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Am J Physiol Heart Circ Physiol 293: H142-H151, 2007. First published February 23, 2007; doi:10.1152/ajpheart.00783.2006
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Cytochrome P-450 epoxygenases protect endothelial cells from apoptosis induced by tumor necrosis factor-{alpha} via MAPK and PI3K/Akt signaling pathways

Shilin Yang,1,,* Li Lin,1,,* Ji-Xiong Chen,1,,* Craig R. Lee,2 John M. Seubert,3 Yan Wang,1 Hong Wang,1 Zhong-Ren Chao,4 De-Ding Tao,1 Jian-Ping Gong,1 Zai-Ying Lu,1 Dao Wen Wang,1 and Darryl C. Zeldin2

1Institute of Hypertension and Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China; 2Division of Intramural Research, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina; 3Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada; and 4Life Science College of Beijing University, Beijing, People's Republic of China

Submitted 21 July 2006 ; accepted in final form 22 February 2007


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Endothelial cells play a vital role in the maintenance of cardiovascular homeostasis. Epoxyeicosatrienoic acids (EETs), cytochrome P-450 (CYP) epoxygenase metabolites of arachidonic acid in endothelial cells, possess potent and diverse biological effects within the vasculature. We evaluated the effects of overexpression of CYP epoxygenases on tumor necrosis factor-{alpha} (TNF-{alpha})-induced apoptosis in bovine aortic endothelial cells. CYP epoxygenase overexpression significantly increased endothelial cell viability and inhibited TNF-{alpha} induction of endothelial cell apoptosis as evaluated by morphological analysis of nuclear condensation, DNA laddering, and fluorescent-activated cell sorting (FACS) analysis. CYP epoxygenase overexpression also significantly inhibited caspase-3 activity and downregulation of Bcl-2 expression induced by TNF-{alpha}. The antiapoptotic effects of CYP epoxygenase overexpression were significantly attenuated by inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK signaling pathways; however, inhibition of endothelial nitric oxide synthase activity had no effect. Furthermore, CYP epoxygenase overexpression significantly attenuated the extent of TNF-{alpha}-induced ERK1/2 dephosphorylation in a time-dependent manner and significantly increased PI3K expression and Akt phosphorylation in both the presence and absence of TNF-{alpha}. Collectively, these results suggest that CYP epoxygenase overexpression, which is known to increase EET biosynthesis, significantly protects endothelial cells from apoptosis induced by TNF-{alpha}. This effect is mediated, at least in part, through inhibition of ERK dephosphorylation and activation of PI3K/Akt signaling.

epoxyeicosatrienoic acid; arachidonic acid

ENDOTHELIAL CELLS PLAY a vital role in the maintenance of cardiovascular homeostasis by synthesizing and releasing endogenous vasodilators such as nitric oxide (NO) and prostacyclin (PGI2). In addition, these substances exhibit potent anti-inflammatory, antithrombotic, and antiapoptotic effects within the vasculature (15, 39, 44). Epoxyeicosatrienoic acids (EETs) are cytochrome P-450 (CYP) epoxygenase-derived metabolites of arachidonic acid that also possess potent vasodilatory properties. These eicosanoids are considered to be the leading candidates for endothelium-derived hyperpolarizing factor (EDHF) (12, 13, 31) because they hyperpolarize vascular smooth muscle cells by activating Ca2+-sensitive K+ channels (11, 30).

CYP epoxygenases of the CYP2C and CYP2J subfamilies actively metabolize arachidonic acid to various EET regio- and stereoisomers (49). In addition to EDHF-like properties, CYP epoxygenase-derived EETs have been shown in multiple in vitro and in vivo studies to possess other potent biological effects in the renal and cardiovascular systems. For example, EETs induce mitogenesis of renal epithelial cells (4, 5), inhibit cytokine-induced vascular cell adhesion molecule expression and leukocyte adhesion to the vascular wall (32), inhibit vascular smooth muscle cell migration (41), and increase tissue plasminogen activator expression (33). More recently, our group has demonstrated that transfection of endothelial cells with various CYP epoxygenases increases EET biosynthesis and significantly upregulates the expression and activity of endothelial NO synthase (eNOS) (45).

The balance between endothelial cell survival and endothelial cell death is critical in various processes, including intravascular inflammation and vascular remodeling. Indeed, endothelial cell apoptosis is hypothesized to be a key initiating event in the development of atherosclerosis and other vascular diseases (1, 7, 40). Recently, Dhanasekaran et al. (8) reported that exogenous EETs increased human endothelial cell surviving ability and attenuated apoptosis. However, the exact role that CYP epoxygenase-derived EETs play in preventing endothelial cell apoptosis remains elusive. In the current study, we utilized cultured bovine aortic endothelial cells (BAECs) transfected with CYP epoxygenases CYP102 F87V, CYP2C11-CYPOR, and CYP2J2 to investigate the role of endogenously formed EETs on tumor necrosis factor-{alpha} (TNF-{alpha})-induced apoptosis. We demonstrate that these CYP epoxygenases markedly attenuate apoptosis and prevent both TNF-{alpha} activation of caspase-3 and TNF-{alpha} reduction of Bcl-2 expression. In addition, our data suggest that this antiapoptotic effect is mediated, at least in part, through activation of the MAPK and PI3K/Akt signaling pathways.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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An expanded MATERIALS AND METHODS section is available as a supplement with the online version of this article.

Cell culture. BAECs were isolated from bovine aortas obtained from a local slaughterhouse by digestion with 0.25% trypsin and were cultured in DMEM supplemented with 5 mM L-glutamine, 10% FBS, and an antibiotic mixture containing penicillin (100 U/ml) and streptomycin (100 µg/ml). Cell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described (10, 23).

Recombinant adeno-associated virus and gene transfection. Plasmids encoding CYP102 F87V, CYP2C11-CYPOR, and CYP2J2 were kindly provided by Drs. Jorge Capdevila (Vanderbilt Univ.) and Darryl Zeldin [National Institute of Environmental Health Sciences (NIEHS)]. The CYP102 F87V is a mutant P-450 from Bacillus megaterium in which phenylalanine 87 was replaced with valine, converting it to a highly regio- and stereoselective epoxygenase that biosynthesizes 14(S),15(R)-EET from arachidonic acid (16). CYP2C11-CYPOR is an active rat P-450 epoxygenase fused with rat NADPH-CYP oxidoreductase that synthesizes a regioisomeric mixture of 5,6-, 8,9-, 11,12-, and 14,15-EETs (36). CYP2J2 is a human P-450 that biosynthesizes primarily 8,9-, 11,12-, and 14,15-EETs in human vascular endothelial cells (32). The recombinant adeno-associated virus (rAAV) vector pXXUF1, packaging plasmid pXX2, adenovirus helper plasmid pXX6, and a rAAV plasmid containing the green fluorescent protein (GFP) cDNA (GFP-pUF1) were generous gifts from Dr. Xiao Xiao (Univ. of Pittsburgh). For rAAV packaging in vitro, epoxygenase cDNAs were subcloned into pXXUF1 and the rAAVs were produced as previously described (45, 47, 48). BAECs were infected with rAAV-CYP102 F87V, rAAV-CYP2C11-CYPOR, rAAV-CYP2J2, or rAAV-GFP (~50 virions/cell) and cultured for 1 wk to obtain maximal expression (the percentage of cells routinely infected by rAAV-GFP is over 50% according to microscope observation). Abundant P-450 expression and increased EET biosynthesis after infection have been confirmed in our previous studies (45, 49). In some experiments, epoxygenase cDNAs were subcloned into the mammalian expression vector pCB6 to produce CYP102 F87V-pCB6, CYP2C11-CYPOR-pCB6, and CYP2J2-pCB6 (45). BAECs were then transfected using Superfect Transfection Reagent (Qiagen, Hilden, Germany) and cultured for 24 h to obtain maximal expression. Cell viability was assessed 48 h and 1 wk following transfection.

14,15-Dihydroxyeicosatrienoic acid detection by ELISA. To reflect activity of metabolizing arachidonic acid into EETs, concentrations of the stable EET metabolite 14,15-dihydroxyeicosatrienoic acid (DHET) in cultured endothelial cells were determined by an ELISA kit (Detroit Research and Development). Briefly, the cells cultured in 100-mm petri dishes were washed with chilled phosphate-buffered saline (PBS) two times and then scraped using 1.0 ml of cold PBS containing triphenylphosphine. The samples were sonicated on ice, and the protein quantity was determined. The eicosanoids were then extracted from the cell samples three times with ethyl acetate after acidification with acetic acid (to convert EETs into DHETs). After evaporation, saponification with 0.4 N KOH in methanol and reextraction were carried out. 14,15-DHET dissolved with 30 µl dimethylformamide was detected following the ELISA kit instructions as we did previously (21).

Treatments of BAECs. Apoptosis was induced by incubating transfected BAECs with TNF-{alpha} (5 ng/ml) and actinomycin D (0.01 µg/µl, to inhibit RNA synthesis) for 24 h. Inhibitor experiments involved 30 min of pretreatment with the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecylcarbonate (Akt-I, 2 µM); the PI3K inhibitor LY-294002 (20 µM), the MAPK inhibitor apigenin (20 µM), or the MEK inhibitor PD-98059 (20 µM) (20). In some experiments, cells were also treated with the P-450 inhibitor 17-octadecynoic acid (17-ODYA; 100 nM) or with synthetic, HPLC-purified 14,15-EET (100 nM) as described (17).

Assessment of apoptosis. Apoptotic responses were assessed by four independent methods following treatment of BAECs with TNF-{alpha} plus actinomycin D. First, DNA fragmentation was assessed by gel electrophoresis as previously described (22). Second, cells were resuspended and stained with fluorescein isothiocyanate-conjugated annexin V and fluorescent dye propidium iodide and analyzed by flow cytometry [fluorescent-activated cell sorting (FACS), Vantage]. The relative increase in apoptotic cells was calculated as a percentage. These percentages were calculated as the ratio of apoptotic cells in rAAV-CYP102 F87V versus rAAV-GFP-infected cells for each signaling molecule inhibitor and then normalized to the corresponding ratio in cells treated with no inhibitor. Third, caspase-3 activity was measured with a colorimetric assay kit using DEVD-p-nitroanilide as a substrate, and an equal amount of cell lysate protein was used to normalize (24). The relative increase percentage of capase-3 activity was also calculated, as completed for the apoptotic cell calculation above.

Immunoblot analyses. Lysates of cell and tissue were subjected to Western blot analysis as described in the online data supplement. Expression was quantified by densitometry and normalized to beta-actin expression, and all groups were then normalized to their respective vehicle or empty vector controls.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Cell viability assays. Viability of BAECs following transfection with CYP102 F87V-, CYP2C11-CYPOR-, or CYP2J2-containing vectors was significantly greater than that following transfection with an empty pCB6 vector or a GFP-containing vector at 48 h (Fig. 1A), and rAAV-mediated transfections of CYP102 F87V, CYP2C11-CYPOR, or CYP2J2 had a very similar effect (data not shown). Treatment with synthetic 14,15-EET also significantly increased cell viability, and the CYP inhibitor 17-ODYA abolished the protective effects of CYP102 F87V transfection (Fig. 1B). The expression levels of transfected target genes in our cell system were previously reported (46). The 14,15-DHET levels in untransfected and GFP transfected cells were 1,225 ± 609 and 923 ± 309 pg/mg cell protein, respectively. Significantly higher 14,15-DHET levels were observed in the CYP102 F87V (7,164 ± 1,382 pg/mg cell protein), CYP2C11-CYPOR (6,018 ± 3,154 pg/mg cell protein), and CYP2J2 (4,886 ± 1,759 pg/mg cell protein) transfected cells (N = 6 repeats, P < 0.05). Importantly, these data indicate that epoxygenase gene overexpression markedly increased EET production in endothelial cells.


Figure 1
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  		Fig. 1. Effect of cytochrome P-450 (CYP) epoxygenase overexpression on bovine aortic endothelial cell (BAEC) viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A: cell viability quantified 48 h after transfection with CYP2J2 (2J2), CYP102 F87V (F87V), or CYP2C11-CYPOR (2C11). Blank, BAECs without any treatment; control, BAECs following transfection with the empty pCB6 vector; GFP, BAECs following transfection with the pCB6 vector containing the green fluorescent protein (GFP) cDNA. Values represent means ± SE of optical density obtained from the MTT assay. Each group was completed in triplicate. *P < 0.05 and **P < 0.01 compared with control. B: cell viability quantified 48 h after transfection with or without the CYP inhibitor 17-octadecynoic acid (17-ODYA; 100 nM) or synthetic 14,15-epoxyeicosatrienoic acid (14,15-EET; 100 nM). *P < 0.05 and **P < 0.01 compared with reference group.

 
CYP epoxygenase overexpression attenuates TNF-{alpha}-induced apoptosis. BAECs infected with rAAV-CYP2J2, rAAV-CYP102 F87V, or rAAV-CYP2C11-CYPOR underwent significantly less apoptosis 24 h after TNF-{alpha} treatment compared with uninfected BAECs. First, DNA fragmentation analysis revealed that CYP epoxygenase transfection reduced TNF-{alpha}-induced DNA degradation in BAECs (Fig. 2). Second, flow cytometry revealed significant attenuation in the percentage of apoptotic cells (annexin V positive and propidium iodide negative) after TNF-{alpha} treatment following transfection with CYP2J2, CYP101 F87V, or CYP2C11 epoxygenases compared with control (17.0 ± 1.1%, 8.3 ± 1.6%, and 12.0 ± 1.9%, respectively, vs. 31.4 ± 2.0%, P < 0.05) (Fig. 3, A and B).


Figure 2
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  		Fig. 2. Effect of CYP epoxygenase overexpression on TNF-{alpha}-induced DNA fragmentation. Cells were infected with recombinant adeno-associated virus (rAAV)-GFP, rAAV-CYP2C11-CYPOR, rAAV-CYP102 F87V, or rAAV-CYP2J2 and, 1 wk later, were incubated with TNF-{alpha} for 24 h in the presence of actinomycin D. DNA (20 µg/lane) extracted from the BAECs was electrophoresed on a 2% agarose gel to detect DNA ladder formation. Representative photo of 6 repeats and control refers to BAECs without infection or with rAAV-GFP infection.

 

Figure 3
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  		Fig. 3. Effect of CYP epoxygenase overexpression on apoptosis induced by TNF-{alpha} as assessed by flow cytometry. A: representative results of 3 independent experiments evaluating TNF-{alpha}-induced apoptosis by annexin-V-FITC/propidium iodide (PI) staining and flow cytometry. Cells were infected with rAAV-CYP2J2, rAAV-CYP102 F87V, or rAAV-CYP2C11-CYPOR and, 1 wk later, were incubated with TNF-{alpha} for 24 h in the presence of actinomycin D. Control, BAECs infected with rAAV-GFP. Cells with negative staining of both PI and annexin V are living. Cells with PI-negative and annexin V-positive staining are early apoptotic cells. Cells with PI-positive and annexin V-positive staining are primarily in a late stage of apoptosis. B: percentage of apoptotic cells from flow cytometry analysis in A, and values are means ± SE from 5 independent experiments. **P < 0.01 compared with control. C: representative results of 3 independent experiments evaluating the effects of signaling molecule inhibitors on TNF-{alpha}-induced apoptosis in CYP epoxygenase-infected BAECs. Cells infected with either rAAV-GFP or rAAV-CYP102 F87V 1 wk prior were incubated with inhibitors of Akt (Akt-I), phosphatidylinositol 3-kinase (PI3K; LY-294002), MAPK (apigenin), MEK (PD-98059), or no inhibitor for 30 min before TNF-{alpha} treatment. D: percentage of apoptotic cells in C. Values shown are means ± SE from 5 independent experiments. *P < 0.05 and **P < 0.01 compared with rAAV-GFP cells without inhibitor treatment; #P < 0.05 and ##P < 0.01 compared with rAAV-CYP102 F87V cells without inhibitor treatment.

 
To identify the signaling pathways responsible for the observed antiapoptotic effects of CYP epoxygenase overexpression, we also characterized TNF-{alpha}-induced apoptosis in BAECs pretreated with various cell signaling pathway inhibitors. Pretreatment of rAAV-GFP-infected BAECs with Akt-I, LY-294002, apigenin, and PD-98059 significantly enhanced TNF-{alpha}-induced apoptosis compared with rAAV-GFP cells without inhibitor treatment (Fig. 3, C and D). Likewise, TNF-{alpha}-induced apoptosis was significantly increased in cells infected with rAAV-CYP102 F87V when treated with these signaling pathway inhibitors compared with rAAV-CYP102 F87V-infected cells without inhibitor treatment (Fig. 3, C and D). Importantly, inhibition of the MAPK and PI3K/Akt signaling pathways increased TNF-{alpha}-induced apoptosis to a significantly greater extent in rAAV-CYP102 F87V- versus rAAV-GFP-infected BAECs, demonstrating that the antiapoptotic effect of CYP epoxygenase overexpression is mediated, at least in part, through these two pathways.

CYP epoxygenase overexpression inhibits caspase-3 activity. Treatment of BAECs with TNF-{alpha} significantly increased caspase-3 activity; however, infection with rAAV-CYP2J2, rAAV-CYP102 F87V, and rAAV-CYP2C11-CYPOR significantly attenuated the TNF-{alpha}-induced increase in caspase-3 activity compared with uninfected and rAAV-GFP-infected cells (Fig. 4A). TNF-{alpha} also induced a significant increase in caspase-3 activity in both rAAV-GFP- and rAAV-CYP102 F87V-infected BAECs when incubated with Akt-I, LY-294002, apigenin, and PD-98059 compared with the respective cells without inhibitor treatment (Fig. 4B). However, inhibition of the MAPK and PI3K/Akt signaling pathways enhanced the TNF-{alpha}-induced increase in caspase-3 activity to a significantly greater extent in rAAV-CYP102 F87V- versus rAAV-GFP-infected BAECs, further demonstrating the role of these signaling pathways in the antiapoptotic effect of CYP epoxygenase overexpression.


Figure 4
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  		Fig. 4. Effect of CYP epoxygenase overexpression on caspase-3 activity. A: caspase-3 activity measurements in cells infected with rAAV-GFP, rAAV-CYP2J2, rAAV-CYP102 F87V, or rAAV-CYP2C11-CYPOR and, 1 wk later, incubated with TNF-{alpha} for 24 h. Control, BAECs without infection as 100%, and values of other samples were normalized to the untreated controls and expressed as means ± SE of percentage according to values from 4 independent experiments. *P < 0.05 and **P < 0.01 compared with untreated control; ##P < 0.01 compared with control treated with TNF-{alpha}. B: caspase-3 activity measurements evaluating the effects of cell signaling molecule inhibitors on TNF-{alpha}-induced apoptosis. Cells infected with rAAV-CYP102 F87V or rAAV-GFP 1 wk prior were incubated with an inhibitor of Akt (Akt-I), PI3K (LY-294002), MAPK (apigenin), MEK (PD-98059), or no inhibitor 30 min before TNF-{alpha} treatment. Control without inhibitor treatment was assigned 100%, and values of other samples are expressed as percentages of the control (all values are from 6 independent experiments). **P < 0.01 compared with control; ##P < 0.01 compared with rAAV-CYP102 F87V cells without inhibitor treatment.

 
CYP epoxygenase overexpression inhibits Bcl-2 downregulation. Overexpression of CYP2C11-CYPOR, CYP102 F87V, or CYP2J2 in BAECs caused a significant increase in Bcl-2 protein expression at baseline but also significantly inhibited the TNF-{alpha}-induced downregulation of Bcl-2 protein expression, even though TNF-{alpha} incubation downregulated Bcl-2 expression (data not shown). Further experiments demonstrated that TNF-{alpha} treatment resulted in a time-dependent downregulation of Bcl-2 protein expression over 48 h; however, transfection with the three CYP epoxygenases significantly inhibited this Bcl-2 downregulation at each time point (Fig. 5, A and B).


Figure 5
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  		Fig. 5. Effect of CYP epoxygenase overexpression on Bcl-2 expression. A: Bcl-2 and beta-actin expression by immunoblot after stimulation with TNF-{alpha} for 0, 24, or 48 h. Control, transfection with the empty pCB6 vector. B: densitometry of Bcl-2 protein expression relative to beta-actin expression, normalized to control of the individual time points. Values shown represent means ± SE from 3 independent experiments. **P < 0.01 compared with corresponding control. C: Bcl-2 and beta-actin expression by immunoblot in pCB6- and CYP102 F87V-transfected BAECs after incubation with an inhibitor of Akt (Akt-I), PI3K (LY-294002), MAPK (apigenin), or MEK (PD-98059) 30 min before TNF-{alpha} treatment. D: densitometry of Bcl-2 protein expression relative to beta-actin expression, normalized to control. Values shown are means ± SE from 4 or 5 independent experiments. **P < 0.01 compared with control cells without inhibitor treatment; ##P < 0.01 compared with CYP102 F87V transfected cells without inhibitor treatment. E: Bcl-2 and beta-actin expression by immunoblot. After transfection with CYP2C11-CYPOR or CYP102 F87V for 24 h, NG-monomethyl-L-arginine (L-NMMA; 2 mM) was incubated for 2 h before TNF-{alpha} treatment. F: densitometry of Bcl-2 protein expression relative to beta-actin expression, normalized to control. Values shown are means ± SE from 3 independent experiments. Molecular weights of Bcl-2 and beta-actin are 28K and 43K, respectively. #P < 0.05 and ##P < 0.01 compared with corresponding treatment control group; **P < 0.01 compared with corresponding L-NMMA (–) and TNF-{alpha} (–) control group.

 
Pretreatment of BAECs with Akt-I, LY-294002, apigenin, or PD-98059 before TNF-{alpha} treatment significantly reduced Bcl-2 protein expression in both control and CYP102 F87V-transfected cells (Fig. 5, C and D). Moreover, this signaling inhibitor-mediated reduction in Bcl-2 expression was significantly more pronounced in the CYP102 F87V-transfected cells compared with control cells, suggesting that the MAPK and PI3K/Akt signaling pathways mediate, at least in part, the effect of CYP epoxygenase overexpression on Bcl-2 expression. In addition, incubation of control cells with the eNOS inhibitor, NG-monomethyl-L-arginine (L-NMMA), before TNF-{alpha} treatment significantly decreased Bcl-2 protein expression compared with treatment with TNF-{alpha} alone; however, L-NMMA pretreatment did not significantly attenuate the increase in Bcl-2 expression observed in TNF-{alpha}-treated cells transfected with CYP2C11-CYPOR or CYP102 F87V (Fig. 5, E and F). Together, these findings suggest that the antiapoptotic effect of CYP epoxygenase overexpression in BAECs is likely not mediated via CYP epoxygenase-induced upregulation of eNOS expression.

Effects of CYP epoxygenase overexpression on ERK1/2 and PI3K/Akt signaling. Treatment of BAECs with TNF-{alpha} induced a significant time-dependent dephosphorylation of ERK1/2 over 2–4 h in control cells; however, transfection with CYP2C11-CYPOR or CYP102 F87V significantly attenuated the extent of ERK1/2 dephosphorylation at these time points (Fig. 6, A and B). Moreover, phosphorylation of ERK1/2 was significantly increased in cells transfected with the CYP epoxygenases compared with control cells before TNF-{alpha} treatment. Overexpression of CYP2J2, CYP102 F87V, and CYP2C11-CYPOR also significantly increased PI3K protein expression in both the presence (Fig. 7, A and B) and absence (Fig. 7, C and D) of TNF-{alpha} treatment compared with uninfected and rAAV-GFP-infected control cells. Similarly, phosphorylation of Akt was significantly increased in BAECs transfected with the three CYP epoxygenases in both the presence (Fig. 7, E and F) and absence (Fig. 7, G and H) of TNF-{alpha} treatment. Collectively, these findings demonstrate that CYP epoxygenase overexpression stimulates phosphorylation of ERK1/2 and PI3K/Akt signaling molecules in BAECs, and this may contribute to the observed antiapoptotic effects of CYP epoxygenase overexpression in endothelial cells.


Figure 6
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  		Fig. 6. Effect of CYP epoxygenase overexpression on ERK1/2 phosphorylation. A: phospho (p)- and total ERK1/2 expression by immunoblot. Cells were infected with rAAV-GFP, rAAV-CYP2C11-CYPOR, or rAAV-CYP102 F87V. One week later, they were incubated with TNF-{alpha} for 0, 1, 2, or 4 h and the cells were lysed. Control refers to BAECs infected with rAAV-GFP. B: densitometry of p-ERK protein expression relative to total ERK expression, normalized to the ratio of control-to-0 h. Values shown are means ± SE of 3 independent experiments. *P < 0.05 and **P < 0.01 compared with corresponding groups at 0 h; &P < 0.05 and &&P < 0.01 compared with control at 0-h time point; #P < 0.05 and ##P < 0.01 compared with control at 1-h time point, $P < 0.05 compared with control at 2-h time point.

 

Figure 7
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  		Fig. 7. Effect of CYP epoxygenase overexpression on PI3K expression and Akt phosphorylation. A and C: PI3K and beta-actin expression by immunoblot. Cells were infected with rAAV-GFP, rAAV-CYP2J2, rAAV-CYP102 F87V, or rAAV-CYP2C11-CYPOR, respectively, and, 1 wk later, incubated with (A) or without (C) TNF-{alpha} for 24 h. Control, BAECs without transfection. B and D: densitometry of PI3K protein expression relative to beta-actin expression normalized to control after incubation with (B) or without (D) TNF-{alpha} for 24 h. E and G: p-Akt and total Akt expression by immunoblot after incubation with (E) or without (G) TNF-{alpha} for 24 h. F and H: densitometry of p-Akt protein expression relative to total Akt expression normalized to control after incubation with (F) or without (H) TNF-{alpha} for 24 h. Values are means ± SE from 3 independent experiments. *P < 0.05 and **P < 0.01 compared with control.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Endothelial cells play an integral role in the regulation of vascular function via the synthesis and release of several vasoactive factors, including NO and prostacyclin. EETs are epoxygenase metabolites of arachidonic acid synthesized in the endothelium that possess potent vasodilatory properties (12, 13, 31). These CYP epoxygenases, which are known to increase the synthesis of EETs, also play critical roles in the regulation of vascular and bronchial smooth muscle tone, cellular proliferation, peptide hormone secretion, and ionic transport (49). Importantly, EETs possess potent effects within the vasculature via regulation of intravascular inflammation and homeostasis (32, 33); however, their role in regulating endothelial cell apoptosis has not been characterized.

Dhanasekaran et al. (8) recently demonstrated the capacity of EETs to enhance human endothelial cell survival by inhibiting pathways of apoptosis in cell lines of human lung microvascular endothelial cells and coronary artery endothelial cells and found that an application of the PI3K inhibitor wortmannin abolished the protective effect, suggesting that the activation of PI3K/Akt signaling pathway involves the EETs-mediated cytoprotective effect. However, it is not clear whether endogenous EETs have the antiapoptotic role on endothelial cells. Dhanasekaran's study demonstrated the protective effect of EETs in a serum-deprivation-induced apoptosis model, but, in other models, especially in the apoptosis induced by inflammatory cytokines, diversely known injury factors (such as TNF-{alpha}), the antiapoptotic effect of EETs, as well as its mechanisms, remains elusive.

We sought to evaluate the potential influence of CYP epoxygenase overexpression and increased EET biosynthesis on endothelial cell apoptosis induced by the inflammatory cytokine TNF-{alpha}. Our findings demonstrate that CYP epoxygenase overexpression significantly increases BAEC viability, as observed previously by our group (46). Moreover, CYP epoxygenase overexpression significantly protects BAECs from TNF-{alpha}-induced apoptosis, as evaluated by morphological analysis of nuclear condensation, DNA laddering, and FACS analysis. Moreover, CYP epoxygenase overexpression significantly inhibits caspase-3 activity and the downregulation of Bcl-2 expression induced by TNF-{alpha} in this model of endothelial injury. Collectively, our data implicate the CYP epoxygenase pathway as an important regulator of endothelial cell apoptosis and as a potent antiapoptotic factor within the vasculature. Although the observed antiapoptotic effects related to epoxygenase overexpression could be due to increased synthesis of a non-EET product, we demonstrated that increased epoxygenase expression was associated with increased 14,15-DHET production (a stable EET metabolite), consistent with our previous studies using these transfected epoxygenases. Moreover, our findings parallel the findings from other studies where exogenous EETs were administered. Importantly, our findings also demonstrate the important role of ERK dephosphorylation and activation of PI3K/Akt signaling in the antiapoptotic effects of epoxygenase overexpression.

Apoptosis is an enzymatically controlled and energy-dependent form of programmed cell death, and caspases are a group of enzymes vital to the regulation of this process (42). A diverse array of intrinsic or extrinsic stimuli regulates endothelial cell apoptosis by modulating the balance between the proapoptotic caspases and various antiapoptotic proteins, such as Bcl-2 (1, 7, 40). We observed that CYP epoxygenase overexpression significantly inhibited caspase-3 activity and the time-dependent downregulation of Bcl-2 expression induced by TNF-{alpha}, suggesting that EETs not only inhibit activity of proapoptotic proteins but also maintain levels of antiapoptotic proteins. Moreover, these antiapoptotic effects were significantly attenuated by the inhibition of the PI3K/Akt and MAPK signaling pathways, suggesting that they were mediated, at least in part, by the activation of these pathways. Previous studies have indicated that increased eNOS expression also has antiapoptotic effects (9, 37). Our prior work has demonstrated that exogenous EET treatment or CYP expoxygenase overexpression significantly increases eNOS expression and activity in BAECs (45). To determine whether upregulation of eNOS expression mediated the antiapoptotic effects of CYP epoxygenase overexpression, we preincubated BAECs with the eNOS inhibitor L-NMMA. We found that eNOS inhibition did not attenuate the inhibitory effect of CYP epoxygenase overexpression on TNF-{alpha}-induced Bcl-2 downregulation, suggesting that the observed antiapoptotic effects of CYP epoxygenase overexpression are independent of eNOS.

It has been reported that EETs participate in the regulation of several intracellular signaling pathways (2, 33, 35, 49). For example, 14,15-EET activates ERK during EET-induced mitogenesis (6). Moreover, transfection of porcine coronary arteries with CYP2C8 resulted in increased ERK1/2 phosphorylation compared with untreated cells (14). In endothelial cells, MAPK (ERK1/2)-dependent phosphorylation stabilized Bcl-2 and prevented its proteasome-dependent degradation, increasing cell survival (7, 38). In the current study, phosphorylation of ERK1/2 was increased following CYP epoxygenase overexpression compared with control cells, similar to our previous observations (46), suggesting a role for MAPK in the observed apoptosis-suppressive effect. Whereas CYP expoxygenase transfection activated ERK1/2, it did not prevent the time-dependent dephosphorylation of ERK1/2 induced by TNF-{alpha}, implying the existence of other important signaling pathways. One such antiapoptotic pathway involves signaling through PI3K/Akt (18, 19, 34), resulting in increased Bcl-2 expression (25) and the prevention of mitochondrial cytochrome-c release (27, 28, 43). Interestingly, CYP epoxygenase overexpression in BAECs also increased PI3K expression and Akt phosphorylation, as has been previously observed to occur in cancer cells (21). Moreover, inhibition of PI3K and Akt signaling significantly attenuated the CYP epoxygenase-mediated increase in Bcl-2 expression, indicating a role for the PI3K/Akt pathway in EET-mediated antiapoptosis in endothelial cells. Collectively, our results demonstrate that CYP epoxygenase overexpression significantly attenuates TNF-{alpha}-induced apoptosis in BAECs via both ERK1/2 and PI3K/Akt signaling.

Chen et al. (3) reported that 14,15-EET inhibits apoptosis induced by serum withdrawal or H2O2 in LLCPKcl4 cells, a renal proximal tubule-like epithelial cell line. In the current study, we provide evidence that endogenously synthesized EETs prevent endothelial cell apoptosis induced by TNF-{alpha}. Endothelial cell apoptosis represents a form of endothelial injury that may significantly influence endothelial function, as well as vascular inflammation and homeostasis. Indeed, endothelial cell apoptosis may be an important initiating event in a variety of cardiovascular diseases, such as atherosclerosis and hypertension (1, 7, 40). TNF-{alpha}, a multifunctional cytokine, promotes endothelial cell apoptosis and elicits inflammatory responses by increasing expression and secretion of adhesion molecules, such as E-selectin, ICAM-1, and VCAM-1 (1, 26, 29, 40), which have been implicated in the pathogenesis of atherosclerosis. Our results suggest that CYP epoxygenase overexpression and increased EET biosynthesis may also inhibit the atherogenic effects of TNF-{alpha} by preventing endothelial cell apoptosis, demonstrating yet another potent vascular effect of EETs independent of their EDHF properties. Thus it appears that CYP epoxygenase-derived EETs may have atheroprotective effects in the endothelium via multiple synergistic mechanisms. Even so, however, it is still necessary in further experiments to investigate whether the protective roles are induced at least partially by some other metabolites of epoxygenases rather than EETs because it is likely that these enzymes catalyze more than one substrate and generate more than one product.

In conclusion, the current study reveals a novel role for CYP epoxygenase-derived EETs in endothelial cell survival. The antiapoptotic effect of EETs markedly attenuated TNF-{alpha}-induced apoptosis of endothelial cells and increased cell viability. These protective effects appear to involve both attenuation of the TNF-{alpha}-mediated decrease in the antiapoptotic protein Bcl-2 and activation of the proapoptotic caspase-3, as well as activation of the prosurvival PI3K/Akt and ERK signaling pathways. Thus it appears that increased EET biosynthesis contributes to the survival of endothelial cells and the maintenance of cardiovascular homeostasis. The apoptosis-suppressive effect of EETs may have important clinical implications, and the modulation CYP epoxygenase-mediated EET biosynthesis may represent a novel therapeutic strategy for the treatment and/or prevention of cardiovascular diseases.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by grants from the National Natural Science Foundation Committee (30270561 and 30430320), National "973" grant (2006CB503801), and the Intramural Research Program of the NIEHS.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Jorge Capdevila for providing the CYP102 F87V and CYP2C11-CYPOR cDNAs and corresponding polyclonal antibodies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. W. Wang, Inst. of Hypertension and Dept. of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong Univ. of Science and Technology, 1095# Jie Fang Da Dao (Ave.), Wuhan 430030, People's Republic of China (e-mail: dwwang{at}tjh.tjmu.edu.cn)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* S. Yang, L. Lin, and J.-X. Chen contributed equally to this work. Back


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 DISCUSSION
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 REFERENCES
 

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