Investigating the pathogenesis of Moyamoya Disease using patient derived induced pluripotent stem cells

使用患者来源的诱导多能干细胞研究烟雾病的发病机制

• 批准号: 10373587
• 负责人: GARY K STEINBERG
• 金额: $ 23.61万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10373587
• 关键词: 3-Dimensional; Adult; Affect; Angiogenic Factor; Animal Model; Arteries; Biological Assay; Blood Vessels; Brain hemorrhage; Bypass; Caliber; Cell Communication; Cell Culture Techniques; Cell Differentiation process; Cell Proliferation; Cell Survival; Cell model; Cells; Cerebral Infarction; Cerebrospinal Fluid; Cerebrovascular Disorders; Cerebrovascular system; Characteristics; Child; Chronic; Clinical; Coculture Techniques; Complex; Cues; Data; Development; Disease; Disease Progression; Disease model; Endothelial Cells; Environmental Risk Factor; Etiology; Extracellular Matrix; Future; Gene Expression; Genetic; Grant; Growth; High-Throughput Nucleotide Sequencing; Human; Hypoxia; In Vitro; Inflammation; Ischemic Stroke; Knowledge; Lead; Length; Measures; Mediating; Mediator of activation protein; Methods; Modeling; Molecular; Molecular Target; Moyamoya Disease; Nervous System Physiology; Operative Surgical Procedures; Organoids; Paralysed; Pathogenesis; Pathogenicity; Pathologic; Pathology; Pathway Analysis; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Phenotype; Platelet-Derived Growth Factor; Play; Process; Proliferating; Property; Reporting; Research; Role; Sampling; Serum; Signal Transduction; Smooth Muscle Myocytes; Stroke; Structure; Techniques; Technology; Thinness; Transient Ischemic Attack; Translating; Tube; Up-Regulation; Vascular Proliferation; Vascular Smooth Muscle; angiogenesis; base; cell type; cerebral artery; clinically relevant; functional disability; induced pluripotent stem cell; insight; migration; novel therapeutics; phenotypic biomarker; revascularization surgery; transcriptome; transcriptome sequencing; vascular smooth muscle cell proliferation

PROJECT SUMMARY Moyamoya Disease (MMD) is a rare, chronic cerebrovascular disease that affects the blood vessels of the brain, causing occlusion of major cerebral arteries and formation of fragile vessels in the vicinity. Clinical manifestations of MMD are transient ischemic attacks and cerebral infarctions, often leading to ischemic or hemorrhagic stroke. Invasive revascularization surgery is the only current treatment available. There may be a combination of genetic, circulating and environmental factors involved in the pathogenesis of MMD, however, the molecular mechanisms underlying MMD is largely unknown, mainly due to the lack of established MMD-specific cellular or animal models. In this proposal we aim to understand the pathogenesis of MMD by using MMD patient- derived iPSCs cellular models in combination with functional assays and high throughput sequencing approaches. The main histopathological finding in MMD is the fibro-cellular thickening of the innermost layer of the vessel (intima) which causes narrowing and occlusion of the vessel. This is likely due to an increase in proliferating vascular smooth muscle cells (VSMCs) or endothelial cells (ECs) and extracellular matrix components. Cues from ECs could cause VSMCs to switch to a phenotype that is proliferative and migrates from media to the intima, thus contributing to the thickening of the intima. Thus, we hypothesize that dysregulated signaling between VSMCs and ECs drive MMD pathology. Using MMD patient iPSC-derived ECs and VSMCs, we have established co-culture model and 3D cellular model by generating vascular organoids. Preliminary co-culture data show that both MMD ECs and VSMCs are functionally impaired when compared to healthy controls, with respect to cell proliferation and in vitro angiogenic tube stabilization. In Aim 1, we aim to characterize the functional properties of MMD iPSC-derived ECs and VSMCs in co-cultures by assessing their ability in cell proliferation, migration and tube formation in normal and hypoxic conditions. VSMC phenotype switching will be examined using specific phenotypic markers and contractility assay. We will also characterize vessel structural characteristics using our established vascular organoids generated from MMD iPSCs. In Aim 2, we will use RNA sequencing technology to investigate the transcriptome of VSMCs and ECs and identify potential molecular mediators involved in MMD. Top targets will be validated using quantitative PCR and their expression pattern will be investigated in our cellular models using immunostaining. Our study will elucidate cell-type specific factors that may drive MMD pathology. Vascular organoids from MMD may be an efficient human in vitro MMD model and provide invaluable information on MMD mechanisms. Data from our studies will advance the knowledge in MMD pathogenesis and open up new avenues of research to yield clinically relevant drug-based methods to treat MMD.

项目概要 烟雾病 (MMD) 是一种罕见的慢性脑血管疾病，会影响大脑血管， 导致主要脑动脉闭塞并在附近形成脆弱血管。临床表现 MMD 的主要症状是短暂性脑缺血发作和脑梗塞，通常导致缺血性或出血性中风。 侵入性血运重建手术是目前唯一可用的治疗方法。可能是遗传因素的结合， 循环和环境因素参与MMD的发病机制，然而，分子机制 潜在的 MMD 在很大程度上是未知的，主要是由于缺乏已建立的 MMD 特异性细胞或动物 模型。在本提案中，我们的目标是通过使用 MMD 患者来源的 iPSC 来了解 MMD 的发病机制 细胞模型与功能测定和高通量测序方法相结合。 MMD 的主要组织病理学发现是血管最内层的纤维细胞增厚 （内膜）导致血管变窄和闭塞。这可能是由于增殖的增加 血管平滑肌细胞（VSMC）或内皮细胞（EC）和细胞外基质成分。提示 来自 EC 的细胞可能会导致 VSMC 转变为增殖性表型并从培养基迁移到 内膜，从而促进内膜增厚。因此，我们假设信号传导失调 VSMC 和 EC 之间驱动 MMD 病理。使用 MMD 患者 iPSC 衍生的 EC 和 VSMC，我们有 通过生成血管类器官建立了共培养模型和3D细胞模型。初步共培养 数据显示，与健康对照相比，MMD EC 和 VSMC 均出现功能受损， 关于细胞增殖和体外血管生成管稳定。在目标 1 中，我们的目标是描述 通过评估 MMD iPSC 衍生的 EC 和 VSMC 在细胞中的能力在共培养中的功能特性 正常和低氧条件下的增殖、迁移和管形成。 VSMC表型转换将 使用特定表型标记和收缩性测定进行检查。我们还将表征船舶结构 使用我们建立的从 MMD iPSC 生成的血管类器官来分析特征。在目标 2 中，我们将使用 RNA 测序技术研究 VSMC 和 EC 的转录组并鉴定潜在的分子 参与 MMD 的调解者。主要目标将使用定量 PCR 及其表达模式进行验证 将使用免疫染色在我们的细胞模型中进行研究。我们的研究将阐明细胞类型的特异性因素 这可能会导致 MMD 病理。 MMD 血管类器官可能是一种有效的人类体外 MMD 模型 并提供有关 MMD 机制的宝贵信息。我们的研究数据将推进我们的知识 MMD 发病机制并开辟新的研究途径，以产生临床相关的基于药物的方法 治疗MMD。