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SHEAR STRESS MODULATION OF ENDOTHELIAL WOUND HEALING

内皮伤口愈合的剪切应力调节

基本信息

批准号：
6615107
负责人：
MARIA LUIZA C ALBUQUERQUE
金额：
$ 15.77万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-08-01 至 2004-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6615107
关键词：
RNase protection assay biological signal transduction biomechanics blocking antibody cadherins fibroblast growth factor immunocytochemistry integrins northern blottings phosphorylation tyrosine vascular endothelium western blottings wound healing

项目摘要

Endothelial damage can occur in vivo in various conditions as a result of pathological disease entities such as chronic hypertension, atherosclerosis, or during invasive medical procedures. Wounding injuries to the vascular endothelium require a number of proteins to interact with the cell cytoskeleton for appropriate cellular movement. In addition to biochemical signaling, endothelial cells, interfaced between the flow of blood and the vascular wall, require biomechanical forces such as shear stress to direct their cellular movement. Biomechanical forces can directly influence endothelial cell structure and function, acutely and chronically, therefore constituting a novel paradigm of endothelial cell activation. However, the mechanisms by which shear forces affect adhesion proteins and growth factors, such as basic fibrobast growth factor (bFGF), for cellular movement remain elusive. We have preliminary data to suggest that laminar shear stress enhances human umbilical vein and coronary artery endothelial cells wound closure by mechanisms involving cell migration, spreading, and enhancement of specific adhesion proteins. In this proposal, the P.I. addresses the overall hypothesis that shear stress enhance the rate of endothelial cell wound closure by mechanisms involving expression of cell-cell (cadherin) and cell-matrix (integrin) adhesion proteins, and bFGF production. Four specific aims are proposed. In Specific Aim 1, we will characterize the relationship between shear stress and wound closure in endothelial cell monolayers. Measurements of wound width, cell area, and internuclear distances will indicate cell spreading and migration at the wound edge. In Specific Aim 2, we will determine the functional significance of the relationship between shear stress and adhesion molecules during wound closure. Western blots of beta1 integrin chain and VE-cadherin protein expression and Northern blots of mRNA levels will be used to determine the adaptation response to shear stress during wound closure. Immunocytochemistry will be used to detect changes in the pattern of adhesion proteins during the wound healing process. In Specific Aim 3, the mechanisms by which integrins and cadherins influence bFGF expression during wound closure will be investigated. Western blots for bFGF production, ribonuclease protection assays for bFGF mRNA will be used to determine the adaption response to shear stress. Effects of activating and blocking antibodies to beta1 integrin and VE-cadherin on bFGF expression will be assessed during wound closure. In Specific Aim 4, we will examine the effect of shear stress on proteins related to actin-associated signaling. Tyrosine phosporylation and translocation of the p120 armadillo protein for enhancement of bFGF production will be assessed. Investigation of the mechanisms underlying endothelial repair during shear stress may lead to better strategies for the management of patients suffering from a variety of cardiovascular disorders.

内皮损伤可在体内由于病理性疾病实体如慢性高血压、动脉粥样硬化或在侵入性医疗程序期间在各种条件下发生。血管内皮的创伤需要许多蛋白质与细胞细胞骨架相互作用以进行适当的细胞运动。除了生物化学信号传导之外，在血流和血管壁之间连接的内皮细胞需要生物力学力（例如剪切应力）来引导其细胞运动。生物力学力可以直接影响内皮细胞的结构和功能，急性和慢性，因此构成了一个新的范式内皮细胞活化。然而，剪切力影响粘附蛋白和生长因子，如碱性成纤维细胞生长因子（bFGF），细胞运动的机制仍然难以捉摸。我们有初步的数据表明，层流剪切应力增强人脐静脉和冠状动脉内皮细胞伤口愈合的机制，涉及细胞迁移，蔓延，并增强特定的粘附蛋白。在这份提案中，P.I.阐述了剪切应力通过涉及细胞-细胞（钙粘蛋白）和细胞-基质（整联蛋白）粘附蛋白的表达以及bFGF产生的机制来提高内皮细胞伤口闭合速率的总体假设。提出了四个具体目标。在具体目标1中，我们将描述内皮细胞单层中剪切应力和伤口闭合之间的关系。伤口宽度、细胞面积和核间距的测量将指示伤口边缘的细胞扩散和迁移。在具体目标2中，我们将确定伤口闭合过程中剪切应力和粘附分子之间关系的功能意义。将使用β 1整联蛋白链和VE-钙粘蛋白蛋白表达的Western印迹和mRNA水平的北方印迹来确定伤口闭合期间对剪切应力的适应性反应。免疫细胞化学将用于检测伤口愈合过程中粘附蛋白模式的变化。在具体目标3中，将研究创伤闭合过程中整合素和钙粘蛋白影响bFGF表达的机制。将使用bFGF产生的Western印迹、bFGF mRNA的核糖核酸酶保护测定来确定对剪切应力的适应性反应。将在伤口闭合期间评估β 1整联蛋白和VE-钙粘蛋白的激活和阻断抗体对bFGF表达的影响。在具体目标4中，我们将研究剪切应力对与肌动蛋白相关信号传导相关的蛋白质的影响。将评估p120犰狳蛋白的酪氨酸磷酸化和易位以增强bFGF的产生。研究切应力作用下内皮细胞修复的机制可能会为治疗各种心血管疾病提供更好的策略。