Role of mitochondrial dysfunction in hyperoxia-induced pulmonary vascular endothelial injury

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More than 250,000 veterans are placed on mechanical ventilation annually. These patients often require high fractions of oxygen (hyperoxia) which significantly exacerbates the injury that triggered mechanical ventilation initially. Pulmonary endothelial cells (PECs) are particularly sensitive to hyperoxia, exhibiting increased production rates of mitochondrially-derived reactive oxygen species (mtROS), mitochondrial (mt) dysfunction, pulmonary edema and ultimately increased morbidity/mortality in critically ill patients, but mechanisms are incompletely understood. Cells adapt to stress by increasing both mitochondrial fission and fusion. Our data identify for the first time hyperoxia-enhanced mt-fragmentation in PECs, and decreased expression of mt- fusion and increased expression of mt-fission promoting proteins which underlie the increased mt- fragmentation. In addition, we show that mitochondrial targeted endonuclease repair protein (mt-ENDO-III) protects from hyperoxic PEC loss. Finally, we have demonstrated that inhaled 2% hydrogen gas (H2) can protect against hyperoxia-induced lung injury, and that this protection can be identified by single photon emission computed tomography (SPECT) imaging. The molecular basis of hyperoxia-associated mt- fragmentation and subsequent pulmonary microvascular permeability is the focus of this proposal. Our hypothesis is that hyperoxia-induced pulmonary edema results from mtDNA damage which signals a shift to pro-fission protein expression and mt-fragmentation, leading to increased microvascular permeability and edema. Furthermore, we believe that 2% H2 in atmospheric gases will counteract hyperoxia-evoked pulmonary edema with diminished mtDNA damage and mt-fragmentation. Using novel tools including Dendra- 2 mice, which express a fluorescent protein targeted to the mitochondrial membrane in endothelial cells to quantify mt-fragmentation in intact tissue, recombinant adeno- and lentivirus, siRNA, unique genetically modified rodents, vertical experimental designs from cultured cells to intact animals and human tissue, our work will determine mechanisms linking hyperoxia-induced mtDNA damage, mt-fragmentation and pulmonary edema. Specific Aims: 1) To determine if hyperoxia-induced pulmonary endothelial mtDNA damage modifies expression/activation ratios of specific mt-fission and fusion proteins, thereby enhancing mt-fragmentation, and increasing microvascular permeability. We will use mt-ENDO-III to repair mtDNA damage in cultured PECs and in vivo and measure hyperoxia-induced changes in pro-fission or fusion protein expression, mt-fragmentation, mt-function, monolayer transendothelial electrical resistance (TEER) or filtration coefficient (Kf) as measures of endothelial permeability. 2) To test if pulmonary endothelial mt-fragmentation, independent of mtDNA damage, increases microvascular permeability. Using genetically modified rodents, siRNA and overexpression of pro-fission or fusion protein in cultured PECs and in vivo, we will measure pulmonary endothelial mt-fragmentation, mt-function, and Kf in intact lungs or TEER in cultured PECs. 3) To determine if (i) H2 protects from hyperoxia-induced mtDNA damage, increased mt-fragmentation, or increased microvascular permeability, and (ii) SPECT imaging can identify protection secondary to limited mtDNA damage, diminished mt-fragmentation, or diminished microvascular permeability in vivo. In this translational aim, we will assess the effect of 2% H2 on mtDNA integrity, shifts in pro-fission/fusion proteins, and mt- fragmentation. Genetic manipulations of fission/fusion proteins or ENDO-III will be employed to modify mt- fragmentation, and in vivo SPECT imaging markers of death or oxidoreductive state will identify clinically relevant endpoints associated with changes in pulmonary endothelial mt-fragmentation. Key results will be confirmed in human tissue. This work will provide critical new information about the role of mitochondrial damage in mediating hyperoxia-induced changes in pulmonary microvascular permeability and is expected to lead to mechanism-based approaches to the prevention and treatment of hyperoxia-induced lung disease.