Major Arachidonic Acid Metabolite Research Articles

Introduction of a carboxylic acid group into pyrazolylpyridine derivatives increased selectivity for inhibition of the 20-HETE synthase CYP4A11/4F2

20-Hydroxyeicosatetraenoic acid (20-HETE) is a lipid mediator and one of the major arachidonic acid metabolites whose formation is mainly catalyzed by the enzymes cytochrome P450 (CYP) 4F2 and CYP4A11. Several studies have suggested that 20-HETE is involved in the pathogenesis of renal diseases, including diabetic nephropathy and autosomal dominant polycystic kidney disease, and we previously reported compound 1 as a dual inhibitor of CYP4A11/4F2 with therapeutic potential against renal fibrosis. Subsequent studies revealed that compound 1, the dual CYP4A11/4F2 inhibitor, however, exhibited low selectivity over another CYP4F subtype, CYP4F22, which catalyzes ω-hydroxylation of ultra-long-chain fatty acids (ULCFAs); ULCFAs are important for the formation of acylceramides, which play a role in skin barrier formation. Therefore, we sought to develop a CYP4A11/4F2 inhibitor that would show greater CYP4A11/4F2 selectivity against CYP4F22, to avoid potential dermatological side effects. We re-evaluated a series of compounds from our 20-HETE program and identified pyrazolylpyridine derivatives containing a carboxylic acid group showing only weak CYP4F22 inhibition. Subsequent optimization studies from these derivatives led to identification of compound 15, which showed CYP4A11/4F2 inhibition with improved selectivity against CYP4F22. Compound 15 inhibited 20-HETE production in both human and rat renal microsomes and did not inhibit ω-hydroxylation of ULCFAs in human keratinocytes. Compound 15 also significantly inhibited renal 20-HETE production after oral administration.

Abstract MP155: Oxidized Lipids Diet Causes Pulmonary Hypertension Through T-cell Dependent Endothelial Cell Apoptosis

Background: Pulmonary hypertension (PH) is a fatal disease characterized by an increased mean pulmonary arterial pressure above 25mmHg. This increased pressure is, at least in part, due to thickening of the distal pulmonary arteries. Recently, numerous studies demonstrated an increased plasma concentration of oxidized lipids in PH and in diseases where secondary PH developed. Furthermore, 15-hydroxyeicosatetraenoic acid (15-HETE) an oxidized lipid and a major metabolite of arachidonic acid in the lung, has been implicated in dysregulation of major biological pathways in PH. However, the mechanisms involved in the causal role of 15-HETE in pulmonary hypertension development are not known. Methods and Results: To study the role of 15-HETE in PH development, we fed C57BL6/J mice a diet supplemented with 15-HETE for 3 weeks with no other insults. After 3 weeks on the diet with added 15-HETE, C57BL6/J mice had increased concentrations of not only 15-HETE but also of other oxidized lipids (5-, 11- and 12-HETE) in plasma and lung, and they developed PH. RNA-seq analysis revealed the activation of pathways involved in antigen processing and presentation, and with evidence of T cell mediated cytotoxicity in lungs of mice fed 15-HETE. Transcriptomic profiling of lung tissues obtained from patients with pulmonary arterial hypertension (PAH) demonstrated activation of pathways similar to those seen mice. In mice fed a 15-HETE diet, there was an increase in the number of CD8/CD69 double positive cells, as well as an increase in pulmonary arterial endothelial cell (PAEC) apoptosis. Furthermore, PAEC exposed to 15-HETE were more prone to apoptosis when exposed to CD8 cells. Adding Tg6F, an apoA-I mimetic peptide to the 15-HETE diet prevented and rescued PH in C57BL6/J mice, in part, by inhibiting PAEC apoptosis. Conclusions: 15-HETE diet induced PH in C57Bl6/J mice by triggering PAEC death in a T-cell dependent mechanism. The apoA-I mimetic peptide Tg6F was able to prevent and rescue PH induced by 15-HETE.

Angiotensin II upregulates CYP4A isoform expression in the rat kidney through angiotensin II type 1 receptor

Angiotensin II (AngII) stimulates the renal production and release of 20-hydroxyeicosatetraenoic acids (20-HETE), which is a major metabolite of arachidonic acid catalyzed by CYP4A isoforms. However, the effects of AngII on CYP4A isoform expression in the kidney and its mechanism remains unclear. To clarify the regulation of CYP4A isoform expression by AngII, we examined the chronic effects of AngII and AngII type 1 receptor (AT1-R) blockade on CYP4A isoform expression. Sprague-Dawley rats were infused with vehicle or AngII for 1 week, and the AngII-infused rats were also treated with or without the AT1-R blocker, candesartan. AngII increased CYP4A isoform protein expression in the renal cortex (CO) and outer medulla (OM) in a dose-dependent manner, and candesartan inhibited the AngII-increased CYP4A expression in a dose-dependent manner. AngII increased the CYP4A isoform mRNA expression in the CO and OM, and candesartan inhibited AngII-increased CYP4A isoform mRNA expression. These results indicated that AngII chronically increased the CYP4A isoform expression in the rat kidney. The AngII-induced CYP4A isoform expression was mediated by AT1-R.

The 15-LO-1/15-HETE system promotes angiogenesis by upregulating VEGF in ischemic brains

Objectives: Angiogenesis promotes neurobehavioral recovery after cerebral ischemic stroke. 15(S)-hydroxyeicosatetraenoic acid (15-HETE) is one of the major metabolites of arachidonic acid by 15-lipoxygenase (15-LO) and stimulates the production of vascular endothelial growth factor (VEGF), thus, inducing autocrine-mediated angiogenesis. The present study aimed to investigate the role of 15-LO/15-HETE system on VEGF expression and angiogenesis in brain ischemia.Methods: Rat cerebral arterial vascular endothelial cells were used to set up a cell injury model of oxygen-glucose deprivation and reoxygenation (OGD/R), mimicking a condition of brain ischemia. A mouse model of middle cerebral artery occlusion (MCAO) was established.Results: Oxygen-glucose deprivation increased cellular expression of 15-LO-1 and VEGF. Transfection of 15-LO-1 siRNA depleted cells of 15-LO-1, and sequentially induced downregulation of VEGF expression; while, incubation of 15-HETE increased the expression of VEGF. Incubation of 15-HETE attenuated the reduction in cell viability induced by oxygen-glucose deprivation, and promoted cell migration, while transfection of 15-LO-1 siRNA showed an opposite effect. In animal experiments, the density of microvessels in hypoxic regions of brains was significantly increased after MCAO, while intracerebroventricular delivery of 15-LO-1 siRNA significantly reduced the density of microvessels, and downregulates VEGF expression.Discussion: The results indicate that the 15-LO-1/15-HETE system promotes angiogenesis in ischemic brains by upregulation of VEGF, representing a potential target for improving neurobehavioral recovery after cerebral ischemic stroke.

Deactivation of 12‐HETE by Peroxisomal β‐Oxidation by Alternatively Activated Macrophages

Polarization of macrophages to pro‐inflammatory M1 and to anti‐inflammatory or alternatively activated M2 state has important physiological implications in development of experimental hypertension and inflammation. 12‐ and 15‐hydroxyeicosatetraenoic acids (12‐ and 15‐HETE), the two major arachidonic acid metabolites of 12/15‐lipoxygenase, are essential in the process since the disruption of the encoding gene, Alox15, renders the mice unsusceptible to variety of types of experimental hypertension. The objective of this study was to test the hypothesis that alternatively activated M2 macrophages catabolize 12‐HETE into products that do not enhance angiotensin II‐mediated arterial constriction.We used cultures of M1‐ or M2‐polarized macrophages to study the metabolism of externally added [14C]‐12‐HETE. The 12‐HETE metabolites were separated by a reverse phase HPLC and identified by mass spectrometry.M2 polarized peritoneal macrophages converted most of the 12‐HETE into more polar metabolites within 2 hours of incubation. M1 macrophages, however, had little effect on the 12‐HETE catabolism. The same effect was observed in cultures of M1 or M2 polarized bone marrow‐derived macrophages. The major metabolite peaks were identified by mass‐spectrometry as β‐oxidation products. The conversion of 12‐HETE was inhibited by 50 μM thioridazine, a peroxisomal β‐oxidation inhibitor. Mitochondrial β‐oxidation inhibitors, 4‐pentenoic acid and etomoxir, had no effect on conversion. qPCR analysis of peroxisomal and mitochondrial β‐oxidation markers confirmed that only peroxisomal markers were upregulated upon M2 polarization. The major 12‐HETE metabolite peaks were collected and their activities were tested on abdominal aortic rings. While 12‐HETE (100 nM) pre‐incubation enhanced constrictions to angiotensin II (1–100 nM) from 15 % to 25 %, the metabolites lacked such an activity.Our results indicate that alternatively activated M2 macrophages are capable of degrading 12‐HETE and eliminating 12‐HETE's enhancement of angiotensin II‐induced vascular constriction, while M1 macrophages are not. These data support an antihypertensive role of M2 macrophages.Support or Funding InformationThis research was supported by the grant: NIH HL103673

Prenatal exposure to the organophosphate insecticide chlorpyrifos enhances brain oxidative stress and prostaglandin E2 synthesis in a mouse model of idiopathic autism.

BackgroundAutism spectrum disorders (ASD) are emerging as polygenic and multifactorial disorders in which complex interactions between defective genes and early exposure to environmental stressors impact on the correct neurodevelopment and brain processes. Organophosphate insecticides, among which chlorpyrifos (CPF), are widely diffused environmental toxicants associated with neurobehavioral deficits and increased risk of ASD occurrence in children. Oxidative stress and dysregulated immune responses are implicated in both organophosphate neurodevelopmental effects and ASD etiopathogenesis. BTBR T+tf/J mice, a well-studied model of idiopathic autism, show several behavioral and immunological alterations found in ASD children, and we recently showed that CPF gestational exposure strengthened some of these autistic-like traits. In the present study, we aimed at investigating whether the behavioral effects of gestational CPF administration are associated with brain increased oxidative stress and altered lipid mediator profile.MethodsBrain levels of F2-isoprostanes (15-F2t-IsoP), as index of in vivo oxidative stress, and prostaglandin E2 (PGE2), a major arachidonic acid metabolite released by immune cells and by specific glutamatergic neuron populations mainly in cortex and hippocampus, were assessed by specific enzyme-immuno assays in brain homogenates from BTBR T+tf/J and C57Bl6/J mice, exposed during gestation to either vehicle or CPF. Measures were performed in mice of both sexes, at different postnatal stages (PNDs 1, 21, and 70).ResultsAt birth, BTBR T+tf/J mice exhibited higher baseline 15-F2t-IsoP levels as compared to C57Bl6/J mice, suggestive of greater oxidative stress processes. Gestational treatment with CPF-enhanced 15-F2t-IsoP and PGE2 levels in strain- and age-dependent manner, with 15-F2t-IsoP increased in BTBR T+tf/J mice at PNDs 1 and 21, and PGE2 elevated in BTBR T+tf/J mice at PNDs 21 and 70. At PND 21, CPF effects were sex-dependent being the increase of the two metabolites mainly associated with male mice. CPF treatment also induced a reduction of somatic growth, which reached statistical significance at PND 21.ConclusionsThese findings indicate that the autistic-like BTBR T+tf/J strain is highly vulnerable to environmental stressors during gestational period. The results further support the hypothesis that oxidative stress might be the link between environmental neurotoxicants such as CPF and ASD. The increased levels of oxidative stress during early postnatal life could result in delayed and long-lasting alterations in specific pathways relevant to ASD, of which PGE2 signaling represents an important one.

An EP2 Agonist Facilitates NMDA-Induced Outward Currents and Inhibits Dendritic Beading through Activation of BK Channels in Mouse Cortical Neurons.

Prostaglandin E2 (PGE2), a major metabolite of arachidonic acid produced by cyclooxygenase pathways, exerts its bioactive responses by activating four E-prostanoid receptor subtypes, EP1, EP2, EP3, and EP4. PGE2 enables modulating N-methyl-D-aspartate (NMDA) receptor-mediated responses. However, the effect of E-prostanoid receptor agonists on large-conductance Ca2+-activated K+ (BK) channels, which are functionally coupled with NMDA receptors, remains unclear. Here, we showed that EP2 receptor-mediated signaling pathways increased NMDA-induced outward currents (I NMDA-OUT), which are associated with the BK channel activation. Patch-clamp recordings from the acutely dissociated mouse cortical neurons revealed that an EP2 receptor agonist activated I NMDA-OUT, whereas an EP3 receptor agonist reduced it. Agonists of EP1 or EP4 receptors showed no significant effects on I NMDA-OUT. A direct perfusion of 3,5′-cyclic adenosine monophosphate (cAMP) through the patch pipette facilitated I NMDA-OUT, which was abolished by the presence of protein kinase A (PKA) inhibitor. Furthermore, facilitation of I NMDA-OUT caused by an EP2 receptor agonist was significantly suppressed by PKA inhibitor. Finally, the activation of BK channels through EP2 receptors facilitated the recovery phase of NMDA-induced dendritic beading in the primary cultured cortical neurons. These results suggest that a direct activation of BK channels by EP2 receptor-mediated signaling pathways plays neuroprotective roles in cortical neurons.

Mass spectrometry-based metabolomic profiling identifies alterations in salivary redox status and fatty acid metabolism in response to inflammation and oxidative stress in periodontal disease

Periodontal diseases represent the most common chronic inflammatory diseases in humans and a major cause of tooth loss. Combining mass spectrometry-based ionomics and targeted lipidomics on fatty acid metabolites, we identified significant alterations in redox status and fatty acid metabolism in saliva in response to chronic inflammation and oxidative stress in periodontal disease in a cohort of nonsmoker subjects with chronic periodontitis. For the first time, ionomic profiling of around 30 ions in saliva revealed significantly decreased levels of redox-active metal ions including Mn, Cu, and Zn in the periodontal group, which is consistent with decreased levels of superoxide dismutases in saliva and serum. A targeted lipidomic approach was employed to monitor the major metabolites of arachidonic acid and linoleic acid in saliva. We observed increased levels of cyclooxygenase products including PGE2, PGD2, and PGF2α and TXB2, but decreased level of PGI2 in the periodontal group. A unique pattern of the lipoxygenase products of arachidonic acid and linoleic acid was observed with increased level of 5-HETE but decreased levels of 13-HODE and 9-HODE. Levels of salivary F2-isoprostanes, free radical lipid peroxidation products, and a gold standard for oxidative stress in vivo were also significantly elevated. Taking these data together, our study using multiple powerful omics techniques demonstrates that local redox alteration contributes significantly to periodontitis through the modulation of fatty acid metabolism in response to inflammation and oxidative stress. This study highlights the importance of redox status in periodontitis and provides a rationale for preventing periodontal disease by dietary interventions aiming to restore redox balance.

Regulation of prostaglandin F2α against β amyloid clearance and its inflammation induction through LXR/RXR heterodimer antagonism in microglia

Alzheimer's disease (AD) is characterized by extracellular deposit of β-amyloid (Aβ) and accumulation of intracellular neurofibrillary tangles in the brain. Prostaglandin F2α (PGF2α) is one of the major metabolites of arachidonic acid (AA), and plays essential roles in a series of key physiological processes like luteolysis and parturition. Additionally, PGF2α is also involved in the regulation of chronic and acute inflammation processes. Recent clinical studies have revealed the high content of PGF2α metabolite, 15-keto-dihydro-PGF2α in AD patients, implying the activation of in vivo PGF2α biosynthesis. However, the mechanism underlying the involvement of PGF2α in the progression of AD still remains unclear. Here we discovered that PGF2α selectively antagonized LXR (liver X receptors)/RXR (retinoid X receptor α) and RXR/RXR dimers. Cell based assays indicated that PGF2α effectively antagonized the activation of LXR agonist (t0901317) on Aβ clearance via inhibiting apolipoprotein E (apoE) expression, and cell apoptosis alleviation by accelerating inflammatory response to Aβ or Lipopolysaccharide (LPS) in microglia. Therefore, our current findings have addressed the potential association of PGF2α with AD progression, and highlighted that inhibition of PGF2α biosynthesis might be a useful therapeutic strategy against AD.

Impact of IL-33 on PGD2 Generation by Activated Human Cord Blood-Derived Mast Cell: Lack of Effect on Tryptase Release

Mast cells are important not only in allergic reactions, but also in inflammation and are involved in a variety of responses including the immediate release of potent inflammatory mediators after activation by cross-linking of FcεRI molecules. Prostaglandin D2 (PGD2) is a major cyclooxygenase metabolite of arachidonic acid produced by mast cells and it is released following allergen challenge in allergic diseases. IL-33 is an iflammatory cytokine which is critically involved in the regulation of in vitro and in vivo cyclooxygenase production, providing a potential therapeutic target for inflammatory disorders. In this study, using human derived umbelical cord blood mast cells, we show that IL-33 (50 ng/ml), and calcium ionophore A 23187 (0.5 μg/ml), compound 48/80 (10−5 M) or anti-IgE (10 μg/ml), enhaced the production of PGD2 and this effect was inhibited by indomethacin. However, IL-33 was unable to induce tryptase release in these cells. These effects confirm the inflammatory property of IL-33 by stimulating PGD2 but not tryptase in human mast cells. The inhibitory effect of this new cytokine may have a potential therapeutic response in allergic and inflammatory diseases.

Abstract 3483: Activation of thromboxane A2 receptor-alpha promotes tumor growth and angiogenesis through expression of VEGF in prostate cancer

Abstract The primary objective of this study is to investigate the role of TXA2R signaling in prostate cancer progression and metastasis. Thromboxane A2 (TXA2) is a major arachidonic acid metabolite that signals through TXA2 receptors to induce platelet aggregation and smooth muscle contraction. TXA2 receptors (TP) are expressed as two different isoforms in humans- TP-alpha (TPα) and TP-beta (TPβ), which share both common and distinct signaling pathways. Previous studies have established the important role played by TXA2R isoforms in several cancers such as bladder, lung and breast cancer. But the role played by TXA2R signaling in prostate cancer (PCa) progression is not well understood. Our data indicates that human prostate cancer cell lines such as PC3, DU145, PC3M and LNCaP only express the TPα isoform but not the TPβ isoform. Also expression of TPα was higher in PCa cells compared to normal prostate epithelial cells. Results from the angiogenesis array indicated that activation of TXA2 receptors using TXA2 mimetic-IBOP resulted in an increase in angiogenesis related proteins such as VEGF, MMP9 and uPA in DU145 cells. To further confirm the role of TPα activation on VEGF, which is one of the most potent and predominant angiogenic factor, Real-time PCR and ELISA for human VEGF was performed that showed an increased expression of VEGF in response to IBOP in DU145 cells. To further understand the mechanisms behind TPα mediated VEGF expression, immunoblotting was performed on DU145 cells lysates treated with IBOP in order to analyze the phosphorylation status of downstream targets with phospho-specific antibodies. Activation of TXA2 signaling by IBOP did result in phosphorylation of downstream targets such as Src kinase, EGFR kinase and ERK. To verify whether EGFR kinase is involved in TPα induced VEGF expression, we pre-treated these cells with an EGFR kinase inhibitor AG1478 before treatment with IBOP. Results from Real-time PCR and ELISA suggest that inhibition of EGFR kinase did result in reduced VEGF mRNA and protein expression that confirmed its role in TPα mediated angiogenesis. Over expression of TPα in another PCa cell line, PC3 (PC3-TPα) that has lower endogenous expression of TPα also resulted in an increase in VEGF expression. Increased angiogenesis was further verified by a preliminary in vivo study that showed mice injected with PC3-TPα cells exhibited greater tumor growth and increased neovascularization compared to mice with PC3-Neo cells. Taken together, these data suggests that, activation of TPα receptor in PCa cells induced the expression of pro angiogenic factors such as VEGF and hence might play an important role in prostate cancer progression and metastasis. Acknowledgements: This research was supported by NIH Grant 1R01 CA114051-01A1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3483. doi:10.1158/1538-7445.AM2011-3483

5-Oxo-ETE is a major oxidative stress-induced arachidonate metabolite in B lymphocytes

B lymphocytes convert arachidonic acid (AA) to the 5-lipoxygenase products leukotriene B4 (LTB4) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) when subjected to oxidative stress. 5-HETE has little biological activity, but can be oxidized by a selective dehydrogenase in some cells to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent eosinophil chemoattractant. We found that CESS cells, a B lymphocyte cell line, convert AA to 5-oxo-ETE and this is selectively stimulated by oxidative stress. In the presence of H2O2, 5-oxo-ETE is a major AA metabolite in these cells (5-oxo-ETE≈5-HETE>LTB4). The cyclooxygenase product 12-hydroxy-5,8,10-heptadecatrienoic acid is also formed, but is not affected by H2O2. Diamide had effects similar to those of H2O2 and both substances had similar effects on human tonsillar B cells. H2O2 also stimulated 5-oxo-ETE formation from its direct precursor 5-HETE in tonsillar B and CESS cells, and this was inhibited by the glutathione reductase inhibitor carmustine. H2O2 concomitantly induced rapid increases in GSSG and NADP+ and reductions in GSH and NADPH. We conclude that oxidative stress stimulates 5-oxo-ETE synthesis in B lymphocytes by two mechanisms: activation of 5-lipoxygenase and increased oxidation of 5-HETE by NADP+-dependent 5-hydroxyeicosanoid dehydrogenase. B lymphocyte-derived 5-oxo-ETE could contribute to eosinophilic inflammation in asthma and other allergic diseases.

Inhibition of carcinoma cell motility by epoxyeicosatrienoic acid (EET) antagonists

Cytochrome P450 (CYP) epoxygenases, CYP2C8, 2C9 and 2J2 mRNA and proteins, were expressed in prostate carcinoma (PC-3, DU-145 and LNCaP) cells. 11,12-Epoxyeicosatrienoic acid (11,12-EET) was the major arachidonic acid metabolite in these cells. Blocking EET synthesis by a selective CYP epoxygenase inhibitor (N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide [MS-PPOH]) inhibited tonic (basal) invasion and migration (motility) while exogenously added EET induced cell motility in a concentration-dependent manner. An epidermal growth factor receptor (EGFR) kinase inhibitor (AG494) or a PI3 kinase inhibitor (LY294002) inhibited cell migration and reduced 11,12-EET-induced cell migration. Importantly, synthetic EET antagonists (14,15-epoxyeicosa-5(Z)-enoic acid [14,15-EEZE], 14,15-epoxyeicosa-5(Z)-enoic acid 2-[2-(3-hydroxy-propoxy)-ethoxy]-ethyl ester [14,15-EEZE-PEG] and 14,15-epoxyeicosa-5(Z)-enoic-methylsulfonylimide [14,15-EEZE-mSI]) inhibited EET-induced cell invasion and migration. 11,12-EET induced cell stretching and myosin-actin microfilament formation as well as increased phosphorylation of EGFR and Akt (Ser473), while 14,15-EEZE inhibited these effects. These results suggest that EET induce and EET antagonists inhibit cell motility, possibly by putative EET receptor-mediated EGFR and PI3K/Akt pathways, and suggest that EET antagonists are potential therapeutic agents for prostate cancer.

Src-dependent STAT-3-mediated Expression of Monocyte Chemoattractant Protein-1 Is Required for 15(S)-Hydroxyeicosatetraenoic Acid-induced Vascular Smooth Muscle Cell Migration

To understand the role of human 15-lipoxygenase 1 (15-LOX1) in vascular wall remodeling, we have studied the effect of the major 15-LOX1 metabolite of arachidonic acid, 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), on vascular smooth muscle cell (VSMC) migration both in vitro and in vivo. Among 5(S)-HETE, 12(S)-HETE, and 15(S)-HETE, 15(S)-HETE potentially stimulated more vascular smooth muscle cell (VSMC) migration. In addition, 15(S)-HETE-induced VSMC migration was dependent on Src-mediated activation of signal transducer and activator of transcription-3 (STAT-3). 15(S)-HETE also induced monocyte chemoattractant protein-1 (MCP-1) expression via Src-STAT-3 signaling, and neutralizing anti-MCP-1 antibodies completely negated 15(S)-HETE-induced VSMC migration. Cloning and characterization of a 2.6-kb MCP-1 promoter revealed the presence of four putative STAT-binding sites, and the site that is proximal to the transcription start site was found to be essential for 15(S)-HETE-induced Src-STAT-3-mediated MCP-1 expression. Rat carotid arteries that were subjected to balloon injury and transduced with Ad-15-LOX1 upon exposure to [(3)H]arachidonic acid ex vivo produced 15-HETE as a major eicosanoid and enhanced balloon injury-induced expression of MCP-1 in smooth muscle cells in Src and STAT-3-dependent manner in vivo. Adenovirus-mediated delivery of 15-LOX1 into rat carotid artery also led to recruitment and homing of macrophages to medial region in response to injury. In addition, transduction of Ad-15-LOX1 into arteries enhanced balloon injury-induced smooth muscle cell migration from media to intima and neointima formation. These results show for the first time that 15-LOX1-15(S)-HETE axis plays a major role in vascular wall remodeling after balloon angioplasty.

Mast Cells and Arachidonic Acid Cascade in Inflammation

Prostaglandin D2 PGD2 is a major cyclooxygenase metabolite of arachidonic acid produced by mast cells and it is released following allergen challenge in diseases, such as allergic diseases. PGD2 may act as a neuromodulator and as an allergic and inflammatory mediator. In allergic diseases, activated mast cell synthesizes prostaglandin D2 (first cyclo-oxygenate mediator) which has bronchoconstrictive and vasodilating effects and attracts several leukocytes. It has been found that activated mast cells, challenged with physiological and non- physiological secretagogues, release elevated histamine and tryptase and chymase, leukotrienes B4, C4 and D4, 5-hydroxyeicosatetraenoic acid, PGD2, Platelet Activating Factor (PAF), heparin, and high-molecular-weight neutrophil chemotactic factor and cytokines/chemokines. PGD2 exerts its biological activity through the DP and CRTH2 receptors and their cDNA cloning which were characterized 15 years ago. In this report, we revisited the biological effects of arachidonic acid compounds released by activated mast cells in allergic and inflammatory states.

A bioluminescent enzyme immunoassay for prostaglandin E2 using Cypridina luciferase

Prostaglandin E(2) is one of the major cyclooxygenase metabolites of arachidonic acid. We developed a competitive immunosorbent assay for prostaglandin E(2) utilizing a bioluminescent enzyme Cypridina luciferase. The prostaglandin E(2) amount could be quantified over the concentration ranging from 7.8 to 500 pg/mL. The amount of unlabeled prostaglandin E(2) required to displace 50% of the maximal binding of Cypridina luciferase-labeled prostaglandin E(2) (B/B(0)) was approximately 35 pg/mL. The results show a great potential of Cypridina luciferase as a new labeling enzyme for enzyme-linked immunosorbent assay.

Cytosolic phospholipase A 2-driven PGE 2 synthesis within unsaturated fatty acids-induced lipid bodies of epithelial cells

Cytoplasmic lipid bodies (also known as lipid droplets) are intracellular deposits of arachidonic acid (AA), which can be metabolized for eicosanoid generation. PGE 2 is a major AA metabolite produced by epithelial cells and can modulate restoration of epithelium homeostasis after injury. We studied lipid body biogenesis and their role in AA metabolic pathway in an epithelial cell line derived from normal rat intestinal epithelium, IEC-6 cells. Lipid bodies were virtually absent in confluent IEC-6 cells. Stimulation of confluent IEC-6 cells with unsaturated fatty acids, including AA or oleic acid (OA), induced rapid lipid body assembly that was independent on its metabolism to PGE 2, but dependent on G-coupled receptor-driven signaling through p38, PKC, and PI3K. Newly formed lipid bodies compartmentalized cytosolic phospholipase (cPL)A 2-α, while facilitated AA mobilization and synthesis of PGE 2 within epithelial cells. Thus, both lipid body-related events, including highly regulated biogenesis and functional assembly of cPLA 2-α-driven enhanced AA mobilization and PGE 2 production, may have key roles in epithelial cell-driven inflammatory functions, and may represent relevant therapeutic targets of epithelial pathologies.

5-LOX, 12-LOX and 15-LOX in immature forms of human leukemic blasts

Several reports have demonstrated an important role of leukotriene B 4 (LTB 4) in the immune system. We investigated whether leukemic blasts from acute myeloid leukemic (AML) and acute lymphoid leukemic (ALL) patients produced LTB 4, 12- and 15-hydroxyeicosatetraenoic acids (12-HETE and 15-HETE) and whether these compounds affected blast proliferation and apoptosis. Leukemic blasts from AML M 0-2 and ALL patients expressed 5-LOX, 12-LOX and 15-LOX transcripts. Quantitative polymerase chain reaction indicated that 5-LOX transcripts were far more abundant than 12-LOX and 15-LOX ones. Leukemic blasts expressed 5-LOX activating protein (FLAP) transcripts and produced LTB 4 in response to calcium ionophore. In contrast no 15-HETE production was found. Calcium ionophore-stimulated leukemic blasts produced 12-HETE but also released thromboxane A 2 suggesting that contaminating platelets accounted for the release of these compounds. No significant effect of LTB 4, 12-HETE or 15-HETE could be documented on leukemic blast growth and on their apoptose rate. Results of the present study indicate that immature form of leukemic blasts produce LTB 4. However, the three major lipoxygenase metabolites of arachidonic acid; i.e., LTB 4, 12-HETE or 15-HETE, had no evident effect on their growth and apoptosis. We may speculate that LTB 4-derived blast cells might initiate, augment or prolong tissue inflammation and damages by affecting the marrow and blood cytokine network.

Increased Cortical Cell Loss and Prolonged Hemodynamic Depression after Traumatic Brain Injury in Mice Lacking the IP Receptor for Prostacyclin

Prostacyclin is the major arachidonic acid metabolite of the vascular endothelium and is produced mainly via the cyclooxygenase-2 pathway. By acting on the prostacyclin (IP) receptor on platelets and vascular smooth muscle cells, prostacyclin exerts vasodilatory and antiaggregative/antiadhesive effects. Previous studies have shown that prostacyclin production increases after brain trauma, but the importance of prostacyclin for posttraumatic hemodynamic alterations and neuron survival has not been investigated. This study evaluated if endogenous prostacyclin plays a role in the pathophysiologic process in the brain after brain trauma. This was performed by comparing prostacyclin (IP) receptor-deficient (IP(-/-)) mice and mice with functional IP receptor (IP(+/+)) after a controlled cortical injury regarding contusion volume, cerebral blood flow ([(14)C]iodoantipyrine autoradiography), number of perfused capillaries (fluorescein isothiocyanate-dextran fluorescence technique), the transfer constant (K(i)) for [(51)Cr]EDTA, and brain water content (wet vs dry weight) in the injured and contralateral cortex. Contusion volume was increased in IP(-/-) mice compared with IP(+/+) mice. Three hours after trauma, cortical blood flow was decreased in the injured cortex of both groups and the reduction in blood flow in the cortex of the IP(-/-) mice persisted from 3 to 24 h, whereas blood flow approached normal values in the IP(+/+) mice after 24 h. No differences could be detected between the two genotypes regarding other hemodynamic parameters. We conclude that the prostacyclin IP receptor is beneficial for neuron survival after brain trauma in mice, an effect that may be mediated by improved cortical perfusion.

Microsomal prostaglandin E synthase-1 and blood pressure regulation

Prostaglandin E (PGE)(2) is a major arachidonic acid metabolite in a wide variety of tissues and is implicated in the control of inflammatory as well as physiological responses. At least three major forms of PGE synthase (PGES) have recently been cloned and characterized: membrane-associated PGES (mPGES)-1, mPGES-2, and cytosolic PGES (cPGES). Among them, mPGES-1 is highly inducible by cytokine and is critically involved in pain and inflammatory responses. Emerging evidence suggests that mPGES-1 may also participate in blood pressure (BP) regulation through an impact on renal and vascular functions. Within the kidney, mPGES-1 predominates in the distal nephron where its expression is highly inducible by salt loading. Mice lacking mPGES-1 exhibit blunted natriuretic response paralleled with remarkably suppressed nitric oxide production, leading to salt-sensitive hypertension. These mice also exhibit an exaggerated hypertensive response to angiotensin II infusion. Together, these results suggest that mPGES-1 may be an important physiological regulator of BP.