Determination of the Contribution of Cysteinyl Leukotrienes and Leukotriene B4 in Acute Inflammatory Responses Using 5-Lipoxygenase- and Leukotriene A4 Hydrolase-Deficient Mice
150
Citation
52
Reference
10
Related Paper
Citation Trend
Abstract:
Arachidonic acid metabolism by 5-lipoxygenase leads to production of the potent inflammatory mediators, leukotriene (LT) B4 and the cysteinyl LT. Relative synthesis of these subclasses of LT, each with different proinflammatory properties, depends on the expression and subsequent activity of LTA4 hydrolase and LTC4 synthase, respectively. LTA4 hydrolase differs from other proteins required for LT synthesis because it is expressed ubiquitously. Also, in vitro studies indicate that it possesses an aminopeptidase activity. Introduction of cysteinyl LT and LTB4 into animals has shown LTB4 is a potent chemoattractant, while the cysteinyl LT alter vascular permeability and smooth muscle tone. It has been impossible to determine the relative contributions of these two classes of LT to inflammatory responses in vivo or to define possible synergy resulting from the synthesis of both classes of mediators. To address this question, we have generated LTA4 hydrolase-deficient mice. These mice develop normally and are healthy. Using these animals, we show that LTA4 hydrolase is required for the production of LTB4 in an in vivo inflammatory response. We show that LTB4 is responsible for the characteristic influx of neutrophils accompanying topical arachidonic acid and that it contributes to the vascular changes seen in this model. In contrast, LTB4 influences only the cellular component of zymosan A-induced peritonitis. Furthermore, LTA4 hydrolase-deficient mice are resistant to platelet-activating factor, identifying LTB4 as one mediator of the physiological changes seen in systemic shock. We do not identify an in vivo role for the aminopeptidase activity of LTA4 hydrolase.Keywords:
Leukotriene B4
Zymosan
Leukotriene C4
Proinflammatory cytokine
Epoxide hydrolase 2
Hydrolase
Soluble epoxide hydrolase is a class of α/β-fold hydrolase enzymes that exist in numerous organs and tissues, including the liver, kidney, brain, and vasculature. This homodimer enzyme is responsible for degrading epoxyeicosatrienoic acids to the less active vicinal diols, dihydroxyeicosatrienoic acids by adding a molecule of water to an epoxide in the cytochrome P450 pathway. Soluble epoxide hydrolase was firstly assayed and characterized by Hammock and colleagues about 40 years ago. Upholding high epoxyeicosatrienoic acid blood levels by inhibiting soluble epoxide hydrolase has been proposed as a hopeful strategy to treat renal and cardiovascular diseases, inflammation, and pain. Therefore, developing novel soluble epoxide hydrolase inhibitors has been an attractive research topic for many years. Regarding this issue, some carbamates, heterocycles, amides, and ureas have been proposed; however, rapid metabolism, low solubility, high melting point, and weak pharmacokinetic characteristics are challenges posed to the researchers. In this review, we have focused on the role of the soluble epoxide hydrolase in the metabolic pathway of arachidonic acid, and categorized the most representative soluble epoxide hydrolase inhibitors into two main classes of synthetic and natural compounds. The structures have been evaluated and an exemplary structure-activity relationship has been provided for further development of potent inhibitors at the end. According to our findings, urea-based inhibitors were preferred to the amide-based scaffolds due to the better fitting into the active site. An aromatic linker is a suitable bridge to connect primary and secondary pharmacophores compared with aliphatic linkers.
Epoxide hydrolase 2
Hydrolase
Epoxyeicosatrienoic acid
Microsomal epoxide hydrolase
Cite
Citations (2)
Epoxide hydrolase 2
Microsomal epoxide hydrolase
Hydrolase
Styrene oxide
Clofibrate
Cite
Citations (17)
Epoxide hydrolase 2
Microsomal epoxide hydrolase
Hydrolase
Cite
Citations (34)
The effect of antipileptic drug valpromide (VPM) on the activity of epoxide hydrolase was studied in human adult and foetal liver, kidneys, lungs, intestine and in placenta. The activity of the epoxide hydrolase was measured with both styrene oxide and benzo(a)pyrene‐4,5‐oxide as substrates. VPM inhibited the epoxide hydrolase obtained from all organs studied. The degree of inhibition was independent of the substrate used. A lowering of the epoxide hydrolase activity by 50% was observed when the concentration of VPM was similar to that of the substrates. VPM competitively inhibited the activity of adult liver epoxide hydrolase with styrene oxide as substrate.
Styrene oxide
Epoxide hydrolase 2
Hydrolase
Cite
Citations (20)
Epoxide hydrolase 2
Microsomal epoxide hydrolase
Hydrolase
Cite
Citations (432)
Epoxide hydrolase 2
Cite
Citations (8)
The roles of CYP lipid-metabolizing pathways in endothelial cells are poorly understood. Human endothelial cells expressed CYP2J2 and soluble epoxide hydrolase (sEH) mRNA and protein. The TLR-4 agonist LPS (1 μg/ml; 24 h) induced CYP2J2 but not sEH mRNA and protein. LC-MS/MS analysis of the stable commonly used human endothelial cell line EA.Hy926 showed active epoxygenase and epoxide hydrolase activity: with arachidonic acid (stable epoxide products 5,6-DHET, and 14,15-DHET), linoleic acid (9,10-EPOME and 12,13-EPOME and their stable epoxide hydrolase products 9,10-DHOME and 12,13-DHOME), docosahexaenoic acid (stable epoxide hydrolase product 19,20-DiHDPA) and eicosapentaenoic acid (stable epoxide hydrolase product 17,18-DHET) being formed. Inhibition of epoxygenases using either SKF525A or MS-PPOH induced TNFα release, but did not affect LPS, IL-1β, or phorbol-12-myristate-13-acetate (PMA)-induced TNFα release. In contrast, inhibition of soluble epoxide hydrolase by AUDA or TPPU inhibited basal, LPS, IL-1β and PMA induced TNFα release, and LPS-induced NFκB p65 nuclear translocation. In conclusion, human endothelial cells contain a TLR-4 regulated epoxygenase CYP2J2 and metabolize linoleic acid>eicosapentaenoic acid > arachidonic acid>docosahexaenoic acid to products with anti-inflammatory activity.
Epoxide hydrolase 2
Epoxygenase
Epoxyeicosatrienoic acid
Cite
Citations (43)
Growing male rats received rations containing 5.18 and 33% of protein during 2 months. In contrast to other microsomal enzymes, epoxide hydrolase activity was maximal in the liver of the animals which received the ration with 5% of protein, and minimal--with 18% of protein. Epoxide hydrolase in the liver of rats that received the rations with 5 and 18% of protein showed equally expressed capacity for induction and activation in vivo and in vitro.
Epoxide hydrolase 2
Microsomal epoxide hydrolase
Hydrolase
Xenobiotic
Cite
Citations (0)
Epoxide hydrolases (EHs) of the α/β hydrolase fold enzyme family hydrolyze epoxides to the corresponding vicinal diols. In mammals, epoxides are mainly formed within the body through epoxidation of xenobiotic or endogenous substrates by cytochrome P450-dependent monooxygenases (CYPs). Two of the five known mammalian EHs are well characterized. The microsomal epoxide hydrolase (mEH) is primarily involved in detoxification of carcinogenic epoxides derived from xenobiotic compounds and the soluble epoxide hydrolase (sEH) primarily regulates endogenous signaling epoxides derived from fatty acids. Although some substrates are known for the epoxide hydrolase 3 (EH3), its function remains unclear. For the epoxide hydrolase 4 (EH4) and mesoderm specific transcript (MEST) no substrates are known, but knockout of MEST in mice leads to growth retardation and a behavioral phenotype. Another EH with activity in the human body is the CFTR inhibitory factor (Cif), a virulence factor secreted by the opportunistic pathogen Pseudomonas aeruginosa. Cif reduces the surface expression of the chloride channel CFTR in human airway endothelial cells, resulting in increased mucus viscosity facilitating bacterial colonization. The enzyme activity of Cif is crucial for the effect on CFTR but the molecular target remains to be identified.
In this project, we aimed to identify the physiologically relevant substrate(s) of the aforementioned EHs. For the bacterial EH Cif, a substrate screening was performed using recombinantly expressed enzyme, but kinetic analysis suggested that the found substrates 14,15-epoxyeicosatrienoic acid (14,15-EET), 17,18-epoxyeicosatetraenoic acid, and 19,20-epoxydocosapentaenoic acid are not relevant in vivo. To identify relevant substrates in vivo, an unbiased approach was developed that takes advantage of the characteristic two-step reaction mechanism of α/β hydrolase fold EHs. By introducing a point mutation, trapping mutants of the six EHs were constructed which are able to nucleophilically attack and bind their substrates but cannot perform the second hydrolytic step, thus trapping their substrates with a covalent ester bond. Using adeno-associated viruses (AAVs), these trapping mutants were expressed in mice, where they should encounter and bind their substrates in a physiologic environment. For expression in peripheral organs of mice, the serotype AAV-rh10 was used, while the serotype AAV-PHP.B was used to target the brain after the intravenous injection of the virus into the tail vein. Successful expression in the liver was confirmed for all trapping EHs except EH3, and in the brain for mEH, sEH and EH4 by western blot analysis.
Mass spectrometry was used to identify the substrates that were trapped by the enzymes. For this, mouse tissue was lyzed and the virally expressed trapping mutants were enriched using His-tag affinity chromatography. After digestion with trypsin, the samples were analyzed on a TripleTOF mass spectrometer using SWATH, a data independent scan mode which provides time-resolved recording of the fragment ions of all precursors. Since the sequence and therefore the mass of the peptide which carries the trapped substrate is known, the mass of the substrate can be determined.
With mass spectrometry we confirmed the presence of virally expressed mEH, sEH, Cif and MEST in the liver and mEH and sEH in the brain by detecting peptides specific for these enzymes. However, no substrate modified peptide was detected for any enzyme in any of the tissues. As proof of principle, the peptide-substrate complex of recombinantly expressed sEH incubated with 14,15-EET was detected in an in vitro trapping experiment confirming that the ester intermediate is stable enough for detection by mass spectrometry.
Analysis of the sample preparation procedure with synthetic peptides revealed a selective and substantial loss of the sEH, EH3, and EH4 peptides. This was probably the main problem in the analysis of the sEH expressing tissue. For mEH and MEST, only the unmodified peptide was detected, indicating that the trapping mutants were not able to bind their substrates, probably due to protein misfolding. EH3, EH4, and Cif peptides were likely not detected due to their low tissue concentrations, probably caused by a shorter half-life of these proteins. Nevertheless, successful trapping and analysis was shown in vitro and required measures to improve the method were identified. Establishment of the in vivo trapping approach and identification of EH substrates could bring fundamental insights into the role of EHs in mammalian physiology and new strategies for therapeutic intervention.
Unexpectedly, mice expressing trapping mEH in the brain developed a striking trembling phenotype which was milder in mEH KO mice and absent when wild type instead of trapping mEH was expressed. Further analysis is required but preliminary experiments suggest a loss of dopaminergic cells in the substantia nigra causing a Parkinson’s disease-like pathology Weniger anzeigen
Epoxide hydrolase 2
Microsomal epoxide hydrolase
Hydrolase
Xenobiotic
Cite
Citations (0)
Epoxyeicosatrienoic acids are cytochrome P-450 metabolites of arachidonic acid with multiple biological functions, including the regulation of vascular tone, renal tubular transport, cellular proliferation, and inflammation. Epoxyeicosatrienoic acids are converted by soluble epoxide hydrolase into the corresponding dihydroxyeicosatrienoic acids, and epoxyeicosatrienoic acid hydration is regarded as one mechanism whereby their biological effects are eliminated. Previous animal studies indicate that soluble epoxide hydrolase plays an important role in the regulation of renal eicosanoid levels and systemic blood pressure. To begin to elucidate the mechanism of these effects, we determined the cellular localization of soluble epoxide hydrolase in human kidney by examining biopsies taken from patients with a variety of non-end-stage renal diseases, as well as those without known renal disease. Immunohistochemical staining of acetone-fixed kidney biopsy samples revealed that soluble epoxide hydrolase was preferentially expressed in the renal vasculature with relatively low levels in the surrounding tubules. Expression of soluble epoxide hydrolase was evident in renal arteries of varying diameter and was localized mostly in the smooth muscle layers of the arterial wall. Western blot analysis and functional assays confirmed the expression of soluble epoxide hydrolase in the human kidney. There were no obvious differences in soluble epoxide hydrolase expression between normal and diseased human kidney tissue in the samples examined. Our results indicate that soluble epoxide hydrolase is present in the human kidney, being preferentially expressed in the renal vasculature, and support an essential role for this enzyme in renal hemodynamic regulation and its potential utility as a target for therapeutic intervention.
Epoxide hydrolase 2
Cite
Citations (67)