Phenotyping, Human Tissue and Biomarkers Core

脑海绵状血管畸形(CCM)是一种常见的出血性血管畸形，由生殖系引起。 和/或三个已知基因的内皮体细胞突变。它影响了100多万美国人， 使他们面临中风和癫痫的终生风险。病变在手术后再次出血的可能性高得惊人。 既往有症状性出血，目前尚无治疗方法可阻止其发生或临床进展 损伤的数量。在过去的五年中，我们的计划项目通过以下方式阐明了CCM发病的关键机制 四个实验室之间的出色协作和协同作用。这些措施包括确定 在CCM丢失的情况下，MEKK3/KLF2/4信号是病变发展的主要触发因素 RhoA激酶(ROCK)的激活，以及微生物组驱动的TLR4/CD14信号转导，肠粘蛋白丢失， 低氧、膳食补充剂、凝血酶敏感蛋白-1、血栓调节蛋白与突变的克隆扩增 内皮细胞。芝加哥大学的科学核心优化了病变负荷的高通量评估 Micro-CT，并用非血红素铁Perls染色密度计量法对慢性出血进行定量。我们 熟悉数据结构以获得最佳样本量和统计数据，并遵循前瞻性原则 在不同的实验中阐明结果和致盲。协议是统一的，模型， 样本和数据在项目和站点之间共享。Core生产的转录文库 突变的细胞、线虫和神经血管单位，来自人类和小鼠的病变，具有不同的基因和 处于不同的发展阶段。我们简化了基因分型人CCM的收集和分发 从切除的手术标本到项目现场的病变。我们评估了人类的外周血 研究对象为与信号异常相关的蛋白质水平和microRNAs。然而，关键的知识差距 仍然存在，因为试点数据激发了关于CCM缺陷的内皮在病变发生中的作用的假说 与成熟，PIK3信号，ADAMTS蛋白水解酶，激活的蛋白C抗凝剂和 细胞保护途径。应继续提供核心服务，并通过旨在促进 皮损的单细胞RNA和DNA分析，急性出血的形态计量评估(作为临床 不同于慢性出血的相关表型特征)，以及血浆的微量采样和处理 在老鼠身上发现生物标记物。每个项目都将得到拟议方案的协助和利用 表型，人类组织和生物标志物核心，旨在(1)表型评估和人类损伤 解剖；(2)生物标志物的发现和验证。正在探索的新假说将确定小说 治疗和由此产生的生物标记物将有助于患者的风险分层和疾病监测。在一个 在战略意义上，拟议的核心将继续加强项目地点之间的协作和协同作用， 确保严谨，促进创新。核心应允许老鼠和人之间的交叉验证 样本，进一步利用将发现转化为人类受试者。

The cerebral cavernous malformation (CCM) is a common hemorrhagic vascular anomaly, caused by germ line and/or endothelial somatic mutations in three known genes. It affects more than a million Americans, predisposing them to a lifetime risk of stroke and epilepsy. A lesion is alarmingly more likely to rebleed after a prior symptomatic hemorrhage, and there is currently no therapy to prevent the genesis or clinical progression of lesions. In the past 5 years, our program project has clarified key mechanisms of CCM pathogenesis through exceptional collaboration and synergy among four laboratories. These included the identification of MEKK3/KLF2/4 signaling as a primary trigger of lesion development in the setting of Ccm loss, downstream activation of RhoA kinase (ROCK), and roles of microbiome driven TLR4/CD14 signaling, intestinal mucin loss, hypoxia, dietary supplements, thrombospondin-1, thrombomodulin and clonal expansion of mutated endothelium. The Scientific Core at U Chicago optimized the high throughput assessment of lesion burden using micro-CT, and the quantification of chronic hemorrhage using densitometry of non-heme iron Perls staining. We developed familiarity with data structure for optimal sample sizes and statistics, and a discipline of prospective articulation of outcomes and blinding in the various experiments. Protocols were harmonized, and models, specimens and data were shared across projects and sites. The Core produced transcriptomic libraries of mutated cells, C. elegans, and neurovascular units from human and murine lesions with various genotypes and at different stages of development. We streamlined the collection and distribution of genotyped human CCM lesions from excised surgical specimens to the project sites. And we assessed the peripheral blood of human subjects for protein levels and microRNAs related to the signaling aberrations. Yet critical knowledge gaps remain, as pilot data has motivated hypotheses about the roles of Ccm deficient endothelium in lesion genesis versus maturation, PIK3 signaling, ADAMTS proteolysis of versican, and activated protein C anticoagulant and cytoprotective pathways. Core services shall continue, further enhanced by new techniques aimed at facilitating single-cell RNA and DNA analyses from lesions, morphometric assessment of acute hemorrhage (as a clinically relevant phenotypic feature distinct from chronic bleeding), and micro-sampling and processing for plasma biomarker discovery in mice. Each of the projects will be assisted by and take advantage of the proposed Phenotyping, Human Tissue and Biomarkers Core, aimed at (1) phenotype assessment and human lesion dissection, and (2) biomarker discovery and validation. The new hypotheses being probed will identify novel therapies, and the resulting biomarkers will facilitate risk stratification and disease monitoring in patients. In a strategic sense, the proposed Core will continue to enhance collaboration and synergy among the project sites, ensure rigor, and facilitate innovation. The Core shall allow cross-validation between mouse and human samples, further leveraging the translation of discoveries to human subjects.