INFLAMMATORY CYTOKINE EFFECTS ON CELL ADHESION IN PULMONARY VASCULAR EPITHELIUM

炎症细胞因子对肺血管上皮细胞粘附的影响

• 批准号: 6202501
• 负责人: LEWIS H ROMER
• 金额: $ 19.62万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-12-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6202501
• 关键词: biological signal transduction; cell adhesion; cell adhesion molecules; cytokine; extracellular matrix proteins; gel mobility shift assay; gene expression; genetic promoter element; genetic transcription; human tissue; immunoelectron microscopy; immunogenetics; inflammation; laboratory mouse; laboratory rabbit; nuclear runoff assay; protein kinase; pulmonary artery; receptor binding; respiratory epithelium; respiratory infections; selectins; tissue /cell culture; transcription factor; vascular endothelium

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

拟议的研究将检查炎症对 内皮P-选择素、PECAM-1和整合素介导的粘附。 粘附分子将在培养的人内皮细胞中进行研究 暴露于重组人细胞因子或培养基中 由病毒感染的人支气管上皮细胞调节。 这些体外炎症模型将允许研究粘连 与生长因子的变化分离的分子反应， 细胞因子水平和底物递送，使整个器官和 全动物研究 转录，翻译后加工， 细胞骨架相互作用和相关的信号转导途径 与这些主要内皮细胞粘附分子的关系将被研究。 初步数据表明TNF α诱导P-选择素转录 在人类内皮细胞中。 具体目标1是表征 细胞因子诱导人P-选择素表达动力学及机制 血管内皮 这将通过刺激人类 脐静脉和肺动脉内皮细胞炎症 介质和确定P-选择素上调的动力学 使用北方印迹、原位杂交和蛋白质 测定。 刺激特异性启动子区和转录因子 对于P-选择素诱导，然后将使用截短的 启动子/报告基因构建体，凝胶迁移率测定，和核 连续研究。 我们以前的工作和初步数据表明，炎症 细胞因子改变细胞间定位和细胞骨架 PECAM-1在人内皮细胞中的结合。 具体目标二是 描述再分配的机制。 PECAM-1 将使用免疫荧光在空间上定义再分布， 免疫电镜 PECAM-1的细胞骨架结合 从暴露于炎症介质的内皮细胞中， 定量，并与特定的细胞骨架配体的相互作用将被 测定 细胞因子诱导的PECAM-1磷酸化变化和 细胞骨架配体结合亲和力的相关变化将 测定了 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粘附 proteins. 炎性细胞因子作用过程中粘着斑激酶的激活状态 暴露将与FAK和其他蛋白的酪氨酸磷酸化进行比较。 细胞骨架蛋白 将通过以下方法测量粘着斑动力学： FAK正常和改变的内皮细胞的共聚焦显微镜 细胞因子治疗期间的活性。 之间的直接相互作用 将研究酪氨酸应答转录因子和FAK表达的变化。 通过凝胶迁移率分析测量。 该研究计划的长期目标是确定机制 血管内皮细胞粘附发生的潜在变化 病毒性呼吸道感染 这些调查可能导致 治疗性调节由以下原因引起的炎性肺损伤 儿童呼吸道病毒感染