Arachidonic Acid Epoxygenase Metabolites Stimulate Endothelial Cell Growth. and Angiogenesis via MAP kinase and PI3 Kinase/Akt Signaling Pathways

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1 JPET This Fast article Forward. has not been Published copyedited and on formatted. April 19, The 2005 final version as DOI: /jpet may differ from this version. JPET #83477 Arachidonic Acid Epoxygenase Metabolites Stimulate Endothelial Cell Growth and Angiogenesis via MAP kinase and PI3 Kinase/Akt Signaling Pathways Yan Wang, Xin Wei, Xiao Xiao, Rutai Hui, Jeffrey W. Card, Michelle A. Carey, Dao Wen Wang, and Darryl C. Zeldin The Institute of Hypertension and Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan , People s Republic of China (Y.W., X.W., X.X., D.W.W); Departments of Molecular Genetics and Biochemistry & Gene Therapy Center, University of Pittsburgh, Pittsburgh, PA, USA (X.X.); Sino-German Laboratory for Molecular Medicine and Center for Molecular Cardiology, Fuwai Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences (R.T.H.); and Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA(J.W.C., M.A.C, D.C.Z.). 1 Copyright 2005 by the American Society for Pharmacology and Experimental Therapeutics.

2 JPET #83477 Running Title: Epoxygenases and Angiogenesis Corresponding Author: Dao Wen Wang The Institute of Hypertension and Department of Internal Medicine Tongji Hospital, Tongji Medical College Huazhong University of Science and Technology 1095# Jiefang Ave. Wuhan , People s Republic of China Tel & Fax (86-27) dwwang@tjh.tjmu.edu.cn Number of text pages: 21 Number of tables: 0 Number of figures: 7 Number of references: 44 Number of words in the Abstract: 250 Number of words in Introduction: 583 Number of words in Discussion: 791 ABBREVIATIONS: AA, arachidonic acid; Akt, cellular homolog of the v-alt oncogene, an S/T protein kinase, PKB; Annexin-V-FITC, Annexin-V-fluorescein isothiocyanate; BAECs, bovine aortic endothelial cells; CAM, chicken embryo chorioallantoic membrane; CYP, cytochrome P450; CYPOR, NADPH- 2

3 JPET #83477 cytochrome P450 oxidoreductase; DMEM, Dulbecco s modified Eagle s medium; ECL, enhanced chemiluminescence; EDHF, endothelium-derived hyperpolarizing factor; EDRF, endothelium-derived relaxing factor; EET, epoxyeicosatrienoic acids; EGF, epidermal growth factor; enos: endothelial nitric oxide synthase; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HEPES, N-[2-Hydroxyethyl]piperazine-N -[2-ethanesulfonic acid]; H-7, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine; HETE, hydroxyeicosatetraenoic acid; ICAM, intercellular adhesion molecular-1; L- NMMA, N G -monomethyl-l-arginine; LOX, lipoxygenase; LY294002, 2-(4- morpholinyl)-8-phenyl-4h-1-benzopyran-4-one; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide; NADPH, nicotinamide adenine dinucleotide phosphate; NO, nitric oxide; PGHS, prostaglandin endoperoxide H synthase; PI, propidium iodide; 17-ODYA, 17-octadecynoic acid; PI3K, phosphatidylinositol 3-kinase; PMSF, phenylmethylsulfonyl fluoride; p-na, DEVD-p-nitroanilide; PKC, protein kinase C; raav, recombinant adenoassociated virus; PD98059, 2-amino-3-methoxyflavone; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SMC, smooth muscle cells; TNFα, tumor necrosis factor alpha; VCAM-1, vascular cell adhesion molecular-1. 3

4 JPET #83477 ABSTRACT Cytochrome P450 arachidonic acid (AA) epoxygenase metabolites, the epoxyeicosatrienoic acids (EETs), dilate arteries via hyperpolarization of smooth muscle cells and also have non-vasodilatory effects within the vasculature. The present study investigated the angiogenenic effects of endogenous and exogenous EETs and the relevant signaling mechanisms involved. Bovine aortic endothelial cells (BAECs) were incubated with synthetic EETs or infected with recombinant adeno-associated viruses (raav) containing CYP2C11-CYPOR, CYP2J2 or CYP102 F87V mutant to increase endogenous levels of EETs. The following endpoints were measured: BAEC proliferation, migration, capillary formation and in vivo angiogenesis. The potential involvement of various signaling pathways was explored using selective inhibitors. The results showed that transfection with either raav-cyp2c11-cypor, raav-cyp2j2 or raav-cyp102 F87V, or incubation with EETs promoted BAEC proliferation, increased migration of BAECs as assessed by transwell analysis and wound healing assays, and enhanced capillary tubule formation as determined by chicken embryo chorioallantoic membrane assays (CAM) and tube formation tests on matrigel. The effects of EETs on proliferation, migration and capillary tubule formation were attenuated by inhibitors of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3 kinase)/akt pathways, and partially attenuated by an enos inhibitor, but not by a protein kinase C (PKC) inhibitor. In a rat ischemic hind limb model, raav-mediated AA epoxygenase transfection induced angiogenesis. We conclude that AA epoxygenase metabolites 4

5 JPET #83477 can promote angiogenesis, which may provide protection to ischemic tissues. The results also suggest that the angiogenic effects of EETs involve the MAPK and PI3 kinase/akt signaling pathways, and to some extent, the enos pathway. 5

6 JPET #83477 INTRODUCTION Arachidonic acid (AA) is esterified to cell membrane glycerophospholipids and liberated by phospholipase A 2 in response to various stimuli. AA can be activated by three different enzyme pathways; the cyclooxygenase (COX), the lipoxygenase (LOX) and the cytochrome P450 monooxygenase (CYP) pathways(muller, 1991).While the COX and LOX pathways have been extensively studied, much less is known about the CYP pathway. Metabolism of AA via the CYP pathway produces two major groups of metabolites; the epoxyeicosatrienoic acids (EETs) formed by CYP epoxygenases and ω-terminal hydroxyeicosatetraenoic acids (HETEs) formed by CYP ω-oxidases(campbell et al., 1991). CYP epoxygenases produce four different EET regioisomers; 5,6-, 8,9-, 11,12-, and 14,15-EET. Over the last decade, accumulating evidence has suggested that EETs play crucial and diverse roles in cardiovascular homeostasis (Node et al., 1999; Fleming, 2001; Chen et al., 2003). EETs activate vascular smooth muscle cell large conductance Ca 2+ -activated K + channels leading to hyperpolarization of the resting membrane potential and resulting in vasorelaxation and lowering of blood pressure. Indeed, 11,12-EET has been proposed to be identical to endothelium derived hyperpolarizing factor (EDHF) (Bauersachs et al., 1997; Bolz et al., 2000; Matoba et al., 2000; Hamilton et al., 2001; Lacza et al., 2002; Matoba et al., 2002; Matoba and Shimokawa, 2003; Matoba et al., 2003; Miura et al., 2003; Morikawa et al., 2003; Tanaka et al., 2003; Yada et al., 2003). Exogenous application of EETs inhibits vascular smooth muscle cell migration, platelet aggregation, nuclear factor-κb (NF- 6

7 JPET #83477 κb) activation and vascular cell adhesion molecule-1 (VCAM-1) expression (Node et al., 1999; Fleming et al., 2001; Sun et al., 2002; Krotz et al., 2004), suggesting an overall beneficial role for EETs within the vasculature and a protective role in the development of atherosclerosis. Endothelial cells play a central role in the cardiovascular system through regulation of blood circulation and fluidity, vascular tone, coagulation, inflammatory responses and angiogenesis. The role of EETs in mediating endothelial cell functions has been a subject of particular interest in the cardiovascular field within the last decade. Addition of EETs or overexpression of the AA epoxygenase CYP2J2 in endothelial cells decreased cytokine-induced endothelial cell adhesion molecule expression, and prevented leukocyte adhesion to the vascular wall via inhibition of NF-κB and IκB kinase. These studies also demonstrated that these effects of EETs were independent of their membrane-hyperpolarizing effects, suggesting an important nonvasodilatory role for EETs within the vasculature (Node et al., 1999). We previously demonstrated that EETs or overexpression of AA epoxygenases significantly upregulated endothelial nitric oxide synthase (enos) expression and enhanced enos phosphorylation through activation of extracellular signal-regulated kinase (ERK) and protein kinase C (PKC) pathways (Wang et al., 2003b). Furthermore, we found that transfection of three different epoxygenase cdnas protected endothelial cells from apoptosis induced by tumor necrosis factor alpha (TNFα), an effect which was related to activation of the ERK and PI3 kinase/akt signaling pathways, but independent of NO production in endothelial cells (Wang et 7

8 JPET #83477 al., 2002). Angiogenesis is a neovascularization process which is essential for the successful repair of wounds and tissues damaged by ischemia, and is also important for tumor growth and metastasis. Endothelial cells play an important role in angiogenesis, but the role that epoxygenases and EETs play in this process remains enigmatic. Hence, the purpose of the present study was to investigate whether addition of synthetic EETs and/or overexpression of AA epoxygenase cdnas affects endothelial proliferation, migration of vascular endothelial cells and neovascularization, and to begin to investigate the signaling mechanisms involved. 8

9 MATERIALS AND METHODS Experimental Reagents: All cell culture reagents were obtained from GibcoBRL (Life Technologies, Inc., Grand Island, NY) including Dulbecco s modified Eagle s medium (DMEM), trypsin and fetal bovine serum (FBS). PD98059, Apigenin and H-7 were supplied by Calbiochem Novabiochem Corporation (Darmstadt, Germany). Rabbit anti-pi3k antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, California), Rabbit anti-erk1/2 (also called p42/44 MAPK) and anti-phospho-erk1/2 antibodies were purchased from New England Biolabs (Beverly, MA). Anti-CYP2C11 and anti- CYP102 antibodies were a generous gift from Dr. Jorge Capdevila (Vanderbilt University, Nashville, TN). Anti-CYP2J2 antibodies were prepared as previously described (Wu et al., 1996). Enhanced chemiluminescent substrate (SuperSignal West Pico Chemiluminescent Substrate) was purchased from Pierce (Rockford, IL). Hybrisol solution was purchased from Intergen (Purchase, NY). PVDF and nylon membranes were purchased from Schleicher & Schuell (Dassel, Germany). Rabbit polyclonal antibodies specific for epidermal growth factor receptor (EGFR) and phosphorylated EGFR were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Vascular endothelial growth factor was from Collaborative Biomedical Products (Becton Dickinson Labware, Bedford, MA). 8,9-EET, 11,12-EET, 14,15-EET, HEPES, PD98059, LY294002, Tween-20, PMSF, aprotinin, 17-ODYA, N G -methyl-l-arginine,3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and collagen IV were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO). Matrigel was purchased from B&D Biosciences (Heidelberg, Germany). All other chemicals and reagents were purchased from Sigma-Aldrich Chemical Company unless otherwise specified.

10 JPET #83477 Construction and Preparation of Recombinant Adeno-associated Virus: The recombinant adeno-associated virus (raav) vector pxxuf 1, packaging plasmid pxx 2, adenovirus helper plasmid pxx 6, and a raav plasmid containing the GFP cdna (GFPpUF 1 ) were from Dr. Xiao Xiao (University of Pittsburgh, Pittsburgh, PA). The CYP102 F87V mutant cdna and a cdna encoding rat CYP2C11 fused to rat NADPHcytochrome P450 oxide reductase (CYPOR) (Helvig and Capdevila, 2000) were kindly provided by Dr. Jorge Capdevila (Vanderbilt University, Nashville, TN). The CYP2J2 cdna was from Dr. Darryl Zeldin (NIEHS, Research Triangle Park, NC). These epoxygenase cdnas or GFP were subcloned into the raav vector pxxuf 1 downstream of the CMV promoter and raav-cyp102 F87V, raav-cyp2c11-cypor, raav-cyp2j2 and raav-gfp were packed in human 293 cells (American Type Culture Collection, Rockville, MD) and purified by a single-step gravity-flow column purification method as described previously (Wang et al., 2003b)(Xiao et al., 1996; Xiao et al., 1998). Purified raav viruses were titered using a dot blot method (Wang et al., 2004). Isolation and Culture of Endothelial Cells: Bovine aortic endothelial cells (BAECs) were isolated and cultured as described previously (Wang et al., 2003b). Briefly, fresh bovine thoracic aortas were obtained from a local slaughterhouse, and BAECs were harvested using trypsin (0.25%) and grown to confluence in a growth medium containing DMEM supplemented with 5 mm L-glutamine, 10% FBS and an antibiotic mixture of penicillin (100 units/ml) and streptomycin (100 µg/ml). Purity of the BAEC preparation was determined by cell morphology using phase-contrast microscopy and by immunofluorescent staining for CD31. All passages were performed using 0.05% trypsin and 0.02% EDTA. Only the cells passaged less than five times were 10

11 JPET #83477 used for experiments. Protein Extraction and Western Blotting: BAEC protein was extracted as previously described (Wang et al., 2003b). Briefly, the media in 6-well plate was discarded and cells were gently washed three times with cooled PBS. Lysis buffer (500 mm Tris-Cl, ph 8.0, 150 mm NaCl, 0.02% sodium azide, 0.1% SDS, 100 µg/ml PMSF, 1 µg/ml aprotinin, 1% NP-40, 0.5% sodium deoxycholate) was added to the cells (0.25 ml/well). After incubation on ice for 30 minutes, the lysate was centrifuged at 12,000 x g at 4 0 C for 10 minutes. The protein concentration of the supernatant was determined using the Bradford method. Lysates (25 µg protein/lane) were resolved by SDS-PAGE (12%), transferred to nitrocellulose membranes, and blocked with 5% nonfat dry milk in TBS-T (10 mm Tris-Cl, ph 7.5, 100 mm NaCl, 0.1% Tween 20). The membranes were then incubated with the primary rabbit antibody (1:150 dilution) overnight at 4 C, followed by peroxidase-conjugated secondary antibody for 2-3 hours. The ECL system was used to visualize the separated proteins. Blots were stripped and reprobed with β- actin as a loading control. Effects of EETs and AA Epoxygenase Transfection on BAEC Proliferation: The effects of EETs and AA epoxygenase transfection on BAEC proliferation were evaluated using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) colorimetric assay and by direct cell counting. BAECs were seeded in triplicate 24-well plates (1 x 10 4 cells/well), placed in reduced serum (0.5%) media for 12 h after cell attachment, and then stimulated with different concentrations of EETs (10 nm, 50 nm and 250 nm) at 0 hours and again at 12 hours. The second dose at 12 hours was added in order to minimize reduction in levels of EETs due to autooxidation. After 24 11

12 JPET #83477 hours, the cells were washed with PBS, trypsinized and counted. For the MTT assays, BAECs were seeded in triplicate 96-well plates (1 x 10 4 cells/well) and treated in an identical manner. Twenty four hours after treatment with EETs, viable cell numbers were estimated by the MTT assay as described (Law et al., 1996). Briefly, medium was removed and replaced with medium containing 5 mg/ml MTT and incubated for 4 hours. The medium was then aspirated, and the product was solubilized with dimethyl sulfoxide (DMSO). Absorbance was measured at 570 nm for each well using a microplate reader (Bio-Tek Instrument, USA) according to the manufacturer's protocol. In order to confirm a linear relationship between optical density and cell number, we performed correlational analysis. To study the effects of AA epoxygenase overexpression on BAEC proliferation, BAECs were infected with raav-cyp102 F87V, raav-cyp2c11-cypor, raav-cyp2j2 or raav-gfp (~50 virions/cell) in 6-well plates in triplicate, and 5 days later the cells were trypsinized and seeded in 96-well plates in triplicate (1 x 10 4 cells/well). After attachment, the cells were exposed to DMEM with 0.5% FBS for 48 hours and then processed for cell counting and MTT assays as described above. Flow Cytometry Analysis: In order to further examine the proliferationstimulating potential of EETs and AA epoxygenases in endothelial cells, we analyzed cell cycle distribution after treatment with EETs and after transfection with different AA epoxygenase cdnas. Cells (1.5 x 10 6 cells) were cultured as described above and 12 hours later, they were fixed with 70% ethanol and incubated with 4 mm phosphatecitrate buffer (4 mm citric acid, 192 mm Na 2 HPO 4 ) for 30 min at room temperature. After centrifugation, cell pellets were resuspended in PBS containing PI/RNase (10 µg/ml 12

13 JPET #83477 each) and incubated for 20 min at room temperature. Quantification and ratio of sub-g1 DNA content was determined using the CELLQuest program in a FACStar-Plus flow cytometer (Becton-Dickinson). Cell Migration: Migration was assessed by a cell-wounding assay. Briefly, BAECs were grown to confluence in 60 mm-diameter dishes and synchronized in 0.5% FBS for 6 hours. Round cell-free areas were made by abrasion with a sterile pipet tip and cells were then stimulated with different EETs (100 nm) for 36 hours. Cells that migrated into the cell-free area were visualized using a Nikon TE 2000 microscope, and cell-free surface area was calculated using a Scion Image Analysis System (Scion Corp, NIH Image). Each experiment was performed in triplicate for each EET and experiments were repeated four times. Data are presented as a ratio of cell-covered surface area to initial cell-free surface area. In addition, cell migration was also assayed using a modified Boyden chamber technique (transwell analysis). Porous filters (8 µm pores) were coated with type,9 collagen on the both sides via passive adsorption by incubating with 10 µg/ml collagen in coating buffer for 24 hours. Serum-free medium containing individual EETs (100 µm) was added into the lower chamber as a chemoattractant and cells (1 x 10 4 ) were plated in the upper chamber and allowed to migrate for 24 hours. Nonmigrating cells were removed from the upper chamber with a cotton swab, filters were stained with Diff-Quik Stain, and migrating cells adherent to the underside of the filter were enumerated using an ocular micrometer and by counting a minimum of 10 high powered fields (HPF). Data are presented as relative migration 13

14 JPET #83477 (number of cells/hpf) and represent mean ± standard error of quadruplicate experiments. Tube Formation: In vitro formation of capillary-like tube structures was examined on Matrigel. Matrigel (0.5 ml) was polymerized on 24-well plates and cells were then plated in full-growth media for 1 hour. Once the cells were seeded, the media was replaced with media containing 0.5% serum with or without individual EETs. Tube formation was visualized using an inverted microscope (Nikon TE 2000) equipped with digital imaging. For each treatment, 10 HPF images were captured and the area of endothelial tubes and networks formed was quantified using the Scion Image Analysis System with background subtraction. To examine effects of AA epoxygenase transfection on tube formation, BAECs were first infected with different raav viruses and 4 days later, they were plated in 24-well plates with matrigel followed by tube formation analysis as described above. Chicken Embryo Chorioallantoic Membrane (CAM) Assay: Fertilized chicken eggs were incubated at 37 C in an 80% humidified atmosphere. On day 6 of development, a window was made in the eggshell on the large side of the egg and a small piece (2 2 mm 2 ) of nitrocellulose membrane containing raav ( virions/membrane) was put on the CAM and then the window was sealed with sterile plastic tape. Incubation of the eggs continued for 9 days, after which the tape was removed and the CAM around nitrocellulose membrane was fixed in 4% paraformaldehyde for 30 min at room temperature. The area containing the nitrocellulose membrane was then removed for further analysis. Photos of each CAM 14

15 JPET #83477 were taken under a stereomicroscope (Nikon SMZ800) using a digital camera (Nikon Coolpix 950). Two observers quantified the small vessels (first- and second-order). The result was taken as the mean number from the two observers. A minimum of 6 eggs was used for each treatment, and the experiments were repeated at least twice. Evaluation of Signaling Pathways: In order to examine the signaling mechanisms through which EETs and AA epoxygenases enhanced endothelial cell proliferation, migration and angiogenesis, inhibitors of ERK (also called MAPK) (apigenin), MEK (PD98059), PI3 kinase (LY294002), PKC (H-7), enos (L-NMMA) and AA epoxygenases (17-ODYA) were added to cultured BAECs or CAM, and their effects on cell proliferation, cell cycle, cell migration and tube formation were observed. In vivo Study in Rat Ischemic Hind limb Model: This study was approved by the Institutional Animal Research Committee of Tongji Medical Center. Animals were cared for according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. For these experiments, thirty 12-week old male normotensive Wistar rats were used. The right femoral arteries were occluded using a 3-0 silk suture under pentobarbital anesthesia (50 mg/kg, intra-peritoneal injection). The ligature was placed on the femoral artery 0.5 cm proximal to the bifurcation of the saphenous and popliteal arteries. A total volume of 200 µl of saline (containing of ~3h10 11 virions of raav-cyp2j2, raav-cyp102 F87V, raav-cyp2c11-cypor or raav-gfp) was injected at 4-5 sites in the adductor and surrounding muscles 6 days after surgery. Six weeks after gene delivery, microangiographic analysis was completed by assessment of capillary density as previously described by Silvestre and co-workers (Silvestre et al., 2000). Briefly animals were sacrificed under anesthesia and their hind limb skeletal 15

16 JPET #83477 muscles were excised and fixed in formalin. After paraffin embedding, 3 µm thick sections were cut from each sample with the muscle fibers were oriented in a transverse direction and immunostained with an antibody specific for platelet endothelial cell adhesion molecule (PECAM). The number of capillaries were counted at 400 h magnification (mean number of capillaries/mm 2 ) in 20 randomly chosen fields in a blinded fashion. To ensure that analysis of capillary density was not overestimated due to muscle atrophy, capillary density was also evaluated as a function of the number of muscle fibers in the histological section (capillary-to-fiber ratio). Statistical Analysis: Continuous data were expressed as mean ± S.E. Comparisons between groups were performed by a Student's paired two-tailed t test. Two-way analysis of variance was used to examine differences in response to treatments and between groups, with post hoc analyses performed by Student- Newman-Keuls method. P-values of <0.05 were considered statistically significant. 16

17 JPET #83477 RESULTS Effects of EETs and AA Epoxygenase Transfection on BAEC Proliferation To determine the effects of AA epoxygenase products on BAEC proliferation, a necessary process for angiogenesis, we conducted experiments involving either direct addition of synthetic EETs or production of endogenous EETs via overexpression of AA epoxygenases, and then determined proliferative effects using cell counting and MTT viability assays. Since EETs and overexpression of AA epoxygenases upregulate enos at the mrna, protein and enzyme activity levels in BAECs (Wang et al., 2003b), we also stimulated BAECs with EETs in the presence or absence of L-NMMA to study whether NO was involved in the effect of EETs on BAEC proliferation. Addition of individual EETs significantly promoted proliferation of endothelial cells as assessed by cell counts, and this effect was significantly inhibited by L-NMMA (Fig. 1A). Furthermore, the effects of EETs occurred at concentrations as low as 10 nm and were concentrationdependent up to 250 nm (Fig. 1B). MTT assays gave similar results (Fig. 1C and 1D). Addition of the AA epoxygenase inhibitor 17-ODYA (10 µm) into the cultures significantly inhibited BAEC proliferation, measured by both cell counts (Fig. 1A) and MTT assay (Fig. 1C), lending further support to the concept that AA epoxygenase products promote proliferation of endothelial cells. Transfection of BAECs with raav-cyp102 F87V, raav-cyp2c11-cypor or raav-cyp2j2 resulted in expression of the corresponding epoxygenase proteins (Fig. 1E) and significantly enhanced BAEC proliferation compared to transfection with the control raav-gfp (Fig. 1F). The proliferative effects of AA epoxygenase transfection were significantly inhibited by addition of 17-ODYA (Fig. 1F). In order to verify the 17

18 JPET #83477 reliability of the MTT assay, we examined the relationship between cell count and OD values; results demonstrated that these parameters were linearly related (Fig. 1G). To investigate the signaling mechanisms through which EETs and AA epoxygenases promote proliferation of endothelial cells, we applied various signaling pathway inhibitors to BAEC cultures in the presence of EETs or following transfection with AA epoxygenases. Inhibitors of ERK or MAPK (apigenin), MEK (PD89059) and PI3 kinase (LY294002) inhibited proliferation induced by EETs and by AA epoxygenase transfection, whereas the PKC inhibitor H-7 did not (Fig. 2A and 2B). This suggests the involvement of the MAPK and PI3 kinase/akt signaling pathways in EET-mediated proliferation of BAECs. Immunoblot analysis revealed that EETs significantly increased phosphorylation of ERK (Fig. 2C) and the level of PI3K (Fig. 2D), and also increased the phosphorylation level of EGFR (Fig. 2E). Treatment with the AA epoxygenase inhibitor 17-ODYA resulted in reduced phosphorylation of ERK, decreased levels of PI3K, and reduced phosphorylation of EGFR (Fig. 2C, 2D and 2E). To determine the role of the enos pathway in mediating the effects of EETs, we determined levels of phosphorylated ERK and PI3 kinase in BAECs in the presence and absence of L-NMMA. Inhibition of enos attenuated the EET-induced increase in PI3 kinase (Fig. 2F) suggesting a role for NO in EET-mediated activation of this pathway. In contrast, inhibition of enos had no effect on the EET-induced increase in phosphorylated ERK (Fig. 2G). Flow Cytometry Analysis Cell cycle analysis by flow cytometry was performed one week after infection of BAECs with raav-cyp2j2, raav-cyp2c11-cypor, raav-cyp102 F87V or raav-gfp, 18

19 JPET #83477 or after treatment with synthetic EETs for 12 hours. Infection with raav-cyp2j2, raav- CYP2C11-CYPOR or raav-cyp102 F87V resulted in a significant increase in the proportion of cells in S/G2/M phase (45.67 ± 0.22%, ± 0.17%, and ± 1.34%, respectively) compared with noninfected and raav-gfp infected cells (26.83 ± 1.65% and ± 0.25%, respectively; p<0.01) (Fig. 3A and 3B). In contrast, infection with raav-cyp2j2, raav-cyp2c11-cypor or raav-cyp102 F87V dramatically reduced the proportion of cells in G0/G1 phase (54.45 ± 0.22%, ± 0.98%, and ± 1.35%, respectively) compared with noninfected and raav-gfp infected groups (73.32 ± 1.67% and ± 0.26%, respectively, p<0.01 ). Treatment with individual EETs produced similar results (Fig. 3C and 3D). Combined, these data provide further evidence that CYP epoxygenases and their eicosanoid products promote proliferation of endothelial cells. Effect of EETs and AA Epoxygenase Transfection on Migration of BAECs Migration of endothelial cells is an important process in angiogenesis and vessel sprouting. We determined the effects of EETs and raav-mediated AA epoxygenase gene transfection using wound healing and Boyden chamber assays. In wound healing studies, EETs markedly enhanced BAEC migration, an effect that was significantly attenuated by inhibition of enos with L-NMMA (Fig. 4A and 4B). The effects of EETs were dose-dependent and occurred at concentrations as low as 10 nm (Fig. 4C). Similarly, infection of raav-cyp102 F87V, raav-cyp2c11-cypor or raav-cyp2j2 stimulated BAEC migration, an effect that was attenuated by the epoxygenase inhibitor 17-ODYA (Fig. 4D). In transwell Boyden chamber assays, EETs increased cell migration (Fig. 4E and 4F), and this effect was concentration-dependent (Fig. 4G). 19

20 JPET #83477 Importantly, addition of L-NMMA also attenuated the EET-induced migration of BAECs in Boyden chamber assays (Fig. 4E and 4F). Similar to their effects on EET-induced BAEC proliferation, inhibitors of ERK or MAPK (apigenin), MEK (PD89059) and PI3 kinase (LY294002) markedly attenuated the EET-stimulated migration of BAECs and delayed wound repair, whereas the PKC inhibitor H-7 did not (Fig. 4H and 4I). These data suggest that the ERK and PI3 kinase/akt pathways play important roles in EET-stimulated migration of BAECs. Effects of EETs and AA Epoxygenase Transfection on Angiogenesis The possibility that EETs could promote angiogenesis was assessed using a variety of experimental approaches. First, we employed the CAM assay, which allows for investigation of the ongoing angiogenic process in vivo. Infection with raav-cyp102 F87V, raav-cyp2c11-cypor or raav-cyp2j2 dramatically increased capillary formation and novel capillary branch number relative to that observed in noninfected or raav-gfp infected groups (p<0.01) (Fig. 5 A and 5B). We next examined whether EETs or AA epoxygenase transfection promoted tubule formation. Matrigel tests demonstrated that treatment with EETs significantly increased tubule formation (Fig. 6A and 6B), and that this effect was attenuated by inhibitors of ERK or MAPK (apigenin), MEK (PD89059), PI3 kinase (LY294002) and enos (L-NMAA), but not PKC (H-7) (Fig. 6C). raav-mediated AA epoxygenase gene transfection also increased tubule formation. Importantly, 17-ODYA inhibited tubule formation in this model (Fig. 6D). These data suggest that EETs and AA epoxygenases enhance capillary tubule formation through mechanisms involving the MAPK, PI3 kinase/akt and enos signaling pathways. 20

21 JPET #83477 Finally, an ischemic rat hind limb skeletal muscle model was employed to determine the effect of AA epoxygenase transfection on angiogenesis in vivo. Skeletal muscle sections were immunostained with an antibody against CD-31 to quantify capillary formation. Muscle capillary density was significantly increased following AA epoxygenase transfection; capillary numbers in raav-cyp102 F87V, raav-cyp2c11- CYPOR and raav-cyp2j2 infected groups were 1260 ± 62/mm 2, 1096 ± 53/mm 2 and 905 ± 43/mm 2, respectively. In comparison, capillary number in the raav-gfp transfected group was 706 ± 24/mm 2 (p<0.01). 21

22 JPET #83477 DISCUSSION Mounting evidence supports the concept that EETs possess a variety of activities in the vasculature, in addition to their recognized ability to relax smooth muscle cells via activation of Ca 2+ -sensitive K + channels. These non-vasodilatory activities affect the function and viability of vascular endothelial cells, and include upregulation of enos at the levels of mrna, protein, posttranslational modification, protection from apoptosis induced by TNFα, and reduction of expression of cytokine-induced endothelial cell adhesion molecules, thereby preventing leukocyte adhesion to the vascular wall (Fisslthaler et al., 1999; Node et al., 1999; Wang et al., 2003a; Wang et al., 2003b). Recently, astrocyte-derived EETs were shown to induce cerebral capillary endothelial cell mitogenesis and tube formation (Munzenmaier and Harder, 2000), and adenovirusmediated CYP2C9 gene transfection and exogenous 14,15-EET were shown to exert similar effects in human lung endothelial cell lines (Medhora et al., 2003). Additionally, CYP2C9 overexpression in endothelial cells was shown to induce endothelial tube formation via stimulating cyclooxygenase-2 expression and prostacyclin production (Michaelis UR, 2005). Collectively, these data suggest that CYP-derived EETs may represent a class of important endogenous angiogenic factors. In the current study, we utilized both in vitro and in vivo approaches to demonstrate that both exogenous and endogenously formed EETs enhance endothelial cell proliferation, migration and capillary tube formation, and stimulate angiogenesis. Vascular endothelial growth factor (VEGF) is an angiogenic factor that has been successfully used to treat severe myocardial ischemia and occlusive peripheral artery disease (Kliche and Waltenberger, 2001; Bliznakov, 2002). Additionally, fibroblast 22

23 JPET #83477 growth factor and NO exert angiogenic effects by increasing proliferation and migration of endothelial cells and capillary tube formation (Cooke, 2003; Penny and Hammond, 2004). NO and EDHF represent the two major endothelial autacoids involved in the local control of vascular tone (Bauersachs et al., 1997). Importantly, the angiogenic effects of NO have been shown to be mediated by activation of the PI3-kinase/Akt pathway (Kawasaki et al., 2003). The present study demonstrated that addition of synthetic EETs or overexpression of AA epoxygenases stimulated proliferation and migration of BAECs and angiogenesis, similar to the effects of VEGF. We previously reported that EETs or overexpression of AA epoxygenases upregulated enos at the levels of mrna, protein and enzyme activity in BAECs via activation of MAPK (Wang et al., 2003b). To determine whether the effects of EETs and AA epoxygenases observed in the present study were related to upregulation of enos, we examined the influence of the enos inhibitor L-NMMA. Addition of L-NMMA to cultures of BAECs attenuated the EET- and AA epoxygenase-induced migration and proliferation of these cells, suggesting that enos upregulation may mediate, at least in part, the angiogenic effects of EETs. We also demonstrated that inhibitors of the PI3 kinase/akt pathway significantly attenuated the EET-mediated angiogenic effects, consistent with the concept that the PI3 kinase/akt pathway may mediate the angiogenic effects of NO (Kawasaki et al., 2003). The present study also revealed that EETs or epoxygenase gene transfection activate the MAPK pathway in BAECs, suggesting that EET-stimulated angiogenic processes may be only partially dependent on NO (Wang et al., 2003b). Our previous studies demonstrated that EETs and AA epoxygenase overexpression enhance 23

24 JPET #83477 phosphorylation of EGFR and activate MAPK in LLC-PK1 pig kidney epithelial cells (Burns et al., 1995; Chen et al., 1999), indicating that EETs function as second messengers of EGFR. Here, we found that EETs induced proliferation and migration of BAECs and promote angiogenesis, and that these effects were reduced by inhibitors of MAPK and MEK. Furthermore, both endogenous and exogenous EETs promoted phosphorylation of EGFR. Together, these data indicate that the EET-mediated effects on BAEC proliferation, migration and angiogenesis likely occur via NO-dependent mechanisms as well as via activation of PI3 kinase/akt, MAPK, and possibly EGFR pathways as well. Interestingly, a recent study indicated that EETs inhibit vascular smooth muscle cell migration but not proliferation, and that this effect is mediated by an EET-induced increase in intracellular camp and activation of the PKA signaling pathway (Sun et al., 2002). The differential response to EETs in vascular smooth muscle cells and endothelial cells may be due to tissue specificity of EET activity. Indeed, camp is elevated in the former cell types whereas cgmp is elevated in the latter cell types due to G-protein stimulation and activation of MAPK and Akt(Kawasaki et al., 2003). Taken together, our data demonstrate that EETs and AA epoxygenase overexpression promote an angiogenic phenotype, including endothelial cell proliferation, migration and capillary tubule formation. All of these processes are requisite steps in new vessel formation. The mechanisms underlying these effects may include activation of the MAPK pathway and of the PI3 kinase/akt pathway, with NO formation contributing as well. Our observations also suggest the possibility that AA epoxygenase products may promote development of collateral circulation in ischemic tissues. 24

25 JPET #83477 ACKNOWLEDGMENTS We thank Dr. Jorge Capdevila (Vanderbilt University) for providing CYP102 F87V and CYP2C11-CYPOR cdnas and related antibodies. 25

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28 JPET # : Lacza Z, Puskar M, Kis B, Perciaccante JV, Miller AW and Busija DW (2002) Hydrogen peroxide acts as an EDHF in the piglet pial vasculature in response to bradykinin. Am J Physiol Heart Circ Physiol 283:H Law RE, Meehan WP, Xi XP, Graf K, Wuthrich DA, Coats W, Faxon D and Hsueh WA (1996) Troglitazone inhibits vascular smooth muscle cell growth and intimal hyperplasia. J Clin Invest 98: Matoba T and Shimokawa H (2003) Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in animals and humans. J Pharmacol Sci 92:1-6. Matoba T, Shimokawa H, Kubota H, Morikawa K, Fujiki T, Kunihiro I, Mukai Y, Hirakawa Y and Takeshita A (2002) Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in human mesenteric arteries. Biochem Biophys Res Commun 290: Matoba T, Shimokawa H, Morikawa K, Kubota H, Kunihiro I, Urakami-Harasawa L, Mukai Y, Hirakawa Y, Akaike T and Takeshita A (2003) Electron spin resonance detection of hydrogen peroxide as an endothelium-derived hyperpolarizing factor in porcine coronary microvessels. Arterioscler Thromb Vasc Biol 23: Matoba T, Shimokawa H, Nakashima M, Hirakawa Y, Mukai Y, Hirano K, Kanaide H and Takeshita A (2000) Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in mice. J Clin Invest 106: Medhora M, Daniels J, Mundey K, Fisslthaler B, Busse R, Jacobs ER and Harder DR (2003) Epoxygenase-driven angiogenesis in human lung microvascular endothelial cells. Am J Physiol Heart Circ Physiol 284:H

29 JPET #83477 Michaelis UR FJ, Schmidt R, Fleming I (2005) Cytochrome P4502C9-derived epoxyeicosatrienoic acid induce the expression of cyclooxygenase-2 in endothelial cells. Arterioscler Thromb Vasc Biol 25: Miura H, Bosnjak JJ, Ning G, Saito T, Miura M and Gutterman DD (2003) Role for hydrogen peroxide in flow-induced dilation of human coronary arterioles. Circ Res 92:e Morikawa K, Shimokawa H, Matoba T, Kubota H, Akaike T, Talukder MA, Hatanaka M, Fujiki T, Maeda H, Takahashi S and Takeshita A (2003) Pivotal role of Cu,Znsuperoxide dismutase in endothelium-dependent hyperpolarization. J Clin Invest 112: Muller B (1991) Pharmacology of thromboxane A2, prostacyclin and other eicosanoids in the cardiovascular system. Therapie 46: Munzenmaier DH and Harder DR (2000) Cerebral microvascular endothelial cell tube formation: role of astrocytic epoxyeicosatrienoic acid release. Am J Physiol Heart Circ Physiol 278:H Node K, Huo Y, Ruan X, Yang B, Spiecker M, Ley K, Zeldin DC and Liao JK (1999) Antiinflammatory properties of cytochrome P450 epoxygenase-derived eicosanoids. Science 285: Penny WF and Hammond HK (2004) Clinical use of intracoronary gene transfer of fibroblast growth factor for coronary artery disease. Curr Gene Ther 4: Silvestre J-S, Mallat Z, Duriez M, Tamarat R, Bureau MF, Scherman D, Duverger N, Branellec D, Tedgui A and Levy BI (2000) Antiangiogenic Effect of Interleukin-10 in Ischemia-Induced Angiogenesis in Mice Hindlimb. Circ Res 87:

30 JPET #83477 Sun J, Sui X, Bradbury JA, Zeldin DC, Conte MS and Liao JK (2002) Inhibition of vascular smooth muscle cell migration by cytochrome p450 epoxygenasederived eicosanoids. Circ Res 90: Tanaka M, Kanatsuka H, Ong BH, Tanikawa T, Uruno A, Komaru T, Koshida R and Shirato K (2003) Cytochrome P-450 metabolites but not NO, PGI2, and H2O2 contribute to ACh-induced hyperpolarization of pressurized canine coronary microvessels. Am J Physiol Heart Circ Physiol 285:H Wang A, Nomura M, Patan S and Ware JA (2002) Inhibition of protein kinase Calpha prevents endothelial cell migration and vascular tube formation in vitro and myocardial neovascularization in vivo. Circ Res 90: Wang D, Borrego-Conde LJ, Falck JR, Sharma KK, Wilcox CS and Umans JG (2003a) Contributions of nitric oxide, EDHF, and EETs to endothelium-dependent relaxation in renal afferent arterioles. Kidney Int 63: Wang H, Lin L, Jiang J, Wang Y, Lu ZY, Bradbury JA, Lih FB, Wang DW and Zeldin DC (2003b) Up-regulation of endothelial nitric-oxide synthase by endotheliumderived hyperpolarizing factor involves mitogen-activated protein kinase and protein kinase C signaling pathways. J Pharmacol Exp Ther 307: Wang T, Li H, Zhao C, Chen C, Li J, Chao J, Chao L, Xiao X and Wang DW (2004) Recombinant adeno-associated virus-mediated kallikrein gene therapy reduces hypertension and attenuates its cardiovascular injuries. Gene Ther. Xiao X, Li J and Samulski RJ (1996) Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. J Virol 70:

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32 JPET #83477 Footnotes This work was supported by grants from National Nature Science Foundation Committee of China (No , and ), National 973 project (No. G ) and the NIEHS Division of Intramural Research. Address correspondence to: Dao Wen Wang The Institute of Hypertension and Department of Internal Medicine Tongji Hospital, Tongji Medical College Huazhong University of Science and Technology 1095# Jiefang Ave. Wuhan , People s Republic of China Tel & Fax (86-27) dwwang@tjh.tjmu.edu.cn 32

33 JPET #83477 Legends For Figures Figure 1. EETs and AA epoxygenase transfection promote proliferation of BAECs. (A) and (C), BAECs were incubated with individual EETs (50 nm), the AA epoxygenase inhibitor 17-ODYA (10 µm) in the presence or absence of the enos inhibitor L-NMMA (100 µm) for 24 hours. VEGF (1 µg/ml) was used as a positive control. (A) and (C) represent results from direct cell counts and MTT assays, respectively. (B) and (D), BAECs were stimulated with different concentrations of individual EETs (10 nm, 50 nm and 250 nm) for 24 hours to show dose-dependent effects on cell proliferation. (B) and (D) represent the results of direct cell counts and MTT assays, respectively. (E) Western blots showing AA epoxygenase levels in BAECs infected with raav-cyp102 F87V, raav-cyp2c11-cypor, raav-cyp2j2 and raav-gfp (50 virions/cell) for 1 week. (F) MTT assay results 7 days after transfection with AA epoxygenases or GFP in the presence or absence of the epoxygenase inhibitor 17-ODYA (10 µm). (G) standard curve showing linear relationship between cell number and OD values in the MTT assay. Each experiment was performed at least in triplicate. *, p<0.05 vs. control group. #, p<0.05 vs. no L-NMMA or no 17-ODYA group. Figure 2. EETs and AA epoxygenase transfection promote endothelial cell proliferation via PI3 kinase, MAPK and EGFR signaling pathways, but not via PKC. (A) and (B), MTT assays showing effects of treatment with inhibitors of ERK or MAPK (Apigenin, 25 µm), PKC (H-7, 12 µm), PI3 kinase (LY294002, 15 µm) and MEK (PD98059, 20 µm) on EET- (50 nm) or AA epoxygenase-induced BAEC proliferation. (C), (D) and (E) show effects of EETs (50 nm) or AA epoxygenase inhibitor (17-ODYA, 10 µm) on levels of phospho-erk, PI3K, and phospho-egfr, respectively, in BAECs. The 33

34 JPET #83477 upper panels show representative blots and the lower panels show the densitometric analysis. Blots are representative of three separate experiments. (F), Effects of enos inhibitor L-NMMA (100 µm) on EET-induced increase in PI3 kinase. (G) Effects of enos inhibitor L-NMMA (100 µm) on EET-induced increase in phospho-erk. Data are representative of 3 separate experiments. *, p<0.05 vs. control group. #, p<0.05 vs. no signaling pathway inhibitor group. Figure 3. Flow cytometry analysis showing that EETs and AA epoxygenase transfection enhance cell cycle progression in BAECs. (A) and (C) show representative histograms of flow cytometry analysis for raav-mediated AA epoxygenase gene transfected BAECs and EET treated (50 nm) BAECs, respectively. The x- and y-axes represent the intensity of PI fluorescence and cell number, respectively. (B) and (D) show the proportion of BAECs in S and G 2 /M phases from (A) and (B), respectively. Each experiment was performed in triplicate. *, p<0.01 vs. control group. Figure 4. EETs and AA epoxygenase transfection enhance migration of BAECs. (A), BAECs were cultured to confluency, treated with individual EETs (50 nm), VEGF (1 µg/ml) or vehicle in the presence or absence of L-NMMA (100 µm), and the monolayer was then wounded by means of a pipet tip. Cellular wounding healing was observed under a microscope. (B), Summary of cellular wound healing results showing the ratio of cell-covered surface area to initial cell-free area ( S/S). (C), BAECs were stimulated with different concentrations of individual EETs (10 nm, 50 nm and 250 nm) to show dose-dependent effects on wound healing. (D), Transfection with AA epoxygenases enhanced cellular wound healing of BAECs, an effect that was inhibited by incubation with 17-ODYA (10 µm). (E) and (F) show the effects of EETs (100 nm), 17-ODYA (10 34

35 JPET #83477 µm) and VEGF (1 µg/ml) in the presence or absence of L-NMMA (100 µm) on BAEC migration through the membrane filters in a transwell analysis. (E) is a representative photomicrograph of filters and (F) shows migrated cell number per high power field (HPF) from 5 individual experiments. (G) shows dose-dependent increase in the number of migrated BAECs through the membrane filter in response to treatment with different concentrations of EETs (10 nm, 50 nm and 250 nm) for 24 hours. (H) shows the effects of inhibitors of MAPK or ERK (Apigenin, 25 µm), PKC (H-7, 12 µm), PI3- kinase (LY294002, 15 µm) and MEK (PD98059, 20 µm) on EET-enhancement of wound healing in cellular wound assay. (I) shows effects of signaling pathway inhibitors on EET-enhancement of migration of BAECs through membrane filter. Original Magnification 40x (A), 200x (E). *, p<0.05 vs. control group. #, p<0.05 vs. no L-NMMA, no 17-ODYA or no signaling pathway inhibitor group. Figure 5. raav-mediated AA epoxygenase transfection stimulates angiogenesis in a CAM assay. (A), Representative pictures of CAM assay showing that transfections with AA epoxygenases increase formation of microvessels. (B), Graph showing quantitation of the small vessels (first- and second-order). N=6 per group. *, p<0.01 vs. control group. Figure 6. EETs and AA epoxygenase transfection enhance formation of tubulelike structures on Matrigel. (A), Representative photomicrographs showing effects of EETs on tubule formation. (B), Quantitative analysis of the effects of EETs on BAECs tubule formation. (C), Effect of apigenin (25 µm), H-7 (12 µm), LY (15 µm), PD98059 (20 µm), and L-NMMA (100 µm) on EET-induced tube formation. (D), Quantitative analysis of the effects of infections with raav-cyp102 F87V, raav- 35

36 JPET #83477 CYP2C11-CYPOR, raav-cyp2j2 or raav-gfp on BAEC tubule formation on Matrigel. Expression of AA epoxygenases significantly enhanced tubule formation, and incubation with 17-ODYA (10 µm) inhibited tube formation. *, p<0.05 vs. control group; #, p<0.05 vs. no L-NMMA or no signaling pathway inhibitor group. Figure 7. AA epoxygenase transfection increases angiogenesis in rat ischemic hind limb in vivo model. (A), number of capillaries per mm 2 are increased in the ischemic hind limb following AA epoxygenase transfection. (B), capillary/muscle fiber ratio were significantly increased following AA epoxygenase transfection in ischemic hind limb at day 42. * p< 0.01 vs. control. 36

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