Novel regulators, components and function of endothelial diaphragms

内皮隔膜是与血管内皮亚细胞结构相关的蛋白质屏障，如 作为窗孔，跨内皮细胞通道(TEC)、小窝和囊泡-空泡细胞器参与了 血管通透性和免疫反应。窗孔是血管内皮细胞内的圆形跨细胞小孔 细胞在维持基础通透性方面起着关键作用。人类和小鼠的最新数据 证明窗孔及其横隔膜在维持正常内皮细胞中起关键作用 屏障功能、血液动态平衡和最终生存。我们小组证明了质膜 囊泡相关蛋白(PLVAP或PV1)是横隔膜组装的关键蛋白，而在 小鼠和人类由于多个血管缺陷而导致异常的窗孔和围产儿死亡。PV1是 在免疫反应中也很重要，基于PV1生物学的治疗策略正在开发中 心肌梗塞、免疫性疾病和癌症。虽然对横隔膜和横隔膜的兴趣正在显现 在临床上，我们对这些成分的了解存在着很大的差距 窗孔和横隔膜形态发生的调控途径，在本提案中讨论。危急地 对于这个提议，PV1是唯一已知的横隔膜标记，我们将在我们的 调节横隔膜形态发生的研究。根据我们的初步数据和文献，我们的中央 假说是多条信号通路在血管床上以特定的方式协同作用于隔膜和 栅栏队形。具体地说，生长因子和层粘连蛋白结合整合素受体之间的串扰是 对PV1的表达以及窗孔和横隔膜的形态发生至关重要。这一假设是基于我们的关键 新的观察结果包括：1)不同血管床中窗孔的不同节段和空间控制； 2)筛选参与PV1调控的基因表达因子，鉴定出SCF、成纤维细胞生长因子5、血管内皮生长因子A和 特定的层粘连蛋白通过其各自的c-kit、FGFR1、VEGFR2和ITGB1受体进行信号转导 PV1以ERK1/2依赖的方式表达；3)层粘连蛋白与干细胞因子、成纤维细胞生长因子-5和血管内皮生长因子的结合。 A在PV1的强健表达和新的窗孔的形成中的结果我们将使用 经功能验证的内皮细胞培养系统与基因工程动物的结合 使用功能丧失、功能获得和重建方法的模型。我们的目标将定义角色和 新发现的生长因子、层粘连蛋白及其整合素受体的下游信号通路 调节PV1表达和窗孔形态发生。我们在关键和关键两方面都处于独特的地位 完成这项工作所需的专业知识。这些调查将确定监管因素和途径 控制复杂和生理相关的细胞结构的组装，提供有价值的 目前所缺乏的洞察力。

Endothelial diaphragms are protein barriers associated with vascular endothelial subcellular structures, such as fenestrae, transendothelial channels (TEC), caveolae and vesiculo-vacuolar organelles implicated in vascular permeability and immune response. Fenestrae are circular transcellular pores in vascular endothelial cells playing critical roles in the maintenance of basal permeability. Recent data in humans and mice demonstrate that fenestrae and their diaphragms play critical roles in the maintenance of normal endothelial barrier function, blood homeostasis and ultimately survival. Our group demonstrated that Plasmalemma Vesicle Associated Protein (PLVAP or PV1) is critical for diaphragm assembly, and that absence of PV1 in mice and humans causes abnormal fenestrae and perinatal lethality due to multiple vascular defects. PV1 is also critical in the immune response, and therapeutic strategies based on PV1 biology are being developed for myocardial infarction, immune diseases and cancer. While there is emerging interest in diaphragms and fenestrae in the clinic, there is a significant knowledge gap in our understanding of the components and regulatory pathways governing fenestrae and diaphragm morphogenesis, addressed in this proposal. Critically for this proposal, PV1 is the only known marker for diaphragms, and we will exploit this property in our investigation of regulation diaphragm morphogenesis. Based on our preliminary data and literature, our central hypothesis is that multiple signaling pathways synergize in vascular bed specific manner for diaphragm and fenestrae formation. Specifically, crosstalk between growth factors and laminin binding integrin receptors are critical for PV1 expression and fenestrae and diaphragm morphogenesis. This hypothesis is based on our key new observations including: 1) differential segmental and spatial control of fenestrae in different vascular beds; 2) Gene expression screens for factors involved in PV1 regulation, identified SCF, FGF-5, VEGF-A and specific laminins signaling via their respective c-KIT, FGFR1, VEGFR2, and ITGB1 receptors as necessary for PV1 expression in ERK1/2 dependent manner; and 3) the combination of laminin with SCF, FGF-5 and VEGF- A results in robust PV1 expression and de novo fenestrae formation We will test our hypothesis using a combination of functionally validated endothelial cell culture systems and genetically engineered animal models using loss of function, gain of function and reconstitution approaches. Our Aims will define the role and downstream signaling pathways of newly identified growth factors and laminins and their integrin receptors regulating PV1 expression and fenestrae morphogenesis. We are uniquely positioned both in terms of key and necessary expertise to complete this work. These investigations will define regulatory factors and pathways controlling the assembly for an intricate and physiologically relevant cellular structure, providing valuable insight that is currently lacking.