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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：
10591483
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/10591483
关键词：
Adult Affect Angiopoietin-2 Angiopoietins Binding Biochemical Blood - brain barrier anatomy Blood Vessels Blood brain barrier dysfunction 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 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 Surgical Replantation TIE-2 Receptor Testing Therapeutic Effect Tissues Vesicle Visualization Work angiogenesis autosome blood-brain barrier function brain endothelial cell caveolin 1 cerebral cavernous malformations cerebrovascular effective therapy experimental study germinal center kinases 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1/100至200的一般人群，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携带三个基因之一的功能缺失突变：CCM 1，CCM 2和CCM 3（Pdcd 10）。我们专注于CCM 3（Pdcd 10），因为CCM 3缺失的人和小鼠表现出更多的 CCM 1、CCM 2缺失者的表型比CCM 1、CCM 2缺失者严重。为什么人类CCM病变主要是局限于脑血管系统，尽管CCM蛋白的普遍表达，仍然不清楚。我们最近使用新型脑EC（BEC）特异性建立了可诱导的Ccm 3缺失 Cre系（Pdcd 10 BECKO）促进脑内CCM病变。重要的是，Pdcd 10 BECKO小鼠存活长达6-12个月，使我们能够通过实时成像可视化血管病变形成，定义CCM发病机制，并在成年期测试治疗方法。我们之前的研究表明 CCM 3抑制Unc 13 B依赖性胞吐介导的促血管生成素-2分泌。最近的研究表明，小窝囊泡及其核心蛋白Caveolin-1（Cav 1）是由CCM 3严格控制，并且在Ccm 3 - 100的脑微血管EC中显著增加。缺陷小鼠由于增加的小窝与增加的BBB功能障碍有关，我们推测，CCM 3通过控制脑微血管内皮细胞中的胞内囊泡，调节血脑屏障的完整性; CCM 3的缺失诱导异常囊泡运输，特别是小窝- 介导的胞吞转运和蛋白运输，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）确定小窝和Cav 1介导的信号传导对脑血管畸形的贡献。我们将确定Cav 1遗传缺陷是否可预防Pdcd 10 BECKO小鼠中的CCM病变，表征BBB 功能和小窝介导的转胞吞作用，表征EC-周细胞结合和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）分析结构和功能; 2）确定机制 CCM 3通过其调节Cav 1-Tie 2信号传导和血管稳定。我们将执行生物化学和成像实验以确定CCM 3调节的Tie 2是否是小窝特异性的，确定CCM 3-Cav 1-Tie 2信号传导在调节EC连接、EC-周细胞通过体外模型确定联合和血管稳定性; 3）确定治疗效果 Cav 1-Tie 2信号在CCM疾病进展中的作用。我们将测试Tie 2的治疗效果抑制剂，Tie 2基因缺失对CCM疾病的影响，定义Tie 2介导的基因表达，通过scRNA-seq分析Tie 2 high信号通路，并确定Tie 2 high EC在CCM损伤形成中的作用。