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。在目前的资助期内，我们完成了小说 以新的三维理论模型为指导的实验 单次灌流节段的通透性 微血管对交接链超微结构的影响 裂隙内邻近的内皮细胞和纤维基质成分 在内皮表面。主要焦点一直是分析 低分子量示踪剂分子在表面的三维扩散 确定连接链的管腔一侧的大小和频率 在这股毛孔中。因为尾流比毛孔大得多， 这种新的方法提供了检测连接链的可能性 中断和细小的毛孔超出了 常规的透射电子显微镜。这些研究导致了 在对当前关于水和溶质路径的想法的重大修订中 通过连接股。提出了三个具体目标来 研究本建议书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)由以下因素形成的可视尾迹 连接链开口侧的小电子致密示踪剂 短时间后，在相距很远的不连续面处形成灌流。 在连接线上；和(2)肉眼可见的在肉肠上唤醒 在较长时间的灌流后，连接链的一侧由 弥散通过分布在 绞线的长度。直到稍后检测到此唤醒时， 组织中的示踪剂浓度已经增加到接近 检测阈值。在特定目标2中假设是分子 较大分子量示踪剂的筛选仅限于细纤维 裂隙入口区的母体层。中的假设 具体目标3是渗透率的变化不是由于 小静脉内相邻内皮细胞间间隙的形成 毛细血管是血管的大小和频率改变的结果。 连接链中的不连续性和 在管腔表面有分子筛。我们的方法提供了新的方法 研究连接和基质结构的这种细微变化。在……里面 水和溶质运移的拟议研究、理论模型 通过内皮间裂隙和邻近组织将被发展 为了进一步解释文献[1]中提出的时间相关尾迹实验 具体目标1和3，并分析实验结果 在特定目标2和3中提出的更大的溶质分子。所有实验 将在个别灌流的微血管上进行手术 利用微灌注技术和新型共聚焦技术研究渗透性 方法可视化示踪剂在微血管周围的分布。 这种理论和实验相结合的方法是最直接的- 对结点和光纤性质的新认识 调节微血管通透性的基质结构。

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.