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调节血液和组织之间流体平衡的局部斯塔林力。用于过滤和重吸收的经典斯塔林力是等离子体和组织之间流体静力和胶体渗透压的整体差。在这个更新的应用中，我们将评估一个假设，即局部流体静力和胶体渗透力决定了穿过内皮表面糖萼（初级分子过滤器）而不是整个壁的斯塔林力。在特异性目标1中要验证的假设是，糖萼上的渗透压差大于血液到组织的渗透压差，这是两个过程的结果：(1)糖萼中蛋白质的筛分；(2)通过连接链断裂的高流速减少了组织蛋白向糖萼下游空间的反向扩散。(假设1)。两种假设将在特定目标2中进行测试：一种假设认为，细胞内cAMP水平的变化导致连接链断裂的大小和频率的变化，通过改变组织蛋白反扩散的阻力（因此产生有效浓度差异）来改变渗透压，而不是通过改变初级分子过滤器。（假设2）第二个有待检验的假设是，酶降解糖萼导致内皮细胞糖萼厚度和/或组织的减少，通过直接改变初级分子过滤器，降低了施加在微血管壁上的渗透压差的大小（假设3）。在Specific Aim 3中需要验证的假设是，连接相关分子occludin是决定连接链断裂大小和频率的关键调节分子之一（假设4）。单独灌注的哺乳动物微血管和培养的哺乳动物内皮细胞将用于测试这些假设。所有实验的设计和解释都将以通过内皮间隙的溶质和水流耦合的详细三维模型为指导，同时考虑到测量的水流穿过微血管壁的实际连接链的超微结构。这一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。生物物理、超微结构、数学和分子方法的结合有望为正常组织和损伤后调节体液平衡的机制提供新的认识。新的信息可能导致策略，以减少水肿的形成和促进损伤和手术后的组织恢复。

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.