SHEAR STRESS EFFECTS ON ENDOTHELIAL TRANSPORT

描述：（改编自申请人摘要）内皮细胞 从主动脉到毛细血管的血管壁内层（EC 提供了水和溶质之间运输的主要障碍 血液和皮下组织 EC持续暴露于机械 由流动的血液施加在其表面上的剪切力（剪切应力）。 我们 最近已经表明，剪切应力对运输有很大的影响， EC层在明确定义的细胞培养模型中的性质，这具有 在不同的活体动物容器中得到证实。 剪切相关EC 运输对正常的 微血管，其必须按血液的比例将物质输送到组织 在需求区域流动。 在动脉中， 大分子如低密度脂蛋白被假设为 在动脉粥样硬化病变的定位中起关键作用。 由于其 生理意义，我们建议使用一个完善的细胞 培养模型在工程剪切装置研究的现象， 剪切依赖EC运输。 拟议研究的具体目标 1）确定稳定和振荡剪切应力对 渗透系数（Lp）和大分子渗透率（Pe） 牛主动脉内皮细胞（BAEC）单层。 一种独特的装置将 可以使用，这允许明确定义的稳定或振荡剪切应力， 施加在生长至多孔聚碳酸酯上汇合的BAEC单层上 同时测量Lp和Pe。 2)确定 受剪切应力影响的物理运输途径，使用四种 技术：（i）正面荧光显微镜以检测 内皮间隙或内皮间连接渗漏，（ii） ZO-1蛋白的免疫荧光标记，以确定 紧密连接，（iii）胶体的电子显微镜观察 金标记示踪剂，以评估囊泡转运与细胞旁转运 运输，和（iv）EC糖萼的部分链霉蛋白酶消化，以探测 EC表面糖蛋白对转运屏障功能贡献 和剪切响应。 3)确定生化机制 介导EC传输特性的剪切依赖性响应。 的作用 重要的第二信使系统（cAMP，cGMP，Ca++）耦合到 通过G蛋白依赖性受体的质膜将被探测， 各种抑制剂和其他调节剂来操纵剪切依赖性 EC运输的响应。 将评估细胞骨架的作用 用特定的药剂破坏三种主要成分： 微丝、微管和中间丝。

DESCRIPTION: (Adapted from the applicant's abstract) The endothelial cell layer (EC) which lines blood vessel walls from the aorta to the capillaries provides the principal barrier to transport of water and solutes between blood and underlying tissue. ECs are continuously exposed to the mechanical shearing force (shear stress) imposed by flowing blood on their surface. We have shown recently that shear stress has an acute effect on transport properties of EC layers in a well defined cell culture model, and this has been confirmed in different vessels of live animals. Shear-dependent EC transport has important implications for the function of normal microvessels, which must deliver material to tissue in proportion to blood flow in the region of demand. In arteries, shear dependent permeability of macromolecules such as low density lipoprotein has been hypothesized to play a key role in the localization of atherosclerotic lesions. Because of its physiological significance, we propose to use a well-established cell culture model in an engineered shearing device to study the phenomena of shear-dependent EC transport. The specific aims of the proposed research are: 1) To determine the effect of steady and oscillatory shear stress on the hydraulic conductivity (Lp) and macromolecular permeability (Pe) of bovine aortic endothelial cell (BAEC) monolayers. A unique apparatus will be used which allows well-defined steady or oscillatory shear stress to be imposed on BAEC monolayers grown to confluence on porous, polycarbonate filters while Lp and Pe are measured simultaneously. 2) To determine the physical transport pathways that are affected by shear stress, using four techniques: (i) en face fluorescence microscopy to detect the presence of endothelial gaps or leaky interendothelial junctions, (ii) immunofluorescence labeling for ZO-1 protein to determine the status of tight junctions, (iii) electron microscopic observation of colloidal gold-labeled tracers to assess vesicular transport versus paracellular transport, and (iv) partial pronase digestion of the EC glycocalyx to probe the contribution of EC surface glycoproteins to transport barrier function and its shear response. 3) To determine the biochemical mechanism(s) mediating the shear-dependent response of EC transport properties. The role of important second messengers systems (cAMP, cGMP, Ca++) coupled to the plasma membrane through G-protein-dependent receptors will be probed with various inhibitors and other modulators to manipulate the shear-dependent response of EC transport. The role of the cytoskeleton will be assessed with specific agents which disrupt the three major components: microfilaments, micro tubules and intermediate filaments.