GPR39: An orphan receptor begging for ligands.

Progress in the understanding of the receptor GPR39 is held up by inconsistent pharmacological data. First, the endogenous ligand(s) remain(s) contentious. Data pointing to zinc ions (Zn2+) and/or eicosanoids as endogenous ligands are a matter of debate. Second, there are uncertainties in the specificity of the widely used synthetic ligand (agonist) TC-G 1008. Third, activation of GPR39 has been often proposed as a novel treatment strategy, but new data also support that inhibition might be beneficial in certain disease contexts. Constitutive activity/promiscuous signaling suggests the need for antagonists/inverse agonists in addition to (biased) agonists. Here, we scrutinize data on the signaling and functions of GPR39 and critically assess factors that might have contributed to divergent outcomes and interpretations of investigations on this important receptor.

Lower serum 15-HETE level predicts nasal ILC2 accumulation during COX-1 inhibition in AERD.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is associated with high levels of cysteinyl leukotrienes, prostaglandin D2, and low levels of prostaglandin E2. Further, 15-hydroxyeicosatetraenoic acid (15-HETE) levels may have predictive value in therapeutic outcomes of aspirin desensitization. Accumulation of nasal group 2 innate lymphoid cells (ILC2s) has been demonstrated during COX-1 inhibition in AERD, although the relationships between tissue ILC2 accumulation, reaction symptom severity, and novel lipid biomarkers are unknown. OBJECTIVE: We sought to determine whether novel lipid mediators are predictive of nasal ILC2 accumulation and symptom scores during COX-1 inhibitor challenge in patients with AERD. METHODS: Blood and nasal scraping samples from patients with AERD were collected at baseline and COX-1 inhibitor reaction and then processed for flow cytometry for nasal ILC2s and serum for lipidomic analysis. RESULTS: Eight patients with AERD who were undergoing aspirin desensitization were recruited. Of the 161 eicosanoids tested, 42 serum mediators were detected. Baseline levels of 15-HETE were negatively correlated with the change in numbers of airway ILC2s (r = -0.6667; P = .0428). Docosahexaenoic acid epoxygenase metabolite 19,20-dihydroxy-4Z,7Z,10Z,13Z,16Z-docosapentaenoic acid (19,20-diHDPA) was positively correlated with both changes in airway ILC2s (r = 0.7143; P = .0305) and clinical symptom scores (r = 0.5000; P = .0081). CONCLUSION: Low levels of baseline 15-HETE predicted a greater accumulation of airway ILC2s in patients with AERD who were receiving COX-1 inhibition. Further, increases in the cytochrome P pathway metabolite 19,20-dihydroxy-4Z,7Z,10Z,13Z,16Z-docosapentaenoic acid (19,20-diHDPA) were associated with increased symptoms and nasal ILC2 accumulation. Future studies to assess how these mediators might control ILC2s may improve the understanding of AERD pathogenesis.

Roles of hydroxyeicosatetraenoic acids in diabetes (HETEs and diabetes).

Hydroxyeicosatetraenoic acids (HETEs) are metabolites of arachidonate acid (AA) oxidized by lipoxygenases or cytochrome P450 enzymes (CYP450). Since lipoxygenases and CYP450 enzymes widely exist in different organs and tissues, HETEs play significant roles in normal physiological and pathophysiological conditions. Mounting evidence has shown that HETEs play roles in modulation of inflammation during diabetes development. And accumulating evidence suggests that in prediabetic conditions, HETEs have already impacted on adipose tissue, kidney, heart, and islet. In the current review, we focused on the role of specific HETEs, namely 5-HETE, 12-HETE, 15-HETE and 20-HETE in diabetes, and highlighted their effects in the development of diabetes and diabetes-related complications. In conclusion, elucidation of HETEs' impacts on different organs that contribute to the development of diabetes leads to identification of novel therapeutic modalities.

Arachidonate 15-lipoxygenase type B: Regulation, function, and its role in pathophysiology.

As a lipoxygenase (LOX), arachidonate 15-lipoxygenase type B (ALOX15B) peroxidizes polyenoic fatty acids (PUFAs) including arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and linoleic acid (LA) to their corresponding fatty acid hydroperoxides. Distinctive to ALOX15B, fatty acid oxygenation occurs with positional specificity, catalyzed by the non-heme iron containing active site, and in addition to free PUFAs, membrane-esterified fatty acids serve as substrates for ALOX15B. Like other LOX enzymes, ALOX15B is linked to the formation of specialized pro-resolving lipid mediators (SPMs), and altered expression is apparent in various inflammatory diseases such as asthma, psoriasis, and atherosclerosis. In primary human macrophages, ALOX15B expression is associated with cellular cholesterol homeostasis and is induced by hypoxia. Like in inflammation, the role of ALOX15B in cancer is inconclusive. In prostate and breast carcinomas, ALOX15B is attributed a tumor-suppressive role, whereas in colorectal cancer, ALOX15B expression is associated with a poorer prognosis. As the biological function of ALOX15B remains an open question, this review aims to provide a comprehensive overview of the current state of research related to ALOX15B.

Involvement of Proinflammatory Arachidonic Acid (ARA) Derivatives in Crohn's Disease (CD) and Ulcerative Colitis (UC).

Recently, an increase in the incidence of inflammatory bowel disease (IBD) has been observed, especially among children and adolescents. Currently, few studies focus on the differentiation of inflammation in IBD subunits, i.e., Crohn's Disease (CD) and Ulcerative Colitis (UC). The aim of this study was to compare the concentrations of proinflammatory mediators of arachidonic acid (ARA) and linoleic acid (LA) in patients with CD (n = 34) and UC (n = 30), in order to identify differences in inflammation in both diseases and within the same entity, according to disease activity. Sixty-four adolescents with a mean age of 13.76 ± 2.69 and 14.15 ± 3.31, for CD and UC, respectively, were enrolled in the study. Biochemical analysis of ARA and LA derivatives was performed using a liquid chromatography. A trend was observed in the concentration of 15S-HETE (hydroxyeicosatetraenoic acids) in CD relative to UC. The active phase of both diseases showed a higher 15S-HETE concentration in active CD relative to active UC. Comparing patients with CD with active and inactive disease showed a trend of increased levels of thromboxane B2, leukotriene B4 and 9S-HODE (hydroxyoctadecadienoic acid) in the active versus the inactive disease. We also observed statistically significantly higher levels of 12S-HETE in inactive CD relative to active CD. In the UC group, on the other hand, statistically significantly higher levels of prostaglandin E2 and 16RS-HETE were observed in active UC relative to inactive UC. Moreover, significantly higher concentrations of LTX A4 5S, 6R were observed in inactive UC relative to the active phase. In conclusion, the present study indicated the activity of the 15-LOX pathway in CD. Further studies involving lipid mediators in patients with IBD may contribute to the development of new therapies for the treatment of IBD. The identification of differences in the course of inflammation may help to target therapy in CD and UC, and perhaps allow the introduction of an additional diagnostic marker between the two main IBD subtypes.

Levels of 15-HETE and TXB2 in exhaled breath condensates as markers for diagnosis of childhood asthma and its therapeutic outcome.

BACKGROUND: Dysregulation of eicosanoids is associated with asthma and a composite of oxylipins, including exhaled leukotriene B4 (LTB4 ), characterizes childhood asthma. While fractional exhaled nitric oxide (FeNO) has been used as the standard for monitoring steroid responsiveness, the potential utility of eicosanoids in monitoring the therapeutic outcomes remains unclear. We aimed to examine the levels of major eicosanoids representing different metabolic pathways in exhaled breath condensates (EBCs) of children with asthma during exacerbation and after treatment. METHODS: Levels of 6 exhaled eicosanoid species in asthmatic children and healthy subjects were evaluated using ELISA. RESULTS: In addition to those previously reported, including LTB4 , the levels of exhaled 15-hydroxyeicosatetraenoic acid (15-HETE), but not thromboxane B2 (TXB2 ), showed significant difference between asthmatics (N = 318) and healthy controls (N = 97), particularly the severe group showed the lowest levels of exhaled 15-HETE. Receiver operating characteristic (ROC) curve analyses revealed similar distinguishing power for the levels of 15-HETE, FEV1 (forced expiratory volume in the first second), and FeNO, while the 15-HETE/LTB4 ratio was significantly lower in subjects with asthma as compared to that of healthy controls (p < 0.0001). Analysis of asthmatics (N = 75) during exacerbation and convalescence showed significant improvement in lung function (FEV1 , p < .001), but not FeNO, concomitant with significantly increased levels of 15-HETE (p < .001) and reduced levels of TXB2 (p < .05) at convalescence, particularly for those who at the top 30% level during exacerbation. Further, decreased LTB4 and lipoxin A4 (LXA4 ) at convalescence were noted only in those at the top 30 percentile during exacerbation. CONCLUSION: The exhaled 15-HETE was found to discriminate childhood asthma while decreased levels of exhaled TXB2 and increased levels of 15-HETE were prominent at convalescence.

The anti-inflammatory effects of 15-HETE on osteoarthritis during treadmill exercise.

AIMS: Investigate the involvement of 15-hydroxyeicosatetraenoic acid (15-HETE), an anti-inflammatory molecule, on the beneficial effects of exercise therapy for osteoarthritis (OA). MAIN METHODS: 15-HETE (10 µM, twice a week) and monosodium iodoacetate (MIA) (1 mg) were injected into rat knee joints. Treadmill exercise was applied on OA rat. Primary rat chondrocytes were treated with 15-HETE, LY294002 and interleukin (IL)-1ß. Rats undergo a 1 hour single session treadmill exercise once. 15-HETE levels in the knee joint were evaluated using ELISA after a single session of treadmill exercise on healthy and OA rats. Matrix metalloproteinase (MMP)3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS)-5, p-Akt, Akt, and collagen type 2 (COL2) expression were evaluated using RT-PCR and western blotting. OA degree was evaluated using X-ray, scored by Osteoarthritis Research Society International (OARSI) and Mankin scores. COL2 and MMP-13 expression in articular was evaluated using immunohistochemistry. KEY FINDINGS: Medium intensity exercise alleviated OA. 15-HETE levels after exercise was increased. 15-HETE inhibited IL-1ß-induced inflammation in primary chondrocytes and increased p-Akt levels. LY294002 blocked the effect of 15-HETE in vitro. Finally, 15-HETE alleviated cartilage damage, inhibited MMP-13 expression, and increased COL2 expression in joint cartilage tissue. SIGNIFICANCE: Treadmill exercise alleviates OA and increases 15-HETE levels in the knee joint, which suppresses inflammation in chondrocytes via PI3k-Akt signalling in vitro and in vivo.

Effect of renal replacement therapy on selected arachidonic acid derivatives concentration.

BACKGROUND: Platelet activation is an important side effect of dialysis, resulted in a subsequent release of arachidonic acid (AA) from activated platelets. AA is involved in many pathologic conditions, such as inflammation, asthma, cancer, diabetes, hypertension, and the pathogenesis of kidney disease. The aim of this study was to define whether the dialysis type affects the concentration of AA derivatives in patients with chronic kidney disease. METHODS: 117 patients were qualified to the study group. Based on the type of renal replacement therapy, patients were divided into the following groups: hemodialysis (HD A - before/HD B - after hemodialysis), peritoneal dialysis (PD), kidney transplant patients (TE - before/TE A - after transplantation) and conservative treatment (CT) (30; 30; 27; 30 patients, respectively). The control group consisted of 30 healthy volunteers (NK). The ELISA methods were used to measure the concentrations of TXB2, 5-HETE, 12-HETE, and 15-HETE in the blood serum. RESULTS: Renal replacement therapy significantly influences the concentration of TXB2 (mean ± SD [ng/mL]: HD A- 34.6 ± 9; HD B- 28.3 ± 15.2; PD- 28.3 ± 15.2; CT- 34.2 ± 8.0; TE- 36.7 ± 42.9; TE A- 27.9 ± 8.8; NK- 19.6 ± 15; p = 0.010), 5-HETE (mean ± SD [ng/mL]: HD A- 284.2 ± 428.4; HD B- 304.8 ± 516.2; PD - 530.0 ± 553.3; CT- 318.7 ± 366.0; TE- 525.6 ± 358.0; TE A - 409.8 ± 377.1; NK 838.1 ± 497.8; p < 0.001) and 15-HETE (HD A-18.1 ± 8.7; HD B- 42.2 ± 14; PD - 36.3 ± 13.8; CT- 33.7 ± 14.0; TE- 19.5 ± 10.2; TE A - 34.4 ± 16.3; NK 22.2 ± 17.8; p < 0,001). There was a significant relationship between the type of renal replacement therapy and the duration of dialysis, and the concentration of TXB2, 12-HETE acid, and 15-HETE. CONCLUSIONS: The type of renal replacement therapy significantly affects the concentration of AA derivatives. Peritoneal dialysis is the best method of dialysis, taking into account the concentration of arachidonic acid derivatives.

The functional link between microsomal prostaglandin E synthase-1 (mPGES-1) and peroxisome proliferator-activated receptor γ (PPARγ) in the onset of inflammation.

Many years have elapsed since the discovery of anti-inflammatories as effective therapeutics for the treatment of inflammatory-related diseases, but we are still uncovering their various mechanisms of action. Recent biochemical and pharmacological studies have shown that in different tissues and cell types lipid mediators from thearachidonic acid cascade, play a crucial role in the initiation and resolution of inflammation by shifting from pro-inflammatory prostaglandin (PG)E2 to anti-inflammatory PGD2 and PGJ2. Considering that until now very little is known about the biological effects evoked by microsomal prostaglandin E synthase-1 (mPGES-1) and contextually by peroxisome proliferator-activated receptor γ (PPARγ) modulation (key enzymes involved in PGE2 and PGD2/PGJ2metabolism), in this opinion paper we sought to define the coordinate functional regulation between these two enzymes at the "crossroads of phlogistic pathway" involved in the induction and resolution of inflammation.

Brozopine Inhibits 15-LOX-2 Metabolism Pathway After Transient Focal Cerebral Ischemia in Rats and OGD/R-Induced Hypoxia Injury in PC12 Cells.

The goal of this study was to elucidate the mechanisms of protection of Sodium (±)-5-bromo-2-(α-hydroxypentyl) benzoate (trade name: Brozopine, BZP) against cerebral ischemia in vivo and in vitro. To explore the protective effect of BZP on focal cerebral ischemia-reperfusion injury, we evaluated the effects of various doses of BZP on neurobehavioral score, cerebral infarction volume, cerebral swelling in MCAO rats (ischemia for 2 h, reperfusion for 24 h). In addition, the effects of various doses of BZP on OGD/R-induced-PC12 cells injury (hypoglycemic medium containing 30 mmol Na2S2O4 for 2 h, reoxygenation for 24 h) were evaluated. Four in vivo and in vitro groups were evaluated to characterize targets of BZP: Control group, Model group, BZP group (10 mg/kg)/BZP group (30 µmol/L), C8E4 group (10 mg/kg)/C8E4 group (30 µmol/L). An ELISA kit was used to determine the levels of 15-HETE (a 15-LOX-2 metabolite) in vivo and in vitro. Rat nuclear factor κB subunit p65 (NF-κB p65), tumor necrosis factor (TNF-α), interleukin-6 (IL-6), and intercellular adhesion molecule-1 (ICAM-1) were also quantified in vivo and in vitro. The results showed that BZP improved focal cerebral ischemia-reperfusion injury in rats and PC12 cells treated with Na2S2O4 in dose/concentration-dependent manners through inhibition of production of 15-HETE and expression of NF-κB, IL-6, TNF-α, and ICAM-1. In conclusion, BZP exerted protective effects against cerebral ischemia via inhibition of 15-LOX-2 activity.

Contributions of 12/15-Lipoxygenase to Bleeding in the Brain Following Ischemic Stroke.

Ischemic strokes are caused by one or more blood clots that typically obstruct one of the major arteries in the brain, but frequently also result in leakage of the blood-brain barrier and subsequent hemorrhage. While it has long been known that the enzyme 12/15-lipoxygenase (12/15-LOX) is up-regulated following ischemic strokes and contributes to neuronal cell death, recent research has shown an additional major role for 12/15-LOX in causing this hemorrhagic transformation. These findings have important implications for the use of 12/15-LOX inhibitors in the treatment of stroke.

15-HETE protects pulmonary artery smooth muscle cells against apoptosis via SIRT1 regulation during hypoxia.

15-Hydroxyeicosatetraenoic acid (15-HETE) is produced by the catalytic metabolism of arachidonic acid by the enzyme 15-lipoxygenase. It is produced during hypoxia, and participates in the remodeling of pulmonary artery smooth muscle (PASM). Previous research has revealed that sirtuin 1 (SIRT1) involved in apoptosis in various cells and tissues. Herein, we attempted to determine whether 15-HETE counteracts SIRT1-promoted cell death in murine PASM cells (PASMCs). To verify this theory, we investigated changes in SIRT1 concentration in response to the counteraction of cell death by 15-HETE. We used western blotting and a terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay, and investigated the survival, nuclear morphology, and mitochondrial potential of the cells. Our results revealed that 15-HETE promotes the transcription and translation of SIRT1. Moreover, 15-HETE increases viability and impaired mitochondrial depolarization, and promotes the expression of Bcl-2 and Bcl-xL in PASMCs without serum. The reactions mentioned above were eliminated by SIRT1 inhibitors (EX 527 and SIRT1 inhibitor IV). Our findings suggest that 15-HETE is crucial for the protection of PASMCs against cell death, and the SIRT1 pathway may provide a new strategy for pulmonary artery hypertension therapy.

MMP-2 and MMP-9 contribute to the angiogenic effect produced by hypoxia/15-HETE in pulmonary endothelial cells.

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.

Role of endoplasmic reticulum stress in 12/15-lipoxygenase-induced retinal microvascular dysfunction in a mouse model of diabetic retinopathy.

AIMS/HYPOTHESIS: Our earlier studies have established the role of 12/15-lipoxygenase (LO) in mediating the inflammatory reaction in diabetic retinopathy. However, the exact mechanism is still unclear. The goal of the current study was to identify the potential role of endoplasmic reticulum (ER) stress as a major cellular stress response in the 12/15-LO-induced retinal changes in diabetic retinopathy. METHODS: We used in vivo and in vitro approaches. For in vivo studies, experimental diabetes was induced in wild-type (WT) mice and 12/15-Lo (also known as Alox15) knockout mice (12/15-Lo-/-); ER stress was then evaluated after 12-14 weeks of diabetes. We also tested the effect of intravitreal injection of 12-hydroxyeicosatetraenoic acid (HETE) on retinal ER stress in WT mice and in mice lacking the catalytic subunit of NADPH oxidase, encoded by Nox2 (also known as Cybb) (Nox2-/- mice). In vitro studies were performed using human retinal endothelial cells (HRECs) treated with 15-HETE (0.1 µmol/l) or vehicle, with or without ER stress or NADPH oxidase inhibitors. This was followed by evaluation of ER stress response, NADPH oxidase expression/activity and the levels of phosphorylated vascular endothelial growth factor receptor-2 (p-VEGFR2) by western blotting and immunoprecipitation assays. Moreover, real-time imaging of intracellular calcium (Ca2+) release in HRECs treated with or without 15-HETE was performed using confocal microscopy. RESULTS: Deletion of 12/15-Lo significantly attenuated diabetes-induced ER stress in mouse retina. In vitro, 15-HETE upregulated ER stress markers such as phosphorylated RNA-dependent protein kinase-like ER-regulated kinase (p-PERK), activating transcription factor 6 (ATF6) and protein disulfide isomerase (PDI) in HRECs. Inhibition of ER stress reduced 15-HETE-induced-leucocyte adhesion, VEGFR2 phosphorylation and NADPH oxidase expression/activity. However, inhibition of NADPH oxidase or deletion of Nox2 had no effect on ER stress induced by the 12/15-LO-derived metabolites both in vitro and in vivo. We also found that 15-HETE increases the intracellular calcium in HRECs. CONCLUSIONS/INTERPRETATION: ER stress contributes to 12/15-LO-induced retinal inflammation in diabetic retinopathy via activation of NADPH oxidase and VEGFR2. Perturbation of calcium homeostasis in the retina might also play a role in linking 12/15-LO to retinal ER stress and subsequent microvascular dysfunction in diabetic retinopathy.

Arachidonic acid metabonomics study for understanding therapeutic mechanism of Huo Luo Xiao Ling Dan on rat model of rheumatoid arthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Huo Luo Xiao Ling Dan (HLXLD), a traditional Chinese medicine (TCM), is commonly used for the treatment of rheumatoid arthritis (RA). AIM OF THE STUDY: To explore the potential therapeutic mechanism of HLXLD on anti-inflammatory activity. MATERIALS AND METHODS: A metabolomic approach based on UFLC-MS/MS to profile arachidonic acid (AA) metabolic changes was used. The cyclooxygenase (COX) and lipoxygenase (LOX) catalyzed metabolites in plasma were quantified on 7, 14, 21, and 28 days after the rats injected with Complete Freund's adjuvant and orally administrated with HLXLD, methotrexate and dexamethasone in parallel as the positive control drugs. RESULTS: Nineteen metabolites involved in COX and LOX pathways in RA model group were significant increased compared with normal group (P < 0.05), including 12-hydroxyeicosatetraenoic acid (12-HETE), 15-HETE, 8-HETE, leukotriene B4(LTB4), prostaglandin E2 (PGE2), PGI2, PGD2, PGF2α, thromboxane B2 (TXB2), etc. From day 7 to day 28, the trajectory direction of HLXLD group and positive control groups gradually moved towards the initial space, and the concentrations of AA and its metabolites after HLXLD treatment were significantly reduced in dual pathways compared to control groups. CONCLUSION: HLXLD induced a substantial change in the AA metabolic profiles through refrain the expression of COX and LOX. The present investigation also highlights that distinct ingredients of this formula tend to inhibit different target to achieve a therapeutic effect.

Targeting of 12/15-Lipoxygenase in retinal endothelial cells, but not in monocytes/macrophages, attenuates high glucose-induced retinal leukostasis.

AIMS: Our previous studies have established a role for 12/15-lipoxygenase (LO) in mediating the inflammatory response in diabetic retinopathy (DR). However, the extent at which the local or systemic induction of 12/15-LO activity involved is unclear. Thus, the current study aimed to characterize the relative contribution of retinal endothelial versus monocytic/macrophagic 12/15-LO to inflammatory responses in DR. MATERIALS & METHODS: We first generated a clustered heat map for circulating bioactive lipid metabolites in the plasma of streptozotocin (STZ)-induced diabetic mice using liquid chromatography coupled with mass-spectrometry (LC-MS) to evaluate changes in circulating 12/15-LO activity. This was followed by comparing the in vitro mouse endothelium-leukocytes interaction between leukocytes isolated from 12/15-LO knockout (KO) versus those isolated from wild type (WT) mice using the myeloperoxidase (MPO) assay. Finally, we examined the effects of knocking down or inhibiting endothelial 12/15-LO on diabetes-induced endothelial cell activation and ICAM-1 expression. RESULTS: Analysis of plasma bioactive lipids' heat map revealed that the activity of circulating 12/15-LO was not altered by diabetes as evident by no significant changes in the plasma levels of major metabolites derived from 12/15-lipoxygenation of different PUFAs, including linoleic acid (13-HODE), arachidonic acid (12- and 15- HETEs), eicosapentaenoic acid (12- and 15- HEPEs), or docosahexaenoic acid (17-HDoHE). Moreover, leukocytes from 12/15-LO KO mice displayed a similar increase in adhesion to high glucose (HG)-activated endothelial cells as do leukocytes from WT mice. Furthermore, abundant proteins of 12-LO and 15-LO were detected in human retinal endothelial cells (HRECs), while it was undetected (15-LO) or hardly detectable (12-LO) in human monocyte-like U937 cells. Inhibition or knock down of endothelial 12/15-LO in HRECs blocked HG-induced expression of ICAM-1, a well-known identified important molecule for leukocyte adhesion in DR. CONCLUSION: Our data support that endothelial, rather than monocytic/macrophagic, 12/15-LO has a critical role in hyperglycemia-induced ICAM-1 expression, leukocyte adhesion, and subsequent local retinal barrier dysfunction. This may facilitate the development of more precisely targeted treatment strategies for DR.

(5Z,11Z,15R)-15-Hydroxyeicosa-5,11-dien-13-ynoic acid: A stable isomer of 15(S)-HETE that retains key vasoconstrictive and antiproliferative activity.

15(S)-Hydroxyeicosa-(5Z,8Z,11Z,13E)-tetraenoic acid (15(S)-HETE) is a metabolite of arachidonic acid that elicits a number of biological effects including vasoconstriction and angiogenesis. (5Z,11Z,15R)-15-Hydroxyeicosa-5,11-dien-13-ynoic acid (HETE analog 1) is a synthetic isomer of 15(S)-HETE that is much more stable to autoxidation. Using isometric recording of isolated pulmonary arteries from male and female rabbits, HETE analog 1 and 15(S)-HETE were found to elicit concentration-dependent contractions that were slightly greater in females compared to males. The maximal response in females was greater with 15(S)-HETE. HETE analog 1 and 15(S)-HETE increased [(3)H]-thymidine incorporation in vascular smooth muscle cells cultured from male rabbit pulmonary arteries; both the maximal response and potency were greater with 15(S)-HETE. In contrast, HETE analog 1 produced a concentration-dependent inhibition in proliferation and migration of human hormone-independent prostate carcinoma PC-3 cells. The protocol for synthesis of HETE analog 1 is reported. The stability of this substance and its similar biological profile to 15(S)-HETE support future studies in eicosanoid research.

Hormetic and regulatory effects of lipid peroxidation mediators in pancreatic beta cells.

Nutrient sensing mechanisms of carbohydrates, amino acids and lipids operate distinct pathways that are essential for the adaptation to varying metabolic conditions. The role of nutrient-induced biosynthesis of hormones is paramount for attaining metabolic homeostasis in the organism. Nutrient overload attenuate key metabolic cellular functions and interfere with hormonal-regulated inter- and intra-organ communication, which may ultimately lead to metabolic derangements. Hyperglycemia and high levels of saturated free fatty acids induce excessive production of oxygen free radicals in tissues and cells. This phenomenon, which is accentuated in both type-1 and type-2 diabetic patients, has been associated with the development of impaired glucose tolerance and the etiology of peripheral complications. However, low levels of the same free radicals also induce hormetic responses that protect cells against deleterious effects of the same radicals. Of interest is the role of hydroxyl radicals in initiating peroxidation of polyunsaturated fatty acids (PUFA) and generation of α,ß-unsaturated reactive 4-hydroxyalkenals that avidly form covalent adducts with nucleophilic moieties in proteins, phospholipids and nucleic acids. Numerous studies have linked the lipid peroxidation product 4-hydroxy-2E-nonenal (4-HNE) to different pathological and cytotoxic processes. Similarly, two other members of the family, 4-hydroxyl-2E-hexenal (4-HHE) and 4-hydroxy-2E,6Z-dodecadienal (4-HDDE), have also been identified as potential cytotoxic agents. It has been suggested that 4-HNE-induced modifications in macromolecules in cells may alter their cellular functions and modify signaling properties. Yet, it has also been acknowledged that these bioactive aldehydes also function as signaling molecules that directly modify cell functions in a hormetic fashion to enable cells adapt to various stressful stimuli. Recent studies have shown that 4-HNE and 4-HDDE, which activate peroxisome proliferator-activated receptor δ (PPARδ) in vascular endothelial cells and insulin secreting beta cells, promote such adaptive responses to ameliorate detrimental effects of high glucose and diabetes-like conditions. In addition, due to the electrophilic nature of these reactive aldehydes they form covalent adducts with electronegative moieties in proteins, phosphatidylethanolamine and nucleotides. Normally these non-enzymatic modifications are maintained below the cytotoxic range due to efficient cellular neutralization processes of 4-hydroxyalkenals. The major neutralizing enzymes include fatty aldehyde dehydrogenase (FALDH), aldose reductase (AR) and alcohol dehydrogenase (ADH), which transform the aldehyde to the corresponding carboxylic acid or alcohols, respectively, or by biding to the thiol group in glutathione (GSH) by the action of glutathione-S-transferase (GST). This review describes the hormetic and cytotoxic roles of oxygen free radicals and 4-hydroxyalkenals in beta cells exposed to nutritional challenges and the cellular mechanisms they employ to maintain their level at functional range below the cytotoxic threshold.

Activation of ERK pathway is required for 15-HETE-induced angiogenesis in human umbilical vascular endothelial cells.

Angiogenesis plays a critical role in the progression of cardiovascular disease, retinal ischemia, or tumorigenesis. The imbalance of endothelial cell proliferation and apoptosis disturbs the establishment of the vasculogenesis, which is affected by several arachidonic acid metabolites. 15-Hydroxyeicosatetraenoic acid (15-HETE) is one of the metabolites. However, the underlying mechanisms of angiogenesis induced by 15-HETE in human umbilical vascular endothelial cells (HUVECs) are still poorly understood. Since extracellular signal-regulated kinase (ERK) is a critical regulator of cell proliferation, there may be a crosstalk between 15-HETE-regulating angiogenic process and ERK-proliferative effect in HUVECs. To test this hypothesis, we study the effect of 15-HETE on cell proliferation, angiogenesis, and apoptosis using cell viability measurement, cell cycle analysis, western blot, scratch-wound, tube formation assay, and nuclear morphology determination. We found that 15-HETE promoted HUVEC angiogenesis, which were mediated by ERK. Moreover, 15-HETE-induced proliferation and cell cycle transition from the G(0)/G(1) phase to the G(2)/M + S phase. All these effects were reversed after blocking ERK with PD98059 (an ERK inhibitor). In addition, HUVEC apoptosis was relieved by 15-HETE through the ERK pathway. Thus, ERK is necessary for the effects of 15-HETE in the regulation of HUVEC angiogenesis, which may be a novel potential target for the treatment of angiogenesis-related diseases.

On the biosynthesis of 15-HETE and eoxin C4 by human airway epithelial cells.

Several lines of evidence indicate that 15-lipoxygenase type 1 (15-LO-1) plays a pathophysiological role in asthma. The aim for this study was to investigate the 15-LO-1 expression and activity in primary human airway epithelial cells cultivated on micro-porous filters at air-liquid interface. Incubation of human airway epithelial cells with arachidonic acid led to the formation of 15(S)-hydroxy-eicosatetraenoic acid (15-HETE) and exposing the cells to bacteria or physical injury markedly increased their production of 15-HETE. The cells were also found to convert arachidonic acid to eoxin C4 (EXC4). Subcellular fractionation revealed that the conversion of EXA4 to EXC4 was catalyzed by a soluble glutathione transferase (GST). The GST P1-1 enzyme was found to possess the highest activity of the investigated soluble GSTs. Following IL-4 treatment of airway epithelial cells, microarray analysis confirmed high expression of 15-LO-1 and GST P1-1, and immunohistochemical staining of bronchial biopsies revealed co-localization of 15-LO-1 and GST P1-1 in airway epithelial cells. These results indicate that respiratory infection and cell injury may activate the 15-LO pathway in airway epithelial cells. Furthermore, we also demonstrate that airway epithelial cells have the capacity to produce EXC4.