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.

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： 血管完整性的遗传和实验分析 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。我们目前正在利用我们生成的 rhoaa 等位基因突变系列以及一系列用于操纵该途径的其他特定工具和试剂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揭示对于维持血管屏障功能重要的新分子机制。 我们还使用体内分析工具来检查脑内皮的转录特征，以识别参与血脑屏障建立和维持的新基因。 研究内皮细胞和免疫血管串扰在皮肤伤口愈合过程中的作用 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疾病中会受到显着损害。我们开发了新的斑马鱼皮肤伤口愈合模型，现在正在使用这些模型来研究指导成年鱼血管修复和再生的解剖学、细胞和分子机制，使用我们还开发的用于插管和长期成像的新方法。成年斑马鱼以及血管和免疫细胞特异性体内基因表达谱分析。 我们的目标是确定伤口愈合过程中新血管生成所必需的关键信号通路和分子，长期目标是确定潜在的新型治疗靶点。 研究内皮细胞和免疫血管串扰在创伤性脑损伤 (TBI) 恢复中的作用 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。尽管脑膜中存在的细胞类型及其功能和发育起源很重要，但仍不清楚。 脑膜也是创伤性脑损伤 (TBI) 后损伤和炎症的主要部位，其中血管破裂、免疫-血管相互作用以及血管破裂相关炎症在 TBI 最具破坏性的后遗症中发挥着关键但尚未明确的作用。 。 成年斑马鱼薄而透明的头骨使其成为对完整活体动物的脑膜和脑膜细胞进行高分辨率光学成像的理想选择。 我们开发了新的脑膜 TBI 斑马鱼模型，现在使用这些模型来研究成年鱼对脑膜损伤的细胞和分子反应，重点关注血管、免疫细胞和血管-免疫串扰在反应中的作用脑膜 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传导途径和分子，长期目标是确定潜在的新型治疗靶点。