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

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. 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. 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 CCl4- 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. These results demonstrate that autophagy in HSCs attenuates liver fibrosis by inhibiting the release of fibrogenic EVs. 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.

Gao J ，Wei B ，de Assuncao TM ，Liu Z ，Hu X ，Ibrahim S ，Cooper SA ，Cao S ，Shah VH ，Kostallari E ... -  《-》

Hepatic stellate cell-derived platelet-derived growth factor receptor-alpha-enriched extracellular vesicles promote liver fibrosis in mice through SHP2.

Liver fibrosis is characterized by the activation and migration of hepatic stellate cells (HSCs), followed by matrix deposition. Recently, several studies have shown the importance of extracellular vesicles (EVs) derived from liver cells, such as hepatocytes and endothelial cells, in liver pathobiology. While most of the studies describe how liver cells modulate HSC behavior, an important gap exists in the understanding of HSC-derived signals and more specifically HSC-derived EVs in liver fibrosis. Here, we investigated the molecules released through HSC-derived EVs, the mechanism of their release, and the role of these EVs in fibrosis. Mass spectrometric analysis showed that platelet-derived growth factor (PDGF) receptor-alpha (PDGFRα) was enriched in EVs derived from PDGF-BB-treated HSCs. Moreover, patients with liver fibrosis had increased PDGFRα levels in serum EVs compared to healthy individuals. Mechanistically, in vitro tyrosine720-to-phenylalanine mutation on the PDGFRα sequence abolished enrichment of PDGFRα in EVs and redirected the receptor toward degradation. Congruently, the inhibition of Src homology 2 domain tyrosine phosphatase 2, the regulatory binding partner of phosphorylated tyrosine720, also inhibited PDGFRα enrichment in EVs. EVs derived from PDGFRα-overexpressing cells promoted in vitro HSC migration and in vivo liver fibrosis. Finally, administration of Src homology 2 domain tyrosine phosphatase 2inhibitor, SHP099, to carbon tetrachloride-administered mice inhibited PDGFRα enrichment in serum EVs and reduced liver fibrosis. PDGFRα is enriched in EVs derived from PDGF-BB-treated HSCs in an Src homology 2 domain tyrosine phosphatase 2-dependent manner and these PDGFRα-enriched EVs participate in development of liver fibrosis. (Hepatology 2018;68:333-348).

Kostallari E ，Hirsova P ，Prasnicka A ，Verma VK ，Yaqoob U ，Wongjarupong N ，Roberts LR ，Shah VH ... -  《-》

Therapeutic role of FNDC5/irisin in attenuating liver fibrosis via inhibiting release of hepatic stellate cell-derived exosomes.

Cleavage of fibronectin type III domain-containing protein 5 (FNDC5), a membrane-bound precursor protein, would cleave into a myokine, irisin, which is also expressed in the liver. FNDC5/Irisin has been reported to play a critical role in maintaining glucose and lipid homeostasis in the liver and in combating liver fibrosis. Recently, several studies have shown that extracellular vesicles (EVs) derived from hepatic stellate cells (HSCs) could modulate liver fibrosis; however, there is a large gap in understanding whether inhibition of fibrogenic EVs derived from HSCs could alleviate the progression of liver fibrosis. Here, we investigated the role of FNDC5/irisin in liver fibrosis and the mechanism of its inhibitory role in the release of HSC-derived fibrogenic EVs. Experiments were performed in wild-type and FNDC5-/- mice, primary mouse HSCs, and human hepatic stellate cell line (LX2). Mice were treated with carbon tetrachloride (CCl4) or bile duct ligation (BDL) to induce liver fibrosis. EVs derived from HSCs were purified and injected intraperitoneally into mice. Our results showed that FNDC5 deficiency exacerbated CCl4-induced liver fibrosis and activation of HSCs in mice. Moreover, fibrogenic EVs derived from PDGF-BB-treated HSCs promoted HSC migration in vitro and liver fibrosis in vivo. However, administration of irisin, a cleavage of FNDC5, inhibited the release of fibrogenic EVs and activation of HSCs by promoting ubiquitylation degradation of Rab27b. In vivo, the promoting role of HSC-derived fibrogenic EVs in liver fibrosis was also reversed by irisin. All these results demonstrate that FNDC5/irisin is a novel therapeutic agent for chronic liver fibrosis.

Liao X ，Luo Y ，Gu F ，Song W ，Nie X ，Yang Q ... -  《-》

Coordinated signaling of activating transcription factor 6α and inositol-requiring enzyme 1α regulates hepatic stellate cell-mediated fibrogenesis in mice.

Xue F ，Lu J ，Buchl SC ，Sun L ，Shah VH ，Malhi H ，Maiers JL ... -  《-》

Human induced pluripotent stem cell-derived extracellular vesicles reduce hepatic stellate cell activation and liver fibrosis.

Povero D ，Pinatel EM ，Leszczynska A ，Goyal NP ，Nishio T ，Kim J ，Kneiber D ，de Araujo Horcel L ，Eguchi A ，Ordonez PM ，Kisseleva T ，Feldstein AE ... -  《JCI Insight》