HEMODYNAMICS--HETEROGENEOUS ENDOTHELIAL GENE EXPRESSION

血流动力学--异质内皮基因表达

• 批准号: 6591067
• 负责人: Peter Francis Davies
• 金额: $ 17.52万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6591067
• 关键词: antisense nucleic acid; atherosclerotic plaque; atomic force microscopy; genetically modified animals; hemodynamics; human tissue; immunocytochemistry; in situ hybridization; laboratory mouse; low density lipoprotein receptor; micromanipulator; surface property; tissue /cell culture; vascular endothelium

The initiation and progression of focal atherosclerotic lesions has long been associated with regions of disturbed blood flow, yet the relationships linking hemodynamics to vessel wall biology are poorly understood. Recent in vitro and in vivo studies from this laboratory have demonstrated major variations in the lumenal 3-dimensional surface geometry from cell to cell within the nominally homogeneous endothelial monolayer. Thus the detailed distribution of hemodynamic shear stresses over the lumenal cell surface varies significantly. The predicted consequences of these differences is the hypothesis to be addressed in this proposal: that throughout the arterial circulation there is significant heterogeneity of endothelial gene expression regulated by differential shear stress as a consequence of topographic differences from cell to cell. Both single cell and regional heterogeneities will be addressed. The hypothesis will be tested by analysis of gene expression in single cells and groups of cells, removed from the precise hemodynamic locations in vitro and in vivo and analyzed by single cell amplified antisense RNA technology. Disturbed flow regions will be identified in the mouse arterial circulation and in cultures of human endothelial cells in vitro. Topographic measurements of luminal endothelial surfaces will be performed by atomic force microscopy enabling force concentrations to be mapped for individual cells. Micro- manipulation/dissection will isolate single cells (and small groups of cells) for mRNA amplification. Microarray hybridization technology will be used to determine the expression profile of known stress-responsive genes and other known and unknown human and mouse genes resulting in cell-specific mRNA expression "fingerprints" related to the local hemodynamic forces. The arteries of atherosclerosis-susceptible LDL Receptor-Edit transgenic mice will be probed by in situ hybridization and immunocytochemistry (after antibody generation) to evaluate protein expression. Genes uniquely and prominently associated with specific hemodynamic stress profiles will be identified, a selective number of which will be studied by the generation of transgenic mice. In vitro flow systems will be manipulated to study the complex spatial and temporal characteristics of gene expression in controlled conditions of disturbed laminar flow. In a larger sense, expression profiles from disturbed flow regions in vivo will integrate gene discovery, hemodynamics, and focal atherosclerosis at the sites at which lesions develop.

局灶性动脉粥样硬化病变的发生和发展由来已久。 与血液流动紊乱的区域有关，但 将血流动力学与血管壁生物学联系起来的关系很差 明白了。本实验室最新的体外和体内研究 已经显示出管腔三维表面的主要变化 名义上均质内皮内细胞间的几何构型 单层。因此，血液动力学切应力的详细分布 在管腔细胞表面上差异很大。被预测的 这些差异的后果是要在 这个提议是：在整个动脉循环中， 内皮基因表达的显著异质性受 地形差异导致的差异剪应力 从一个细胞到另一个细胞。单细胞和区域异质性都将是 地址。这一假设将通过对基因表达的分析来检验 在单个单元格和单元组中，从精确的 体内外血流动力学定位及单细胞分析 扩增的反义RNA技术。扰流区域将被 在小鼠动脉循环和人类培养中鉴定 体外培养内皮细胞。管腔的地形测量 内皮细胞表面将由原子力显微镜进行处理 从而能够绘制单个细胞的力密度图。微型机 操作/解剖将分离单个细胞(和一小群 细胞)，用于mRNA扩增。微阵列杂交技术将 用来确定已知的应激反应基因的表达谱 基因和其他已知和未知的人类和老鼠基因导致 细胞特异性mRNA表达的“指纹”与局部相关 血液动力学力量。动脉粥样硬化易感低密度脂蛋白的动脉 受体编辑转基因小鼠将通过原位杂交进行探测 和免疫细胞化学(在抗体产生后)来评估蛋白质 表情。与特定基因唯一和显著相关的基因 血液动力学应力分布将被识别，有选择的数量 这将通过转基因小鼠的产生来研究。离体 流动系统将被操纵来研究复杂的空间和 受控条件下基因表达的时间特征 扰动层流。从更广泛的意义上讲，来自 体内紊乱的流动区域将整合基因发现， 血流动力学和病变部位的局灶性动脉粥样硬化 发展。