Regulation of Vascular Integrity

血管完整性的调节

基本信息

批准号：
10266523
负责人：
Brant Weinstein
金额：
$ 55.12万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10266523
关键词：
Alleles Animal Model Animals Arteries Basement membrane Behavior Blood Circulation Blood Vessels Brain Brain hemorrhage Caliber Cell Communication Cell physiology Cells Cephalic Cerebrospinal Fluid Clinical Clustered Regularly Interspaced Short Palindromic Repeats Connective Tissue Defect Development Embryo Endothelium Ethylnitrosourea Fishes Fresh Water Generations Genes Genetic Models Genetic Screening Goals Location Lymphatic Endothelial Cells Maintenance Meningeal Meninges Modeling Molecular Molecular Cloning Morbidity - disease rate Morphogenesis Morphology Mutation Nature Neurocognitive Deficit Neurodegenerative Disorders Nutrient Optics Organ Oxygen Pathology Pathway interactions Pericytes Play Population Regulation Reporting Role Sclerotome Series Signal Transduction Smooth Muscle Myocytes Stroke Structure Surface Tissues Transgenic Organisms Trauma Tube Vascular Diseases Vascular Endothelial Growth Factors Vascular Endothelium Vascular Smooth Muscle Veins Vertebrates Zebrafish base cell type experimental analysis genetic analysis genetic regulatory protein growth differentiation factor 6 insight interest macrophage mortality mutant next generation sequencing novel rhoA GTP-Binding Protein screening tool transdifferentiation wasting

项目摘要

As described in the goals and objectives section of this report, this project consists of three specific aims: Studying genes regulating vascular integrity We have used forward-genetic screens and CRISPR gene editing to generate zebrafish mutants that disrupt cranial vascular integrity in the zebrafish, using next-generation sequencing to perform rapid molecular cloning of the defective genes from mutants. We previously characterized the role of GDF6 (BMP13) in vascular integrity, demonstrating that this gene promotes maintenance of vascular integrity by suppressing excess VEGF signaling. We are currently characterizing the molecular nature of the defects in the important intracellular regulatory protein RhoA, for which we have generated an allelic series of mutations. These and other new vascular integrity mutants promise to bring to light new pathways important in the maintenance of vascular barrier function. Studying the acquisition and function of supporting vascular smooth muscle cells The vascular smooth cells (VSMC) that surround the endothelial tube play a critical role in regulating vascular tone and vascular integrity. We have recently been examining the acquisition and function of these cells using the zebrafish. We have developed a number of useful transgenic tools to visualize and experimentally manipulate this cell population in the zebrafish, and are using these and other tools to, among other things, (i) examine the origins of these cells from early sclerotome, (ii) demonstrate that VSMC interaction with the endothelium is required to maintain the vascular basement membrane and restrict vessel diameter, and (iii) elucidate the molecular pathways responsible for the selective acquisition of VSMC by arteries (as opposed to veins). Studying the vasculature and vascular-associated cells in the meninges The meninges are an external enveloping connective tissue that encases the brain, producing cerebrospinal fluid, acting as a cushion against trauma, nourishing the brain via nutrient circulation, and removing waste. Despite its importance, the cell types present in the meninges and the function and embryonic origins of this tissue are still not well understood. We recently discovered a novel perivascular cell population closely associated with blood vessels on the zebrafish brain. Based on similarities in their morphology, location, and highly unusual scavenger behavior, these cells appear to be the zebrafish equivalent of mammalian Fluorescent Granular Perithelial cells (FGPs), macrophage-like cells about which very little is known that likely play important roles in brain function and in a variety of CNS pathologies. Despite their macrophage-like morphology and their perivascular location these cells are molecularly most similar to lymphatic endothelial cells and they transdifferentiate from primitive endothelium deep inside the brain before migrating to the brain surface. Using forward-genetic screening of transgenic zebrafish for ENU-induced recessive mutations, we recently identified a mutant that appears to be specifically deficient in FGPs, providing a genetic model that we are using to further explore the important functional role of these cells. Our findings thus far provide the first report of a non-vessel forming, perivascular cell population in the brain that emerges by transdifferentiation from vascular endothelium. We are further exploring the structure, cellular composition, and functional roles of meningeal vascular-associated cell populations using the zebrafish model.

正如本报告的目的和目标部分所述，该项目包含三个具体目标：研究调节血管完整性的基因我们使用正向遗传筛选和 CRISPR 基因编辑来生成破坏斑马鱼颅血管完整性的斑马鱼突变体，并使用下一代测序对突变体的缺陷基因进行快速分子克隆。我们之前描述了 GDF6 (BMP13) 在血管完整性中的作用，证明该基因通过抑制过量的 VEGF 信号传导来促进血管完整性的维持。我们目前正在表征重要的细胞内调节蛋白 RhoA 缺陷的分子性质，为此我们已经生成了一系列等位基因突变。这些和其他新的血管完整性突变体有望揭示在维持血管屏障功能中重要的新途径。研究支持血管平滑肌细胞的获取和功能围绕内皮管的血管平滑细胞（VSMC）在调节血管张力和血管完整性方面发挥着关键作用。我们最近一直在使用斑马鱼检查这些细胞的获取和功能。我们开发了许多有用的转基因工具来可视化和实验操作斑马鱼中的细胞群，并使用这些工具和其他工具，除其他外，（i）检查这些细胞来自早期骨节的起源，（ii）证明 VSMC 与内皮细胞的相互作用是维持血管基底膜和限制血管直径所必需的，并且 (iii) 阐明了动脉（而不是静脉）选择性获取 VSMC 的分子途径。研究脑膜中的脉管系统和血管相关细胞脑膜是外部包封结缔组织，包围大脑，产生脑脊液，充当创伤缓冲垫，通过营养循环滋养大脑并清除废物。尽管脑膜很重要，但脑膜中存在的细胞类型以及该组织的功能和胚胎起源仍不清楚。我们最近发现了一种与斑马鱼大脑血管密切相关的新型血管周围细胞群。基于其形态、位置和极不寻常的清道夫行为的相似性，这些细胞似乎是斑马鱼中哺乳动物荧光颗粒状上皮细胞（FGP）的等价物，这种巨噬细胞样细胞人们知之甚少，但它们可能在大脑中发挥重要作用。功能和各种中枢神经系统病理。尽管它们具有巨噬细胞样形态和血管周围位置，但这些细胞在分子上与淋巴内皮细胞最相似，并且它们在迁移到大脑表面之前从大脑深处的原始内皮细胞转分化。通过对转基因斑马鱼进行 ENU 诱导的隐性突变的正向遗传筛选，我们最近发现了一种似乎在 FGP 中特别缺乏的突变体，这提供了一个遗传模型，我们正在使用该模型来进一步探索这些细胞的重要功能作用。迄今为止，我们的研究结果首次报告了大脑中通过血管内皮转分化而产生的非血管形成血管周围细胞群。我们正在利用斑马鱼模型进一步探索脑膜血管相关细胞群的结构、细胞组成和功能作用。