Role of End Binding 3 in Mechanism of vascular permeability

描述（由申请人提供）：肺血管通透性增加导致富含蛋白质的组织水肿，这是成人呼吸窘迫综合征的重要特征。微管（MT）细胞骨架在内皮通透性增加机制中的作用尚不清楚。已知MTs在响应促炎介质时经历重组，因此可能有助于内皮通透性增加的机制。拟议的研究将解决末端结合蛋白-3 （EB3）在调节肺血管通透性增加中的核心作用，EB3是一种MT +末端结合因子。我们将验证以下假设：(i) EB3是血管内皮(VE)-钙粘蛋白粘附复合物与MT细胞骨架之间串扰的主要成分；（ii） EB3介导的MT动力学控制对于维持肺微血管的基础通透性和炎症介质引起的通透性增加至关重要。这些研究将解决以下具体目标：(1)ve -cadherin介导的信号在EB3磷酸化和抑制MT生长的机制中的作用，从而在建立肺内皮基础通透性中的“外向内”信号的作用；(2) EB3在调节Ca2+信号传导中起关键作用，从而介导内皮通透性增加和肺水肿的发生。我们期望，通过了解ve -钙粘蛋白粘附如何发出EB3磷酸化信号，以及EB3如何由此引发屏障通透性增加，将为肺液体稳态失调的机制提供新的见解。我们将利用最先进的技术，包括活细胞成像、突变结构的表达、基因转移和肺部炎症的小鼠模型来实现特定的目标。

DESCRIPTION (provided by applicant): Increases in lung vascular permeability result in protein rich tissue edema, an important feature of adult respiratory distress syndrome. The role of the microtubule (MT) cytoskeleton in the mechanism of increased endothelial permeability is not well understood. MTs are known to undergo re-organization in response to pro- inflammatory mediators, and may thus contribute to the mechanism of increased endothelial permeability. The proposed studies will address the central role of End Binding protein-3 (EB3), a MT plus-end binding factor, in regulating increased lung vascular permeability. We will test the hypotheses that (i) EB3 is a major component of cross-talk between Vascular Endothelial (VE)-cadherin adhesion complexes and the MT cytoskeleton and (ii) EB3-mediated control of MT dynamics is critical for maintenance of basal permeability of lung microvessels and for permeability increase caused by inflammatory mediators. These studies will address the following Specific Aims: (1) role of VE-cadherin-mediated signaling in the mechanism of EB3 phosphorylation and inhibition of MT growth and, thereby the role of "outside-in" signaling in establishing basal permeability of lung endothelia; and (2) critical role of EB3 in regulating Ca2+ signaling, thus in mediating increased endothelial permeability and development of lung edema. It is our expectation that by understanding how VE-cadherin adhesion signals EB3 phosphorylation and how EB3 thereby elicits the barrier permeability increase will provide novel insights into the mechanisms of dysregulation of lung fluid homeostasis. We will exploit state of the art technologies including live cell imaging, expression of mutant constructs, gene transfer, and murine models of lung inflammation to accomplish the specific aims. PUBLIC HEALTH RELEVANCE: The focus of the work is the lung endothelium, in which the planned studies will establish the relevance of EB3, a microtubule-binding protein, to the pathophysiology of ALI/ ARDS. The work leading up to this proposal has allowed us to design a potential therapeutic peptide that by inhibiting EB3 function prevents lung edema and lethality in sepsis.