Regulation of Vascular Integrity

血管完整性的调节

• 批准号: 10915322
• 负责人: Brant Weinstein
• 金额: $ 77.79万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 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/10915322
• 关键词: Adult; Aging; Alleles; Anatomy; Animals; Blood - brain barrier anatomy; Blood Vessels; Brain; Brain hemorrhage; Cells; Cephalic; Cerebrospinal Fluid; Circulation; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Connective Tissue; Defect; Development; Diabetes Mellitus; Embryo; Endothelium; Fishes; Fresh Water; Gene Expression Profile; Gene Expression Profiling; Generations; Genes; Genetic Screening; Goals; Growth; Image; Immune; Immune response; Impairment; Inflammation; Intubation; Larva; Maintenance; Meningeal; Meninges; Methods; Modeling; Molecular; Molecular Cloning; Morbidity - disease rate; Mutation; Natural regeneration; Neurocognitive Deficit; Neurodegenerative Disorders; Nutrient; Optics; Organ; Oxygen; Pathology; Pathway interactions; Play; Process; Rapid screening; Reagent; Regulation; Reporting; Resolution; Role; Rupture; Series; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Skin wound healing; Stroke; Thinness; Tissues; Trauma; Traumatic Brain Injury; Traumatic Brain Injury recovery; Vascular Diseases; Vascular regeneration; Vertebrates; Zebrafish; angiogenesis; cell type; cranium; experimental analysis; genetic analysis; genetic regulatory protein; healing; in vivo; insight; interest; model organism; mortality; mutant; new therapeutic target; next generation sequencing; optical imaging; repaired; response; rhoA GTP-Binding Protein; tool; wasting; wound healing

As described in the goals and objectives section of this report, this project consists of three specific aims: Genetic and experimental analysis of 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 are currently characterizing the role of the important intracellular regulatory protein RhoA and its downstream pathways in vascular integrity and angiogenesis using an allelic series of mutations in rhoaa that we have generated and a battery of additional specific tools and reagents for manipulating this pathway. Studying RhoA and its downstream players promises to uncover new molecular mechanisms important for the maintenance of vascular barrier function. We are also using in vivo profiling tools to examine the transcriptional signature of brain endothelium to identify new genes involved in establishment and maintenance of the blood-brain barrier. Studying the role of the endothelium and immune-vascular cross-talk during cutaneous wound healing Proper vascular regeneration and repair is essential for wound healing, but is significantly impaired in aging and in pathologies such as diabetes. We have developed new zebrafish models of cutaneous wound healing and are now using these models to study the anatomical, cellular, and molecular mechanisms guiding vascular repair and regeneration in adult fish, using new methods we have also developed for intubation and long-term imaging of adult zebrafish and for vascular- and immune-cell specific in vivo gene expression profiling. Our goal is to identify key signaling pathways and molecules essential for neoangiogenesis during wound healing, with the long-term goal of identifying potential novel therapeutic targets. Studying the role of the endothelium and immune-vascular cross-talk in recovery from traumatic brain injury (TBI) 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 their function and developmental origins are still not well understood. The meninges are also the major site of damage and inflammation following traumatic brain injury (TBI), with vascular rupture, immune-vascular interactions, and vascular rupture-associated inflammation playing a key but as yet poorly characterized role in the most damaging sequelae to TBI. The thin, transparent skull of the adult zebrafish makes it ideal for high-resolution optical imaging of the meninges and meningeal cells in intact, living animals. We have developed new zebrafish models of meningeal TBI and we are now using these models=to study the cellular and molecular responses to meningeal damage in adult fish, focusing on the role of vessels, immune cells, and vascular-immune cross talk in the response to meningeal TBI. Our goal is to identify key endothelial and immune cell signaling pathways and molecules that facilitate healing and recovery from TBI, with the long-term goal of identifying potential novel therapeutic targets.

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： 血管完整性的遗传和实验分析 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。我们目前正在使用我们已经生成的Rhoaa中的一系列等位基因突变来表征重要的细胞内调节蛋白RhoA及其下游途径在血管完整性和血管生成中的作用。研究Rhoa及其下游参与者有望发现新的分子机制对于维持血管屏障功能很重要。 我们还使用体内分析工具来检查脑内皮的转录特征，以识别与血脑屏障的建立和维持有关的新基因。 在皮肤伤口愈合过程中研究内皮和免疫血管串扰的作用 适当的血管再生和修复对于伤口愈合至关重要，但在衰老和糖尿病等病理中受到显着损害。我们开发了皮肤伤口愈合的新型斑马鱼模型，现在正在使用这些模型来研究指导成人鱼的血管修复和再生的解剖，细胞和分子机制，我们还使用新方法开发了成人斑马鱼的intubation和长期成像，以实现成人斑马和血管 - 和免疫细胞和免疫细胞的特定表达。 我们的目标是确定伤口愈合过程中新血管生成至关重要的关键信号通路和分子，其长期目标是确定潜在的新型治疗靶标。 研究内皮和免疫血管串扰在创伤性脑损伤中恢复（TBI）中的作用 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。尽管它很重要，但脑膜中存在的细胞类型及其功能和发育起源仍然不太了解。 这些脑膜也是创伤性脑损伤（TBI）后的损伤和炎症的主要部位，血管破裂，免疫 - 血管相互作用以及血管破裂相关的炎症起着关键，但在最有害的序列中的作用却较差，但其作用却较差。 成年斑马鱼的稀薄，透明的头骨使其非常适合完整活跃动物中脑膜和脑膜细胞的高分辨率光学成像。 我们已经开发了脑膜化TBI的新型斑马鱼模型，现在我们正在使用这些模型=研究成年鱼类中脑膜损伤的细胞和分子反应，重点是血管，免疫细胞和血管免疫交叉的作用，以响应脑膜TBI。 我们的目标是确定促进TBI愈合和恢复的关键内皮和免疫细胞信号通路和分子，其长期目标是确定潜在的新型治疗靶标。