Hepatic stellate cell autophagy inhibits extracellular vesicle release to attenuate liver fibrosis

Abstract Background and aim In chronic liver injury, platelet-derived growth factor (PDGF)-stimulated hepatic stellate cells (HSCs) release fibrogenic extracellular vesicles (EVs). EVs include multivesicular body (MVB)-derived exosomes and Rho-associated kinase (ROCK)-dependent plasma membrane budding-derived microvesicles. Autophagy plays substantial roles in maintaining liver homeostasis and its deregulation has been associated with liver disease. However, the effect of autophagy in regulating fibrogenic EVs and amplifying pro-fibrotic signals among HSCs remains unknown. Here, we aim to understand the role of autophagy in HSC-derived fibrogenic EV release in liver fibrosis. Methods Liver fibrosis in mice was induced by carbon tetrachloride (CCl4) administration or bile duct ligation (BDL). Small EVs were purified by differential ultracentrifugation. Results In vitro, PDGF and its downstream molecule SHP2 (Src homology 2-containing protein tyrosine phosphatase 2) inhibited autophagy and increased HSC-derived EV release. We used this PDGF/SHP2 model to further investigate how autophagy affects fibrogenic EV release. RNA-seq identified an mTOR (mammalian target of rapamycin) signaling molecule to be regulated by SHP2 and PDGF. Disruption of mTOR signaling abolished PDGF-dependent EV release. Activation of mTOR signaling induced the release of MVB-derived exosomes by inhibiting autophagy as well as microvesicles through activation of ROCK1 signaling. These mTOR-dependent EVs promoted in vitro HSC migration. To assess the importance of this mechanism in vivo, SHP2 was selectively deleted in HSCs, which attenuated CCl4 or BDL-induced liver fibrosis. Furthermore, administration of circulating EVs from mice with HSC-specific SHP2 deletion to mice undergoing CCl4-mediated fibrogenesis demonstrated less fibrosis than EVs derived from control mice. Congruently, SHP2 was upregulated in patients with liver cirrhosis. Conclusion These results demonstrate that in HSCs, autophagy inhibits fibrogenic EV release which can attenuate liver fibrosis. (274 words)