The Mechanistic Role of Extracellular Vesicles as Therapeutic and Diagnostic Vehicles for Single-Ventricle Defects Restricted; Files & ToC

Abstract

Single-ventricle defects represent a severe subset of congenital heart disease that requires staged surgical palliation, culminating in the Fontan procedure. While lifesaving, this intervention results in an abnormal physiology which leads to chronic and multi-system complications, such as heart failure and Fontan-associated liver disease. This dissertation investigates the mechanistic role of small extracellular vesicles (sEVs) as both targeted therapeutic agents and non-invasive diagnostic biomarkers to address the progressive morbidities inherent to the Fontan circulation. First, to overcome the translational barriers posed by cargo heterogeneity in cell-free therapeutics, we characterized the molecular landscape of the cardiac progenitor cell sEV (CPC-EV) population. Through a multiple co-inertia analysis, laminin subunit alpha-4 was identified as a critical functional driver and surface marker for a highly angiogenic CPC-EV subpopulation. Isolation of this enriched fraction significantly enhanced endothelial cell migration and vascular tube formation, which establishes a precision strategy to refine sEV therapies for cardiac repair. Second, to elucidate systemic molecular changes in the Fontan population, we analyzed the circulating sEV transcriptome during the perioperative period of the Glenn and Fontan procedures. Our analysis revealed a distinct upregulation of pro-angiogenic RNA cargo following surgery. Partial least squares regression identified hsa-miR-340-5p, hsa-miR-199b-5p, MAPK6, and GLE1 as key mediators of venovenous collaterals. Additionally, tissue deconvolution indicated a significant postoperative shift in sEV origin, marked by decreased cardiac secretion and increased release from brain tissue. Finally, we leveraged a translational ovine Fontan model to develop a noninvasive risk stratification tool for Fontan-associated liver disease. Comprehensive profiling of circulating sEVs demonstrated that the proteome changes following the Fontan palliation, whereas sEV small RNA cargo stratifies according to the severity of hepatic fibrosis. These small RNA cargos were then used in an ordinal logistic regression model to construct specific RNA biomarkers that predict hepatic stiffness accurately. Collectively, this work demonstrates that sEVs can be manipulated to maximize regenerative efficacy and leveraged to provide a non-invasive surveillance of systemic complications in the single-ventricle population.

Table of Contents

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About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Bioinformatics Biology, Cell
Keyword	Fontan Proteomics Single-ventricle defects Extracellular Vesicle Transcriptomics
Committee Chair / Thesis Advisor	Michael Davis, Emory University
Committee Members	Jennifer Kwong, Emory University David Gorkin, Emory University Joshua Chandler, Emory University Nael McCarty, Emory University

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