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Activation of Endothelial NADPH Oxidase as the Source of a Reactive Oxygen Species in Lung Ischemia* FREE TO VIEW

A. B. Fisher, MD; A. B. Al-Mehdi, MD, PhD; V. Muzykantov, MD, PhD
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*From the Institute for Environmental Medicine, University of Pennsylvania, Philadelphia, PA. Supported by grant HL41939. PR39 was provided by C. Ross, Kansas State University, and NADPH knock-out mice were provided by M. Dinauer, University of Indiana.

Correspondence to: Aron B. Fisher, MD, Univ of Pennsylvania, I John Morgan Bldg/6068, 3620 Hamilton Walk, Philadelphia, PA 19104

Chest. 1999;116(suppl_1):25S-26S. doi:10.1378/chest.116.suppl_1.25S
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We have used the isolated perfused rat lung to simulate lung ischemia due to vascular obstruction. In this model, ventilation to the ischemic lung continues and tissue oxygenation and cellular adenosine triphosphate content are maintained at normal levels. Nevertheless, lung ischemia leads to increased reactive oxygen species (ROS) generation resulting in tissue lipid and protein oxidation and endothelial injury, as indicated by shedding of angiotensin-converting enzyme. In this study, we evaluated potential pathways as the source of ROS. ROS production with ischemia was unaffected by pretreatment of lungs with allopurinol (100 μM) but was abolished with diphenyliodonium (DPI; 100 μM). (DPI is a flavoprotein inhibitor.) PR-39 showed a concentration-dependent effect with 80% inhibition of ROS production at 10 μM. (PR-39 is a proline/arginine-rich, 39-amino-acid peptide that binds to SH3 domains and has been demonstrated to inhibit assembly of nicotinamide adenine dinucleotide phosphate [NADPH] oxidase in neutrophils by binding to p47phox.) Similar studies in mice with NADPH oxidase “knock-out” (deletion of gp91phox, the membrane-bound cytochrome of the enzyme complex) demonstrated essentially no production of ROS with lung ischemia, in contrast to wild-type mice in which ROS production was similar to that observed with rat lungs. Thus, the results using inhibition at three different sites with DPI, PR-39, and “knock-out” mice are compatible with activation of NADPH oxidase as the source of ROS. Contrasting results were obtained in a model of anoxia/reoxygenation produced by ventilating lungs sequentially with N2 and O2. With anoxia/reoxygenation, ROS production was inhibited by allopurinol but was unaffected by DPI, PR-39, or “knock-out” of NADPH oxidase. In order to localize the cellular site of ROS generation in the ischemia model, we used an in situ fluorescence imaging technique. Dichlorofluorescin fluorescence images from the subpleural region of the lung were obtained with a microscope and a computer-controlled, cooled charge-coupled device camera with graphics control software (Metamorph Imaging Systems; West Chester, PA). Increased dichlorofluorescein fluorescence, indicating increased ROS generation, colocalized with diacetylated low-density lipoprotein (an endothelial specific marker) and was clearly distinct from alveolar epithelial cells (type II) labeled with Nile red. These results indicate that ROS generation in ischemia/reperfusion and anoxia/reoxygenation occur through different pathways and that the ROS-generating pathway with lung ischemia is endothelial NAPDH oxidase.




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