The NOTCH Signaling Pathway in Large Vessel Vasculitis

项目摘要 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。在老龄化人口中， 需要长期管理LVV的患者一直在稳步上升，而治疗设备 一直严格限制在高剂量皮质类固醇。在过去的十年里， 提示先天性和适应性免疫系统参与LVV的免疫发病机制。但有一个 我们的知识的关键差距，为什么疾病的目标是主动脉及其主要分支，以及如何免疫， 动脉壁中的基质交通引发并促进血管炎。病原性免疫反应 具有抗原诱导的克隆扩增的特征，但我们最近发现共刺激信号 来源于组织小生境中的驻留细胞在驱动组织破坏性免疫中同样重要。GCA 动脉表达大量的NOTCH受体和配体，提供了一个分子平台， 细胞间通讯阻断NOTCH信号传导可有效抑制血管炎。来自GCA的CD 4 T细胞 患者组成型表达NOTCH 1受体，使他们能够与表达NOTCH配体的细胞相互作用， 血管平滑肌细胞（VSMC）和内皮细胞（EC）。此应用程序旨在揭示如何 Notch通路参与免疫-内皮和免疫-基质通讯，以及Notch- 依赖性信号传导形成血管生成性T细胞应答和适应不良的VSMC和EC行为。的 该项目建立在一系列有利资源的基础上，包括GCA患者的临床表型队列; 一种新的人体动脉壁的三维模型系统，它允许从 可堆叠的单元，其填充有限定的细胞群;以及携带发炎的人源化小鼠模型， 人体动脉Notch受体和配体的进入可以通过配体竞争性阻断。 可以通过RNAi技术使抗体/融合蛋白和细胞的Notch信号传导缺陷。具体 目的1在机制水平上研究VSMC和EC上的NOTCH配体如何调节VSMC和EC的效应器功能。 血管生成性CD 4 T细胞;调节其生长、组织侵袭能力和细胞因子产生。具体 目的2寻求鉴定可用于抑制NOTCH 1表达或靶向NOTCH 1的信号传导网络。 致病性T细胞中的NOTCH依赖性存活信号。小分子抑制剂破坏Notch衍生的 将在嵌合体模型中测试信号的抗血管炎潜力。具体目标3侧重于 VSMC作为信号发送和信号接收细胞的作用，并决定了NOTCH-NOTCH配体 相互作用影响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细胞调节细胞增殖的分子机制。 EC的功能行为，并研究这些T细胞如何调节EC促炎功能， 血管生成能力、EC屏障的渗透性和渗漏性。

Project Summary 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.