HEMODYNAMICS--HETEROGENEOUS ENDOTHELIAL GENE EXPRESSION

局灶性动脉粥样硬化病变的发生和发展与动脉粥样硬化的发生和发展有很长的关系。 与血流紊乱的区域有关，然而， 血液动力学与血管壁生物学之间的关系很差， 明白本实验室最近的体外和体内研究 已经证明了在内腔三维表面中的主要变化 在名义上均匀的内皮细胞内， 单层因此，血流动力学剪应力的详细分布 在细胞表面的分布有很大的不同。预测 这些差异的后果是要解决的假设， 这个建议：在整个动脉循环中， 内皮细胞基因表达的显著异质性受 地形差异引起的剪应力差 从细胞到细胞。单细胞和区域异质性都将是 处理。这一假设将通过分析基因表达来检验 在单个细胞和细胞群中，从精确的 体外和体内血流动力学位置并通过单细胞分析 扩增反义RNA技术。扰动流区域将 在小鼠动脉循环和人类培养物中鉴定出 体外培养内皮细胞。管腔的地形测量 内皮表面将通过原子力显微镜进行 使得能够针对各个单元绘制力集中。微型 操作/解剖将分离单个细胞（和小的细胞群）。 细胞）用于mRNA扩增。微阵列杂交技术将 可用于确定已知的应激反应的表达谱， 基因和其他已知和未知的人类和小鼠基因， 细胞特异性mRNA表达“指纹”相关的局部 血液动力学力动脉粥样硬化易感性LDL 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（抗体生成后）以评价蛋白质 表情基因独特和显著地与特定的 将识别血流动力学应力曲线，选择一些 这将通过转基因小鼠的产生来研究。体外 流动系统将被操纵，以研究复杂的空间和 在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说， 体内扰动流动区域将整合基因发现， 血流动力学和病变部位的局部动脉粥样硬化 开发.

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.