Several recent studies demonstrated that hypoxia induces the expression of CYP epoxygenases, particularly CYP2C9, and EET production in several cell types. Our exciting preliminary results suggest several lines of evidence for important roles of CYP-derived EETs and possible signajing pathways in eliciting cardioprotection. In cells, hypoxia induces the production of EETs. Preliminary data suggest that exogenously administered EETs increased the activity (phosphorylation) of Epidermal Growth Factor Receptor (EGFR). Furthermore, EETs increased hypoxia-inducible factor-1 (a subunit, HIF-1a), as detected by Western blots in the nuclear fraction. These results suggest several signaling pathways that are important in cardioprotection. For example, EETs may transactivate the EGFR. The activation of HIF-1a by EETs indicates a possible positive feedback of hypoxia-induced CYP-derived EETs, and in turn EETs upregulate HIF-1a which leads to cardioprotection. Other evidence from our studies which suggest mechanisms that might be involved in CYP epoxygenase and EET-induced cardioprotection include reactive oxygen species (ROS), Protein Kinase C-epsilon, the PI3K/Akt signaling pathway, glycogen synthase kinase-beta (GSK3- beta) and the sarcolemmal and mitochondria! ATP-regulated K channels and the mitochondrial permeability transition pore (MPTP). Specifically, we will test the following aims:
Specific Aim 1 : Demonstrate whether CYP epoxygenases and EETs are upregulated by hypoxia in cardiomyocytes. We will determine whether hypoxia induces expression of CYP epoxygenases (isoforms) and production of EETs (isomers). We will detect CYP epoxygenases in hypoxic cardiomyocytes and ischemic heart tissue.
Specific Aim 2 : Demonstrate the functions of EETs in cardiomyocytes using pharmacological agents and exogenous EETs. We will test the effects of exogenous EETs (11,12- and 14,15-EET) to increase the activity of EGFRandHIF-1a.
Specific Aim 3 : Elucidate the EET signaling pathways that lead to cardioprotection. We will investigate the transactivation of EGFR by EETs and the role of ROS in activating EGFR. Furthermore, we will investigate the down stream signaling pathways of EGFR activation by EETs, including the PKC/Akt/TK, p38, or ERK1/2 pathways. We will also investigate whether exogenous EETs activate HIF-1a, and whether it is through the activation of ROS and EGFR. Thus, in the ongoing research plan, we will investigate the mechanistic actions of EETs in cellular systems such as cardiomyocytes (HL-1 cells, rat cardiomyocytes) and in gene knockout or overexpressing mice.

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Specific Aim 1: Demonstrate whether CYP epoxygenases and EETs are upregulated by hypoxia in cardiomyocytes. We will determine whether hypoxia induces expression of CYP epoxygenases (which isoforms) and production of EETs (which isomers). We will detect CYP epoxygenases in hypoxic cardiomyocytes and ischemic heart tissue. These results will be compared with our previous results from in vivo studies. Specific Aim 2: Demonstrate the functions of EETs in cardiomyocytes using pharmacological agents and exogenous EETs. We will test the effects of exogenous EETs (11,12- and 14,15-EET) to increase the activity of EGFR and HIF-1a. These results will be compared with our previous results from in vivo studies. We will also determine the effects of EETs on cell proliferation and migration in isolated cells. Specific Aim 3: Elucidate the EET signaling pathways that lead to cardioprotection. These studies will be divided into two parts, (a) Investigate the transactivation of EGFR by EETs and the role of ROS in activating EGFR. Our preliminary results and previous results by others indicate that the phosphorylation of EGFR is enhanced by treatment of cells by exogenous EETs and that ROS may play an important role in the signaling pathway. Furthermore, we will investigate the down stream signaling pathways of EGFR activation by EETs, including the PKC/Akt/TK, p38, or ERK1/2 pathways, (b) We will investigate whether exogenous EETs activate HIF-1a, and whether it is through the activation of ROS and EGFR. This activation of HIF-1 will indicate the positive feedback of hypoxia-induced CYP epoxygenases (EETs) and EETs in turn activates the HIF-1 that promotes cardioprotection.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37HL074314-10
Application #
8282847
Study Section
Special Emphasis Panel (NSS)
Program Officer
Schwartz, Lisa
Project Start
2003-07-01
Project End
2013-06-30
Budget Start
2012-06-01
Budget End
2013-06-30
Support Year
10
Fiscal Year
2012
Total Cost
$395,477
Indirect Cost
$126,980
Name
Medical College of Wisconsin
Department
Pharmacology
Type
Schools of Medicine
DUNS #
937639060
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
Gross, Garrett J; Hsu, Anna; Gross, Eric R et al. (2013) Factors mediating remote preconditioning of trauma in the rat heart: central role of the cytochrome p450 epoxygenase pathway in mediating infarct size reduction. J Cardiovasc Pharmacol Ther 18:38-45
Gross, Eric R; Hsu, Anna K; Urban, Travis J et al. (2013) Nociceptive-induced myocardial remote conditioning is mediated by neuronal gamma protein kinase C. Basic Res Cardiol 108:381
Gross, Garrett J; Hsu, Anna; Pfeiffer, Adam W et al. (2013) Roles of endothelial nitric oxide synthase (eNOS) and mitochondrial permeability transition pore (MPTP) in epoxyeicosatrienoic acid (EET)-induced cardioprotection against infarction in intact rat hearts. J Mol Cell Cardiol 59:20-9
Du, Lili; Gao, Zhan-Guo; Nithipatikom, Kasem et al. (2012) Protection from myocardial ischemia/reperfusion injury by a positive allosteric modulator of the A? adenosine receptor. J Pharmacol Exp Ther 340:210-7
Gross, Garrett J; Hsu, Anna; Nithipatikom, Kasem et al. (2012) Eribis peptide 94 reduces infarct size in rat hearts via activation of centrally located ýý opioid receptors. J Cardiovasc Pharmacol 59:194-7
Auchampach, John A; Maas, Jason E; Wan, Tina C et al. (2011) Are we putting too much stock in mice? J Mol Cell Cardiol 50:584-5
Gumina, Richard J; Newman, Peter J; Gross, Garrett J (2011) Effect on ex vivo platelet aggregation and in vivo cyclic flow with Na+/H+ exchange inhibition: Gumina, NHE-1 inhibition and platelet aggregation. J Thromb Thrombolysis 31:431-5
Gross, Garrett J; Baker, John E; Moore, Jeannine et al. (2011) Abdominal surgical incision induces remote preconditioning of trauma (RPCT) via activation of bradykinin receptors (BK2R) and the cytochrome P450 epoxygenase pathway in canine hearts. Cardiovasc Drugs Ther 25:517-22
Maas, Jason E; Wan, Tina C; Figler, Robert A et al. (2010) Evidence that the acute phase of ischemic preconditioning does not require signaling by the A 2B adenosine receptor. J Mol Cell Cardiol 49:886-93
Nithipatikom, Kasem; Gross, Garrett J (2010) Review article: epoxyeicosatrienoic acids: novel mediators of cardioprotection. J Cardiovasc Pharmacol Ther 15:112-9

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