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：
10180204
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/10180204
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 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/antagonist 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.

脑血管畸形会影响100至200的普通人群中的1个中风，癫痫发作和局灶性神经系统缺陷的风险。遗传常染色体显性的患者 CCM在三个基因之一中携带功能突变的丧失：CCM1，CCM2和CCM3（PDCD10）。我们专注于CCM3（PDCD10），因为人类和CCM3损失的小鼠都表现出更多严重的表型比损失CCM1或CCM2的表型。为什么人类CCM病变主要是尽管CCM蛋白无处不在，但局限于脑脉管系统仍不清楚。我们最近使用新型的脑EC（BEC）特异性建立了可诱导的CCM3缺失 CRE系（PDCD10BECKO）促进了大脑中的CCM病变。重要的是，PDCD10BECKO小鼠生存长达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发病机理，并在成年期测试治疗剂。我们以前的作品显示 CCM3抑制了UNC13B依赖性胞吐介导的牛血管素-2的分泌。最近的研究表明，小囊泡及其核心蛋白可窝蛋白1（CAV1）是由CCM3紧密控制，并在CCM3-的脑微血管EC中急剧增加不足的小鼠。自从Caveolae增加与BBB功能障碍增加有关我们假设CCM3通过控制脑微血管EC中的细胞内囊泡，调节BBB完整性； CCM3的损失会引起异常的囊泡运输，尤其是小窝。介导的转胞病和蛋白质运输，导致BBB功能障碍和血管畸形。我们提出以下三个特定目的和研究：1）确定小窝和CAV1介导的信号对脑血管畸形的贡献。我们将确定CAV1遗传缺陷是否阻止PDCD10BECKO小鼠中的CCM病变，表征BBB 功能和小窝介导的转介症，表征Ec-Ec-Ec-Ec-Ec-Ec Lumen DKO的扩张，并表征与BBB相关的新型基因表达和信号通路单细胞RNA-SEQ（SCRNA-SEQ）分析的结构和功能； 2）定义机制 CCM3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是否特定于小窝确定CCM3-CAV1-TIE2信号在调节EC连接的作用体外模型的关联和血管稳定； 3）确定治疗效果 CCM疾病进展中的CAV1-TIE2信号传导。我们将测试TIE2的治疗作用抑制剂，TIE2对CCM疾病的遗传缺失，定义TIE2介导的基因表达和通过SCRNA-SEQ信号通路，并确定TIE2高EC在CCM病变形成中的作用。