Abstract 4368563: Progression of Fontan Associated Liver Disease (FALD) Leads to a Metabolic Transformation in Hepatocytes

Abstract

Background: Single-ventricle congenital heart disease is a rare and lethal cardiac abnormality that disrupts normal circulation in neonates. The cavopulmonary redirection of blood in these patients during the staged Fontan procedure leads to chronic central venous congestion that causes progressive fibrosis within the liver parenchyma. Beyond the progressive fibrosis, little is known about the metabolic implications of FALD. Hypothesis: Progressive advancement of FALD causes changes the metabolic function of hepatocytes. Aims: Identify unique metabolic profiles of hepatocytes with FALD progression. Materials and Methods: Single-cell spatial transcriptomic (CosMxTM Spatial Molecular Imaging with in-situ hybridization of 6000 genes) was performed on liver explant tissue sections from FALD patients (n=2) identified at the University of Pittsburgh Medical Center (UPMC) with early and advanced fibrosis. Unbiased clustering and dimensionality reduction was performed to identify cell populations across normal liver, early, and advanced FALD. Gene set enrichment analysis (GSEA) identified pathways enriched in specific cell populations. Results were confirmed in 20 FALD tissue samples using immunofluorescence. Results: We analyzed 60,339 and 291,840 high-quality cells from normal and FALD livers, respectively. Unbiased clustering resulted in 12 liver cell types, including five subtypes of hepatocytes. While the population of pericentral hepatocytes involved with glycolysis and lipogenesis decreased with the progression of FALD, periportal hepatocytes involved with gluconeogenesis and fatty acid oxidation increased. Additionally, with FALD progression, hepatocytes adopted an ambiguous metabolic phenotype that subsequently caused an increase in markers of cell stress and exhaustion (JUN, HSPA1B, and ATF3) with a 4-fold enrichment. WNT2 secreted by liver endothelial cells activated beta-catenin signaling in stressed hepatocytes, likely through chronic hypoxia and increased central venous pressures. Immunofluorescence confirmed our CosMx findings in 20 FALD samples. Conclusions: We generated the first single-cell spatial transcriptomic profile in human FALD using the CosMxTM 6k platform to identify a unique population of stressed hepatocytes in advanced FALD. Cross-talk between ECs and hepatocytes perturbed the metabolic profile of the hepatocytes, subsequently increasing cell-stress markers.