Project Grant R35GM155165

TRANSLATIONAL APPROACHES TO UNRAVEL ORGAN-SPECIFIC MICROVASCULAR ENDOTHELIAL RESPONSES IN SEPSIS. - TRANSLATIONAL APPROACHES TO UNRAVEL ORGAN-SPECIFIC MICROVASCULAR ENDOTHELIAL RESPONSES IN SEPSIS. PROGRAM SUMMARY: SEPSIS IS DEFINED AS LIFE-THREATENING ORGAN DYSFUNCTION CAUSED BY A DYSREGULATED HOST RESPONSE TO AN INFECTION AND IS ASSOCIATED WITH HIGH MORTALITY. SYSTEMIC INFLAMMATION AND ENDOTHELIAL ACTIVATION WITH RESULTANT MICROVASCULAR LEAK, THROMBOSES, AND HYPOXIC TISSUE INJURY ARE HALLMARKS OF SEPSIS. YET, IN OVER 100 TRIALS, DRUGS AIMED AT MODULATING CASCADES OF INFLAMMATION AND COAGULATION HAVE NOT PROVEN TO BE EFFICACIOUS. THE PRIMARY REASON FOR THIS FAILURE IS ATTRIBUTED TO CLINICAL AND BIOLOGICAL VARIABILITY AMONG CRITICALLY ILL PATIENTS. MORE RECENTLY, HIGH-THROUGHPUT APPROACHES INCLUDING GENE-EXPRESSION PROFILING HAVE SHOWN PROMISE IN DISENTANGLING PATIENT- LEVEL HETEROGENEITY IN THE HOST IMMUNE RESPONSE. IN CONTRAST, SAMPLING CHALLENGES COUPLED WITH CELL AND ORGAN LEVEL HETEROGENEITY HAVE IMPEDED A SIMILAR UNDERSTANDING OF THE HOST ENDOTHELIAL RESPONSE. IT FOLLOWS THAT TRANSLATIONAL APPROACHES THAT SHED LIGHT ON HUMAN MICROVASCULAR PATHOBIOLOGY MAY LEAD TO THE DISCOVERY OF TARGETED THERAPIES THAT RESTORE TISSUE HOMEOSTASIS AND SHIFT SEPSIS CARE PARADIGMS TOWARD ORGAN RECOVERY. MY RESEARCH PROGRAM SEEKS TO ADDRESS KEY KNOWLEDGE GAPS THAT CURRENTLY IMPEDE SCIENTIFIC PROGRESS THROUGH PROJECTS SPANNING 3 DOMAINS. 1) DISCOVERY: THE ENDOTHELIUM IN PATIENTS REMAINS INACCESSIBLE. MOREOVER, WHILE ENDOTHELIAL HETEROGENEITY AND ORGAN-SPECIFICITY ARE INCREASINGLY RECOGNIZED, THEIR CONTRIBUTION TO THE EVOLUTION OF ORGAN DYSFUNCTIONS REMAINS POORLY UNDERSTOOD. TO ADDRESS THIS, WE WILL ENRICH CIRCULATING ENDOTHELIAL CELLS FROM WHOLE BLOOD OF CHILDREN WITH SEPTIC SHOCK WITH THE PRIMARY OBJECTIVE OF DEVELOPING A TRANSCRIPTOMIC ATLAS AT SINGLE-CELL RESOLUTION. BY COMPARING SIGNATURES OF CIRCULATING ENDOTHELIAL SUBSETS FROM PATIENTS, RELATIVE TO PUBLISHED DATASETS OF TISSUE-RESIDENT ENDOTHELIAL CELLS, WE WILL IDENTIFY ORGAN-SPECIFIC TARGETS FOR FUTURE HYPOTHESES TESTING. 2) DISEASE MODELS: THE REDUCTIONIST NATURE OF CURRENT PRECLINICAL MODELS HAS IMPEDED TRANSLATION OF BASIC SCIENCE DISCOVERIES INTO IMPROVED SEPSIS OUTCOMES. THUS, THERE IS A SIGNIFICANT NEED FOR ADVANCED DISEASE MODELS THAT RECAPITULATE HUMAN SEPSIS PATHOBIOLOGY. MY LABORATORY WILL DEVELOP HUMAN INDUCED PLURIPOTENT STEM CELL DERIVED MICROVASCULAR ORGANOIDS AND STIMULATE THEM WITH BIOMARKER RISK- STRATIFIED PLASMA FROM PATIENTS TO MODEL ENDOTHELIAL DYSFUNCTION IN HUMAN SEPSIS. OUR MODELS ARE EXPECTED TO SERVE AS ROBUST TESTBEDS FOR HYPOTHESES TESTING. 3) DISEASE MECHANISMS: FINALLY, WE WILL ESTABLISH CUTTING- EDGE CRISPR-CAS9 GENE-EDITING TOOLS IN VITRO TO EFFICIENTLY STUDY THE MECHANISTIC BASIS OF CANDIDATE GENES IDENTIFIED THROUGH OUR STUDIES. THIS TRANSDISCIPLINARY PROPOSAL IS MADE FEASIBLE BY A LONG-STANDING STUDY OF PEDIATRIC SEPTIC SHOCK WITH UNPARALLELED ACCESS TO BIOSPECIMENS, INSTITUTIONAL SHARED RESOURCES, AND ONGOING COLLABORATIONS WITH A RICH NETWORK OF SCIENTISTS WITH TECHNICAL EXPERTISE. THE APPROACHES ESTABLISHED THROUGH THIS PROPOSAL ARE EXPECTED TO ENHANCE OUR UNDERSTANDING OF ORGAN-SPECIFIC MICROVASCULAR ENDOTHELIAL RESPONSES AND HOLD POTENTIAL TO INFORM DEVELOPMENT OF PRECISION THERAPIES. FUNDING THROUGH THE NIGMS ESI- MIRA WILL THUS MAXIMIZE MY LABORATORY'S RESEARCH EFFORTS TO IMPROVE THE OUTCOMES OF CHILDREN WITH SEPSIS.