PARs and S1P receptors in endothelial biology

描述(申请人提供)：我们将描述两个调节血管通透性和完整性的信号系统：凝血酶和蛋白酶激活的受体，以及鞘氨醇-1-磷酸和S1P受体。我们将测试两个系统都能感觉到血浆外溢并触发适当的内皮细胞反应的假设，我们将探索这些系统之间的相似之处和可能的联系。我们会问：1)S1P对于调节血管通透性和完整性有多重要？我们产生了不能向血浆供应S1P的成年小鼠，发现血管通透性和完整性出现了惊人的异常。我们将确定a)这些“pS1Pless”小鼠屏障功能的改变是由于未能代谢鞘氨醇而导致内皮细胞自主代谢/毒性效应，还是由于未能向血浆供应S1P并激活内皮细胞和其他细胞上的S1P受体，b)它们血管泄漏增加的解剖学基础以及它是否显示出组织或血管类型特异性，c)在这种情况下，内皮细胞是否是血浆S1P信号的主要靶点，如果是，这种信号是否连续，或者血浆S1P是否提供了使内皮细胞能够感知和帮助终止泄漏的动态信号，和d)缺乏血浆S1P的长期影响，以及它们是否由于屏障功能失调所致。2)PARs对于调节血管通透性和完整性有多重要？我们为所有的PARs和相关的转基因基因建立了基因敲除小鼠，并将使用这些小鼠来确定a)不同的内皮PARs的激活对体内血管通透性和完整性的影响，以及pS1Pless小鼠是否提供了一种敏化系统来揭示PARs的此类角色，b)PAR信号是否平行、部分冗余或依赖于S1P信号。3)顶端和基础S1P和PAR功能的差异是否与它们在屏障调节中的作用有关？我们的初步研究提出了一个模型，该模型将允许S1P信号提供动态检漏功能，并通过类比提出关于PAR的新问题。我们将确定a)内皮细胞S1P1受体在体外和体内是否表现出顶端-基底端的极性，以实现我们假设的动态渗漏检测功能，以及b)顶端和基础的差异，如EPCR结扎，是否调节PAR在任一表面的激活效应。互补的遗传和药理学方法将用于小鼠模型和细胞培养。初步研究揭示了血浆S1P在体内调节内皮屏障功能中的重要作用，对不同PARs激活的不同屏障反应，S1P和PAR信号的操纵对过敏模型生存的显著影响，以及体内屏障功能改变的长期影响。拟议的研究将提供有关血管生理学和病理生理学的新信息。与公共健康相关：我们的研究将揭示新的分子和细胞机制，血管内皮细胞通过这些机制感知和调节渗漏。内皮通透性的改变在过敏反应、癌症新血管生长、心脏病发作和中风、动脉粥样硬化以及包括脓毒症在内的炎症状态中发挥着重要作用。因此，了解如何处理内皮细胞渗漏可能会使患有一系列疾病的患者受益。

DESCRIPTION (provided by applicant): We will characterize two signaling systems that regulate the permeability and integrity of blood vessels: coagulation proteases and protease-activated receptors, and sphingosine-1-phosphate and S1P receptors. We will test the hypothesis that both systems sense extravasation of plasma and trigger appropriate endothelial cell responses, and we will explore parallels and possible connections between these systems. We shall ask: 1) How is S1P important for regulation of vascular permeability and integrity? We generated adult mice that fail to supply S1P to plasma and found striking abnormalities in vascular permeability and integrity. We shall determine a) whether altered barrier function in these "pS1Pless" mice is due to failure to metabolize sphingosine with consequent endothelial cell-autonomous metabolic/toxic effects or to failure to supply S1P to plasma and S1P receptor activation on endothelial and other cells, b) the anatomic basis for their increased vascular leak and whether it shows tissue or vessel-type specificity, c) whether endothelial cells are the main target of plasma S1P signaling in this context, and if so, whether such signaling is continuous or whether plasma S1P provides a dynamic signal that enables endothelial cells to sense and help terminate leaks, and d) the long-term effects of lack of plasma S1P and whether they are due to dysregulated barrier function. 2) How are PARs important for regulation of vascular permeability and integrity? We generated knockout mice for all the PARs and relevant transgenics and will use these to determine a) the effects of activation of different endothelial PARs on vascular permeability and integrity in vivo and whether pS1Pless mice provide a sensitized system for uncovering such roles for PARs, b) whether PAR signaling is parallel to, partially redundant with, or dependent upon S1P signaling. 3) Do differences in apical and basal S1P and PAR function contribute to their roles in barrier regulation? Our preliminary studies suggest a model that would permit S1P signaling to serve a dynamic leak detector function and raise new questions regarding PARs by analogy. We will determine a) whether endothelial cell S1P1 receptors display apical-basal polarity in vitro and in vivo to enable the dynamic leak detecting function we posit, and b) whether apical and basal differences, such as EPCR ligation, modulate the effects of PAR activation on either surface. Complementary genetic and pharmacological approaches will be used in mouse models and in cell culture. Preliminary studies reveal an important role for plasma S1P in regulating endothelial barrier function in vivo, distinct barrier responses to activation of different PARs, dramatic effects of manipulation of S1P and PAR signaling on survival in models of anaphylaxis, and long-term effects of altered barrier function in vivo. The proposed studies will provide new information regarding vascular physiology and pathophysiology. PUBLIC HEALTH RELEVANCE: Our studies will reveal new molecular and cellular mechanisms by which the endothelial cells that line blood vessels sense and regulate leakiness. Alterations in endothelial permeability play an important role in allergic reactions, new blood vessel growth in cancer, heart attacks and strokes, atherosclerosis, and inflammatory states including sepsis. Thus, understanding of how to manipulate the endothelial leakiness might benefit patients with a range of disorders.