Ionizing irradiation-induced pulmonary damage limits effective radiation dose escalation in the treatment of lung and esophageal cancer. The C57BL/6J mouse model of irradiation-induced late organizing alveolitis/fibrosis closely follows the parameters of human pulmonary irradiation damage. We have demonstrated that bone marrow origin macrophages migrate to the mouse lung at the time of up regulation of endothelial cell VCAM-1 and ICAM-1 at 120-140 days after pulmonary irradiation, followed by TGF2-mediated migration and proliferation of bone marrow origin myofibroblast progenitor cells (bone marrow stromal cells/mesenchymal stem cells), which contribute to pulmonary fibrosis. Both migrations are significantly reduced by intrapulmonary manganese superoxide dismutase-plasmid liposome (MnSOD-PL) gene therapy. We now propose to elucidate the cellular and molecular mechanism(s) of initiation of the late pulmonary lesion and optimize its amelioration by MnSOD-PL. We will use female C57BL/6J mice and HSV-TK (Herpes Simplex Virus-Thymidine Kinase)-CCSP+ transgenic mice (which have gancyclovir sensitive Clara Cell Secretory Protein positive lung stem cells) and are chimeric for male GFP+ bone marrow. The first specific aim tests the hypothesis that inhalation MnSOD-PL gene therapy facilitates lung irradiation through enhancement of migration of marrow origin reparative alveolar and bronchial stem cell progenitors. The second specific aim tests the hypothesis that periodic repeat MnSOD-PL inhalation gene therapy decreases late pulmonary fibrosis through decreased migration of marrow origin myofibroblasts. The third specific aim tests the hypothesis that adding inhalation of mitochondrially targeted catalase plasmid liposomes to MnSOD-PL will enhance lung radiation protection. Methods include nebulizer-inhalation of mt-catalase-PL, and MnSOD-PL in pulmonary irradiated chimeric mice, continuous BUDR labeling by mini-osmotic pump, cell sorting, and histopathology. These studies should define critical steps in irradiation pulmonary fibrosis and identify new targets for therapeutic intervention, thereby decreasing patient side effects and facilitating dose escalation in the initial treatment or retreatment of recurrent thoracic cancers. Project Narrative This irradiation application addresses a critical problem in managing the side effects of thoracic radiotherapy for lung and esophagus cancer.