VASCULAR CELL INTERACTIONS AND FLOW IN VIVO

该项目的重点是确定白细胞与微血管壁（内皮细胞，EC）在体内相互作用的机制。虽然大量的信息有关的当地生物物理和生物现象是可用的，相对较少的这已被直接耦合到事件发生在血液灌注微血管原位。因此，我们的主要重点是了解流体动力学现象，或指定的白细胞群体，在这个“完整”的环境中与血管壁相互作用。大部分工作将在麻醉小鼠的血液灌注微血管中进行。假设1A：粘附分子的表达在不同的小静脉类别之间没有差异，但是由于个体内皮细胞之间的表达差异，在个体小静脉内局部变化是显著的。假设1B：粘附分子局部分布的不均匀性与中性滚动和粘附行为相关。免疫荧光标记的粘附分子的分布将使用共聚焦显微镜在血液灌注的小静脉中测定。我们将询问在对照组和TNF α激活后，粘附分子表达的异质性是否映射到同一群体的单个EC。神经元滚动和粘附将与每个粘附分子的高免疫荧光强度区域相关，并与使用0.5 μ荧光珠确定的局部壁剪切速率相关，以从堆叠的共聚焦图像中定义径向速度分布。通过项目5，将探索白细胞-EC相互作用中的局部流体动力学参与。假设2A：炎症介质对内皮细胞的钙依赖性激活是中性粒细胞粘附和渗出所必需的。假设2B：炎症介质仅在某些内皮细胞中增加内皮细胞钙，并且这些细胞优先与中性粒细胞迁移相关。我们将使用 Fluo-4负载EC以确定在局部暴露于炎症介质期间，EC Ca 2+是否在单个EC或整个小静脉中发生增加，并将EC Ca 2+的反应与单个EC中粘附分子的表达以及血流动力学环境相关联。我们将确定是否白细胞本身，独立的炎症介质的作用，可以启动Ca 2+依赖的信号在内皮细胞在其天然环境中的小静脉。在对照和激活的小静脉中，以及在用BAPTA缓冲EC Ca 2+变化期间，神经元粘附密度将在空间和时间上与Fluo-4强度的变化相关。

This project is centered on determining mechanisms underlying leukocyte interactions with the microvascular wall (endothelial cells, ECs) in vivo. While a great deal of information about relevant local biophysical and biological phenomena is available, relatively little of this has been directly coupled to events occuring in blood perfused microvessels in situ. Our primary focus therefore is on understanding how hydrodynamic phenomena, or specified leukocyte populations, interact with the vessel wall in this "intact" environment. The majority of the work will be undertaken in the blood perfused microvasculature of anesthetized mice. Hypothesis 1A: Adhesion molecule expression is not different among different venule classes, but within individual venules local variation is significant, due to differences in expression among individual endothelial cells. Hypothesis 1B: Non-uniformities in local distribution of adhesion molecules correlate with neutrophiI rolling and adhesive behavior. Distributions of immunofluorescently labeled adhesion molecules will be determined in blood perfused venules using confocal microscopy. We will ask whether heterogeneity in adhesion molecule expression maps to the same population of individual ECs, in both controls and after activation with TNFalpha. Neutrophil rolling and adhesion will be correlated with regions of high immunofluorescence intensity for each adhesion molecule, and with local wall shear rate determined using 0.5mu fluorescent beads to define radial velocity profiles from stacked confocal images. With Project 5, the local hydrodynamic involvement in leukocyte-EC interactions will be explored. Hypothesis 2A: Calcium dependent activation of endothelial cells by inflammatory mediators is necessary for neutrophil adhesion and diapedesis. Hypothesis 2B: Inflammatory mediators increase endothelial cell calcium only in some endothelial cells, and these cells are preferentially associated with neutrophil transmigration. We will use Fluo-4 loaded ECs to determine whether EC Ca 2+ increases occur in individual ECs, or in the venule as a whole, during local exposure to inflammatory mediators, and to relate the responses in EC Ca 2+ to expression of adhesion molecules in individual ECs, and to the hemodynamic environment. We will determine whether leukocytes themselves, independently of the action of inflammatory agents, can initiate Ca 2+ dependent signaling in ECs in venules in their native environment. Neutrophil adhesion density will be spatially and temporally correlated with changes in Fluo-4 intensity, in both control and activated venules, and during buffering of EC Ca2+ changes with BAPTA.