INFLAMMATORY CYTOKINE EFFECTS ON CELL ADHESION IN PULMONARY VASCULAR EPITHELIUM

拟议的调查将检查炎症对 内皮P-选择素、PECAM-1和整合素介导的黏附。 将在培养的人内皮细胞中研究黏附分子 暴露在重组人细胞因子或培养液中的 由感染病毒的人支气管上皮细胞调节。 这些体外炎症模型将使粘连研究成为可能。 从生长因子的变化中分离出来的分子反应， 细胞因子水平和底物传递使整个器官和 完整的动物研究。转录，翻译后处理， 细胞骨架相互作用及相关的信号转导途径 有了这些主要的内皮细胞黏附分子将被研究。 初步数据表明，肿瘤坏死因子α可诱导P-选择素转录 在人类内皮细胞中。具体目标1是描述 P-选择素在人体内的细胞因子诱导动力学及机制 血管内皮细胞。这将通过刺激人类来实现 炎性脐静脉和肺动脉内皮细胞 介质与P-选择素上调动力学的测定 Northern印迹、原位杂交和蛋白质转录 化验。刺激特异性启动子区域和转录因子 对于P-选择素的诱导，将使用截断 启动子/报告结构、凝胶移位迁移率分析和核 连续的研究。 我们之前的工作和初步数据表明，炎症性 细胞因子改变细胞间定位和细胞骨架 PECAM-1在人内皮细胞中的相关性。具体目标2是 描述重新分配的机制。PECAM-1 将使用免疫荧光在空间上定义重新分布和 免疫电子显微镜。PECAM-1的细胞骨架结合 从内皮细胞暴露于炎症介质将是 与特定细胞骨架配体的相互作用将被量化 下定决心。细胞因子诱导的PECAM-1磷酸化和 细胞骨架配体结合亲和力的相关变化将是 量过了。 具体目标3是检查炎症介质对 FAK信号转导与内皮细胞与细胞外基质的黏附 蛋白质。FAK在炎性细胞因子中的激活状态 暴露将与FAK和其他酪氨酸磷酸化进行比较 细胞骨架蛋白。焦点粘连动力学将通过以下方法测量 FAK正常和改变的内皮细胞的共聚焦显微镜观察 细胞因子治疗期间的活性。之间的直接交互作用 细胞因子反应性转录因子和FAK的表达 通过凝胶迁移率分析进行测量。 研究计划的长期目标是确定机制 血管内皮细胞黏附的潜在变化 在病毒呼吸道感染期间。这些调查可能会导致 慢性阻塞性肺疾病炎性肺损伤的治疗调节 儿童呼吸道病毒感染

The proposed investigations will examine the effects of inflammation on endothelial P-selectin, PECAM-1, and integrin-mediated adhesion. Adhesion molecules will be studied in cultured human endothelial cells that are exposed to recombinant human cytokines or culture medium conditioned by human bronchial epithelial cells infected by viruses. These in vitro models of inflammation will allow the study of adhesion molecule responses in isolation from the variations in growth factors, cytokine levels, and substrate delivery that complicate whole organ and whole animal studies. The transcription, post-translational processing, cytoskeletal interactions, and signal transduction pathways associated with these major endothelial cell adhesion molecules will be studied. Preliminary data indicate that TNFalpha induces P-selectin transcription in human endothelial cells. Specific aim 1 is to characterize the kinetics and mechanisms of cytokine induction of P-selectin in human vascular endothelium. This will be accomplished by stimulating human umbilical vein and pulmonary artery endothelial cells with inflammatory mediators and determining the kinetics of upregulation of P-selectin transcription using northern blotting, in situ hybridization and protein assays. Stimulus-specific promoter regions and transcription factors for P-selectin induction will then be identifies using truncated promoter/reporter constructs, gel shift mobility assays, and nuclear run-on studies. Our previous work and preliminary data demonstrated that inflammatory cytokines alter the intercellular localization and cytoskeletal association of PECAM-1 in human endothelial cells. Specific aim 2 is to characterize the mechanisms of the redistribution. PECAM-1 redistribution will be spatially defined using immunofluorescence and immuno-electron microscopy. The cytoskeletal association of PECAM-1 from endothelial cells exposed to inflammatory mediators will be quantified, and interactions with specific cytoskeletal ligands will be determined. Cytokine-induced changes in PECAM-1 phosphorylation and associated changes in cytoskeletal ligand-binding affinities will be measured. Specific aim 3 is to examine the effects of inflammatory mediators on FAK signalling and endothelial cell adhesion to extracellular matrix proteins. The activation state of FAK during inflammatory cytokine exposure will be compared with tyrosine phosphorylation of FAK and other cytoskeletal proteins. Focal adhesion dynamics will be measured by confocal microscopy in endothelial cells with normal and altered FAK activity during cytokine treatment. Direct interactions between cytokine-responsive transcription factors and FAK expression will be measured by gel shift mobility analysis. The long term goal of the research program is to define the mechanisms underlying changes in vascular endothelial cell adhesion that occur during viral respiratory infections. These investigations may lead to the therapeutic modulation of inflammatory lung injury due to respiratory viral infection in childhood