Stiffness is associated with hepatic stellate cell heterogeneity during liver fibrosis

The fibrogenic wound-healing response in liver increases stiffness. Stiffness mechano-transduction in turn amplifies fibrogenesis. Here, we aimed to understand the distribution of stiffness in fibrotic liver, how it impacts hepatic stellate cell (HSC) heterogeneity and identify mechanisms by which stiffness amplifies fibrogenic responses. Magnetic resonance elastography and atomic force microscopy demonstrated a heterogenous distribution of liver stiffness at macroscopic and microscopic levels, respectively, in a carbon tetrachloride (CCl4) mouse model of liver fibrosis as compared to controls. High stiffness was mainly attributed to extracellular matrix dense areas. To identify a stiffness-sensitive HSC sub-population, we performed scRNA-seq on primary HSCs derived from healthy versus CCl4-treated mice. A sub-cluster of HSCs was matrix-associated with the most upregulated pathway in this sub-population being focal adhesion signaling, including a specific protein termed four and a half LIM domains protein 2 (FHL2). In vitro, FHL2 expression was increased in primary human HSCs cultured on stiff matrix as compared to HSCs on soft matrix. Moreover, FHL2 knockdown inhibited fibronectin and collagen 1 expression, whereas its overexpression promoted matrix production. In summary, we demonstrate stiffness heterogeneity at the whole organ, lobular, and cellular level which drives an amplification loop of fibrogenesis through specific focal adhesion molecular pathways.

Tenofovir disoproxil fumarate directly ameliorates liver fibrosis by inducing hepatic stellate cell apoptosis via downregulation of PI3K/Akt/mTOR signaling pathway

Background Antifibrotic agent for the treatment of liver fibrosis has not been developed so far. Long term treatment of chronic hepatitis B patients with antiviral drugs tenofovir disoproxil fumarate (TDF) and entecavir (ETV) results in the regression of liver fibrosis, but the underlying mechanism has not been clarified. Therefore, we aimed to investigate the direct impact of TDF and ETV on liver fibrosis. Methods Activated hepatic stellate cell (HSC) cell lines were used to evaluate the effects of TDF and ETV. After treatment with each antiviral agent, cell viability, morphology, apoptotic features, autophagy and antifibrosis signalling pathways were examined. Then, collagen deposition, fibrosis markers and activated HSCs were measured in liver tissues of the liver fibrosis model mice. Results After TDF treatment, the viabilities of LX2 and HSC-T6 cells were decreased, and the cells exhibited apoptotic features, but ETV did not induce these effects. Cleavage of PARP and Caspase-3 and the inhibition of the antiapoptotic gene Bcl-xl indicated activated HSC apoptosis following TDF treatment. TDF simultaneously increased autophagy, which also regulated apoptosis through crosstalk. TDF inactivated the PI3K/Akt/mTOR signalling pathway, which was associated with the activation of both apoptosis and autophagy. In the liver fibrosis mouse model, the fibrotic area and activated HSC markers were decreased by TDF but not ETV treatment. Additionally, apoptotic cells were concentrated in the periportal fibrotic area after TDF treatment, which indicated the specific antifibrotic effect of TDF. Conclusions TDF directly ameliorates liver fibrosis by downregulating the PI3K/Akt/mTOR signalling pathway, which results in the apoptosis of activated HSCs. The antifibrotic effects of TDF indicate that it may be a therapeutic agent for the treatment of liver fibrosis.

Dysregulation of miR-21-associated miRNA regulatory networks by chronic ethanol consumption impairs liver regeneration

Impaired liver regeneration has been considered as a hallmark of progression of alcohol-associated liver disease. Our previous studies demonstrated that in vivo inhibition of the microRNA (miRNA) miR21 can restore regenerative capacity of the liver in chronic ethanol-fed animals. The present study focuses on the role of microRNA regulatory networks that are likely to mediate the miR-21 action. Rats were chronically fed an ethanol-enriched diet along with pair-fed control animals and treated with AM21 (anti-miR-21), a locked nucleic acid antisense to miR-21. Partial hepatectomy (PHx) was performed and miRNA expression profiling over the course of liver regeneration was assessed. Our results showed dynamic expression changes in several miRNAs post PHx, notably with altered miRNA expression profiles between ethanol versus control groups. We found that in vivo inhibition of miR-21 led to correlated differential expression of miR-340-5p, and anti-correlated expression of miR-365, let-7a, miR-1224 and miR-146a across all sample groups post PHx. Gene set enrichment analysis identified a miRNA signature significantly associated with hepatic stellate cell activation within whole-liver tissue data. We hypothesized that at least part of the PHx-induced miRNA network changes responsive to miR-21 inhibition is localized to hepatic stellate cells. We validated this hypothesis using AM21 and TGF-β treatments in LX-2 human hepatic stellate cells in culture, and measured expression levels of select miRNAs by quantitative RT-PCR. Based on the in vivo and in vitro results, we propose a hepatic stellate cell miRNA regulatory network as contributing to the restoration of liver regenerative capacity by miR-21 inhibition.

Divergent Effect of Birinapant, BV6 SMAC Mimetic on TNFα induced NF-κB Signaling and Cell Viability in Activated Hepatic Stellate Cell

Background: Tumor necrosis factor-α (TNFα) is a pleiotropic cytokine involved in nuclear factor kappa B (NF-κB) mediated cell survival as well as cell death. High serum TNFα levels correlate with liver fibrosis and participate in enhancing hepatic stellate cell (HSC) viability. However, the regulatory role of cellular inhibitor of apoptosis-1/2 (cIAP1/2) in TNFα induced NF-κB signaling in activated HSCs is largely unknown. Methods and Results: Activated HSCs treated with cIAP1/2 inhbitior SMAC mimetic BV6 and Birinapant in presence of TNFα and macrophage secretome. TNFα cytokine increased cIAP2 expression in activated HSCs and enhanced cell viability through canonical NF-κB signaling in activated HSCs. cIAP2 inhibition via BV6 decreased the TNFα induced canonical NF-κB signaling and reduced cell viability in activated HSCs. SMAC mimetic, Birinapant alone did not affect the cell viability and was incompetent in inducing cell death. However, pre-treatment of TNFα sensitized HSCs and Birinapant was capable in inducing cell death. Birinapant mediated cIAP2 ablation was unable to decrease the TNFα induced canonical NF-κB signaling in comparison to BV6. Secreted TNFα from M1 polarized macrophages sensitized activated HSCs to BV6 or Birinapant mediated cell death. However, M2 polarized macrophages rescued the activated HSCs from BV6 and Birinapant mediated cytotoxicity. Conclusions: In this study, we describe regulatory role of cIAP2 in TNFα induced NF-κB signaling in activated HSCs. Targeting cIAP2 may be a promising approach for liver fibrosis treatment via modulating NF-κB signaling in activated HSCs.