NEW APPROACH TO ENDOTHELIAL CLEFT STRUCTURE

我们研究的总体目标是开发一种组合工程， 用超微结构和生物物理方法研究其作用机制 血管内皮细胞和细胞间裂隙调节微血管 渗透性。冷冻断口研究和超薄系列切片 证明内皮细胞是由一系列连接的 每隔一段时间中断，允许水和水通过的绳索 溶质。细胞化学和渗透性研究也表明，所有或 裂隙的一部分可能被纤维基质填充。我们描述的是小说 以新的概念理论框架指导的实验 单独灌流的微血管节段的通透性， 具有已知的渗透性属性，对细胞的超微结构 相邻内皮细胞与纤维基质之间的连接链 在裂缝里。该提案的具体目标是：1)评估 建议微血管壁对水的渗透性和 小的溶质由小的不连续的频率决定 连接链；以及2)评估 与连接链和纤维基质相关联的结构以 微血管壁上的分子过滤器。我们新计划的主要重点是 方法是对三维对流扩散的分析 示踪剂分子在白蛋白一侧的扩散 结合链排列。这种扩散类似于 在雷诺数很低的情况下，小物体下游的尾流。 亚当森博士的连续切片的初步结果表明 需要一种方法来研究其几何形状和分布。 假设连接链上的小孔或缝隙不能 通过常规的30-40 nm切片进行分辨。理论和实践相结合 这里采用的实验方法利用了最近的一种 Weinbaum博士开发了高精度的解析解 含有连接股的裂隙中的三维粘性流动 具有离散的毛孔和细长的交叉桥纤维组件。模型 来解释示踪剂在土壤中的三维分布 在具体目标1中提出的尾流实验，并分析了 在特定目标2中提出了较大溶质分子的实验。 在这两个实验设计中，测量将在个体上进行 已精确知道通透性特性的灌流微血管 这些技术已经在库里博士的实验室里得到了广泛的开发。 我们相信，这种理论和实验相结合的方法将大有可为。 超越过去和现在描绘连接和纤维基质的尝试 调节微血管通透性的结构。这些研究是 以最直接的方式重新理解人类社会的本质 决定正常组织和微血管通透性的阻力 在伤病恢复期间。

The overall aim of our research is to develop a combined engineering, ultrastructural and biophysical approach to the mechanisms whereby endothelial cells and the clefts between the cells modulate microvessel permeability. Freeze fracture studies and ultrathin serial sections have demonstrated that endothelial cells are joined by an array of junctional strands which are interrupted at intervals allowing passage of water and solutes. Cytochemical and permeability studies also indicate that all or part of the cleft may be filled with a fibrous matrix. We describe novel experiments guided by a new conceptual theoretical framework to relate permeability properties of segments of individually perfused microvessels, having known permeability properties, to the ultrastructure of the junctional strands between adjacent endothelial cells and a fiber matrix within the cleft. The specific aims of the proposal are 1) to evaluate the proposal that the permeability properties of microvessel walls to water and small solutes are determined by the frequency of small discontinuities in the junctional strand; and 2) to evaluate the relative contribution of structures associated with the junctional strand and the fiber matrix to the molecular filter at the microvessel wall. The primary focus of our new approach is the analysis of the three-dimensional convective-diffusive spread of tracer molecules on the albuminal side of the interruptions in the junction strand arrays. This spread is analogous to the growth of a wake on the downstream side of a small object at very low Reynolds numbers. Preliminary results from serial sections by Dr. Adamson indicate that this approach is necessary to investigate the geometry and distribution of hypothetical small pores or slits in the junction strands which cannot be resolved by conventional 30-40nm sections. The combined theoretical and experimental approach adopted herein takes advantage of a recently developed highly accurate analytic solution by Dr. Weinbaum for the three-dimensional viscous flow in a cleft, which contains junction strands with discrete pores and slender cross-bridging fiber components. The model is needed to interpret the three-dimensional distribution of tracer in the wake experiments proposed in Specific Aim 1, and to analyze the results of the experiments with larger solute molecules proposed in Specific Aim 2. In both experimental designs measurements will be performed on individual perfused microvessels of precisely known permeability properties using techniques that have been extensively developed in Dr. Curry's laboratory. We believe this combined theoretical and experimental approach goes far beyond past and current attempts to delineate the junction and fiber matrix structures which modulate microvessel permeability. These studies are the most direct approach to a new understanding of the nature of the resistances which determine microvessel permeability in normal tissue and during recovery from injury.