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To rigorously explore the role of omega-3 polyunsaturated fatty acids (PUFA) in the treatment of diabetic peripheral neuropathy (DPN), we have created a transgenic mouse utilizing a Cre-lox promoter to control overexpression of human 15-lipoxygenase-1 (15-LOX-1). In this study, we sought to determine the effect of treating type 2 diabetic wild-type mice and transgenic mice ubiquitously overexpressing 15-LOX-1 with menhaden oil on endpoints related to DPN. Wild-type and transgenic mice on a C57Bl/6J background were divided into three groups. Two of each of these groups were used to create a high-fat diet/streptozotocin model for type 2 diabetes. The remaining mice were control groups. Four weeks later, one set of diabetic mice from each group was treated with menhaden oil for twelve weeks and then evaluated using DPN-related endpoints. Studies were also performed using dorsal root ganglion neurons isolated from wild-type and transgenic mice. Wild-type and transgenic diabetic mice developed DPN as determined by slowing of nerve conduction velocity, decreased sensory nerve fibers in the skin and cornea, and impairment of thermal and mechanical sensitivity of the hindpaw compared to their respective control mice. Although not significant, there was a trend for the severity of these DPN-related deficits to be less in the diabetic transgenic mice compared to the diabetic wild-type mice. Treating diabetic wild-type and transgenic mice with menhaden oil improved the DPN-related endpoints with a trend for greater improvement or protection by menhaden oil observed in the diabetic transgenic mice. Treating dorsal root ganglion neurons with docosahexanoic acid but not eicosapentaenoic acid significantly increased neurite outgrowth with greater efficacy observed with neurons isolated from transgenic mice. Targeting pathways that will increase the production of the anti-inflammatory metabolites of omega-3 PUFA may be an efficacious approach to developing an effective treatment for DPN.

Many enzyme reactions present instantaneous disorder. These dynamic fluctuations in the enzyme-substrate Michaelis complexes generate a wide range of energy barriers that cannot be experimentally observed, but that determine the measured kinetics of the reaction. These individual energy barriers can be calculated using QM/MM methods, but then the problem is how to deal with this dispersion of energy barriers to provide kinetic information. So far, the most usual procedure has implied the so-called exponential average of the energy barriers. In this paper, we discuss the foundations of this method, and we use the free energy perturbation theory to derive an alternative equation to get the Gibbs free energy barrier of the enzyme reaction. In addition, we propose a practical way to implement it. We have chosen four enzyme reactions as examples. In particular, we have studied the hydrolysis of a glycosidic bond catalyzed by the enzyme Thermus thermophilus β-glycosidase, and the mutant Y284P Ttb-gly, and the hydrogen abstraction reactions from C13 and C7 of arachidonic acid catalyzed by the enzyme rabbit 15-lipoxygenase-1.

Nucleic acids Research 49(6), 3507-3523 (2021). doi:10.1093/nar/gkab116, Published by Oxford Univ. Press, Oxford

Lipoxygenases are widespread enzymes that catalyze oxidation of polyunsaturated fatty acids (linoleic, linolenic, and arachidonic acid) to produce hydroperoxides. Lipoxygenase reactions can be desirable, but also lipoxygenases can react in undesirable ways. Most of the products of lipoxygenase reactions are aromatic compounds that can affect food properties, especially during long-term storage. Lipoxygenase action on unsaturated fatty acids could result in off-flavor/off-odor development, causing food spoilage. In addition, lipoxygenases are present in the human body and play an important role in stimulation of inflammatory reactions. Inflammation is linked to many diseases, such as cancer, stroke, and cardiovascular and neurodegenerative diseases. This review summarized recent research on plant families and species that can inhibit lipoxygenase activity.

N-Arachidonoyl-ethanolamine (AEA) is an endocannabinoid (eCB) and endogenous lipid mimicking many of the effects of Δ9-tetrahydrocannabinol, notably on brain functions, appetite, pain and inflammation. The eCBs and eCB-like compounds contain fatty acids, the main classes being the monoacylglycerols and the N-acyl-ethanolamines (NAEs). Thus, each long chain fatty acid likely exists under the form of a monoacylglycerol and NAE, as it is the case for arachidonic acid (AA) and linoleic acid (LA). Following their biosynthesis, AA and AEA can be further metabolized into additional eicosanoids, notably by the 15-lipoxygenase pathway. Thus, we postulated that NAEs possessing a 1Z,4Z-pentadiene motif, near their omega end, would be transformed into their 15-lipoxygenase metabolites. As a proof of concept, we investigated N-linoleoyl-ethanolamine (LAE). We successfully synthesized LEA and LEA-d4 as well as their 15-lipoxygenase-derived derivatives, namely 13-hydroxy-9Z,11E-octadecadienoyl-N-ethanolamine (13-HODE-EA) and 13-HODE-EA-d4, using Novozyme 435 immobilized on acrylic resin and soybean lipoxygenase respectively. We also show that both human 15-lipoxygenase-1 and -2 can biosynthesize 13-HODE-EA. Co-incubation of LEA and LA with either human 15-lipoxygenase led to the biosynthesis of 13-HODE-EA and 13-HODE in a ratio equal to or greater than 3:1, indicating that LEA is preferred to LA by these enzymes. Finally, we show that 13-HODE-EA is found in human saliva and skin and is a weak although selective TRPV1 agonist. The full biological importance of 13-HODE-EA remains to be explored.

Here we report the inhibitory effects of nine non-steroidal anti-inflammatory drugs (NSAIDs) on soybean 15-lipoxygenase (15-LOX) enzyme (EC 1.13.11.12) by three different methods; UV-absorbance, colorimetric and chemiluminescence methods. Only two drugs, Ibuprofen and Ketoprofen, exhibited enzyme inhibition by UV-absorbance method but none of the drug showed inhibition through colorimetric method. Chemiluminescence method was found highly sensitive for the identification of 15-LOX inhibitors and it was more sensitive and several fold faster than the other methods. All tested drugs showed 15-LOX-inhibition with IC50 values ranging from 3.52 ± 0.08 to 62.6 ± 2.15 µM by chemiluminescence method. Naproxen was the most active inhibitor (IC50 3.52 ± 0.08 µM) followed by Aspirin (IC50 4.62 ± 0.11 µM) and Acetaminophen (IC50 6.52 ± 0.14 µM). Ketoprofen, Diclofenac and Mefenamic acid showed moderate inhibitory profiles (IC50 24.8 ± 0.24 to 39.62 ± 0.27 µM). Piroxicam and Tenoxicam were the least active inhibitors with IC50 values of 62.6 ± 2.15 µM and 49.5 ± 1.13 µM, respectively. These findings are supported by expression analysis, molecular docking studies and density functional theory calculations. The expression analysis and flow cytometry apoptosis analysis were carried out using mononuclear cells (MNCs) which express both human 15-LOX and 5-LOX. Selected NSAIDs did not affect the cytotoxic activity of MNCs at IC50 concentrations and the cell death showed dose dependent effect. However, MNCs apoptosis increased only at the higher concentrations, demonstrating that these drugs may not induce loss of immunity in septic and other inflammatory conditions at the acceptable inhibitory concentrations. The data collectively suggests that NSAIDs not only inhibit COX enzymes as reported in the literature but soybean 15-LOX and MNCs LOXs are also inhibited at differential values. A comparison of the metabolomics studies of arachidonic acid pathway after inhibition of either COX or LOX enzymes may reconfirm these findings.

Chronic inflammation is now widely recognized to play important roles in many commonly occurring diseases, including COVID-19. The resolution response to this chronic inflammation is an active process governed by specialized pro-resolving mediators (SPMs) like the lipid mediators known as lipoxins. The biosynthesis of lipoxins is catalyzed by several lipoxygenases (LOXs) from arachidonic acid. However, the molecular details of the mechanisms involved are not well known yet. In this paper, we have combined molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations to analyze how reticulocyte 15-LOX-1 catalyzes the production of lipoxins from 5(

Background Lipid-metabolizing enzymes and their metabolites affect inflammation and fibrosis, but their roles in chronic kidney disease (CKD) have not been completely understood. Methods To clarify their role in CKD, we measured the mRNA levels of major lipid-metabolizing enzymes in 5/6 nephrectomized (Nx) kidneys of C57BL/6 J mice. Mediator lipidomics was performed to reveal lipid profiles of CKD kidneys. Results In 5/6 Nx kidneys, both mRNA and protein levels of Alox15 were higher when compared with those in sham kidneys. With respect to in situ hybridization, the mRNA level of Alox15 was higher in renal tubules of 5/6 Nx kidneys. To examine the role of Alox15 in CKD pathogenesis, we performed 5/6 Nx on Alox15−/− mice. Alox15−/− CKD mice exhibited better renal functions than wild-type mice. Interstitial fibrosis was also inhibited in Alox15−/− CKD mice. Mediator lipidomics revealed that Alox15−/− CKD mouse kidneys had significantly higher levels of PGD2 than the control. To investigate the effects of PGD2 on renal fibrosis, we administered PGD2 to TGF-β1-stimulated NRK-52E cells and HK-2 cells, which lead to a dose-dependent suppression of type I collagen and αSMA in both cell lines. Conclusion Increased PGD2 in Alox15−/− CKD mouse kidneys could inhibit fibrosis, thereby resulting in CKD improvement. Thus, Alox15 inhibition and PGD2 administration may be novel therapeutic targets for CKD. Supplementary Information The online version contains supplementary material available at 10.1007/s10157-021-02021-y.

Orientador: Katia Borgia Barbosa Pagnano Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: A Leucemia Mielóide Crônica (LMC) é uma neoplasia mieloproliferativa crônica caracterizada pela presença do cromossomo Filadélfia (Ph) e produção da fusão proteica BCR-ABL que possui atividade tirosina quinase. O tratamento da LMC é realizado com inibidores de tirosinoquinase (ITKs) e apesar das altas taxas de respostas obtidas com imatinibe, alguns pacientes são resistentes ou intolerantes ao tratamento, sendo necessário a troca para um ITK de segunda geração (nilotinibe ou dasatinibe). A identificação dos mecanismos de resistência pode contribuir para o desenvolvimento de novas estratégias para o tratamento dos casos resistentes. Em um trabalho anterior do nosso grupo foram identificados diversos genes e RNAs longos não codificantes diferencialmente expressos em pacientes responsivos e não responsivos ao dasatinibe, entre eles os genes 15-LOX-2 e PAR-4, ambos descritos como supressores tumorais em diversos tipos de cânceres. No entanto, o papel desses genes na LMC é pouco conhecido. O objetivo desse trabalho foi avaliar o perfil de expressão de 15-LOX-2 e PAR-4 na LMC e em doadores saudáveis e o papel desses genes na resistência aos ITKs. A expressão dos genes 15-LOX-2 e PAR-4 foram avaliadas através da técnica de q-PCR em amostras de doadores saudáveis e pacientes com LMC ao diagnóstico e após tratamento com ITKs (imatinibe e dasatinibe), em grupos resistentes ou responsivos ao tratamento. Células K562 foram transfectadas com vetor vazio (NEO) e vetor contendo PAR-4. Células K562 normais e transfectadas foram utilizadas para estudos funcionais. As análises de expressão gênica e expressão protéica foram realizadas através da técnica de q-PCR e Western blot. Citometria de fluxo foi utilizada para análises de ciclo celular por PI e de apoptose (Anexina V-PI). A técnica de MTT foi utilizada para avaliação de crescimento celular e citotoxicidade. Nossos resultados mostraram pela primeira vez que o gene 15-LOX-2 estava hiperexpresso nos pacientes com resposta ótima ao imatinibe/dasatinibe em relação aos pacientes resistentes aos ITKs. A cloroquina foi capaz de aumentar a expressão de 15-LOX-2 e PPAR-gama em células K562. Dessa forma, 15-LOX-2 pode ter um papel na resistência da LMC, uma vez que sua baixa expressão pode estar relacionada com a manutenção das células-tronco leucêmicas quiescentes através da regulação de PPAR-gamma. Embora novos estudos sejam necessários, os inibidores de autofagia, como a cloroquina, podem ser empregados para o aumento da expressão de 15-LOX-2 e ter um potencial terapêutico para pacientes com células leucêmicas remanescentes. Em relação ao PAR-4, observamos uma menor expressão na LMC em comparação com doadores saudáveis e maior expressão em pacientes responsivos ao imatinibe ou dasatinibe em relação aos pacientes resistentes. No entanto, o aumento da expressão de PAR-4 em células leucêmicas K562 não foi suficiente para alterar a sobrevida, viabilidade e sensibilidade dessas células aos ITKs ou à cloroquina. Nossa hipótese é que nessas células existam mecanismos para inibição da ação da PAR-4, como por exemplo, a fosforilação de AKT. A ativação de AKT pode inibir a atividade de PAR-4, e consequentemente manter a sobrevida e viabilidade das células leucêmicas. Demonstramos ainda que a cloroquina é capaz de aumentar a expressão de PAR-4 em células PAR-4 positivas. No presente estudo foi avaliado somente o aumento de PAR-4 intracelular, mas outros estudos mostraram a eficácia de PAR-4 secretado por células normais na indução de apoptose em células cancerígenas. Futuros estudos são necessários para melhor compreensão do papel de PAR-4 na resistência da LMC Abstract: Chronic myeloid leukemia (CML) is a chronic myeloproliferative neoplasm characterized by the presence of Philadelphia chromosome (Ph) and production of BCR-ABL fusion protein that has tyrosine kinase activity. Currently, CML is treated with tyrosine kinase inhibitors (TKIs). Despite the high rates of responses obtained with imatinib, some patients are resistant or intolerant to treatment and need to switch to second-generation TKIs (dasatinib or nilotinib). Identification of resistance mechanisms may contribute to the development of new strategies for the treatment of resistant cases. In a previous report of our group were identified several genes and long non-coding RNAs differentially expressed in CML patients¿ responders and non-responders to dasatinib treatment, including 15-LOX-2 and PAR-4 genes, both described as tumor suppressors in various types of cancers. However, the role of these genes in CML is poorly understood. This work aimed to evaluate the expression profile of 15-LOX-2 and PAR-4 in CML patients and the role of these genes in resistance to ITKs. Expression of the 15-LOX-2 and PAR-4 genes were assessed by the q-PCR technique in samples from healthy donors and patients with CML at diagnosis and responders or resistant to ITKs (imatinib and dasatinib). K562 cells were transfected with empty vector (NEO) and a vector containing PAR-4. Normal and transfected K562 cells were used for functional studies. Gene expression and protein expression analyses were performed using the q-PCR and Western blot technique. Flow cytometry was used for cell cycle analysis using PI and apoptosis (Annexin V-PI). The MTT technique was used for the evaluation of cell growth and cytotoxicity. Our results showed for the first time that the 15-LOX-2 gene was overexpressed in patients with optimal response to imatinib/dasatinib compared to patients resistant to ITKs. Chloroquine was able to increase the expression of 15-LOX-2 and PPAR-gamma in K562 cells. Thus, 15-LOX-2 may play a role in CML resistance; its low expression may be related to the maintenance of quiescent leukemic stem cells through the regulation of PPAR-gamma. Although further studies are necessary, autophagy inhibitors, such as chloroquine, may be employed to increase 15-LOX-2 expression and have a therapeutic potential for CML patients with remaining leukemic cells. In relation to PAR-4, we observed low expression in CML compared to healthy donors and high expression in patients responsive to imatinib or dasatinib in relation with patients resistant. However, increased PAR-4 expression in leukemic cells was not enough to alter the survival, viability and the sensitivity of these cells to ITKs or chloroquine. We hypothesize that in these cells there are mechanisms to inhibit the action of PAR-4, such as phosphorylation of AKT. Activation of AKT can inhibit PAR-4 activity and thereby maintain the survival and viability of leukemic cells. We also demonstrated that chloroquine was able to increase PAR-4 expression in PAR-4 positive cells. In the present study, we evaluated only the increase of intracellular PAR-4, while other studies have shown the efficacy of PAR-4 secreted by normal cells in inducing apoptosis in cancer cells. Further studies are needed to evaluate the role of PAR-4 in CML resistance Doutorado Clínica Médica Doutora em Ciências CAPES 8881.134556/2016-01

The significance of tunneling contributions to the rate constants of enzymatic reactions has been described at length using experimental evidence as well as theoretical computations. Within the frame of variational transition state theory (VTST), tunneling corrections are included using the so-called ground-state tunneling transmission coefficient. For the calculation of those enzymatic rate constants using the ensemble-averaged extension of VTST on QM/MM potential energy surfaces, the transmission coefficient at a given temperature is averaged over a set of coefficient values, each one obtained from an individual minimum energy path (MEP). However, the calculation of accurate QM/MM MEPs for tunneling calculations, also using a reliable QM method like DFT, is highly costly in enzyme models. For this reason, more affordable methodologies have been used. In this paper, we validate a feasible computational strategy to compute multidimensional tunneling corrections that describes better than cheaper alternatives the physics of the hydrogen abstraction from linoleic acid catalyzed by the enzyme 15-rLOX-1. Our recommendations to obtain better values of kinetic isotope effects and, especially, of rate constants are based on multidimensional small-curvature tunneling (SCT) coefficients derived from electrostatic embedding QM(DFT)/MM MEPs. The MEPs used must be calculated with a small enough step-size. Also, the number of gradients and Hessians along the reaction path must be checked to cover the whole tunneling region and to obtain converged adiabatic potential energy profiles. Distinguished reaction coordinates (DCPs) that are commonly used to describe enzyme reaction mechanisms are not adequate for tunneling calculations in such biological systems.

The oxylipins, 5S,12S-dihydroxy-6E,8Z,10E,14Z-eicosatetraenoic acid (5S,12S-diHETE) and 5S,15S-dihydroxy-6E,8Z,11Z,13E-eicosatetraenoic acid (5S,15S-diHETE), have been identified in cell exudates and have chemotactic activity toward eosinophils and neutrophils. Their biosynthesis has been proposed to occur by sequential oxidations of arachidonic acid (AA) by lipoxygenase enzymes, specifically through oxidation of AA by h5-LOX followed by h12-LOX, h15-LOX-1, or h15-LOX-2. In this work, h15-LOX-1 demonstrates altered positional specificity when reacting with 5S-HETE, producing 90% 5S,12S-diHETE, instead of 5S,15S-diHETE, with kinetics 5-fold greater than that of h12-LOX. This is consistent with previous work in which h15-LOX-1 reacts with 7S-HDHA, producing the noncanonical, DHA-derived, specialized pro-resolving mediator, 7S,14S-diHDHA. It is also determined that oxygenation of 5S-HETE by h15-LOX-2 produces 5S,15S-diHETE and its biosynthetic k(cat)/K(M) flux is 2-fold greater than that of h15-LOX-1, suggesting that h15-LOX-2 may have a greater role in lipoxin biosynthesis than previously thought. In addition, it is shown that oxygenation of 12S-HETE and 15S-HETE by h5-LOX is kinetically slow, suggesting that the first step in the in vitro biosynthesis of both 5S,12S-diHETE and 5S,15S-diHETE is the production of 5S-HETE.

Hunting small molecules as anti-inflammatory agents/drugs is an expanding and successful approach to treat several inflammatory diseases such as cancer, asthma, arthritis, and psoriasis. Besides other methods, inflammatory diseases can be treated by lipoxygenase inhibitors, which have a profound influence on the development and progression of inflammation. In the present study, a series of new N-alkyl/aralky/aryl derivatives (7a-o) of 2-(4-phenyl-5-(1-phenylcarbamoyl)piperidine-4H-1,2,4-triazol-3-ylthio)acetamide was synthesized and screened for their inhibitory potential against the enzyme 15-lipoxygenase. The simple precursor ethyl piperidine-4-carboxylate (a) was successively converted into phenylcarbamoyl derivative (1), hydrazide (2), semicarbazide (3) and N-phenylated 5-(1-phenylcarbamoyl)piperidine-1,2,4-triazole (4), then in combination with electrophiles (6a-o) through further multistep synthesis, final products (7a-o) were generated. All the synthesized compounds were characterized by FTIR, 1H, 13C NMR spectroscopy, EIMS, and HREIMS spectrometry. Almost all the synthesized compounds showed excellent inhibitory potential against the tested enzyme. Compounds 7c, 7f, 7d, and 7g displayed potent inhibitory potential (IC50 9.25 ± 0.26 to 21.82 ± 0.35 µM), followed by the compounds 7n, 7h, 7e, 7a, 7b, 7l, and 7o with IC50 values in the range of 24.56 ± 0.45 to 46.91 ± 0.57 µM. Compounds 7c, 7f, 7d exhibited 71.5 to 83.5% cellular viability by MTT assay compared with standard curcumin (76.9%) when assayed at 0.125 mM concentration. In silico ADME studies supported the drug-likeness of most of the molecules. In vitro inhibition studies were substantiated by molecular docking wherein the phenyl group attached to the triazole ring was making a π-δ interaction with Leu607. This work reveals the possibility of a synthetic approach of compounds in relation to lipoxygenase inhibition as potential lead compounds in drug discovery.

Background Eosinophilic chronic rhinosinusitis with nasal polyps (ECRSwNP) exhibits a poorer outcome compared with non-eosinophilic chronic rhinosinusitis with nasal polyps (nonECRSwNP), so it is significant to identify effective markers to differentiate ECRSwNP in guiding the treatment strategies of these patients. Although arachidonate 15-lipoxygenase (ALOX15) is positioned as a marker of eosinophilic inflammation, its study in differentiating ECRSwNP has not been reported. The aim of this study is to assess the potential of ALOX15 in distinguishing and predicting ECRSwNP. Methods Forty-eight patients with chronic rhinosinusitis with nasal polyps (CRSwNP), including 30 ECRSwNP and 18 nonECRSwNP patients, were enrolled. ALOX15 mRNA level was determined in polyps by real-time polymerase chain reaction (RT-PCR). The patients’ baseline characteristics were evaluated and analyzed for correlations with ALOX15. Receiver operating characteristic (ROC) curve was used to assess the predictive significance of the potential predictors for ECRSwNP. Results ALOX15 mRNA level was significantly higher in ECRSwNP patients than in nonECRSwNP patients (P r = 0.565, P r = 0.395, P = 0.006), olfaction scores (r = 0.400, P = 0.005), total visual analogue scale (VAS) symptom scores (r = 0.383, P = 0.007), ethmoid/maxillary sinus (E/M) ratio (r = 0.463, P = 0.001), and endoscopy scores (r = 0.409, P = 0.004). Logistic regression analysis showed ALOX15 mRNA level and percentage of blood eosinophils to be predictive factors for ECRSwNP (P = 0.004 and P = 0.036, respectively). ROC curve indicated ALOX15 to have high predictive accuracy for ECRSwNP (area under the curve (AUC) = 0.909), which was further improved by combination of ALOX15 with percentage of blood eosinophils (AUC = 0.933). Conclusions The relative ALOX15 mRNA level alone or in combination with blood eosinophils might be a reliable biomarker for predicting a diagnosis of ECRSwNP.

Aim: Immune responsive 12/15 lipoxygenase (12/15LOX)-orchestrate biosynthesis of essential inflammation-resolution mediators during acute inflammatory response in post-myocardial infarction (MI). Lack of 12/15LOX dampens proinflammatory mediator 12-(S)-hydroxyeicosatetraenoic acid (12-(S)-HETE), improves post-MI survival, through the biosynthesis of endogenous mediators epoxyeicosatrienoic acids (EETs; cypoxins) to resolve post-MI inflammation. However, the mechanism that amplifies cypoxins-directed cardiac repair in acute heart failure (AHF) and chronic HF (CHF) remains of interest in MI-directed renal inflammation. Therefore, we determined the role of EETs in macrophage-specific receptor activation in facilitating cardiac repair in 12/15LOX deficient mice experiencing HF. Methods and Results: Risk-free young adult (8 -12 week-old) male C57BL/6J wild-type mice (WT; n = 43) and 12/15LOX-/- mice (n = 31) were subjected to permanent coronary artery ligation and monitored at day (d)1, d5 (as acute HF), and d28 to d56 (8 weeks; chronic HF) post-surgery maintaining no-MI mice that served as d0 naive controls. Left ventricle (LV) infarcted area of 12/15LOX-/- mice displayed an increase in expression of prostanoid receptor EP4 along with monocyte chemoattractant protein-1 CCL2 in AHF and CHF. The transcriptome analysis of isolated leukocytes (macrophages/neutrophils) from infarcted LV revealed a higher expression of EP4 on reparative macrophages expressing MRC-1 in 12/15LOX-/- mice. Deletion of 12/15LOX differentially modulated the miRNA levels, downregulating miR-23a-3p (~20 fold; p < 0.05) and upregulating miR-125a-5p (~160 fold; p < 0.05) in AHF which promoted polarization of the macrophages towards reparative phenotype. Furthermore, 12/15LOX deletion markedly attenuated renal inflammation with reduced levels of NGAL and KIM-1 and apoptotic markers in the kidney during CHF. Conclusion: In risk-free mice during physiological cardiac repair, absence of 12/15LOX promoted reparative macrophages with marked activation of EP4 signaling thereby improving post-MI survival and limiting renal inflammation in acute and advanced HF. The future studies are warranted to advance the role of EETs in macrophage receptor biology.

Grapes contain many flavonoid and non-flavonoid compounds with anticancer effects. In this work we fully characterized the polyphenolic profile of two grape skin extracts (GSEs), Autumn Royal and Egnatia, and assessed their effects on Polyunsaturated Fatty Acid (PUFA) membrane levels of Caco2 and SW480 human colon cancer cell lines. Gene expression of 15-lipoxygenase-1 (15-LOX-1), and peroxisome proliferator-activated receptor gamma (PPAR-&gamma, ), as well as cell morphology, were evaluated. The polyphenolic composition was analyzed by Ultra-High-Performance Liquid Chromatography/Quadrupole-Time of Flight mass spectrometry (UHPLC/QTOF) analysis. PUFA levels were evaluated by gas chromatography, and gene expression levels of 15-LOX-1 and PPAR-&gamma, were analyzed by real-time Polymerase Chain Reaction (PCR). Morphological cell changes caused by GSEs were identified by field emission scanning electron microscope (FE-SEM) and photomicrograph examination. We detected a different profile of flavonoid and non-flavonoid compounds in Autumn Royal and Egnatia GSEs. Cultured cells showed an increase of total PUFA levels mainly after treatment with Autumn Royal grape, and were richer in flavonoids when compared with the Egnatia variety. Both GSEs were able to affect 15-LOX-1 and PPAR-&gamma, gene expression and cell morphology. Our results highlighted a new antitumor mechanism of GSEs that involves membrane PUFAs and their downstream pathways.

Made available in DSpace on 2020-12-12T02:01:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-28 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Ethnopharmacological relevance: Terminalia catappa L. (Combretaceae), known as “amendoeira da praia” in Brazil, has been recognized as a medicinal plant in folk medicine for the treatment of gastrointestinal disorders and other inflammatory conditions. The present study aimed to investigate the preventive and healing effects of the infusion of leaves of T. catappa (ILTC) against gastric lesions caused by ischemia and reperfusion (I/R) injury and characterize its mechanism of action in the gastric mucosa of rats. Materials and methods: Different doses (30, 100, and 300 mg/kg) of ILTC were orally administered as acute and subacute treatments against I/R-induced gastric lesion in rats. After treatment, the stomach of rats was collected to measure the lesion area, redox parameters malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) and inflammatory parameters myeloperoxidase activity (MPO), interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α). The activities of matrix metalloproteinases 2 and 9 (MMPs 2 and 9) were assessed by zymography method to clarify the mechanisms of the healing acceleration promoted by ILTC. Results: Pretreatment with ILTC (100 mg/kg) was effective in preventing the aggravation of lesions in the acute model by reducing MPO activity by 38% relative to control group, despite the lack of clarity of this action at the macroscopical level at the lesion area (p < 0.05). After three days of treatment with ILTC (30 and 100 mg/kg), this infusion significantly reduced the lesion area by 95% and 89%, respectively, compared the control (p < 0.05). The gastric healing effect of all doses of ILTC was followed by a reduction in MPO activity (decrease by 70–78%). Compared to the negative control, an improvement in gastric healing owing to treatment with ILTC was observed and this was followed by an increase in MMP-2 (20–47%) (p < 0.05). Conclusion: Three days of treatment with ILTC could accelerate the healing process in I/R-induced lesions in rats. By decreasing MPO levels, ILTC enabled the action of MMP-2, which led to tissue recovery in the gastric mucosa. Department of Physiology Biosciences Institute UNESP-São Paulo State University, São Paulo Snyder Institute for Chronic Diseases Cumming School of Medicine University of Calgary Biosciences Institute UNESP-São Paulo State University, São Vicente, São Paulo Institute of Chemistry UNESP-São Paulo State University, São Paulo Laboratory of Extracellular Matrix UNESP- São Paulo State University, São Paulo Department of Physiology Biosciences Institute UNESP-São Paulo State University, São Paulo Biosciences Institute UNESP-São Paulo State University, São Vicente, São Paulo Institute of Chemistry UNESP-São Paulo State University, São Paulo Laboratory of Extracellular Matrix UNESP- São Paulo State University, São Paulo FAPESP: 2015/14797-3 CNPq: 301397/2017-1 CNPq: 306209/2016-0 CAPES: 88882.183585/2018-01 CAPES: 88882.183586/2018-01

Different fatty acids have distinct effects on the survival of breast cancer cells, which could be mediated by fatty acid binding proteins (FABPs), a family of lipid chaperones. Due to the diverse structures of the members of FABP family, each FABP demonstrates distinct binding affinities to different fatty acids. Of note, FABP7 is predominantly expressed in triple negative breast cancer (TNBC), the most aggressive subtype of breast cancer. Yet, the role of FABP7 in modulating the effects of fatty acids on TNBC survival was unclear. In contrast to the high expression of FABP7 in human TNBC tumours, FABP7 protein was undetectable in TNBC cell lines. Hence, a FABP7 overexpression model was used for this study, in which the transduced TNBC cell lines (MDA-MB-231 and Hs578T) were treated with various mono- and polyunsaturated fatty acids. Oleic acid (OA), docosahexaenoic acid (DHA) and arachidonic acid (AA) inhibited TNBC cell growth at high concentrations, with no differences resulted from FABP7 overexpression. Interestingly, overexpression of FABP7 augmented linoleic acid-induced cell death in MDA-MB-231 cells. The increased cell death may be explained by a decrease in 13-HODE, a pro-tumorigenic oxidation product of linoleic acid. The phenotype was, however, attenuated with a rescue treatment using 25 nM 13-HODE. The decrease in 13-HODE was potentially due to fatty acid partitioning modulated by FABP7, as demonstrated by a 3-fold increase in fatty acid oxidation. Our findings suggest that linoleic acid could be a potential therapeutic strategy for FABP7-overexpressing TNBC patients.

The two oxylipins 7S,14S-dihydroxydocosahexaenoic acid (diHDHA) and 7S,17S-diHDHA [resolvin D5 (RvD5)] have been found in macrophages and infectious inflammatory exudates and are believed to function as specialized pro-resolving mediators (SPMs). Their biosynthesis is thought to proceed through sequential oxidations of DHA by lipoxygenase (LOX) enzymes, specifically, by human 5-LOX (h5-LOX) first to 7(S)-hydroxy-4Z,8E,10Z,13Z,16Z,19Z-DHA (7S-HDHA), followed by human platelet 12-LOX (h12-LOX) to form 7(S),14(S)-dihydroxy-4Z,8E,10Z,12E,16Z,19Z-DHA (7S,14S-diHDHA) or human reticulocyte 15-LOX-1 (h15-LOX-1) to form RvD5. In this work, we determined that oxidation of 7(S)-hydroperoxy-4Z,8E,10Z,13Z,16Z,19Z-DHA to 7S,14S-diHDHA is performed with similar kinetics by either h12-LOX or h15-LOX-1. The oxidation at C14 of DHA by h12-LOX was expected, but the noncanonical reaction of h15-LOX-1 to make over 80% 7S,14S-diHDHA was larger than expected. Results of computer modeling suggested that the alcohol on C7 of 7S-HDHA hydrogen bonds with the backbone carbonyl of Ile399, forcing the hydrogen abstraction from C12 to oxygenate on C14 but not C17. This result raised questions regarding the synthesis of RvD5. Strikingly, we found that h15-LOX-2 oxygenates 7S-HDHA almost exclusively at C17, forming RvD5 with faster kinetics than does h15-LOX-1. The presence of h15-LOX-2 in neutrophils and macrophages suggests that it may have a greater role in biosynthesizing SPMs than previously thought. We also determined that the reactions of h5-LOX with 14(S)-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-DHA and 17(S)-hydroperoxy-4Z,7Z,10Z,13Z,15E,19Z-DHA are kinetically slow compared with DHA, suggesting that these reactions may be minor biosynthetic routes in vivo. Additionally, we show that 7S,14S-diHDHA and RvD5 have anti-aggregation properties with platelets at low micromolar potencies, which could directly regulate clot resolution.

Human reticulocyte 15-lipoxygenase-1 (h15-LOX-1 or ALOX15) and platelet 12-lipoxygenase (h12-LOX or ALOX12) catalysis of docosahexaenoic acid (DHA) and the maresin precursor, 14S-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid (14S-HpDHA), were investigated to determine their product profiles and relative rates in the biosynthesis of the key maresin intermediate, 13S,14S-epoxy-4Z,7Z,9E,11E,16Z,19Z-docosahexaenoic acid (13S,14S-epoxy-DHA). Both enzymes converted DHA to 14S-HpDHA, with h12-LOX having a 39-fold greater k(cat)/K(M) value (14.0 ± 0.8 s(−1) μM(−1)) than that of h15-LOX-1 (0.36 ± 0.08 s(−1) μM(−1)) and a 1.8-fold greater 14S-HpDHA product selectivity, 81 and 46%, respectively. However, h12-LOX was markedly less effective at producing 13S,14S-epoxy-DHA from 14S-HpDHA than h15-LOX-1, with a 4.6-fold smaller k(cat)/K(M) value, 0.0024 ± 0.0002 and 0.11 ± 0.006 s(−1) μM(−1), respectively. This is the first evidence of h15-LOX-1 to catalyze this reaction and reveals a novel in vitro pathway for maresin biosynthesis. In addition, epoxidation of 14S-HpDHA is negatively regulated through allosteric oxylipin binding to h15-LOX-1 and h12-LOX. For h15-LOX-1, 14S-HpDHA (K(d) = 6.0 μM), 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12S-HETE) (K(d) = 3.5 μM), and 14S-hydroxy-7Z,10Z,12E,16Z,19Z-docosapentaenoic acid (14S-HDPA(ω-3)) (K(d) = 4.0 μM) were shown to decrease 13S,14S-epoxy-DHA production. h12-LOX was also shown to be allosterically regulated by 14S-HpDHA (K(d) = 3.5 μM) and 14S-HDPA(ω-3) (K(d) = 4.0 μM); however, 12S-HETE showed no effect, indicating for the first time an allosteric response by h12-LOX. Finally, 14S-HpDHA inhibited platelet aggregation at a submicrololar concentration, which may have implications in the benefits of diets rich in DHA. These in vitro biosynthetic pathways may help guide in vivo maresin biosynthetic investigations and possibly direct therapeutic interventions.

Type 2-associated goblet cell hyperplasia and mucus hypersecretion are well known features of asthma. 15-Lipoxygenase-1 (15LO1) is induced by the type 2 cytokine IL-13 in human airway epithelial cells (HAECs) in vitro and is increased in fresh asthmatic HAECs ex vivo. 15LO1 generates a variety of products, including 15-hydroxyeicosatetraenoic acid (15-HETE), 15-HETE-phosphatidylethanolamine (15-HETE-PE), and 13-hydroxyoctadecadienoic acid (13-HODE). In this study, we investigated the 15LO1 metabolite profile at baseline and after IL-13 treatment, as well as its influence on goblet cell differentiation in HAECs. Primary HAECs obtained from bronchial brushings of asthmatic and healthy subjects were cultured under air-liquid interface culture supplemented with arachidonic acid and linoleic acid (10 μM each) and exposed to IL-13 for 7 days. Short interfering RNA transfection and 15LO1 inhibition were applied to suppress 15LO1 expression and activity. IL-13 stimulation induced expression of 15LO1 and preferentially generated 15-HETE-PE in vitro, both of which persisted after removal of IL-13. 15LO1 inhibition (by short interfering RNA and chemical inhibitor) decreased IL-13-induced forkhead box protein A3 (FOXA3) expression and enhanced FOXA2 expression. These changes were associated with reductions in both mucin 5AC and periostin. Exogenous 15-HETE-PE stimulation (alone) recapitulated IL-13-induced FOXA3, mucin 5AC, and periostin expression. The results of this study confirm the central importance of 15LO1 and its primary product, 15-HETE-PE, for epithelial cell remodeling in HAECs.

To produce high concentrations of 13-hydroxy-14,15-epoxy-eicosatrienoic acid (14,15-hepoxilin BThe products obtained from the bioconversion of ARA by recombinant Escherichia coli cells containing Archangium violaceum 15-LOX without and with Myxococcus xanthus EH were identified as 14,15-HXBThese are the highest concentrations, productivities, and yields of hepoxilin and trioxilin from ARA reported thus far.

Lipid mediators play important roles in regulating inflammatory responses and tissue homeostasis. Since 12/15-lipoxygenase (12/15-LOX)-derived lipid mediators such as lipoxin A4 (LXA4 ) and protectin D1 (PD1) protect against corneal epithelial cell damage, the major cell types that express 12/15-LOX and contribute to the corneal wound healing process are of particular interest. Here, we found that eosinophils were the major cell type expressing 12/15-LOX during the corneal wound healing process. Eosinophils were recruited into the conjunctiva after corneal epithelium wounding, and eosinophil-deficient and/or eosinophil-specific 12/15-LOX knockout mice showed delayed corneal wound healing compared with wild-type mice. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based mediator lipidomics revealed that a series of 12/15-LOX-derived mediators were significantly decreased in eosinophil-deficient mice and topical application of 17-hydroxydocosahexaenoic acid (17-HDoHE), a major 12/15-LOX-derived product, restored the phenotype. These results indicate that 12/15-LOX-expressing eosinophils, by locally producing pro-resolving mediators, significantly contribute to the corneal wound healing process in the eye.

Autoimmune diseases are defined by an immune response against a specific autoantigen, driven by antigen-specific T cells or antibodies. While the mechanisms resolving brief episodes of acute inflammation elicited by microbial components or tissue injury are well understood, the mechanisms resolving tissue inflammation in autoimmune diseases are still largely elusive. We have, therefore, addressed the mechanisms of resolution in IgG-mediated autoimmune diseases using a mouse model of the pemphigoid disease ""bullous pemphigoid-like epidermolysis bullosa acquisita"" (BP-like EBA) as prototypical example. We found that 12/15-LO is induced in skin lesions of BP-like EBA and is predominantly expressed in eosinophils. Dependent on the expression of 12/15-LO, large amounts of proresolving lipid mediators, are biosynthesized in the skin by the point disease peaks. Their production is timely correlated to the gradual reversal of tissue inflammation. Genetic deficiency in Alox15, the gene encoding 12/15-LO, disrupts this process significantly protracting and aggravating disease. This protraction is associated reduced recruitment of regulatory T cells (Tregs) into lesional skin. Intriguingly, Alox15−/− mice also exhibit reduced recruitment of eosinophils into the skin, and the chemotaxis of cultured Alox15−/− eosinophils towards CCL11/eotaxin-1 is compromised. Finally, we demonstrate that 15-lipoxygenase-1, the human homologue of 12/15-LO is induced in granulocytes in lesional skin of patients suffering from a pemphigoid disease. Collectively, our result uncover key mechanisms resolving IgG-mediated skin inflammation. These mechanisms are orchestrated by 12/15-LO expressed in eosinophils promoting the recruitment of eosinophils and Tregs, which in turn inhibit neutrophils.

Ferroptotic death is the penalty for losing control over three processes-iron metabolism, lipid peroxidation and thiol regulation-that are common in the pro-inflammatory environment where professional phagocytes fulfill their functions and yet survive. We hypothesized that redox reprogramming of 15-lipoxygenase (15-LOX) during the generation of pro-ferroptotic signal 15-hydroperoxy-eicosa-tetra-enoyl-phosphatidylethanolamine (15-HpETE-PE) modulates ferroptotic endurance. Here, we have discovered that inducible nitric oxide synthase (iNOS)/NO•-enrichment of activated M1 (but not alternatively activated M2) macrophages/microglia modulates susceptibility to ferroptosis. Genetic or pharmacologic depletion/inactivation of iNOS confers sensitivity on M1 cells, whereas NO• donors empower resistance of M2 cells to ferroptosis. In vivo, M1 phagocytes, in comparison to M2 phagocytes, exert higher resistance to pharmacologically induced ferroptosis. This resistance is diminished in iNOS-deficient cells in the pro-inflammatory conditions of brain trauma or the tumour microenvironment. The nitroxygenation of eicosatetraenoyl (ETE)-PE intermediates and oxidatively truncated species by NO• donors and/or suppression of NO• production by iNOS inhibitors represent a novel redox mechanism of regulation of ferroptosis in pro-inflammatory conditions.

15-lipoxygenase (15-LOX) is a critical enzyme that allows the direction of arachidonic acid metabolism to change from inflammation into the resolution. This study aims to reveal how the immunomodulation properties of mesenchymal stem cells (MSC) alter by the 15-LOX overexpression. For this purpose, peripheral blood mononuclear cells (PBMCs) isolated from seven healthy volunteers, and both MSCs and 15-LOX overexpressing MSCs (15-LOXMSCs) were co-cultured at different cell ratios (1/1, 1/5 and 1/10). Alterations of CD4+Tbet+, CD4+Gata3+, CD4+RoRC2+, and CD4+FoxP3+ lymphocyte frequencies were detected by flow cytometry, and IFN-?, IL-4, IL-6, IL-10, IL-17a, TGF-? and LXA4 levels of medium supernatants were measured by ELISA method. According to our findings, MSC and 15-LOXMSCs have a suppressive effect on PHA activated PBMCs. However, as the ratio of PBMCs increased, the effects of 15-LOXMSCs increased significantly, while the effects of MSCs decreased. The most notable effect of the 15-LOX modification was the significant reduction in IL-6, IL-10 and IL-17a expression and the accompanying increase in TGF-? and LXA4 levels. We also observed a similar situation between CD4+RoRC2+ and CD4+FoxP3+ cell frequencies. These data suggested that the effects of MSCs on the balance of Th17 / Treg could change by the 15-LOX overexpression, and this might be in favor of the Treg cells. © 2021, 16-TIP-041, The Scientific Research Projects Commission of Ege University financially supported the project for this study, with the reference number 16-TIP-041 .

Fungal keratitis (FK) is one of the main causes of blindness in China. People with diabetes are susceptible to corneal epithelial disease, even fungal keratitis. At present, there are few studies on this disease. Resolvins (Rv) has been reported as a mediators that exert crucial anti-inflammatory and immune regulation roles in serval diseases. In order to investigate the roles and underlying mechanism of Resolvins D1 (RvD1) on the Aspergillus fumigatus (A. fumigatus) keratitis in diabetes, we established in vivo and in vitro models of A. fumigatus keratitis, which were then exposed to high glucose. The expression levels of RvD1, 5-lipoxygenase (5-LOX), and 15-lipoxygenase (15-LOX) in A. fumigatus keratitis patients with diabetes were determined through Enzyme Linked Immunosorbent Assay (ELISA), Western blot and immunohistochemistry. Reactive Oxygen Species (ROS) production, ELISA, flow cytometry, Hematoxylin-Eosin (HE) staining and fungal loading determination were conducted to evaluate the severity of A. fumigatus infection. Lymphangiogenesis and angiogenesis were examined by immunofluorescence assay. Western blot was applied to detect the proteins of the MAPK-NF-κB pathway. The results showed that RvD1 diminished the high glucose-induced oxidative stress and inflammatory response, as evidenced by the reduction of ROS production, Interleukin-6 (IL-6), Interleukin-8 (IL-8), Heme Oxygenase-1 (HMOX-1), and the elevation of Cyclooxygenase-2 (COX2), Superoxide Dismutase (SOD-1), and Glutathione Peroxidase-2 (GPX2) levels in A. fumigatus-infected Human Corneal Endothelial Cells (HCECs). Additionally, lymphangiogenesis and angiogenesis prominently decreased after intervention with RvD1. Furthermore, RvD1 significantly reduced the levels of p-MEK1/2 and p-ERK1/2, and restrained the NF-κB and GPR32 activation. The above results showed that RvD1 protects against A. fumigatus keratitis in diabetes by suppressing oxidative stress, inflammatory response, fungal growth, and immunoreaction via modulating MAPK-NF-κB pathway. RvD1 provides clues for the therapeutic targets of Fungal keratitis complicated with diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disease caused by proinflammatory T cell infiltration associated cytokine secretion and reactive oxygen species (ROS) production. In this study, our objective was to determine whether co-delivery of sunitinib, a member of tyrosine kinase inhibitor (TKI) family, and siRNA against Alox15 (siAlox15) could reverse the new-onset of T1D in non-obese diabetic (NOD)/ShiLtJ female mice which spontaneously develop diabetes. A cationic copolymer methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol-graft-tetraethylenepentamine) (mPEG-b-PCC-g-DC-g-TEPA) was used to encapsulate sunitinib with 8.32-9.39% loading efficacy and form complex with siAlox15 at the N/P ratio of 16:1. These micellar formulations exhibited sustained release of sunitinib for around 50% at 24 h and protected siAlox15 from serum degradation for at least 10 h. Further, both sunitinib and siAlox15 inhibited human peripheral blood mononuclear cell (PBMC) proliferation and activation by downregulating the phosphorylation of Akt, platelet-derived growth factor receptor (PDGFR) at protein levels and Alox15 in mRNA levels, respectively. Besides, siAlox15 treatment reversed the cytokine induced ROS upregulation in rat INS-1E β cells. Finally, systemic administration of these micelles carrying sunitinib and siAlox15 efficiently reversed T1D in NOD mice as indicated by decreased blood glucose concentration to below 250 mg/dL. The mice showed fast response to high glucose which was determined by intraperitoneal glucose tolerance test and promoted serum insulin production. Tissue staining indicated that sunitinib and siAlox15 combination could inhibit T cell infiltration to mouse islets, thereby promoting islet survival and function. In summary, our results demonstrated that combination therapy of sunitinib and siAlox15 could be a novel, reliable and safe approach for patients who were recently diagnosed with T1D.

The reaction of 5 S,15 S-dihydroperoxyeicosatetraenoic acid (5,15-diHpETE) with human 5-lipoxygenase (LOX), human platelet 12-LOX, and human reticulocyte 15-LOX-1 was investigated to determine the reactivity and relative rates of producing lipoxins (LXs). 5-LOX does not react with 5,15-diHpETE, although it can produce LXA4 when 15-HpETE is the substrate. In contrast, both 12-LOX and 15-LOX-1 react with 5,15-diHpETE, forming specifically LXB4. For 12-LOX and 5,15-diHpETE, the kinetic parameters are kcat = 0.17 s-1 and kcat/ KM = 0.011 μM-1 s-1 [106- and 1600-fold lower than those for 12-LOX oxygenation of arachidonic acid (AA), respectively]. On the other hand, for 15-LOX-1 the equivalent parameters are kcat = 4.6 s-1 and kcat/ KM = 0.21 μM-1 s-1 (3-fold higher and similar to those for 12-HpETE formation by 15-LOX-1 from AA, respectively). This contrasts with the complete lack of reaction of 15-LOX-2 with 5,15-diHpETE [Green, A. R., et al. (2016) Biochemistry 55, 2832-2840]. Our data indicate that 12-LOX is markedly inferior to 15-LOX-1 in catalyzing the production of LXB4 from 5,15-diHpETE. Platelet aggregation was inhibited by the addition of 5,15-diHpETE, with an IC50 of 1.3 μM; however, LXB4 did not significantly inhibit collagen-mediated platelet activation up to 10 μM. In summary, LXB4 is the primary product of 12-LOX and 15-LOX-1 catalysis, if 5,15-diHpETE is the substrate, with 15-LOX-1 being 20-fold more efficient than 12-LOX. LXA4 is the primary product with 5-LOX but only if 15-HpETE is the substrate. Approximately equal proportions of LXA4 and LXB4 are produced by 12-LOX but only if LTA4 is the substrate, as described previously [Sheppard, K. A., et al. (1992) Biochim. Biophys. Acta 1133, 223-234].

The reaction specificity of lipoxygenases (ALOX) impacts the biological activity of these enzymes and also constitutes the decisive parameter for enzyme classification. ALOX15 orthologs, commonly known as 12/15-lipoxygenases, have recently been suggested to exhibit mainly arachidonic acid 12-lipoxygenating specificity in mammals ranked in evolution lower than gibbons and 15-lipoxygenating specificity in the higher ranking primates (Adel et al., PNAS 113, E4266–75). This hypothesis was worked out on the basis of the functional characteristics of 15 mammalian ALOX15 orthologs with a focus on three sequence determinants affecting substrate binding in the active site (Triad Concept). This review is aimed at summarizing the currently available functional data on the reaction specificity of mammalian ALOX15 orthologs and at analyzing the primary structures of 97 mammalian ALOX15 isoforms that have been deposited in public sequence databases. Our conclusions suggest that the reaction specificity of ALOX15 orthologs can be predicted by analyzing their amino acid sequence and that 98% of all mammalian ALOX15 orthologs follow the Evolutionary Hypothesis.

The green microalga Lobosphaera incisa is an oleaginous eukaryotic alga that is rich in arachidonic acid (20:4). Being rich in this polyunsaturated fatty acid (PUFA), however, makes it sensitive to oxidation. In plants, lipoxygenases (LOXs) are the major enzymes that oxidise these molecules. • Here, we describe, to our best knowledge, the first characterisation of a cDNA encoding a LOX (LiLOX) from a green alga. To obtain first insights into its function, we expressed it in E. coli, purified the recombinant enzyme and analysed its enzyme activity. • The protein sequence suggests that LiLOX and plastidic LOXs from bryophytes and flowering plants may share a common ancestor. The fact that LiLOX oxidises all PUFAs tested with a consistent oxidation on the carbon n-6, suggests that PUFAs enter the substrate channel through their methyl group first (tail first). Additionally, LiLOX form the fatty acid hydroperoxide in strict S configuration. • LiLOX may represent a good model to study plastid LOX, because it is stable after heterologous expression in E. coli and highly active in vitro. Moreover, as the first characterised LOX from green microalgae, it opens the possibility to study endogenous LOX pathways in these organisms. peerReviewed

Background It has been shown that dietary protein supplementation during lactation boosts immunity in Nippostrongylus brasiliensis-infected periparturient rats. It is not known whether body protein reserves accumulated during gestation have a similar effect during lactation. Objective This study aimed to quantify the impact of body protein reserves and dietary protein supplementation on maternal performance and immune responses to N. brasiliensis during lactation. Methods Multiparous female Sprague-Dawley rats were administered a primary infection of N. brasiliensis before mating and were restriction-fed either 60 g [low-protein diet gestation (Lge)] or 210 g [high-protein diet gestation (Hge)] crude protein (CP) per kilogram of dry matter (DM) until parturition. From parturition onward, dams were restriction-fed either 100 g [low-protein diet lactation (Lla)] or 300 g [high-protein diet lactation (Hla)] CP per kilogram of DM, generating 4 different dietary treatments. A subset of rats was sampled before parturition; postparturition, dams were secondarily infected with N. brasiliensis and samples were collected at days 5 and 11 postparturition. Results Maternal performance until parturition, as measured by pup weight, was better in Hge rats than in Lge rats [Lge: 4.84 g; Hge: 6.15 g; standard error of the difference (SED): 0.19]. On day 11, pup weights of dams with reduced protein reserves fed protein during lactation (Lge-Hla; 20.28 g) were higher than their counterparts from Hge-Lla dams (17.88 g; SED: 0.92). Worm counts were significantly different between Lge-Lla-fed (253; 95% CI: 124, 382) and Hge-Hla-fed (87; 95% CI: 22, 104) dams on day 11 (P = 0.024). The expression of splenic interleukin 13 (Il13) and arachidonate 15-lipoxygenase (Alox15) was significantly higher (P

Bioactive lipid mediators derived from n-3 and n-6 fatty acids are known to modulate leukocytes. Metabolic transformation of essential fatty acids to endogenous bioactive molecules plays a major role in human health. Here we tested the potential of substrates; linoleic acid (LA) and docosahexaenoic acid (DHA) and their bioactive products; resolvin D1 (RvD1) and 12- S-hydroxyeicosatetraenoic acids (HETE) to modulate macrophage plasticity and cardiac fibroblast phenotype in presence or absence of lipid metabolizing enzyme 12/15-lipoxygenase (LOX). Peritoneal macrophages and cardiac fibroblasts were isolated from wild-type (C57BL/6J) and 12/15LOX -/- mice and treated with DHA, LA, 12(S)-HETE, and RvD1 for 4, 8, 12, and 24 hr. LA, DHA, 12(S)-HETE, and RvD1 elicited mRNA expression of proinflammatory markers; tumor necrosis factor-α ( Tnf-α), interleukin 6 ( IL-6), chemokine (C-C motif) ligand 2 (Ccl2), and IL-1β in wild type (WT) and in 12/15LOX -/- macrophages at early time point (4 hr). Bioactive immunoresolvent RvD1 lowered the levels of Tnf-α, IL-6, and IL-1β at 24 hr time point. Both DHA and RvD1 stimulated the proresolving markers such as arginase 1 ( Arg-1), chitinase-like protein 3 ( Ym-1), and mannose receptor C-type 1 in WT macrophage. RvD1 induced proresolving phenotype Arg-1 expression in both WT 12/15LOX -/- macrophages even in presence of 12(S)-HETE. RvD1 peaked 5LOX expression in both WT and 12/15LOX -/- at 24 hr time point compared with DHA. RvD1 diminished cyclooxygenase-2 but upregulated 5LOX expression in fibroblast compared with DHA. In summary, the feed-forward enzymatic interaction with fatty acids substrates and direct mediators (RvD1 and 12(S)-HETE) are responsive in determining macrophages phenotype and cardiac fibroblast plasticity. Particularly, macrophages and fibroblast phenotypes are responsive to milieu and RvD1 governs the milieu-dependent chemokine signaling in presence or absence of 12/15LOX enzyme to resolve inflammation.

15-Lipoxygenase-2 (15-LOX-2) and 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) have been considered as latent mediators of diverse biological processes including cancer. However, their functions in lung adenocarcinoma (LAC) are unclear. In this study, we aimed to determine whether 15-LOX-2/15(S)-HETE is involved in the proliferation and migration of A549 cells and to identify the signaling pathways that participate in this process. We used immunohistochemistry to identify the expression levels of 15-LOX-2 in lung cancer tissue samples. The effects of 15(S)-HETE on the proliferation and migration of A549 cells under hypoxic conditions were assessed by cell viability assays, immunofluorescence, western blotting, scratch wound assays and transwell assays. We found that the expression of 15-LOX-2 was significantly up-regulated in LAC tissue samples compared with adjacent normal tissue samples. The content of 15(S)-HETE in A549 cells was increased under hypoxic conditions. Moreover, 15(S)-HETE could stimulate the expression of PCNA, cyclin A and cyclin D. In addition, siRNA of 15-LOX-2 inhibited the proliferation and migration of A549 cells in vitro. Our data also provide novel evidence demonstrating that the STAT3 pathway participates in the 15(S)-HETE-induced proliferation and migration of A549 cells. This study may provide a greater understanding of LAC metastasis and shed new light on the mechanisms by which the 15(S)-HETE/STAT3 pathway is related to this disease.

We studied the effect of SIRT1 deacetylase and PPARγ receptor activators on proinflammatory (M1), anti-inflammatory (M2) polarization of RAW264.7 macrophages and their modulating effects on insulin sensitivity of adipocytes. In M1 macrophages, the expression of TNFα and CXCL9, secretion of CXCL11, ROS generation, and content of dendritic-like cells were elevated. In M2 macrophages, expression of IGF-1 and ALOX15 factors was enhanced. SIRT1 activator (DCHC) and PPARγ receptor ligand (rosiglitazone) reduced expression of inflammatory markers TNFα and CXCL9 and increased expression of IGF-1 and ALOX15. SIRT1 inhibitor Ex527 increased the proportion of dendritic cells in macrophage populations. The paracrine effect of M1-macrophage-conditioned media attenuated insulin-dependent phosphorylation of threonine (Thr308) in Akt kinase and enhanced phosphorylation of serine (Ser473). This effect was attenuated by DCHC and rosiglitazone.

Lipoxygenases (ALOXs) are involved in the regulation of cellular redox homeostasis. They also have been implicated in the biosynthesis of pro- and anti-inflammatory lipid mediators and play a role in the pathogenesis of inflammatory diseases, which constitute a major health challenge owing to increasing incidence and prevalence in all industrialized countries around the world. To explore the pathophysiological role of Alox15 (leukocyte-type 12-LOX) in mouse experimental colitis we tested the impact of systemic inactivation of the Alox15 gene on the extent of dextrane sulfate sodium (DSS) colitis. We found that in wildtype mice expression of the Alox15 gene was augmented during DSS-colitis while expression of other Alox genes (Alox5, Alox15b) was hardly altered. Systemic Alox15 (leukocyte-type 12-LOX) deficiency induced less severe colitis symptoms and suppressed in vivo formation of 12-hydroxyeicosatetraenoic acid (12-HETE), the major Alox15 (leukocyte-type 12-LOX) product in mice. These alterations were paralleled by reduced expression of pro-inflammatory gene products, by sustained expression of the zonula occludens protein 1 (ZO-1) and by a less impaired intestinal epithelial barrier function. These results are consistent with in vitro incubations of colon epithelial cells, in which addition of 12S-HETE compromised enantioselectively transepithelial electric resistance. Consistent with these data transgenic overexpression of human ALOX15 intensified the inflammatory symptoms. In summary, our results indicate that systemic Alox15 (leukocyte-type 12-LOX) deficiency protects mice from DSS-colitis. Since exogenous 12-HETE compromises the expression of the tight junction protein ZO-1 the protective effect has been related to a less pronounced impairment of the intestinal epithelial barrier function.

Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) are the predominant gelatinases in the developing lung. Studies have shown that the expression of MMP-2 and MMP-9 is upregulated in hypoxic fibroblasts, 15-hydroxyeicosatetraenoic acid (15-HETE) regulated fibroblasts migration via modulating MMP-2 or MMP-9, and that hypoxia/15-HETE is a predominant contributor to the development of pulmonary arterial hypertension (PAH) through increased angiogenesis. However, the roles of MMP-2 and MMP-9 in pulmonary arterial endothelial cells (PAECs) angiogenesis as well as the molecular mechanism of hypoxia-regulated MMP-2 and MMP-9 expression have not been identified. The aim of this study was to investigate the role of MMP-2 and MMP-9 in PAEC proliferation and vascular angiogenesis and to determine the effects of hypoxia-induced 15-HETE on the expression of MMP-2 and MMP-9. Western blot, immunofluorescence, and real-time PCR were used to measure the expression of MMP-2 and MMP-9 in hypoxic PAECs. Immunohistochemical staining, flow cytometry, and tube formation as well as cell proliferation, viability, scratch-wound, and Boyden chamber migration assays were used to identify the roles and relationships between MMP-2, MMP-9, and 15-HETE in hypoxic PAECs. We found that hypoxia increased MMP-2 and MMP-9 expression in pulmonary artery endothelium both in vivo and in vitro in a time-dependent pattern. Moreover, administration of the MMP-2 and MMP-9 inhibitor MMI-166 significantly reversed hypoxia-induced increases in right ventricular systemic pressure (RVSP), right ventricular function, and thickening of the tunica media. Furthermore, up-regulation of MMP-2 and MMP-9 expression was induced by 15-HETE, which regulates PAEC proliferation, migration, and cell cycle transition that eventually leads to angiogenesis. Our study demonstrated that hypoxia increases the expression of MMP-2 and MMP-9 through the 15-lipoxygenase/15-HETE pathway, and that MMP-2 and MMP-9 promote PAEC angiogenesis. These findings suggest that MMP-2 and MMP-9 may serve as new potential therapeutic targets for the treatment of PAH.

We have previously shown that 15-lipoxygenase (15-LOX) and its metabolite 15-hydroxyeicosanoid (15-HETE) play a critical role on hypoxia-triggered pulmonary artery smooth muscle cell (PASMC) phenotype alteration through multifactorial pathways, like extracellular signal-regulated kinase and p38 mitogen-activated protein kinases. Here, we hypothesize that activator protein 1 (AP-1) was also involved in the PASMC phenotype alteration. Hypoxia elevated AP-1 expression in pulmonary arterials and in cultured PASMCs with a time-dependent manner. Both the gene disruption and pharmacological inactivation of 15-lipoxygenase (15-LOX) significantly attenuated the hypoxia-elevated AP-1 expression. Silencing of AP-1 with small interference RNA abrogated the hypoxia- and 15-HETE-increased cell viability, proliferating cell nuclear antigen expression, and Ki67 and α-tubulin staining. Moreover, AP-1 knockdown prevented hypoxia- and 15-HETE-promoted cyclin D1 expression and subsequent cell cycle progression into G2/M+S phase. Interestingly, AP-1 knockdown also inhibited the expression of 15-LOX, indicating a feedback regulation of 15-LOX/15-HETE signaling by AP-1. These findings shed light on the involvement of AP-1 in the PASMCs phenotype alteration via the hypoxia/15-LOX/15-HETE signaling.

The tumor immune landscape gained considerable interest based on the knowledge that genetic aberrations in cancer cells alone are insufficient for tumor development. Macrophages are basically supporting all hallmarks of cancer and owing to their tremendous plasticity they may exert a whole spectrum of anti-tumor and pro-tumor activities. As part of the innate immune response, macrophages are armed to attack tumor cells, alone or in concert with distinct T cell subsets. However, in the tumor microenvironment, they sense nutrient and oxygen gradients, receive multiple signals, and respond to this incoming information with a phenotype shift. Often, their functional output repertoire is shifted to become tumor-supportive. Incoming and outgoing signals are chemically heterogeneous but also comprise lipid mediators. Here, we review the current understanding whereby arachidonate metabolites derived from the cyclooxygenase and lipoxygenase pathways shape the macrophage phenotype in a tumor setting. We discuss these findings in the context of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) expression and concomitant prostaglandin E2 (PGE2) formation. We elaborate the multiple actions of this lipid in affecting macrophage biology, which are sensors for and generators of this lipid. Moreover, we summarize properties of 5-lipoxygenases (ALOX5) and 15-lipoxygenases (ALOX15, ALOX15B) in macrophages and clarify how these enzymes add to the role of macrophages in a dynamically changing tumor environment. This review will illustrate the potential routes how COX-2/mPGES-1 and ALOX5/-15 in macrophages contribute to the development and progression of a tumor.

12/15 lipoxygenase (LOX) directs inflammation and lipid remodeling. However, the role of 12/15LOX in post-myocardial infarction (MI) left ventricular remodeling is unclear. To determine the role of 12/15LOX, 8–12 week-old C57BL/6 J wild-type (WT; n = 93) and 12/15LOX−/− (n = 97) mice were subjected to permanent coronary artery ligation and monitored at day (d)1 and d5 post-operatively. Post-MI d28 survival was measured in male and female mice. No-MI surgery mice were maintained as d0 naive controls. 12/15LOX−/− mice exhibited higher survival rates with lower cardiac rupture and improved LV function as compared with WT post-MI. Compared to WT, neutrophils and macrophages in 12/15LOX−/− mice were polarized towards N2 and M2 phenotypes, respectively, with increased of expression mrc-1, ym-1, and arg-1 post-MI. 12/15LOX−/− mice exhibited lower levels of pro-inflammatory 12-(S)-hydroperoxyeicosatetraenoic acid (12(S)-HETE) and higher CYP2J-derived epoxyeicosatrienoic acids (EETs) levels. CYP2J-derived 5,6-, 8,9-, 11,12-, and 14,15-EETs activated macrophage-specific hemeoxygenase (HO)-1 marked with increases in F4/80+/Ly6Clow and F4/80+/CD206high cells at d5 post-MI in 12/15LOX−/− mice. In contrast, inhibition of HO-1 led to total mortality in 12/15LOX−/− mice by post-MI d5. 12/15LOX−/− mice exhibited reduced collagen density and lower α-smooth muscle actin (SMA) expression at d5 post-MI, indicating delayed or limited fibroblast-to-myofibroblast differentiation. In conclusion, genetic deletion of 12/15LOX reduces 12(S)-HETE and activates CYP2J-derived EETs to promote effective resolution of inflammation post-MI leading to reduced cardiac rupture, improved LV function, and better survival.

The 12-lipoxygenase (12LO) pathway is a promising target to reduce islet dysfunction, adipose tissue (AT) inflammation and insulin resistance. Optimal pre-clinical models for the investigation of selective12LO inhibitors in this context have not yet been identified. The objective of this study was to characterize the time course of 12LO isoform expression and metabolite production in pancreatic islets and AT of C57BLKS/J-db/db obese diabetic mouse in a pre-diabetic state in order to establish a suitable therapeutic window for intervention with selective lipoxygenase inhibitors. Mice have 2 major 12LO isoforms -the leukocyte type (12/15LO) and the platelet type (p12LO) and both are expressed in islets and AT. We found a sharp increase in protein expression of 12/15LO in the pancreatic islets of 10-week old db-/- mice compared to 8- week old counterparts. Immunohistochemistry showed that the increase in islet 12/15LO parallels a decline in islet number. Analysis of 12- and 15-hydroperoxytetraeicosanoid acids (HETE)s showed a 2-3 fold increase especially in 12(S)-HETE that mirrored the increase in 12/15LO expression in islets. Analysis of AT and stromal vascular fraction (SVF) showed a significant increase of platelet 12LO gene expression along with 12- and 15- HETEs. The data demonstrate that the db/db mouse is a suitable model for investigation of 12/15LO inhibitors in the development of inflammatory mediated type 2 diabetes, with a narrow window of therapeutic intervention prior to 8 weeks of age.

Although autacoids primarily derived from the cyclooxygenase-2 and 5-lipoxygenase (LOX) pathways are essential mediators of inflammation, endogenous specialized proresolving mediators (SPMs) act as robust agonists of resolution. SPM biosynthesis is initiated by the conversion of arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid primarily via the 12/15-LOX pathway. Although 12/15-LOX activity is prominent in the cornea, the role of SPM pathway activation during infection remains largely unknown and is the focus of the current study. Pseudomonas keratitis was induced in resistant BALB/c and susceptible C57BL/6 (B6) mice. Biosynthetic pathways for proinflammatory autacoids and SPMs were assessed. Divergent lipid mediator profiles demonstrate the importance of 15-LOX pathways in the pathogenesis of ocular infectious disease. Results indicate that an imbalance of LOX enzymatic pathways contributes to susceptibility observed in B6 mice where deficient activation of SPM circuits, as indicated by reduced 15-hydroxy-eicosatetraenoic acid and 17-hydroxydocosahexaenoic acid levels, prevented transition toward resolution and led to chronic inflammation. In sharp contrast, BALB/c mice demonstrated a well-balanced axis of 5-LOX/12-LOX/15-LOX pathways, resulting in sufficient proresolving bioactive metabolite formation and immune homeostasis. Furthermore, a novel immunoregulatory role for 15-LOX was revealed in inflammatory cells (polymorphonuclear leukocytes and macrophages), which influenced phagocytic activity. These data provide evidence that SPM circuits are essential for host defense during bacterial keratitis.-Carion, T. W., Greenwood, M., Ebrahim, A. S., Jerome, A., Suvas, S., Gronert, K., Berger, E. A. Immunoregulatory role of 15-lipoxygenase in the pathogenesis of bacterial keratitis.

One of the leading causes of male infertility is defective sperm function, a pathology that commonly arises from oxidative stress in the germline. Lipid peroxidation events in the sperm plasma membrane result in the generation of cytotoxic aldehydes such as 4-hydroxynonenal (4HNE), which accentuate the production of reactive oxygen species (ROS) and cause cellular damage. One of the key enzymes involved in the metabolism of polyunsaturated fatty acids to 4HNE in somatic cells is arachidonate 15-lipoxygenase (ALOX15). Although ALOX15 has yet to be characterized in human spermatozoa, our previous studies have revealed a strong link between ALOX15 activity and the levels of oxidative stress and 4HNE in mouse germ cell models. In view of these data, we sought to assess the function of ALOX15 in mature human spermatozoa and determine whether the pharmacological inhibition of this enzyme could influence the level of oxidative stress experienced by these cells. By driving oxidative stress in vitro with exogenous H2O2, our data reveal that 6,11-dihydro[1]benzothiopyrano[4,3-b]indole (PD146176; a selective ALOX15 inhibitor) was able to significantly reduce several deleterious, oxidative insults in spermatozoa. Indeed, PD146176 attenuated the production of ROS, as well as membrane lipid peroxidation and 4HNE production in human spermatozoa. Accordingly, ALOX15 inhibition also protected the functional competence of these cells to acrosome react and bind homologous human zonae pellucidae. Together, these results implicate ALOX15 in the propagation of oxidative stress cascades within human spermatozoa and offer insight into potential therapeutic avenues to address male in fertility that arises from oxidative stress.

Inflammations, sensitivities, and some cancers in mammals are intimately linked to the activity of lipo-oxygenase enzymes. Owing to the importance of these enzymes, mechanistic studies, product analysis, and synthesis of inhibitors have expanded. In this study, a series of hydroxycoumarins, methoxy-3-hydroxy coumarins, and 7-alkoxy-3-hydroxy coumarins were synthesized and evaluated as potential inhibitors of human 15-LOX-1. Among the synthetic coumarins, 7-methoxy-3-hydroxycoumarin derivative demonstrated potent inhibitory activity and the compound, 5f, showed the best result. Radical scavenging assessment, IC50 , HNMR, and DPPH bleaching results indicate that the electronic properties are the major factors for the lipo-oxygenase inhibition potency of the synthetic coumarins. Based on the theoretical studies, it was suggested that the mesomeric effect of the substituent at the seventh position of the benzene ring is one of the major factors in the stability of the oxy-radical intermediate.

Vascular structure and integrity are at the forefront of blood pressure regulation. However, there are many factors that affect the responses of the vessels. One of these is the inflammatory processes associated with high cholesterol and its modification. 15-lipoxygenase (15-LOX) is the critical enzyme in cholesterol oxidation, but this enzyme is also responsible for the synthesis of specialized proresoving lipid mediators (SPMs) called Lipoxin (Lxs) and Resolvin (Rvs). In this study, we determined serum LXA4, RvD1 and RvE1 levels in newly diagnosed hypertension (HT) and normotension (NT) cases. We evaluated how the presence of hypercholesterolemia (HC) in the follow-up changes the levels of these SPMs. We found that the three SPMs we measured decreased significantly in the presence of HC. In addition, we found a negative and significant correlation with systolic blood pressure and total cholesterol levels for the three SPMs. In conclusion, HT and HC are independent risk factors for cardiovascular death. However, the presence of HC may be an important factor for the development of HT. Increasing cholesterol levels may cause 15-LOX to shift towards LDL oxidation, thus leading to inflammation. This situation may negatively affect the vascular functions in the regulation of blood pressure. Serum LXA4, RvD1 and RvE1 measurements may provide clues that represent a shift of 15-LOX enzyme activity towards cholesterol.