CELLULAR MECHANISMS OF PULMONARY ENDOTHELIAL INJURY

肺内皮细胞损伤是成人呼吸窘迫的典型特征 综合征(ARDS)。革兰氏阴性细菌败血症是最常见的 发生ARDS的临床环境，可能是内毒素的结果- 致肺损伤。在此续期申请中，我们建议延长我们的 内毒素对培养的肺内皮细胞影响的观察 达到分子水平。我们建议检验内毒素的假设 暴露会导致内皮细胞内氧化应激增加， 导致白介素1(IL-1)的产生增加导致 抗氧化剂锰超氧化物歧化酶的生产 (MnSOD)(我们已经证明了这个序列中的第一步也是最后一步 发生)。此外，我们认为前列腺素的内毒素刺激 内皮细胞的产生也是由氧化剂介导的， 微血管内皮细胞的主要产物前列腺素E2(PGE2) 细胞，调节IL-1，从而调节MnSOD的诱导。我们建议 不同器官和不同来源内皮细胞的反应差异 肺内不同部位可以用不同的 锰超氧化物歧化酶和前列腺素E_2原生产物及其基因工程 内皮细胞产生更多的MnSOD将使 抵抗内毒素损伤的细胞。为了检验这些假设，我们 将在培养的大大小小的内皮细胞中进行研究 肺部和人脐静脉中的血管。我们将1)澄清 诱导IL-1和MnSOD基因改变的时程 细胞暴露于内毒素后的表达，并确定 这些反应是否被进入细胞的抗氧化剂抑制 内部，2)确定内毒素效应是否由 细胞因子TNFpha(与内毒素一样，通过以下途径刺激IL-1的产生 内皮细胞)或外源性IL-1，3)决定外源性 前列腺素E_2对IL-1和MnSOD基因表达的影响，4)比较 不同物种、器官和部位细胞的内毒素暴露，5) 确定内皮细胞是否通过基因工程产生 增加MnSOD的量可以抵抗内毒素的影响和6) 猪传染性支气管炎内毒素诱导分子机制研究进展 MnSOD基因在内皮细胞中的表达。这些研究顺理成章地 这笔赠款支持的工作在过去五年中得到了支持，并承诺 了解内毒素如何影响内皮细胞以及如何影响内皮细胞 内毒素的有害影响是可以预防的。这样的信息 将有助于开发新的干预措施，可能会发现 最终在临床环境中应用。

Pulmonary endothelial injury typifies the adult respiratory distress syndrome (ARDS). Gram negative bacterial sepsis is the most frequent clinical setting in which ARDS occurs, presumably a result of endotoxin- induced lung injury. In this renewal application, we propose to extend our observations of effects of endotoxin on lung endothelial cells in culture to a molecular level. We propose to test the hypothesis that endotoxin exposure results in increased oxidant stress within endothelial cells which causes increased production of interleukin-1 (IL-1) resulting in increased production of the antioxidant enzyme, manganous superoxide dismutase (MnSOD) (we have demonstrated that the first and last step in this sequence occur). Further, we propose that endotoxin stimulation of prostanoid production by endothelial cells is also oxidant mediated and that prostaglandin E2 (PGE2), a major product of microvascular endothelial cells, modulates IL-1 and thus MnSOD induction. We propose that differences in response of endothelial cells from different organs and from different sites within the lung can be explained by differences in constitutive production of MnSOD and PGE2 and that genetic engineering of endothelial cells to produce increased amounts of MnSOD will render the cells resistant to endotoxin-induced injury. To test these hypotheses we will conduct studies in cultured endothelial cells from large and small vessels in the lungs and from human umbilical veins. We will 1) elucidate the time course of induction of alteration in IL-1 and MnSOD gene expression following exposure of the cells to endotoxin and determine whether the responses are inhibited by antioxidants which access the cell interior, 2) determine whether the endotoxin effect are mimicked by the cytokine TNFalpha (which, like endotoxin, stimulates IL-1 production by endothelial cells) or exogenous IL-1, 3) determine effects of exogenous PGE2 on IL-1 and MnSOD gene expression, 4) compare these responses to endotoxin exposure in cells from different species, organs and sites, 5) determine whether endothelial cells genetically engineered to produce increased amounts of MnSOD are resistant to endotoxin effect and 6) initiate studies of the molecular mechanisms of endotoxin induction of MnSOD gene expression in endothelial cells. These studies follow logically the work supported by this grant over the past five years and promise a new level of understanding of how endotoxin affects endothelial cells and how the deleterious effects of endotoxin can be prevented. Such information will contribute to the development of novel interventions which may find eventual application in the clinical setting.