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: 6273176
• 负责人: LEWIS H ROMER
• 金额: $ 19.06万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-12-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6273176
• 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

拟议的调查将检查炎症对