GAS6/TAM Axis as Therapeutic Target in Liver Diseases.

TAM (TYRO3, AXL, and MERTK) protein tyrosine kinase membrane receptors and their vitamin K-dependent ligands GAS6 and protein S (PROS) are well-known players in tumor biology and autoimmune diseases. In contrast, TAM regulation of fibrogenesis and the inflammation mechanisms underlying metabolic dysfunction-associated steatohepatitis (MASH), cirrhosis, and, ultimately, liver cancer has recently been revealed. GAS6 and PROS binding to phosphatidylserine exposed in outer membranes of apoptotic cells links TAMs, particularly MERTK, with hepatocellular damage. In addition, AXL and MERTK regulate the development of liver fibrosis and inflammation in chronic liver diseases. Acute hepatic injury is also mediated by the TAM system, as recent data regarding acetaminophen toxicity and acute-on-chronic liver failure have uncovered. Soluble TAM-related proteins, mainly released from activated macrophages and hepatic stellate cells after hepatic deterioration, are proposed as early serum markers for disease progression. In conclusion, the TAM system is becoming an interesting pharmacological target in liver pathology and a focus of future biomedical research in this field.

Relevance of SIRT1-NF-κB Axis as Therapeutic Target to Ameliorate Inflammation in Liver Disease.

Inflammation is an adaptive response in pursuit of homeostasis reestablishment triggered by harmful conditions or stimuli, such as an infection or tissue damage. Liver diseases cause approximately 2 million deaths per year worldwide and hepatic inflammation is a common factor to all of them, being the main driver of hepatic tissue damage and causing progression from non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH), cirrhosis and, ultimately, hepatocellular carcinoma (HCC). The metabolic sensor SIRT1, a class III histone deacetylase with strong expression in metabolic tissues such as the liver, and transcription factor NF-κB, a master regulator of inflammatory response, show an antagonistic relationship in controlling inflammation. For this reason, SIRT1 targeting is emerging as a potential strategy to improve different metabolic and/or inflammatory pathologies. In this review, we explore diverse upstream regulators and some natural/synthetic activators of SIRT1 as possible therapeutic treatment for liver diseases.

Ileal FGF15 contributes to fibrosis-associated hepatocellular carcinoma development.

Fibroblast growth factor 15 (FGF15), FGF19 in humans, is a gut-derived hormone and a key regulator of bile acids and carbohydrate metabolism. FGF15 also participates in liver regeneration after partial hepatectomy inducing hepatocellular proliferation. FGF19 is overexpressed in a significant proportion of human hepatocellular carcinomas (HCC), and activation of its receptor FGFR4 promotes HCC cell growth. Here we addressed for the first time the role of endogenous Fgf15 in hepatocarcinogenesis. Fgf15(+/+) and Fgf15(-/-) mice were subjected to a clinically relevant model of liver inflammation and fibrosis-associated carcinogenesis. Fgf15(-/-) mice showed less and smaller tumors, and histological neoplastic lesions were also smaller than in Fgf15(+/+) animals. Importantly, ileal Fgf15 mRNA expression was enhanced in mice undergoing carcinogenesis, but at variance with human HCC it was not detected in liver or HCC tissues, while circulating FGF15 protein was clearly upregulated. Hepatocellular proliferation was also reduced in Fgf15(-/-) mice, which also expressed lower levels of the HCC marker alpha-fetoprotein (AFP). Interestingly, lack of FGF15 resulted in attenuated fibrogenesis. However, in vitro experiments showed that liver fibrogenic stellate cells were not direct targets for FGF15/FGF19. Conversely we demonstrate that FGF15/FGF19 induces the expression of the pro-fibrogenic and pro-tumorigenic connective tissue growth factor (CTGF) in hepatocytes. These findings suggest the existence of an FGF15-triggered CTGF-mediated paracrine action on stellate cells, and an amplification mechanism for the hepatocarcinogenic effects of FGF15 via CTGF production. In summary, our observations indicate that ileal FGF15 may contribute to HCC development in a context of chronic liver injury and fibrosis.

Gas6/Axl pathway is activated in chronic liver disease and its targeting reduces fibrosis via hepatic stellate cell inactivation.

BACKGROUND & AIMS: Liver fibrosis, an important health concern associated to chronic liver injury that provides a permissive environment for cancer development, is characterized by accumulation of extracellular matrix components mainly derived from activated hepatic stellate cells (HSCs). Axl, a receptor tyrosine kinase and its ligand Gas6, are involved in cell differentiation, immune response and carcinogenesis. METHODS: HSCs were obtained from WT and Axl(-/-) mice, treated with recombinant Gas6 protein (rGas6), Axl siRNAs or the Axl inhibitor BGB324, and analyzed by western blot and real-time PCR. Experimental fibrosis was studied in CCl4-treated WT and Axl(-/-) mice, and in combination with Axl inhibitor. Gas6 and Axl serum levels were measured in alcoholic liver disease (ALD) and hepatitis C virus (HCV) patients. RESULTS: In primary mouse HSCs, Gas6 and Axl levels paralleled HSC activation. rGas6 phosphorylated Axl and AKT prior to HSC phenotypic changes, while Axl siRNA silencing reduced HSC activation. Moreover, BGB324 blocked Axl/AKT phosphorylation and diminished HSC activation. In addition, Axl(-/-) mice displayed decreased HSC activation in vitro and liver fibrogenesis after chronic damage by CCl4 administration. Similarly, BGB324 reduced collagen deposition and CCl4-induced liver fibrosis in mice. Importantly, Gas6 and Axl serum levels increased in ALD and HCV patients, inversely correlating with liver functionality. CONCLUSIONS: The Gas6/Axl axis is required for full HSC activation. Gas6 and Axl serum levels increase in parallel to chronic liver disease progression. Axl targeting may be a therapeutic strategy for liver fibrosis management.

Mitochondrial glutathione: features, regulation and role in disease.

BACKGROUND: Mitochondria are the powerhouse of mammalian cells and the main source of reactive oxygen species (ROS) associated with oxygen consumption. In addition, they also play a strategic role in controlling the fate of cells through regulation of death pathways. Mitochondrial ROS production fulfills a signaling role through regulation of redox pathways, but also contributes to mitochondrial damage in a number of pathological states. SCOPE OF REVIEW: Mitochondria are exposed to the constant generation of oxidant species, and yet the organelle remains functional due to the existence of an armamentarium of antioxidant defense systems aimed to repair oxidative damage, of which mitochondrial glutathione (mGSH) is of particular relevance. Thus, the aim of the review is to cover the regulation of mGSH and its role in disease. MAJOR CONCLUSIONS: Cumulating evidence over recent years has demonstrated the essential role for mGSH in mitochondrial physiology and disease. Despite its high concentration in the mitochondrial matrix, mitochondria lack the enzymes to synthesize GSH de novo, so that mGSH originates from cytosolic GSH via transport through specific mitochondrial carriers, which exhibit sensitivity to membrane dynamics. Depletion of mGSH sensitizes cells to stimuli leading to oxidative stress such as TNF, hypoxia or amyloid ß-peptide, thereby contributing to disease pathogenesis. GENERAL SIGNIFICANCE: Understanding the regulation of mGSH may provide novel insights to disease pathogenesis and toxicity and the opportunity to design therapeutic targets of intervention in cell death susceptibility and disease. This article is part of a Special Issue entitled Cellular functions of glutathione.

Induction of the Inflammasome Pathway by Tyrosine Kinase Inhibitors Provides an Actionable Therapeutic Target for Hepatocellular Carcinoma.

During the last decade, tyrosine kinase inhibitors (TKIs) sorafenib and regorafenib have been standard systemic treatments for advanced hepatocellular carcinoma (HCC). Previous data associated sorafenib with inflammasome activation. However, the role of the inflammasome in sorafenib and regorafenib signaling has not been described in liver cancer patients. For this purpose, we analyzed inflammasome-related transcriptomic changes in a murine HCC model. Our data confirmed inflammasome activation after both TKI treatments, sharing a similar pattern of increased gene expression. According to human database results, transcriptional increase of inflammasome genes is associated with poorer prognosis for male liver cancer patients, suggesting a sex-dependent role for inflammasome activation in HCC therapy. In biopsies of HCC and its surrounding tissue, we detected durable increases in the inflammasome activation pattern after sorafenib or regorafenib treatment in male patients. Further supporting its involvement in sorafenib action, inflammasome inhibition (MCC950) enhanced sorafenib anticancer activity in experimental HCC models, while no direct in vitro effect was observed in HCC cell lines. Moreover, activated human THP-1 macrophages released IL-1ß after sorafenib administration, while 3D Hep3B spheres displayed increased tumor growth after IL-1ß addition, pointing to the liver microenvironment as a key player in inflammasome action. In summary, our results unveil the inflammasome pathway as an actionable target in sorafenib or regorafenib therapy and associate an inflammasome signature in HCC and surrounding tissue with TKI administration. Therefore, targeting inflammasome activation, principally in male patients, could help to overcome sorafenib or regorafenib resistance and enhance the efficacy of TKI treatments in HCC.

Dynamic changes in immune cell populations by AXL kinase targeting diminish liver inflammation and fibrosis in experimental MASH.

Background and aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant health concern with limited treatment options. AXL, a receptor tyrosine kinase activated by the GAS6 ligand, promotes MASH through activation of hepatic stellate cells and inflammatory macrophages. This study identified cell subsets affected by MASH progression and the effect of AXL inhibition. Methods: Mice were fed chow or different fat-enriched diets to induce MASH, and small molecule AXL kinase inhibition with bemcentinib was evaluated. Gene expression was measured by qPCR. Time-of-flight mass cytometry (CyTOF) used single cells from dissociated livers, acquired on the Fluidigm Helios, and cell populations were studied using machine learning. Results: In mice fed different fat-enriched diets, liver steatosis alone was insufficient to elevate plasma soluble AXL (sAXL) levels. However, in conjunction with inflammation, sAXL increases, serving as an early indicator of steatohepatitis progression. Bemcentinib, an AXL inhibitor, effectively reduced proinflammatory responses in MASH models, even before fibrosis appearance. Utilizing CyTOF analysis, we detected a decreased population of Kupffer cells during MASH while promoting infiltration of monocytes/macrophages and CD8+ T cells. Bemcentinib partially restored Kupffer cells, reduced pDCs and GzmB- NK cells, and increased GzmB+CD8+ T cells and LSECs. Additionally, AXL inhibition enhanced a subtype of GzmB+CD8+ tissue-resident memory T cells characterized by CX3CR1 expression. Furthermore, bemcentinib altered the transcriptomic landscape associated with MASH progression, particularly in TLR signaling and inflammatory response, exhibiting differential cytokine expression in the plasma, consistent with liver repair and decreased inflammation. Conclusion: Our findings highlight sAXL as a biomarker for monitoring MASH progression and demonstrate that AXL targeting shifted liver macrophages and CD8+ T-cell subsets away from an inflammatory phenotype toward fibrotic resolution and organ healing, presenting a promising strategy for MASH treatment.

Mitochondrial cholesterol: a connection between caveolin, metabolism, and disease.

Caveolin (CAV) is an essential component of caveolae, cholesterol-enriched invaginations of the plasma membrane of most mammalian cells. However, CAV is not restricted to plasma membrane caveolae, and pools of CAV are present in myriad intracellular membranes. CAV proteins tightly bind cholesterol and contribute to regulation of cholesterol fluxes and distributions within cells. In this context, we recently showed that CAV1 regulates the poorly understood process controlling mitochondrial cholesterol levels. Cholesterol accumulates in mitochondrial membranes in the absence of CAV1, promoting the organelle's dysfunction with important metabolic consequences for cells and animals. In this article, we suggest a working hypothesis that addresses the role of CAV1 within the homeostatic network that regulates the influx/efflux of mitochondrial cholesterol.

Cathepsin B overexpression due to acid sphingomyelinase ablation promotes liver fibrosis in Niemann-Pick disease.

Niemann-Pick disease (NPD) is a lysosomal storage disease caused by the loss of acid sphingomyelinase (ASMase) that features neurodegeneration and liver disease. Because ASMase-knock-out mice models NPD and our previous findings revealed that ASMase activates cathepsins B/D (CtsB/D), our aim was to investigate the expression and processing of CtsB/D in hepatic stellate cells (HSCs) from ASMase-null mice and their role in liver fibrosis. Surprisingly, HSCs from ASMase-knock-out mice exhibit increased basal level and activity of CtsB as well as its in vitro processing in culture, paralleling the enhanced expression of fibrogenic markers α-smooth muscle actin (α-SMA), TGF-ß, and pro-collagen-α1(I) (Col1A1). Moreover, pharmacological inhibition of CtsB blunted the expression of α-SMA and Col1A1 and proliferation of HSCs from ASMase-knock-out mice. Consistent with the enhanced activation of CtsB in HSCs from ASMase-null mice, the in vivo liver fibrosis induced by chronic treatment with CCl(4) increased in ASMase-null compared with wild-type mice, an effect that was reduced upon CtsB inhibition. In addition to liver, the enhanced proteolytic processing of CtsB was also observed in brain and lung of ASMase-knock-out mice, suggesting that the overexpression of CtsB may underlie the phenotype of NPD. Thus, these findings reveal a functional relationship between ASMase and CtsB and that the ablation of ASMase leads to the enhanced processing and activation of CtsB. Therefore, targeting CtsB may be of relevance in the treatment of liver fibrosis in patients with NPD.

Impaired liver regeneration in Ldlr-/- mice is associated with an altered hepatic profile of cytokines, growth factors, and lipids.

BACKGROUND & AIMS: It is widely recognized that in the early stages of liver regeneration after partial hepatectomy, the hepatocytes accumulate a significant amount of lipids. The functional meaning of this transient steatosis and its effect on hepatocellular proliferation are not well defined. In addition, the basic mechanisms of this lipid accumulation are not well understood although some studies suggest the participation of the Low Density Lipoprotein Receptor (Ldlr). METHODS: To address these questions, we studied the process of liver regeneration in Ldlr null mice and wild type mice following partial hepatectomy. RESULTS: Ldlr deficiency was associated with a significant decrease in serum albumin concentration, during early stages of liver regeneration, and a delayed hepatic regeneration. Remnant livers of Ldlr(-)(/)(-) showed a time-shifted expression of interleukin-6 (IL6) and a defective activation of tumor necrosis factor-α (TNFα) and hepatocyte growth factor (HGF) expression in early phases of liver regeneration. Unexpectedly, Ldlr(-)(/)(-) showed no significant differences in the content of lipid droplets after partial hepatectomy compared to wild type mice. However, lipidomic analysis of the regenerating liver from Ldlr(-)(/)(-) revealed a lipid profile compatible with liver quiescence: high content of cholesterol esters and ceramide, and low levels of phosphatidylcholine. CONCLUSIONS: Ldlr deficiency is associated with significant changes in the hepatic lipidome that affect cytokine-growth factor signaling and impair liver regeneration. These results suggest that the analysis of the hepatic lipidome may help predict the success of liver regeneration in the clinical environment, specifically in the context of pre-existing liver steatosis.

ASMase is required for chronic alcohol induced hepatic endoplasmic reticulum stress and mitochondrial cholesterol loading.

BACKGROUND & AIMS: The pathogenesis of alcohol-induced liver disease (ALD) is poorly understood. Here, we examined the role of acid sphingomyelinase (ASMase) in alcohol induced hepatic endoplasmic reticulum (ER) stress, a key mechanism of ALD. METHODS: We examined ER stress, lipogenesis, hyperhomocysteinemia, mitochondrial cholesterol (mChol) trafficking and susceptibility to LPS and concanavalin-A in ASMase(-)(/-) mice fed alcohol. RESULTS: Alcohol feeding increased SREBP-1c, DGAT-2, and FAS mRNA in ASMase(+/+) but not in ASMase(-/-) mice. Compared to ASMase(+/+) mice, ASMase(-/-) mice exhibited decreased expression of ER stress markers induced by alcohol, but the level of tunicamycin-mediated upregulation of ER stress markers and steatosis was similar in both types of mice. The increase in homocysteine levels induced by alcohol feeding was comparable in both ASMase(+/+) and ASMase(-/-) mice. Exogenous ASMase, but not neutral SMase, induced ER stress by perturbing ER Ca(2+) homeostasis. Moreover, alcohol-induced mChol loading and StARD1 overexpression were blunted in ASMase(-/-) mice. Tunicamycin upregulated StARD1 expression and this outcome was abrogated by tauroursodeoxycholic acid. Alcohol-induced liver injury and sensitization to LPS and concanavalin-A were prevented in ASMase(-/-) mice. These effects were reproduced in alcohol-fed TNFR1/R2(-/-) mice. Moreover, ASMase does not impair hepatic regeneration following partial hepatectomy. Of relevance, liver samples from patients with alcoholic hepatitis exhibited increased expression of ASMase, StARD1, and ER stress markers. CONCLUSIONS: Our data indicate that ASMase is critical for alcohol-induced ER stress, and provide a rationale for further clinical investigation in ALD.

Inflammasome activation under high cholesterol load triggers a protective microglial phenotype while promoting neuronal pyroptosis.

BACKGROUND: Persistent inflammatory response in the brain can lead to tissue damage and neurodegeneration. In Alzheimer's disease (AD), there is an aberrant activation of inflammasomes, molecular platforms that drive inflammation through caspase-1-mediated proteolytic cleavage of proinflammatory cytokines and gasdermin D (GSDMD), the executor of pyroptosis. However, the mechanisms underlying the sustained activation of inflammasomes in AD are largely unknown. We have previously shown that high brain cholesterol levels promote amyloid-ß (Aß) accumulation and oxidative stress. Here, we investigate whether these cholesterol-mediated changes may regulate the inflammasome pathway. METHODS: SIM-A9 microglia and SH-SY5Y neuroblastoma cells were cholesterol-enriched using a water-soluble cholesterol complex. After exposure to lipopolysaccharide (LPS) plus muramyl dipeptide or Aß, activation of the inflammasome pathway was analyzed by immunofluorescence, ELISA and immunoblotting analysis. Fluorescently-labeled Aß was employed to monitor changes in microglia phagocytosis. Conditioned medium was used to study how microglia-neuron interrelationship modulates the inflammasome-mediated response. RESULTS: In activated microglia, cholesterol enrichment promoted the release of encapsulated IL-1ß accompanied by a switch to a more neuroprotective phenotype, with increased phagocytic capacity and release of neurotrophic factors. In contrast, in SH-SY5Y cells, high cholesterol levels stimulated inflammasome assembly triggered by both bacterial toxins and Aß peptides, resulting in GSDMD-mediated pyroptosis. Glutathione (GSH) ethyl ester treatment, which recovered the cholesterol-mediated depletion of mitochondrial GSH levels, significantly reduced the Aß-induced oxidative stress in the neuronal cells, resulting in lower inflammasome activation and cell death. Furthermore, using conditioned media, we showed that neuronal pyroptosis affects the function of the cholesterol-enriched microglia, lowering its phagocytic activity and, therefore, the ability to degrade extracellular Aß. CONCLUSIONS: Changes in intracellular cholesterol levels differentially regulate the inflammasome-mediated immune response in microglia and neuronal cells. Given the microglia-neuron cross-talk in the brain, cholesterol modulation should be considered a potential therapeutic target for AD treatment, which may help to block the aberrant and chronic inflammation observed during the disease progression.

Sex-based differences in natural killer T cell-mediated protection against diet-induced steatohepatitis in Balb/c mice.

BACKGROUND: Metabolic dysfunction-associated steatotic liver disease (MASLD) is prevalent in Western countries, evolving into metabolic dysfunction-associated steatohepatitis (MASH) with a sexual dimorphism. Fertile women exhibit lower MASLD risk than men, which diminishes post-menopause. While NKT-cell involvement in steatohepatitis is debated, discrepancies may stem from varied mouse strains used, predominantly C57BL6/J with Th1-dominant responses. Exploration of steatohepatitis, encompassing both genders, using Balb/c background, with Th2-dominant immune response, and CD1d-deficient mice in the Balb/c background (lacking Type I and Type II NKT cells) can clarify gender disparities and NKT-cell influence on MASH progression. METHODS: A high fat and choline-deficient (HFCD) diet was used in male and female mice, Balb/c mice or CD1d-/- mice in the Balb/c background that exhibit a Th2-dominant immune response. Liver fibrosis and inflammatory gene expression were measured by qPCR, and histology assessment. NKT cells, T cells, macrophages and neutrophils were assessed by flow cytometry. RESULTS: Female mice displayed milder steatohepatitis after 6 weeks of HFCD, showing reduced liver damage, inflammation, and fibrosis compared to males. Male Balb/c mice exhibited NKT-cell protection against steatohepatitis whereas CD1d-/- males on HFCD presented decreased hepatoprotection, increased liver fibrosis, inflammation, neutrophilic infiltration, and inflammatory macrophages. In contrast, the NKT-cell role was negligible in early steatohepatitis development in both female mice, as fibrosis and inflammation were similar despite augmented liver damage in CD1d-/- females. Relevant, hepatic type I NKT levels in female Balb/c mice were significantly lower than in male. CONCLUSIONS: NKT cells exert a protective role against experimental steatohepatitis as HFCD-treated CD1d-/- males had more severe fibrosis and inflammation than male Balb/c mice. In females, the HFCD-induced hepatocellular damage and the immune response are less affected by NKT cells on early steatohepatitis progression, underscoring sex-specific NKT-cell influence in MASH development.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a common liver condition today. In its more advanced form, called metabolic dysfunction-associated steatohepatitis (MASH), adult men are more often affected than women, though this difference vanishes after menopause. Various factors contribute to MASH, including a specific immune cell type called NKT cells, which has not been deeply researched yet. To explore the role of NKT cells in steatohepatitis, we used male and female mice with or without NKT cells (CD1d^−/− mice), feeding them a high-fat diet that induces steatohepatitis. Our findings revealed that female mice had less severe steatohepatitis compared to males. Interestingly, we observed a protective role of NKT cells during steatohepatitis, as male mice without these cells had more damage, inflammation, and fibrosis than those with NKT cells. However, in females, even though those lacking NKT cells showed more liver damage and immune alterations, NKT did not seem to play a major role in early steatohepatitis progression. Notably, females had much fewer NKT cells in their livers compared to males, possibly explaining this difference. In conclusion, NKT cells seem to slow down steatohepatitis progression, especially in male mice. In females, their impact on early steatohepatitis advance appears more limited.

Critical role of tumor necrosis factor receptor 1, but not 2, in hepatic stellate cell proliferation, extracellular matrix remodeling, and liver fibrogenesis.

UNLABELLED: Tumor necrosis factor (TNF) has been implicated in the progression of many chronic liver diseases leading to fibrosis; however, the role of TNF in fibrogenesis is controversial and the specific contribution of TNF receptors to hepatic stellate cell (HSC) activation remains to be established. Using HSCs from wild-type, TNF-receptor-1 (TNFR1) knockout, TNF-receptor-2 (TNFR2) knockout, or TNFR1/R2 double-knockout (TNFR-DKO) mice, we show that loss of both TNF receptors reduced procollagen-α1(I) expression, slowed down HSC proliferation, and impaired platelet-derived growth factor (PDGF)-induced promitogenic signaling in HSCs. TNFR-DKO HSCs exhibited decreased AKT phosphorylation and in vitro proliferation in response to PDGF. These effects were reproduced in TNFR1 knockout, but not TNFR2 knockout, HSCs. In addition, matrix metalloproteinase 9 (MMP-9) expression was dependent on TNF binding to TNFR1 in primary mouse HSCs. These results were validated in the human HSC cell line, LX2, using neutralizing antibodies against TNFR1 and TNFR2. Moreover, in vivo liver damage and fibrogenesis after bile-duct ligation were reduced in TNFR-DKO and TNFR1 knockout mice, compared to wild-type or TNFR2 knockout mice. CONCLUSION: TNF regulates HSC biology through its binding to TNFR1, which is required for HSC proliferation and MMP-9 expression. These data indicate a regulatory role for TNF in extracellular matrix remodeling and liver fibrosis, suggesting that targeting TNFR1 may be of benefit to attenuate liver fibrogenesis.

Hepatocellular Carcinoma: Molecular Pathogenesis and Therapeutic Advances.

Hepatocellular carcinoma (HCC), the most common form of liver cancer, continues to be a serious medical problem with poor prognosis, without major therapeutic improvement for years and increasing incidence. Fortunately, advances in systemic treatment options are finally arriving for HCC patients. After a decade of sorafenib as a standard therapy for advanced HCC, several tyrosine kinase inhibitors (TKIs), antiangiogenic antibodies, and immune checkpoint inhibitors have reached the clinic. Although infections by hepatitis B virus and hepatitis C virus remain principal factors for HCC development, the rise of non- alcoholic steatohepatitis from diabetes mellitus or metabolic syndrome is impeding HCC decline. Knowledge of specific molecular mechanisms, based on the etiology and the HCC microenvironment that influence tumor growth and immune control, will be crucial for physician decision-making among a variety of drugs to prescribe. In addition, markers of treatment efficacy are needed to speed the movement of patients towards other potentially effective treatments. Consequently, research to provide scientific data for the evidence-based management of liver cancer is guaranteed in the coming years and discussed here.

Mitochondrial free cholesterol loading sensitizes to TNF- and Fas-mediated steatohepatitis.

The etiology of progression from steatosis to steatohepatitis (SH) remains unknown. Using nutritional and genetic models of hepatic steatosis, we show that free cholesterol (FC) loading, but not free fatty acids or triglycerides, sensitizes to TNF- and Fas-induced SH. FC distribution in endoplasmic reticulum (ER) and plasma membrane did not cause ER stress or alter TNF signaling. Rather, mitochondrial FC loading accounted for the hepatocellular sensitivity to TNF due to mitochondrial glutathione (mGSH) depletion. Selective mGSH depletion in primary hepatocytes recapitulated the susceptibility to TNF and Fas seen in FC-loaded hepatocytes; its repletion rescued FC-loaded livers from TNF-mediated SH. Moreover, hepatocytes from mice lacking NPC1, a late endosomal cholesterol trafficking protein, or from obese ob/ob mice, exhibited mitochondrial FC accumulation, mGSH depletion, and susceptibility to TNF. Thus, we propose a critical role for mitochondrial FC loading in precipitating SH, by sensitizing hepatocytes to TNF and Fas through mGSH depletion.

Cholesterol and peroxidized cardiolipin in mitochondrial membrane properties, permeabilization and cell death.

Mitochondria are known to actively regulate cell death with the final phenotype of demise being determined by the metabolic and energetic status of the cell. Mitochondrial membrane permeabilization (MMP) is a critical event in cell death, as it regulates the degree of mitochondrial dysfunction and the release of intermembrane proteins that function in the activation and assembly of caspases. In addition to the crucial role of proapoptotic members of the Bcl-2 family, the lipid composition of the mitochondrial membranes is increasingly recognized to modulate MMP and hence cell death. The unphysiological accumulation of cholesterol in mitochondrial membranes regulates their physical properties, facilitating or impairing MMP during Bax and death ligand-induced cell death depending on the level of mitochondrial GSH (mGSH), which in turn regulates the oxidation status of cardiolipin. Cholesterol-mediated mGSH depletion stimulates TNF-induced reactive oxygen species and subsequent cardiolipin peroxidation, which destabilizes the lipid bilayer and potentiates Bax-induced membrane permeabilization. These data suggest that the balance of mitochondrial cholesterol to peroxidized cardiolipin regulates mitochondrial membrane properties and permeabilization, emerging as a rheostat in cell death.

Acidic sphingomyelinase controls hepatic stellate cell activation and in vivo liver fibrogenesis.

The mechanisms linking hepatocellular death, hepatic stellate cell (HSC) activation, and liver fibrosis are largely unknown. Here, we investigate whether acidic sphingomyelinase (ASMase), a known regulator of death receptor and stress-induced hepatocyte apoptosis, plays a role in liver fibrogenesis. We show that selective stimulation of ASMase (up to sixfold), but not neutral sphingomyelinase, occurs during the transdifferentiation/activation of primary mouse HSCs into myofibroblast-like cells, coinciding with cathepsin B (CtsB) and D (CtsD) processing. ASMase inhibition or genetic down-regulation by small interfering RNA blunted CtsB/D processing, preventing the activation and proliferation of mouse and human HSCs (LX2 cells). In accordance, HSCs from heterozygous ASMase mice exhibited decreased CtsB/D processing, as well as lower levels of alpha-smooth muscle actin expression and proliferation. Moreover, pharmacological CtsB inhibition reproduced the antagonism of ASMase in preventing the fibrogenic properties of HSCs, without affecting ASMase activity. Interestingly, liver fibrosis induced by bile duct ligation or carbon tetrachloride administration was reduced in heterozygous ASMase mice compared with that in wild-type animals, regardless of their sensitivity to liver injury in either model. To provide further evidence for the ASMase-CtsB pathway in hepatic fibrosis, liver samples from patients with nonalcoholic steatohepatitis were studied. CtsB and ASMase mRNA levels increased eight- and threefold, respectively, in patients compared with healthy controls. These findings illustrate a novel role of ASMase in HSC biology and liver fibrogenesis by regulating its downstream effectors CtsB/D.

Growth arrest-specific protein 6 is hepatoprotective against murine ischemia/reperfusion injury.

UNLABELLED: Growth arrest-specific gene 6 (GAS6) promotes growth and cell survival during tissue repair and development in different organs, including the liver. However, the specific role of GAS6 in liver ischemia/reperfusion (I/R) injury has not been previously addressed. Here we report an early increase in serum GAS6 levels after I/R exposure. Moreover, unlike wild-type (WT) mice, Gas6(-/-) mice were highly sensitive to partial hepatic I/R, with 90% of the mice dying within 12 hours of reperfusion because of massive hepatocellular injury. I/R induced early hepatic protein kinase B (AKT) phosphorylation in WT mice but not in Gas6(-/-) mice without significant changes in c-Jun N-terminal kinase phosphorylation or nuclear factor kappa B translocation, whereas hepatic interleukin-1ß (IL-1ß) and tumor necrosis factor (TNF) messenger RNA levels were higher in Gas6(-/-) mice versus WT mice. In line with the in vivo data, in vitro studies indicated that GAS6 induced AKT phosphorylation in primary mouse hepatocytes and thus protected them from hypoxia-induced cell death, whereas GAS6 diminished lipopolysaccharide-induced cytokine expression (IL-1ß and TNF) in murine macrophages. Finally, recombinant GAS6 treatment in vivo not only rescued GAS6 knockout mice from severe I/R-induced liver damage but also attenuated hepatic damage in WT mice after I/R. CONCLUSION: Our data have revealed GAS6 to be a new player in liver I/R injury that is emerging as a potential therapeutic target for reducing postischemic hepatic damage.