BACKGROUND AND AIMS:Hepatic stellate cell (HSC) activation is central to liver fibrosis, but emerging evidence suggests HSC homeostatic activity. We compared HSC functions in parenchymal injury (CCl 4 -driven) versus metabolic dysfunction-associated steatohepatitis (MASH; choline-deficient, high-fat diet-CD-HFD) and identified therapeutic targets preserving HSC homeostatic functions. APPROACH AND RESULTS:Inducible HSC ablation was performed in Lrat-iDTR mice during active fibrogenesis. Multi-parametric analyses were conducted to assess roles of HSCs in 2 established fibrosis models, with a focus on elucidating the cellular origins of myofibroblasts and the alterations in regeneration and ductular reaction. RNA-seq from human biopsies validated mechanisms. HSC depletion in the CD-HFD model not only exacerbated MASH but also elevated a-SMA + myofibroblasts derived from PDGFRα + portal fibroblasts, impaired hepatocyte function (metabolic zonation and regeneration), and enhanced the ductular reaction. Conversely, HSC depletion in the CCl 4 model attenuated fibrosis without affecting hepatic regeneration or metabolic zonation. Strikingly, 85.5% of quiescent HSC-enriched genes remained upregulated in MASH-associated HSCs, unlike in CCl 4 fibrosis. RNA-seq followed by in vivo studies identified extracellular matrix protein 1 (ECM1) as a master regulator of HSC quiescence, and HSC-specific ECM1 overexpression suppressed CCl 4 -induced fibrosis. In human biopsies (MASH, HBV, PBC, PSC), ECM1 expression inversely correlated with fibrosis stage. CONCLUSIONS:HSCs exhibit dual roles contingent on disease context: in MASH with moderate inflammation, they maintain homeostasis, whereas in massive CCl 4 -driven injury, activated HSCs promote fibrogenesis. ECM1 enforces HSC quiescence and facilitates fibrosis resolution. Anti-fibrotic therapies based on general HSC ablation may be harmful.

Cholestatic liver injury (CLI) is a severe liver disorder caused by impaired bile flow, for which effective therapeutic options remain limited. Although the aryl hydrocarbon receptor (AHR) has been implicated in protection against CLI, the mechanisms underlying its dysregulation and functional role during cholestasis remain incompletely understood. Here, we investigated the role of AHR in cholestatic liver injury and the mechanisms contributing to impaired AHR activation using multi-omics approaches. Using mouse models of CLI induced by a 0.1

Elastin stabilization has been correlated with the reversibility of fibrosis. Fibulin-1 can participate in elastin assembly, which promotes its stabilization. However, the role of Fibulin-1 in liver fibrosis remains unknown. Here, we performed a proteomics analysis to identify notable changes in Fibulin-1 expression during continuous fibrosis progression and regression. Fibulin-1 expression was dramatically increased in the plasma of patients with cirrhosis as well as in liver fibrosis models and hepatic stellate cells (HSCs) treated with TGF-β1, and significant accumulation of Fibulin-1 was observed in chronic hepatitis B (CHB)- and metabolic dysfunction-associated steatohepatitis (MASH)-related cirrhosis. Functional studies demonstrated that Fibulin-1 silencing inhibited HSC activation, while the opposite effects were observed for Fibulin-1 overexpression in vitro. Furthermore, transcriptomic analysis revealed that Fibulin-1 mediated p38 MAPK pathway activation, which was confirmed by the addition of a p38 MAPK inhibitor. More importantly, Fibulin-1 depletion in a CCl4-induced liver fibrosis model substantially ameliorated fibrosis progression, which was accompanied by decreased profibrogenic gene expression and decreased levels of insoluble elastin. Moreover, activation of the p38 MAPK pathway was inhibited in vivo. The expression of Fibulin-1D, rather than Fibulin-1C, was elevated during liver fibrogenesis, which suggested a major role for Fibulin-1D in liver fibrosis. Next, we established Fibulin-1D/elastin-coated culture models with LX-2 cells. LX-2 cells with extracellular elastin and Fibulin-1D deposition showed more significant profibrotic phenotypic alterations than those with elastin alone. Fibulin-1 deficiency alleviated liver fibrosis by reducing insoluble elastin and HSC activation, and finally, the p38 MAPK pathway might be involved in the effect of Fibulin-1 on HSCs.

BACKGROUND AND OBJECTIVE:Liver fibrosis is a pathological process driven by chronic liver injury, characterized by excessive extracellular matrix (ECM) deposition due to hepatic stellate cell (HSC) activation. Integrins are critical regulators of ECM remodeling and HSC activation, yet the role of integrin α8(ITGA8) in liver fibrosis remains unclear. This study aims to investigate the function and underlying mechanisms of HSC-derived ITGA8 in liver fibrosis and evaluate the therapeutic potential of ITGA8-targeted intervention. METHODS:A CCl4-induced mouse liver fibrosis model and public database analysis were used to assess ITGA8 expression and localization in liver fibrosis. AAV2/6-shItga8 was utilized to selectively silence HSC-derived ITGA8, and its effects on HSC activation and ECM accumulation were examined. In addition, in vitro ITGA8 knockdown combined with proteomic analysis was performed to explore the molecular mechanisms linking ITGA8 to ECM remodeling. RESULTS:ITGA8 expression was significantly upregulated in fibrotic liver tissues across different etiologies, with a strong colocalization with HSCs. Silencing ITGA8 using AAV2/6-shItga8 effectively reduced liver fibrosis, as indicated by decreased hepatic inflammation, lower serum ALT levels, reduced inflammatory cell infiltration, and downregulated expression of pro-inflammatory cytokines. Fibrosis markers, including Sirius Red staining, type I collagen deposition, and α-SMA expression, were all reduced upon Itga8 silencing. Proteomic analysis revealed that ITGA8 regulates liver fibrosis through the ECM-receptor interaction pathway, with COL11A1 identified as a key downstream target. ITGA8 knockdown significantly suppressed COL11A1 expression, and reduced HSC-mediated collagen contraction, suggesting that ITGA8 contributes to ECM cross-linking and fibrosis progression via COL11A1 regulation. CONCLUSION:This study demonstrates that HSC-derived ITGA8 promotes ECM accumulation and liver fibrosis progression by regulating COL11A1. Targeted silencing of ITGA8 via AAV2/6-shItga8 effectively alleviates liver fibrosis, providing new insights into ITGA8 as a potential therapeutic target for antifibrotic treatment.

BACKGROUND & AIMS:Hepatic stellate cells (HSCs) are the major source of excessive production of extracellular matrix (ECM) proteins and act as a hub for intrahepatic fibrosis signaling. Although extensive crosstalk between HSCs and liver sinusoidal endothelial cells (LSECs) significantly influences disease progression, the detailed mechanisms remain poorly understood. Here, we investigated the role of lysyl oxidase-like 1 (LOXL1), a pivotal enzyme in ECM cross-linking, in crosstalk between HSCs and LSECs during liver fibrosis. METHODS:Coculture systems (EA.hy926/LX2 or LSECs/HSCs) were used to investigate signaling crosstalk. HSC-specific Loxl1 knockout mice were generated, and experimental liver fibrosis was induced using a high-fat choline-deficient amino acid-defined (HFCDAA) diet or chronic carbon tetrachloride (CCl4) exposure. Liver samples were assessed by histology, scanning electron microscopy, immunostaining, and quantitative polymerase chain reaction (qPCR). Liver tissue and HSCs were analyzed by RNA sequencing to study LOXL1's mechanisms regulating liver fibrosis. RESULTS:LOXL1 in HSCs modulated ECM composition changes in endothelial cells in coculture studies. LOXL1 loss in HSCs suppressed HSC activation, LSEC capillarization, and macrophage infiltration, improved ECM remodeling, and attenuated liver fibrosis in HFCDAA-fed and CCl4-exposed mice. Our RNA sequencing data, corroborated by public database analyses, indicated RUNX family transcription factor 1 (RUNX1) was implicated in HSC activation and LOXL1-mediated angiogenesis. We propose that LOXL1 enhances HSC activation and LSEC capillarization through the RUNX 1/vascular endothelial growth factor A signaling axis CONCLUSIONS: Our study reveals novel mechanistic insights into liver fibrosis, highlighting HSC-derived LOXL1 as a central modulator of disease initiation and progression. Targeting the LOXL1/RUNX1/vascular endothelial growth factor A axis offers a promising therapeutic strategy for liver fibrosis.

Non-alcoholic steatohepatitis (NASH) is characterized by hepatic lipid accumulation and fibrosis, yet the molecular mechanisms linking metabolic dysfunction to extracellular matrix (ECM) remodeling remain poorly defined. This study investigated the role of laminin γ2 (LAMC2, encoded by Lamc2), a basement membrane component, in NASH pathogenesis. Using a high-fat choline-deficient l-amino acid-defined (HF-CDAA) diet-induced murine NASH model and AML12 hepatocytes, we assessed LAMC2 expression via qPCR, immunohistochemistry, and lipidomics. Functional studies included LAMC2 overexpression (adenovirus) and TGFβ pathway inhibition (SB431542). LAMC2 was markedly upregulated in steatotic hepatocytes and localized adjacent to lipid droplets. Laminin-332 (Ln-332), which contains the γ2 chain (LAMC2), directly amplified lipogenesis by increasing Srebf1 and Mlxipl gene expression. TGFβ1 signaling via TGFBR1/Fra2 drove LAMC2 expression. Crucially, LAMC2 amplified both lipogenesis and fibrogenesis, thereby forming a feedforward loop that exacerbated hepatic fibrosis in this predominantly fibrotic NASH model. In vivo, LAMC2 overexpression exacerbated hepatic lipid accumulation and collagen deposition in the HF-CDAA diet-induced NASH model compared to controls. Fra2 silencing via AAV-shRNA attenuated NASH progression. LAMC2 bridges metabolic and fibrotic reprogramming in NASH through TGFβ1/Fra2-dependent mechanisms. Targeting this ECM-metabolism axis, particularly LAMC2 or Fra2, offers novel therapeutic strategies for fibrosis-dominant NASH, addressing a critical unmet clinical need.

Microfibrillar-associated protein 2 (MFAP-2) is a crucial component of the extracellular matrix (ECM) microfibrils, yet its role in liver fibrosis remains elusive. Methods: Human tissue arrays and mouse models of fibrosis progression and resolution were used to investigate MFAP-2 expression patterns. Mfap2 deficiency (Mfap2 -/-) or overexpression (ovMfap2) mice were subjected to carbon tetrachloride (CCl4) injection or bile duct ligation (BDL) to induce liver fibrosis. Histological, biochemical, bulk, or single-cell RNA-sequencing (scRNA-seq), proteomics to analyze the matrisome, and in vitro studies were conducted. Results: MFAP-2 was predominantly enriched in activated hepatic stellate cells (HSCs) and upregulated in advanced liver fibrosis. Although Mfap2 ablation had minimal impact on collagen deposition during CCl4 injection, it significantly delayed fibrosis regression after CCl4 cessation. The delayed fibrosis regression due to Mfap2 deficiency was likely linked to aggravated intrahepatic inflammation, ECM stabilization, and activated focal adhesion signaling in HSCs. Mechanically, inhibiting HSC-derived Mfap2 enhanced HSC interactions and increased matrisome protein production, while reducing the interaction between HSCs and liver-resident macrophages by decreasing macrophage migration inhibitory factor secretion from HSCs. Additionally, we validated the role of Mfap2 deletion in liver fibrosis using the BDL mouse model, demonstrating a more pronounced effect on fibrosis progression. Adeno-associated virus vector (serotype 6)-mediated Mfap2 overexpression in HSCs conferred protection against liver fibrosis in both models. Conclusion: This study reveals the compensatory protective effects of HSC-derived MFAP-2 on liver fibrosis and its underlying mechanisms. Enhancing MFAP-2 in HSCs may therefore benefit patients with liver fibrosis.

Background & Aims: Thrombospondin-2 (THBS2) expression is associated with liver fibrosis regardless of etiology. However, the role of THBS2 in the pathogenesis of liver fibrosis has yet to be elucidated. Methods: The in vivo effects of silencing Thbs2 in hepatic stellate cells (HSCs) were examined using an adeno-associated virus vector (serotype 6, AAV6) containing short -hairpin RNAs targeting Thbs2, under the regulatory control of cytomegalovirus, U6 or the a -smooth muscle promoter, in mouse models of carbon tetrachloride or methionine-choline deficient (MCD) dietinduced liver fibrosis. Crosstalk between THBS2 and toll -like receptor 4 (TLR4), as well as the cascaded signaling, was systematically investigated using mouse models, primary HSCs, and human HSC cell lines. Results: THBS2 was predominantly expressed in activated HSCs and dynamically increased with liver fibrosis progression and decreased with regression. Selective interference of Thbs2 in HSCs retarded intrahepatic inflammatory infiltration, steatosis accumulation, and fibrosis progression following carbon tetrachloride challenge or in a dietary model of metabolic dysfunctionassociated steatohepatitis. Mechanically, extracellular THBS2, as a dimer, specifically recognized and directly bound to TLR4, activating HSCs by stimulating downstream profibrotic focal adhesion kinase (FAK)/transforming growth factor beta (TGF-b) pathways. Disruption of the THBS2-TLR4-FAK/TGF-b signaling axis notably alleviated HSC activation and liver fibrosis aggravation. Conclusions: THBS2 plays a crucial role in HSC activation and liver fibrosis progression through TLR4-FAK/TGF-b signaling in an autocrine manner, representing an attractive potential therapeutic target for liver fibrosis. Impact and implications: Thrombospondin-2 (THBS2) is emerging as a factor closely associated with liver fibrosis regardless of etiology. However, the mechanisms by which THBS2 is involved in liver fibrosis remain unclear. Here, we showed that THBS2 plays a prominent role in the pathogenesis of liver fibrosis by activating the TLR4-TGF-b/FAK signaling axis and hepatic stellate cells in an autocrine manner, providing a potential therapeutic target for the treatment of liver fibrosis. (c) 2024 Published by Elsevier B.V. on behalf of European Association for the Study of the Liver (EASL). This is an open access article under the CC BY -NC -ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Background: Lysyl oxidase (LOX) family members (LOX and LOXL1 to 4) are crucial copper-dependent enzymes responsible for cross-linking collagen and elastin. Previous studies have revealed that LOX and LOXL1 are the most dramatically dysregulated LOX isoforms during liver fibrosis. However, the crosstalk between them and the underlying mechanisms involved in the profibrotic behaviors of HSCs, as well as the progression of liver fibrosis, remain unclear. Methods: pCol9GFP-HS4,5Tg mice, Loxl1 fl/fl Gfap Cre mice, human HSC line, and primary HSCs were enrolled to study the dysregulation pattern, profibrotic roles, and the potential mechanisms of LOX and LOXL1 interaction involved in the myofibroblast-like transition of HSCs and liver fibrogenesis. Results: LOX and LOXL1 were synergistically upregulated during liver fibrogenesis, irrespective of etiology, together orchestrating the profibrotic behaviors of HSCs. LOX and LOXL1 coregulated in HSCs, whereas LOXL1 dominated in the coregulation loop. Interestingly, the interaction between LOXL1 and LOX prolonged their half-lives, specifically enhancing the Notch signal-mediated myofibroblast-like transition of HSCs. Selective disruption of Loxl1 in Gfap + HSCs deactivated the Notch signal, inhibited HSC activation, and relieved carbon tetrachloride-induced liver fibrosis. Conclusions: Our current study confirmed the synergistic roles and the underlying mechanisms of LOXL1 and LOX crosstalk in the profibrotic behaviors of HSCs and liver fibrosis progression, providing experimental evidence for further clear mechanism-based anti-LOXL1 strategy development in the therapy of liver fibrosis.

BACKGROUND AIMS:Excessive deposition and crosslinking of extracellular matrix increases liver density and stiffness, promotes fibrogenesis, and increases resistance to fibrinolysis. An emerging therapeutic opportunity in liver fibrosis is to target the composition of the extracellular matrix or block pathogenic communication with surrounding cells. However, the type and extent of extracellular changes triggering liver fibrosis depend on the underlying etiology. Our aim was to unveil matrisome genes not dependent on etiology, which are clinically relevant to liver fibrosis.APPROACH RESULTS:We used transcriptomic profiles from liver fibrosis cases of different etiologies to identify and validate liver fibrosis-specific matrisome genes (LFMGs) and their clinical and biological relevance. Dysregulation patterns and cellular landscapes of LFMGs were further explored in mouse models of liver fibrosis progression and regression by bulk and single-cell RNA sequencing. We identified 35 LFMGs, independent of etiology, representing an LFMG signature defining liver fibrosis. Expression of the LFMG signature depended on histological severity and was reduced in regressive livers. Patients with liver fibrosis, even with identical pathological scores, could be subclassified into LFMG Low and LFMG High , with distinguishable clinical, cellular, and molecular features. Single-cell RNA sequencing revealed that microfibrillar-associated protein 4 + activated HSC increased in LFMG High patients and were primarily responsible for the LFMG signature expression and dysregulation.CONCLUSIONS:The microfibrillar-associated protein 4 + -activated HSC-derived LFMG signature classifies patients with liver fibrosis with distinct clinical and biological characteristics. Our findings unveil hidden information from liver biopsies undetectable using traditional histologic assessments.

Thrombospondin-2 (THBS2) expression is closely associated with liver fibrosis regardless of etiology. However, the role of THBS2 in the pathogenesis of liver fibrosis has not been elucidated yet. Here we report THBS2 is predominantly expressed in activated HSCs and dynamically increases with liver fibrosis progression and decreased in regression. Selective interference of HSC Thbs2 evidently retards fibrosis progression and intrahepatic inflammatory infiltration in liver fibrosis mouse models. Mechanically, extracellular THBS2, as a dimer, specifically recognizes and directly binds to TLR4 receptor, activating HSCs via stimulating downstream profibrotic focal adhesion kinase (FAK)/transforming growth factor beta (TGF-β) pathways. Disruption of THBS2-TLR4-FAK/TGF-β signaling axis notably alleviates HSC activation and liver fibrosis aggravation. In conclusion, THBS2 plays a crucial role in HSC activation and liver fibrosis progression through TLR4-FAK/TGF-β signaling in an autocrine manner. Therapies targeting HSC Thbs2 via AAV6 vector-capsulated shRNA may represent a novel promising strategy to prevent or treat liver fibrosis.