Epoxyeicosatrienoic acids protect rat hearts against tumor necrosis factor-α-induced injury.

Epoxyeicosatrienoic acids (EET), the primary arachidonic acid metabolites of cytochrome P450 2J (CYP2J) epoxygenases, possess potent vasodilatory, anti-inflammatory, antiapoptotic, and mitogenic effects. To date, little is known about the role of CYP2J2 and EETs in tumor necrosis factor (TNF)-α–induced cardiac injury. We utilized cell culture and in vivo models to examine the effects of exogenously applied EETs or CYP2J2 overexpression on TNF-α–induced cardiac apoptosis and cardiac dysfunction. In neonatal rat cardiomyocytes, TNF-α–induced apoptosis was markedly attenuated by EETs or CYP2J2 overexpression, leading to significantly improved cell survival. Further studies showed that TNF-α decreased expression of the antiapoptotic proteins Bcl-2 and Bcl-xL, decreased IκBα and PPARγ, and also inhibited PI3K-dependent Akt and EGFR signaling. Both EETs and CYP2J2 overexpression reversed the effects of TNF-α on these pathways. Furthermore, overexpression of CYP2J2 in rats prevented the decline in cardiac function that is normally observed in TNF-α-challenged animals. These results demonstrate that EETs or CYP2J2 overexpression can prevent TNF-α–induced cardiac cell injury and cardiac dysfunction by inhibiting apoptosis, reducing inflammation, and enhancing PPARγ expression. Targeting the CYP2J2 epoxygenase pathway may represent a novel approach to mitigate cardiac injury in diseases such as heart failure, where increased TNF-α levels are known to occur.


Flow cytometry
Cells were harvested with trypsin/EDTA, resuspended in binding buffer [10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl 2 ], and incubated with FITC-conjugated Annexin V and 1 µg/ml propidium iodide (Annexin V-FITC kit; Bender MedSystems, San Bruno, CA) according to the manufacturer's protocol. Cells were then analyzed with a FACStar-Plus fl ow cytometer (Becton Dickinson, Franklin Lakes, NJ). The annexin V positive (FL1-H) cells were counted for early stages of apoptosis (the lower right quadrant) and late apoptotic or necrotic cells (the upper right quadrant).

Animal treatment and gene delivery
Forty eight male Sprague-Dawley rats weighing 200-220 g were obtained from the Experimental Animal Center of Tongji Medical College. Experimental protocols were approved by the Institutional Animal Research Committee of Tongji Medical College and complied with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Rats were randomized to three groups (16 animals per group) and received intravenous injection of saline control, pcDNA3.1-GFP control, or pcDNA3.1-CYP2J2 at a dose of 2.5 mg/kg body weight ( 25 ) ( 2 ). Two weeks later, 8 rats from each group randomly received a single intravenous injection of TNF-␣ (10 µg/kg). At 6 h and 24 h after TNF-␣ injection, rats were anesthetized by intraperitoneal administration of pentobarbital at 40 mg/kg body weight. A polyethylene catheter ( 26 ) was inserted into the left ventricle (LV) through the right carotid artery for measurement of left ventricular end diastolic pressure (LVEDP) and maximum left ventricular pressure ascending speed (+dp/dt max) ( 26 ).

Human umbilical vein endothelial cell culture and adhesion assays
Human umbilical vein endothelial cells (HUVEC) were isolated from fresh human umbilical cords and cultured. The institutional especially in the context of heart failure. Seubert et al. recently demonstrated a protective role of CYP2J2 overexpression or exogenously administered EETs in ischemia-reperfusion injury in both rodents and dogs via a mechanism that involved opening of sarcolemmal and mitochondrial K + channels and activation of p42/44 MAPK ( 18,19 ). Importantly, the role of CYP2J2-derived EETs in nonischemic cardiac injury and dysfunction remains unknown. In the present study, neonatal rat cardiomyocytes were used to evaluate the effects of CYP2J2 overexpression and exogenous EET supplementation on tumor necrosis factor (TNF)-␣ -induced apoptosis and infl ammation. An in vivo model of TNF-␣ -induced cardiac dysfunction was also utilized to evaluate the hypothesis that CYP2J2 could improve cardiac function by inhibiting myocardial infl ammation and apoptosis.

Primary culture and treatment of neonatal cardiomyocytes
Neonatal cardiomyocytes were obtained from one-day-old rats as described previously ( 22 ). Experimental protocols were approved by the Institutional Animal Research Committee of Tongji Medical College and complied with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The cells were incubated with TNF-␣ (10 ng/ml) in the presence/ absence of exogenous 8,9-, 11,12-, or 14,15-EET (100 nM) or the selective CYP epoxygenase inhibitor PPOH (10 µM) for 24 h. Alternatively, the cells were infected with rAAV-GFP or rAAV-CYP2J2 (MOI = 500) for 48 h prior to treatment with TNF-␣ .
Effect of 11,12-EET and CYP2J2 overexpression on viability of rat heart H9C2 cells H9C2 cells transfected with pcDNA3.1/CYP2J2 had increased CYP2J2 expression and increased DHET levels relative to H9C2 cells transfected with pcDNA3.1/GFP (supplementary Fig. IIIA-C). These results are similar to those previously reported for endothelial cells ( 3,4 ). The results of MTT assays demonstrate that viability of H9C2 cells transfected with pcDNA3.1/CYP2J2 was signifi cantly greater than control cells transfected with pcDNA3.1/GFP in both the presence and absence of TNF-␣ ( Fig. 2A ). The prosurvival effects of CYP2J2 were signifi cantly attenuated by both the EGFR inhibitor AG1478 (100 nM) and the PPAR ␥ inhibitor GW9662 (1 M). Addition of 11,12-EET to H9C2 cells had similar protective effects on cell viability in both the presence and absence of TNF-␣ , and these effects were attenuated by AG1478 and GW9662 ( Fig. 2B ). These results show that the prosurvival effects of CYP2J2 overexpression and 11,12-EET treatment are mediated, at least in part, through activation of EGFR and PPAR ␥ .

Effect of 11,12-EET treatment and CYP2J2 overexpression on apoptosis of rat heart H9C2 cells
We also evaluated the effect of EETs and CYP2J2 overexpression on apoptosis of H9C2 cells by fl ow cytometry. Our results show that cells overexpressing CYP2J2 had signifi cantly reduced apoptosis 24 h after TNF-␣ treatment compared with GFP-transfected cells treated with TNF-␣ ( Fig. 2C ). Exogenous supplementation with 11,12-EET had similar protective effects ( Fig. 2D ). Representative fl ow cytometry scatterplots from all treatment groups are shown in supplementary Fig. IVA, B. The antiapoptotic effects of CYP2J2 overexpression or exogenous 11,12-EET supplementation were attenuated by both AG1478 and GW9662 ( Fig. 2C, D ). These results show that the antiapoptotic effects of CYP2J2 overexpression and 11,12-EET treatment are mediated, at least in part, through activation of EGFR and PPAR ␥ .

Effect of 11,12-EET treatment and CYP2J2 overexpression on caspase-3 activity in rat heart H9C2 cells
TNF-␣ treatment of H9C2 cells signifi cantly increased caspase-3 activity. Transfection with pcDNA3.1/CYP2J2 signifi cantly attenuated the TNF-␣ -induced increase in caspase-3 compared with control and pcDNA3.1/GFPtransfected cells ( Fig. 3A ). This effect was largely inhibited when cells were pretreated with AG1478 or GW9662. Exogenous supplementation with 11,12-EET had similar review board of Tongji Hospital approved this study, and written informed consent was obtained from all participants. Experiments were conducted according to the principles expressed in the Declaration of Helsinki. HUVECs were infected with rAAV-CYP2J2 before incubating with TNF-␣ (10 ng/ml). At 48 h after infection, adhesion assays in peripheral blood mononuclear cells (PBMC) were performed as previously described ( 27,28 ). Briefl y, PBMCs from healthy volunteers (n = 8) were isolated and labeled with Vybrant TM CFDA SE Cell Tracer Kit (Molecular Probes Inc., OR) following the manufacturer's instructions. HUVECs were washed three times with PBS after incubation for 6 h with TNF-␣ (10 ng/ml). Labeled PBMCs were added to the medium (1 × 10 6 /ml) and incubated for 30 min, followed by washing with PBS. A video record of adhesive events was made in at least eight video-microscopic fi elds on each slide after 5 min of washout. For monocytes, the number of adherent cells was counted, averaged per fi eld, and converted to cells per square millimeter using the calibrated fi eld dimensions.

Western blot analysis
Protein concentrations were determined using the Bradford method. Western blots were performed according to methods described previously ( 29 ). Mainly, Western blots were probed with antibodies against Bcl-2, Bcl-xl, and Bax (Santa Cruz Biotechnology, Santa Cruz, CA); PI3-kinase; phosphorylated Akt and Akt (Santa Cruz Biotechnology); phospho-ERK1/2 and ERK1/2 (Santa Cruz Biotechnology); NF-B inhibitor alpha (IkB ␣ ) and PPAR ␥ (Cell Signaling Technology, Beverly, MA); and epidermal growth factor receptor (EGFR) and phosphorylated EGFR (Cell Signaling Technology). Then the membranes were treated with secondary antibody conjugated to horseradish peroxidase (Jackson Immunoresearch Laboratories, West Grove, PA) at room temperature for 2 h. Antibody against ␤ -actin was purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO). Polyclonal antibodies against CYP2J2 were developed as described previously ( 30 ). The horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit and goat anti-mice) were bought from (Jackson Immunoresearch Laboratories).

Statistics
All data were expressed as means ± SEM. Comparisons between groups were performed by a one-way analysis of ANOVA with post hoc analyses performed using the Student-Newman-Keuls method. Statistical signifi cance was defi ned as P < 0.05.

Effect of EETs and CYP2J2 overexpression on apoptosis of primary rat cardiomyocytes
The primary rat cardiomyocytes transfected with rAAVmediated CYP2J2 epoxygenase had increased CYP2J2 expression and increased DHET levels relative to corresponding cells transfected with rAAV-GFP. But the DHET levels were notably decreased by coincubation with TNF-␣ or PPOH (supplementary Fig. IA-C). Flow cytometry analysis showed that 8,9-EET, 11,12-EET, and 14,15-EET signifi cantly decreased apoptosis of primary rat cardiomyocytes induced by TNF-␣ treatment, whereas PPOH, a selective CYP epoxygenase inhibitor, increased apoptosis compared with the corresponding cells without PPOH treatment ( Fig. 1A ). Furthermore, rAAV-mediated CYP2J2 epoxygenase overexpression reduced spontaneous apoptosis and markedly reduced TNF-␣ -induced cardiomyocyte following TNF-␣ treatment ( Fig. 3C ). Overexpression of CYP2J2 had similar effects on Bcl-2, Bcl-xL, and Bax expression ( Fig. 3D ). Furthermore, the effects of 11,12-EET treatment and CYP2J2 overexpression were attenuated by addition of AG1478 or GW9662 ( Fig. 3C, D ), supporting the roles of EGFR and PPAR ␥ in the EET-mediated regulation of apoptosis signaling.

Effect of 11,12-EET treatment and CYP2J2 overexpression on PI3K-dependent Akt phosphorylation and EGFR-ERK pathways in rat heart H9C2 cells
TNF-␣ treatment inhibited expression of PI3K and PI3K-dependent Akt phosphorylation, whereas CYP2J2 overexpression induced PI3K protein expression and Akt protective effects on caspase-3 activity in TNF-␣ -treated H9C2 cells, an effect that was also attenuated by AG1478 and GW9662 ( Fig. 3B ). These results show that the effects of CYP2J2 overexpression and 11,12-EET treatment on caspase-3 activity are mediated, at least in part, through activation of EGFR and PPAR ␥ .

Effect of 11,12-EET treatment and CYP2J2 overexpression on pro-and antiapoptotic protein expression in rat heart H9C2 cells
Incubation of H9C2 cells with exogenous 11,12-EET signifi cantly attenuated the downregulation of antiapoptotic proteins Bcl-2 and Bcl-xL and prevented the upregulation of the proapoptotic protein Bax that is normally observed suggest that CYP2J2 overexpression or exogenous 11,12-EET activates the EGFR-ERK1/2 signaling pathway, a process that may be involved in the antiapoptotic effects observed in cardiac myocytes.

Effect of 11,12-EET treatment and CYP2J2 overexpression on PPAR ␥ and I B ␣ expression in neonatal rat cardiomyocytes
CYP epoxygenase-derived eicosanoids are known to possess potent anti-infl ammatory properties, some of which may be mediated by activation of PPAR ␥ and/or inhibition of I B ␣ degradation ( 2, 31 ). Interestingly, CYP2J2 overexpression or addition of exogenous 11,12-EET signifi cantly upregulated PPAR ␥ and I B ␣ , both in the presence and absence of TNF-␣ ( Fig. 5A , B ). These effects were signifi cantly attenuated by addition of GW9662 or AG1478 prior to TNF-␣ treatment ( Fig. 5A, B ). Therefore, the EET/PPAR ␥ /I B ␣ pathway may represent a novel protective pathway for inhibition of TNF-␣ -induced effects in cardiomyocytes. phosphorylation in both the presence and absence of TNF-␣ ( Fig. 4A ). The effects of CYP2J2 overexpression were apparently inhibited when cells were pretreated with AG1478 or GW9662. Similarly, exogenous treatment with 11,12-EET increased Akt phosphorylation and enhanced PI3K expression, an effect that was blocked by both AG1478 and GW9662 ( Fig. 4B ).

Improvement of hemodynamics and systemic infl ammation in TNF-␣ -treated rats after CYP2J2 gene delivery
Western blot for expression of CYP epoxygenases indicated that CYP2J2 gene delivery induced signifi cant expression of CYP2J2 in vivo in the heart and aorta (supplementary Fig. VA). Overexpression of CYP epoxygenases was associated with a signifi cant increase in urinary 14,15-DHET and serum 14,15-EET levels two weeks after gene delivery compared with levels in rats injected with saline or with pcDNA3.1/GFP ( P < 0.05; supplementary Fig. VB, C). These results indicate that CYP2J2 gene delivery induced signifi cant epoxygenase expression and increases in enzyme activity in vivo.
Hemodynamic assessments showed that TNF-␣ treatment of rats induced a marked deterioration of cardiac function compared with control rats ( Table 1 ). Treatment with pcDNA3.1/CYP2J2 resulted in a signifi cant decrease in LVEDP ( P < 0.05) and a notable increase in +dp/dt max  in primary neonatal cardiomyocytes and in the H9C2 rat cardiac cell line. Furthermore, CYP2J2 overexpression and exogenous addition of 11,12-EET signifi cantly attenuate the TNF-␣ -induced increase in caspase-3 activity and decrease in expression of prosurvival proteins Bcl-2 and Bcl-xL, a process which involves activation of PI3K/Akt, EGF/ERK, and PPAR ␥ signaling pathways. Finally, in vivo overexpression of CYP2J2 signifi cantly inhibits infl ammation and ameliorates the cardiac dysfunction induced by TNF-␣ .
Cardiomyocyte apoptosis and infl ammation induced by cytokines such as TNF-␣ are important in the pathogenesis of acute and chronic myocardial injury and heart failure (32)(33)(34)(35). Apoptosis plays an important role in the basic pathogenic mechanisms that underlie a variety of heart diseases. Owing to the chronic nature of heart failure, a very low level of cellular apoptosis over a period of months or years can be detrimental. Importantly, cardiac myocytes are terminally differentiated and have little potential for division as a rescue mechanism. As a result, it is appealing to develop therapeutic strategies for heart failure that enhance cell viability and inhibit apoptosis.
The cardioprotective effects of 11,12-EET and CYP2J2 overexpression that we observed in this study are consistent with the observation by Seubert et al. that CYP2J2 overexpression in transgenic mice protects against postischemic myocardial dysfunction ( 18 ). EETs and CYP2J2 overexpression have also been shown to attenuate cytokineinduced endothelial cell adhesion molecule expression, and endogenous EETs prevented leukocyte adhesion to the vascular wall by a mechanism involving transcription factor NF-B and I B kinase ( 2 ). The current study also demonstrated inhibitory effects of CYP2J2 overexpression on cytokine-induced leukocyte adhesion to endothelial cells. In vivo experiments showed that CYP2J2 overexpression attenuates production of VCAM, E-selectin, IL-1 ␤ , and IL-6 while enhancing production of the anti-infl ammatory cytokine IL-10. In addition, CYP2J2 gene delivery via intravenous injection signifi cantly increased CYP2J2 expression in vivo in most organs, mainly aorta, heart, liver, and kidney ( 3,5,6,36,37 ), which may also explain some effects of heart protection. These results support the view that CYP2J2-derived EETs protect the heart via their antiinfl ammatory, antiapoptotic, and prosurvival effects.
Additional experiments demonstrate an important role for PPAR ␥ and EGFR signaling in mediating the cardioprotective effects of EETs and CYP2J2 overexpression. Treatment of H9C2 cells with GW9662 (a PPAR ␥ inhibitor) or AG1478 (an EGFR inhibitor) signifi cantly inhibited the antiapoptotic and prosurvival effects of 11,12-EET treatment and CYP2J2 overexpression, with EGFR inhibition being especially detrimental. These results demonstrate that EET-induced EGFR transactivation and PPAR ␥ signaling is an essential event in preventing cardiac myocyte apoptosis and enhancing survival. These results are supported by previous studies in other cell types showing EGFR transactivation in response to EET treatment ( 9,38 ).
PPAR ␥ agonists have consistently been shown to decrease both chemokine secretion and matrix metalloproteinase DISCUSSION In the present study, we show that CYP2J2 transfection or exogenous addition of 11,12-EET signifi cantly increases cell viability and protects against TNF-␣ -induced apoptosis  Values are expressed as mean ± SEM (n = 8 per group). +dp/dt max, maximum left ventricular pressure ascending speed. a P < 0.05 versus control. b P < 0.05 versus GFP + TNF-␣ . activity and Akt/ERK activation, which may be linked to increase in heparin-binding epithelial growth factor (HB-EGF) release ( 9,38 ). Together, these fi ndings suggest that PPAR ␥ activation plays some role in EET-mediated cardioprotection (Scheme 1). In our study, CYP2J2 overexpression or EET activated MAPK1/2, but PPAR ␥ inhibitor GW9662 blocked this effect; However, PPAR ␥ inhibitor GW9662 had much less inhibitory effect on EET-mediated EGFR activation. These results suggest that PPAR ␥ in heart may have cross-talk with EGFR downstream pathway.
In previous studies, we demonstrated that overexpression of epoxygenases can reduce systolic blood pressure in hypertensive rats via enhancing atrial natriuretic peptide production in cardiomyocytes ( 36 ) and can attenuate pulmonary artery hypertension with signifi cantly decreased infl ammatory cytokines in monocrotaline-induced pulmonary artery hypertension model. EETs protect endothelial cells from apoptosis and increased eNOS activity and (MMP) expression by inhibiting NF-B activation (39)(40)(41). PPAR ␥ activation is also known to attenuate infl ammation and apoptosis during cardiac ischemia and injury ( 42 ). A study by Liu et al. showed that PPAR ␥ inhibition abolished the EET/AUDA-mediated anti-infl ammatory effect in endothelial cells subjected to laminar fl ow ( 31 ). In addition, PPAR ␥ is an effector of EETs via competition and direct binding assays, and PPAR ␥ mediated anti-infl ammatory effects of EETs in endothelium ( 31 ). PPAR ␥ agonist reduced lipopolysaccharides (LPS) -induced NF-B activation by antagonizing NF-B DNA activation ( 43 ). In addition, PPAR ␥ agonists similarly inhibit cardiomyocyte infl ammatory responses ( 44,45 ). Consistent with these fi ndings, we found that 11,12-EET and CYP2J2 over expression partially but signifi cantly prevented the loss of PPAR ␥ expression that is normally induced by TNF-␣ . Furthermore, we found that the PPAR ␥ inhibitor GW9662 hindered the 11,12-EET-and CYP2J2-mediated downregulation of caspase-3 expression level ( 46 ). Dr. Liao demonstrated that EETs inhibited leukocyte adhesion to endothelial cells and reduced IL-1 via inhibition of NF-B in endothelial cells ( 2 ). These data indicate that EETs really have systemic effects and that these cardiovascular protective effects should be able to exert benefi cial effects on the heart, especially by decreasing infl ammatory cytokines. In the present study, artery blood pressure was not high; therefore, overexpression of epoxygenase had no hypotensive effect. This study has some limitations. Further studies are needed to fi gure out whether cardioprotective effects of EETs in anti-infl ammation and antiapoptosis involve activation of G-protein coupled receptors and their hyperpolarization role via activating calcium-activated potassium channels, as well as more detailed molecular mechanisms and targets of EETs.
In summary, the present study provides insight on the protective role of EETs against cardiac dysfunction and myocardial injuries. The potency of EET-mediated cardiac protection suggests that abundant CYP2J2 expression and EET production in the myocardium are important protective factors whose mechanisms are likely mediated via EGFR and PPAR ␥ activation. Our data support the development of tools to enhance endogenous EETs and/or to deliver exogenous EETs as a novel approach to the treatment of cardiovascular complications, including heart failure.