Extracellular vesicle cargo and risk of NAFLD and NASH in U.S. youth

The prevalence of pediatric metabolic dysfunction-associated steatotic liver disease (MASLD) is escalating in US youth. MASLD in children is associated with poor long-term outcomes, including diabetes, cardiovascular disease, and higher liver-related morbidity and mortality in adulthood. Despite the clinical significance of pediatric MASLD, the mechanisms underlying its pathogenesis are poorly understood. This lack of knowledge is a major obstacle to the development of effective treatment and prevention strategies. Emerging studies support a role for extracellular vesicles (EVs) in MASLD. Circulating EVs can influence intracellular signaling, tissue injury and repair, and matrix remodeling in liver cells. Plasma EV levels discriminate between adult patients with MASLD and metabolic dysfunction-associated steatohepatitis (MASH) and are positively correlated with histological grade. We conducted a series of proof-of-principle studies that demonstrate: 1) distinctive protein signatures in plasma EVs isolated from children with MASLD, 2) MASLD-specific signatures approximate non-MASLD signatures following lifestyle intervention that reduced liver fat, and 3) isolation of hepatocyte-specific EVs showed enrichment of liver-specific proteins in MASLD. To date, however, investigations of EVs in pediatric MASLD are scarce, and the role of EV-derived cargo in MASLD pathogenesis in youth remains unknown. Here we propose a strategy to characterize the biological role of EVs in pediatric MASLD. In Aim 1, we will apply unbiased methods to study proteins and RNAs carried in circulating and hepatocyte-enriched EVs to derive characteristic signatures associated with MASLD and MASH in youth. In Aim 2, we will measure EV concentration and content in youth with MASLD who experienced changes in hepatic fat fraction following two distinct intervention modalities (i.e., intensive lifestyle and bariatric surgery). Importantly in Aim 3, we propose a series of molecular experiments to obtain mechanistic insight into the functional aspects of EV cargo. The combination of these approaches is innovative; and the strategy comprises a novel and sequentially appropriate set of aims that has not previously been used to address potential mechanisms of pathogenesis in pediatric MASLD. The focus on children is especially impactful due to the growing prevalence of MASLD in this population, the association of pediatric MASLD with poor health outcomes in adulthood, and the expected future economic burden to care for these individuals. The identification of EV-derived cargo associated with pediatric MASLD will enhance our understanding of the biological mechanisms contributing to disease pathogenesis, provide a means to improve diagnostic and therapeutic strategies, and identify new targets for potential drug development.