Human lipoxygenase isoforms form complex patterns of double and triple oxygenated compounds from eicosapentaenoic acid.

Lipoxygenases (ALOX) are lipid peroxidizing enzymes that catalyze the biosynthesis of pro- and anti-inflammatory lipid mediators and have been implicated in (patho-)physiological processes. In humans, six functional ALOX isoforms exist and their arachidonic acid oxygenation products have been characterized. Products include leukotrienes and lipoxins which are involved in the regulation of inflammation and resolution. Oxygenation of n3-polyunsaturated fatty acids gives rise to specialized pro-resolving mediators, e.g. resolvins. However, the catalytic activity of different ALOX isoforms can lead to a multitude of potentially bioactive products. Here, we characterized the patterns of oxygenation products formed by human recombinant ALOX5, ALOX15, ALOX15B and ALOX12 from eicosapentaenoic acid (EPA) and its 18-hydroxy derivative 18-HEPE with particular emphasis on double and triple oxygenation products. ALOX15 and ALOX5 formed a complex mixture of various double oxygenation products from EPA, which include 5,15-diHEPE and various 8,15-diHEPE isomers. Their biosynthetic mechanisms were explored using heavy oxygen isotopes (H218O, 18O2 gas) and three catalytic activities contributed to product formation: i) fatty acid oxygenase activity, ii) leukotriene synthase activity, iii) lipohydroperoxidase activity. For ALOX15B and ALOX12 more specific product patterns were identified, which was also the case when these enzymes reacted in concert with ALOX5. Several double oxygenated compounds were formed from 18-HEPE by ALOX5, ALOX15B and ALOX12 including previously identified resolvins (RvE2, RvE3), while formation of triple oxygenation products, e.g. 5,17,18-triHEPE, required ALOX5. Taken together our data show that EPA can be converted by human ALOX isoforms to a large number of secondary oxygenation products, which might exhibit bioactivity.

Angiography-based quantitative coronary contrast-flow ratio measurements correlate with myocardial ischemia assessed by stress MRI.

Contrast-flow quantitative flow ratio (cQFR) is a new technology for quantitative evaluation of coronary stenosis using computational fluid dynamics based on angiograms. The aim of this study was to assess the sensitivity and specificity of cQFR to detect myocardial ischemia using stress magnetic resonance imaging (MRI) as a reference standard. Patients who received stress MRI and coronary angiography were selected from the hospital database. Relevant ischemia on stress MRI was defined as a perfusion deficit in ≥ 2 of 16 segments. cQFR was quantitated based on 3-dimensional quantitative coronary angiography using QAngio XA3D1.1 software by two blinded and independent investigators. A cQFR of ≤ 0.80 was considered abnormal. Among 87 patients 230 vessels met the criteria for full analysis by cQFR (88%). In vascular territories with a significant perfusion deficit, cQFR was significantly lower compared to areas with normal perfusion (0.72 (0.62-0.78) vs. 0.96 (0.89-0.99); p < 0.001). The sensitivity of cQFR in detecting significant epicardial stenoses of coronary vessels with documented ischemia in stress MRI was 81% (68-90%), the specificity was 88% (82-92%). Diameter stenoses (DS) and area stenoses (AS) in vessels with positive stress MRI were significantly higher than in vessels without ischemia (DS 59.1% (49.4-68.4%) vs. 34.8% (27.1-46.1%) p < 0.001; AS 75.6% (63.0-85.2%) vs. 45.0% (30.8-63.6%), p < 0.001). The analysis reveals a high correlation between coronary stenosis measured by cQFR and ischemic areas detected by stress MRI. The data set the stage to plan randomized studies assessing cQFR measurements with regard to clinical outcomes.

A synthetic epoxyeicosatrienoic acid analogue prevents the initiation of ischemic acute kidney injury.

AIM: Imbalances in cytochrome P450 (CYP)-dependent eicosanoid formation may play a central role in ischemic acute kidney injury (AKI). We reported previously that inhibition of 20-hydroxyeicosatetraenoic acid (20-HETE) action ameliorated ischemia/reperfusion (I/R)-induced AKI in rats. Now we tested the hypothesis that enhancement of epoxyeicosatrienoic acid (EET) actions may counteract the detrimental effects of 20-HETE and prevent the initiation of AKI. METHODS: Male Lewis rats underwent right nephrectomy and ischemia was induced by 45 min clamping of the left renal pedicle followed by up to 48 h of reperfusion. Circulating CYP-eicosanoid profiles were compared in patients who underwent cardiac surgery with (n = 21) and without (n = 38) developing postoperative AKI. RESULTS: Ischemia induced an about eightfold increase of renal 20-HETE levels, whereas free EETs were not accumulated. To compensate for this imbalance, a synthetic 14,15-EET analogue was administered by intrarenal infusion before ischemia. The EET analogue improved renal reoxygenation as monitored by in vivo parametric MRI during the initial 2 h reperfusion phase. The EET analogue improved PI3K- as well as mTORC2-dependent rephosphorylation of Akt, induced inactivation of GSK-3ß, reduced the development of tubular apoptosis and attenuated inflammatory cell infiltration. The EET analogue also significantly alleviated the I/R-induced drop in creatinine clearance. Patients developing postoperative AKI featured increased preoperative 20-HETE and 8,9-EET levels. CONCLUSIONS: Pharmacological interventions targeting the CYP-eicosanoid pathway could offer promising new options for AKI prevention. Individual differences in CYP-eicosanoid formation may contribute to the risk of developing AKI in clinical settings.

Chiral lipidomics of monoepoxy and monohydroxy metabolites derived from long-chain polyunsaturated fatty acids.

A chiral lipidomics approach was established for comprehensive profiling of regio- and stereoisomeric monoepoxy and monohydroxy metabolites of long-chain PUFAs as generated enzymatically by cytochromes P450 (CYPs), lipoxygenases (LOXs), and cyclooxygenases (COXs) and, in part, also unspecific oxidations. The method relies on reversed-phase chiral-LC coupled with ESI/MS/MS. Applications revealed partially opposing enantioselectivities of soluble and microsomal epoxide hydrolases (mEHs). Ablation of the soluble epoxide hydrolase (sEH) gene resulted in specific alterations in the enantiomeric composition of endogenous monoepoxy metabolites. For example, the (R,S)/(S,R)-ratio of circulating 14,15-EET changed from 2.1:1 in WT to 9.7:1 in the sEH-KO mice. Studies with liver microsomes suggested that CYP/mEH interactions play a primary role in determining the enantiomeric composition of monoepoxy metabolites during their generation and release from the ER. Analysis of human plasma showed significant enantiomeric excess with several monoepoxy metabolites. Monohydroxy metabolites were generally present as racemates; however, Ca2+-ionophore stimulation of whole blood samples resulted in enantioselective increases of LOX-derived metabolites (12S-HETE and 17S-hydroxydocosahexaenoic acid) and COX-derived metabolites (11R-HETE). Our chiral approach may provide novel opportunities for investigating the role of bioactive lipid mediators that generally exert their physiological functions in a highly regio- and stereospecific manner.

Functional characterization of a novel arachidonic acid 12S-lipoxygenase in the halotolerant bacterium Myxococcus fulvus exhibiting complex social living patterns.

Lipoxygenases are lipid peroxidizing enzymes, which frequently occur in higher plants and mammals. These enzymes are also expressed in lower multicellular organisms but here they are not widely distributed. In bacteria, lipoxygenases rarely occur and evaluation of the currently available bacterial genomes suggested that <0.5% of all sequenced bacterial species carry putative lipoxygenase genes. We recently rescreened the public bacterial genome databases for lipoxygenase-like sequences and identified two novel lipoxygenase isoforms (MF-LOX1 and MF-LOX2) in the halotolerant Myxococcus fulvus. Both enzymes share a low degree of amino acid conservation with well-characterized eukaryotic lipoxygenase isoforms but they involve the catalytically essential iron cluster. Here, we cloned the MF-LOX1 cDNA, expressed the corresponding enzyme as N-terminal hexa-his-tag fusion protein, purified the recombinant enzyme to electrophoretic homogeneity, and characterized it with respect to its protein-chemical and enzymatic properties. We found that M. fulvus expresses a catalytically active intracellular lipoxygenase that converts arachidonic acid and other polyunsaturated fatty acids enantioselectively to the corresponding n-9 hydroperoxy derivatives. The enzyme prefers C20 - and C22 -polyenoic fatty acids but does not exhibit significant membrane oxygenase activity. The possible biological relevance of MF-LOX1 will be discussed in the context of the suggested concepts of other bacterial lipoxygenases.

Hypoxia-reoxygenation enhances murine afferent arteriolar vasoconstriction by angiotensin II.

We tested the hypothesis that hypoxia-reoxygenation (H/R) augments vasoreactivity to angiotensin II (ANG II). In particular, we compared an in situ live kidney slice model with isolated afferent arterioles (C57Bl6 mice) to assess the impact of tubules on microvessel response. Hematoxylin and eosin staining was used to estimate slice viability. Arterioles in the slices were located by differential interference contrast microscopy, and responses to vasoactive substances were assessed. Cytosolic calcium transients and NADPH oxidase (NOX) mRNA expression were studied in isolated afferent arterioles. SOD activity was measured in live slices. Both experimental models were subjected to control and H/R treatment (60 min). Slices were further analyzed after 30-, 60-, and 90-min hypoxia followed by 10- or 20-min reoxygenation (H/R). H/R resulted in enhanced necrotic tissue damage compared with control conditions. To characterize the slice model, we applied ANG II (10-7 M), norepinephrine (NE; 10-5 M), endothelin-1 (ET-1; 10-7 M), and ATP (10-4 M), reducing the initial diameter to 44.5 ± 2.8, 50.0 ± 2.2, 45.3 ± 2.6, and 74.1 ± 1.8%, respectively. H/R significantly increased the ANG II response compared with control in live slices and in isolated afferent arterioles, although calcium transients remained similar. TEMPOL incubation prevented the H/R effect on ANG II responses. H/R significantly increased NOX2 mRNA expression in isolated arterioles. SOD activity was significantly decreased after H/R. Enhanced arteriolar responses after H/R occurred independently from the surrounding tissue, indicating no influence of tubules on vascular function in this model. The mechanism of increased ANG II response after H/R might be increased oxidative stress and increased calcium sensitivity of the contractile apparatus.

Vasopressin lowers renal epoxyeicosatrienoic acid levels by activating soluble epoxide hydrolase.

Activation of the thick ascending limb (TAL) Na+-K+-2Cl- cotransporter (NKCC2) by the antidiuretic hormone arginine vasopressin (AVP) is an essential mechanism of renal urine concentration and contributes to extracellular fluid and electrolyte homeostasis. AVP effects in the kidney are modulated by locally and/or by systemically produced epoxyeicosatrienoic acid derivates (EET). The relation between AVP and EET metabolism has not been determined. Here, we show that chronic treatment of AVP-deficient Brattleboro rats with the AVP V2 receptor analog desmopressin (dDAVP; 5 ng/h, 3 days) significantly lowered renal EET levels (-56 ± 3% for 5,6-EET, -50 ± 3.4% for 11,12-EET, and -60 ± 3.7% for 14,15-EET). The abundance of the principal EET-degrading enzyme soluble epoxide hydrolase (sEH) was increased at the mRNA (+160 ± 37%) and protein levels (+120 ± 26%). Immunohistochemistry revealed dDAVP-mediated induction of sEH in connecting tubules and cortical and medullary collecting ducts, suggesting a role of these segments in the regulation of local interstitial EET signals. Incubation of murine kidney cell suspensions with 1 µM 14,15-EET for 30 min reduced phosphorylation of NKCC2 at the AVP-sensitive threonine residues T96 and T101 (-66 ± 5%; P < 0.05), while 14,15-DHET had no effect. Concomitantly, isolated perfused cortical thick ascending limb pretreated with 14,15-EET showed a 30% lower transport current under high and a 70% lower transport current under low symmetric chloride concentrations. In summary, we have shown that activation of AVP signaling stimulates renal sEH biosynthesis and enzyme activity. The resulting reduction of EET tissue levels may be instrumental for increased NKCC2 transport activity during AVP-induced antidiuresis.

Renal Ischemia/Reperfusion Injury in Soluble Epoxide Hydrolase-Deficient Mice.

AIM: 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) are cytochrome P450 (CYP)-dependent eicosanoids that play opposite roles in the regulation of vascular tone, inflammation, and apoptosis. 20-HETE aggravates, whereas EETs ameliorate ischemia/reperfusion (I/R)-induced organ damage. EETs are rapidly metabolized to dihydroxyeicosatrienoic acids (DHETs) by the soluble epoxide hydrolase (sEH). We hypothesized that sEH gene (EPHX2) deletion would increase endogenous EET levels and thereby protect against I/R-induced acute kidney injury (AKI). METHODS: Kidney damage was evaluated in male wildtype (WT) and sEH-knockout (KO)-mice that underwent 22-min renal ischemia followed by two days of reperfusion. CYP-eicosanoids were analyzed by liquid chromatography tandem mass spectrometry. RESULTS: Contrary to our initial hypothesis, renal function declined more severely in sEH-KO mice as indicated by higher serum creatinine and urea levels. The sEH-KO-mice also featured stronger tubular lesion scores, tubular apoptosis, and inflammatory cell infiltration. Plasma and renal EET/DHET-ratios were higher in sEH-KO than WT mice, thus confirming the expected metabolic consequences of sEH deficiency. However, CYP-eicosanoid profiling also revealed that renal, but not plasma and hepatic, 20-HETE levels were significantly increased in sEH-KO compared to WT mice. In line with this finding, renal expression of Cyp4a12a, the murine 20-HETE-generating CYP-enzyme, was up-regulated both at the mRNA and protein level, and Cyp4a12a immunostaining was more intense in the renal arterioles of sEH-KO compared with WT mice. CONCLUSION: These results indicate that the potential beneficial effects of reducing EET degradation were obliterated by a thus far unknown mechanism leading to kidney-specific up-regulation of 20-HETE formation in sEH-KO-mice.