Dataset Information

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

ABSTRACT:

Background & aims

Autophagy plays a crucial role in hepatic homeostasis and its deregulation has been associated with chronic liver disease. However, the effect of autophagy on the release of fibrogenic extracellular vesicles (EVs) by platelet-derived growth factor (PDGF)-stimulated hepatic stellate cells (HSCs) remains unknown. Herein, we aimed to elucidate the role of autophagy, specifically relating to fibrogenic EV release, in fibrosis.

Methods

In vitro experiments were conducted in primary human and murine HSCs as well as LX2 cells. Small EVs were purified by differential ultracentrifugation. Carbon tetrachloride (CCl₄) or bile duct ligation (BDL) were used to induce fibrosis in our mouse model. Liver lysates from patients with cirrhosis or healthy controls were compared by RNA sequencing.

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 sequencing identified an mTOR (mammalian target of rapamycin) signaling molecule that was regulated by SHP2 and PDGF. Disruption of mTOR signaling abolished PDGF-dependent EV release. Activation of mTOR signaling induced the release of multivesicular body-derived exosomes (by inhibiting autophagy) and microvesicles (by activating 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 CCl_4- or BDL-induced liver fibrosis. Furthermore, in the CCl₄ model, mice receiving circulating EVs derived from mice with HSC-specific Shp2 deletion had less fibrosis than mice receiving EVs from control mice. Correspondingly, SHP2 was upregulated in patients with liver cirrhosis.

Conclusion

These results demonstrate that autophagy in HSCs attenuates liver fibrosis by inhibiting the release of fibrogenic EVs.

Lay summary

During liver fibrosis and cirrhosis, activated hepatic stellate cells (HSCs) are the key cell type responsible for fibrotic tissue deposition. Recently, we demonstrated that activated HSCs release nano-sized vesicles enriched with fibrogenic proteins. In the current study, we unveil the mechanism by which these fibrogenic vesicles are released, moving a step closer to the long-term goal of therapeutically targeting this process.

SUBMITTER: Gao J

PROVIDER: S-EPMC7572579 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Progression of fibrosis and the development of cirrhosis are responsible for the liver related morbidity and mortality associated with chronic liver diseases. There is currently a great unmet need for effective anti-fibrotic strategies. Stem cells play a central role in wound healing responses to restore liver homeostasis following injury. Here we tested the hypothesis that extracellular vesicles (EVs) isolated from induced pluripotent stem cells (iPSC) modulate hepatic stellate cell (HSCs) activation and may have anti-fibrotic effects. Human iPSCs were generated by reprogramming primary skin fibroblasts. EVs were isolated by differential centrifugation, quantified by flow cytometry (FACS) and characterized by dynamic light scattering (DLS) and electron microscopy (TEM). Primary human HSCs were activated with TGFβ (10 ng/mL) and exposed to iPSC-EVs. Efficacy of iPSC-EVs was tested on HSC in vitro and in two murine models of liver injury (CCl4 and bile duct ligation). Characterization of iPSC-derived EVs by flow cytometry identified a large population of EVs released by iPSC, primarily with a diameter of 300 nm and that could be visualized by TEM as round, cup-shaped objects. Fluorescent tracing assays detected iPSC-EVs in HSC cytosol after a short incubation and EV uptake by HSCs resulted in both decrease of pro-fibrogenic markers αSMA, CollagenIα1, Fibronectin and TIMP-1 and HSC pro-fibrogenic responses such as chemotaxis and proliferation. Genomics analyses of iPSC-EV miRNA cargo revealed 22 highly expressed miRNAs, among which miR-92a-3p resulted the most abundant. Transcriptome analysis identified 60 genes down-modulated and 235 up-regulated in TGF-β-primed HSC in presence or absence of iPSC-EVs. Intravenous injection of iPSC-EVs in CCl4 and bile duct ligation-induced liver fibrosis resulted in anti-fibrotic effects at protein and gene levels. Results of this study identify iPSC-EVs as a novel anti-fibrotic approach that may reduce or reverse liver fibrosis in patients with chronic liver disease.