Investigating the intracellular vesicle-mediated mechanism contributing to cerebral cavernous malformation

研究细胞内囊泡介导的脑海绵状血管瘤机制

基本信息

批准号：
10372136
负责人：
Jenny Huanjiao Zhou
金额：
$ 41.88万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10372136
关键词：
Adult Affect Angiopoietin-2 Angiopoietins Binding Biochemical Blood - brain barrier anatomy Blood Vessels Blood capillaries Brain Brain Vascular Malformation CCM1 gene Caveolae Cavernous Malformation Cells Cellular biology Cerebrovascular system Chronic Clathrin Core Protein Data Defect Disease Disease Progression Endocytosis Endothelial Cells Endothelial Growth Factors Endothelium Exhibits Exocytosis Focal Neurologic Deficits Functional disorder Gene Expression General Population Genes Genetic Human Image Inherited Intercellular Junctions Lesion Mediating Mesenchymal Molecular Mus Mutation Nature Pathogenesis Pathologic Pathway interactions Patients Pericytes Perinatal Permeability Persons Phenotype Primary Lesion Proteins Receptor Signaling Role Seizures Signal Pathway Signal Transduction Stress Fibers Structure Supporting Cell Surface TIE-2 Receptor Testing Therapeutic Effect Tissues Vesicle Work angiogenesis blood-brain barrier function brain endothelial cell caveolin 1 cerebral cavernous malformations cerebrovascular effective therapy experimental study germinal center kinases implantation in vitro Model inhibitor loss of function mutation malformation microscopic imaging mouse model neurovascular unit novel postcapillary venule prevent protein transport single-cell RNA sequencing stroke risk therapeutic evaluation trafficking transcriptome transcytosis two photon microscopy two-photon

项目摘要

Cerebral vascular malformations affect 1 in 100 to 200 of the general population with increased risk for stroke, seizures and focal neurological deficits. Patients with inherited autosomal dominant CCM carry loss of function mutations in one of three genes: CCM1, CCM2 and CCM3 (Pdcd10). We have focused on CCM3 (Pdcd10) as both humans and mice with CCM3 loss exhibit more severe phenotype than those with loss of CCM1 or CCM2. Why human CCM lesions are primarily confined to the brain vasculature, despite ubiquitous expression of CCM proteins, remains unclear. We have recently established an inducible Ccm3 deletion using a novel brain EC (BEC)-specific Cre line (Pdcd10BECKO) that promoted CCM lesions in the brain. Importantly, the Pdcd10BECKO mice survive up to 6-12 months, allowing us to visualize vascular lesion formation by live imaging, to define the CCM pathogenesis, and to test therapeutics in adulthood. Our previous work shows that CCM3 suppresses Unc13B-dependent exocytosis-mediated secretion of angiopoietin-2. More recent study indicates that caveolae vesicle and its core protein caveolin-1 (Cav1) are tightly controlled by CCM3, and dramatically increased in the brain microvascular ECs of Ccm3- deficient mice. Since increased caveolae has been associated with increased BBB dysfunction, we hypothesize that CCM3, by controlling intracellular vesicles in brain microvascular ECs, regulates BBB integrity; loss of CCM3 induces abnormal vesicle trafficking, particularly caveolae- mediated transcytosis and protein trafficking, leading to BBB dysfunction and vascular malformation. We propose the following three specific aims and studies: 1) To determine the contribution of caveolae and Cav1-mediated signaling to brain vascular malformations. We will determine if Cav1 genetic defect prevents CCM lesion in Pdcd10BECKO mice, characterize BBB function and caveolae-mediated transcytosis, characterize EC-pericyte association and EC lumen dilation in DKO, and characterize novel gene expression and signaling pathways related to BBB structure and function by single cell RNA-seq (scRNA-seq) analyses; 2) To define the mechanism by which CCM3 regulates Cav1-Tie2 signaling and vascular stabilization. We will perform biochemical and imaging experiments to determine if CCM3-regulated Tie2 is caveolae-specific, determine the role of CCM3-Cav1-Tie2 signaling in regulating EC junction, EC-pericyte association and vascular stabilization by in vitro models; 3) To determine the therapeutic effects of Cav1-Tie2 signaling in CCM disease progression. We will test therapeutic effects of Tie2 inhibitor, genetic deletion of Tie2 on CCM disease, define Tie2-mediated gene expression and signaling pathways by scRNA-seq, and determine the role of Tie2high ECs in CCM lesion formation.

脑血管畸形影响普通人群中百分之一到两百的人，其发病率增加中风、癫痫发作和局灶性神经功能缺损的风险。常染色体显性遗传患者 CCM 在以下三个基因之一中携带功能丧失突变：CCM1、CCM2 和 CCM3 (Pdcd10)。我们重点关注 CCM3 (Pdcd10)，因为 CCM3 缺失的人类和小鼠都表现出更多比那些失去 CCM1 或 CCM2 的人表现型更严重。为什么人类 CCM 病变主要是尽管 CCM 蛋白普遍表达，但仅限于脑血管系统仍不清楚。我们最近使用一种新型脑 EC (BEC) 特异性建立了可诱导的 Ccm3 缺失 Cre 系 (Pdcd10BECKO) 促进大脑中的 CCM 病变。重要的是，Pdcd10BECKO 小鼠存活长达 6-12 个月，使我们能够通过实时成像观察血管病变的形成，定义 CCM 发病机制，并在成年后测试治疗方法。我们之前的工作表明 CCM3 抑制 Unc13B 依赖性胞吐作用介导的血管生成素 2 的分泌。最近的研究表明，小窝囊泡及其核心蛋白 Caveolin-1 (Cav1) 受CCM3严格控制，并且Ccm3-的脑微血管EC显着增加缺乏的老鼠。由于小凹的增加与 BBB 功能障碍的增加有关，我们假设 CCM3 通过控制脑微血管 EC 中的细胞内囊泡，规范 BBB 完整性； CCM3 的缺失会导致异常的囊泡运输，特别是小窝 - 介导的转胞吞作用和蛋白质运输，导致 BBB 功能障碍和血管畸形。我们提出以下三个具体目标和研究： 1）确定小凹和 Cav1 介导的信号对脑血管畸形的贡献。我们将确定 Cav1 遗传缺陷是否可以预防 Pdcd10BECKO 小鼠的 CCM 病变，表征 BBB 功能和小窝介导的转胞吞作用，表征 EC-周细胞关联和 EC 腔 DKO 中的扩张，并表征与 BBB 相关的新基因表达和信号通路通过单细胞 RNA-seq (scRNA-seq) 分析进行结构和功能； 2）定义机制 CCM3 通过其调节 Cav1-Tie2 信号传导和血管稳定。我们将表演生化和成像实验以确定 CCM3 调节的 Tie2 是否具有小凹特异性，确定 CCM3-Cav1-Tie2 信号在调节 EC 连接、EC 周细胞中的作用体外模型的关联和血管稳定性； 3）确定治疗效果 Cav1-Tie2 信号在 CCM 疾病进展中的作用。我们将测试Tie2的治疗效果抑制剂，CCM 疾病中 Tie2 的基因缺失，定义了 Tie2 介导的基因表达和通过 scRNA-seq 分析信号通路，并确定 Tie2high ECs 在 CCM 病变形成中的作用。