15-oxo-Eicosatetraenoic Acid, a Metabolite of Macrophage 15-Hydroxyprostaglandin Dehydrogenase That Inhibits Endothelial Cell Proliferation
- Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
- Address correspondence to:
Dr. Ian A. Blair, Center for Cancer Pharmacology, University of Pennsylvania, 854 BRB II/III, 421 Ci Boulevard, Philadelphia, PA 19104-6160. E-mail: ianblair{at}mail.med.upenn.edu
Abstract
The formation of 15-oxo-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-oxo-ETE) as a product from rabbit lung 15-hydroxyprostaglandin dehydrogenase (PGDH)-mediated oxidation of 15(S)-hydroperoxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid was first reported more than 30 years ago. However, the pharmacological significance of 15-oxo-ETE formation has never been established. We have now evaluated 15-lipoxygenase (LO)-1-mediated arachidonic acid (AA) metabolism to 15-oxo-ETE in human monocytes and mouse RAW macrophages that stably express human 15-LO-1 (R15L cells). A targeted lipidomics approach was used to identify and quantify the oxidized lipids that were formed. 15-oxo-ETE was found to be a major AA-derived LO metabolite when AA was given exogenously or released from endogenous esterified lipid stores by calcium ionophore (CI) calcimycin (A-23187). This established the R15L cells as a useful in vitro model system. Pretreatment of the R15L cells with cinnamyl-3,4-dihydroxycyanocinnamate significantly inhibited AA- or CI-mediated production of 15(S)-hydroperoxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid [15(S)-HETE] and 15-oxo-ETE, confirming the role of 15-LO-1 in mediating AA metabolite formation. Furthermore, 15(S)-HETE was metabolized primarily to 15-oxo-ETE. Pretreatment of the R15L cells with the 15-hydroxyprostaglandin dehydrogenase (PGDH) inhibitor 5-[[4-(ethoxycarbonyl)phenyl]azo]-2-hydroxy-benzeneacetic acid (CAY10397) reduced AA- and 15(S)-HETE-mediated formation of 15-oxo-ETE in a dose-dependent manner. This confirmed that macrophage-derived 15-PGDH was responsible for catalyzing the conversion of 15(S)-HETE to 15-oxo-ETE. Finally, 15-oxo-ETE was shown to inhibit the proliferation of human vascular vein endothelial cells by suppressing DNA synthesis, implicating a potential antiangiogenic role. This is the first report describing the biosynthesis of 15-oxo-ETE by macrophage/monocytes and its ability to inhibit endothelial cell proliferation.
Footnotes
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This work was supported by the National Institutes of Health National Cancer Institute [Grant R01-CA091016] and the National Institutes of Health National Institute of Environmental Health Sciences [Grant P30-ES013508].
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ABBREVIATIONS: 15-oxo-ETE, 15-oxo-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid; PGDH, hydroxyprostaglandin dehydrogenase; 15-HETE, 15-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid; COX, cyclooxygenase; AA, arachidonic acid; 5-oxo-ETE, 5-oxo-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid; LO, lipoxygenase; 15-LO-1, type 1 human 15-lipoxygenase; 15-HPETE, 15-hydroperoxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid; CAY10397, 5-[[4-(ethoxycarbonyl)phenyl]azo]-2-hydroxy-benzeneacetic acid; CDC, cinnamyl-3,4-dihydroxy-α-cyanocinnamate; CI, calcium ionophore; PFB, 2,3,4,5,6-pentafluorobenzyl; PBS, phosphate-buffered saline; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; IL, interleukin; LC, liquid chromatography; MS, mass spectrometry; ECAPCI, electron capture atmospheric pressure chemical ionization; MRM, multiple reaction monitoring; R15L, mouse macrophage RAW cells stably expressing human 15-LO-1; RMock, mouse macrophage RAW cells transfected with the pcDNA3 plasmid; HUVEC, human umbilical vein endothelial cell; EC, endothelial cell; BrdU, 5-bromo-2-deoxyuridine; ELISA, enzyme-linked immunosorbent assay; rt, retention time; PG, prostaglandin; cPLA2, cytosolic phospholipase A2; GSH, reduced glutathione; 15d-PGJ2, 15-deoxy-Δ12,14-prostaglandin J2; PPAR, peroxisome proliferator-activated receptor; Stat, signal transducer and activator of transcription; A-23187, calcimycin.
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- Accepted June 17, 2009.
- Received May 4, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
