Role of VE-cadherin cleavage as a clinical marker and trigger of endothelial barrier breakdown in sepsis

最近，败血症被定义为一种由于宿主对感染的反应失调而导致的危及生命的器官功能障碍。脓毒症器官功能障碍之前的一个关键事件是微血管内皮屏障功能的丧失。内皮屏障的完整性依赖于完整的黏附连接蛋白血管内皮钙粘附素(VE-cadherin)。以前，我们已经证明VE-钙粘蛋白在炎症刺激下被切割，导致sVE-钙粘蛋白的产生，它由细胞外结构域EC1-5组成。在体外，sVE-钙粘附素的形成与内皮屏障功能的丧失密切相关，并且在严重脓毒症患者的微血管屏障功能障碍中显着增强。我们的初步数据表明，重组sVE-钙粘附素不仅是炎症的结果，而且是不依赖于细胞凋亡的内皮屏障功能障碍的剂量依赖性诱导剂。因此，本研究的目的是研究炎症导致sVE-钙粘蛋白形成的机制，并确定sVE-钙粘蛋白在体外和体内诱导的内皮屏障功能障碍丧失中的作用。在我们前期工作的基础上，我们将通过研究负责sVE-钙粘素生成的脱落酶ADAM10是如何在内皮细胞中调节的来详细阐明sVE-钙粘蛋白形成的细胞机制。由于sve-cadherin呈剂量依赖性地诱导内皮细胞屏障功能丧失，我们将确定其潜在的机制。使用原子力显微镜，我们将测试sve-cadherin是否阻断VE-cadherin介导的黏附。在免疫沉淀中，我们发现SVE-钙粘附素与屏障调节磷酸酶(VE-PTP)有直接的相互作用。因此，我们将测试sVE-cadherin通过与VE-PTP结合是否导致VE-cadherin的磷酸化改变，或者调节依赖于Tie-2的信号通路，而不依赖于炎症刺激，从而导致内皮屏障功能的丧失。在我们的体内脓毒症模型中，我们将研究sVE-钙粘附素形成的时间进程及其与炎症诱导的微血管屏障功能障碍的直接相关性。此外，我们将测试在健康动物中应用sve-cadherin是否会导致微血管屏障功能的丧失和器官功能障碍，与脓毒症时的情况相当。综上所述，这些实验将在脓毒症诱导的微血管屏障功能障碍的复杂背景下确定新的病理生理学方面。

Recently Sepsis was defined as a life-threatening organ dysfunction due to a dysregulated host response to infection. A key event preceding organ dysfunction in sepsis is loss of microvascular endothelial barrier function. The integrity of the endothelial barrier is dependent on intact adherens junction protein vascular endothelial cadherin (VE-cadherin). Previously, we have demonstrated that VE-cadherin is cleaved in response to inflammatory stimuli leading to the generation of sVE-cadherin which consists of the extracellular domains EC1-5. Formation of sVE-cadherin was shown to occur in strong correlation with loss of endothelial barrier function in vitro and was significantly augmented in patients microvascular barrier dysfunction in severe sepsis. Our preliminary data show that recombinant sVE-cadherin is not only the result of inflammation but rather is dose-dependent inducer of endothelial barrier dysfunction independent of apoptosis. Therefore, the aims of this proposal are to characterize the mechanisms underlying sVE-cadherin formation as a result of inflammation and to identify the role of sVE-cadherin-induced loss of endothelial barrier dysfunction in vitro and in vivo.Based on our preliminary work we will elucidate the cellular mechanisms contributing to the formation of sVE-cadherin in detail by investigating how the sheddase ADAM10 which is responsible for sVE-cadherin generation is regulated in endothelial cells. Since sVE-cadherin dose-dependently induces loss of endothelial barrier function we will identify the underlying mechanisms. Using atomic force microscopy we will test whether sVE-cadherin blocks VE-cadherin-mediated adhesion. In immunoprecipitation we have identified a direct interaction of sVE-cadherin with barrier-regulating phosphatase (VE-PTP). Therefore, we will test whether sVE-cadherin via binding to VE-PTP leads to altered phosphorylation of VE-cadherin or modulates Tie-2-dependent signaling pathways independent of inflammatory stimuli leading to loss of endothelial barrier function. In our in vivo sepsis models we will investigate the time course of sVE-cadherin formation and its direct correlation with inflammation-induced microvascular barrier dysfunction. Furthermore we will test whether application of sVE-cadherin to healthy animals induces loss of microvascular barrier function and organ dysfunction comparable to their occurrence in sepsis. Taken together these experiments will identify a novel pathophysiologic aspect in the complex context of sepsis-induced microvascular barrier dysfunction.