ENDOTHELIAL CELL DERIVED NEUTROPHIL CHEMOATTRACTANTS

拟议研究的总体目标是调查 血管内皮细胞调节自身环境的潜力 与肺血管损伤相关的条件，这是 发病率和死亡率。内皮细胞损伤与中性粒细胞 蓄积可能是本病发病机制中的重要事件。 我们已经找到了暴露在组胺中的初步证据， 血管紧张素II、滑石粉或低氧环境刺激内皮细胞 在组织培养中释放刺激中性粒细胞迁移的物质 在改良的博伊登小室试验中。环氧合酶的进一步研究 和脂氧合酶抑制剂以及反相高密度脂氧合酶的初步检测 高效液相色谱分析表明，这些物质 花生四烯酸的脂氧合酶代谢产物。刻画、提纯和 鉴定内皮细胞来源的中性粒细胞趋化活性， 我们将检查：1)热稳定性，2)在水中的溶解度或 非水溶剂，3)对蛋白酶的稳定性，4)结合的能力 ~3H-花生四烯酸代谢物，6)反相进一步纯化 化学诱导剂活性峰在初始高效液相色谱上的解析 7)纯化产物的扫描紫外光谱分析 与已知标准光谱相比较的级分。要定义 与中性粒细胞趋化物质产生相关的条件 将决定：1)内皮细胞类型对产量的影响 使用牛主动脉、牛肺动脉和人脐静脉 2)检测内皮细胞的特异性 血管平滑肌细胞和IMR-90成纤维细胞的作用 内皮细胞单层的融合程度。此外, 我们将比较内皮细胞来源的中性粒细胞的性质 组胺、血管紧张素II、 滑石粉和缺氧--可能通过不同的方式影响内皮细胞 手段，以便提供对可能引发 与中性粒细胞趋化物质释放相关的代谢过程。 更好地了解内皮细胞调节其功能的能力 通过这些研究获得的自己的环境可能会导致更有效的 各种形式血管损伤的预防和治疗方法。

The overall objective of the proposed research is to investigate the potential of the endothelial cell to regulate its own environment in conditions associated with lung vascular injury, an important cause of mobidity and mortality. Endothelial cell injury and neutrophil accumulation may be important events in the pathogenesis of this disorder. We have developed preliminary evidence that exposure to histamine, angiotensin II, talc, or a hypoxic environment stimulates endothelial cells in tissue culture to release substances that stimulate neutrophil migration in a modified Boyden chamber assay. Further studies with cyclo-oxygenase and lipoxygenase inhibitors and initial assays with reverse phase high performance liquid chromatography (HPLC) suggest that these substances are lipoxygenase metabolites of arachidonic acid. To characterize, purify and identify the endothelial cell derived neutrophil chemoattractant activity, we will examine: 1) heat stability, 2) solubility in aqueous or non-aqueous solvents, 3) stability to proteases, 4) ability to incorporate 3H-arachidonic acid metabolites, 6) further purification of reverse phase HPLC of peaks of chemoattractant activity resolved on the initial HPLC analysis, and 7) scanning ultraviolet spectrum analysis of the purified fractions as compared to the spectrum of known standards. To define the conditions associated with generation of neutrophil chemoattractant, we will determine: 1) the effect of endothelial cell type on production by using bovine aortic, bovine pulmonary arterial, and human umbilical vein endothelial cells; 2) the specificity for endothelial cells by testing vascular smooth muscle cells and IMR-90 fibroblasts, and 3) the effect of the degree of confluence of the endothelial cell monolayer. In addition, we will compare the nature of endothelial cell derived neutrophil chemoattractant induced by the four agents - histamine, angiotensin II, talc, and hypoxia - which likely influence endothelial cells by different means in order to provide insights into the events which might initiate the metabolic process associated with release of neutrophil chemoattractants. Better understanding of the ability of the endothelial cell to regulate its own environment obtained by these studies might lead to more effective methods of prevention and treatment of various forms of vascular injury.