Liver sinusoidal endothelial cells induce senescence in activated hepatic stellate cells via extracellular vesicles

Background: Liver sinusoidal endothelial cells (LSECs) have been considered as key gatekeepers in maintaining hepatic stellate cells (HSCs) homeostasis. The intricate intercellular crosstalk between HSCs and LSECs plays a fundamental role 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 govern the deactivation of activated HSCs (aHSCs) remain unclear.
Aim: to investigate the molecular mechanisms of LSECs-mediated HSC regulation.
Methods: Primary rat LSECs and HSCs were isolated from male Wistar or Zucker rats. EVs were isolated from the conditioned medium (CM) through differential centrifugation and characterized by nanoparticle tracking analysis, transmission electron microscopy, and marker determination. EVs were added to target cells for 24/48/72 hours. Additionally, a primary cell co-culture system was established to dissect the intercellular signaling mechanisms. Gene expression was determined by qPCR and protein expression was determined by Western blot and immunofluorescence and proliferation of cells by Xcelligence and BrdU assay.
Results: LSECs induced an upregulation in aHSCs of proinflammatory genes, senescence markers such as senescence-associated β-galactosidase. Importantly, LSEC-derived EVs elicited consistent effects by inhibiting the proliferation of aHSCs, as assessed by Xcelligence and bromodeoxyuridine (BrdU) assays. Furthermore, the TLR4/NF-κB signaling pathways were activated and played a crucial role in the regulation of aHSCs orchestrated by LSECs.
Conclusion: LSECs induce aHSC senescence by secreting EVs, which depends on the activation of the TLR4/NF-κB pathways, thus presenting the inactivation of aHSCs.