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

脑血管畸形影响到100到200人中的1人，而且 中风、癫痫和局灶性神经功能障碍的风险。遗传性常染色体显性遗传患者 CCM携带三个基因之一的功能突变：CCM1、CCM2和CCM3(Pdcd10)。 我们一直关注CCM3(Pdcd10)，因为CCM3缺失的人和小鼠表现出更多 CCM1或CCM2缺失者的表型较重。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，目前仍不清楚。 我们最近利用一种新的脑EC(BEC)特异性基因建立了可诱导的Ccm3缺失 Cre株(Pdcd10BECKO)，促进脑内CCM损伤。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可视化血管病变的形成， 明确CCM的发病机制，并测试成年期的治疗方法。我们之前的工作表明 CCM3抑制Unc13B依赖的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小窝蛋白-1(Cav1)是 CCM3严密调控，脑微血管内皮细胞数量显著增加。 有缺陷的小鼠。由于小凹的增加与血脑屏障功能障碍的增加有关， 我们假设CCM3通过控制脑微血管内皮细胞内的小泡， 调节血脑屏障的完整性；CCM3的丢失导致异常的囊泡运输，特别是小凹- 介导的跨细胞作用和蛋白质转运，导致血脑屏障功能障碍和血管 畸形。我们提出了以下三个具体目标和研究：1)确定 小窝和Cav1介导的信号在脑血管畸形中的作用。我们会 确定Cav1基因缺陷是否能阻止Pdcd10BECKO小鼠CCM损伤，表征BBB 功能和小凹介导的细胞转运，表征EC-周细胞联合和EC管腔 DKO中的扩张性，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 CCM3通过其调控Cav1-Tie2信号转导和血管稳定。我们将表演 生化和成像实验以确定CCM3调节的Tie2是否是小窝特异性的， 确定CCM3-Cav1-Tie2信号在EC连接、EC-周细胞调控中的作用 体外模型的缔合和血管稳定；3)确定疗效 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 CCM疾病中Tie2基因缺失的抑制物，定义Tie2介导的基因表达和 通过scRNA-seq的信号转导途径，确定Tie2High ECs在CCM病变形成中的作用。

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.