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Targeting biosynthetic networks of the proinflammatory and proresolving lipid metabolome

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Nonsteroidal anti-inflammatory drugs interfere with the metabolism of arachidonic acid to proinflammatory prostaglandins and leukotrienes by targeting cyclooxygenases (COXs), 5-lipoxygenase (LOX), or the 5-LOX-activating protein (FLAP). These and related enzymes act in conjunction with marked crosstalk within a complex lipid mediator (LM) network where also specialized proresolving LMs (SPMs) are formed. Here, we present how prominent LM pathways can be differentially modulated in human proinflammatory M1 and proresolving M2 macrophage phenotypes that, upon exposure to Escherichia coli, produce either abundant prostaglandins and leukotrienes (M1) or SPMs (M2). Targeted liquid chromatography-tandem mass spectrometry-based metabololipidomics was applied to analyze and quantify the specific LM profiles. Besides expected on-target actions, we found that: 1) COX or 15-LOX-1 inhibitors elevate inflammatory leukotriene levels, 2) FLAP and 5-LOX inhibitors reduce leukotrienes in M1 but less so in M2 macrophages, 3) zileuton blocks resolution-initiating SPM biosynthesis, whereas FLAP inhibition increases SPM levels, and 4) that the 15-LOX-1 inhibitor 3887 suppresses SPM formation in M2 macrophages. Conclusively, interference with discrete LM biosynthetic enzymes in different macrophage phenotypes considerably affects the LM metabolomes with potential consequences for inflammation-resolution pharmacotherapy. Our data may allow better appraisal of the therapeutic potential of these drugs to intervene with inflammatory disorders.-Werner, M., Jordan, P. M., Romp, E., Czapka, A., Rao, Z., Kretzer, C., Koeberle, A., Garscha, U., Pace, S., Claesson, H.-E., Serhan, C. N., Werz, O., Gerstmeier, J. Targeting biosynthetic networks of the proinflammatory and proresolving lipid metabolome.
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Targeting biosynthetic networks of the proinammatory and proresolving lipid metabolome
Markus Werner, Paul M. Jordan, Erik Romp, Anna Czapka, Zhigang Rao, Christian Kretzer, Andreas Koeberle, Ulrike Garscha,
Simona Pace, Hans-Erik Claesson, Charles N. Serhan, Oliver Werz, Jana Gerstmeier
The FASEB Journal, February 2019, Federation of American Societies For Experimental Biology (FASEB)
DOI: 10.1096/fj.201802509r
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What is it about?
Polyunsaturated fatty acids are precursors for the large family of bioactive lipid
mediators. Some of these lipid mediators (i.e. prostaglandins, leukotrienes) promote
inammatory processes and contribute to inammation-related diseases such as
arthritis, asthma and cardiovascular diseases, while other, newly discovered, lipid
mediators (termed specialized pro-resolving mediators = SPM) have opposing eects
and actively terminate inammation and support tissue repair. All these lipid
mediators are formed by enzymes termed lipoxygenases and cyclooxygenases within
a complex biosynthetic network with marked cross-talk. These enzymes are targeted
by anti-inammatory drugs such as ibuprofen or celecoxib that are used in the clinics
to treat inammatory disorders, fever and pain, apparently due to suppression of
the formation of pro-inammatory lipid mediators. But how interference of these
anti-inammatory drugs with cyclooxygenases and lipoxygenases inuences the
formation of inammation-resolving SPM and how they aect the overall lipid
mediator network is unknown, partly because suitable biological test systems and
analytical tools were not available yet. Here, we present a convenient experimental
approach using bacteria-stimulated immune cells (i.e. macrophages) that occur
either as pro-inammatory subtype (M1) producing mainly prostaglandins and
leukotrienes or as anti-inammatory subtype (M2) generating primarily SPM. Our
results led to important, partly unexpected insights into how these anti-inammatory
drugs modulate the formation of SPM in M2 macrophages. Thus, we document the
pharmacological prole of anti-inammatory drugs and disclose for the rst time
their ability to manipulate inammation-resolving molecules, insights that may allow
better appraisal of the therapeutic potential of these drugs to intervene with
inammatory disorders.
The following have contributed to this page: Dr. Jana Gerstmeier
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... Lipid mediator (LM) analysis using ultra performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) was performed as described previously [8] with some minor modifications [39]. Briefly, freshly thawed human plasma or Bacillus megaterium cultures were first mixed with the same volume of ice-cold methanol containing deuteriumlabeled internal standards (200 nM d8-5S-HETE, d4-LTB4, d5-LXA4, d5-RvD2, d4-PGE2 and 10 µM d8-AA; Cayman Chemical/Biomol GmbH, Hamburg, Germany) to facilitate quantification and sample recovery. ...
... The LM were analyzed with an Acquity ™ UPLC system (Waters, Milford, MA, USA) and a QTRAP 5500 Mass Spectrometer (ABSciex, Darmstadt, Germany), equipped with a Turbo V ™ Source and electrospray ionization. LM were separated using an ACQUITY UPLC ® BEH C18 column (1.7 µm, 2.1 × 100 mm; Waters, Eschborn, Germany) at 50 • C with a flow rate of 0.3 mL/min and a mobile phase consisting of methanol/water/acetic acid of 42/58/0.01 (v/v/v) that was ramped to 86/14/0.01 (v/v/v) over 12.5 min and then to 98/2/0.01 (v/v/v) for 3 min [39]. The QTrap 5500 was operated in negative ionization mode using scheduled multiple reaction monitoring (MRM) coupled with information-dependent acquisition. ...
... Linear calibration curves were obtained for each LM and gave r2 values of 0.998 or higher (for PUFA 0.95 or higher). Additionally, the limit of detection for each targeted LM was determined [39]. ...
Article
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Specialized pro-resolving mediators (SPM) have emerged as crucial lipid mediators that confer the inflammation-resolving effects of omega-3 polyunsaturated fatty acids (n-3 PUFA). Importantly, SPM biosynthesis is dysfunctional in various conditions, which may explain the inconclusive efficacy data from n-3 PUFA interventions. To overcome the limitations of conventional n-3 PUFA supplementation strategies, we devised a composition enabling the self-sufficient production of SPM in vivo. Bacillus megaterium strains were fed highly bioavailable n-3 PUFA, followed by metabololipidomics analysis and bioinformatic assessment of the microbial genomes. All 48 tested Bacillus megaterium strains fed with the n-3 PUFA formulation produced a broad range of SPM and precursors thereof in a strain-specific manner, which may be explained by the CYP102A1 gene polymorphisms that we detected. A pilot study was performed to test if a synbiotic Bacillus megaterium/n-3 PUFA formulation increases SPM levels in vivo. Supplementation with a synbiotic capsule product led to significantly increased plasma levels of hydroxy-eicosapentaenoic acids (5-HEPE, 15-HEPE, 18-HEPE) and hydroxy-docosahexaenoic acids (4-HDHA, 7-HDHA) as well as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in healthy humans. To the best of our knowledge, we report here for the first time the development and in vivo application of a self-sufficient SPM-producing formulation. Further investigations are warranted to confirm and expand these findings, which may create a new class of n-3 PUFA interventions targeting inflammation resolution.
... LM were separated on an ACQUITY UPLC ® BEH C18 column (1.7 µm, 2.1 mm × 100 mm; Waters, Eschborn, Germany) at 50°C with a flow rate of 0.3 mL/min and a mobile phase consisting of methanol-water-acetic acid (starting at 42:58:0.01, v/v/v) that was ramped to 86:14:0.01 over 12.5 minutes and then to 98:2:0.01 for 3 minutes. 43 The QTRAP 5500 was operated in negative mode using scheduled multiple reaction monitoring (MRM) coupled with informationdependent acquisition. The scheduled MRM window was 60 seconds, optimized LM parameters were adopted, 43 and the curtain gas pressure was set to 35 psi. ...
... 43 The QTRAP 5500 was operated in negative mode using scheduled multiple reaction monitoring (MRM) coupled with informationdependent acquisition. The scheduled MRM window was 60 seconds, optimized LM parameters were adopted, 43 and the curtain gas pressure was set to 35 psi. The retention time and at least six diagnostic ions for each LM were confirmed by means of an external standard (Cayman Chemical/Biomol GmbH, Hamburg, Germany). ...
... Linear calibration curves were obtained for each LM and gave r 2 values of 0.99. 43 Determination of Cell Viability by MTT Assay M0 GM-CSF or M0 M-CSF (2×10 5 /mL) in a 96-well plate were pre-incubated with 0.1% vehicle (DMSO), CS, or 1% Triton X-100 that was used as a positive control, for 15 minutes. Then, 100 ng/mL LPS and 20 ng/mL IFNγ or 20 ng/mL IL-4 (for M1 and M2 polarization, respectively) were added. ...
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Background and purpose: Celastrol (CS) is a major active ingredient of the Chinese/Asian herb Tripterygium wilfordii that is frequently used as phytomedicine to treat inflammation and autoimmune diseases. We showed before that short-term exposure to CS (1 µM) favorably impacts the biosynthesis of inflammation-related lipid mediators (LM) in human polarized macrophages by modulating the activities of different lipoxygenases (LOXs). However, whether CS regulates the expression of LOXs and other related LM-biosynthetic enzymes during macrophage polarization is unknown. Here, we investigated how CS affects LM-biosynthetic enzyme expression on the protein level and studied concomitant LM signature profiles during polarization of human monocyte-derived macrophages (MDM) towards M1- and M2-like phenotypes. Methods and results: We used LM metabololipidomics to study the long-term effects of CS on LM profile signatures after manipulation of human monocyte-derived macrophages (MDM) during polarization. Exposure of MDM to low concentrations of CS (ie, 0.2 µM) during polarization to an inflammatory M1 phenotype potently suppressed the formation of pro-inflammatory cyclooxygenase (COX)- and 5-LOX-derived LM, especially prostaglandin (PG)E2. Notably, gene and enzyme expression of COX-2 and microsomal PGE2 synthase (mPGES)-1 as well as M1 markers were strongly decreased by CS during M1-MDM polarization, along with impaired activation of nuclear factor-κB and p38 mitogen-activated protein kinase. During IL-4-induced M2 polarization, CS decreased the capacity of the resulting M2-MDM to generate pro-inflammatory COX and 5-LOX products as well but it also reduced the formation of 12/15-LOX products and specialized pro-resolving mediators, without affecting the levels of liberated fatty acid substrates. Conclusion: Depending on the timing and concentration, CS not only favorably affects LOX activities in macrophages but also the expression of LM-biosynthetic enzymes during macrophage polarization connected to changes of inflammation-related LM which might be of relevance for potential application of CS to treat inflammatory disorders.
... The supernatants were then transferred to 2 ml of ice-cold methanol containing 10 ml of deuterium-labeled internal standards (200 nM d8-5S-hydroxyeicosatetraenoic acid, d4leukotriene B 4 , d5-lipoxin A 4 , d5-resolvin D2, d4-PGE 2 and 10 mM d8-arachidonic acid [AA]) to accomplish quantification and sample recovery. Sample preparation was conducted by adapting published criteria (23,24). Briefly, samples were kept at -20°C for 60 min to allow protein precipitation. ...
... The QTrap 5500 was operated in negative ionization mode using scheduled multiple reaction monitoring coupled with information-dependent acquisition. The scheduled multiple reaction monitoring window was 60 s, optimized LM parameters (i.e., collision energy, entrance potential, declustering potential, collision cell exits potential) were adopted (23,24). The curtain gas pressure was set to 35 psi. ...
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Macrophages are the primary human host cells of intracellular Mycobacterium tuberculosis (M.tb) infection, where the magnitude of inflammatory reactions is crucial for determining the outcome of infection. Previously, we showed that the anti-inflammatory drug sulfasalazine (SASP) significantly reduced the M.tb bactericidal burden and histopathological inflammation in mice. Here, we asked which genes in human inflammatory macrophages are affected upon infection with M.tb and how would potential changes impact the functional state of macrophages. We used a flow cytometry sorting system which can distinguish the dead and alive states of M.tb harbored in human monocyte-derived macrophages (MDM). We found that the expression of cyclooxygenase-2 and microsomal prostaglandin E2 synthase (mPGES)-1 increased significantly in tagRFP+ MDM which were infected with alive M.tb. After exposure of polarized M1-MDM to M.tb (H37Rv strain)-conditioned medium (MTB-CM) or to the M.tb-derived 19-kD antigen, the production of PGE2 and pro-inflammatory cytokines increased 3- to 4-fold. Upon treatment of M1-MDM with SASP, the MTB-CM-induced expression of COX-2 and the release of COX products and cytokines decreased. Elevation of PGE2 in M1-MDM upon MTB-CM stimulation and modulation by SASP correlated with the activation of the NF-κB pathway. Together, infection of human macrophages by M.tb strongly induces COX-2 and mPGES-1 expression along with massive PGE2 formation which is abrogated by the anti-inflammatory drug SASP.
... [18] The site of cascade inhibition (of FLAP vs 5-LO) may also be of biological relevance: inhibition of FLAP increases the biosynthesis of potentially beneficial lipid proresolving mediators, which actively terminate inflammation and promote tissue healing and regeneration, while inhibition of 5-LO blocks proresolving mediator biosynthesis. [19] AZD5718 is a FLAP inhibitor at the first step of leukotriene biosynthesis that suppresses all leukotriene production ( Supplementary Fig. S1). [20] In phase 1 studies (NCT02632526; NCT02963116), multiple oral doses of AZD5718 60-600 mg were generally well tolerated and potently reduced endogenous urine leukotriene E 4 (uLTE 4 ) and ex vivo calcium ionophore-stimulated LTB 4 synthesis in whole blood. ...
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Background Leukotrienes are pro-inflammatory vasoactive lipid mediators implicated in the pathophysiology of atherosclerotic cardiovascular disease. We studied the effect of the 5-lipoxygenase-activating protein inhibitor AZD5718 on leukotriene biosynthesis and coronary microvascular function in a single-blind, phase 2a study. Methods Patients 7–28 days after myocardial infarction (±ST elevation), with <50% left anterior descending coronary artery stenosis and Thrombolysis in Myocardial Infarction flow grade ≥ 2 after percutaneous coronary intervention, were randomized 2:1:2 to once-daily AZD5718 200 mg or 50 mg, or placebo, in 4- and 12-week cohorts. Change in urine leukotriene E4 (uLTE4) was the primary endpoint, and coronary flow velocity reserve (CFVR; via echocardiography) was the key secondary endpoint. Results Of 129 randomized patients, 128 received treatment (200 mg, n = 52; 50 mg, n = 25; placebo, n = 51). Statistically significant reductions in uLTE4 levels of >80% were observed in both AZD5718 groups versus the placebo group at 4 and 12 weeks. No significant changes in CFVR were observed for AZD5718 versus placebo. Adverse events (AEs) occurred in 12/18, 3/6 and 6/13 patients receiving 200 mg, 50 mg and placebo, respectively, in the 4-week cohort, and in 27/34, 14/19 and 24/38 patients, respectively, in the 12-week cohort. Serious AEs in seven patients receiving AZD5718 and four receiving placebo were not treatment-related, and there were no deaths. Conclusions In patients with recent myocardial infarction, AZD5718 was well tolerated, and leukotriene biosynthesis was dose-dependently inhibited. No significant changes in CFVR were detected. ClinicalTrials.gov identifier: NCT03317002.
... Patient's serum samples were also analyzed, and levels of arachidonic acid-derived lipid mediators prostaglandin E 2 (PGE 2 ) and lipoxin A 4 (LXA 4 ); and docosahexaenoic acid-derived pro-resolving lipid mediator resolvin D1 (RvD1) were higher in lepromatous than in tuberculoid patients (Bobosha et al., 2014). LXA 4 and RvD1 are known as specialized pro-resolving mediators (SPMs), and it has been observed that M2 macrophages are the main sources of SPMs (Werz et al., 2018;Werner et al., 2019;Jordan and Werz, 2021). These lipid mediators are known to induce the M2 macrophage phenotype, as well as FOXP3 expression, Treg differentiation and proliferation (Baratelli et al., 2005;Schwab et al., 2007;Garg et al., 2008;Chiurchiù et al., 2016). ...
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The initial infection by the obligate intracellular bacillus Mycobacterium leprae evolves to leprosy in a small subset of the infected individuals. Transmission is believed to occur mainly by exposure to bacilli present in aerosols expelled by infected individuals with high bacillary load. Mycobacterium leprae -specific DNA has been detected in the blood of asymptomatic household contacts of leprosy patients years before active disease onset, suggesting that, following infection, the bacterium reaches the lymphatic drainage and the blood of at least some individuals. The lower temperature and availability of protected microenvironments may provide the initial conditions for the survival of the bacillus in the airways and skin. A subset of skin-resident macrophages and the Schwann cells of peripheral nerves, two M. leprae permissive cells, may protect M. leprae from effector cells in the initial phase of the infection. The interaction of M. leprae with these cells induces metabolic changes, including the formation of lipid droplets, that are associated with macrophage M2 phenotype and the production of mediators that facilitate the differentiation of specific T cells for M. leprae -expressed antigens to a memory regulatory phenotype. Here, we discuss the possible initials steps of M. leprae infection that may lead to active disease onset, mainly focusing on events prior to the manifestation of the established clinical forms of leprosy. We hypothesize that the progressive differentiation of T cells to the Tregs phenotype inhibits effector function against the bacillus, allowing an increase in the bacillary load and evolution of the infection to active disease. Epigenetic and metabolic mechanisms described in other chronic inflammatory diseases are evaluated for potential application to the understanding of leprosy pathogenesis. A potential role for post-exposure prophylaxis of leprosy in reducing M. leprae -induced anti-inflammatory mediators and, in consequence, Treg/T effector ratios is proposed.
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The aim of this in silico study was the identification of NSAID’s amides with ability to bypass the COX inhibition and selectively target mPGES-2 and FLAP. Molecular binding analysis was conducted on selected compounds using molecular docking, MD simulations and MM/GBSA calculations to assess their potential in the prevention of neuroinflammation. Ligand-based virtual screening was performed in the ChEMBL database, thereby 196 compounds were obtained. Resulting compounds were passed through the BBB permeability and druglikeness filters, and six compounds were selected for further analysis. Molecular docking analysis showed that compounds 4 and 6 can avoid the COX inhibition and simultaneously target mPGES-2 and FLAP. These two top docked compounds were subjected to MD simulations, which indicated that complexes of compound 4 with mPGES-2 and FLAP have shown conformational stability throughout the simulation. Calculated MM/GBSA binding energies confirmed that compound 4 demonstrated dual binding affinity against mPGES-2 and FLAP. Based on the docking score, MM/GBSA average binding energy and MD results, compound 4 emerged as the best in silico hit for the prevention of neuroinflammation.
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In this review it is attempted to summarize current studies about formation of eicosanoids and other oxylipins in different human macrophages. There are several reports on M1 and M2 cells, also other phenotypes have been described. The eicosanoids formed in the largest amounts are the COX products TxB2 and PGE2. Thus shortlived bioactive TxA2 is a dominating product both in M1- and in M2-lineages, one exception seems to be MGM-CSF, TGFβ cells. 5-LOX products are produced in both M1 and M2 macrophages, as well as in not fully polarized cells of both lineages. MM-CSF as well as M2 macrophages produced LTC4 more readily compared to M1 lineage cells. In MGM-CSF, TGFβ cells LTB4 is a major eicosanoid, in line with high expression of LTA4 hydrolase. Recent reports described increased formation of leukotrienes in macrophages subjected to trained immunity with inflammatory transcriptional reprogramming. Also in macrophages derived from monocytes collected from post-COVID-19 patients. 15-LOX-1 is strongly upregulated in CD206⁺ M2 cells (M2a), differentiated in presence of IL-4. These macrophages also express 15-LOX-2. In incubations with pathogenic E. coli as well as other stimuli 15(S)-HETE and 17(S)-HDHA were major oxylipins formed. Also, the SPM precursor 5,15-diHETE and the SPM RvD5 were produced in considerable amounts, while other SPMs were less abundant. In M2 macrophages incubated with E. coli or S. aureus the cytosolic 15-LOX-1 enzyme accumulated to punctuate structures in a Ca²⁺ dependent manner with a relatively slow time course, leading to formation of mediators from endogenous substrate. Chalcones, flavone-like anti-inflammatory natural products, induced translocation of 15-LOX-1 in M2 cells, with high formation of 15-LOX derived oxylipins.
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Background Inflammation plays an important role in diabetes mellitus (DM)-related acute ischemic stroke (AIS). The mechanisms of un-resolved inflammation in DM-related AIS are not fully understood. Specialized pro-resolving mediators (SPMs) are key regulators that promote resolution of inflammation. We aimed to examine resolution function in patients with AIS complicated with DM, and explore potential treatment effects of one of the SPMs, resolvin D2 (RvD2) ex vivo and in vivo. Methods Cultured human macrophages, which were derived from peripheral blood mononuclear cells of AIS and none-AIS patients with or without DM, were stimulated with oxidized-low density lipoprotein (ox-LDL). Levels of SPMs and inflammatory markers were analysed, and RvD2 treatment effects were evaluated in these cells. For experiments in vivo, challenges with high fat diet and low-dose streptozotocin (STZ) were used to induce DM in C57BL/6J mice. AIS model was established by permanent middle cerebral artery occlusion (pMCAO) followed by intra-cerebroventricular injection of RvD2. Results Compared with macrophages of AIS patients without DM, the ratios of SPMs to leukotriene B4 (LTB4) were decreased in AIS patients with DM, accompanied by reduced expression of SPM synthesis enzyme, 15-lipoxygenase-1. Moreover, the levels of pro-inflammatory pathway markers were increased, and the macrophages were skewed to M1 polarization in AIS patients with DM. In mice, treatment with RvD2 ameliorated pMCAO-induced brain injury, neurological dysfunction, and inflammatory response. Furthermore, RvD2 rescued resolution of inflammation by promoting macrophage/microglia polarization to pro-resolving M2 phenotype ex vivo and in vivo. Conclusions Our data demonstrate resolution of inflammation is impaired by DM in AIS patients, implicating a novel mechanism of un-resolved inflammation in DM-related AIS. Furthermore, RvD2 promotes inflammation resolution in macrophages/microglia and protects DM-related AIS, and may thus serve as a novel therapeutic target.
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Proinflammatory leukotrienes (LTs) are produced by 5-lipoxygenase (5-LO) aided by 5-LO-activating protein (FLAP). LT biosynthesis inhibitors are currently under clinical investigation as treatments for respiratory and cardiovascular diseases. Here, we have revealed a sex bias in the efficiency of clinically relevant LT biosynthesis inhibitors, showing that their effects are superior in females. We found that androgens cause these sex differences by impeding the LT-biosynthetic 5-LO/FLAP complex assembly. Lower doses of the FLAP inhibitor MK886 were required to reduce LTB4 levels in exudates of female versus male mice and rats. Following platelet-activating factor-induced shock, MK886 increased survival exclusively in female mice, and this effect was abolished by testosterone administration. FLAP inhibitors and the novel-type 5-LO inhibitors licofelone and sulindac sulfide exhibited higher potencies in human blood from females, and bioactive 5-LO/FLAP complexes were formed in female, but not male, human and murine leukocytes. Supplementation of female blood or leukocytes with 5α-dihydrotestosterone abolished the observed sex differences. Our data suggest that females may benefit from anti-LT therapy to a greater extent than males, prompting consideration of sex issues in LT modifier development.
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Unresolved inflammation is central to the pathophysiology of commonly occurring vascular diseases such as atherosclerosis, aneurysm, and deep vein thrombosis - conditions that are responsible for considerable morbidity and mortality. Surgical or catheter-based procedures performed on affected blood vessels induce acute-on-chronic inflammatory responses. The resolution of vascular inflammation is an important driver of vessel wall remodeling and functional recovery in these clinical settings. Specialized pro-resolving lipid mediators (SPMs) derived from omega-3 polyunsaturated fatty acids orchestrate key cellular processes driving resolution and a return to homeostasis. The identification of their potent effects in classic animal models of sterile inflammation triggered interest in their vascular properties. Recent studies have demonstrated that SPMs are locally synthesized in vascular tissues, have direct effects on vascular cells and their interactions with leukocytes, and play a protective role in the injury response. Early translational work has established the potential for SPMs as vascular therapeutics, and as candidate biomarkers in vascular disease. Further investigations are needed to understand the molecular and cellular mechanisms of resolution in the vasculature, to improve tools for clinical measurement, and to better define the potential for "resolution therapeutics" in vascular patients.
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The ability of nonsteroidal anti-inflammatory drugs and cyclooxygenase-2 inhibitors to exacerbate inflammatory bowel disease suggests that prostaglandins are important anti-inflammatory mediators in this context. Prostaglandin D 2 has been suggested to exert anti-inflammatory effects. We investigated the possibility that prostaglandin D 2 derived from cyclooxygenase-2 plays an important role in downregulating colonic inflammation in rats. Colitis was induced by intracolonic administration of trinitrobenzene sulfonic acid. At various times thereafter (from 1 h to 7 days), colonic prostaglandin synthesis and myeloperoxidase activity (index of granulocyte infiltration) were measured. Prostaglandin D 2 synthesis was elevated >4-fold above controls within 1–3 h of induction of colitis, preceding significant granulocyte infiltration. Treatment with a selective cyclooxygenase-2 inhibitor abolished the increase in prostaglandin D 2 synthesis and caused a doubling of granulocyte infiltration. Colonic granulocyte infiltration was significantly reduced by administration of prostaglandin D 2 or a DP receptor agonist (BW-245C). These results demonstrate that induction of colitis results in a rapid increase in prostaglandin D 2 synthesis via cyclooxygenase-2. Prostaglandin D 2 downregulates granulocyte infiltration into the colonic mucosa, probably through the DP receptor.
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PD1n-3 DPA is a specialized pro-resolving lipid mediator that displays potent anti-inflammatory properties and pro-resolving bioactivities. Such naturally occurring compounds are of current interest in biomolecular chemistry and drug discovery. To investigate the involvement of an epoxide intermediate in the biosynthesis of PD1n-3 DPA from n-3 docosapentaenoic acid, the epoxy acid 16(S),17(S)-epoxy-PDn-3 DPA, herein named ePDn-3 DPA, was prepared by stereoselective total synthesis. The synthetic material of ePDn-3 DPA allowed investigations of its role in the biosynthesis of PD1n-3 DPA. The obtained results establish that the biosynthesis of PD1n-3 DPA in neutrophils occurs with ePDn-3 DPA as the intermediate, and that 15-LOX produces ePDn-3 DPA from n-3 docosapentaenoic acid. Furthermore, support for the involvement of a hydrolytic enzyme in the biosynthetic conversion of ePDn-3 DPA to PD1n-3 DPA was found. In addition, ePDn-3 DPA was found to regulate the formation of the potent neutrophil chemoattractant LTB4 with equal potencies to that obtained with PD1n-3 DPA.
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Mammalian lipoxygenases (LOX) have been implicated in cell differentiation and in the pathogenesis of inflammatory, hyperproliferative and neurological diseases. Although the reaction specificity of mammalian LOX with n-6 fatty acids (linoleic acid, arachidonic acid) has been explored in detail little information is currently available on the product patterns formed from n-3 polyenoic fatty acids, which are of particular nutritional importance and serve as substrate for the biosynthesis of pro-resolving inflammatory mediators such as resolvins and maresins. Here we expressed the ALOX15 orthologs of eight different mammalian species as well as human ALOX12 and ALOX15B as recombinant his-tag fusion proteins and characterized their reaction specificity with the most abundantly occurring polyunsaturated fatty acids (PUFAs) including 5,8,11,14,17-eicosapentaenoic acid (EPA) and 4,7,10,13,16,19-docosahexaenoic acid (DHA). We found that the LOX isoforms tested accept these fatty acids as suitable substrates and oxygenate them with variable positional specificity to the corresponding n-6 and n-9 hydroperoxy derivatives. Surprisingly, human ALOX15 as well as the corresponding orthologs of chimpanzee and orangutan, which oxygenates arachidonic acid mainly to 15S–HETE, exhibit a pronounced dual reaction specificity with DHA forming similar amounts of 14- and 17-H(p)DHA. Moreover, ALOX15 orthologs prefer DHA and EPA over AA when equimolar concentrations of n-3 and n-6 PUFA were supplied simultaneously. Taken together, these data indicate that the reaction specificity of mammalian LOX isoforms is variable and strongly depends on the chemistry of fatty acid substrates. Most mammalian ALOX15 orthologs exhibit dual positional specificity with highly unsaturated n-3 polyunsaturated fatty acids.
Article
Introduction: Leukotrienes (LTs) are lipid mediators produced from arachidonic acid with a broad variety of bioactivities in allergy and inflammation. The biosynthesis of LTs mainly involves 5-lipoxygenase (5-LO) and its 5-lipoxygenase-activating protein (FLAP), LTA4 hydrolase and LTC4 synthase that all may represent potential targets for LT biosynthesis inhibitors. Areas covered: We introduce the LT biosynthetic pathway and its cellular regulation, the diverse biological actions of LTs and their receptors, and we briefly describe the pharmacological strategies for suppression of LT formation as well as the classes of current LT biosynthesis inhibitors. The main focus is placed on the comprehensive discussion of recently reported inhibitors of 5-LO, FLAP, LTA4 hydrolase and LTC4 synthase, based on literature search (PubMed and Thomson Innovation Patents Searches), covering 2012-2016. Expert opinion: Although many new series of 5-LO inhibitors have been presented without patenting, essentially by academia, novel FLAP inhibitors (many patented) are most advanced in clinical development and are apparently the focus of pharmaceutical companies. Only few novel inhibitors of LTA4 hydrolase and LTC4 synthase were reported. Major issues in the development of LT synthesis inhibitors are related to loss of potency in biological relevant environment, poor pharmacokinetics, lack of oral efficacy, and side effects.