PROJECT SUMMARY Emphysema is an important pathologic finding in Chronic Obstructive Lung Disease (COPD), a leading cause of death in the United States and worldwide. Emphysema is characterized by increased DNA damage and the consequences of failed DNA repair: apoptosis, cellular senescence, and inflammation. Cigarette smoke (CS) is the most important etiology for DNA damage in emphysema. Yet, emphysema does not occur universally among smokers and there is little understanding of protective mechanisms that underlie this heterogeneity. Therefore, identifying cellular responses to CS that promote DNA repair may lead to new therapies for this disease. This proposal seeks to understand the role of Macrophage Migration Inhibitory Factor (MIF) in promoting DNA repair and antagonizing the development of emphysema. MIF is a cytokine secreted in response to CS and a critical regulator of multiple signaling pathways. We previously demonstrated that circulating MIF is decreased in patients with COPD, and genetic deletion of murine MIF results in increased susceptibility to CS mediated airspace enlargement. The endothelial cells of MIF-KO mice are particularly susceptible to CS-mediated DNA damage and consequent apoptosis and cellular senescence. The source of protective MIF is unknown. Our preliminary data suggests that MIF promotes DNA repair in endothelial cells exposed to CS extract and that endothelial cells may be an importance source of protective MIF. Based on these findings, we propose the following hypotheses and specific aims: Hypotheses 1. MIF, via its receptor CD74, promotes HR and NHEJ following CS-mediated DNA damage. 2. MIF, via CD74, protects lung endothelial cells from CS-mediated cellular senescence and apoptosis by promoting BRCA1 gene expression. 3. Deletion of MIF in the endothelium is necessary and sufficient to increase murine susceptibility to DNA damage and emphysema. Aims 1. Define the DNA repair mechanism(s) promoted by MIF in lung endothelial cells. 2. Determine the role of BRCA1 in mediating MIF's protective effect. 3. Define the protective role of endothelial-derived MIF in CS-mediated emphysema. Experimental Approach To address Aim 1, we will use primary mouse and human endothelial cells and expose them to CS extract, followed by a recovery period to observe DNA repair. We will characterize DNA damage and repair using single- cell gel electrophoresis, imaging-flow cytometry (Amnis) and host cell reactivation assays. We will use silencing RNA and chemical inhibitors of MIF-CD74 binding to determine if MIF promotes DNA repair via its receptor CD74. In Aim 2, we will characterize the effects of CS on BRCA1 gene expression and protein in primary lung endothelial cells and paraffin-embedded, de-identified human lung tissue samples. We will use chromatin precipitation assays to determine the mechanism via which MIF promotes BRCA1 gene expression, and we will measure consequences of CS extract in endothelial cells, such as apoptos...