Liver sinusoidal endothelial cell-derived extracellular vesicles contain catalase that induces senescence of activated hepatic stellate cells

Background and Aims: Liver sinusoidal endothelial cells (LSECs) are key to maintaining hepatic stellate cell (HSC) homeostasis. The intricate intercellular crosstalk between HSCs and LSECs is fundamental in modulating HSC quiescence and activation. Previous studies have identified LSEC-derived extracellular vesicles (EVs) as primary mediators in this regulatory process. However, the mechanisms through which LSECs and their EVs modulate the activated phenotype of HSCs (aHSCs) remain unclear. This study aims to investigate how LSEC-EVs influence HSC activation during liver fibrosis.<br/>Method: Primary rat LSECs and HSCs were isolated from male Wistar rats. A primary cell transwell co-culture system of HSCs and LSECs was established to dissect the intercellular signaling mechanisms. EVs were isolated from LSEC-derived conditioned medium (CM) through differential centrifugation and characterized by nanoparticle tracking analysis, transmission electron microscopy, and EV-marker determination. Proteins present in EVs were identified by proteomic analysis. EVs and CM as well as selected proteins (catalase) in EVs were added to HSCs for 24 - 72 hours. Gene expression was determined by qPCR, protein expression by Western blot and immunofluorescence, and cell proliferation by Xcelligence and BrdU assay.<br/>Results: LSEC-derived EVs induced increased expression of components of the senescence-associated secretory phenotype (SASP), like IL-1 and senescence markers like senescence-associated β-galactosidase activity in aHSCs, while decreasing aHSC proliferation. These effects were also observed in the LSEC-aHSC transwell co-culture system. Catalase was identified in LSEC-derived EVs via proteomics. Since previous studies showed that exogenous catalase inhibits the activation of HSCs, we further investigated the role of catalase. The presence of catalase in LSEC-derived EVs was confirmed by Western blot. Furthermore, the effects of LSEC-derived EVs on HSCs were mimicked by pegylated (cell permeable) catalase. <br/>Conclusion: Catalase-rich extracellular vesicles from liver sinusoidal endothelial cells reduce activation of HSCs by inducing senescence.