SHEAR STRESS EFFECTS ON ENDOTHELIAL TRANSPORT

描述：(改编自申请人的摘要)内皮细胞 从主动脉到毛细血管的血管壁的层(EC) 为水和溶质之间的运输提供了主要障碍 血液和底层组织。ECS不断地暴露在机械的 血液流动对其表面施加的剪切力(剪切力)。我们 最近发现剪切力对运输有很大的影响。 明确定义的细胞培养模型中EC层的属性，这具有 已经在活体动物的不同血管中得到证实。剪切相关EC 运输对正常的功能有重要的影响 微血管，它必须按照血液的比例将物质输送到组织 在需求区域内流动。在动脉中，与切变相关的渗透率 像低密度脂蛋白这样的大分子被认为在 在动脉粥样硬化病变的定位中起着关键作用。因为它的 生理意义，我们建议使用一个成熟的细胞 在工程剪切机上建立培养模型来研究这种现象 与剪切有关的EC运输。拟议研究的具体目标 包括：1)确定恒定和振荡剪应力对 其渗透系数(Lp)和大分子渗透率(Pe) 牛主动脉内皮细胞(BAEC)单层。一种独特的仪器将会 使用允许明确定义的稳定或振荡剪应力 施加于BAEC单层，使其在多孔聚碳酸酯上融合 当同时测量Lp和Pe时，滤光片。2)确定 受剪切应力影响的物理传输路径，使用四个 技术：(I)面部荧光显微镜以检测是否存在 内皮间隙或内皮间连接泄漏，(Ii) 用免疫荧光标记检测ZO-1蛋白的状态 紧密结，(III)胶体的电子显微镜观察 金标记示踪剂评价囊泡转运与细胞旁转运的比较 转运，以及(Iv)部分链霉蛋白酶对EC糖萼的消化以探测 EC表面糖蛋白在转运屏障功能中的作用 以及它的剪切响应。3)生化机制的确定(S) 介导EC输运性质的剪切相关响应。角色 重要的第二信使系统(cAMP、cGMP、Ca++)与 质膜通过G蛋白依赖受体将被用来探测 各种抑制剂和其他调节剂来操纵剪切依赖的 EC运输的响应。我们将评估细胞骨架的作用。 具有破坏三个主要组成部分的特定代理： 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.