Hepatocyte-derived extracellular vesicles promote endothelial dedifferentiation in chronic liver disease through the miR-153-3p – pyroptosis axis

Background & Aims: Liver cells communicate paracrinally through extracellular vesicles (EVs). This study investigated the role of miRNAs embedded in hepatocyte derived EVs (hepEVs) modulating endothelial cell dedifferentiation in chronic liver disease. Methods: EVs were purified from primary hepatocytes isolated from healthy (CT) and cirrhotic (CH) livers in both humans and rats. CT-rats were intravenously treated with fluorescence-labelled hepEVs-CT or hepEVs-CH (200 μg/d, 3 d, n=10/group) to assess biodistribution and liver sinusoidal endothelial cells (LSECs) phenotype. miRNA profiling of human hepEVs-CH was validated in rat models, and commonly altered miRNAs were over-expressed in CT-LSECs. Gene dysregulation was analysed in primary CH-LSECs and liver tissues from rats and humans with cirrhosis, as well as CT-LSECs treated with miRNA-specific upregulated hepEVs. CH-rats were treated with the Caspase-1 inhibitor VX-765 (15 mg/kg/d, 10 doses, n=11/group) to evaluate its effects on liver function, haemodynamics, and endothelial phenotype. Results: In vivo , hepEVs-CH accumulated predominantly in LSECs, disrupting genes involved in fibrosis, inflammation and cell death, while enhancing vWF expression and altering eNOS activity. Profiling of human hepEVs-CH revealed 37 dysregulated miRNAs, with miR-200a-3p and miR-153-3p validated in rat hepEVs-CH. Transfection of miR-153-3p into CT-LSEC dysregulated 771 genes involved in inflammation and cell death, particularly pyroptosis. Increased active Caspase-1 and Gasdermin-D expression was observed in human and rat cirrhotic liver tissue, as well as in CT-LSEC treated with miR-153-3p-hepEVs, indicating enhanced pyroptosis. In vivo , VX-765 treatment reduced hepatic pyroptosis, improving endothelial phenotype and portal hypertension. Conclusions: HepEVs-derived miRNAs, particularly miR-153-3p, contribute to endothelial dysfunction in CLD by triggering pyroptosis through a paracrine mechanism. Inhibiting Caspase-1 may provide a novel therapeutic approach to mitigate endothelial dysfunction in CLD.