SHEAR STRESS MODULATION OF ENDOTHELIAL WOUND HEALING

内皮伤口愈合的剪切应力调节

• 批准号: 6526071
• 负责人: MARIA LUIZA C ALBUQUERQUE
• 金额: $ 15.47万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 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/6526071
• 关键词: RNase protection assay; biological signal transduction; biomechanics; blocking antibody; cadherins; clinical research; fibroblast growth factor; immunocytochemistry; integrins; northern blottings; phosphorylation; shear stress; tyrosine; vascular endothelium; western blottings; wound healing

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.

由于慢性高血压、动脉粥样硬化等病理性疾病实体，或在侵入性医疗过程中，血管内皮细胞损伤可在体内发生。损伤血管内皮细胞需要大量的蛋白质与细胞骨架相互作用，以实现适当的细胞运动。除了生化信号外，连接在血流和血管壁之间的内皮细胞还需要剪切力等生物力学力量来指导其细胞运动。生物力学力可以直接影响内皮细胞的结构和功能，从而构成了一种新的内皮细胞激活范式。然而，剪切力影响黏附蛋白和生长因子(如碱性成纤维细胞生长因子)促进细胞运动的机制仍然不清楚。我们有初步的数据表明，层流切应力通过涉及细胞迁移、扩散和增强特定黏附蛋白的机制来增强人脐静脉和冠状动脉内皮细胞的伤口闭合。在这项建议中，P.I.提出了一个总体假设，即剪切力通过细胞-细胞(钙粘附素)和细胞-基质(整合素)黏附蛋白的表达以及bFGF的产生来提高内皮细胞伤口的闭合率。提出了四个具体目标。在特定的目标1中，我们将表征内皮细胞单层中切应力和伤口闭合之间的关系。伤口宽度、细胞面积和核间距离的测量将显示细胞在伤口边缘的扩散和迁移。在特定的目标2中，我们将确定切应力与黏附分子在伤口闭合过程中的功能意义。用Beta1整合素链和VE-钙粘蛋白蛋白表达的Western印迹和Northern印迹的mRNA水平来确定伤口闭合过程中对剪应力的适应性反应。免疫细胞化学将被用来检测伤口愈合过程中黏附蛋白模式的变化。在具体目标3中，将研究整合素和钙粘附素在伤口闭合过程中影响bFGF表达的机制。免疫印迹法检测bFGF的产生，核糖核酸酶保护实验检测bFGFmRNA的表达，以确定细胞对剪切力的适应性反应。在伤口闭合过程中，将评估激活和封闭β1整合素和VE-钙粘附素抗体对bFGF表达的影响。在特定的目标4中，我们将研究剪切力对肌动蛋白相关信号转导相关蛋白的影响。将评估酪氨酸磷酸化和p120螳螂蛋白转位促进碱性成纤维细胞生长因子的产生。对切应力作用下内皮修复机制的研究可能有助于更好地治疗患有各种心血管疾病的患者。