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• W2004193154 abstract "The cytochrome P450 arachidonic acid epoxygenase metabolites, the epoxyeicosatrienoic acids (EETs) are powerful, nonregioselective, stimulators of cell proliferation. In this study we compared the ability of the four EETs (5,6-, 8,9-, 11,12-, and 14,15-EETs) to regulate endothelial cell proliferation in vitro and angiogenesis in vivo and determined the molecular mechanism by which EETs control these events. Inhibition of the epoxygenase blocked serum-induced endothelial cell proliferation, and exogenously added EETs rescued cell proliferation from epoxygenase inhibition. Studies with selective ERK, p38 MAPK, or PI3K inhibitors revealed that whereas activation of p38 MAPK is required for the proliferative responses to 8,9- and 11,12-EET, activation of PI3K is necessary for the cell proliferation induced by 5,6- and 14,15-EET. Among the four EETs, only 5,6- and 8,9-EET are capable of promoting endothelial cell migration and the formation of capillary-like structures, events that are dependent on EET-mediated activation of ERK and PI3K. Using subcutaneous sponge models, we showed that 5,6- and 8,9-EET are pro-angiogenic in mice and that their neo-vascularization effects are enhanced by the co-administration of an inhibitor of EET enzymatic hydration, presumably because of reduced EET metabolism and inactivation. These studies identify 5,6- and 8,9-EET as powerful and selective angiogenic lipids, provide a functional link between the EET proliferative chemotactic properties and their angiogenic activity, and suggest a physiological role for them in angiogenesis and de novo vascularization. The cytochrome P450 arachidonic acid epoxygenase metabolites, the epoxyeicosatrienoic acids (EETs) are powerful, nonregioselective, stimulators of cell proliferation. In this study we compared the ability of the four EETs (5,6-, 8,9-, 11,12-, and 14,15-EETs) to regulate endothelial cell proliferation in vitro and angiogenesis in vivo and determined the molecular mechanism by which EETs control these events. Inhibition of the epoxygenase blocked serum-induced endothelial cell proliferation, and exogenously added EETs rescued cell proliferation from epoxygenase inhibition. Studies with selective ERK, p38 MAPK, or PI3K inhibitors revealed that whereas activation of p38 MAPK is required for the proliferative responses to 8,9- and 11,12-EET, activation of PI3K is necessary for the cell proliferation induced by 5,6- and 14,15-EET. Among the four EETs, only 5,6- and 8,9-EET are capable of promoting endothelial cell migration and the formation of capillary-like structures, events that are dependent on EET-mediated activation of ERK and PI3K. Using subcutaneous sponge models, we showed that 5,6- and 8,9-EET are pro-angiogenic in mice and that their neo-vascularization effects are enhanced by the co-administration of an inhibitor of EET enzymatic hydration, presumably because of reduced EET metabolism and inactivation. These studies identify 5,6- and 8,9-EET as powerful and selective angiogenic lipids, provide a functional link between the EET proliferative chemotactic properties and their angiogenic activity, and suggest a physiological role for them in angiogenesis and de novo vascularization. Interest in the biochemical mechanism of angiogenesis, the de novo formation of blood vessels from pre-existing vessels, stems from the critical roles played by this process in the pathophysiology of inflammation, cancer, and cardiovascular diseases. Neo-vascularization requires the coordinated contributions of endothelial cell proliferation and migration leading to the formation of nascent capillary structures and finally, to new functional vessels. The eicosanoids prostaglandin E2, prostacyclin (1Fosslien E. Ann. Clin. Lab Sci. 2001; 31: 325-348PubMed Google Scholar, 2Gately S. Li W.W. Semin. Oncol. 2004; 31: 2-11Crossref PubMed Google Scholar), HETEs (3Nie D. Honn K.V. Semin. Thromb. Hemost. 2004; 30: 119-125Crossref PubMed Scopus (39) Google Scholar, 4Pratt P.F. Medhora M. Harder D.R. Curr. Opin. Investig. Drugs. 2004; 5: 952-956PubMed Google Scholar, 5Jiang M. Mezentsev A. Kemp R. Byun K. Falck J.R. Miano J.M. Nasjletti A. Abraham N.G. Laniado-Schwartzman M. Circ. Res. 2004; 94: 167-174Crossref PubMed Scopus (60) Google Scholar, 6Chen P. Guo M. Wygle D. Edwards P.A. Falck J.R. Roman R.J. Scicli A.G. Am. J. Pathol. 2005; 166: 615-624Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 7Amaral S.L. Maier K.G. Schippers D.N. Roman R.J. Greene A.S. Am. J. Physiol. Heart Circ. Physiol. 2003; 284: H1528-H1535Crossref PubMed Scopus (79) Google Scholar), and the epoxyeicosatrienoic acids (EET) 1The abbreviations used are: EET, epoxyeicosatrienoic acid; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; EGF, epidermal growth factor; DHET, dihydroxyeicosatrienoic acid; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; RITC, rhodamine isothiocyanate; DAPI, 4′,6-diamidino-2-phenylindole; HPLC, high pressure liquid chromatography; MS-PPOH, N-methylsulfonyl-6-(2-proparglyloxyphenyl)hexanamide; ACU, adamantyl-cyclohexyl-urea. (8Zhang C. Harder D.R. Stroke. 2002; 33: 2957-2964Crossref PubMed Scopus (100) Google Scholar, 9Medhora M. Daniels J. Mundey K. Fisslthaler B. Busse R. Jacobs E.R. Harder D.R. Am. J. Physiol. Heart Circ. Physiol. 2003; 284: H215-H224Crossref PubMed Scopus (105) Google Scholar, 10Michaelis U.R. Fisslthaler B. Medhora M. Harder D. Fleming I. Busse R. FASEB J. 2003; 17: 770-772Crossref PubMed Scopus (150) Google Scholar, 11Michaelis U.R. Falck J.R. Schmidt R. Busse R. Fleming I. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 321-326Crossref PubMed Scopus (63) Google Scholar) have been characterized as pro-angiogenic molecules. In addition, the roles of cyclooxygenases 1 and 2, as well as cytochrome P450 (P450) 4A and 2C isoforms in these processes have been suggested (4Pratt P.F. Medhora M. Harder D.R. Curr. Opin. Investig. Drugs. 2004; 5: 952-956PubMed Google Scholar, 5Jiang M. Mezentsev A. Kemp R. Byun K. Falck J.R. Miano J.M. Nasjletti A. Abraham N.G. Laniado-Schwartzman M. Circ. Res. 2004; 94: 167-174Crossref PubMed Scopus (60) Google Scholar, 6Chen P. Guo M. Wygle D. Edwards P.A. Falck J.R. Roman R.J. Scicli A.G. Am. J. Pathol. 2005; 166: 615-624Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 7Amaral S.L. Maier K.G. Schippers D.N. Roman R.J. Greene A.S. Am. J. Physiol. Heart Circ. Physiol. 2003; 284: H1528-H1535Crossref PubMed Scopus (79) Google Scholar, 8Zhang C. Harder D.R. Stroke. 2002; 33: 2957-2964Crossref PubMed Scopus (100) Google Scholar, 9Medhora M. Daniels J. Mundey K. Fisslthaler B. Busse R. Jacobs E.R. Harder D.R. Am. J. Physiol. Heart Circ. Physiol. 2003; 284: H215-H224Crossref PubMed Scopus (105) Google Scholar, 10Michaelis U.R. Fisslthaler B. Medhora M. Harder D. Fleming I. Busse R. FASEB J. 2003; 17: 770-772Crossref PubMed Scopus (150) Google Scholar, 11Michaelis U.R. Falck J.R. Schmidt R. Busse R. Fleming I. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 321-326Crossref PubMed Scopus (63) Google Scholar). However, the individual and/or combined contribution of these lipids to in vivo angiogenic responses remains to be determined. The P450 arachidonic acid epoxygenases catalyze the metabolism of endogenous pools of arachidonic acid to 5,6-, 8,9-, 11,12-, and 14,15-EET (12Capdevila J.H. Falck J.R. Harris R.C. J. Lipid Res. 2000; 41: 163-181Abstract Full Text Full Text PDF PubMed Google Scholar, 13Roman R.J. Physiol. Rev. 2002; 82: 131-185Crossref PubMed Scopus (1170) Google Scholar, 14Holla V.R. Makita K. Zaphiropoulos P.G. Capdevila J.H. J. Clin. Investig. 1999; 104: 751-760Crossref PubMed Scopus (110) Google Scholar, 15Tsao C.C. Coulter S.J. Chien A. Luo G. Clayton N.P. Maronpot R. Goldstein J.A. Zeldin D.C. J. Pharmacol. Exp. Ther. 2001; 299: 39-47PubMed Google Scholar, 16DeLozier T.C. Tsao C.C. Coulter S.J. Foley J. Bradbury J.A. Zeldin D.C. Goldstein J.A. J. Pharmacol. Exp. Ther. 2004; 310: 845-854Crossref PubMed Scopus (69) Google Scholar, 17Spector A.A. Fang X. Snyder G.D. Weintraub N.L. Prog. Lipid Res. 2004; 43: 55-90Crossref PubMed Scopus (490) Google Scholar), a reaction catalyzed predominantly by members of the P450 CYP2C gene subfamily (12Capdevila J.H. Falck J.R. Harris R.C. J. Lipid Res. 2000; 41: 163-181Abstract Full Text Full Text PDF PubMed Google Scholar). Furthermore, CYP2C8, CYP2C23, and Cyp2c44 have been identified as predominant stereoselective epoxygenases in several human, rat, and mouse organ tissues (12Capdevila J.H. Falck J.R. Harris R.C. J. Lipid Res. 2000; 41: 163-181Abstract Full Text Full Text PDF PubMed Google Scholar, 13Roman R.J. Physiol. Rev. 2002; 82: 131-185Crossref PubMed Scopus (1170) Google Scholar, 17Spector A.A. Fang X. Snyder G.D. Weintraub N.L. Prog. Lipid Res. 2004; 43: 55-90Crossref PubMed Scopus (490) Google Scholar, 18Zeldin D.C. DuBois R.N. Falck J.R. Capdevila J.H. Arch. Biochem. Biophys. 1995; 322: 76-86Crossref PubMed Scopus (155) Google Scholar). Since the original description of the mitogenic properties of 14,15-EET (12Capdevila J.H. Falck J.R. Harris R.C. J. Lipid Res. 2000; 41: 163-181Abstract Full Text Full Text PDF PubMed Google Scholar, 19Harris R.C. Homma T. Jacobson H.R. Capdevila J. J. Cell. Physiol. 1990; 144: 429-437Crossref PubMed Scopus (91) Google Scholar), several studies have characterized 11,12- and 14,15-EET as powerful mitogens using cultured cells derived from kidney, brain, and endothelium (4Pratt P.F. Medhora M. Harder D.R. Curr. Opin. Investig. Drugs. 2004; 5: 952-956PubMed Google Scholar,8Zhang C. Harder D.R. Stroke. 2002; 33: 2957-2964Crossref PubMed Scopus (100) Google Scholar, 9Medhora M. Daniels J. Mundey K. Fisslthaler B. Busse R. Jacobs E.R. Harder D.R. Am. J. Physiol. Heart Circ. Physiol. 2003; 284: H215-H224Crossref PubMed Scopus (105) Google Scholar, 10Michaelis U.R. Fisslthaler B. Medhora M. Harder D. Fleming I. Busse R. FASEB J. 2003; 17: 770-772Crossref PubMed Scopus (150) Google Scholar, 11Michaelis U.R. Falck J.R. Schmidt R. Busse R. Fleming I. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 321-326Crossref PubMed Scopus (63) Google Scholar, 12Capdevila J.H. Falck J.R. Harris R.C. J. Lipid Res. 2000; 41: 163-181Abstract Full Text Full Text PDF PubMed Google Scholar,18Zeldin D.C. DuBois R.N. Falck J.R. Capdevila J.H. Arch. Biochem. Biophys. 1995; 322: 76-86Crossref PubMed Scopus (155) Google Scholar, 19Harris R.C. Homma T. Jacobson H.R. Capdevila J. J. Cell. Physiol. 1990; 144: 429-437Crossref PubMed Scopus (91) Google Scholar, 20Chen J.K. Wang D.W. Falck J.R. Capdevila J. Harris R.C. J. Biol. Chem. 1999; 274: 4764-4769Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 21Chen J.K. Capdevila J. Harris R.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6029-6034Crossref PubMed Scopus (83) Google Scholar), and a role of 14,15-EET in mediating the mitogenic responses of EGF and HB-EGF has been thoroughly documented in cultured LLCPk cells (20Chen J.K. Wang D.W. Falck J.R. Capdevila J. Harris R.C. J. Biol. Chem. 1999; 274: 4764-4769Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 21Chen J.K. Capdevila J. Harris R.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6029-6034Crossref PubMed Scopus (83) Google Scholar). More recently, 11,12-EET was identified as an angiogenic molecule, and in vitro roles for cyclooxygenase-2 and EGF receptor identified (10Michaelis U.R. Fisslthaler B. Medhora M. Harder D. Fleming I. Busse R. FASEB J. 2003; 17: 770-772Crossref PubMed Scopus (150) Google Scholar, 11Michaelis U.R. Falck J.R. Schmidt R. Busse R. Fleming I. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 321-326Crossref PubMed Scopus (63) Google Scholar). Furthermore, 11,12-EET has also been shown to activate PI3K and tyrosine kinases (10Michaelis U.R. Fisslthaler B. Medhora M. Harder D. Fleming I. Busse R. FASEB J. 2003; 17: 770-772Crossref PubMed Scopus (150) Google Scholar, 22Hoebel B.G. Steyrer E. Graier W.F. Clin. Exp. Pharmacol. Physiol. 1998; 25: 826-830Crossref PubMed Scopus (42) Google Scholar). However, only a few studies have addressed whether activation of these downstream pathways are indeed required to mediate these EET-induced cell responses. Whereas the mitogenic activity of 11,12- and 14,15-EET, and the signaling pathways for 14,15-EET are well documented, less is known regarding the proliferative and/or angiogenic properties of 5,6- and 8,9-EET and their potential mechanisms of action. The biosynthesis of 8,9-EET from endogenous fatty acid pools is well documented (12Capdevila J.H. Falck J.R. Harris R.C. J. Lipid Res. 2000; 41: 163-181Abstract Full Text Full Text PDF PubMed Google Scholar), and that of the labile 5,6-EET has been inferred from the identification of its hydration product, 5,6-dihydroxyeicosatrienoic acid (5,6-DHET), in several biological samples (12Capdevila J.H. Falck J.R. Harris R.C. J. Lipid Res. 2000; 41: 163-181Abstract Full Text Full Text PDF PubMed Google Scholar, 17Spector A.A. Fang X. Snyder G.D. Weintraub N.L. Prog. Lipid Res. 2004; 43: 55-90Crossref PubMed Scopus (490) Google Scholar). Furthermore, these EETs are known to circulate, associated with plasma lipoproteins, in rat and human blood (12Capdevila J.H. Falck J.R. Harris R.C. J. Lipid Res. 2000; 41: 163-181Abstract Full Text Full Text PDF PubMed Google Scholar). We report here (i) the characterization of 5,6-and 8,9-EET as powerful mitogens, and selective chemotactic lipids for primary cultures of mouse pulmonary endothelial cells; (ii) the identification of the intracellular signaling pathways associated with their proliferative and chemotactic activities; and (iii) the demonstration of a role for these two EETs in promoting de novo angiogenesis in vivo. The observation that inhibition of enzymatic EET hydration potentiates the angiogenic activities of 5,6- and 8,9-EET suggests that inhibitors of epoxide hydrolase(s) could serve to promote angiogenesis in pathophysiological conditions in which de novo vascularization is compromised. Synthesis of EETs by Endothelial Cells—Pulmonary murine microvascular endothelial cells were isolated and cultured as previously described (23Pozzi A. Moberg P.E. Miles L.A. Wagner S. Soloway P. Gardner H.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2202-2207Crossref PubMed Scopus (349) Google Scholar). Cells at early passages (1Fosslien E. Ann. Clin. Lab Sci. 2001; 31: 325-348PubMed Google Scholar, 2Gately S. Li W.W. Semin. Oncol. 2004; 31: 2-11Crossref PubMed Google Scholar, 3Nie D. Honn K.V. Semin. Thromb. Hemost. 2004; 30: 119-125Crossref PubMed Scopus (39) Google Scholar) were used for these studies because the expression of CYP2Cs in cultured endothelial cells decreases with increased cell passage (24Fleming I. Pharmacol. Res. 2004; 49: 525-533Crossref PubMed Scopus (122) Google Scholar). To study cellular EET synthase activity, semiconfluent endothelial cells (passage 3) were cultured in serum-free medium in the presence or absence of arachidonic acid (10 μm). After 4 or 24 h, the cells and the medium were removed from the plates, and the suspension centrifuged to obtain a cell pellet and a medium-containing supernatant. The medium was mixed with 10 ng each of 1-14C-labeled DHETs (55 μCi/μmol; 95 atom % enrichment in 14C) or 1-14C-labeled EETs (55 μCi/μmol) and extracted with ethyl acetate. The EETs and DHETs present in the culture medium were purified by reversed-phase HPLC (25Capdevila J.H. Falck J.R. Dishman E. Karara A. Methods Enzymol. 1990; 187: 385-394Crossref PubMed Scopus (89) Google Scholar), converted to the corresponding pentafluorobenzylesters (EETs) or pentafluorobenzylester-trimethylsilylether derivatives, and quantified by NICI/GC/MS exactly as described (26Capdevila J.H. Dishman E. Karara A. Falck J.R. Methods Enzymol. 1991; 206: 441-453Crossref PubMed Scopus (72) Google Scholar). The cell pellets were suspended in PBS, mixed with 10 ng each of 1-14C-labeled EETs and DHETs (55 μCi/μmol), and extracted with CHCl3/CH3OH (2:1). The organic extracts were submitted to alkaline hydrolysis, and EETs and DHETs purified and quantified by NICI/GC/MS exactly as described (26Capdevila J.H. Dishman E. Karara A. Falck J.R. Methods Enzymol. 1991; 206: 441-453Crossref PubMed Scopus (72) Google Scholar). Prior to use, synthetic EETs and DHETs were purified by reversed-phase HPLC (25Capdevila J.H. Falck J.R. Dishman E. Karara A. Methods Enzymol. 1990; 187: 385-394Crossref PubMed Scopus (89) Google Scholar). The labile 5,6-EET was purified by normal phase HPLC on a μSorb silica column (250 × 4.6 mm; 5μ), using hexane containing 0.5% HOAc and 2% 2-propyl alcohol as mobile phase at 2 ml/min. Proliferation Assays—Endothelial cells (5 × 103/96-well plates) were plated in EGM-2-MV (Clonetics) containing 5% fetal calf serum with or without different concentrations of synthetic 5,6-, 8,9-, 11,12-, and 14,15-EETs or their corresponding DHETs. In some experiments cells were incubated in 5% fetal calf serum in the absence or presence of various concentrations of the EET synthase inhibitors N-methylsulfonyl-6-(2-proparglyloxyphenyl)hexanamide (MS-PPOH) and ketoconazole (17Spector A.A. Fang X. Snyder G.D. Weintraub N.L. Prog. Lipid Res. 2004; 43: 55-90Crossref PubMed Scopus (490) Google Scholar, 27Wang M.H. Brand-Schieber E. Zand B.A. Nguyen X. Falck J.R. Balu N. Schwartzman M.L. J. Pharmacol. Exp. Ther. 1998; 284: 966-973PubMed Google Scholar), or adamantyl-cyclohexyl-urea (ACU), an inhibitor of cytosolic epoxide hydrolase (17Spector A.A. Fang X. Snyder G.D. Weintraub N.L. Prog. Lipid Res. 2004; 43: 55-90Crossref PubMed Scopus (490) Google Scholar). Two days after, the medium was replaced with fresh medium containing [3H]thymidine (1 μCi/well), the cells incubated for another 48 h, and their levels of [3H]thymidine incorporation determined as described (23Pozzi A. Moberg P.E. Miles L.A. Wagner S. Soloway P. Gardner H.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2202-2207Crossref PubMed Scopus (349) Google Scholar). In some experiments endothelial cells were plated as above and serum-starved for 24 h. The cells were then cultured in serum-free medium containing [3H]thymidine (1 μCi/well) with or without 5,6-, 8,9-, 11,12-, and 14,15-EETs or their corresponding DHETs. Some cells were also treated with 10 μm MS-PPOH, ketoconazole, or ACU added alone or in combination with each EET (1 μm, each). Twenty-four hours later, cells were collected and proliferation determined as above. To determine the pathways involved in the EET-induced cell proliferation, 24 h serum-starved cells were incubated in serum-free medium containing [3H]thymidine (10 μCi/ml) with or without the individual EET regioisomers (1 μm), and in the presence or absence of either a MEK1 inhibitor (PD98059) (10 μm), a p38 MAPK inhibitor (10 μm) (cat. number 506126) or a PI3K inhibitor (wortmannin) (0.1 μm) (all from Calbiochem). After 24 h, the cells were collected, and [3H]thymidine incorporation was determined as above. At least three independent experiments with quadruplicate samples were performed for each set of experiments described above. The [3H]thymidine incorporation assay was also corroborated by manual cell counting (not shown). Migration Assay—Cell migration was assayed in transwell plates fitted with 8-μm membrane filters (Corning Ware). Lower wells were coated by an overnight incubation with collagen type I (10 μg/ml) at 4 °C and then incubated 1 h at 37 °C with bovine serum albumin (1% in PBS) to inhibit nonspecific cell migration. Serum-free medium with or without 1 μm EETs was then added to the lower wells and 24 h serum-starved endothelial cells (5 × 104 cells in 300 μl of serum-free medium containing 0.1% bovine serum albumin) to the upper wells. To determine the contribution of ERK, p38, and PI3K in EET-induced migration, serum-starved cells were allowed to migrate as indicated above with the difference that PD98059 (10 μm), P38 MAPK inhibitor (10 μm), or wortmannin (100 nm) were added to both upper and lower wells. After 12 h at 37 °C, cells on the top of the filter were removed by wiping, and the filters were then fixed in 4% formaldehyde in PBS. Migrating cells were stained with 1% crystal violet, and five randomly chosen fields from triplicate wells were counted at ×400 magnification. Three independent experiments were performed in duplicate. Matrigel-based Capillary Formation Assay—Capillary-like formation was analyzed as described (28Kubota Y. Kleinman H.K. Martin G.R. Lawley T.J. J. Cell Biol. 1988; 107: 1589-1598Crossref PubMed Scopus (984) Google Scholar). Briefly, 96-well plates were coated with 50 μl of Matrigel and incubated 30 min at 37 °C. Serum-starved endothelial cells (1 × 104) were plated over solidified Matrigel in 200 μl of serum-free medium with or without EETs (1 μm). To determine the contribution of EET synthases in capillary-like formation, endothelial cells were plated on Matrigel in 200 μl of complete medium in the presence or absence of different concentrations of MS-PPOH or ketoconazole. Capillary-like structures were recorded (3 images per gel per treatment) hourly for a period of 10 h, and representative images taken 3 h after plating are shown. To quantify capillary-like network formation, cellular nodes were defined as junctions linking at least three cells, and they were counted from digital images. Four independent experiments were performed with a total of 12 images analyzed per treatment. Western Blot Analysis and Reverse Transcriptase PCR Analysis—To evaluate the effects of EETs on ERK, p38, and Akt phosphorylation, semiconfluent endothelial cells were serum-starved for 24 h and then treated with the EET (1 μm each) for 0, 10, and 40 min. The cells were washed with PBS, collected, suspended in 50 mm HEPES, pH 7.5, 150 mm NaCl, 1% Triton X-100, and centrifuged for 10 min at 14,000 rpm. Cell lysates were resolved by SDS/PAGE (10% gels; 50 μg of total protein/lane) and transferred to Immobilon-P membranes (Millipore, Billerica, MA). Membranes were incubated with a rabbit anti-phospho-ERK, anti-phospho-p38, or anti-phospho-Akt antibody (all from Cell Signaling Technology). Immunoreactive proteins were visualized using a peroxidase-conjugated goat anti-rabbit and an ECL kit (Pierce). Total ERK, p38, and Akt content were verified by stripping the membranes in 50 mm Tris-HCl, pH 6.5, containing 2% SDS and 0.4% β-mercaptoethanol for 1 h at 55 °C, and re-probing with a rabbit anti-ERK, anti-p38, or anti-Akt antibody (Cell Signaling Technology). EET synthase expression in endothelial cell lysates (50 μg of protein/lane) was analyzed as above using a rabbit anti-CYP2C23 or anti-CYP2C11 (14Holla V.R. Makita K. Zaphiropoulos P.G. Capdevila J.H. J. Clin. Investig. 1999; 104: 751-760Crossref PubMed Scopus (110) Google Scholar) antibody, cross-reactive against the murine Cyp2c44and Cyp2c38 EET synthase, respectively. Purified cyp2c44 or cyp2c38 (20 ng/lane) was used as positive controls. Total RNA was purified from primary pulmonary endothelial cells (passage 2) using TRIzol reagent (Invitrogen). RNA samples were reverse-transcribed using a SuperScript II™ kit and oligo(dT) (12-18 bp), and cDNAs amplified using the following mouse Cyp2c isoform-specific PCR primers: Cyp2c38 (300 bp) sense, 5′-tttgtgaatggattaattgc-3′, antisense, 5′-tgccggtgaagttttattct-3′; Cyp2c44 (700 bp) sense, 5′-ttggatcctggcctaccgtg-3′, antisense, 5′-tgtctctgtgcctgccgtaa-3′. These primers are cDNA-specific in that they amplify exons that are separated by intronic sequences in the genomic DNA. The following primers for β-actin (800 bp) were used as positive control: sense, 5′-ccagagcaagagaggtatcctgac-3′, antisense, 5′-aatctccttctgcatcctgtcagc-3′. Immunofluorescence—Frozen sections (7-μm each) of tumors derived from human non-small cell lung cancer cells grown subcutaneously for 3 weeks in athymic nude mice were costained with biotinylated rabbit anti-rat CYP2C23 (1:300) and rat anti-mouse CD31 (1:100, PharMingen) followed by FITC-conjugated streptavidin (1:200, Sigma), RITC-conjugated goat anti-rat IgG (1:200, Jackson), and DAPI (2 ng/ml, Sigma) to visualize cell nuclei. Fluorescence emissions were with an epifluorescence microscope equipped with a triple filter channel. In Vivo Angiogenesis—The subcutaneous sponge model was used to determine the effects of EETs on in vivo angiogenesis (29Shi Y. Reitmaier B. Regenbogen J. Slowey R.M. Opalenik S.R. Wolf E. Goppelt A. Davidson J.M. Am. J. Pathol. 2005; 166: 303-312Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Sterile polyvinyl acetal CF-50 round sponges (8 × 3 mm, a gift from Dr. J. M. Davidson, Vanderbilt University) were implanted under the dorsal skin of 129 SvJ male mice (6 weeks of age, 20 g body weight, n = 5/treatment). The sponges were then injected every second day for 14 days with 50 μl of either vehicle (corn oil), EETs (50 μm), ACU (250 μm), or a mixture of EET and ACU (50 and 250 μm, respectively). Ten minutes before sacrifice, mice were injected intravenously with 50 μl of rhodamine-dextran (Mr 65, 2% in PBS, Sigma) to label blood vessels (30Brantley D.M. Cheng N. Thompson E.J. Lin Q. Brekken R.A. Thorpe P.E. Muraoka R.S. Cerretti D.P. Pozzi A. Jackson D. Lin C. Chen J. Oncogene. 2002; 21: 7011-7026Crossref PubMed Scopus (287) Google Scholar), and the sponges were subsequently collected and analyzed under an epifluorescence microscope. Rhodamine-dextran-positive structures were imaged, the color images converted to black and white pictures using Photoshop (Adobe), and processed as described (23Pozzi A. Moberg P.E. Miles L.A. Wagner S. Soloway P. Gardner H.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2202-2207Crossref PubMed Scopus (349) Google Scholar). Vascularity within sponges was expressed as a percentage of area occupied by rhodamine-dextran-positive structures per microscopic field. Three images/sponge with a total of 15 images per treatment were used for analysis. Statistical Analysis—The Student's t test for comparisons between two groups, and analysis of variance using Sigma-Stat software for statistical differences between multiple groups. p ≤ 0.05 was considered statistically significant. Microvascular Endothelial Cells Express CYP2C Isoforms and Biosynthesize EETs—To establish the presence of a functional epoxygenase in mouse lung endothelial cells, we quantified cellular EETs and DHETs by the isotope ratio GC/MS method (26Capdevila J.H. Dishman E. Karara A. Falck J.R. Methods Enzymol. 1991; 206: 441-453Crossref PubMed Scopus (72) Google Scholar). Lung endothelial cells generate epoxygenase metabolites from endogenous precursors. Whereas EETs were found only in the cell pellet (∼0.37 ng/mg total proteins), their hydrated products, the DHETs, were mostly secreted into the culture medium (∼0.65 ng/mg). Incubation of the cells with exogenous arachidonic acid for 4 or 24 h significantly increased the yield of epoxygenase products. Interestingly, the amount of total cell epoxygenase products (EETs + DHETs) was higher at 4 h than at 24 h (36 versus 24 ng/mg, respectively), suggesting that depletion of the precursor arachidonic acid and further metabolism (i.e. β-oxidization) could account for these changes (17Spector A.A. Fang X. Snyder G.D. Weintraub N.L. Prog. Lipid Res. 2004; 43: 55-90Crossref PubMed Scopus (490) Google Scholar). Furthermore, within the first 4 h, ∼80% of the EETs (∼3.6 ng/mg), and >95% of the DHETs (∼30 ng/mg) formed were secreted into the medium. 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