SHEAR STRESS MODULATION OF ENDOTHELIAL WOUND HEALING

内皮伤口愈合的剪切应力调节

• 批准号: 6386615
• 负责人: MARIA LUIZA C ALBUQUERQUE
• 金额: $ 15.18万
• 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/6386615
• 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

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或侵入性医疗操作期间，内皮损伤可能在多种情况下发生在体内。 血管内皮损伤需要许多蛋白质与细胞骨架相互作用，以实现适当的细胞运动。 除了生化信号传导之外，连接血流和血管壁的内皮细胞还需要剪切应力等生物力学力来引导其细胞运动。生物力学力可以直接或长期地影响内皮细胞的结构和功能，因此构成了内皮细胞激活的新范例。然而，剪切力影响细胞运动的粘附蛋白和生长因子（例如碱性成纤维细胞生长因子（bFGF））的机制仍然难以捉摸。 我们有初步数据表明，层流剪切应力通过涉及细胞迁移、扩散和特定粘附蛋白增强的机制增强人脐静脉和冠状动脉内皮细胞伤口闭合。 在此提案中，P.I.提出了总体假设，即剪切应力通过涉及细胞-细胞（钙粘蛋白）和细胞-基质（整合素）粘附蛋白表达以及 bFGF 产生的机制来提高内皮细胞伤口闭合率。 提出了四个具体目标。 在具体目标 1 中，我们将描述单层内皮细胞​​中剪切应力和伤口闭合之间的关系。 伤口宽度、细胞面积和核间距离的测量将表明细胞在伤口边缘的扩散和迁移。 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。 β1 整联蛋白链和 VE-钙粘蛋白表达的蛋白质印迹以及 mRNA 水平的北方印迹将用于确定伤口闭合过程中对剪切应力的适应反应。 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，将研究整合素和钙粘蛋白在伤口闭合过程中影响 bFGF 表达的机制。 bFGF 产生的蛋白质印迹、bFGF mRNA 的核糖核酸酶保护测定将用于确定对剪切应力的适应反应。 将在伤口闭合期间评估激活和阻断β1整合素和VE-钙粘蛋白抗体对bFGF表达的影响。 在具体目标 4 中，我们将研究剪切应力对肌动蛋白相关信号传导相关蛋白质的影响。 将评估用于增强 bFGF 产生的 p120 犰狳蛋白的酪氨酸磷酸化和易位。 对剪切应力期间内皮修复机制的研究可能会为患有各种心血管疾病的患者提供更好的治疗策略。