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1.  [No title available] 
PMCID: PMC4292900  PMID: 23291334
2.  Pathogen Induced Chemo-attractant Hepoxilin A3 Drives Neutrophils, but not Eosinophils across Epithelial Barriers 
Pathogen induced migration of neutrophils across mucosal epithelial barriers requires epithelial production of the chemotactic lipid mediator, hepoxilin A3 (HXA3). HXA3 is an eicosanoid derived from arachidonic acid. Although eosinophils are also capable of penetrating mucosal surfaces, eosinophilic infiltration occurs mainly during allergic processes whereas neutrophils dominate mucosal infection. Both neutrophils and eosinophils can respond to chemotactic gradients of certain eicosanoids, however, it is not known whether eosinophils respond to pathogen induced lipid mediators such as HXA3. In this study, neutrophils and eosinophils were isolated from human blood and placed on the basolateral side of polarized epithelial monolayers grown on permeable Transwell filters and challenged by various chemotactic gradients of distinct lipid mediators. We observed that both cell populations migrated across epithelial monolayers in response to a leukotriene B4 (LTB4) gradient, whereas only eosinophils migrated towards a prostaglandin D2 (PGD2) gradient. Interestingly, while pathogen induced neutrophil trans-epithelial migration was substantial, pathogen induced eosinophil trans-epithelial migration was not observed. Further, gradients of chemotactic lipids derived from pathogen infected epithelial cells known to be enriched for HXA3 as well as purified HXA3 drove significant numbers of neutrophils across epithelial barriers, whereas eosinophils failed to respond to these gradients. These data suggest that although the eicosanoid HXA3 serves as an important neutrophil chemo-attractant at mucosal surfaces during pathogenic infection, HXA3 does not appear to exhibit chemotactic activity towards eosinophils.
doi:10.1016/j.prostaglandins.2013.11.001
PMCID: PMC4004677  PMID: 24315875
neutrophils; eosinophils; eicosanoids; hepoxilin A3; leukotriene B4; prostaglandin D2
3.  Roles of the epoxygenase CYP2J2 in the endothelium 
Prostaglandins & other lipid mediators  2013;107:10.1016/j.prostaglandins.2013.02.003.
Cytochrome p450 (CYP)2J2 is an epoxygenase enzyme that metabolises arachidonic acid to epoxyeicosatrienoic acids (EETs). EETs are inactivated by soluble epoxide hydrolase (sEH), which converts them in to their corresponding dihydroxyeicosatrienoic acids (DHETs). CYP2J2 is highly expressed in cardiovascular tissue including the heart and vascular endothelial cells. CYP2J2 and the EETs it produces have been shown to have a diverse range of effects on the vasculature, including the regulation of inflammation, vascular tone, cellular proliferation, angiogenesis, and metabolism. This review will examine these established and emerging roles of CYP2J2 in the biology of vascular endothelial cells.
doi:10.1016/j.prostaglandins.2013.02.003
PMCID: PMC3711961  PMID: 23474289
endothelial; epoxygenase; inflammation; metabolism; angiogenesis; dilation
4.  Genetic Deletion of Microsomal Prostaglandin E Synthase-1 Suppresses Mouse Mammary Tumor Growth and Angiogenesis 
Prostaglandins & other lipid mediators  2013;0:10.1016/j.prostaglandins.2013.04.002.
The cyclooxygenase/prostaglandin (COX/PG) signaling pathway is of central importance in inflammation and neoplasia. COX inhibitors are widely used for analgesia and also have demonstrated activity for cancer prophylaxis. However, cardiovascular toxicity associated with this drug class diminishes their clinical utility and motivates the development of safer approaches both for pain relief and cancer prevention. The terminal synthase microsomal PGE synthase-1 (mPGES-1) has attracted considerable attention as a potential target. Overexpression of mPGES-1 has been observed in both colorectal and breast cancers, and gene knockout and overexpression approaches have established a role for mPGES-1 in gastrointestinal carcinogenesis. Here we evaluate the contribution of mPGES-1 to mammary tumorigenesis using a gene knockout approach. Mice deficient in mPGES-1 were crossed with a strain in which breast cancer is driven by overexpression of human epidermal growth factor receptor 2 (HER2/neu). Loss of mPGES-1 was associated with a substantial reduction in intramammary PGE2 levels, aromatase activity, and angiogenesis in mammary glands from HER2/neu transgenic mice. Consistent with these findings, we observed a significant reduction in multiplicity of tumors ≥1mm in diameter, suggesting that mPGES-1 contributes to mammary tumor growth. Our data identify mPGES-1 as a potential anti-breast cancer target.
doi:10.1016/j.prostaglandins.2013.04.002
PMCID: PMC3830707  PMID: 23624019
Mouse; mPGES-1; breast cancer; aromatase; angiogenesis; PGE2
5.  EFFECT OF HUMAN 15-LIPOXYGENASE-1 METABOLITES ON VASCULAR FUNCTION IN MOUSE MESENTERIC ARTERIES AND HEARTS 
Prostaglandins & other lipid mediators  2013;0:10.1016/j.prostaglandins.2013.07.002.
Lipoxygenases regulate vascular function by metabolizing arachidonic acid (AA) to dilator eicosanoids. Previously, we showed that endothelium-targeted adenoviral vector-mediated gene transfer of the human 15-lipoxygenase-1 (h15-LO-1) enhances arterial relaxation through the production of vasodilatory hydroxyepoxyeicosatrienoic acid (HEETA) and trihydroxyeicosatrienoic acid (THETA) metabolites. To further define this function, a transgenic (Tg) mouse line that overexpresses h15-LO-1 was studied. Western blot, immunohistochemistry and RT-PCR results confirmed expression of 15-LO-1 transgene in tissues, especially high quantity in coronary arterial wall, of Tg mice. Reverse-phase HPLC analysis of [14C]-AA metabolites in heart tissues revealed enhanced 15-HETE synthesis in Tg vs. WT mice. Among the 15-LO-1 metabolites, 15-HETE, erythro-13-H-14,15-EETA, and 11(R),12(S),15(S)-THETA relaxed the mouse mesenteric arteries to the greatest extent. The presence of h15-LO-1 increased acetylcholine- and AA-mediated relaxation in mesenteric arteries of Tg mice compared to WT mice. 15-LO-1 expression was most abundant in heart; therefore, we used the Langendorff heart model to test the hypothesis that elevated 15-LO-1 levels would increase coronary flow following a short ischemia episode. Both peak flow and excess flow of reperfused hearts were significantly elevated in hearts from Tg compared to WT mice being 2.03 and 3.22 times greater, respectively. These results indicate that h15-LO-1-derived metabolites are highly vasoactive and may play a critical role in regulating coronary blood flow.
doi:10.1016/j.prostaglandins.2013.07.002
PMCID: PMC3844054  PMID: 23872364
15-lipoxygenase; eicosanoids; vasodilation; coronary flow; ischemia/reperfusion; reactive hyperemia; endothelium-derived hyperpolarizing factor
6.  Sphingosine-1-phosphate receptor-2 mediated NFκB activation contributes to tumor necrosis factor-α induced VCAM-1 and ICAM-1 expression in endothelial cells 
Prostaglandins & other lipid mediators  2013;0:10.1016/j.prostaglandins.2013.06.001.
Sphingosine-1-phosphate (S1P) regulates a wide array of biological functions in endothelial cells. We previously showed that S1P receptor subtype 2 (S1P2) is significantly up-regulated in the atherosclerotic endothelium (J. Biol. Chem. 283:30363, 2008). In this study, we investigated the roles of S1P2-mediated signaling in the proinflammatory responses of endothelial cells. Treatment with tumor necrosis factor-α (TNFα), a proinflammatory cytokine, increased the expression of S1P2 receptors in endothelial cells. TNFα treatment also enhanced sphingosine kinase 1 expression and increased S1P production. Pharmacological inhibition or knockdown of S1P2 receptors completely abrogated the TNFα-induced VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) expression in endothelial cells. In contrast, pharmacological inhibition or knockdown of other S1P receptor subtypes had no effect on the TNFα-stimulated ICAM-1 and VCAM-1 expression. Moreover, ectopic expression of S1P2 receptors increased VCAM-1 and ICAM-1 expression in endothelial cells in response to S1P stimulation. Mechanistically, we show that antagonizing S1P2 signaling markedly inhibited the TNFα-stimulated NFκB activation. Utilizing the NFκB reporter luciferase assay, the S1P/S1P2 signaling was shown to stimulate NFκB activation. Moreover, the S1P/S1P2-stimulated VCAM-1/ICAM-1 expression was completely abolished by the pharmacological inhibitor of NFκB. Collectively, our data suggest that TNFα treatment activates autocrine S1P/S1P2 signaling, which subsequently activates NFκB and leads to the proinflammatory responses in endothelial cells.
doi:10.1016/j.prostaglandins.2013.06.001
PMCID: PMC3844125  PMID: 23770055
sphingosine-1-phosphate; S1P family of G-protein coupled receptor; sphingolipids; sphingosine kinase; vasculature
7.  12- and 15-Lipoxygenases in Adipose Tissue Inflammation 
The lipoxygenases (LOs) are principal enzymes involved in the oxidative metabolism of polyunsaturated fatty acids, including arachidonic acid. 12- and 15-LO and their lipid metabolites have been implicated in the development of insulin resistance and diabetes. Adipose tissue, and in particular visceral adipose tissue, plays a primary role in the development of the inflammation seen in these conditions. 12- and 15-LO and their lipid metabolites act as upstream regulators of many of the cytokines involved in the inflammatory response in adipose tissue. While the role that 12- and 15-LO play in chronically inflamed adipose tissue is becoming clearer, there are still many questions that remain unanswered regarding their activation, signaling pathways, and roles in healthy fat. 12- and 15-LO also generate products with anti-inflammatory properties that are under investigation. Therefore, 12- and 15-LO have the potential to be very important targets for therapeutics aimed at reducing insulin resistance and the comorbid conditions associated with obesity.
doi:10.1016/j.prostaglandins.2012.07.004
PMCID: PMC3526691  PMID: 22951339
12/15-lipoxygenase; adipose tissue; inflammation; insulin resistance; diabetes; adipogenesis; obesity
8.  Dual Modulation of Cyclooxygenase and CYP Epoxygenase Metabolism and Acute Vascular Inflammation in Mice 
Cyclooxygenase (COX)-derived prostaglandins and cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids are important regulators of inflammation; however, functional interactions between these pathways in the regulation of vascular inflammation in vivo have not been studied. We investigated the relative and additive effects of endothelial CYP2J2 overexpression (Tie2-CYP2J2-Tr), global sEH disruption (Ephx2−/−), and pharmacologic COX inhibition with indomethacin on the acute vascular inflammatory response to endotoxin in mice. Compared to vehicle-treated wild-type C57BL/6 controls, induction of myeloperoxidase (MPO) activity in lung and liver was similarly attenuated in Tie2-CYP2J2-Tr mice, Ephx2−/− mice and wild-type mice treated with moderate dose indomethacin. Dual modulation of both pathways, however, did not produce an additive anti-inflammatory effect. These findings demonstrate that both COX and CYP epoxygenase-mediated eicosanoid metabolism are important regulators of the acute vascular inflammatory response in vivo, and suggest that the anti-inflammatory effects of modulating each pathway may be mediated, at least in part, by overlapping mechanisms.
doi:10.1016/j.prostaglandins.2012.09.003
PMCID: PMC3549041  PMID: 23000418
CYP2J2; soluble epoxide hydrolase; cyclooxygenase; vascular inflammation; epoxyeicosatrienoic acid; prostaglandin
9.  Eicosanoid profiling in colon cancer: emergence of a pattern 
Oxidative metabolism of polyunsaturated fatty acids has been linked to tumorigenesis in general and colonic tumorigenesis in particular. Earlier studies showed that cyclooxygenase-2 (COX-2) and 15-lipoxygenase-1 (15-LOX-1) have opposing impacts on colonic tumorigenesis: COX-2 promotes while 15-LOX-1 inhibits colonic tumorigenesis. Advances in liquid chromatography/mass spectrometry have allowed for measurement of various products of oxidative metabolism in a single colonic biopsy specimen. Studies of LOX products in preclinical models and in patients with familial adenomatous polyposis and sporadic colorectal tumorigenesis indicate that LOX pathways are shifted during colonic tumorigenesis and that the main shift is downregulation of 15-LOX-1. This shift occurs during the polyp formation stage and thus offers the opportunity to modulate tumorigenesis early by correcting 15-LOX-1 downregulation.
doi:10.1016/j.prostaglandins.2012.08.004
PMCID: PMC3532570  PMID: 22960430
Eicosanoid profiling; lipoxygenases; cyclooxygenases; 15-LOX-1; colon cancer
10.  Epoxyeicosatrienoic Acids, 20-Hydroxyeicosatetraenoic Acid, and Renal Microvascular Function 
The development of pharmacological, genetic, and biochemical tools have allowed for detailed studies to determine the contribution of cytochrome P450 (CYP) metabolites of arachidonic acid to renal microvascular function. Renal microvessels can generate CYP hydroxylase metabolites including 20-hydroxyeicosatetraenoic acid (20-HETE) and CYP epoxygenase metabolites, epoxyeicosatrienoic acids (EETs). 20-HETE constricts afferent arterioles and contributes to renal blood flow autoregulation. EETs act as endothelium-dependent hyperpolarizing factors (EDHFs) on the renal microcirculation. 20-HETE inhibits whereas EETs activate renal microvascular smooth muscle cell large-conductance calcium-activated K+ channels (KCa). Likewise, 20-HETE renal microvascular actions are pro-hypertensive and EET actions are anti-hypertensive. These findings in the renal microvasculature and those of others have provided impetus for the development of enzymatic inhibitors, agonists, and antagonists for 20-HETE and EETs to determine their potential therapeutic value. Initial genetic studies and experimental studies with soluble epoxide hydrolase inhibitors to increase EETs, EET analogs, and 20-HETE inhibitors have demonstrated improved renal microvascular function in hypertension. These findings have demonstrated the important contributions that 20-HETE and EETs play in the regulation of renal microvascular function.
doi:10.1016/j.prostaglandins.2013.01.002
PMCID: PMC3664103  PMID: 23333581
11.  Regulation of T Helper Cell Subsets by Cyclooxygenases and Their Metabolites 
Cyclooxygenases and their metabolites are important regulators of inflammatory responses and play critical roles in regulating the differentiation of T helper cell subsets in inflammatory diseases. In this review, we highlight new information on regulation of T helper cell subsets by cyclooxygenases and their metabolites. Prostanoids influence cytokine production on both antigen presenting cells and T cells to regulate the differentiation of naïve CD4+ T cells to Th1, Th2 and Th17 cell phenotypes. Cyclooxygenases and PGE2 generally exacerbate Th2 and Th17 phenotypes, while suppressing Th1 differentiation. Thus, cycloxygenases may play a critical role in diseases that involve immune cell dysfunction. Targeting of cyclooxygenases and their eicosanoid products may represent a new approach for treatment of inflammatory diseases, tumors and autoimmune disorders.
doi:10.1016/j.prostaglandins.2012.11.002
PMCID: PMC3620713  PMID: 23201570
Cyclooxygenases; T helper cells; Prostanoids; PGE2
12.  NS-398 reverses hypotension in endotoxemic rats: contribution of eicosanoids, NO, and peroxynitrite 
We have previously demonstrated that inhibition of vasodilator prostanoids, PGI2 and PGE2, and nitric oxide (NO) synthsesis by a selective cyclooxygenase-2 (COX-2) inhibitor, NS-398, restores blood pressure as a result of increased systemic and renal levels of 20-hydroxyeicosatetraenoic acid (20-HETE) in endotoxemic rats. The aim of this study was to further investigate the effects of NS-398 on the changes in expression and/or activity of COX-2, cyctochrome P450 4A1 (CYP4A1), inducible NO synthase (iNOS), and peroxynitrite formation in serum, renal, cardiac, and/or vascular tissues of lipopolysaacharide (LPS)-treated rats. LPS (10 mg/kg, i.p.)-induced decrease in blood pressure was associated with increased protein levels of COX-2, iNOS, and nitrotyrosine in kidney, heart, thoracic aorta, and superior mesenteric artery. The activities of COX-2 and iNOS as well as levels of PGI2, PGE2, and nitrotyrosine were also increased in the systemic circulation and renal, cardiac, and vascular tissues of LPS-treated rats. In contrast, renal, cardiac, and vascular CYP4A1 protein expression as well as systemic and tissue levels of 20-HETE were decreased in endotoxemic rats. These effects of LPS, except COX-2 protein expression, were prevented by NS-398 (10 mg/kg, i.p.), given 1 h after injection of LPS. These data suggest that COX-2-derived vasodilator prostanoids, PGI2 and PGE2, produced during endotoxemia increase iNOS protein expression and activity as well as peroxynitrite formation resulting in decreased CYP4A1 protein expression and 20-HETE synthesis. Taken together, we concluded that an increase in 20-HETE levels associated with a decrease in the production of vasodilator prostanoids and NO participates in the effect of NS-398 to prevent hypotension in the rat model of septic shock.
doi:10.1016/j.prostaglandins.2012.08.007
PMCID: PMC3774147  PMID: 22975359
Endotoxin; Blood Pressure; Cyclooxygenase-2; Prostaglandins; Nitric Oxide; Peroxynitrite; 20-HETE
13.  Beneficial effects of inhibition of soluble epoxide hydrolase on glucose homeostasis and islet damage in a streptozotocin-induced diabetic mouse model 
Soluble epoxide hydrolase (sEH) is an enzyme involved in the metabolism of endogenous inflammatory and anti-apoptotic mediators. In the present study, we determined the effects of the inhibition of sEH on glucose homeostasis and islet damage in mice treated with streptozotocin (STZ), a model of chemical-induced diabetes. STZ increased daily water intake and decreased visceral (spleen and pancreas) weight in mice; sEH inhibition in STZ mice decreased water intake, but did not affect visceral weight. Hyperglycemia induced by STZ treatment in mice was attenuated by inhibiting sEH. The beneficial effects of sEH inhibition were accompanied, after 2 and 4 weeks of initial administration, by improving glucose tolerance. In contrast, sEH inhibition did not affect insulin tolerance. Using LC/MS analysis, neither STZ nor STZ plus sEH inhibition affected pancreatic and plasma ratios of epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs), an index of EETs levels. Western blot analysis showed that mouse cytochrome P450 (CYP) 2C enzymes are the major epoxygenases in islets. On day 5 after initial STZ treatment, STZ induced islet cell apoptosis, while sEH inhibition in STZ mice significantly reduced islet cell apoptosis. These studies provide pharmacological evidence that inhibiting sEH activity provides significant protection against islet β-cell damage and improves glucose homeostasis in STZ-induced diabetes.
doi:10.1016/j.prostaglandins.2012.12.001
PMCID: PMC4052893  PMID: 23247129
CYP-derived eicosanoids; Islets; Glucose homeostasis; Apoptosis
14.  Regulation of Inflammation in Cancer by Eicosanoids 
Inflammation in the tumour microenvironment is now recognized as one of the hallmarks of cancer. Endogenously produced lipid autacoids, locally acting small molecule lipid mediators, play a central role in inflammation and tissue homeostasis, and have recently been implicated in cancer. A well-studied group of autacoid mediators that are the products of arachidonic acid metabolism include: the prostaglandins, leukotrienes, lipoxins and cytochrome P450 (CYP) derived bioactive products. These lipid mediators are collectively referred to as eicosanoids and are generated by distinct enzymatic systems initiated by cyclooxygenase (COX 1 and 2), lipoxygenases (5-LOX, 12-LOX, 15-LOXa, 15-LOXb), and cytochrome P450s, respectively. These pathways are the target of approved drugs for the treatment of inflammation, pain, asthma, allergies, and cardiovascular disorders. Beyond their potent anti-inflammatory and anti-cancer effects, non-steroidal anti-inflammatory drugs (NSAIDs) and COX-2 specific inhibitors have been evaluated in both preclinical tumor models and clinical trials. Eicosanoid biosynthesis and actions can also be directly influenced by nutrients in the diet, as evidenced by the emerging role of omega-3 fatty acids in cancer prevention and treatment. Most research dedicated to using eicosanoids to inhibit tumor-associated inflammation has focused on the COX and LOX pathways. Novel experimental approaches that demonstrate the anti-tumor effects of inhibiting cancer-associated inflammation currently include: eicosanoid receptor antagonism, overexpression of eicosanoid metabolizing enzymes, and the use of endogenous anti-inflammatory lipid mediators. Here we review the actions of eicosanoids on inflammation in the context of tumorigenesis. Eicosanoids may represent a missing link between inflammation and cancer and thus could serve as therapeutic target(s) for inhibiting tumor growth.
doi:10.1016/j.prostaglandins.2011.08.004
PMCID: PMC4051344  PMID: 21864702
Eicosanoids; Inflammation; Cancer; Metastasis; Tumor Microenvironment
15.  COX-2 and PGE2-dependent immunomodulation in breast cancer 
COX-derived prostanoids play multiple roles in inflammation and cancer. This review highlights research examining COX-2 and PGE2-dependent regulation of immune cell polarization and function within the tumor microenvironment, particularly as it pertains to breast cancer. Appreciating PGE2-mediated immunomodulation is important in understanding how tumors evade immune surveillance by reeducating infiltrating inflammatory and immune cells to support tumorigenesis. Elucidation of the multiple and complex influences exerted by tumor stromal components may lead to targeted therapies in breast and other cancers that restrain microenvironmental permissiveness and maintain natural defenses against malignancies.
doi:10.1016/j.prostaglandins.2011.08.005
PMCID: PMC4031099  PMID: 21907301
Breast cancer; COX-2; Macrophage polarization; Microenvironment; PGE2; Regulatory T lymphocytes; Myeloid-derived suppressor cells; Immune suppression
16.  5,14-HEDGE, a 20-HETE mimetic, reverses hypotension and improves survival in a rodent model of septic shock: Contribution of soluble epoxide hydrolase, CYP2C23, MEK1/ERK1/2/IKKβ/IκB-α/NF-κB pathway, and proinflammatory cytokine formation 
We have previously demonstrated that a stable synthetic analog of 20-HETE, N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14-HEDGE), restores vascular reactivity, blood pressure, and heart rate in endotoxemic rats. The aim of this study was to determine whether decreased renal expression and activity of soluble epoxide hydrolase (sEH), MEK1, ERK1/2, IKKβ, IκB-α, and NF-κB as well as systemic and renal proinflammatory cytokine production associated with increased expression and activity of CYP2C23 contributes to the effect of 5,14-HEDGE to prevent hypotension, tachycardia, inflammation, and mortality in response to systemic administration of lipopolysaccharide (LPS). Blood pressure fell by 33 mmHg and heart rate rose by 57 beats/min in LPS (10 mg/kg, i.p.)-treated rats. Administration of LPS also increased mRNA and protein expression of sEH associated with a decrease in CYP2C23 mRNA and protein expression. Increased activity of sEH and p-MEK1, p-ERK1/2, p-IκB-α, NF-κB, and p-NF-κB protein levels as well as TNF-α and IL-8 production by LPS were also associated with a decreased activity of AA epoxygenases. These effects of LPS were prevented by 5,14-HEDGE (30 mg/kg, s.c.; 1 h after LPS). Treatment of endotoxemic mice with 5,14-HEDGE also raised the survival rate of animals from 84% to 98%. A competitive antagonist of vasoconstrictor effects of 20-HETE, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, 20-HEDE (30 mg/kg, s.c.; 1 h after LPS) prevented the effects of 5,14-HEDGE on blood pressure, heart rate, expression and/or activity of sEH, CYP2C23, and ERK1/2 as well as TNF-α and IL-8 levels in rats treated with LPS. These results suggest that decreased expression and/or activity of sEH and MEK1/ERK1/2/IKKβ/IκB-α/NF-κB pathway as well as proinflammatory cytokine production associated with increased CYP2C23 expression and antiinflammatory mediator formation participate in the protective effect of 5,14-HEDGE against hypotension, tachycardia, inflammation, and mortality in the rodent model of septic shock.
doi:10.1016/j.prostaglandins.2013.01.005
PMCID: PMC3739859  PMID: 23454652
Endotoxin; Soluble epoxide hydrolase; CYP2C23; NF-κB; Hypotension; Inflammation; Mortality
19.  Altered Behavioral Phenotypes In Soluble Epoxide Hydrolase Knockout Mice: Effects of Traumatic Brain Injury 
After traumatic brain injury (TBI), arachidonic acid (ArA) is released from damaged cell membranes and metabolized to many bioactive eicosanoids, including several epoxyeicosatrienoic acids (EETs). Soluble epoxide hydrolase (Ephx2, sEH) appears to be the predominant pathway for EET metabolism to less active dihydroxyeicosatrienoates (DHETs). Prior studies indicate that brain levels of EETs increase transiently after TBI and EETs have antiinflammatory and neuroprotective activities which may benefit the injured brain. If the net effect of increased EET levels in the injured brain is beneficial to recovery, then Ephx2 gene disruption would be expected to enhance elevated EET levels and improve recovery in the injured brain. Thus, Ephx2-KO (Ephx2−/− bred onto pure C57Bl/6 background) mice were compared to wild-type controls in a unilateral controlled cortical impact model of TBI.
Before injury, animals behaved comparably in open field activity and neurologic reflexes. Interestingly, the Ephx2-KO mice showed improved motor coordination on a beam walk task, yet showed indications of defective learning in a test of working spatial memory. After surgery, brain-injured Ephx2-KO mice again had less of a deficit in the beam walk than wild-type, and the difference in latency (post – pre) showed a trend of protection for Ephx2-KO mice after TBI. Brain-injured mice showed no genotype differences in working memory. Surprisingly, sham-operated Ephx2-KO mice exhibited an injured phenotype for working memory, compared to sham-operated wild-type mice.
Brain eicosanoid levels were measured using liquid chromatography with tandem mass spectrometry. Of the 20 eicosanoids evaluated, only 8,9-EET was elevated in the Ephx2-KO cerebral cortex (37d post-surgery, in both sham and injured). Tissue DHET levels were below the limit of quantification. These results reflect a significant contribution of sEH deficiency in coordination of ambulatory movements and working spatial memory in the mouse. Further investigation of differential sEH expression and EET levels at earlier time points and across other brain regions may shed light on these behavioral differences.
doi:10.1016/j.prostaglandins.2012.07.005
PMCID: PMC3932494  PMID: 22922090
20.  Loss of neutral ceramidase increases inflammation in a mouse model of inflammatory bowel disease 
Sphingolipids are emerging as important mediators of immune and inflammatory responses. We have previously demonstrated that sphingosine-1-phosphate (S1P) and its synthetic enzyme sphingosine kinase-1 (SK1) play an important role in inflammatory bowel disease. S1P generation is dependent on SK phosphorylation of sphingosine. Generation of sphingosine results only from the breakdown of ceramide by ceramidases (CDase). In this study, we set out to determine the role of neutral CDase (nCDase) in S1P generation and inflammatory bowel disease. To this end, we established nCDase expression is increased in patients with ulcerative colitis. Using the dextran sulfate sodium (DSS)-induced colitis model, we determined nCDase activity increased in colon epithelium, but not submucosa, in wild-type (WT) mice. Following DSS, ceramide levels were elevated in colon epithelium from WT and nCDase−/− mice, while S1P levels were significantly elevated only in the epithelium of nCDase−/− mice. Similarly, cyclooxygenase-2 (Cox-2) levels were significantly elevated only in the epithelium of nCDase−/− mice. Neutral CDase−/− mice also exhibited higher endotoxin levels in circulation, as well as higher circulating levels of S1P. This increase in S1P in nCDase−/− mice was accompanied by a marked leukocytosis, most notably circulating neutrophils and lymphocytes. Taken together these data demonstrate that loss of nCDase results in an unexpected increase in S1P generation in inflammation, and suggests that nCDase may actually protect against inflammation.
doi:10.1016/j.prostaglandins.2012.08.003
PMCID: PMC3661865  PMID: 22940715
ceramide; ceramidase; sphingosine-1-phosphate; colitis; neutrophil; inflammation
21.  Tumor necrosis factor-alpha induces renal cyclooxygenase-2 expression in response to hypercalcemia 
The effect of tumor necrosis factor-alpha (TNF) on cyclooxygenase-2 (COX-2) expression in the renal outer medulla (OM) was determined in a model of dihydrotachysterol (DHT)-induced hypercalcemia. Increases in serum calcium and water intake were observed during ingestion of a DHT-containing diet in both wild type (WT) and TNF deficient mice (TNF−/−). Polyuria and a decrease in body weight were observed in response to DHT treatment in WT and TNF−/− mice. A transient elevation in urinary TNF was observed in WT mice treated with DHT. Moreover, increased urinary levels of prostaglandin E2 (PGE2) and a corresponding increase in COX-2 expression in the OM were observed in WT mice fed DHT. Increased COX-2 expression was not observed in TNF−/− mice fed DHT, and the characteristics of PGE2 synthesis were distinct from those in WT mice. This study demonstrates that COX-2 expression in the OM, secondary to hypercalemia, is TNF-dependent.
doi:10.1016/j.prostaglandins.2012.07.001
PMCID: PMC3429635  PMID: 22800939
COX-2; thick ascending limb; TNF; hypercalcemia; calcium-sensing receptor
22.  Prostaglandin E2 promotes the nuclear accumulation of lymphoid enhancer factor-1 in poorly differentiated breast cancer cells 
Products of the COX reaction are frequently elevated in solid tumors and their roles in the malignant phenotype have been extensively investigated. We have shown that COX-2 is essential for the growth of MDA-MB-231 cells in the fat pad of SCID mice and for their extrapulmonary colonization following injection in the tail vein of SCID mice. The molecular changes that follow shRNA-mediated silencing of COX-2 include a significant downregulation of LEF-1, a transcription factor normally activated during development following the Wnt-induced nuclear translocation of β-catenin. We also report that COX-2-silenced cells have reduced nuclear accumulation of LEF-1 protein and that the COX-2 product PGE2 partially restored nuclear LEF-1 expression in COX-2-silenced cells. Further, we demonstrate that, like parental COX-2 containing MDAMB-231 cells, COX-2-silenced cells maintain nuclear localization of β-catenin.
doi:10.1016/j.prostaglandins.2012.05.002
PMCID: PMC3429715  PMID: 22652293
Cyclooxygenase-2; Prostaglandin E2; Lymphoid Enhancer Factor-1; β-catenin; Wnt; breast cancer
23.  Eicosanoids and Tumor Necrosis Factor-alpha in the kidney 
The thick ascending limb of Henle’s loop (TAL) is capable of metabolizing arachidonic acid (AA) by cytochrome P450 (CYP450) and cyclooxygenase (COX) pathways and has been identified as a nephron segment that contributes to salt-sensitive hypertension. Previous studies demonstrated a prominent role for CYP450-dependent metabolism of AA to products that inhibited ion transport pathways in the TAL. However, COX-2 is constitutively expressed along all segments of the TAL and is increased in response to diverse stimuli. The ability of Tamm-Horsfall glycoprotein, a selective marker of cortical TAL (cTAL) and medullary (mTAL), to bind TNF and localize it to this nephron segment prompted studies to determine the capacity of mTAL cells to produce TNF and determine its effects on mTAL function. The colocalization of calcium-sensing receptor (CaR) and COX-2 in the TAL supports the notion that activation of CaR induces TNF-dependent COX-2 expression and PGE2 synthesis in mTAL cells. Additional studies showed that TNF produced by mTAL cells inhibits 86Rb uptake, an in vitro correlate of natriuresis, in an autocrine- and COX-2-dependent manner. The molecular mechanism for these effects likely includes inhibition of Na+-K+-2Cl− cotransporter (NKCC2) expression and trafficking.
doi:10.1016/j.prostaglandins.2011.11.002
PMCID: PMC3294193  PMID: 22101002
Eicosanoids; COX-2; calcium-sensing receptor; TNF; NKCC2; NFAT5
24.  Contribution of Cytochrome P450 1B1 to Hypertension and Associated Pathophysiology: A Novel Target for Antihypertensive Agents 
The aim of this review is to discuss the contribution of cytochrome P450 (CYP) 1B1 in vascular smooth muscle cell growth, hypertension, and associated pathophysiology. CYP1B1 is expressed in cardiovascular and renal tissues, and mediates angiotensin II (Ang II)-induced activation of NADPH oxidase and generation of reactive oxygen species (ROS), and vascular smooth muscle cell migration, proliferation, and hypertrophy. Moreover, CYP1B1 contributes to the development and/or maintenance of hypertension produced by Ang II-, deoxycorticosterone Nω-nitro-(DOCA)-salt-, and L-arginine methyl ester-induced hypertension and in spontaneously hypertensive rats. The pathophysiological changes, including cardiovascular hypertrophy, increased vascular reactivity, endothelial and renal dysfunction, injury and inflammation associated with Ang II- and/or DOCA-salt induced hypertension in rats, and Ang II-induced hypertension in mice are minimized by inhibition of CYP1B1 activity with 2,4,3′,5′-tetramethoxystilbene or by Cyp1b1 gene disruption in mice. These pathophysiological changes appear to be mediated by increased production of ROS via CYP1B1-dependent NADPH oxidase activity and extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and c-Src.
doi:10.1016/j.prostaglandins.2011.12.003
PMCID: PMC3339277  PMID: 22210049
cytochrome P450 1B1; reactive oxygen species; angiotensin II-/DOCA-salt-hypertension; cardiovascular hypertrophy; renal dysfunction; inflammation
25.  Role of the adenosine 2A receptor-epoxyeicosatrienoic acid pathway in the development of salt-sensitive hypertension 
Activation of rat adenosine 2A receptors (A2A R) dilates preglomerular microvessels, an effect mediated by epoxyeicosatrienoic acids (EETs). High salt (HS) intake increases epoxygenase activity and adenosine levels and greater vasodilator response to a stable adenosine analog, 2-chloroadenosine (2-CA), was seen in kidneys obtained from HS-fed rats which was mediated by increased EET release. Because this pathway is antipressor, we examined the role of the A2A R-EET pathway in a genetic model of salt-sensitive hypertension, the Dahl salt-sensitive (SS) rats. Dahl S resistant (R) rats fed a HS diet demonstrated a greater renal vasodilator response to 2-CA. In contrast, Dahl SS rats did not exhibit a difference in the vasodilator response to 2-CA whether fed normal salt (NS) or HS diet. In Dahl SR but not Dahl SS rats, HS intake significantly increased purine flux, augmented the protein expression of A2A R and cytochrome P450 2C23 and 2C11 epoxygenases, and elevated the renal efflux of EETs. Thus the Dahl SR rat is able to respond to HS intake by recruiting EET formation, whereas the Dahl SS rat appears to have exhausted its ability to increase EET synthesis above the levels observed on NS intake. In vivo inhibition of the A2A R-EET pathway in Dahl SR rats fed a HS diet results in reduced renal EETs levels, diminished natriuretic capacity and hypertension, thus supporting a role for the A2A R-EET pathway in the adaptive natriuretic response to modulate blood pressure during salt loading. An inability of Dahl SS rats to upregulate the A2A R-EET pathway in response to salt loading may contribute to the development of salt-sensitive hypertension.
doi:10.1016/j.prostaglandins.2011.12.002
PMCID: PMC3348415  PMID: 22227265
Cytochrome P450; Epoxyeicosatrienoic acids; Adenosine; Kidney; Salt-sensitive hypertension

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