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Endothelin-1/endothelin A receptor (ET-1/ETAR) pathway plays an important role in the progression of liver fibrosis by activating hepatic stellate cells (HSCs) - a key cell type involved in the pathogenesis of liver fibrosis. Inactivating HSCs by blocking the ET-1/ETAR pathway using a selective ETAR antagonist (ERA) represents a promising therapeutic approach for liver fibrosis. Unfortunately, small-molecule ERAs possess limited clinical potential due to poor bioavailability, short half-life, and rapid renal clearance. To improve the clinical applicability, we conjugated ERA to superparamagnetic iron-oxide nanoparticles (SPIONs) and investigated the therapeutic efficacy of ERA and ERA-SPIONs in vitro and in vivo and analyzed liver uptake by in vivo and ex vivo magnetic resonance imaging (MRI), HSCs-specific localization, and ET-1/ETAR-pathway antagonism in vivo. In murine and human liver fibrosis/cirrhosis, we observed overexpression of ET-1 and ETAR that correlated with HSC activation, and HSC-specific localization of ETAR. ERA and successfully synthesized ERA-SPIONs demonstrated significant attenuation in TGFβ-induced HSC activation, ECM production, migration, and contractility. In an acute CCl4-induced liver fibrosis mouse model, ERA-SPIONs exhibited higher liver uptake, HSC-specific localization, and ET-1/ETAR pathway antagonism. This resulted in significantly reduced liver-to-body weight ratio, plasma ALT levels, and α-SMA and collagen-I expression, indicating attenuation of liver fibrosis. In conclusion, our study demonstrates that the delivery of ERA using SPIONs enhances the therapeutic efficacy of ERA in vivo. This approach holds promise as a theranostic strategy for the MRI-based diagnosis and treatment of liver fibrosis.

Background & Aims: Among the reprogrammed metabolic pathways described in cancer stem cells, aberrant lipid metabolism has recently drawn increasing attention. Our study explored the contribution of fatty acids (FA) in the regulation of stem-like features in intrahepatic cholangiocarcinoma (iCCA). Methods: We previously identified a functional stem-like subset in human iCCA by using a three-dimensional sphere (SPH) model in comparison to parental cells grown as monolayers (MON). In this study, quantification of intracellular free FA and lipidomic analysis (triacylglycerol [TAG] composition, de novo synthesis products) was performed by Liquid chromatography–mass spectrometry (LC–MS); quadrupole time-of-flight liquid chromatography/mass spectrometry (Q-TOF LC/MS), respectively, in both SPH and MON cultures. Results: Stem-like SPH showed a superior content of free FA (citric, palmitic, stearic, and oleic acids) and unsaturated TAG. Molecularly, SPH showed upregulation of key metabolic enzymes involved in de novo FA biosynthesis (AceCS1, ACLY, ACAC, FASN, ACSL1) and the mTOR signalling pathway. In patients with iCCA (n = 68), tissue expression of FASN, a key gene involved in FA synthesis, correlated with 5-year overall survival. Interference with FASN activity in SPH cells through both specific gene silencing (siRNA) or pharmacological inhibition (orlistat) decreased sphere-forming ability and expression of stem-like markers. In a murine xenograft model obtained by injection of iCCA-SPH cells, FASN inhibition by orlistat or injection of FASN-silenced cells significantly reduced tumour growth and expression of stem-like genes. Conclusion: Altered FA metabolism contributes to the maintenance of a stem-like phenotype in iCCA. FASN inhibition may represent a new approach to interfere with the progression of this deadly disease. Impact and implications: Recent evidence indicates that metabolic disorders correlate with an increased susceptibility to intrahepatic cholangiocarcinoma (iCCA). Our investigation emphasises the pivotal involvement of lipid metabolism in the tumour stem cell biology of iCCA, facilitated by the upregulation of crucial enzymes and the mTOR signalling pathway. From a clinical perspective, this underscores the dual role of FASN as both a prognostic indicator and a therapeutic target, suggesting that FASN inhibitors could enhance patient outcomes by diminishing stemness and tumour aggressiveness. These findings pave the way for novel therapeutic strategies for iCCA and shed light on its relationship with metabolic disorders such as diabetes, obesity, metabolic syndrome, and metabolic dysfunction-associated steatotic liver disease.

Abstract The lysophosphatidic acid (LPA) signaling axis is an important but rather underexplored pathway in liver disease. LPA is predominantly produced by Autotaxin (ATX) that has gained significant attention with an impressive number of ATX inhibitors (type I‐IV) reported. Here, we evaluated the therapeutic potential of a (yet unexplored) type IV inhibitor, Cpd17, in liver injury. We first confirmed the involvement of the ATX‐LPA signaling axis in human and murine diseased livers. Then, we evaluated the effects of Cpd17, in comparison with the classic type I inhibitor PF8380, in vitro, where Cpd17 showed higher efficacy. Thereafter, we characterized the mechanism‐of‐action of both inhibitors and found that Cpd17 was more potent in inhibiting RhoA‐mediated cytoskeletal remodeling, and phosphorylation of MAPK/ERK and AKT/PKB. Finally, the therapeutic potential of Cpd17 was investigated in CCl4‐induced acute liver injury and diet‐induced nonalcoholic steatohepatitis, demonstrating an excellent potential of Cpd17 in reducing liver injury in both disease models in vivo. We conclude that ATX inhibition, by type IV inhibitor in particular, has an excellent potential for clinical application in liver diseases.

Abstract Non‐alcoholic steatohepatitis (NASH) and alcohol‐associated steatohepatitis (ASH) are the major cause of liver‐related mortality with limited therapeutic options available. In this study, we investigated the role of Src tyrosine kinase in the pathogenesis of (N)ASH. We examined Src kinase expression in livers from patients with NASH, ASH, cirrhosis and biliary atresia, and from preclinical mouse models: methionine choline deficient (MCD)‐diet‐induced NASH, Lieber–DeCarli ethanol (EtOH)‐diet‐induced ASH, carbon tetrachloride (CCl4)‐ and bile duct ligation (BDL)‐induced liver fibrosis. Functional studies, using Src kinase inhibitor KX2‐391, were performed in NASH and ASH mouse models, and in relevant in vitro models. Transcriptomic analysis revealed increased Src kinase expression in human and mouse diseased livers that positively correlated with disease progression. Src kinase inhibition ameliorated lipid biosynthesis (steatosis); monocytes/macrophages infiltration and inflammatory cytokines expression (inflammation) and hepatic stellate cells (HSCs) activation and collagen expression (fibrosis) in MCD‐diet and EtOH‐diet induced liver disease mouse models. In vitro Src inhibition attenuated FFA‐induced hepatocytic lipid accumulation, LPS/IFNγ‐induced nitric oxide release and expression of several inflammatory signatures in macrophages and TGFβ‐induced HSCs activation, contractility and collagen expression. Moreover, Src inhibition diminished gene expression of inflammatory markers in LPS/IFNγ‐stimulated BMDMs and LPS‐stimulated PCLS, and reduced FA accumulation, macrophage activation and collagen deposition in 3D human liver spheroids. Mechanistic studies further revealed that Src kinase mediated the effects through FAK/PI3K/AKT pathway. Our results demonstrate a multicellular and functional role of Src kinase highlighting Src kinase as a promising therapeutic target in (non)alcoholic steatohepatitis.

Small animals are widely used as disease models in medical research. Noninvasive imaging modalities with functional capability play an important role in studying the disease state and treatment progress. Photoacoustics, being a noninvasive and functional modality, has the potential for small-animal imaging. However, the conventional photoacoustic tomographic systems use pulsed lasers, making it expensive, bulky, and require long acquisition time. In this work, we propose the use of photoacoustic and ultrasound tomographic imaging with LEDs as the light source and acoustic detection using a linear transducer array. We have demonstrated full-view tomographic imaging of a euthanized mouse and a potential application in liver fibrosis research.