Mitochondrial DNA Integrity and Endothelial Free Radical Stress

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In several important lung diseases, including emphysema, vascular remodeling after ARDS, and possibly primary pulmonary hypertension, there is a prominent cytotoxic response of pulmonary microvascular endothelial cells (MV ECs) leading to a diminution in capillary density. While there is no doubt that reactive oxygen species play an important role in this response, the specific target(s) of ROS that serve as a sentinel molecule - triggering cell death when the oxidant stress is so severe as to preclude effective recovery or threaten the organism with mutation - is not known. In this regard, an intriguing target of ROS is mitochondrial (mt) DNA. The mitochondrial genome is at least 30-fold more sensitive to oxidative damage than nuclear DNA, and our work during the initial funding period supports the hypothesis that oxidative mtDNA damage is a proximate trigger for lung EC death. If this hypothesis is valid, then mtDNA repair pathways could emerge as a new target for intervention in oxidant-induced MV EC death and capillary rarefaction. However, there is a stark lack of the information about the details of mtDNA repair in MV ECs and other cells. For example, while it is suspected that the base excision repair mechanism is the dominant pathway defending the mitochondrial genome from oxidative damage, the presence of other DNA repair pathway components suggests that a more complicated repair paradigm could be operative. In addition, neither the identities of the enzymes participating in mitochondrial base excision repair nor the rate limiting determinants are known. Against this background, the Aims of this proposal are to: (1) Identify the dominant pathway repairing oxidative damage to the mitochondrial genome in MV ECs; (2) Determine the rate-limiting functional steps in mtDNA repair; and, (3) Establish the critical operational enzymes repairing mtDNA in MV ECs. Collectively, these studies will provide the first detailed understanding of pathways defending the mitochondrial genome in this important lung cell population and determine the suitability of mtDNA repair enzymes to serve as isolated targets for intervention. Importantly, the outcome of these studies also will set the stage for pre-clinical, translational experiments on the ability of augmented mtDNA repair to suppress capillary rarefaction in relevant animal models.