ASK1 Enhances Angiotensin II-Induced Liver Fibrosis In Vitro by Mediating Endoplasmic Reticulum Stress-Dependent Exosomes

Abstract

Background. Apoptosis signal-regulating kinase 1 (ASK1) has been reported to induce fibrotic signaling in the setting of oxidative stress. However, the role of ASK1 and its mechanism of action in angiotensin II- (Ang II-) induced liver fibrosis remain largely unknown. Methods. Human hepatic LX-2 stellate cells were treated with Ang II alone or cotreated with Ang II plus an ASK1 inhibitor (GS-4997) or siRNA-targeting ASK1. Immunofluorescent staining, real-time PCR, and western blotting were used to determine the expressionof α-SMA, Col I, and Col III expression. Cell viability was assessed by the CCK-8 assay. The concentrations of IL-1β, IL-18, and TNF-α in conditioned medium were determined by ELISA. The levels of intracellular ROS in LX-2 cells were analyzed using a ROS assay kit. Exosome size was determined by electron microscopy. Results. Ang II markedly increased the expression of extracellular matrix (ECM) proteins (α-SMA, Col I, and Col III) and proinflammatory cytokines (IL-1β, IL-18, and TNF-α). Ang II also increased the expression of endoplasmic reticulum stress (ERS) markers (GRP78, p-PERK, and CHOP) and p-ASK1. Results also showed that pretreatment with GS-4997 or siRNA could abolish all the abovementioned effects on LX-2 cells. Furthermore, we found that exosome release caused by ASK1-mediated ERS was involved in the activation of LX-2 cells by Ang II. The activation of LX-2 cells could be blocked by treating the exosomes with annexin. Conclusions. In summary, we found that ASK1 mediates Ang II-activated ERS in HSCs and the subsequent activation of HSCs, suggesting a promising strategy for treating liver fibrosis.

Extracellular Vesicles in Organ Fibrosis: Mechanisms, Therapies, and Diagnostics

“…Additionally, exosome release from activated HSC was HIF-1 -dependent and the resultant exosomes contained HIF-1 -dependent glucose transporter 1 (GLUT1) and pyruvate kinase M2 (PKM2) which were subsequently delivered to quiescent HSC, KCs, or luminal sinusoidal endothelial cells (LSECs) [ 264 ]. Furthermore, profibrotic CCN2 in activated HSC was packaged into their secreted EVs which, upon binding to target HSC via cell surface integrins and heparan sulfate proteoglycans, caused elevated intracellular CCN2 and αSMA levels [ 36 , 265 ], while stimulation by Ang II of HSC fibrogenesis and endoplasmic reticulum stress in vitro was mediated by apoptosis signaling regulating kinase 1 and resulted in the liberation of pro-fibrogenic EVs [ 266 ].…”

Liver fibrosis: Extracellular vesicles mediated intercellular communication in perisinusoidal space

“…Annexin treatments prevent the activation of HSCs when incubated with exosomes released from angiotensin II–induced LX‐2 cells. [ 70 ] Second, as qHSC‐derived EVs display antifibrotic properties, they can be developed as a treatment to suppress the proliferation of aHSCs in the fibrotic livers. EVs released from qHSCs, normal hepatocytes and mesenchymal stem cells (MSCs) can reduce inflammation, inhibit the activation of HSCs, and reduce the progression of fibrosis.…”

“…qHSCs express α‐SMA consequently to their incubation with exosomes derived from Ang II–induced aHSCs. [ 70 ] The increased glycolysis, mitochondrial respiration, and the Warburg effect are associated with the HSC activation. [ 71 ] Indeed, HSC exosomes contain glucose transporter (GLUT1) and pyruvate kinase M2 (PKM2).…”

Interactions between endoplasmic reticulum stress and extracellular vesicles in multiple diseases

“…Angiotensin I (Ang II) signaling plays an important role in the pathogenesis of fibrosis. Using an in vitro model of hepatic fibrosis in LX-2 cells, researchers reported that treatment with Ang II increased hematopoietic stem cell (HSC) activity, inflammation, ER stress, ROS production, and expression of ECM-related proteins (α-SMA, Col I, and Col III) ( 65 ). Ang II treatment also increased the expression of apoptosis signal-regulating kinase 1 (ASK1), which can mediate ER stress and lead to EV release from Ang II-activated HSCs.…”