxyeicosatetraenoic acid (HETE) induces cardiac hypertrophy through the CYP1B1 in enantioselective manners

Cardiac cellular hypertrophy is the increase in the size of individual cardiac cells. Cytochrome P450 1B1 (CYP1B1) is an extrahepatic inducible enzyme that is associated with toxicity, including cardiotoxicity. We previously reported that 19-hydroxyeicosatetraenoic acid (19-HETE) inhibited CYP1B1 and prevented cardiac hypertrophy in enantioselective manner. Therefore, our aim is to investigate the effect of 17-HETE enantiomers on cardiac hypertrophy and CYP1B1. Human adult cardiomyocyte (AC16) cells were treated with 17-HETE enantiomers (20 µM); cellular hypertrophy was evaluated by cell surface area and cardiac hypertrophy markers. In addition, CYP1B1 gene, protein and activity were assessed. Human recombinant CYP1B1 and heart microsomes of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats were incubated with 17-HETE enantiomers (10–80 nM). Our results demonstrated that 17-HETE induced cellular hypertrophy, which is manifested by increase in cell surface area and cardiac hypertrophy markers. 17-HETE enantiomers allosterically activated CYP1B1 and selectively upregulated CYP1B1 gene and protein expression in AC16 cells at uM range. In addition, CYP1B1 was allosterically activated by 17-HETE enantiomers at nM range in recombinant CYP1B1 and heart microsomes. In conclusion, 17-HETE acts as an autocrine mediator, leading to the cardiac hypertrophy through induction of CYP1B1 activity in the heart.

Eight Zhes Decoction ameliorates the lipid dysfunction of nonalcoholic fatty liver disease using integrated lipidomics, network pharmacology and pharmacokinetics

Nonalcoholic fatty liver disease (NAFLD) has developed into the most common chronic liver disease and can lead to liver cancer. Our laboratory previously developed a novel prescription for NAFLD, “Eight Zhes Decoction” (EZD), which has shown good curative effects in clinical practice. However, the pharmacodynamic material basis and mechanism have not yet been revealed. A strategy integrating lipidomics, network pharmacology and pharmacokinetics was used to reveal the active components and mechanisms of EZD against NAFLD. The histopathological results showed that EZD attenuated the degrees of collagen deposition and steatosis in the livers of nonalcoholic steatofibrosis model mice. Furthermore, glycerophospholipid metabolism, arachidonic acid metabolism, glycerolipid metabolism and linoleic acid metabolism with phospholipase A2 group IVA (PLA2G4A) and cytochrome P450 as the core targets and 12,13-cis-epoxyoctadecenoic acid, 12(S)-hydroxyeicosatetraenoic acid, leukotriene B4, prostaglandin E2, phosphatidylcholines (PCs) and triacylglycerols (TGs) as the main lipids were found to be involved in the treatment of NAFLD by EZD. Importantly, naringenin, artemetin, canadine, and bicuculline were identified as the active ingredients of EZD against NAFLD; in particular, naringenin reduces PC consumption by inhibiting the expression of PLA2G4A and thus promotes sufficient synthesis of very-low-density lipoprotein to transport excess TGs in the liver. This research provides valuable data and theoretical support for the application of EZD against NAFLD.

Membrane Lipid Derivatives: Roles of Arachidonic Acid and Its Metabolites in Pancreatic Physiology and Pathophysiology.

One of the most important constituents of the cell membrane is arachidonic acid. Lipids forming part of the cellular membrane can be metabolized in a variety of cellular types of the body by a family of enzymes termed phospholipases: phospholipase A2, phospholipase C and phospholipase D. Phospholipase A2 is considered the most important enzyme type for the release of arachidonic acid. The latter is subsequently subjected to metabolization via different enzymes. Three enzymatic pathways, involving the enzymes cyclooxygenase, lipoxygenase and cytochrome P450, transform the lipid derivative into several bioactive compounds. Arachidonic acid itself plays a role as an intracellular signaling molecule. Additionally, its derivatives play critical roles in cell physiology and, moreover, are involved in the development of disease. Its metabolites comprise, predominantly, prostaglandins, thromboxanes, leukotrienes and hydroxyeicosatetraenoic acids. Their involvement in cellular responses leading to inflammation and/or cancer development is subject to intense study. This manuscript reviews the findings on the involvement of the membrane lipid derivative arachidonic acid and its metabolites in the development of pancreatitis, diabetes and/or pancreatic cancer.

Sex- and enantiospecific differences in the formation rate of hydroxyeicosatetraenoic acids in rat organs.

Hydroxyeicosatetraenoic acids (HETEs) are hydroxylated arachidonic acid (AA) metabolites that are classified into midchain, subterminal, and terminal HETEs. Hydroxylation results in the formation of R and S enantiomers for each HETE, except for 20-HETE. HETEs have multiple physiological and pathological effects. Several studies have demonstrated sex-specific differences in AA metabolism in different organs. In this study, microsomes from the heart, liver, kidney, lung, intestine, and brain of adult male and female Sprague-Dawley rats were isolated and incubated with AA. Thereafter, the enantiomers of all HETEs were analyzed by liquid chromatography-tandem mass spectrometry. We found significant sex- and enantiospecific differences in the formation levels of different HETEs in all organs. The majority of HETEs, especially midchain HETEs and 20-HETE, showed significantly higher formation rates in male organs. In the liver, the R enantiomer of several HETEs showed a higher formation rate than the corresponding S enantiomer (e.g., 8-, 9-, and 16-HETE). On the other hand, the brain and small intestine demonstrated a higher abundance of the S enantiomer. 19(S)-HETE was more abundant than 19(R)-HETE in all organs except the kidney. Elucidating sex-specific differences in HETE levels provides interesting insights into their physiological and pathophysiological roles and their possible implications for different diseases.

Unsaturated oxidated fatty acid 12(S)-HETE attenuates TNF-α expression in TNF-α/IFN-γ-stimulated human keratinocytes

Chronic skin inflammatory diseases are associated with abnormal immune responses characterized by skin barrier dysfunction. Keratinocytes participate in immune homeostasis regulated by immune cells. Immune homeostasis dysfunction contributes to the pathogenesis of skin diseases mediated by pro-inflammatory cytokines and chemokines, such as tumor necrosis factor (TNF)-α, which are produced by activated keratinocytes. 12(S)-Hydroxy eicosatetraenoic acid [12(S)-HETE], an arachidonic acid metabolite, has anti-inflammatory properties. However, the role of 12(S)-HETE in chronic skin inflammatory diseases has not been elucidated yet. In this study, we investigated the effect of 12(S)-HETE on TNF-α/interferon (IFN)-γ-induced pro-inflammatory cytokine and chemokine expression. Our data showed that 12(S)-HETE modulates TNF-α mRNA and protein expression in TNF-α-/IFN-γ-treated human keratinocytes. Molecular docking analyses demonstrated that 12(S)-HETE bound to extracellular signal-regulated kinase (ERK)1/2, thus preventing ERK activation and downregulating phosphorylated ERK expression. We also demonstrated that 12(S)-HETE treatment inhibited IκB and ERK phosphorylation and nuclear factor (NF)-κB, p65/p50, and CCAAT/enhancerbindingproteinβ (C/EBPβ) translocation. Overall, our results showed that 12(S)-HETE attenuated TNF-α expression and secretion by inhibiting the mitogen-activated protein kinase ERK/NF-κB and C/EBPβ signaling pathways. Overall, these results suggest that 12(S)-HETE effectively resolved TNF-α-induced inflammation.

Vascular Inflammatory Markers as Predictors of Peripheral Arterial Disease Patients' Quality-of-Life Changes after Endovascular Treatment.

The association between chronic inflammation and depression, anxiety, anhedonia, and quality of life (QoL) has been recently emphasized. However, the pathophysiology of this relationship remains unsolved. This study aims to assess the dependence between vascular inflammation represented by eicosanoid concentration and quality of life in patients with peripheral arterial disease (PAD). A total of 175 patients undergoing endovascular treatment due to lower limbs ischemia were covered with eight years of observation after the endovascular procedure, including ankle-brachial index (ABI), color Doppler ultrasound examination, urinary leukotriene E4 (LTE4), thromboxane B2 (TXB2) and 5-Hydroxyeicosatetraenoic acid (5-HETE) measurement and quality-of-life assessment with VascuQol-6. The baseline concentrations of LTE4 and TXB2 reversely correlated with preoperative VascuQol-6 and were predictive of the postoperative values of VascuQol-6 at each follow-up. At every follow-up timepoint, the results of VascuQol-6 reflected the LTE4 and TXB2 concentrations. Higher concentrations of LTE4 and TXB2 were correlated with lower life quality during the next follow-up meeting. Changes in VascuQol-6 at eight years vs. preoperative values were reversely related to the preoperative concentrations of LTE4 and TXB2. This is the first study to confirm that changes in life quality in PAD patients undergoing endovascular treatment are highly dependent on eicosanoid-based vascular inflammation.

Twelve Weeks of High-Intensity Interval Training Alters Adipose Tissue Gene Expression but Not Oxylipin Levels in People with Non-Alcoholic Fatty Liver Disease.

Lifestyle modifications, including increased physical activity and exercise, are recommended for non-alcoholic fatty liver disease (NAFLD). Inflamed adipose tissue (AT) contributes to the progression and development of NAFLD and oxylipins such as hydroxyeicosatetraenoic acids (HETE), hydroxydocosahexanenoic acids (HDHA), prostaglandins (PEG2), and isoprostanoids (IsoP), which all may play a role in AT homeostasis and inflammation. To investigate the role of exercise without weight loss on AT and plasma oxylipin concentrations in NAFLD subjects, we conducted a 12-week randomized controlled exercise intervention. Plasma samples from 39 subjects and abdominal subcutaneous AT biopsy samples from 19 subjects were collected both at the beginning and the end of the exercise intervention. In the AT of women, a significant reduction of gene expression of hemoglobin subunits (HBB, HBA1, HBA2) was observed within the intervention group during the 12-week intervention. Their expression levels were negatively associated with VO2max and maxW. In addition, pathways involved in adipocyte morphology alterations significantly increased, whereas pathways in fat metabolism, branched-chain amino acids degradation, and oxidative phosphorylation were suppressed in the intervention group (p < 0.05). Compared to the control group, in the intervention group, the ribosome pathway was activated, but lysosome, oxidative phosphorylation, and pathways of AT modification were suppressed (p < 0.05). Most of the oxylipins (HETE, HDHA, PEG2, and IsoP) in plasma did not change during the intervention compared to the control group. 15-F2t-IsoP significantly increased in the intervention group compared to the control group (p = 0.014). However, this oxylipin could not be detected in all samples. Exercise intervention without weight loss may influence the AT morphology and fat metabolism at the gene expression level in female NAFLD subjects.

Renoprotective Effect of TP0472993, a Novel and Selective 20-Hydroxyeicosatetraenoic Acid Synthesis Inhibitor, in Mouse Models of Renal Fibrosis.

Kidney fibrosis is considered the essential pathophysiological process for the progression of chronic kidney disease (CKD) toward renal failure. 20-Hydroxyeicosatetraenoic acid (20-HETE) has crucial roles in modulating the vascular response in the kidney and the progression of albuminuria. However, the roles of 20-HETE in kidney fibrosis are largely unexplored. In the current research, we hypothesized that if 20-HETE has important roles in the progression of kidney fibrosis, 20-HETE synthesis inhibitors might be effective against kidney fibrosis. To verify our hypothesis, this study investigated the effect of a novel and selective 20-HETE synthesis inhibitor, TP0472993, on the development of kidney fibrosis after folic acid- and obstructive-induced nephropathy in mice. Chronic treatment with TP0472993 at doses of 0.3 and 3 mg/kg twice a day attenuated the degree of kidney fibrosis in the folic acid nephropathy and the unilateral ureteral obstruction (UUO) mice, as demonstrated by reductions in Masson's trichrome staining and the renal collagen content. In addition, TP0472993 reduced renal inflammation, as demonstrated by markedly reducing interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) levels in the renal tissue. Chronic treatment with TP0472993 also reduced the activity of extracellular signal-regulated kinase 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3) in the kidney of UUO mice. Our observations indicate that inhibition of 20-HETE production with TP0472993 suppresses the kidney fibrosis progression via a reduction in the ERK1/2 and STAT3 signaling pathway, suggesting that 20-HETE synthesis inhibitors might be a novel treatment option against CKD. SIGNIFICANCE STATEMENT: In this study, we demonstrate that the pharmacological blockade of 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis using TP0472993 suppresses the progression of kidney fibrosis after folic acid- and obstructive-induced nephropathy in mice, indicating that 20-HETE might have key roles in the pathogenesis of kidney fibrosis. TP0472993 has the potential to be a novel therapeutic approach against chronic kidney disease.

Precursors of specialized pro-resolving mediators (SPMs) are decreased in hypertension, and treatment with SPMs improve vascular relaxation via formyl peptide-receptor-2

Specialized pro-resolving mediators (SPMs), derived from essential fatty acids, play an immunoregulatory role. Formyl peptide receptor-1 is a pattern recognition receptor that mediate host-defense and inflammation. On the other hand, FPR2 binds SPMs to induce resolution of inflammation. It is well known that low-grade chronic inflammation is present in hypertension, but it is unknown whether this phenomenon occurs due to dysregulation of resolution of inflammation. Also, it is unclear if arteries can sense SPM. We hypothesized that the resolution of inflammation is decreased in hypertension, and treatment with SPM will improve vascular function via FPR-2. Forty-five eicosanoids, precursors of SPMs were detected in the plasma of male Wistar Kyoto rats (WKY) (14 weeks old, n=10) and spontaneously hypertensive rats (SHR; 14 weeks old, n=16). Analysis revealed a significant decrease (%) in 5 different eicosanoids in SHR (1) free eicosapentaenoic acid (60%), (2) free adrenic acid (25%), (3) 20-carboxy-arachidonic acid (52%) (4) 5-hydroxyeicosatetraenoic acid (20%), and (5) 14,15-epoxyeicosa-5,8,11-trienoic acid (14,15-EET) (48%). Additionally, mesenteric resistance arteries (MRA, lumen diameter &lt; 300 μm) from SHR rats showed significant oxidative degradation of lipids measured by the presence of 4-hydroxynonenal (4-HNE) (4-HNE; AU: WKY: 3.8±0.6 vs. SHR: 14.4±3.8; p=0.02). MRA were mounted in a wire myograph to evaluate vascular function, and concentration-response curves (CRC) to acetylcholine (ACh 10−9 to 10−5 mol/L) were performed. As expected, relaxation was impaired in MRA from SHR compared to WKY. This was noted by a shift to the right in the CRC, increasing (%) area under the curve (ΔAUC: 20± 4%). On the other hand, SPMs (10 nM) restored this response by shifting the CRC to the left and reducing the AUC compared to arteries from SHR treated with vehicle [SPMs: Resolvin D1 (RvD1) (ΔAUC:19±4%), Resolvin E1 (RvE1) (ΔAUC: 8.5 ± 9%), and lipoxin A4 (ΔAUC: 20±7%,)]. Specific antagonism of FPR-2 (WRW4, 1μM) abolished the improvement in relaxation induced by SPMs. Superoxide indicator dihydroethidium (DHE) was used in primary MRA endothelial cell culture. SPMs reduced the formation of reactive oxygen species after an inflammatory stimulus [Control: 99±2.5%; TNFα: 272± 50%; RvD1: 109±25%; RvE1: 111±22%; LXA4: 101±10%, A.U.). However, WRW4 did not change these responses. Total FPR2 and lipoxygenase 5 (LOX-5) protein expression was similar in arteries from WKY and SHR. However, there was an increase in LOX-5 serine 523 phosphorylation in SHR, suggesting a compensatory mechanism to increase local LXA4. Overall, SPMs play an important role to maintain vascular heath, and their effects occur both dependent and independent of FPR2 activation. Improvement of resolution of inflammation maybe a new treatment for for hypertension. National Institutes of Health - R00GM118885, R01HL149762, R00HL151889, and RO1DK132948. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sex Differences in Gut Microbiota Fatty Acid Metabolism in Human Salt Sensitivity of Blood Pressure

Salt sensitivity is a trait in which high dietary sodium (Na+) intake causes an increase in blood pressure. We previously demonstrated that in the gut, elevated dietary Na+ causes dysbiosis. The mechanistic interplay between excess dietary Na+-induced alteration in the gut microbiome and sex differences is less understood. The goal of this study was to identify novel metabolites in salt sensitivity and sex differences in response to a high dietary Na+ intake. We hypothesized that excess dietary salt-induced dysbiosis is associated with distinct metabolomic changes in men and women. We performed plasma metabolomics analysis and measured blood pressure of human volunteers who completed a validated 3-day food record to estimate dietary Na+ intake. Based on the recommendations by the American Heart Association, we classified daily Na+ intake of &lt;2.3g as normal salt and high salt for subjects consuming ≥ 2.3g Na+. Spearman correlation was used to assess the relationship between Na+ intake and blood pressure. We also performed RNA sequencing on human (N=11) monocytes treated with high salt in vitro. The relationship between dietary Na+ intake and mean arterial pressure was different in women (r=0.3422, p=0.0022, N=81) and men (r=0.4552, p=0.0010, N=49). Network analysis of human plasma untargeted metabolome revealed fatty acids as top subnetworks differentially changed with salt intake. In subjects consuming a high Na+ diet, levels of linoleate (1.211 ± 0.330 vs. 0.869 ± 0.170; p&lt;0.001); palmitate (1.155 ± 0.292 vs. 0.924 ± 0.233; p= 0.003); arachidonic acid (1.119 ± 0.242 vs. 0.965 ± 0.201; p=0.015); and 12-hydroxyeicosatetraenoic acid (1.329 ± 0.925 vs. 0.902 ± 0.520; p=0.0469) were higher in women (28) than men (25). In contrast, we observed no sex differences in these parameters between men and women consuming a normal salt diet. RNA sequencing analysis revealed that exposure of human monocytes to high salt in vitro upregulated transcription of fatty acid receptors and arachidonic acid-related genes. Specifically, ALOX12 (277.8 ± 13.8 vs. 169.5 ± 16.7, p&lt;0.0001) which encodes for arachidonate 12-lipoxygenase, ALOX5AP (6459 ± 1553 vs. 2598 ± 30, p=0.0310) which encodes for 5-lipoxygenase activating protein, and FFR2 (418.1 ± 97.8 vs. 115.9 ± 29.4, p=0.0078) which encodes for free fatty acids receptor 2 were increased in human monocytes, and this was associated with a pro-inflammatory phenotype. These findings suggest that excess dietary Na+ is associated with fatty acid metabolism dysregulation, and that the arachidonic acid pathway may potentially play a role in sex differences of salt-sensitive hypertension. This work was supported by American Heart Association grants POST9034281 (to JAI) and 15SDG24890015 (to JFF) and NIH grants K01HL130497 (to AK) as well as Venture Pilot Funding (to AK) from Vanderbilt Microbiome Initiative and Vanderbilt Institute for Infection, Immunology, and Inflammation (VI4) and a P&amp;F award (to JFF) from Vanderbilt University Medical Center's Digestive Disease Research Center supported by NIH grant P30DK058404. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.