Regulation of Vascular Integrity

如本报告的目标和目的部分所述，该项目包括三个具体目标： 血管完整性的遗传学和实验分析 我们使用正向遗传筛选和CRISPR基因编辑来产生破坏斑马鱼脑血管完整性的斑马鱼突变体，使用下一代测序来快速分子克隆突变体中的缺陷基因。我们目前正在利用我们在ROAA中产生的一系列等位基因突变和一系列额外的特定工具和试剂来操纵这一途径，从而表征重要的细胞内调节蛋白RhoA及其下游通路在血管完整性和血管生成中的作用。对RhoA及其下游成员的研究有望发现对维持血管屏障功能重要的新的分子机制。我们还在使用体内图谱工具来检查脑内皮细胞的转录签名，以确定与血脑屏障的建立和维护有关的新基因。 内皮细胞和免疫血管信号在皮肤创面愈合中的作用研究 适当的血管再生和修复是伤口愈合的关键，但在衰老和糖尿病等病理情况下，血管再生和修复会显著受损。我们已经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、细胞和分子机制，使用我们还开发的新方法，用于成年斑马鱼的插管和长期成像，以及体内血管和免疫细胞特异性基因表达谱。我们的目标是确定在伤口愈合过程中对新生血管生成至关重要的关键信号通路和分子，长期目标是确定潜在的新治疗靶点。 血管内皮细胞和免疫血管信号在创伤性脑损伤康复中的作用 脑膜是包裹大脑的外部结缔组织，产生脑脊液，起到抵御创伤的缓冲作用，通过营养循环滋养大脑，并清除废物。尽管它很重要，但脑膜中存在的细胞类型及其功能和发育来源仍不清楚。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-血管相互作用和血管破裂相关的炎症在TBI最具破坏性的后遗症中起着关键作用，但目前尚不清楚。成年斑马鱼的薄而透明的头骨使其成为对完好的活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。我们已经开发了新的斑马鱼脑膜脑损伤模型，现在我们正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，重点是血管、免疫细胞和血管免疫串扰在脑膜脑损伤反应中的作用。我们的目标是确定关键的内皮细胞和免疫细胞信号通路和分子，促进创伤性脑损伤的愈合和恢复，长期目标是确定潜在的新治疗靶点。

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.