NEW APPROACH TO ENDOTHELIAL CLEFT STRUCTURE

描述(由申请人提供)：我们提出了一个新的假说来解释施加于内皮细胞糖唇和内皮细胞膜上的剪应力对单独灌流的哺乳动物微血管中溶质和水交换的调节。总的假设是，内皮细胞糖基化的三维有序特性使其能够作为血浆蛋白的初级分子筛发挥作用，这也决定了它作为剪应力传感器调节内皮屏障通透性的功能。目的1是在不改变切应力的情况下，测量单独灌流的微血管中小的溶质和大的溶质的渗透系数，同时保持糖萼的结构。将测量血浆蛋白的跨血管水通量和有效渗透压。具体的假设是，作为机械换能器和主要分子筛的完整的糖基催化剂将增加小的溶质渗透率，而对大分子的渗透率和选择性不变。在目标2中，对糖萼的结构进行了修改，以减少其作为分子筛的作用，并允许剪切应力更接近内皮表面。具体的假设是，完整的糖萼通常保护内皮细胞膜上的剪切传感机械传感器，该传感器调节水和所有溶质的共同途径。目标3是在有或没有完整的糖萼的情况下，以非常低的切变率长时间地灌流微血管。具体的假设是，持续的剪切力是维持内皮细胞表型所必需的，在这种表型中，剪切力的急剧变化会导致小溶质渗透率的快速调节，而水不变，而大的溶质渗透率保持不变。实验设计和解释将以我们目前关于水和溶质通过糖萼和内皮间裂隙运输的模型以及从糖萼到皮质细胞骨架的力传递的新模型为指导。微灌流、生物物理、超微结构和建模相结合的方法将提供对正常微血管中剪应力调节通透性的新理解，并可能导致改善营养物质输送和促进损伤或手术后组织恢复的策略。我们的实验评估了内皮细胞表面调节水、溶质、白细胞和红细胞通量的内皮糖萼的常见机械性能。

DESCRIPTION (provided by applicant): We propose a new hypothesis to explain the regulation of solute and water exchange in individually perfused mammalian microvessels by shear stress applied to the endothelial cell glycocalyx and at the endothelial cell membrane. The overall hypothesis is that the three-dimensional ordered properties of the endothelial glycocalyx which enable it to function as the primary molecular sieve for plasma proteins also determine its function as a shear stress sensor to regulate endothelial barrier permeability. Aim 1 is to measure both small solute and large solute permeability coefficients in individually perfused microvessels when applied shear stress is varied, and the structure of the glycocalyx is maintained. Transvascular water flux and effective osmotic pressures of plasma proteins will be measured. The specific hypothesis is that an intact glycocalyx acting as a mechanotransducer and the principal molecular sieve will increase small solute permeability with no change in the permeability and selectivity to large molecules. In Aim 2, the structure of the glycocalyx is modified to reduce its action as a molecular sieve and to allow shear stress to be applied closer to the endothelial surface. The specific hypothesis is that the intact glycocalyx normally protects shear-sensing mechanotransducers at the endothelial cell membrane that regulate a common pathway for water and all solutes. Aim 3 is to perfuse microvessels for long periods at very low shear with and without an intact glycocalyx. The specific hypothesis is that continuous shear stress is required to maintain an endothelial phenotype in which an acute change in shear stress causes rapid regulation of small solute permeability, with no change in water and large solute permeability. Experimental design and interpretation will be guided by our current model of water and solute transport through the glycocalyx and inter-endothelial cleft and by a new model of force transduction from the glycocalyx to the cortical cytoskeleton. The combined microperfusion, biophysical, ultrastructural, and modeling approaches will provide new understanding of the permeability regulation by shear stress in normal microvessels and may lead to strategies to improve nutrient delivery and enhance tissue recovery after injury or surgery. Our experiments evaluate the common mechanical properties of the endothelial glycocalyx that modulate water, solute, leukocyte, and red cell fluxes at the endothelial cell surface.