Role Of Arachidonic Acid Metabolites Research Articles

Sexual dimorphism in renal heme oxygenase-1 and arachidonic acid metabolizing enzymes in spontaneously hypertensive rats versus normotensive Wistar Kyoto rats

Numerous studies have demonstrated a sexual dimorphism in blood pressure (BP) control in spontaneously hypertensive rats (SHR), however the mechanisms remain to be further elucidated. Based on the established role of arachidonic acid metabolites and heme oxygenase (HO) in BP control, we hypothesize that higher BP in male SHR is associated with differential expression in renal HO and arachidonic acid metabolizing enzymes vs. female SHR. Higher BP in male SHR coincided with significant increases in renal cortical superoxide production and thiobarbituric acid reactive substances (TBARs) levels as measures of oxidative stress compared to normotensive female WKY and female SHR. The elevations in BP and oxidative stress in male SHR were also associated with a decrease in cortical heme oxygenase-1 (HO-1) expression when compared to normotensive female WKY. Although there was no sex or strain differences in cortical expression of the epoxyeicosatrienoic acids (EETs) producing enzyme, cytochrome P450 epoxygenase (CYP2C23), in male and female SHR and WKY, SHR had greater expression of the EETs metabolizing enzyme, soluble epoxide hydrolase (sEH) vs. WKY. Cortical expression of the 20–hydroxyeicosatetraenoic acid (20-HETE) producing enzyme, cytochrome P450 hydroxylase (CYP4A), was less in female WKY and SHR compared to strain-matched males and cortical 20-HETE levels were also less in female SHR vs. male SHR. Cortical cyclooxygenase-2 (COX-2) expression was significantly greater in female SHR and WKY vs. males and cortical prostaglandin E2 (PGE2) levels in female SHR was significantly greater than male WKY. In conclusion, our data suggest that sex differences in renal oxidative stress, HO-1 and arachidonic acid metabolizing enzymes could contribute to sexual dimorphism in hypertension in young SHR.

A Protective Role for Arachidonic Acid Metabolites against Advanced Colorectal Adenoma in a Phase III Trial of Selenium

Oxylipins derived from arachidonic acid (ARA) have been implicated in the development of colorectal adenomas and colorectal cancer. The primary purpose of this work was to determine the relationship between plasma levels of oxylipins and colorectal adenoma characteristics at study entry, as well as with the development of a new adenoma during follow-up within a Phase III adenoma prevention clinical trial with selenium (Sel). Secondarily, we sought to determine whether the selenium intervention influenced plasma oxylipin levels. Four oxylipins were quantified in stored plasma samples from a subset of Sel study subjects (n = 256) at baseline and at 12-months. There were significantly lower odds of an advanced adenoma at baseline with higher prostaglandin E2 (PGE2), with an OR (95% CI) of 0.55 (0.33–0.92), and with 5-hydroxyeicosatetraenoic acid (5-HETE) ((0.53 (0.33–0.94)); and of a large adenoma with higher PGE2 ((0.52 (0.31–0.87)). In contrast, no associations were observed between any oxylipin and the development of a new adenoma during follow-up. Selenium supplementation was associated with a significantly smaller increase in 5-HETE after 12 months compared to the placebo, though no other results were statistically significant. The ARA-derived oxylipins may have a role in the progression of non-advanced adenoma to advanced, but not with the development of a new adenoma.

Combined treatment with epoxyeicosatrienoic acid analog and 20-hydroxyeicosatetraenoic acid antagonist provides substantial hypotensive effect in spontaneously hypertensive rats.

The global morbidity and mortality related to hypertension and associated disorders increases continuously and novel therapeutic strategies are still in high demand. Increasing evidence suggests the important role in blood pressure regulation of cytochrome P-450-dependent metabolites of arachidonic acid. Epoxyeicosatrienoic acids (EETs) induce vasodilation and natriuresis, and have renoprotective and anti-inflammatory properties. 20-HETE is an arachidonic acid metabolite with both prohypertensive and antihypertensive activities. To explore the pathophysiological role of arachidonic acid metabolites in more detail, we examined the antihypertensive efficiency of EET-A, a stable analog of 14,15-EET, and of AAA, a novel antagonist of the 20-HETE receptors. Male spontaneously hypertensive rats (SHR) were treated for 5 weeks with EET-A, AAA or the combination; age-matched untreated SHR and normotensive Wistar-Kyoto rats served as controls. EET-A and AAA were administered in drinking water at 10 mg/kg/day each. SBP was measured by telemetry and urine, blood, and tissue samples were collected for relevant analyses. EET-A or AAA given alone had no significant effect on SHR blood pressure. In contrast, combined treatment with AAA and EET-A was significantly antihypertensive, causing a decrease in SBP from 180 ± 3 to 160 ± 5 mmHg (P < 0.05). Additionally, the combined treatment attenuated cardiac hypertrophy, decreased kidney ANG II level, increased natriuresis, and increased the excretion of nitric oxide metabolites. Considering the beneficial impact of the combined treatment with EET-A and AAA on SHR blood pressure and cardiovascular and renal function, we suggest that the treatment is a promising therapeutic strategy for human hypertension.

Eicosanoid mediator profiles in different phenotypes of nonsteroidal anti-inflammatory drug-induced urticaria.

The role of arachidonic acid metabolites in NSAID-induced hypersensitivity has been studied in depth for NSAID-exacerbated respiratory disease (NERD) and NSAID-exacerbated cutaneous disease (NECD). However, no information is available for NSAID-induced urticarial/angioedema (NIUA), despite it being the most frequent clinical entity induced by NSAID hypersensitivity. We evaluated changes in leukotriene and prostaglandin metabolites for NIUA patients, using patients with NECD and single-NSAID-induced urticaria/angioedema or anaphylaxis (SNIUAA) for comparison. Urine samples were taken from patients with confirmed NSAID-induced urticaria and healthy controls, at baseline and at various time intervals after ASA administration. Eicosanoid measurement was performed using high-performance liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. No differences were found between groups at baseline. Following ASA administration, LTE4 and 9α,11β-PGF2 levels were increased in both NIUA and NECD patients compared to baseline, rising initially, before decreasing toward initial levels. In addition, the levels of these metabolites were higher in NIUA and NECD when compared with the SNIUAA and control groups after ASA administration. No changes were found with respect to baseline values for SNIUAA and control groups. We present for the first time data regarding the role of COX-1 inhibition in NIUA. Patients with this entity show a similar pattern eicosanoid levels following ASA challenge to those with NECD. Further studies will help ascertain the cell populations involved and the underlying molecular mechanisms.

Arachidonic acid pathway alterations in cerebrospinal fluid of dogs with naturally occurring spinal cord injury.

BackgroundCanine intervertebral disc πherniation causes a naturally-occurring spinal cord injury (SCI) that bears critical similarities to human SCI with respect to both injury pathomechanisms and treatment. As such, it has tremendous potential to enhance our understanding of injury biology and the preclinical evaluation of novel therapies. Currently, there is limited understanding of the role of arachidonic acid metabolites in canine SCI.ResultsThe CSF concentrations of PLA2 and PGE2 were higher in SCI dogs compared to control dogs (p = 0.0370 and 0.0273, respectively), but CSF LCT4 concentration in SCI dogs was significantly lower than that in control dogs (p < 0.0001). Prostaglandin E2 concentration in the CSF was significantly and positively associated with increased severity of SCI at the time of sampling (p = 0.041) and recovery 42 days post-injury (p = 0.006), as measured by ordinal behavioral scores.ConclusionArachidonic acid metabolism is altered in dogs with SCI, and these data suggest that these AA metabolites reflect injury severity and recovery, paralleling data from other model systems.

Abstract 181: Therapeutic approaches to metastasis induced by mesenchymal stem cells in the tumor microenvironment

Abstract Metastasis is the primary cause of death in non-hematological cancers yet there are no specific therapeutics against it because of a lack of validated targets. The Weinberg lab demonstrated that bone-marrow derived mesenchymal stem cells (MSCs) home to the stroma of breast tumors and induce metastasis. We verified these findings in MDA-MB-231 (MDA) xenografts in which co-injection of human MSCs increased MDA thoracic metastasis by 5-fold. In vitro, MSC co-culture induced GFP-labeled MDA cells to migrate 3-fold faster in a modified ‘wound-healing’ assay, mirroring metastasis in vivo. To identify therapeutic targets within MSC-induced metastasis, we performed gene-expression analysis of MSC-MDA co-cultures separated by flow cytometry compared with cells grown alone. The interferon pathway was found to be the most activated pathway upon co-culture, increasing mainly in MSCs. We studied the relevance of these genes in human cancers by first analyzing 3 different gene-expression datasets comparing human breast cancer stroma with normal stroma. Genes upregulated in breast cancer stroma were then studied in a meta-analysis of 19 whole tumor gene-expression datasets correlating gene-expression changes with survival. We identified 103 genes that are upregulated in MSCs by MSC-MDA interactions, are increased in human breast cancer stroma and are significantly associated with poor survival. To determine if they are necessary for MSC-induced metastatic behavior, we performed shRNA knockdown in MSC-MDA co-cultures measuring effects on in vitro migration. Knockdown of a number of interferon-associated genes significantly reduced migration supporting an unexpected functional role of interferons in metastasis. The top interferon gene, ISG15, is an attractive candidate for therapeutic targeting because it is a secreted ubiquitin-like factor that conjugates a number of cytoskeletal proteins involved in motility. In parallel, we conducted a small-molecule screen with 1600 compounds on the migration of MSC-MDA co-cultures to identify small-molecule inhibitors of metastasis. Counter screens on highly motile endothelial cells excluded compounds that non-specifically inhibit normal cell migration. Only 1 compound, RSL3, specifically blocked MSC-induced MDA migration with cytotoxicity at &amp;gt;10-fold higher concentrations. RSL3 inhibits the glutathione peroxidase 4 (GPX4) enzyme that metabolizes lipid peroxides including arachidonic acid metabolites participating in inflammatory cascades like interferon gamma signaling. RSL3 activity was completely abrogated by co-treatment with the 5-lipoxygenase inhibitor zileuton consistent with the role of arachidonic acid metabolites in MSC-MDA migration. In summary, targeting components of interferon and arachidonic acid pathways have been discovered as novel therapeutic approaches against microenvironment-induced breast cancer metastasis. Citation Format: Shrikanta Chattopadhyay, Cherrie Huang, Siddhartha Mukherjee, Rushdia Z. Yusuf, Vasanthi Viswanathan, Ben S. Wittner, Jeff Gentry, Alykhan Shamji, Sridhar Ramaswamy, David T. Scadden, Stuart L. Schreiber. Therapeutic approaches to metastasis induced by mesenchymal stem cells in the tumor microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 181. doi:10.1158/1538-7445.AM2014-181

Role of the non-enzymatic metabolite of eicosapentaenoic acid, 5-epi-5-F3t-isoprostane in the regulation of [ (3)H]D-aspartate release in isolated bovine retina.

We have evidence that F2-isoprostanes (F2-IsoPs) regulate the release of excitatory neurotransmitters in isolated bovine retina. Although 5-F3-IsoPs are generated in mammals, in vivo, their pharmacological actions on neurotransmitter release remain unknown. In this study, we investigated the effect of 5-epi-5-F3t-IsoP on K(+)-evoked [(3)H]D-aspartate release in isolated bovine retina using the superfusion method. Furthermore, we examined the role of arachidonic acid metabolites in the regulation of the neurotransmitter release by this novel IsoP. In the concentration range, 0.01nM-0.1µM, 5-epi-5-F3t-IsoP inhibited K(+)-evoked [(3)H]D-aspartate release in a concentration-dependent manner, achieving a maximum inhibition of 46.9% at 0.1µM (IC30=1nM). The prostanoid receptor antagonists, AH 6809 (EP1-3/DP; 10µM), SC 51322 (EP1; 10µM) and SC 19220 (EP1; 1µM) partially reversed 5-epi-5-F3t-IsoP-mediated inhibition of K(+)-induced [(3)H]D-aspartate release. Pretreatment of retinal tissues with the cyclooxygenase (COX) inhibitor, flurbiprofen (3μM) unmasked a biphasic action of 5-epi-5-F3t-IsoP that was inhibitory at lower (0.1-10pM) and stimulatory at higher concentrations (≥0.1nM). The prostanoid pathway antagonists, BAY-u3405 (10μM; TP/DP-receptors), SQ 29548 (10μM; TP-receptor) and ozagrel (10μM; Tx-synthase inhibitor) abolished the stimulatory action of the 5-epi-5-F3t-IsoP (0.1μM) on neurotransmitter release. In conclusion, 5-epi-5-F3t-IsoP attenuates K(+)-induced [(3)H]D-aspartate release in a concentration-dependent manner by mechanisms that are partially dependent on activation of pre-junctional prostanoid EP1-receptors. Moreover, blockade of the COX-pathway unmasks a biphasic action for 5-epi-5-F3t-IsoP that is inhibitory at low concentrations and stimulatory at higher concentrations. Products of the thromboxane synthase pathway may partially account for the stimulatory action of this F3-IsoP on isolated bovine retina.

Inflammation, Cancer and Oxidative Lipoxygenase Activity are Intimately Linked.

Cancer and inflammation are intimately linked due to specific oxidative processes in the tumor microenvironment. Lipoxygenases are a versatile class of oxidative enzymes involved in arachidonic acid metabolism. An increasing number of arachidonic acid metabolites is being discovered and apart from their classically recognized pro-inflammatory effects, anti-inflammatory effects are also being described in recent years. Interestingly, these lipid mediators are involved in activation of pro-inflammatory signal transduction pathways such as the nuclear factor κB (NF-κB) pathway, which illustrates the intimate link between lipid signaling and transcription factor activation. The identification of the role of arachidonic acid metabolites in several inflammatory diseases led to a significant drug discovery effort around arachidonic acid metabolizing enzymes. However, to date success in this area has been limited. This might be attributed to the lack of selectivity of the developed inhibitors and to a lack of detailed understanding of the functional roles of arachidonic acid metabolites in inflammatory responses and cancer. This calls for a more detailed investigation of the activity of arachidonic acid metabolizing enzymes and development of more selective inhibitors.

Role of arachidonic acid metabolites on the control of non-differentiated intestinal epithelial cell growth

Increasingly evidence indicates that enzymes, receptors and metabolites of the arachidonic acid (AA) cascade play a role in intestinal epithelial cell proliferation and colorectal tumorigenesis. However, the information available does not provide a complete picture and contains a number of discrepancies. For this reason it might be appropriate a thorough study into the impacts of the AA cascade on intestinal epithelial cell growth. Our data show that non-differentiated Caco-2 cells cultured with 10% fetal bovine serum (FBS) synthesize appreciable amounts of prostaglandin E2 (PGE2), leukotriene B4 (LTB4) and 5-, 12 and 15-hydroxyeicosatetraenoic acid (HETE) but not LTD4, 20-HETE and epoxyeicosatrienoic acids. We also found that inhibitors of PGE2, LTB4 and 5-, 12-, 15-HETE synthesis as well as receptor antagonists of PGE2 and LTB4 blocked Caco-2 cell growth and DNA synthesis induced by 10% FBS without cytotoxic or apoptotic activity. Interestingly, PGE2, LTB4 and 5-, 12- and 15-HETE at concentrations reached in 10% FBS Caco-2 cultures (1–10nM) were able to induce Caco-2 cell growth and DNA synthesis. This was due to the interaction of PGE2 with EP1 and EP4 receptors and LTB4 and HETEs with BLT1 and BLT2 receptors. Moreover, we provide evidence that PGE2 stimulates several cell signaling pathways such as ERK, P38α, CREB and GSKβ/β-catenin involved in the regulation of Caco-2 growth. Finally, we provide evidence that the mitogenic effects of LTB4 and HETEs can be dependent, at least in part, on PGE2 synthesis.

Role of arachidonic acid metabolites in Trypanosoma cruzi infection

Event Abstract Back to Event Role of arachidonic acid metabolites in Trypanosoma cruzi infection Néstor A. Guerrero1, Manuel Fresno1 and Núria Gironès1* 1 Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Spain Cardiac inflammation plays an important role in the pathophysiology of Chagas disease, caused by Trypanosoma cruzi. Lipid mediators derived from arachidonic acid (AA), like prostaglandins and leukotrienes, are considered regulators of homeostasis and inflammation. These molecules are produced by a biosynthetic pathway controlled by enzymes as cyclooxygenases and lipoxygenases. The role of cyclooxygenase-2 (COX-2) in immunosuppression during the acute phase of T. cruzi infection has been described using non-steroidal anti-inflammatory drugs, which are inhibitors of this enzyme. In this study, we first investigated the expression of enzymes involved in AA metabolism during T. cruzi infection. Susceptible and non-susceptible mouse models of infection were used to further analyze the role of lipid mediators in T. cruzi infection. Our results confirm the expression of several of these enzymes in T. cruzi-infected heart, in particular COX-2. CD68+ heart-infiltrating macrophages were the major cell type expressing COX-2. CD11b+ heart-infiltrating myeloid cells were purified and they were able to produce prostaglandins PGE2 and PGF2α. Studies using gene-deficient mouse models indicated that regulation of COX-2 and PGE2, through PGE2 receptor 2 signaling, was in part responsible of cardiac inflammation in T. cruzi infection. The key role of lipid mediators in Chagas disease inflammation and the availability of drugs that inhibit their synthesis and their receptors could be useful for the treatment of this neglected disease. Acknowledgements This work was supported by grants: SAF2007-61716, SAF2005-02220, PS09/00538, RECAVA RD06/0014/1013, RICET RD06/0021/0016, HEALTH-FE-2008-22303,ChagasEpiNet, CC08-UAM/SAL-4440/08, AECID A/025417/09 and ‘‘Fundacion Ramon Areces". M. Fresno and N. Girones are professors at the UAM. N.A.G. was a recipient of a fellowship from ISCIII and RICET. We thank Beatriz Barrocal, Maria A. Chorro, M.C. Maza and Carlos Chillon for their technical assistance. Keywords: Trypanosoma cruzi, Cyclooxigenase-2, Prostaglandin E2 receptor 2, Cardiac inflammation, in vivo infection Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Host-pathogen interactions Citation: Guerrero NA, Fresno M and Gironès N (2013). Role of arachidonic acid metabolites in Trypanosoma cruzi infection. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01131 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Jul 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Núria Gironès, Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, 28049, Spain, ngirones@cbm.csic.es Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Néstor A Guerrero Manuel Fresno Núria Gironès Google Néstor A Guerrero Manuel Fresno Núria Gironès Google Scholar Néstor A Guerrero Manuel Fresno Núria Gironès PubMed Néstor A Guerrero Manuel Fresno Núria Gironès Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Lack of effect of BW755c on glucose-induced insulin secretion in the rat in-vivo.

Glucose-induced elevations in plasma immunoreactive insulin (IRI) were examined in anaesthetized rats pretreated with BW755c, sodium meclofenamate or vehicle. Neither drug influenced the glucose-induced hyperinsulinaemia or the glucose disappearance rate. The results do not support a physiological role for arachidonic acid metabolites in the regulation of glucose-induced insulin secretion.

Role of arachidonic acid lipoxygenase metabolites in acetylcholine-induced relaxations of mouse arteries

Arachidonic acid (AA) metabolites function as EDHFs in arteries of many species. They mediate cyclooxygenase (COX)- and nitric oxide (NO)-independent relaxations to acetylcholine (ACh). However, the role of AA metabolites as relaxing factors in mouse arteries remains incompletely defined. ACh caused concentration-dependent relaxations of the mouse thoracic and abdominal aorta and carotid, femoral, and mesentery arteries (maximal relaxation: 57 ± 4%, 72 ± 4%, 82 ± 3%, 80 ± 3%, and 85 ± 3%, respectively). The NO synthase inhibitor nitro-L-arginine (L-NA; 30 μM) blocked relaxations in the thoracic aorta, and L-NA plus the COX inhibitor indomethacin (10 μM) inhibited relaxations in the abdominal aorta and carotid, femoral, and mesenteric arteries (maximal relaxation: 31 ± 10%, 33 ± 5%, 41 ± 8%, and 73 ± 3%, respectively). In mesenteric arteries, NO- and COX-independent relaxations to ACh were inhibited by the lipoxygenase (LO) inhibitors nordihydroguaiaretic acid (NDGA; 10 μM) and BW-755C (200 μM), the K(+) channel inhibitor apamin (1 μM), and 60 mM KCl and eliminated by endothelium removal. They were not altered by the cytochrome P-450 inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (20 μM) or the epoxyeicosatrienoic acid antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 μM). AA relaxations were attenuated by NDGA or apamin and eliminated by 60 mM KCl. Reverse-phase HPLC analysis revealed arterial [(14)C]AA metabolites that comigrated with prostaglandins, trihydroxyeicosatrienoic acids (THETAs), hydroxyepoxyeicosatrienoic acids (HEETAs), and hydroxyeicosatetraenoic acids (HETEs). Epoxyeicosatrienoic acids were not observed. Mass spectrometry confirmed the identity of 6-keto-PGF(1α), PGE(2), 12-HETE, 15-HETE, HEETAs, 11,12,15-THETA, and 11,14,15-THETA. AA metabolism was blocked by NDGA and endothelium removal. 11(R),12(S),15(S)-THETA relaxations (maximal relaxation: 73 ± 3%) were endothelium independent and blocked by 60 mM KCl. Western immunoblot analysis and RT-PCR of the aorta and mesenteric arteries demonstrated protein and mRNA expression of leukocyte-type 12/15-LO. Thus, in mouse resistance arteries, 12/15-LO AA metabolites mediate endothelium-dependent relaxations to ACh and AA.

The role of arachidonic acid metabolites in DR

Abstract Purpose The inducible enzyme cyclooxygense‐2 (COX‐2) and its metabolic products are important mediators for angiogenesis. We investigated the expression of COX‐2 and its downstream enzymes microsomal prostaglandin‐E synthase (mPGES)‐1, cytosolic PGES (cPGES) and thromboxane synthase (TXS) and correlated it with vascular endothelial growth factor (VEGF) expression and level of vascularization in proliferative diabetic retinopathy (PDR) epiretinal membranes. Methods Fourteen membranes were studied by immunohistochemistry. Results Vascular endothelial cells expressed COX‐2, mPGES‐1 and VEGF in 75.6%, 64.3% and 50% of the membranes, respectively. TXS was expressed in stromal cells in 85.7% of the membranes. There was no immunoreactivity for cPGES. There were significant correlations between number of blood vessels expressing CD34 and the numbers of blood vessels expressing COX‐2 (rs = 0.858; p&lt;0.001), mPGES‐1 (rs = 0.743; p = 0.002) and VEGF (rs = 0.845; p = 0.001) and the number of cells expressing TXS (rs = 0.74; p = 0.002). Number of blood vessels expressing VEGF correlated significantly with the numbers of blood vessels expressing COX‐2 (rs = 0.879; p&lt;0.001) and mPGES‐1 (rs = 0.942; p&lt;0.001) and the number of cells expressing TXS (rs = 0.702; p = 0.011). Conclusion COX‐2 and its metabolic products might contribute to PDR angiogenesis.

Novel Findings Support a Patophysiological Role of the Arachidonic Cascade in Hodgkin Lymphoma.

Lipoxygenases (LO) are a family of structurally related enzymes which catalyze the conversion of the fatty acid arachidonic acid to biologically active metabolites. The key enzyme in leukotriene synthesis, 5- LO, catalyzes the first step in the metabolism of arachidonic acid to leukotriene (LT) C4. This compound and its metabolites LTD4 and LTE4, collectively called cysteinyl leukotrienes (cysLT), can bind to two high-affinity receptors, named cysLT1 and cysLT2. A sister enzyme to 5-LO is 15-LO-1 which also can catalyze the formation of bioactive metabolites. Arachidonic acid metabolites have traditionally been linked to inflammation and asthma but several studies also indicate a role of these metabolites in carcinogenesis In our studies of arachidonic acid metabolism in human lymphomas, we have identified the expression of 15-LO-1 and receptors for cysLT in primary as well as cultured Hodgkin-Reed Sternberg (H-RS) cells. In tumor tissue, H-RS cells positive for 15-LO-1 and the cysLT1 receptor were detected immunohistochemically in 13/15 and 12/16 cases, respectively. The presence of mRNA for 15-LO-1 and the cysLT1 and 2 receptors was confirmed by microarray analysis of laser dissected H-RS cells. Studies of 15-LO-1 gene transcription revealed that STAT6 activation and chromatin remodeling by DNA demethylation and histone acetylation are crucial for transcriptional activation of this gene in cultured H-RS cells. Incubation of the Hodgkin lymphoma-derived cell lines KMH2 and L1236 with LTD4 led to a robust calcium response that was completely abolished in presence of the cysLT1 receptor inhibitor zafirlukast. In L1236 cells, LTD4 induced increased DNA synthesis, interleukin-13 transcription and release of TNF-alpha, interleukin-6 and interleukin-8 in a dose-dependent manner, all of which were inhibited by zafirlukast. Thus, as the H-RS cells lack the expression of 5-LO, we hypothesize that that tumor-associated cysLT-producing inflammatory cells, such as eosinophils and mast cells, contribute to the pathogenesis of Hodgkin lymphoma through induction of tumor cell proliferation and cytokine release. Furthermore, immunohistochemical studies of 20 non-Hodgkin lymphomas revealed the expression of cysLT1 receptors also in primary mediastinal B cell lymphomas (PMBCL), and the functionality of these receptors was confirmed in the PMBCL-derived cell line MedB1. Taken together, these novel findings support a role for arachidonic acid metabolites in the pathogenesis of lymphoma.

15(S)-HETE Production in Human Retinal Microvascular Endothelial Cells by Hypoxia: Novel Role for MEK1 in 15(S)-HETE–Induced Angiogenesis

To examine for the expression of 15-lipoxygenase 1 (15-LOX1) and 15-LOX2 in human retinal microvascular endothelial cells (HRMVECs) and study the role of arachidonic acid metabolites of these enzymes in angiogenesis. Quantitative RT-PCR and reverse-phase HPLC analyses were used to determine 15-LOX1/2 expression and their arachidonic acid metabolites in HRMVECs. The role of MEK1 in 15(S)-HETE-induced angiogenesis was studied using HRMVEC migration, tube formation, and basement membrane matrix plug angiogenesis. HRMVECs expressed both 15-LOX1 and 15-LOX2. Hypoxia induced the expression of 15-LOX1 and the production of its arachidonic acid metabolites 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) and 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). 15(S)-HETE stimulated HRMVEC migration and tube formation as potently as 20 ng/mL fibroblast growth factor-2 (FGF-2). In addition, 15(S)-HETE stimulated the phosphorylation of ERK1/2, JNK1, p38 MAPK, and MEK1 in a time-dependent manner in these cells. Inhibition of MEK1 by pharmacologic and dominant-negative mutant approaches attenuated 15(S)-HETE-induced phosphorylation of ERK1/2 and JNK1 but not p38 MAPK. Blockade of ERK1/2 and JNK1 activation suppressed 15(S)-HETE-induced HRMVEC migration and tube formation and basement membrane matrix plug angiogenesis. Inhibition of p38 MAPK attenuated 15(S)-HETE-induced HRMVEC migration only. Inhibition of MEK1 also blocked 15(S)-HETE-induced HRMVEC migration and tube formation and basement membrane matrix plug angiogenesis. These results suggest that hypoxia, through the induction of 15-LOX1 expression, leads to the production of 15(S)-HETE in HRMVECs. In addition, 15(S)-HETE, through MEK1-dependent activation of ERK1/2 and JNK1, stimulates the angiogenic differentiation of HRMVECs and basement membrane matrix plug angiogenesis.

ACh-induced relaxations of rabbit small mesenteric arteries: role of arachidonic acid metabolites and K+

ACh-induced endothelium-dependent relaxation in rabbit small mesenteric arteries is resistant to N-nitro-L-arginine (L-NA) and indomethacin but sensitive to high K+, indicating the relaxations are mediated by endothelium-derived hyperpolarizing factors (EDHFs). The identity of the EDHFs in this vascular bed remains undefined. Small mesenteric arteries pretreated with L-NA and indomethacin were contracted with phenylephrine. ACh (10(-10) to 10(-6) M) caused concentration-dependent relaxations that were shifted to the right by lipoxygenase inhibition and the Ca(2+)-activated K+ channel inhibitors apamin (100 nM) or charybdotoxin (100 nM) and eliminated by the combination of apamin plus charybdotoxin. Relaxations to ACh were also blocked by a combination of barium (200 microM) and apamin but not barium plus charybdotoxin. Addition of K+ (10.9 mM final concentration) to the preconstricted arteries elicited small relaxations. K+ addition before ACh restored the charybdotoxin-sensitive component of relaxations to ACh. K+ (10.9 mM) also relaxed endothelium-denuded arteries, and the relaxations were inhibited by barium but not by charybdotoxin and apamin. With the use of whole cell patch-clamp analysis, ACh (10(-7) M) stimulated voltage-dependent outward K+ current from endothelial cells, which was inhibited by charybdotoxin, indicating K+ efflux. Arachidonic acid (10(-7) to 10(-4) M) induced concentration-related relaxations that were inhibited by apamin but not by charybdotoxin and barium. Addition of arachidonic acid after K+ (10.9 mM) resulted in more potent relaxations to arachidonic acid compared with control without K+ (5.9 mM). These findings suggest that, in rabbit mesenteric arteries, ACh-induced, L-NA- and indomethacin-resistant relaxation is mediated by endothelial cell K+ efflux and arachidonic acid metabolites, and a synergism exists between these two separate mechanisms.

Stimulatory effects of eicosanoids on ovarian angiogenesis in early luteal phase in cyclooxygenase-2 inhibitor-treated rats

Our previous study demonstrated that impaired ovarian vasculature is responsible for the decrease in serum progesterone observed in cyclooxygenase (COX)-2 inhibitor-treated rats. To explore the role of arachidonic acid metabolites in the formation of the corpus luteum, we determined in the present study the effects of prostaglandin (PG) and thromboxane (TX) receptor agonists together with vascular endothelial growth factor (VEGF) on progesterone secretion and angiogenesis in the newly formed corpus luteum in NS-398 (a selective inhibitor of COX-2)-treated rats. Uterine injection of PGE 2 or U-46619 (TXA 2 receptor agonist) prevented decreased levels of serum progesterone and ovarian hemoglobin, an indicator of amounts of vasculature in NS-398-treated rats. Luteal capillary vessel establishment was inhibited by NS-398, as determined by histological examination of ovarian vascular plexuses, while administration of PGE 2 reversed the effect. VEGF enhanced the levels of serum progesterone and ovarian hemoglobin, and increased the density of ovarian capillaries. However, VEGF-induced angiogenesis was inhibited by NS-398 treatment. These results suggest that PGE 2 and TXA 2 stimulate angiogenesis in the newly formed corpus luteum and that there is a possibility that these eicosanoids are involved in VEGF-induced progesterone production and the increase in luteal blood flow.

Dual Effect of Isoprostanes on the Release of [3H]D-Aspartate from Isolated Bovine Retinae: Role of Arachidonic Acid Metabolites

The effect of 8-isoprostanes on potassium (K+)-depolarization-evoked release of [3H]D-aspartate from bovine isolated retinae was investigated. Isolated bovine retinae were prepared for studies of K(+)-evoked release of [3H]D-aspartate using the Superfusion Method. Low concentrations of 8-isoPGF(2alpha) (1-100 nM) inhibited whereas higher concentrations of this 8-isoprostane (100 nM-30 microM) enhanced K(+)-induced [3H]D-aspartate overflow. The excitatory effect of 8-isoPGF(2alpha) was mimicked by thromboxane receptor agonist, U-46619 and blocked by thromboxane receptor antagonist, SQ 29,548 (10 microM). Pretreatment of tissues with the cyclooxygenase (COX) inhibitor, flurbiprofen unmasked an inhibitory effect of high concentrations of 8-isoPGF(2alpha) (1-30 microM) on [3H]D-aspartate release that was attenuated by AH 6809 (10 microM). In conclusion, 8-isoPGF(2alpha) exhibits a dual regulatory effect on K(+)-induced [3H]D-aspartate release in isolated bovine retinae. The inhibitory action caused by 8-isoPGF(2alpha) is due to the activation of EP1/EP2 receptors while the excitatory effects are due to the activation of thromboxane receptors.

ATVB in focus: Diabetic vascular disease: pathophysiological mechanisms in the diabetic milieu and therapeutic implications.

In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology begins a series of review articles on recent research advances into the mechanisms of diabetic vascular complications. The goal of this series is to review basic mechanisms by which diabetes accelerates vascular disease, an intense area of current interest. Research into diabetic vascular complications has received increased attention over the last 10 years as indicated by the number of articles published annually in ATVB that featured the term “diabetes” in the title, abstract, or text (Figure). Looking at the Figure, you might wonder what accounts both for the increase, and for the apparent lack of interest before this. This burst of activity was preceded by a greater realization that both type I and type II diabetes dramatically increase the risk for the development of macrovascular disease and its complications. The increase in published articles is also undoubtedly caused by many interdependent factors. First, it is now generally accepted that risk factors such as diabetes, hypertension, and hyperlipidemia have relatively acute effects on vascular cells to which their chronic adverse effects on vascular structure and function can be ascribed. For instance, in the case of diabetes, this principle is based on results from short-term cellular and physiological models in which vascular or blood cells in both animal models and in vitro are exposed to elevated concentrations of glucose for relatively brief periods of time. These are reasonably easy experiments to do, and bolstered by the fact that elevated …

Arachidonic acid metabolites and peroxide-induced inhibition of [3H]D-aspartate release from bovine isolated retinae

We have previously shown that hydrogen peroxide (H 2 O 2) can inhibit K + -depolarization-evoked [ 3 H]D-aspartate release from bovine isolated retinae. In the present study, we investigated the role of arachidonic acid metabolites in the inhibitory response elicited by H 2 O 2 in the bovine retinae. Furthermore, we examined the direct effect of H 2 O 2 on the production of prostaglandins and isoprostanes in this tissue. Isolated bovine retinae were prepared for studies of [ 3 H]D-aspartate release using the Superfusion Method. Release of [ 3 H]D-aspartate was elicited by Krebs solution containing an iso-osmotic concentration of KCl (50mM). A direct action of H 2 O 2 on prostaglandin E 2 (PGE2) and 8-isoprostane F 2a (8-iso-PGF 2a) was also measured by enzyme-linked immunosorbant assay (ELISA). The cyclooxygenase inhibitor, flurbiprofen (3µM), or the thromboxane-receptor antagonist, SQ 29548 (10µM) had no significant (p > 0.05) effect on K + -evoked [ 3 H]D-aspartate release. On the other hand, both flurbiprofen (3µM) and SQ 29548 (10µM) blocked the inhibition of K + -evoked [ 3 H]D-aspartate induced by H 2 O 2 (30µM). In concentrations up to 100µM, H 2 O 2 caused an increase in PGE 2 and 8-iso-PGF 2a over basal levels. For instance, H 2 O 2 (100µM) increased PGE 2 and 8-iso-PGF 2a over basal levels by 348 ± 41% and 185 ± 26 (n = 4), respectively. We conclude that the peroxide-mediated inhibition of [ 3 H]D-aspartate may involve the production of prostaglandins and isoprostanes in the bovine isolated retinae.