Regulation of Vascular Integrity

血管完整性的调节

基本信息

批准号：
10007504
负责人：
Brant Weinstein
金额：
$ 47.26万
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/10007504
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.

如本报告的目标和目标部分所述，该项目包括三个具体目标：研究调节血管完整性的基因我们已经使用了前遗传筛选和CRISPR基因编辑来产生斑马鱼突变体，这些突变体破坏了斑马鱼中颅血管完整性，使用下一代测序来从突变体中对缺陷基因进行快速分子克隆。我们先前表征了GDF6（BMP13）在血管完整性中的作用，表明该基因通过抑制过量的VEGF信号传导来促进维持血管完整性。我们目前正在表征重要的细胞内调节蛋白RhoA中缺陷的分子性质，为此我们产生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光滑细胞（VSMC）在调节血管张力和血管完整性方面起着至关重要的作用。最近，我们一直在使用斑马鱼检查这些细胞的获取和功能。我们已经开发了许多有用的转基因工具来可视化和实验操纵斑马鱼中的细胞种群，并使用这些和其他工具除其他外，（i）检查这些细胞的起源，（ii）证明，VSMC与Endophium的相互作用以维持血管层膜和限制性的毛孔膜片和限制性的毛孔膜片和限制性疾病，并证明II II II型毛囊膜片以及（II II II II II II II;通过动脉（而不是静脉）对VSMC的选择性获取。研究脑膜中的脉管系统和血管相关细胞脑膜是一种包围大脑的外部包裹的结缔组织，产生脑脊液，充当抵抗创伤的缓冲，通过营养循环滋养大脑，并消除废物。尽管它的重要性，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未得到充分了解。我们最近发现了一个与斑马鱼大脑上血管紧密相关的新型周围细胞群。基于它们的形态，位置和高度不寻常的清道夫行为的相似性，这些细胞似乎是斑马鱼等效的哺乳动物荧光颗粒细胞（FGP），巨噬细胞样细胞，巨噬细胞样细胞众所周知，这些细胞可能在大脑功能和各种CNS病理学中起着重要的作用。尽管它们具有巨噬细胞状的形态及其血管周地点，但这些细胞与淋巴内皮细胞最相似，并且它们从脑表面迁移到大脑表面之前从原始的内皮中脱节。我们使用转基因斑马鱼对ENU诱导的隐性突变进行筛选，我们最近确定了一种突变体，该突变体似乎在FGP中特别缺乏，提供了一种遗传模型，我们正在使用该突变体来进一步探索这些细胞的重要功能作用。迄今为止，我们的发现提供了第一个报告，即大脑中非血管形成的，血管周细胞群，该报告是由于血管内皮转差而出现的。我们正在使用斑马鱼模型进一步探索脑膜血管相关细胞种群的结构，细胞组成和功能作用。