MECHANISM OF RADIATION-INDUCED LUNG INJURY

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The application for an RO1 proposes a new paradigm in normal tissue radiation biology suggesting vascular dysfunction and associated reactive oxygen and nitrogen species (ROS/RNS) production to play an important role in development and progression of radiation-induced lung injury. In the past we have successfully demonstrated: 1. The temporal onset of hypoxia in lungs after both single and fractionated irradiation is associated with a significant decrease in lung perfusion and increased macrophage accumulation and activity. 2. Hypoxia amplifies and perpetuates continuous macrophage associated production of ROS/RNS and profibrogenic/proangiogenic cytokines leading to pulmonary tissue damage and fibrosis. 3. Targeting chronic ROS/RNS production after radiation exposure with long term administration of catalytic manganese porphyrin mimetics of SOD results in reduction of tissue hypoxia, oxidative stress, cytokine production, and development of lung injury. 4. Hypoxia is preceded by early activation of TGF-_, induction of iNOS, vascular dysfunction, increase in HIF1_ regulated proteins (CA9, VEGF), and development of oxidative/nitrasative stress. The present proposal will focus on the mechanisms of early events (within 4 weeks after RT), which we believe are responsible for the incipient cascade of events that eventually lead to permanent pulmonary damage. These events lead to hypoxia, inflammation, fibroproliferation and late vascular damage. Specifically we will determine the mechanism of acute ROS/RNS production in lungs after RT and its role in 1) early vascular dysfunction and 2) activation of transcription factors and signaling pathways that could mediate the development of RT-induced lung injury. The overall goal is to identify new therapeutic targets and plasma markers that can be used to prevent, predict and monitor the development of radiation induced lung injury. Following are the specific aims. Specific Aim 1: To determine the mechanism of early ROS/RNS production and its effect on vascular changes and activation of transcription factors and signaling pathways leading to hypoxia, inflammation, fibroproliferation, and angiogenesis. Hypothesis 1: Radiation induced temporary imbalance of redox regulation in endothelial cells results in sustained increase in O2.- and NO from NADPH oxidase activation, mitochondrial respiration, and iNOS induction. The rapid reaction between these two radicals results in formation of peroxynitrite (ONOO) and depletion of NO, thereby causing vascular dysfunction. Hypothesis 2: Formation of peroxynitrite (ONOO) results in oxidative modification of key signaling proteins (e.g. tyrosine nitration, oxidation of protein sulfhydryls by either S- nitrosylation or oxidation to sulfenic acid) important for the stabilization of HIF-1_, activation of MAPK, NF_B and Wnt/_-catenin signaling, all of which are key components of angiogenesis, inflammation, and fibroproliferation. Specific Aim 2: To determine whether targeting O2.-, NO., ONOO- directly and/or indirectly by inhibiting metabolic sources of their production will reduce vascular dysregulation and activation of transcription factors and signaling pathways. Hypothesis 1: Targeting O2.-, NO., and ONOO- with MnTE-2-PyP5+ and/or specific inhibitors of NADPH oxidase (apocynin) and iNOS (L-Name, aminoguanidine) will reduce vascular dysregulation and inhibit nitration and activation of signaling pathways and transcription factors. Hypothesis 2: Targeting upstream activators of NADPH oxidase (TGF, Angiotensin II (AngII) and Endothelin-1 (ET-1) will reduce vascular dysregulation and oxidative/nitrosative stress. -Apocyanin alone -Radiation plus L-Name to target iNOS -L-Name alone -Radiation plus SOD Mimetic  MnTE-2-PyP5+ plus TGF-_ antibody -Radiation plus SOD Mimetic  MnTE-2-PyP5+ plus Apocyanin or L-Name (most effective from solely application)Treatment will be started directly after irradiation with continuous administration over 2 weeks with an osmotic pump (based on results of our previous funding period). The effects of these different treatments will be studied using the same parameters as described in Specific Aim 1. Time points: Relevant time points from results of specific aim 1, approximately 6 different time points Estimation of DSA studies at CIVM 12 time points single dose plus 13 time points fractionated radiation = 25 time points 5 animals per time point 125 scans Specific aim 2: 11 treatment arms Max 6 time points 5 animals per time point 330 scans Total: 455 scans in 4 years Per year 115 scans per year, 5 scans per day: 23 scan days per year Per month 10 scans per month, 2 scan days per month