Regulation of Vascular Integrity

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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.