Hypoxic pulmonary vasoconstriction (HPV) helps to optimize lung gas exchange, but it contributes to pulmonary hypertension in hypoxic lung disease. 2 opposing models have emerged to explain the underlying mechanism of O2 sensing in HPV. 1 proposes that hypoxia decreases reactive oxygen species (ROS) generation, shifting the cytosol to a more reduced state. The other proposes that hypoxia stimulates ROS, generating an oxidant signal in the cytosol. Resolution of this debate has been hindered by a lack of tools to assess intracellular redox.
In Aim 1 we will use novel redox-dependant Fluorescence Resonance Energy Transfer (HSP-FRET) and RoGFP1 probes to assess redox in normoxic and hypoxic pulmonary vascular cells. We hypothesize that increased ROS come from the mitochondrial electron transport chain (ETC). We will target overexpression of antioxidant enzymes to mitochondrial matrix or the cytosol to determine which compartments participate in redox signaling.
Aim 2 will determine which ETC complexes contribute to ROS generation by using short hairpin interfering RNA (shRNA) to suppress expression of critical ETC subunits. We predict that oxidant signals will be attenuated when the subunits required for ROS generation are suppressed.
Aim 3 will test the relationship between mitochondrial ROS generation and functional responses to hypoxia in pulmonary artery (PA) myocytes (increase in cytosolic Ca2+) and in PA endothelial cells (increased activation of Hypoxia Inducible Factor-1 and increased expression of endothelin-1). We predict that inhibiting the propagation of ROS signals from mitochondria to cytosol (by targeted overexpression of antioxidant enzymes) or preventing their generation (by shRNA suppression of critical ETC subunits) will abrogate the functional responses to hypoxia in both cell types. Collectively, these studies will test whether a common O2 sensing mechanism functions in PA myocytes and endothelial cells to trigger their diverse responses in HPV.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL079650-01A1
Application #
6966821
Study Section
Respiratory Integrative Biology and Translational Research Study Section (RIBT)
Program Officer
Denholm, Elizabeth M
Project Start
2005-07-01
Project End
2010-06-30
Budget Start
2005-07-01
Budget End
2006-06-30
Support Year
1
Fiscal Year
2005
Total Cost
$341,446
Indirect Cost
Name
Northwestern University at Chicago
Department
Pediatrics
Type
Schools of Medicine
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
Smith, Kimberly A; Waypa, Gregory B; Schumacker, Paul T (2017) Redox signaling during hypoxia in mammalian cells. Redox Biol 13:228-234
Waypa, Gregory B; Smith, Kimberly A; Schumacker, Paul T (2016) O2 sensing, mitochondria and ROS signaling: The fog is lifting. Mol Aspects Med 47-48:76-89
Waypa, Gregory B; Marks, Jeremy D; Guzy, Robert D et al. (2013) Superoxide generated at mitochondrial complex III triggers acute responses to hypoxia in the pulmonary circulation. Am J Respir Crit Care Med 187:424-32
Waypa, Gregory B; Osborne, Scott W; Marks, Jeremy D et al. (2013) Sirtuin 3 deficiency does not augment hypoxia-induced pulmonary hypertension. Am J Respir Cell Mol Biol 49:885-91
Sabharwal, Simran S; Waypa, Gregory B; Marks, Jeremy D et al. (2013) Peroxiredoxin-5 targeted to the mitochondrial intermembrane space attenuates hypoxia-induced reactive oxygen species signalling. Biochem J 456:337-46
Farrow, Kathryn N; Lee, Keng Jin; Perez, Marta et al. (2012) Brief hyperoxia increases mitochondrial oxidation and increases phosphodiesterase 5 activity in fetal pulmonary artery smooth muscle cells. Antioxid Redox Signal 17:460-70
Mungai, Paul T; Waypa, Gregory B; Jairaman, Amit et al. (2011) Hypoxia triggers AMPK activation through reactive oxygen species-mediated activation of calcium release-activated calcium channels. Mol Cell Biol 31:3531-45
Loor, Gabriel; Kondapalli, Jyothisri; Iwase, Hirotaro et al. (2011) Mitochondrial oxidant stress triggers cell death in simulated ischemia-reperfusion. Biochim Biophys Acta 1813:1382-94
Schumacker, Paul T (2011) SIRT3 controls cancer metabolic reprogramming by regulating ROS and HIF. Cancer Cell 19:299-300
Wedgwood, Stephen; Lakshminrusimha, Satyan; Fukai, Tohru et al. (2011) Hydrogen peroxide regulates extracellular superoxide dismutase activity and expression in neonatal pulmonary hypertension. Antioxid Redox Signal 15:1497-506

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