Mechanisms of Endothelial Cell Dysfunction in Critically Ill Children

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Project Summary Candidate. I am an Instructor in pediatric critical care medicine at Yale School of Medicine and will be promoted to Assistant Professor with academic promotion per Yale School of Medicine's Clinician Scholar track in July 2018. My K08 application will allow me to acquire additional mentored research experience so that I may become an independent investigator with expertise in endothelial cell signaling and its disruption in critically ill children. This research will build on my fellowship training that focused on the signals that regulate changes in vascular permeability and their associated clinical consequences and will further develop my laboratory skills required to successfully conduct vascular research, such as genetic modification of human cells, interrogation of intracellular signaling pathways, and bioinformatic analysis. I will take graduate courses on vascular biology, bioinformatics, and immunology research methods and will participate in national conferences. I have an outstanding mentor, Dr. Jordan Pober, a world-class vascular biologist, along with advisors Dr. Mustafa Khokha, expert in genetics, and Dr. Vince Faustino, expert biostatistician and clinical trialist. This multidisciplinary mentorship team, along with the resources available through the VBT program at Yale, will allow me to develop the necessary skills to function as an extramurally funded clinician scientist focused on improving the care of critically ill children. Research Project. Endothelial cells (ECs) actively regulate vessel permeability that is essential for organ function and patient survival. During cardiopulmonary failure, loss of EC permselectivity results in capillary leak that contributes to morbidity and mortality in children. The unique regulation of the signaling events that produce permeability changes in capillary EC is largely unknown. Likewise, how critical illness induces EC dysfunction remains unknown, due in large part to our inability to directly assess EC changes in critically ill patients. I recently demonstrated the importance of the regulation of the small GTPase RhoB in a pediatric patient with systemic capillary leak syndrome caused by a single gene mutation. In Aim 1, I test the hypothesis that pathological capillary permeability is regulated by small GTPases and that these pathways are influenced by transcriptional changes induced by inflammatory cytokines. I will interrogate intracellular signaling pathways in capillary ECs cultured from different organs (skin and lung) to determine the effect of their modulation on trans-endothelial electrical resistance. In Aim 2, I will test the hypothesis that ECs undergo transcriptional changes in response to critical illness. I will analyze the entire transcriptome of individual venous ECs collected directly from critically ill children using single cell RNA sequencing. I will assess the functions of identified candidate gene products using pharmacologic, genetic, and immunologic techniques to manipulate and analyze their effect on the intracellular signaling pathways that control the initiation and resolution of capillary leak. Better understanding the pathways involved in the development and resolution of capillary leak is the first step in developing therapies for this common condition associated with serious morbidity and mortality.