NEW APPROACH TO ENDOTHELIAL CLEFT STRUCTURE

内皮裂隙结构的新方法

• 批准号: 2221511
• 负责人: FITZ-ROY E CURRY
• 金额: $ 32.14万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2221511
• 关键词: Rana; biological fluid transport; capillary; computer simulation; confocal scanning microscopy; diffusion; extracellular matrix; fluorescent dye /probe; horseradish peroxidase; lanthanum; mathematical model; membrane structure; model design /development; molecular weight; perfusion; solute; transmission electron microscopy; vascular endothelium; vascular endothelium permeability

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. During the current grant period we completed novel experiments guided by new three-dimensional theoretical models to relate the permeability properties of segments of individually perfused microvessels to the ultrastructure of the junctional strands between adjacent endothelial cells and fiber matrix components within-the cleft or at the endothelial surface. The primary focus has been the analysis of the three dimensional spread of low molecular weight tracer molecules on the abluminal side of the junction strand to determine the size and frequency of the pores in this strand. Because the wakes are much larger than pores, this new approach offers the possibility of detecting junction strand interruptions and small pores that lie beyond the resolution of conventional transmission electron microscopy. These studies have resulted in major revision of the current ideas about pathways for water and solute through junctional strands. Three Specific Aims are proposed to investigate new or revised themes in this proposal. The hypotheses to be tested under Specific Aim 1 are: (1) that the visible wakes formed by small electron-dense tracers on the abluminal side of the junction strand after short time perfusions are formed at widely separated discontinuities in the junctional strand; and (2) that the visible wakes on the abluminal side of the junctional strand after longer time perfusions are formed by diffusion through a population of very small pores distributed along the length of the strand. This wake is not detected until later times when the tracer concentration in the tissue has increased to a level close to a detection threshold. The hypothesis in Specific Aim 2 is that molecular sieving of larger molecular weight tracers is confined to the thin fiber matrix layer at the entrance region of the cleft. The hypothesis in Specific Aim 3 is that changes in permeability which are not due to the formation of gaps between adjacent endothelial cells in venular capillaries are the result of changes in the size and frequency of the discontinuities in the junctional strand and the structure of the molecular sieve at the luminal surface. Our approach provides new methods to investigate such subtle changes in junctional and matrix structure. In the proposed studies, theoretical modeling of water and solute transport through the interendothelial cleft and adjacent tissue will be developed further to interpret the time dependent wake experiments proposed in Specific Aims 1 and 3, and to analyze the results of the experiments with larger solute molecules proposed in Specific Aims 2 and 3. All experiments will be performed on individual perfused microvessels of precisely known permeability properties using microperfusion techniques and novel confocal methods to visualize tracer distribution around perfused microvessels. This combined theoretical and experimental approach is the most direct- approach to a new understanding of the nature of the junction and fiber matrix structures which modulate microvessel permeability.

我们研究的总体目标是开发一种组合工程， 超微结构和生物物理方法的机制， 内皮细胞和细胞之间的裂缝调节微血管 磁导率在目前的赠款期间，我们完成了小说 新的三维理论模型指导下的实验， 单独灌注的节段的渗透性特性 微血管之间的连接股的超微结构 裂隙内邻近的内皮细胞和纤维基质成分， 在内皮表面。主要重点是分析 低分子量示踪剂分子的三维扩散 连接链的近腔侧以确定大小和频率 这条线上的小孔因为尾流比毛孔大得多， 这种新方法提供了检测连接链的可能性， 中断和小孔，超出了分辨率 常规透射电子显微镜。这些研究导致 在对目前关于水和溶质的途径的想法进行重大修订时， 通过连接股。提出了三个具体目标， 研究本提案中的新主题或修订主题。假设是 在具体目标1下测试的是：（1）由以下物质形成的可见尾流 连接链近腔侧的小电子密度示踪剂 在短时间灌注后， 在连接链中;以及（2）可见物质在近腔上醒来 在较长时间灌注后，连接链的一侧通过以下方式形成： 通过沿着表面分布的非常小的孔群扩散 链的长度。这种唤醒直到稍后的时间才被检测到， 组织中的示踪剂浓度已经增加到接近 检测阈值具体目标2中的假设是， 较大分子量示踪剂的筛分仅限于细纤维 在裂缝的入口区域处的基质层。假设在 具体目标3是渗透性的变化，这不是由于 微静脉内皮细胞间隙的形成 毛细血管的大小和频率的变化的结果， 连接链中的不连续性和 分子筛在管腔表面。我们的方法提供了新方法 来研究连接和基质结构的这种微妙变化。在 提出的研究，水和溶质运移的理论模型， 通过内皮间隙和邻近组织将被开发 进一步解释时间相关的尾流实验中提出的 具体目的1和3，并分析实验结果， 具体目标2和3中提出的较大溶质分子。所有实验 将在精确已知的单个灌注微血管上进行 使用微灌注技术和新的共聚焦显微镜 方法可视化周围灌注微血管示踪剂分布。 这种理论和实验相结合的方法是最直接的- 对结和纤维性质的新认识 调节微血管通透性的基质结构。