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1.  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.
PMCID: PMC3844125  PMID: 23770055
sphingosine-1-phosphate; S1P family of G-protein coupled receptor; sphingolipids; sphingosine kinase; vasculature
2.  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.
PMCID: PMC4052893  PMID: 23247129
CYP-derived eicosanoids; Islets; Glucose homeostasis; Apoptosis
3.  ER-tethered Transcription Factor CREBH Regulates Hepatic Lipogenesis, Fatty Acid Oxidation, and Lipolysis upon Metabolic Stress 
Hepatology (Baltimore, Md.)  2012;55(4):1070-1082.
CREBH is a liver-specific transcription factor that is localized in the endoplasmic reticulum (ER) membrane. Our previous work demonstrated that CREBH is activated by ER stress or inflammatory stimuli to induce an acute-phase hepatic inflammation. Here we demonstrate that CREBH is a key metabolic regulator of hepatic lipogenesis, fatty acid (FA) oxidation, and lipolysis under metabolic stress. Saturated FA, insulin signals, or an atherogenic high-fat diet can induce CREBH activation in the liver. Under the normal chow diet, CrebH knockout mice display a modest decrease in hepatic lipid contents but an increase in plasma triglycerides (TG). After feeding an atherogenic high-fat diet, massive accumulation of hepatic lipid metabolites and significant increase in plasma TG levels were observed in the CrebH knockout mice. Along with the hypertriglyceridemia phenotype, the CrebH null mice displayed significantly reduced body weight gain, diminished abdominal fat, and increased non-alcoholic steatohepatitis (NASH) activities under the atherogenic high-fat diet. Gene expression analysis and chromatin-immunoprecipitation (ChIP) assay indicated that CREBH is required to activate expression of the genes encoding functions involved in de novo lipogenesis, TG and cholesterol biosynthesis, FA elongation and oxidation, lipolysis, and lipid transport. Supporting the role of CREBH in lipogenesis and lipolysis, forced expression of an activated form of CREBH protein in the liver significantly increases accumulation of hepatic lipids but reduces plasma TG levels in mice. All together our study shows that CREBH plays a key role in maintaining lipid homeostasis by regulating expression of the genes involved in hepatic lipogenesis, FA oxidation, and lipolysis under metabolic stress. The identification of CREBH as a stress-inducible metabolic regulator has important implications in the understanding and treatment of metabolic disease.
PMCID: PMC3319338  PMID: 22095841
4.  12/15-Lipoxygenase-Derived Lipid Metabolites Induce Retinal Endothelial Cell Barrier Dysfunction: Contribution of NADPH Oxidase 
PLoS ONE  2013;8(2):e57254.
The purpose of the current study was to evaluate the effect of 12/15- lipoxygenase (12/15-LOX) metabolites on retinal endothelial cell (REC) barrier function. FITC-dextran flux across the REC monolayers and electrical cell-substrate impedance sensing (ECIS) were used to evaluate the effect of 12- and 15-hydroxyeicosatetreanoic acids (HETE) on REC permeability and transcellular electrical resistance (TER). Effect of 12- or 15-HETE on the levels of zonula occludens protein 1 (ZO-1), reactive oxygen species (ROS), NOX2, pVEGF-R2 and pSHP1 was examined in the presence or absence of inhibitors of NADPH oxidase. In vivo studies were performed using Ins2Akita mice treated with or without the 12/15-LOX inhibitor baicalein. Levels of HETE and inflammatory mediators were examined by LC/MS and Multiplex Immunoassay respectively. ROS generation and NOX2 expression were also measured in mice retinas. 12- and 15- HETE significantly increased permeability and reduced TER and ZO-1expression in REC. VEGF-R2 inhibitor reduced the permeability effect of 12-HETE. Treatment of REC with HETE also increased ROS generation and expression of NOX2 and pVEGF-R2 and decreased pSHP1 expression. Treatment of diabetic mice with baicalein significantly decreased retinal HETE, ICAM-1, VCAM-1, IL-6, ROS generation, and NOX2 expression. Baicalein also reduced pVEGF-R2 while restored pSHP1 levels in diabetic retina. Our findings suggest that 12/15-LOX contributes to vascular hyperpermeability during DR via NADPH oxidase dependent mechanism which involves suppression of protein tyrosine phosphatase and activation of VEGF-R2 signal pathway.
PMCID: PMC3577708  PMID: 23437353
Experimental cell research  2010;316(10):1706-1715.
12-lipoxygenase, an arachidonic acid metabolizing enzyme of the lipoxygenase pathway, has been implicated as a major factor in promoting prostate cancer progression and metastasis. The ability of 12-LOX to aggravate the disease was linked to its proangiogenic role. Recent studies clearly demonstrated that 12-LOX enhances the expression and secretion of the angiogenic factor, vascular endothelial growth factor (VEGF) thus providing a direct link between this enzyme and its angiogenic properties. In the present study we have investigated the relationship between 12-LOX and hypoxia inducible factor-1α (HIF-1α), a transcription factor involved in the regulation of VEGF expression under hypoxic conditions in solid tumors. Our findings have revealed that HIF-1 is one of the target transcription factors regulated by 12-LOX and 12(S)-HETE, in hypoxic tumor cells of the prostate. Regulation of HIF-1α by 12-LOX adds to the complexity of pathways mediated by this enzyme in promoting prostate cancer angiogenesis and metastasis. We have evidence that 12-LOX increases the protein level, mRNA, and functional activity of HIF-1α under hypoxic conditions, one of the mechanisms by which it upregulates VEGF secretion and activity.
PMCID: PMC3420817  PMID: 20303950
12-Lipoxygenase; Hypoxia Inducible Factor-1α (HIF-1α); angiogenesis; prostate cancer; hypoxia
6.  Increased Expression and Activity of 12-Lipoxygenase in Oxygen-Induced Ischemic Retinopathy and Proliferative Diabetic Retinopathy 
Diabetes  2011;60(2):614-624.
Arachidonic acid is metabolized by 12-lipoxygenase (12-LOX) to 12-hydroxyeicosatetraenoic acid (12-HETE) and has an important role in the regulation of angiogenesis and endothelial cell proliferation and migration. The goal of this study was to investigate whether 12-LOX plays a role in retinal neovascularization (NV).
Experiments were performed using retinas from a murine model of oxygen-induced ischemic retinopathy (OIR) that was treated with and without the LOX pathway inhibitor, baicalein, or lacking 12-LOX. We also analyzed vitreous samples from patients with and without proliferative diabetic retinopathy (PDR). Western blotting and RT-PCR were used to assess the expression of 12-LOX, vascular endothelial growth factor (VEGF), and pigment epithelium–derived factor (PEDF). Liquid chromatography–mass spectrometry was used to assess the amounts of HETEs in the murine retina and human vitreous samples. The effects of 12-HETE on VEGF and PEDF expression were evaluated in Müller cells (rMCs), primary mouse retinal pigment epithelial cells, and astrocytes.
Retinal NV during OIR was associated with increased 12-LOX expression and 12-, 15-, and 5-HETE production. The amounts of HETEs also were significantly higher in the vitreous of diabetic patients with PDR. Retinal NV was markedly abrogated in mice treated with baicalein or mice lacking 12-LOX. This was associated with decreased VEGF expression and restoration of PEDF levels. PEDF expression was reduced in 12-HETE–treated rMCs, astrocytes, and the retinal pigment epithelium. Only rMCs and astrocytes showed increased VEGF expression by 12-HETE.
12-LOX and its product HETE are important regulators of retinal NV through modulation of VEGF and PEDF expression and could provide a new therapeutic target to prevent and treat ischemic retinopathy.
PMCID: PMC3028363  PMID: 21228311
7.  Toxicity of Prolonged High Dose Inhaled PGE1 in Ventilated Neonatal Pigs 
To study the toxicity of inhaled PGE1 (IPGE1) in healthy ventilated piglets.
Mechanically ventilated anesthetized piglets received either high dose IPGE1 (IPGE1 group) or nebulized saline (control group) continuously for 24 hours. Cardio-respiratory parameters, complete blood counts and serum electrolytes were monitored. Lung histology was evaluated by a masked pathologist for the severity (minimal, moderate, and severe) and extent (focal, multifocal, and diffuse) of histologic injury.
Ten neonatal pigs were instrumented. Four received nebulized saline and six received high dose IPGE1. There was no evidence of adverse cardio-respiratory effects, bronchial irritation or hypernatremia related to IPGE1. Diffuse/multifocal alveolar edema and focal polymorphonuclear infiltration was observed in both the control and IPGE1 groups suggesting that alveolar alterations may be secondary to effects of mechanical ventilation. The most distinct histomorphological abnormalities observed in the IPGE1 animals were focal ulceration, flattening of the bronchial epithelium and loss of cilia of moderate to severe degree in the trachea and bronchi.
In healthy piglets, inhalation of high dose IPGE1 was not associated with adverse cardiorespiratory effects, bronchial irritation, or hypernatremia and produced minimal signs of pulmonary toxicity even after 24 hours. Prolonged inhalation of high dose PGE1 therefore appears safe in newborn piglets.
PMCID: PMC2443360  PMID: 18343700
Pulmonary toxicity; inhaled; PGE1/Alprostadil; neonatal; piglet/animal; histomorphology; nebulizer; aerosol
8.  Jet Nebulization of Prostaglandin E1 During Neonatal Mechanical Ventilation: Stability, Emitted Dose and Aerosol Particle Size 
We have previously reported the safety of aerosolized PGE1 in neonatal hypoxemic respiratory failure. The aim of this study is to characterize the physicochemical properties of PGE1 solution, stability, emitted dose and the aerodynamic particle size distribution (APSD) of PGE1 aerosol in a neonatal ventilator circuit.
PGE1 was diluted in normal saline and physicochemical properties of the solution characterized. Chemical stability and emitted dose were evaluated during jet nebulization in a neonatal conventional (CMV) or high frequency (HFV) ventilator circuit by a High Performance Liquid Chromatography - Mass Spectrometry method. The APSD of the PGE1 aerosol was evaluated with a six-stage cascade impactor during CMV.
PGE1 solution in normal saline had a low viscosity (0.9818 cP) and surface tension (60.8 mN/m) making it suitable for aerosolization. Little or no degradation of PGE1 was observed in samples from aerosol condensates, the PGE1 solution infused over 24 h, or the residual solution in the nebulizer. The emitted dose of PGE1 following jet nebulization was 32–40% during CMV and 0.1% during HFV. The PGE1 aerosol had a mass median aerodynamic diameter of 1.4 µm and geometric standard deviation of 2.9 with 90% of particles being < 4.0 µm in size.
Nebulization of PGE1 during neonatal CMV or HFV is efficient and results in rapid nebulization without altering the chemical structure. On the basis of the physicochemical properties of PGE1 solution and the APSD of the PGE1 aerosol, one can predict predominantly alveolar deposition of aerosolized PGE1.
PMCID: PMC2265206  PMID: 17997106
aerosol; aerosol particle size distribution; cascade impactor; chemical stability; emitted dose; jet nebulizer; LC-MS; mechanical ventilation; nebulizer; neonatal hypoxemic respiratory failure; prostaglandin E1[PGE1]/ Alprostadil; pulmonary deposition; respirable fraction

Results 1-8 (8)