NEW APPROACH TO ENDOTHELIAL CLEFT STRUCTURE

在这个应用中，我们提出了一个新的假设来解释当地的Starling部队，调节血液和组织之间的液体平衡。 用于过滤和重吸收的经典Starling力是血浆和组织之间的流体静压和胶体渗透压的整体差。 在本更新申请中，我们将评估局部流体静力学和胶体渗透力决定跨内皮表面糖萼（主要分子过滤器）而不是整个壁的Starling力的假设。 在具体目标1中要检验的假设是，由于两个过程，跨糖萼的渗透压差大于血液与组织的渗透压差：（1）糖萼中蛋白质的筛分;以及（2）组织蛋白质通过连接处的断裂处的高水流速度向糖萼下游空间的反向扩散减少，搁浅。 （假设1）。将在具体目标2中检验两个假设：一个假设指出，细胞内cAMP水平变化导致的连接链断裂的大小和频率变化通过改变组织蛋白反向扩散的阻力（因此有效浓度差异）而不是通过改变初级分子过滤器来改变渗透压。（假设2）待检验的第二个假设是，由糖萼的酶促降解引起的内皮细胞糖萼厚度和/或组织的减少通过直接修饰初级分子过滤器降低了跨微血管壁施加的渗透压差的大小（假设3）。在特定目的3中待检验的假设是，连接相关分子occludin是决定连接链断裂大小和频率的关键调控分子之一（假设4）。将使用单独灌注的哺乳动物微血管和培养的哺乳动物内皮细胞来检验这些假设。 所有实验的设计和解释将由通过内皮间裂隙的溶质和水流对的详细三维模型指导，考虑到测量的穿过连接链的微血管壁实际超微结构的水流。 这种策略已经成功地用于在青蛙肠系膜毛细血管中测试这些想法。 结合生物物理，超微结构，数学和分子的方法，预计将提供新的理解的机制，调节液体平衡在正常组织和损伤后。 新的信息可能导致减少水肿形成和增强损伤和手术后组织恢复的策略。

In this application we propose a new hypothesis to explain the local Starling forces which regulate fluid balance between blood and tissue. The classical Starling forces for filtration and reabsorption are the global difference of hydrostatic and colloid osmotic pressure between plasma and tissue. In this renewal application we will evaluate the hypothesis that the local hydrostatic and colloid osmotic forces determine the Starling forces across the endothelial surface glycocalyx, the primary molecular filter, and not the entire wall. The hypothesis is to be tested in Specific Aim 1 is that the osmotic pressure difference across the glycocalyx is alrger than the blood-to- tissue osmotic pressure difference as the result of two processes: (1) sieving of the proteins in the glycocalyx; and (2) the reduction in the back diffusion of the tissue proteins into the space downstream of the glycocalyx by high water velocities through the breaks in the junctional strand. (Hypothesis 1). Tow hypotheses will be tested in Specific Aim 2: One states that changes in the size and frequency of the breaks in the junction strand resulting from changes in intracellular cAMP levels modify the osmotic pressure by changing the resistance to back diffusion of tissue protein (hence the effective concentration differences) not by changing the primary molecular filter. (Hypothesis 2) The second hypothesis to be tested is that a decrease in the thickness and/or organization of the endothelial cell glycocalyx caused by enzymatic degradation of the glycocalyx decreases the magnitude of the osmotic pressure difference exerted across the microvessel wall by directly modifying the primary molecular filter (Hypothesis 3). The hypothesis to be tested in Specific Aim 3 is that the junction associated molecule occludin is one of the key regulatory molecules determining the size and frequency of breaks in the junctional strands (Hypothesis 4). Individually perfused mammalian microvessels and mammalian endothelial cells in culture will be used to test these hypotheses. The design and interpretation of all experiments will be guided by a detailed 3 dimensional model of couple solute and water flows through the interendothelial cleft, taking into account the measured water flows across the microvessel wall actual ultrastructure of the junctional strands. This strategy has already been successfully used to test these ideas in frog mesenteric capillaries. The combined biophysical, ultrastructural, mathematical, and molecular approaches are expected to provide new understanding of the mechanisms which regulate fluid balance in normal tissue and after injury. The new information may lead to strategies to reduce edema formation and enhanced tissue recovery after injury and surgery.