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.
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.
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