Liver inflammation leads to the activation of hepatic stellate cells (HSCs), resulting in the development of liver fibrosis. The present study aimed to investigate the effects of prostaglandin E₂ (PGE₂), which is biosynthesized by Kupffer cells, hepatocytes, and HSCs during inflammation, on HSC activation, including its combinatory effect with caffeine. HSCs isolated from mice were activated by culturing in a medium supplemented with 10% fetal bovine serum for 7 days on plastic plates. The activation of HSCs was evaluated by immunofluorescence of α-smooth muscle actin in HSCs. Comprehensive gene expression analysis was performed using mRNA-sequencing to compare HSCs cultured for 1 or 7 days, with or without PGE₂, caffeine, or both. PGE₂ (1 μM) facilitated the activation of HSCs but inhibited the HSC activation in the presence of caffeine (3 mM). Comprehensive gene expression analysis revealed that HSCs treated with PGE₂ in the presence of caffeine were classified in the same class as HSCs cultured for 1 day, i.e., quiescent HSCs. In contrast, PGE₂ did not exhibit an inhibitory effect on HSC activation when co-treated with any isoform-specific phosphodiesterase inhibitors. Although the adenylate cyclase inhibitor 2',5'-dideoxyadenosine suppressed the elevation of intracellular cAMP level induced by PGE₂ in the presence of caffeine, it had no effect on the inhibition of HSC activation by PGE₂ plus caffeine. The effect of PGE₂ on HSC activation is changed from facilitatory to inhibitory when combined with caffeine, suggesting that caffeine may effectively suppress liver fibrosis during inflammation.

Although quiescent hepatic stellate cells (HSCs) have been suggested to regulate hepatic blood flow, there is no direct evidence that quiescent HSCs display contractile abilities. Here, we developed a new method to quantitatively measure the contraction of single isolated HSCs and evaluated whether endothelin-1 (ET-1) induced contraction of HSCs in a non-activated state. HSCs isolated from mice were seeded on collagen gel containing fluorescent beads. The beads around a single HSC were observed gravitating toward the cell upon contraction. By recording the movement of each bead by fluorescent microscopy, the real-time contraction of HSCs was quantitatively evaluated. ET-1 induced a slow contraction of non-activated HSCs, which was inhibited by the non-muscle myosin II inhibitor blebbistatin, the calmodulin inhibitor W-7, and the ET_A receptor antagonist ambrisentan. ET-1-induced contraction was also largely reduced in Ca²⁺-free conditions, but sustained contraction still remained. The tonic contraction was further diminished by the Rho-kinase inhibitor H-1152. The mRNA expression of P/Q-type voltage-dependent Ca²⁺ channels (VDCC), as well as STIM and Orai, constituents of store-operated channels (SOCs), was observed in mouse non-activated HSCs. ET-1-induced contraction was not affected by amlodipine, a VDCC blocker, whereas it was partly reduced by Gd³⁺ and amiloride, non-selective cation channel blockers. However, neither YM-58483 nor SKF-96365, which inhibit SOCs, had any effects on the contraction. These results suggest that ET-1 leads to Ca²⁺-influx through cation channels other than SOCs and produces myosin II-mediated contraction of non-activated HSCs via ET_A receptors, as well as via mechanisms involving Ca²⁺-calmodulin and Rho kinase.

Myofibroblast-like activated hepatic stellate cells (aHSCs), which produce collagen, a major cause of liver fibrosis, are specific target cells for antifibrotic treatment. Recently, several reports have indicated that extracellular vesicles (EVs) play important roles in cell-to-cell communication through their tropism for specific cells or organs. Therefore, the present study aimed to identify aHSC-directed EVs by focusing on cell-to-cell interactions in the liver under pathological conditions. EVs were derived from the hepatocyte cell line AML12 treated with or without palmitic acid (PA) and evaluated for their physical properties and uptake by the aHSC cell line LX-2. AML12-derived EVs had a mean particle diameter of 110–130 nm, negative charge, and expressed the exosomal makers CD9 and CD63. PA-treated AML12 cells released larger EVs with higher protein levels than those without PA treatment. The intracellular uptake efficacy of EVs derived from PA-treated AML12 cells into activated LX-2 cells was significantly higher than those without PA treatment. Our study revealed that PA treatment induces hepatocytes to release EVs with aHSC-tropism. These findings may contribute to the development of an EV-based drug delivery system (DDS) for aHSC-targeted therapy and provide new insights into the role of steatotic hepatocyte-derived EVs in physiological or pathophysiological functions.