Recent data demonstrate that the cardioprotective effects of late preconditioning (PC) are mediated by COX-2, an enzyme that is generally thought to be detrimental. These findings reveal a novel, unsuspected function of COX-2. At present, the regulation of this protein in intact heart and its actions during myocardial ischemia/reperfusion are essentially unknown. The objective of this proposal is to elucidate the molecular mechanisms underlying the newly discovered cardioprotective role of COX-2. The applicant will test the hypothesis that diverse PC stimuli upregulate COX-2 through a complex signal transduction cascade that includes PKC-epsilon, Src/Lck, and multiple transcription factors. Three different forms of late PC elicited by a pathological (ischemia), physiological (exercise) and pharmacological (NO donor) stimulus will be interrogated in an effort to develop a comprehensive pathophysiologic paradigm. A broad multidisciplinary approach will be used that will combine diverse techniques (integrative physiology, protein chemistry, mass spectrometry, biochemistry, cell biology, molecular biology, gene targeting, and transgenesis) and will integrate genetic and biochemical information at the molecular level with the physiological information at the whole animal level. Twenty-three genetically engineered mouse lines will be studied. Unequivocal evidence for or against an obligatory role of three specific kinases (PKC-epsilon, Src, Lck) in COX-2 upregulation will be provided by the use of two novel PKC-epsilon transgenic mouse lines and Src and Lck knockout mice. The role of PKC-epsilon in recruiting COX-2 will be conclusively established by determining the effects of specific transgenic inhibition of this isozyme. The role of Src PTKs in the transcriptional versus posttranslational modulation of COX-2 will be discerned by comparing inhibition of these kinases during the PC stimulus (on day 1) versus 24 hours after the PC stimulus (on day 2). targeted disruption of the Src and Lck genes will be employed to conclusively establish the specific function of individual PTKs. The role of NF-kappaB in COX-2 regulation will be unequivocally determined by targeted disruption of three specific NF-kappaB subunits (p50, RelB, c-Rel) and by the use of a novel transdominant IkappaBa mutant mouse with cardiac-specific repression of NF-kappaB activity. The specific modulatory proteins that govern COX-2 gene expression will be systematically identified by targeted genetic ablation of the main factors known to bind to the COX-2 promoter (AP-1, STAT5a, STAT5b, CREB, NF-IL-6, CEBPd, and Ets-1). The Src PTK-dependent posttranslational modulation of COX-2 24 hours after PC will be elucidated by identifying the precise phosphorylation site(s) on COX-2 with HPLC coupled-electrospray ionization mass spectrometry. Finally, the specific prostanoid receptor subtype(s) responsible for COX-2-dependent protection will be systematically interrogated by targeted genetic disruption of all known prostaglandin receptors (DP, EP1, EP2, EP3, EP4, F, and IP receptors). this project should produce important new insights into the molecular mechanisms that underlie COX-2-dependent cytoprotection, late PC, and the role of COX-2 in cardiac pathophysiology in general.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Cardiovascular and Renal Study Section (CVB)
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Liang, Isabella Y
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University of Louisville
Internal Medicine/Medicine
Schools of Medicine
United States
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Tang, Xian-Liang; Li, Qianhong; Rokosh, Gregg et al. (2016) Long-Term Outcome of Administration of c-kit(POS) Cardiac Progenitor Cells After Acute Myocardial Infarction: Transplanted Cells Do not Become Cardiomyocytes, but Structural and Functional Improvement and Proliferation of Endogenous Cells Persist for at L Circ Res 118:1091-105
Tokita, Yukichi; Tang, Xian-Liang; Li, Qianhong et al. (2016) Repeated Administrations of Cardiac Progenitor Cells Are Markedly More Effective Than a Single Administration: A New Paradigm in Cell Therapy. Circ Res 119:635-51
Tang, Xian-Liang; Rokosh, Gregg; Sanganalmath, Santosh K et al. (2015) Effects of Intracoronary Infusion of Escalating Doses of Cardiac Stem Cells in Rats With Acute Myocardial Infarction. Circ Heart Fail 8:757-65
Bolli, Roberto; Tang, Xian-Liang; Sanganalmath, Santosh K et al. (2013) Intracoronary delivery of autologous cardiac stem cells improves cardiac function in a porcine model of chronic ischemic cardiomyopathy. Circulation 128:122-31
Sanganalmath, Santosh K; Bolli, Roberto (2013) Cell therapy for heart failure: a comprehensive overview of experimental and clinical studies, current challenges, and future directions. Circ Res 113:810-34
Guo, Yiru; Flaherty, Michael P; Wu, Wen-Jian et al. (2012) Genetic background, gender, age, body temperature, and arterial blood pH have a major impact on myocardial infarct size in the mouse and need to be carefully measured and/or taken into account: results of a comprehensive analysis of determinants of infarc Basic Res Cardiol 107:288
Tang, Xian-Liang; Rokosh, Gregg; Sanganalmath, Santosh K et al. (2010) Intracoronary administration of cardiac progenitor cells alleviates left ventricular dysfunction in rats with a 30-day-old infarction. Circulation 121:293-305
Dai, Shujing; Yuan, Fangping; Mu, Jingyao et al. (2010) Chronic AMD3100 antagonism of SDF-1alpha-CXCR4 exacerbates cardiac dysfunction and remodeling after myocardial infarction. J Mol Cell Cardiol 49:587-97
Tang, Xian-Liang; Rokosh, D Gregg; Guo, Yiru et al. (2010) Cardiac progenitor cells and bone marrow-derived very small embryonic-like stem cells for cardiac repair after myocardial infarction. Circ J 74:390-404
Li, Qianhong; Guo, Yiru; Ou, Qinghui et al. (2009) Gene transfer of inducible nitric oxide synthase affords cardioprotection by upregulating heme oxygenase-1 via a nuclear factor-{kappa}B-dependent pathway. Circulation 120:1222-30

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