Glucocorticoids regulate lipid mediator networks by reciprocal modulation of 15-lipoxygenase isoforms affecting inflammation resolution.

Glucocorticoids (GC) are potent anti-inflammatory agents, broadly used to treat acute and chronic inflammatory diseases, e.g., critically ill COVID-19 patients or patients with chronic inflammatory bowel diseases. GC not only limit inflammation but also promote its resolution although the underlying mechanisms are obscure. Here, we reveal reciprocal regulation of 15-lipoxygenase (LOX) isoform expression in human monocyte/macrophage lineages by GC with respective consequences for the biosynthesis of specialized proresolving mediators (SPM) and their 15-LOX-derived monohydroxylated precursors (mono-15-OH). Dexamethasone robustly up-regulated pre-mRNA, mRNA, and protein levels of ALOX15B/15-LOX-2 in blood monocyte-derived macrophage (MDM) phenotypes, causing elevated SPM and mono-15-OH production in inflammatory cell types. In sharp contrast, dexamethasone blocked ALOX15/15-LOX-1 expression and impaired SPM formation in proresolving M2-MDM. These dexamethasone actions were mimicked by prednisolone and hydrocortisone but not by progesterone, and they were counteracted by the GC receptor (GR) antagonist RU486. Chromatin immunoprecipitation (ChIP) assays revealed robust GR recruitment to a putative enhancer region within intron 3 of the ALOX15B gene but not to the transcription start site. Knockdown of 15-LOX-2 in M1-MDM abolished GC-induced SPM formation and mono-15-OH production. Finally, ALOX15B/15-LOX-2 upregulation was evident in human monocytes from patients with GC-treated COVID-19 or patients with IBD. Our findings may explain the proresolving GC actions and offer opportunities for optimizing GC pharmacotherapy and proresolving mediator production.

Loss of 12-Lipoxygenase Improves the Post-Transfusion Function of Stored Platelets.

BACKGROUND: Platelets for transfusion are stored for 5 to 7 days. Previous studies have shown that HETE levels in the storage bag negatively correlate with platelet performance in vivo, suggesting that the dysregulation of bioactive lipid mediators may contribute to the storage lesion. In the current study, we sought to understand how genetic deletion and pharmacological inhibition of 12-LOX (12-lipoxygenase) affects platelets during storage and after transfusion. METHODS: Platelets from 12-LOX+/+ (wild-type [WT]) and 12-LOX-/- mice were stored for 24 and 48 hours and profiled using liquid chromatography-tandem mass spectrometry-multiple reaction monitoring or transfused into thrombocytopenic hIL4R (human interleukin 4 receptor)-transgenic mice. Platelet function was assessed by flow cytometry and in vivo thrombosis and hemostasis models. To test the role of the COX-1 (cyclooxygenase-1) pathway, donor mice were treated with acetylsalicylic acid. Human platelets were treated with the 12-LOX inhibitor, VLX-1005, or vehicle, stored, and transfused to NOD/SCID (nonobese diabetic/severe combined immunodeficiency) mice. RESULTS: Polyunsaturated fatty acids increased significantly in stored platelets from 12-LOX-/- mice, whereas oxylipin concentrations were significantly higher in WT platelets. After transfusion to thrombocytopenic mice, we observed significantly more baseline αIIbß3 integrin activation in 12-LOX-/- platelets than in WT platelets. Stored platelets from 12-LOX-/- mice occluded vessels significantly faster than stored WT platelets. In hemostasis models, significantly more stored 12-LOX-/- than WT platelets accumulated at the site of venous injury leading to reduced blood loss. Inhibition of COX-1 abrogated both increased integrin activation and thromboxane generation in stored 12-LOX-/- platelets, highlighting the critical role of this pathway for improved post-transfusion function. Consistent with our mouse studies, human platelets stored with VLX-1005, showed increased integrin activation compared with vehicle-treated platelets after transfusion. CONCLUSIONS: Deleting 12-LOX improves the post-transfusion function of stored murine platelets by increasing thromboxane generation through COX-1-dependent arachidonic acid metabolism. Future studies should determine the feasibility and safety of 12-LOX-inhibited platelets transfused to humans.

Single and multiple inhibitors of the biosynthesis of 5-, 12-, 15-lipoxygenase products derived from cinnamyl-3,4-dihydroxy-α-cyanocinnamate: Synthesis and structure-activity relationship.

The involvement of lipoxygenases in various pathologies, combined with the unavailability of safe and effective inhibitors of the biosynthesis of their products, is a source of inspiration for the development of new inhibitors. Based on a structural analysis of known inhibitors of lipoxygenase products biosynthesis, a comprehensive structure-activity study was carried out, which led to the discovery of several novel compounds (16a-c, 17a) demonstrating promising potency to inhibit the biosynthesis of products of 5-, 12- and 15-LO. Compounds 16b and 16c outperformed zileuton (1), the only FDA-approved 5-LO inhibitor, as well as known inhibitors such as caffeic acid phenethyl ester (CAPE (2)) and cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC (4)). However, the introduction of a cyano group at the α-position of the carbonyl abolished the activity. Compounds 16a and 17a also inhibited the biosynthesis of 12- and 15-LO products. Compounds 16a, 17a far surpassed baicalein, a known 12-LO inhibitor, as inhibitors of 12-LO products biosynthesis. Compound 17a and CDC (4) showed equivalent inhibition of LO products, proposing that the double bond in the ester moiety is not necessary for the inhibitory activity. The introduction of the cyano group, as in compound 17a, at the α-position of the carbonyl in compound 16a significantly reduced the inhibitory activity against the biosynthesis of 15-LO products. In addition to the interactions with residues His372 and Phe421 also found with zileuton and CAPE, compounds 16a and 16c each interact with residue His367 as shown by molecular docking. This new interaction may explain their high affinity with the 5-LO active site.

Synthesis and In Vitro Biological Evaluation of p-Carborane-Based Di-tert-butylphenol Analogs.

Targeting inflammatory mediators and related signaling pathways may offer a rational strategy for the treatment of cancer. The incorporation of metabolically stable, sterically demanding, and hydrophobic carboranes in dual cycloxygenase-2 (COX-2)/5-lipoxygenase (5-LO) inhibitors that are key enzymes in the biosynthesis of eicosanoids is a promising approach. The di-tert-butylphenol derivatives R-830, S-2474, KME-4, and E-5110 represent potent dual COX-2/5-LO inhibitors. The incorporation of p-carborane and further substitution of the p-position resulted in four carborane-based di-tert-butylphenol analogs that showed no or weak COX inhibition but high 5-LO inhibitory activities in vitro. Cell viability studies on five human cancer cell lines revealed that the p-carborane analogs R-830-Cb, S-2474-Cb, KME-4-Cb, and E-5110-Cb exhibited lower anticancer activity compared to the related di-tert-butylphenols. Interestingly, R-830-Cb did not affect the viability of primary cells and suppressed HCT116 cell proliferation more potently than its carbon-based R-830 counterpart. Considering all the advantages of boron cluster incorporation for enhancement of drug biostability, selectivity, and availability of drugs, R-830-Cb can be tested in further mechanistic and in vivo studies.

Omega-3 fatty acids protect from colitis via an Alox15-derived eicosanoid.

An increased omega-3 polyunsaturated fatty acid (n-3 PUFA) tissue status can lead to a significant formation of anti-inflammatory lipid mediators and effective reduction in inflammation and tissue injury in murine colitis. Arachidonic acid lipoxygenases (ALOX) have been implicated in the pathogenesis of inflammatory bowel disease as well as in the formation of pro- and anti-inflammatory lipid mediators. To explore the role of Alox15 in the protective response found in fat1 transgenic mice with endogenously increased n-3 PUFA tissue status fat1 transgenic mice were crossed with Alox15-deficient animals and challenged in the dextran sulfate sodium (DSS)- and the 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis model. Transgenic fat1 mice rich in endogenous n-3 PUFAs were protected from colitis. However, additional systemic inactivation of the Alox15 gene counteracted this protective effect. To explore the molecular basis for this effect Alox15 lipid metabolites derived from n-3 PUFA were analyzed in the different mice. Alox15 deficiency suppressed the formation of n-3 PUFA-derived 15-hydroxy eicosapentaenoic acid (15-HEPE). In contrast, treating mice with intraperitoneal injections of 15S-HEPE protected wild-type mice from DSS- and TNBS-induced colitis. These data suggest that the anti-colitis effect of increased n-3 PUFA in the transgenic fat1 mouse model is mediated in part via Alox15-derived 15-HEPE formation.

Involvement of ischemia-driven 5-lipoxygenase-resolvin-E1-chemokine like receptor-1 axis in the resolution of post-coronary artery bypass graft inflammation in coronary arteries.

AIMS: Expression status of pro-resolvin lipid mediators (PLM) and receptors in the post-Coronary artery bypass grafting (CABG) coronary arteries are largely unknown. Here, we aim to investigate the expression of the enzymes involved in PLM synthesis and their receptors in the atherosclerotic post-CABG swine (AS) left anterior descending (LAD) compared to without CABG (LAD-AS), and in isolated coronary artery smooth muscle cells (CASMCs) cultured under ischemia. METHODOLOGY: The arteries of interest were harvested from post-CABG atherosclerotic swine and the histomorphology and the expression status of key PLM mediators were quantified using immunostaining. Smooth muscle cells (SMCs) were cultured under ischemia and confirmed the expression on PLM mediators at transcript and protein level. RESULTS: The histomorphometric analysis revealed considerable alterations in the tissue architecture in LAD-CABG and LAD-AS arteries compared to control. PLM synthetic enzyme 5-lipoxygenases (5LO) was significantly upregulated in LAD-CABG and LAD-AS whereas the other enzymes including 12LO, 15LO, and cyclooxygenase-2, and the receptors including Chemokine like receptor 1 (ChemR23), 7-transmembrane G-protein coupled receptor-18 (GPCR18), GPCR120 were decreased in LAD-CABG than control. LO enzymes and PLM receptors were upregulated in ischemic CASMCs with respect to control. Western blot showed the upregulation of 5LO, and ChemR23. Additionally, higher level of resolvin-E1 (RvE1) was observed in ischemic control CASMCs which was decreased following reperfusion. CONCLUSION: These findings suggest that the CASMCs withstand the ischemia-triggered proinflammatory episodes by increasing the secretion of RvE1 mediated through 5LO and ChemR23 signaling.

Genome-wide identification and classification of Lipoxygenase gene family and their roles in sorghum-aphid interaction.

KEY MESSAGE: This report shows detailed characterization of LOX gene family in sorghum and provides new insight of sorghum LOX genes in genetic structure and their roles in plant response to infestation by sugarcane aphids. Lipoxygenases (LOXs) are monomeric, nonheme iron-containing dioxygenases that initiate the fatty acid oxidation pathway creating oxylipins and plant hormone jasmonate both have a key role in plant development and defense. To date, a comprehensive and systematic analysis of sorghum LOXs is still deficient. Thus, we performed a genome-wide analysis of the sorghum LOXs genome and identified nine LOXs genes. Detailed examination of protein sequences and phylogenetic analysis categorized the sorghum LOXs into two subclasses, 9-LOXs (SbLOX1, SbLOX3, SbLOX4, SbLOXm, and SbLOXo), 13-LOXs (SbLOX9, SbLOX5, and SbLOX2), and the unclassified SbLOX8. This classification was further supported by sequence similarity/identity matrix and subcellular localization analysis. The lipoxygenase domains, motifs, and vital amino acids were highly conserved in all sorghum LOX genes. In silico analysis of the promoter region of SbLOXs identified different hormones responsive cis-elements. Furthermore, to explore the roles of sorghum LOXs during sugarcane aphid feeding and exogenous MeJA application, expression analysis was conducted for all the eight LOXs in resistant (Tx2783) and susceptible (Tx7000) sorghum lines, respectively. As detailed in this report, the data generated from both genome-wide identification and expression analysis of lipoxygenase genes suggest the putative functions of two 13-LOXs (SbLOX9 and SbLOX5) and three 9-LOXs (SbLOX1, SbLOX3, and SbLOXo) in biosynthesis of jasmonic acid, green leaf volatiles and death acids, and all of them are involved in defense-related functions in plants. Furthermore, this report represents the first genome-wide analysis of the LOX gene family in sorghum, which will facilitate future studies to characterize the roles of each individual LOXs gene in aphid resistance and defense responses to other stresses.

Maize biochemistry in response to root herbivory was mediated by domestication, spread, and breeding.

MAIN CONCLUSION: With domestication, northward spread, and breeding, maize defence against root-herbivores relied on induced defences, decreasing levels of phytohormones involved in resistance, and increasing levels of a phytohormone involved in tolerance. We addressed whether a suite of maize (Zea mays mays) phytohormones and metabolites involved in herbivore defence were mediated by three successive processes: domestication, spread to North America, and modern breeding. With those processes, and following theoretical predictions, we expected to find: a change in defence strategy from reliance on induced defences to reliance on constitutive defences; decreasing levels of phytohormones involved in herbivore resistance, and; increasing levels of a phytohormone involved in herbivore tolerance. We tested those predictions by comparing phytohormone levels in seedlings exposed to root herbivory by Diabrotica virgifera virgifera among four plant types encompassing those processes: the maize ancestor Balsas teosinte (Zea mays parviglumis), Mexican maize landraces, USA maize landraces, and USA inbred maize cultivars. With domestication, maize transitioned from reliance on induced defences in teosinte to reliance on constitutive defences in maize, as predicted. One subset of metabolites putatively involved in herbivory defence (13-oxylipins) was suppressed with domestication, as predicted, though another was enhanced (9-oxylipins), and both were variably affected by spread and breeding. A phytohormone (indole-3-acetic acid) involved in tolerance was enhanced with domestication, and with spread and breeding, as predicted. These changes are consistent with documented changes in herbivory resistance and tolerance, and occurred coincidentally with cultivation in increasingly resource-rich environments, i.e., from wild to highly enriched agricultural environments. We concluded that herbivore defence evolution in crops may be mediated by processes spanning thousands of generations, e.g., domestication and spread, as well as by processes spanning tens of generations, e.g., breeding and agricultural intensification.

Relation between ABCB1 overexpression and COX2 and ALOX5 genes in human erythroleukemia cell lines.

This study aimed to characterize the relationship between the COX2 and ALOX5 genes, as well as their link with the multidrug resistance (MDR) phenotype in sensitive (K562) and MDR (K562-Lucena and FEPS) erythroleukemia cells. For this, the inhibitors of 5-LOX (zileuton) and COX-2 (acetylsalicylic acid-ASA) and cells with the silenced ABCB1 gene were used. The treatment with ASA caused an increase in the gene expression of COX2 and ABCB1 in both MDR cell lines, and a decrease in the expression of ALOX5 in the FEPS cells. Silencing the ABCB1 gene induced a decrease in COX2 expression and an increase in the ALOX5 gene. Treatment with zileuton did not alter the expression of COX2 and ABCB1. Cytometry data showed that there was an increase in ABCB1 protein expression after exposure to ASA. In addition, the increased activity of ABCB1 in the K562-Lucena cell line indicates that ASA may be a substrate for this efflux pump, corroborating the molecular docking that showed that ASA can bind to ABCB1. Regardless of the genetic alteration in COX2 and ABCB1, the direct relationship between these genes and the inverse relationship with ALOX5 remained in the MDR cell lines. We assume that ABCB1 can play a regulatory role in COX2 and ALOX5 during the transformation of the parental cell line K562, explaining the increased gene expression of COX2 and decreased ALOX5 in the MDR cell lines.

Expression profile of lipoxygenases in gingival tissues of human periodontitis.

OBJECTIVES: This study aimed to clarify the expression profile and significance of lipoxygenases in periodontitis. MATERIALS AND METHODS: The mRNA levels of lipoxygenases in gingival tissues from 14 patients with periodontitis and 14 healthy individuals were determined by real-time PCR, and validated in datasets, GSE16134 and GSE10334, and by Western blotting. Correlation of differentially expressed lipoxygenases with clinical parameters and expression of tumor necrosis factor-α (TNF-α), interleukin-1ß, matrix metalloproteinase (MMP)-8, MMP-9, and receptor activator of nuclear factor-κB ligand (RANKL) was investigated in patients with periodontitis by Spearman's correlation analysis. RESULTS: The expression of ALOX5 (2.1-fold, p < .05), ALOX12B (2.9-fold, p < .001), and ALOX15B (9.4-fold, p < .001) was upregulated in gingival tissues from patients with periodontitis, which was validated by dataset analysis and Western blotting. Positive correlations were observed between ALOX5 and probing depth, and ALOX15B and probing depth and clinical attachment loss. Furthermore, ALOX5 expression was positively correlated with TNF-α, MMP-8, MMP-9, and RANKL expression, and ALOX15B was positively correlated with MMP-8 and RANKL. CONCLUSIONS: Our findings indicated the upregulation of ALOX5 and ALOX15B in periodontitis and suggested that ALOX5 and ALOX15B may be involved in periodontitis pathogenesis, including inflammation, connective tissue destruction, and abnormal bone metabolism.

In Vitro Effects of Selective COX and LOX Inhibitors and Their Combinations with Antineoplastic Drugs in the Mouse Melanoma Cell Line B16F10.

The constitutive expression or overactivation of cyclooxygenase (COX) and lipoxygenase (LOX) enzymes results in aberrant metabolism of arachidonic acid and poor prognosis in melanoma. Our aim is to compare the in vitro effects of selective COX-1 (acetylsalicylic acid), COX-2 (meloxicam), 5-LOX (MK-886 and AA-861), 12-LOX (baicalein) and 15-LOX (PD-146176) inhibition in terms of proliferation (SRB assay), mitochondrial viability (MTT assay), caspase 3-7 activity (chemiluminescent assay), 2D antimigratory (scratch assay) and synthesis of eicosanoids (EIA) in the B16F10 cell line (single treatments). We also explore their combinatorial pharmacological space with dacarbazine and temozolomide (median effect method). Overall, our results with single treatments show a superior cytotoxic efficacy of selective LOX inhibitors over selective COX inhibitors against B16F10 cells. PD-146176 caused the strongest antiproliferation effect which was accompanied by cell cycle arrest in G1 phase and an >50-fold increase in caspases 3/7 activity. When the selected inhibitors are combined with the antineoplastic drugs, only meloxicam provides clear synergy, with LOX inhibitors mostly antagonizing. These apparent contradictions between single and combination treatments, together with some paradoxical effects observed in the biosynthesis of eicosanoids after FLAP inhibition in short term incubations, warrant further mechanistical in vitro and in vivo scrutiny.

Biochemical Characterization of 13-Lipoxygenases of Arabidopsis thaliana.

13-lipoxygenases (13-LOX) catalyze the dioxygenation of various polyunsaturated fatty acids (PUFAs), of which α-linolenic acid (LeA) is converted to 13-S-hydroperoxyoctadeca-9, 11, 15-trienoic acid (13-HPOT), the precursor for the prostaglandin-like plant hormones cis-(+)-12-oxophytodienoic acid (12-OPDA) and methyl jasmonate (MJ). This study aimed for characterizing the four annotated A. thaliana 13-LOX enzymes (LOX2, LOX3, LOX4, and LOX6) focusing on synthesis of 12-OPDA and 4Z,7Z,10Z)-12-[[-(1S,5S)-4-oxo-5-(2Z)-pent-2-en-1yl] cyclopent-2-en-1yl] dodeca-4,7,10-trienoic acid (OCPD). In addition, we performed interaction studies of 13-LOXs with ions and molecules to advance our understanding of 13-LOX. Cell imaging indicated plastid targeting of fluorescent proteins fused to 13-LOXs-N-terminal extensions, supporting the prediction of 13-LOX localization to plastids. The apparent maximal velocity (Vmax app) values for LOX-catalyzed LeA oxidation were highest for LOX4 (128 nmol·s-1·mg protein-1), with a Km value of 5.8 µM. A. thaliana 13-LOXs, in cascade with 12-OPDA pathway enzymes, synthesized 12-OPDA and OCPD from LeA and docosahexaenoic acid, previously shown only for LOX6. The activities of the four isoforms were differently affected by physiologically relevant chemicals, such as Mg2+, Ca2+, Cu2+ and Cd2+, and by 12-OPDA and MJ. As demonstrated for LOX4, 12-OPDA inhibited enzymatic LeA hydroperoxidation, with half-maximal enzyme inhibition at 48 µM. Biochemical interactions, such as the sensitivity of LOX toward thiol-reactive agents belonging to cyclopentenone prostaglandins, are suggested to occur in human LOX homologs. Furthermore, we conclude that 13-LOXs are isoforms with rather specific functional and regulatory enzymatic features.

Conformational Heterogeneity and Cooperative Effects of Mammalian ALOX15.

Arachidonic acid lipoxygenases (ALOXs) have been suggested to function as monomeric enzymes, but more recent data on rabbit ALOX15 indicated that there is a dynamic monomer-dimer equilibrium in aqueous solution. In the presence of an active site ligand (the ALOX15 inhibitor RS7) rabbit ALOX15 was crystalized as heterodimer and the X-ray coordinates of the two monomers within the dimer exhibit subtle structural differences. Using native polyacrylamide electrophoresis, we here observed that highly purified and predominantly monomeric rabbit ALOX15 and human ALOX15B are present in two conformers with distinct electrophoretic mobilities. In silico docking studies, molecular dynamics simulations, site directed mutagenesis experiments and kinetic measurements suggested that in aqueous solutions the two enzymes exhibit motional flexibility, which may impact the enzymatic properties.

Novel Cardiovascular Risk Factors in Patients with Diabetic Kidney Disease.

Patients with diabetic kidney disease (DKD) are at very high risk for cardiovascular events. Only part of this increased risk can be attributed to the presence of diabetes mellitus (DM) and to other DM-related comorbidities, including hypertension and obesity. The identification of novel risk factors that underpin the association between DKD and cardiovascular disease (CVD) is essential for risk stratification, for individualization of treatment and for identification of novel treatment targets.In the present review, we summarize the current knowledge regarding the role of emerging cardiovascular risk markers in patients with DKD. Among these biomarkers, fibroblast growth factor-23 and copeptin were studied more extensively and consistently predicted cardiovascular events in this population. Therefore, it might be useful to incorporate them in risk stratification strategies in patients with DKD to identify those who would possibly benefit from more aggressive management of cardiovascular risk factors.

Identification of a Bromodomain-like Region in 15-Lipoxygenase-1 Explains Its Nuclear Localization.

Lipoxygenase (LOX) activity provides oxidative lipid metabolites, which are involved in inflammatory disorders and tumorigenesis. Activity-based probes to detect the activity of LOX enzymes in their cellular context provide opportunities to explore LOX biology and LOX inhibition. Here, we developed Labelox B as a potent covalent LOX inhibitor for one-step activity-based labeling of proteins with LOX activity. Labelox B was used to establish an ELISA-based assay for affinity capture and antibody-based detection of specific LOX isoenzymes. Moreover, Labelox B enabled efficient activity-based labeling of endogenous LOXs in living cells. LOX proved to localize in the nucleus, which was rationalized by identification of a functional bromodomain-like consensus motif in 15-LOX-1. This indicates that 15-LOX-1 is not only involved in oxidative lipid metabolism, but also in chromatin binding, which suggests a potential role in chromatin modifications.

Mutation in ALOX12B likely cause of POI and also ichthyosis in a large Iranian pedigree.

Premature ovarian insufficiency (POI) is a clinically and etiologically heterogeneous disorder characterized by menstrual irregularities and elevated levels of FSH before age of 40 years. Genetic anomalies are among the recognized causes of POI. Here, we aimed to identify the genetic cause of POI in an inbred pedigree with nine POI and two ichthyosis-affected members. Inheritance of POI and ichthyosis were, respectively, dominant and recessive. Reproduction-related information and measurements of relevant hormones were obtained. Genetic studies included homozygosity mapping, linkage analysis, exome sequencing, and screening of candidate variants. A mutation within ALOX12B, which is a known ichthyosis causing gene, was identified as cause of ichthyosis. ALOX12B encodes a protein involved in steroidogenesis and lipid metabolism. Considering the importance of steroidogenesis in reproduction functions, the possibility that the ALOX12B mutation is also cause of POI was considered. Screenings showed that the mutation segregated with POI status. Linkage analysis with respect to POI identified a single strongly linked locus (LOD > 3) that includes ALOX12B. Exome sequencing on POI-affected females identified the mutation in ALOX12B and also a sequence variation in SPNS2 within the linked locus. A possible contribution of the SPNS2 variation to POI was not strictly ruled out, but various data presented in the text including reported association of variations in related gene ALOX12 with menopause-age and role of ALOX12B in atretic bovine follicle formation argue in favor of ALOX12B. It is, therefore, concluded that the mutation in ALOX12B is the likely cause of POI in the pedigree.

Eicosanoid blood vessel regulation in physiological and pathological states.

Arachidonic acid can be metabolized in blood vessels by three primary enzymatic pathways; cyclooxygenase (COX), lipoxygenase (LO), and cytochrome P450 (CYP). These eicosanoid metabolites can influence endothelial and vascular smooth muscle cell function. COX metabolites can cause endothelium-dependent dilation or constriction. Prostaglandin I2 (PGI2) and thromboxane (TXA2) act on their respective receptors exerting opposing actions with regard to vascular tone and platelet aggregation. LO metabolites also influence vascular tone. The 12-LO metabolite 12S-hydroxyeicosatrienoic acid (12S-HETE) is a vasoconstrictor whereas the 15-LO metabolite 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA) is an endothelial-dependent hyperpolarizing factor (EDHF). CYP enzymes produce two types of eicosanoid products: EDHF vasodilator epoxyeicosatrienoic acids (EETs) and the vasoconstrictor 20-HETE. The less-studied cross-metabolites generated from arachidonic acid metabolism by multiple pathways can also impact vascular function. Likewise, COX, LO, and CYP vascular eicosanoids interact with paracrine and hormonal factors such as the renin-angiotensin system and endothelin-1 (ET-1) to maintain vascular homeostasis. Imbalances in endothelial and vascular smooth muscle cell COX, LO, and CYP metabolites in metabolic and cardiovascular diseases result in vascular dysfunction. Restoring the vascular balance of eicosanoids by genetic or pharmacological means can improve vascular function in metabolic and cardiovascular diseases. Nevertheless, future research is necessary to achieve a more complete understanding of how COX, LO, CYP, and cross-metabolites regulate vascular function in physiological and pathological states.

Leukocytes from obese individuals exhibit an impaired SPM signature.

Specialized proresolving mediators (SPMs) biosynthesized from docosahexaenoic acids (DHAs) including resolvins (Rvs), protectins, and maresins are potent endogenous autacoids that actively resolve inflammation, protect organs, and stimulate tissue regeneration. Our hypothesis was that failure of resolution programs may lead to unremitting inflammation in obesity, contributing to the development of metabolic comorbidities in this condition. Obese individuals with persistent low-grade systemic inflammation showed reduced leukocyte production of the DHA-derived monohydroxy fatty acid 17-hydroxy-DHA (HDHA) and unbalanced formation of SPMs (in particular D-series Rvs) accompanied by enhanced production of proinflammatory lipid mediators such as leukotriene B4. Mechanistic studies attributed this impairment to reduced 15-lipoxygenase (LOX) activity rather than altered DHA cellular uptake. Moreover, leukocytes from obese individuals exhibited decreased 5-LOX levels and reduced 5-LOX Ser271 phosphorylation and distinct intracellular 5-LOX redistribution. However, 15-LOX appears to be the most critical factor for the deficient production of SPMs by obese leukocytes because the formation of D-series Rvs was completely rescued by incubation with the intermediate precursor 17-HDHA. These data provide proof of concept that administration of intermediate precursors of SPM biosynthesis (e.g., 17-HDHA) could be more efficient in overriding impaired formation of these proresolving lipid mediators in conditions characterized by dysfunctional LOX activity, such as obesity.-López-Vicario, C., Titos, E., Walker, M. E., Alcaraz-Quiles, J., Casulleras, M., Durán-Güell, M., Flores-Costa, R., Pérez-Romero, N., Forné, M., Dalli, J., Clària, J. Leukocytes from obese individuals exhibit an impaired SPM signature.

Enzymatic studies with 3-oxa n-3 DPA.

Cyclooxygenase-2 and several lipoxygenases convert polyunsaturated fatty acids into a large variety of products. During inflammatory processes, these enzymes form several distinct families of specialized pro-resolving lipid mediators possessing potent anti-inflammatory and pro-resolving effects. These mediators have attracted a great interest as leads in drug discovery and have recently been the subject of biosynthetic pathway studies using docosahexaenoic and n-3 docosapentaenoic acid as substrates. Herein we present enzymatic studies with cyclooxygenase-2 and 5-, 12- and 15-lipoxygenase enzymes using 3-oxa n-3 DPA as a synthetic mimic of n-3 docosapentaenoic acid. Structural elucidation based on data from RP-HPLC UV and LC/MS-MS experiments enabled the identification of novel enzymatically formed products. These findings constitute the basis for further biosynthetic studies towards understanding the mechanisms regulating substrate utilization in the biosynthesis of specialized pro-resolving lipid mediators.

Fatty Acid Mediators in the Tumor Microenvironment.

Patients with cancer frequently overexpress inflammatory cytokines with an associated neutrophilia both of which may be downregulated by diets with high omega-3 polyunsaturated fatty acids (ω-3 PUFA). The anti-inflammatory activity of dietary ω-3 PUFA has been suggested to have anticancer properties and to improve survival of cancer patients. Currently, the majority of dietary research efforts do not differentiate between obesity and dietary fatty acid consumption as mediators of inflammatory cell expansion and tumor microenvironmental infiltration, initiation, and progression. In this chapter, we discuss the relationships between dietary lipids, inflammation, neoplasia and strategies to regulate these relationships. We posit that dietary composition, notably the ratio of ω-3 vs. ω-6 PUFA, regulates tumor initiation and progression and the frequency and sites of metastasis that, together, impact overall survival (OS). We focus on three broad topics: first, the role of dietary lipids in chronic inflammation and tumor initiation, progression, and regression; second, lipid mediators linking inflammation and cancer; and third, dietary lipid regulation of murine and human tumor initiation, progression, and metastasis.