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，并且与组织实质密切接触 细胞控制发育和病理疾病状态。它们由共组装的内皮组成 细胞（EC）带有相关周细胞的管网络。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，以及机制和EC衍生的分子 控制周细胞募集，增殖和毛细管地下膜沉积，这一过程 Quires Ec-Pricyte相互作用。血管畸形，例如动脉畸形（AVM）和 海绵状畸形（CMS）（最常在脑组织中观察到，称为CCMS），构成一组 EC管形态发生异常异常的病理，伴随着壁细胞间缺乏症 tions。一个关键点是，根本缺乏对基本分子基础的理解 从EC或周细胞的角度开始这些畸形的发展。为了解决这些问题， 我们开发了两个新型的体外体外血管畸形模型，这是一种使用人ECS的AVM样模型 使用表达K-RASV12的ECS表达K-RAS激活突变（即K-RASV12）和类似CM的模型 和T抗原（TAG）（使细胞周期失调）。在类似AVM的情况下，ECS明显加速管 与对照EC相比，形成的形成是，周细胞募集和地下膜沉积是 与对照组相比，大大减少了。在类似CM的情况下，修饰的EC形成大囊肿（没有发芽 - 行为）具有明显的EC扩散，而周细胞表现出反应性或对EC的招募 衬有衬里的囊肿（在体内强烈模仿CMS）。因此，我们的两个体外模型都概括了 带有AVM和CM的体内以及其他初步数据进一步支持了这些结论。调查和 与体内发现相关，我们正在利用删除CCM1的CCM疾病的小鼠模型（SELEC-- 在ECS内）以可诱导的方式在产后小鼠中，有或没有EC共同诱导激活muta- K-RAS或PI3激酶中的tions。初步数据表明，这种激活突变可以显着增强 CCM在体内发育与CCM1的EC缺失结合使用，这支持我们的体外观察结果。 我们提出了三个特定的目的，以进一步研究血管层的基本分子基础 形成并为这些疾病开发新的治疗选择；他们是： AIM＃1：定义K-RASV12在EC中的表达如何导致EC管形成加速，但减少了 - 周日 cyte-ec相互作用导致动静脉样畸形。 AIM＃2：定义EC中K-RASV12的表达如何与CCM基因丢失和EC细胞周期调节群的丧失相结合。 导致海绵状畸形，明显不足的周细胞募集。 目标＃3：定义促炎性介体如何影响周围EC的相互作用以调节形成或 K-RASV12依赖性动脉静脉样和海绵状畸形的稳定性。