Lipid Modulators of Pulmonary Vascular Tone

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DESCRIPTION (provided by applicant): Our studies have focused on the role of Cyp4 and its lipid product, 20-HETE in pulmonary vascular function and biology. Over the last 4 years, we identified a unique role for Cyp4/20-HETE in regulating pulmonary arterial endothelial cell (PAEC) eNOS, mediating VEGF-induced relaxation of small pulmonary arteries. In addition to effects on vascular tone, Cyp4 promotes angiogenesis in systemic vascular beds. Our preliminary data demonstrate a novel, anti-apoptotic activity of 20-HETE in PAECs, with 20-HETE protecting against starvation-evoked increases in caspase 3 activity. Beyond their well recognized capacity to inflict oxidative injury, reactive oxygen species (ROS) are now understood to play key roles in signaling vital physiologic processes including cell growth and angiogenesis. Therefore we explored the potential of Cyp4/20-HETE to impact ROS production and endothelial cell growth in PAECs. Our data demonstrate for the first time 20-HETE/Cyp4-induced increases in ROS in isolated PAECs as well as in intact lungs. Both NADPH oxidase and mitochondrial respiration appear to be important ROS sources in pulmonary endothelium treated with 20-HETE in our experiments. Inhibition of ROS eliminates the protective effect of 20-HETE on caspase 3 activity. These observations raise the suggestion that at least one role of Cyp4/20- HETE may be to protect pulmonary arterial endothelial cells against apoptosis via stimulation of ROS production. By such actions, 20-HETE/Cyp4 may contribute to sustaining the integrity of the pulmonary vascular bed. We will test the overall hypothesis that 20-HETE/Cyp4 increases ROS in PAECs and protects against apoptosis in a ROS-dependent manner. In the first aim, we will investigate the mechanisms through which Cyp4/20-HETE increases ROS production of PAECs, both in vitro and in vivo, with a focus on NADPH oxidase and mitochondrial effects. In the second aim, we will study the functional implications of 20-HETE stimulated ROS production, probing the role of ROS in protection against apoptosis of endothelium in vivo and in vitro. Together with our collaborators and expertise available in the Free Radical and Vascular Biology Centers, we will use a combination of state of the art techniques to study these exciting and novel functions of Cyp4/20-HETE in lung endothelium.