The NOTCH Signaling Pathway in Large Vessel Vasculitis

描述（由申请人提供）：大血管炎（LVV），如巨细胞动脉炎（GCA），可导致失明、中风、主动脉弓综合征、主动脉瘤、高血压和心肌功能不全。在老龄化人群中，需要长期管理LVV的患者数量一直在稳步上升，而治疗手段仍严格限于高剂量皮质类固醇。在过去的十年里，先天性和适应性免疫系统在LVV的免疫发病机制中的作用取得了令人兴奋的进展。然而，在我们的知识中，为什么疾病以主动脉及其主要分支为目标，以及动脉壁中的免疫基质通讯如何引发和促进血管炎，存在着一个关键的空白。病原性免疫应答具有抗原诱导的克隆扩增的特征，但我们最近发现，来自组织生态位中驻留细胞的共刺激信号在驱动组织损伤免疫中同样重要。GCA动脉表达大量的NOTCH受体和配体，为极好的细胞间通讯提供了分子平台。阻断NOTCH信号传导可有效抑制血管炎。来自GCA患者的CD 4 T细胞组成型表达NOTCH 1受体，使它们能够与表达NOTCH配体的血管平滑肌细胞（VSMC）和内皮细胞（EC）相互作用。本申请旨在揭示Notch途径如何参与免疫-内皮和免疫-基质通讯，以及Notch依赖性信号传导如何塑造血管生成T细胞应答和适应不良的VSMC和EC行为。该项目建立在一系列使能资源的基础上;包括GCA患者的临床表型队列;一种新型的人体动脉壁三维模型系统，该系统允许从填充有定义细胞群的可堆叠单元组装定制血管;以及携带发炎人类动脉的人源化小鼠模型。可通过配体竞争性抗体/融合蛋白阻断对Notch受体和配体的接近，并且可通过RNAi技术使细胞缺乏Notch信号传导。具体目标1在机制水平上检查VSMC和EC上的NOTCH配体如何调节血管生成性CD 4 T细胞的效应子功能;调节它们的生长、组织侵袭能力和细胞因子产生。具体目标2旨在鉴定可用于抑制病原性T细胞中的NOTCH 1表达或靶向NOTCH依赖性存活信号的信号传导网络。将在嵌合体模型中测试破坏Notch衍生信号的小分子抑制剂的抗血管炎潜力。具体目标3集中在VSMC作为信号发送和信号接收细胞的作用，并确定NOTCH-NOTCH配体相互作用如何影响VSMC存活，迁移，基质产生，收缩性和ROS释放。具体目标4揭示了患者来源的CD 4 + NOTCH 1 + T细胞调节EC功能行为的分子机制，并研究了此类T细胞如何调节EC促炎功能、血管生成能力、EC屏障的通透性和渗漏。

DESCRIPTION (provided by applicant): Large vessel vasculitides (LVV), such as giant cell arteritis (GCA) cause blindness, stroke, aortic arch syndrome, aortic aneurysm, hypertension and myocardial insufficiency. In an aging population the number of patients requiring chronic management for LVV has been steadily rising, while the therapeutic armamentarium has remained strictly limited to high-dose corticosteroids. The last decade has seen exciting progress in implicating the innate and adaptive immune system in the immunopathogenesis of LVV. However, there is a critical gap in our knowledge why the disease targets the aorta and its major branches and how immuno- stromal communications in the arterial wall initiate and promote vasculitis. The pathogenic immune response has a signature of antigen-induced clonal expansion, but we have recently seen that costimulatory signals deriving from resident cells in the tissue niche are equally important in driving tissue-damaging immunity. GCA arteries express abundant levels of NOTCH receptors and ligands, providing a molecular platform for superb cell-to-cell communication. Blocking of NOTCH signaling effectively inhibits vasculitis. CD4 T cells from GCA patients constitutively express NOTCH1 receptor, enabling them to interact with NOTCH ligand expressing vascular smooth muscle cells (VSMC) and endothelial cells (EC). This application is designed to uncover how the Notch pathway participates in immuno-endothelial and immuno-stromal communications and how NOTCH- dependent signaling shapes vasculitogenic T cell responses and maladaptive VSMC and EC behavior. The project builds on a series of enabling resources; including a clinically phenotyped cohort of GCA patients; a novel 3-D model system of human arterial walls which permits assembly of custom-made vessels from stackable units populated with defined cell populations; and a humanized mouse model carrying inflamed human arteries. Access to Notch receptor and ligands can be blocked through ligand-competing antibodies/fusion proteins and cells can be rendered Notch signaling deficient by RNAi technology. Specific Aim 1 examines on a mechanistic level how NOTCH ligands on VSMC and EC regulate effector functions of vasculitogenic CD4 T cells; modulate their growth, tissue invasion capacity and cytokine production. Specific Aim 2 seeks to identify signaling networks that can be utilized to either suppress NOTCH1 expression or target NOTCH-dependent survival signals in pathogenic T cells. Small molecule inhibitors disrupting Notch-derived signals will be tested in the chimera model for their anti-vasculitic potential. Specific Aim 3 is focused on the role of VSMC as signal-sending and signal-receiving cells and determines how NOTCH-NOTCH ligand interactions affect VSMC survival, migration, matrix production, contractility and ROS release. Specific Aim 4 unravels the molecular mechanisms through which patient-derived CD4+NOTCH1+ T cells regulate the functional behavior of ECs and investigates how such T cells modulate EC proinflammatory functions, angiogenic capacity, adhesiveness and leakiness of the EC barrier.