Lipid Modulators of Pulmonary Vascular Tone

描述（由申请人提供）：我们的研究集中在Cyp 4及其脂质产物20-HETE在肺血管功能和生物学中的作用。在过去的4年中，我们确定了Cyp 4/20-HETE在调节肺动脉内皮细胞（PAEC）eNOS，介导VEGF诱导的小肺动脉舒张中的独特作用。除了对血管张力的影响外，Cyp 4还促进全身血管床中的血管生成。我们的初步数据表明，20-HETE在PAEC中具有一种新的抗凋亡活性，20-HETE可防止饥饿诱发的caspase 3活性增加。除了公认的造成氧化损伤的能力之外，活性氧（ROS）现在被认为在信号传导重要的生理过程（包括细胞生长和血管生成）中发挥关键作用。因此，我们探索了Cyp 4/20-HETE影响PAEC中ROS产生和内皮细胞生长的潜力。我们的数据首次证明了20-HETE/Cyp 4诱导的分离PAEC以及完整肺中ROS的增加。在我们的实验中，NADPH氧化酶和线粒体呼吸似乎是20-HETE处理的肺内皮细胞中重要的ROS来源。ROS的抑制消除了20-HETE对胱天蛋白酶3活性的保护作用。这些观察结果提示Cyp 4/20- HETE的至少一个作用可能是通过刺激ROS产生来保护肺动脉内皮细胞免于凋亡。通过这些作用，20-HETE/Cyp 4可能有助于维持肺血管床的完整性。我们将测试20-HETE/Cyp 4增加PAEC中的ROS并以ROS依赖性方式保护免于凋亡的总体假设。在第一个目标中，我们将研究Cyp 4/20-HETE通过其增加PAEC的ROS产生的机制，在体外和体内，重点是NADPH氧化酶和线粒体效应。在第二个目标中，我们将研究20-HETE刺激ROS产生的功能意义，探索ROS在体内和体外保护内皮细胞免受凋亡的作用。我们将与自由基和血管生物学中心的合作者和专业知识一起，使用最先进的技术来研究Cyp 4/20-HETE在肺内皮中的这些令人兴奋的新功能。

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.