Skeletal Muscle Atrophy is Exacerbated By Steatotic and Fibrotic Liver-Derived TNF-Alpha in Senescence-Accelerated Mice

Although liver diseases, including non-alcoholic steatohepatitis (NASH), are associated with skeletal muscle atrophy, the mechanism behind their association has not been fully elucidated. In this study, the effects of aging and NASH on the skeletal muscle and the interaction between the liver and muscle were investigated using a diet-induced NASH model in senescence-accelerated mice (SAM). A total of four groups of SAM and its control mice were fed either an NASH-inducing or control diet. In the SAM/NASH group, the histopathology of NASH and markers of oxidative stress were significant. Skeletal muscles were also markedly atrophied. The expression of the ubiquitin ligase Murf1 in the muscle was significantly increased with muscle atrophy, while that of Tnfa was not significantly different. In contrast, the hepatic Tnfa expression and serum TNF-α levels were significantly increased in the SAM/NASH group. These results suggest that liver-derived TNF-α might promote muscle atrophy associated with steatohepatitis and aging through Murf-1. The metabolomic analysis of skeletal muscle indicated higher spermidine and lower tryptophan levels in the NASH-diet group. The findings of this study revealed an aspect of liver-muscle interaction, which might be important in developing treatments for sarcopenia associated with liver diseases.

Nano delivery of simvastatin targets liver sinusoidal endothelial cells to remodel tumor microenvironment for hepatocellular carcinoma

Background Hepatocellular carcinoma (HCC) developed in fibrotic liver does not respond well to immunotherapy, mainly due to the stromal microenvironment and the fibrosis-related immunosuppressive factors. The characteristic of liver sinusoidal endothelial cells (LSECs) in contributing to fibrosis and orchestrating immune response is responsible for the refractory to targeted therapy or immunotherapy of HCC. We aim to seek a new strategy for HCC treatment based on an old drug simvastatin which shows protecting effect on LSEC. Method The features of LSECs in mouse fibrotic HCC model and human HCC patients were identified by immunofluorescence and scanning electron microscopy. The effect of simvastatin on LSECs and hepatic stellate cells (HSCs) was examined by immunoblotting, quantitative RT-PCR and RNA-seq. LSEC-targeted delivery of simvastatin was designed using nanotechnology. The anti-HCC effect and toxicity of the nano-drug was evaluated in both intra-hepatic and hemi-splenic inoculated mouse fibrotic HCC model. Results LSEC capillarization is associated with fibrotic HCC progression and poor survival in both murine HCC model and HCC patients. We further found simvastatin restores the quiescence of activated hepatic stellate cells (aHSCs) via stimulation of KLF2-NO signaling in LSECs, and up-regulates the expression of CXCL16 in LSECs. In intrahepatic inoculated fibrotic HCC mouse model, LSEC-targeted nano-delivery of simvastatin not only alleviates LSEC capillarization to regress the stromal microenvironment, but also recruits natural killer T (NKT) cells through CXCL16 to suppress tumor progression. Together with anti-programmed death-1-ligand-1 (anti-PD-L1) antibody, targeted-delivery of simvastatin achieves an improved therapeutic effect in hemi-splenic inoculated advanced-stage HCC model. Conclusions These findings reveal an immune-based therapeutic mechanism of simvastatin for remodeling immunosuppressive tumor microenvironment, therefore providing a novel strategy in treating HCC. Graphical Abstract

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.

METTL3 facilitates hepatic fibrosis progression via m6A-YTHDF2 dependent silencing of GPR161

Hepatic fibrosis (HF) is a very common condition seen in millions of patients with various liver diseases. N6-methyladenosine (m6A) plays critical roles in various biological and pathological processes. However, the role of m6A and its main methyltransferase METTL3 in HF remains obscure. Here, we reported that METTL3 expression was elevated in HSCs from CCl4 induced fibrotic liver. METTL3 knockdown in HSCs mediated by recombinant adeno-associated-virus serotype 9 packed short hairpin RNA against METTL3 alleviated liver injury and fibrosis compared to empty carrier group. Mechanistically, the decreased liver fibrosis in CCl4-treated HSC-specific METTL3 knockdown mice was due to the increased GPR161 that is a suppressor of Hedgehog pathway, a well-known pathway to activate in liver injury and regeneration. As expect, GPR161 transferred into HSCs alleviated liver fibrosis and HSC activation. Forced GPR161 expression inhibited Gli3 activated form nuclear accumulation and subsequently suppressed fibrosis-associate gene expression. Conclusion, HSC-specific deletion of METTL3 inhibits liver fibrosis via elevated GPR161 expression, which subsequently suppressed Hedgehog pathway activation and fibrosis-associated genes expression, providing novel therapeutic targets for HF therapy.

The role of Mesothelin signaling in Portal Fibroblasts in the pathogenesis of cholestatic liver fibrosis

Liver fibrosis develops in response to chronic toxic or cholestatic injury, and is characterized by apoptosis of damaged hepatocytes, development of inflammatory responses, and activation of Collagen Type I producing myofibroblasts that make liver fibrotic. Two major cell types, Hepatic Stellate Cells (HSCs) and Portal Fibroblasts (PFs) are the major source of hepatic myofibroblasts. Hepatotoxic liver injury activates Hepatic Stellate Cells (aHSCs) to become myofibroblasts, while cholestatic liver injury activates both aHSCs and Portal Fibroblasts (aPFs). aPFs comprise the major population of myofibroblasts at the onset of cholestatic injury, while aHSCs are increasingly activated with fibrosis progression. Here we summarize our current understanding of the role of aPFs in the pathogenesis of cholestatic fibrosis, their unique features, and outline the potential mechanism of targeting aPFs in fibrotic liver.

Recovery from Liver Failure and Fibrosis in a Rat Portacaval Anastomosis Model after Neurointermediate Pituitary Lobectomy

Liver diseases, including cirrhosis, viral hepatitis, and hepatocellular carcinoma, account for approximately two million annual deaths worldwide. They place a huge burden on the global healthcare systems, compelling researchers to find effective treatment for liver fibrosis-cirrhosis. Portacaval anastomosis (PCA) is a model of liver damage and fibrosis. Arginine vasopressin (AVP) has been implicated as a proinflammatory-profibrotic hormone. In rats, neurointermediate pituitary lobectomy (NIL) induces a permanent drop (80%) in AVP serum levels. We hypothesized that AVP deficiency (NIL-induced) may decrease liver damage and fibrosis in a rat PCA model. Male Wistar rats were divided into intact control (IC), NIL, PCA, and PCA+NIL groups. Liver function tests, liver gene relative expressions (IL-1, IL-10, TGF-β, COLL-I, MMP-9, and MMP-13), and histopathological assessments were performed. In comparison with those in the IC and PCA groups, bilirubin, protein serum, and liver glycogen levels were restored in the PCA+NIL group. NIL in the PCA animals also decreased the gene expression levels of IL-1 and COLL-I, while increasing those of IL-10, TGF-β, and MMP-13. Histopathology of this group also showed significantly decreased signs of liver damage with lower extent of collagen deposition and fibrosis. Low AVP serum levels were not enough to fully activate the AVP receptors resulting in the decreased activation of cell signaling pathways associated with proinflammatory-profibrotic responses, while activating cell molecular signaling pathways associated with an anti-inflammatory-fibrotic state. Thus, partial reversion of liver damage and fibrosis was observed. The study supports the crucial role of AVP in the inflammatory-fibrotic processes and maintenance of immune competence. The success of the AVP deficiency strategy suggests that blocking AVP receptors may be therapeutically useful to treat inflammatory-fibrotic liver diseases.

Pan-lysyl oxidase inhibitor PXS-5505 ameliorates multiple-organ fibrosis by inhibiting collagen crosslinks in rodent models of systemic sclerosis

Background/Purpose: Systemic sclerosis (SSc) is characterised by progressive multiple-organ fibrosis leading to morbidity and mortality. Lysyl oxidases play a vital role in the cross-linking of collagens and subsequent build-up of fibrosis in the extracellular matrix. As such, their inhibition provides a novel treatment paradigm for SSc. Experimental Approach: Lysyl oxidases are upregulated in preclinical models of fibrosis in skin, lung, heart, kidney and liver. A novel small molecule pan-lysyl oxidase inhibitor, PXS-5505, currently in clinical development for bone fibrosis treatment was evaluated in in vivo rodent models resembling the fibrotic conditions in SSc. Key Results: Both lysyl oxidase and lysyl oxidase-like 2 (LOXL2) expression was elevated in the skin and lung of SSc patients. Once-a-day oral application of PXS-5505 inhibited lysyl oxidase activity in the skin and LOXL2 activity in the lung. PXS-5505 exhibited anti-fibrotic effects in the SSc skin mouse model, reducing dermal thickness and α-smooth muscle actin compared to the disease controls. Similarly, in the bleomycin-induced mouse lung model, PXS-5505 reduced tissue fibrosis toward normal levels. The anti-fibrotic efficacy of PXS-5505 in the bleomycin exposed lungs was mediated by its ability to normalise collagen/elastin crosslink formation, a direct consequence of lysyl oxidase inhibition. PXS-5505 also reduced area of fibrosis in rodent models of the ischaemia-reperfusion heart, the unilateral ureteral obstruction kidney and the CCl4-induced fibrotic liver. Conclusion/Implication: PXS-5505 consistently demonstrates potent anti-fibrotic efficacy in multiple models of organ fibrosis relevant to the pathogenesis of SSc, suggesting that it may be efficacious as a novel approach for treating SSc.

Targeting SYK of monocyte-derived macrophages regulates liver fibrosis via crosstalking with Erk/Hif1α and remodeling liver inflammatory environment

Liver fibrosis is a danger signal indicating a huge risk of liver cancer occurrence, but there is still no effective clinical means to regulate the progress of liver fibrosis. Although a variety of drugs targeting SYK have been developed for tumors and autoimmune diseases, the mechanism and specific efficacy of SYK’s role in liver fibrosis are not yet clear. Our studies based on chronic CCL4, bile duct ligation, and subacute TAA mouse models show that SYK in monocyte-derived macrophages (MoMFs) is fully dependent on phosphorylation of Erk to up-regulate the expression of Hif1α, thereby forming the crosstalk with SYK to drive liver fibrosis progress. We have evaluated the ability of the small molecule SYK inhibitor GS9973 in a variety of models. Contrary to previous impressions, high-frequency administration of GS9973 will aggravate CCL4-induced liver fibrosis, which is especially unsuitable for patients with cholestasis whose clinical features are bile duct obstruction. In addition, we found that inhibition of MoMFs SYK impairs the expression of CXCL1, on one hand, it reduces the recruitment of CD11bhiLy6Chi inflammatory cells, and on the other hand, it promotes the phenotype cross-dress process of pro-resolution MoMFs, thereby remodeling the chronic inflammatory environment of the fibrotic liver. Our further findings indicate that on the basis of the administration of CCR2/CCR5 dual inhibitor Cenicriviroc, further inhibiting MoMFs SYK may give patients with fibrosis additional benefits.