Elucidating mechanisms of hereditary hemorrhagic telangiectasia 2 gene-mediated arteriovenous malformation development via eNOS signaling using in vivo two-photon imaging in a preclinical mouse model

项目摘要 这个项目的长期目标是确定一个合适的临床前小鼠模型来表征病因 遗传性出血性毛细血管扩张症（HHT），并开发新的治疗方案的疾病。了HHT 一种以动静脉畸形（AVM）为全身核心病理的遗传性疾病。反车辆地雷 从动脉到静脉的异常直接连接，没有毛细血管介入。HHT的症状包括 毛细血管扩张、鼻出血、严重头痛、癫痫、中风、胃肠道出血和脑、肺 和肝脏。HHT的全球估计发病率为1：50002，但90%的HHT患者未被诊断。 目前对HHT的主要临床表现尚无防治方法，需要开发 更好的治疗策略。TGFβ I型受体激活素A受体样1（ACVRL 1或Alk 1）基因 与HHT 2型（HHT 2）的病因学有关，该基因的突变估计是25- 30岁儿童HHT发病的基础。 所有HHT病例的57% 3 -7。Alk 1主要在内皮细胞中表达，是血管内皮细胞的重要组成部分。 发展8 -10.先前基于Alk 1缺失的HHT 2小鼠模型表现出某些疾病， 方面11 -14.然而，现有的动物模型不是理想的HHT动物模型，因为动物要么死亡， 早期出生后第5天（纯合子缺失）或发展血管病变，潜伏期长， 缺乏关键HHT症状的渗透（杂合缺失），e. G.流鼻血15.因此，有一个关键的 需要开发更好的临床前动物模型，以更好地反映HHT 2中存在的血管病变 患者，帮助努力揭示疾病进展的机制， HHT治疗的预防和治疗策略。为了解决这些知识差距，我们的实验室创建了 一种新的小鼠模型，其中Alk 1在出生后动脉内皮细胞中缺失。初步数据表明 这些小鼠存活超过出生后27天，多个器官中有HHT样血管病变， HHT 2的动物模型，以研究疾病的过程和潜在的机制。我将描述 通过检查大体病理学、组织病理学、动物健康状况以及 动物的行为。此外，我将通过显微镜检查血管结构和功能， 成像，包括双光子活体成像。最后，我将描述的氧化应激概况， 小鼠通过测量超氧化物和NO水平，同时还用超氧化物歧化酶模拟物处理小鼠， TEMPOL和一种合成的BH 4化合物，sepiapterin，以确定氧化应激在形成 和预防AVM。该项目的成功完成，将为我国的 研究Alk 1介导的HHT形成的机制，并开发新的 HHT 2患者的预防性和治愈性治疗。

Project Summary The long-term goal of this project is to identify an appropriate preclinical mouse model to characterize the etiology of Hereditary hemorrhagic telangiectasia (HHT) and to develop novel treatment options for the disease. HHT is a genetic disorder with arteriovenous malformations (AVMs) as a core pathology throughout the body. AVMs are abnormal direct connections from arteries to veins without intervening capillaries. Symptoms of HHT include telangiectasias, epistaxis, severe headache, epilepsy, stroke, gastrointestinal bleeding, and AVMs in brain, lung, and liver. The worldwide estimated incidence of HHT is 1:50002, but 90% of people with HHT are undiagnosed. Currently, there is no prevention or cure for the major clinical manifestations of HHT, calling for the development of better treatment strategies. The TGFβ type I receptor activin A receptor-like type 1 (ACVRL1 or Alk1) gene has been implicated in the etiology of HHT type 2 (HHT2), and mutation of this gene is estimated to underlie 25- 57% of all HHT cases3–7. Alk1 is primarily expressed in endothelial cells and is essential for vascular development8–10. Previous mouse models of HHT2, based on deletion of Alk1, exhibited certain disease aspects11–14. However, the existing animal models are not ideal animal models for HHT, as animals either die early by postnatal day 5 (homozygous deletion) or develop vascular lesions with long latency and incomplete penetration (heterozygous deletion) lacking key HHT symptoms, e. g. nosebleed15. Therefore, there is a critical need to develop a better preclinical animal model that better reflects the vascular lesions presented in HHT2 patients, aiding the efforts to uncover the mechanisms of the disease progression and develop potential preventative and treatment strategies for HHT therapy. To resolve these gaps in knowledge, our lab has created a novel mouse model in which Alk1 is deleted postnatally in arterial endothelial cells. Preliminary data suggest that these mice survived beyond postnatal day 27 with HHT-like vascular lesions in multiple organs, offering a valuable animal model of HHT2 to study the disease processes and underlying mechanisms. I will characterize the HHT-like lesions present in these mice by examining the gross pathology, histopathology, animal health, and behavior of the animals. Further, I will examine the vascular structure and function through microscopic imagining, including two-photon in vivo live imaging. Finally, I will characterize the oxidative stress profile of the mice by measuring superoxide and NO levels, while also treating the mice with a superoxide dismutase mimetic, TEMPOL and a synthetic BH4 compound, sepiapterin to determine the effects of oxidative stress in the formation and prevention of AVMs in this model. The successful completion of this project will provide a novel tool for the investigation of mechanism underlying Alk1-mediated HHT formation and for the development of novel preventative and curative therapies for patients with HHT2.