Molecular basis for defective pericyte-endothelial cell interactions regulating vascular malformations

调节血管畸形的有缺陷的周细胞-内皮细胞相互作用的分子基础

• 批准号: 10619624
• 负责人: George E Davis
• 金额: $ 38.03万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10619624
• 关键词: 3-Dimensional; Acceleration; Address; Affect; Antigens; Arteriovenous malformation; Automobile Driving; Basement membrane; Behavior; Biological; Blood Vessels; Blood capillaries; Brain hemorrhage; CCM1 gene; Cavernous Malformation; Cell Communication; Cell Culture Techniques; Cell Cycle; Cell Cycle Regulation; Cell Line; Cell Proliferation; Cells; Cerebrum; Clinical; Coculture Techniques; Coupled; Cyst; Cystic Lesion; DTR gene; Data; Deposition; Development; Disease; Endothelial Cells; Endothelin-1; Event; Genes; Genetic; Growth; Human; In Vitro; Inflammation Mediators; Interleukin-1 beta; KRAS2 gene; Knock-out; Laboratories; Large T Antigen; Lesion; Link; Modeling; Molecular; Morphogenesis; Mus; Mutation; PIK3CA gene; PIK3CG gene; Pathogenesis; Pathogenicity; Pathologic; Pathology; Pericytes; Phenotype; Platelet-Derived Growth Factor; Process; Proliferating; Reporting; Role; Signal Transduction; Site; Small Interfering RNA; Specimen; Stains; Stroke; System; TNF gene; TP53 gene; Thrombin; Tissues; Transforming Growth Factor beta; Tube; Up-Regulation; Venous Malformation; Work; brain arteriovenous malformations; brain endothelial cell; cell immortalization; cell type; cerebral cavernous malformations; comparison control; in vitro Model; in vivo; interest; malformation; mouse model; novel; novel therapeutics; postnatal; recruit; synergism

Capillaries are by far the most abundant blood vessels and are critically interfaced with tissue parenchymal cells to control both development and pathologic disease states. They consist of co-assembled endothelial cells (EC) tube networks with associated pericytes. For many years, our laboratory has been investigating the molecular basis for EC lumen and tube assembly, as well as the mechanisms and EC-derived molecules that control pericyte recruitment, proliferation, and capillary basement membrane deposition, a process that re- quires EC-pericyte interactions. Vascular malformations, such as arteriovenous malformations (AVMs) and cavernous malformations (CMs) (most often observed in cerebral tissue, termed CCMs), constitute a group of pathologies with marked abnormalities in EC tube morphogenesis coupled to deficiencies in mural cell interac- tions. A critical point is that there is a fundamental lack of understanding of the underlying molecular basis for the development of these malformations from either the EC or pericyte perspective. To address these issues, we have developed two novel in vitro models of vascular malformations, an AVM-like model using human ECs expressing a k-Ras activating mutation (i.e. k-RasV12) and a CM-like model using ECs expressing k-RasV12 and T antigen (TAg) (to dysregulate the cell cycle). In the AVM-like case, the ECs markedly accelerate tube formation compared to control ECs, however, pericyte recruitment and basement membrane deposition is strongly reduced compared to controls. In the CM-like case, the modified ECs form large cysts (with no sprout- ing behavior) with evident EC proliferation, while pericytes show responsiveness or no recruitment to the EC- lined cysts (strongly mimicking CMs in vivo). Thus, both of our in vitro models recapitulate what is observed in vivo with AVMs and CMs, and other preliminary data further supports these conclusions. To investigate and correlate in vitro with in vivo findings, we are utilizing mouse models of CCM disorder that delete CCM1 (selec- tively within ECs) in an inducible manner in postnatal mice with or without EC co-induction of activating muta- tions in k-Ras or PI3 kinase. Preliminary data suggests that such activating mutations can markedly enhance CCM development in vivo in conjunction with EC deletion of CCM1, which support our in vitro observations. We propose three specific aims to further investigate the underlying molecular basis for vascular mal- formations and to develop new therapeutic options for these diseases; and they are: Aim #1: Define how k-RasV12 expression in ECs results in accelerated EC tube formation, but reduced peri- cyte-EC interactions leading to arteriovenous-like malformations. Aim #2: Define how k-RasV12 expression in ECs coupled with loss of CCM genes and EC cell cycle regula- tion leads to cavernous-like malformations with markedly deficient pericyte recruitment. Aim #3: Define how pro-inflammatory mediators affect pericyte-EC interactions to regulate the formation or stability of k-RasV12-dependent arteriovenous-like and cavernous-like malformations.

毛细血管是迄今为止最丰富的血管，与组织实质的连接至关重要 细胞控制发育和病理疾病状态。它们由共同组装的内皮细胞组成 细胞（EC）管网络与相关周细胞。多年来，我们的实验室一直在研究 EC 管腔和管组件的分子基础，以及其机制和 EC 衍生分子 控制周细胞募集、增殖和毛细血管基底膜沉积，这一过程重新 需要 EC-周细胞相互作用。血管畸形，例如动静脉畸形（AVM）和 海绵状血管瘤 (CM)（最常在脑组织中观察到，称为 CCM），构成一组 EC管形态发生明显异常的病理与壁细胞相互作用的缺陷相结合 系统蒸发散。关键的一点是，人们对潜在的分子基础缺乏根本性的了解。 从 EC 或周细胞的角度观察这些畸形的发展。为了解决这些问题， 我们开发了两种新型的血管畸形体外模型，一种使用人类 EC 的类 AVM 模型 表达 k-Ras 激活突变（即 k-RasV12）和使用表达 k-RasV12 的 EC 的 CM 样模型 和 T 抗原 (TAg)（使细胞周期失调）。在类似 AVM 的情况下，EC 显着加速管 然而，与对照 ECs 相比，周细胞募集和基底膜沉积是 与对照相比大大减少。在类似 CM 的情况下，修饰后的 EC 形成大的囊肿（没有发芽） 行为），具有明显的 EC 增殖，而周细胞表现出对 EC 的反应性或没有招募 内衬囊肿（强烈模仿体内的 CM）。因此，我们的两个体外模型都概括了在 vivo 的 AVM 和 CM 以及其他初步数据进一步支持了这些结论。调查并 为了将体外与体内的发现联系起来，我们正在利用删除 CCM1 的 CCM 疾病小鼠模型（选择 在有或没有 EC 共同诱导的出生后小鼠中，以诱导方式激活突变 k-Ras 或 PI3 激酶的作用。初步数据表明，这种激活突变可以显着增强 CCM 体内发育与 CCM1 的 EC 缺失相结合，支持我们的体外观察。 我们提出了三个具体目标，以进一步研究血管疾病的潜在分子基础。 形成并为这些疾病开发新的治疗方案；他们是： 目标#1：定义 EC 中的 k-RasV12 表达如何导致加速 EC 管形成，但减少周边 细胞-EC相互作用导致动静脉样畸形。 目标#2：定义 EC 中 k-RasV12 的表达如何与 CCM 基因的丢失和 EC 细胞周期调节相结合 化导致海绵状畸形，周细胞募集明显不足。 目标#3：定义促炎介质如何影响周细胞-EC 相互作用以调节形成或 k-RasV12 依赖性动静脉样和海绵状畸形的稳定性。