Regulation of the Endothelial Cell Glycocalyx

描述(申请人提供)：近年来，内皮细胞糖基化被证明通过调节流变学、通透性和白细胞与内皮细胞的相互作用，在微血管功能中发挥关键作用。我们现在建议扩展这些观察，假设糖萼是一个动态的结构，它是受调节的，病理生理刺激可以导致它的降解和解体，这将有助于增加内膜通透性，血小板黏附，加速白细胞结合，并从血管系统中迁移白细胞。在活体内，视频显微镜使我们能够跟踪微循环对刺激的反应的变化，微灌流使我们能够标记糖基化反应，并选择性地治疗微循环的局部小段。我们提出了两个实验目标，这两个目标将使糖萼在内皮细胞功能中的作用建立在更加坚实的基础上，并将为理解糖萼在病理生理学中的作用奠定基础。 具体目标#1-测试和探索假设，即糖唇是血管壁的一个适应性成分，并且它是一个关键的潜在损伤部位。以下问题将被回答以检验这一假设。1.血管活性物质是否通过改变壁切应力来改变糖萼？2.炎性刺激对糖萼的大小和通透性有共同的影响吗？ 具体目的#2--验证一种假说，即对缺血/再灌流作出反应的关键事件序列之一是腺苷受体的激活及其对糖萼的主要和/或次要作用。以下问题将被回答以检验这一假设。1.能否通过激活腺苷A2a受体来减轻缺血/再灌流对糖萼的影响？2.能否通过激活A3受体来模拟缺血/再灌注的作用？3.大剂量的腺苷是否通过作用于肥大细胞、白细胞或内皮细胞来介导其有害作用？该提案是基于最近开发的高效和选择性腺苷阻滞剂以及一组基因工程动物的使用。这些实验为理解微血管调节的新水平提供了可能性，并为开发策略来理解导致糖基化修饰的细胞内信号提供了可能性。

DESCRIPTION (provided by applicant): In recent years the endothelial cell glycocalyx has been shown to play a pivotal role in microvessel function by modulating rheology, permeability, and leukocyte-endothelial cell interactions. We now propose to extended those observations with the hypothesis that the glycocalyx is a dynamic structure, which is regulated and that pathophysiological stimuli can cause its degradation and disorganization, which will contribute to enhanced intimal permeability, platelet adhesion, accelerated white cell binding, and emigration of white cells from the vasculature. In vivo video microscopy allows us to follow changes in the microcirculation in response to stimuli, and microperfusion allows us to mark the glycocalyx and to selectively treat small, localized segments of the microcirculation. We propose two experimental aims which will place the role of the glycocalyx in endothelial cell function on a much firmer footing, and which will set the groundwork for an understanding of the role for the glycocalyx in pathophysiology. Specific Aim #1 - to test and explore the hypothesis that the glycocalyx is an adaptive component of the vascular wall, and that it is a key potential site for damage. The following questions will be answered to test the hypothesis. 1. Do vasoactive substances alter the glycocalyx by modifying wall shear stress? 2. Do inflammatory stimuli exert a common set of effects on the size and permeability of the glycocalyx? Specific Aim #2 - to test the hypothesis that one of the key sequences of events in response to ischemia/reperfusion is the activation of adenosine receptors and their primary and/or secondary effects on the glycocalyx. The following questions will be answered to test the hypothesis. 1. Can the effects of ischemia/reperfusion on the glycocalyx be reduced by activation of the adenosine A2A receptor? 2. Can the effects of I/R be mimicked by activation of A3 receptors? 3. Are the detrimental effects of large doses of adenosine mediated though actions on mast cells, leukocytes, or endothelial cells? The proposal is based on the use of recently developed highly potent and selective adenosine blockers, and a group of genetically engineered animals. The experiments offer the potential for understanding a new level of microvascular regulation, and for developing strategies to understand the intracellular signaling that leads to modification of the glycocalyx.