Fibrogenic Genes Research Articles

Inhibiting xCT/SLC7A11 induces ferroptosis of myofibroblastic hepatic stellate cells but exacerbates chronic liver injury

The outcome of liver injury is dictated by factors that control accumulation of myofibroblastic (activated) hepatic stellate cells (MF-HSCs) but therapies that specifically block this process have not been discovered. We evaluated the hypothesis that MF-HSCs and liver fibrosis could be safely reduced by inhibiting the cysteine/glutamate antiporter xCT. xCT activity was disrupted in both HSC lines and primary mouse HSCs to determine its effect on HSC biology. For comparison, xCT expression and function were also determined in primary mouse hepatocytes. Finally, the roles of xCT were assessed in mouse models of liver fibrosis. We found that xCT mRNA levels were almost a log-fold higher in primary mouse HSCs than in primary mouse hepatocytes. Further, primary mouse HSCs dramatically induced xCT as they became MF, and inhibiting xCT blocked GSH synthesis, reduced growth and fibrogenic gene expression and triggered HSC ferroptosis. Doses of xCT inhibitors that induced massive ferroptosis in HSCs had no effect on hepatocyte viability in vitro, and xCT inhibitors reduced liver fibrosis without worsening liver injury in mice with acute liver injury. However, TGFβ treatment up-regulated xCT and triggered ferroptosis in cultured primary mouse hepatocytes. During chronic liver injury, xCT inhibitors exacerbated injury, impaired regeneration and failed to improve fibrosis, confirming that HSCs and hepatocytes deploy similar mechanisms to survive chronic oxidative stress. Inhibiting xCT can suppress myofibroblastic activity and induce ferroptosis of MF-HSCs. However, targeting xCT inhibition to MF-HSCs will be necessary to exploit ferroptosis as an anti-fibrotic strategy.

Abstract SY19-01: TGFβ and immune evasion in metastatic cancer

Abstract TGF-β and Immune Evasion in Metastatic Cancer Transforming growthfactor-β (TGF-β) is a major regulator of development,immune homeostasis, tissue regeneration, and cancer. TGF-β signaling suppresses tumor initiation but promotestumor progression through mechanisms that are increasingly understood. TGF-β inhibitsthe expansion and function of many components of the immune system and itspresence in the tumor microenvironment limits the effectiveness of current immunotherapies. To betterdefine the potential benefit of targeting TGF-β in cancer, we investigated therole of TGF-β in metastasis. Metastasis, the main cause of deathfrom cancer, is driven by stem-like disseminated cancer cells (“metastasisinitiating cells”, MICs) capable of reversible quiescence, immune evasion, organ-specificoutgrowth, and drug resistance. We developed models oflung adenocarcinoma (LUAD) metastatic dormancy and outbreak by isolating MICs thatrecapitulate the developmental and regenerative phenotypes of carcinoma cellsin human metastases. We find that TGF-β in thetumor microenvironment has different tumorigenic effects on early-stageprogenitor MICs and their progenies. In SOX2-rich early progenitor MICs, TGF-βinduces dormancy by imposing a growth-inhibited epithelial to mesenchymaltransition (EMT). Pharmacologic inhibition of TGF-β in this context causes MICsto exit from this immune evasive dormancy and become exposed to immune attack. Incontrast, SOX9-rich MIC progeny cells respond to TGF-β with an EMT that iscoupled to expression of fibrogenic cytokines. Deletion of TGF-β receptor orits fibrogenic target genes in the carcinoma cells prevents intratumoralfibrosis and inhibits metastatic outgrowth. These insights suggest that targetingTGF-β in metastatic cancer can improve treatment outcomes by forcing MICs outof immune evasive quiescence and suppressing intra-tumoral fibrosis, inaddition to relieving innate and adaptive immunity from TGF-β mediated inhibition. Citation Format: Joan Massagué. TGFβ and immune evasion in metastatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr SY19-01.

Expression of fibrosis-related genes in liver allografts: Association with histology and long-term outcome after pediatric liver transplantation.

Unexplained graft fibrosis and inflammation are common after pediatric liver transplantation (LT). We investigated the graft expression of fibrogenic genes and correlated the findings with transplant histopathology and outcome. Liver biopsies from 29 recipients were obtained at a median of 13.1 (IQR: 5.0-18.4) years after pediatric LT. Control samples were from six liver-healthy subjects. Hepatic expression of 40 fibrosis-related genes was correlated to histological findings: normal histology, fibrosis with no inflammation, and fibrosis with inflammation. Liver function was evaluated after a subsequent follow-up of 9.0years (IQR: 8.0-9.4). Patients with fibrosis and no inflammation had significantly increased gene expression of profibrotic TGF-β3 (1.17 vs. 1.02 p=.005), CTGF (1.64 vs. 0.66 p=.014), PDGF-α (1.79 vs. 0.98 p=.049), PDGF -β (0.99 vs. 0.76 p=.006), integrin-subunit-β1 (1.19 vs. 1.02 p=.045), α-SMA (1.12 vs. 0.58 p=.013), type I collagen (0.82 vs. 0.53 p=.005) and antifibrotic decorin (1.15 vs. 0.99 p=.045) compared to patients with normal histology. mRNA expression of VEGF A (0.84 vs. 1.06 p=.049) was lower. Only a few of the studied genes were upregulated in patients with both fibrosis and inflammation. The gene expression levels showed no association with later graft outcome. Altered hepatic expression of fibrosis-related genes is associated with graft fibrosis without concurrent inflammation.

Inhibiting HDAC3 (Histone Deacetylase 3) Aberration and the Resultant Nrf2 (Nuclear Factor Erythroid-Derived 2-Related Factor-2) Repression Mitigates Pulmonary Fibrosis.

[Figure: see text].

Sugar kelp (Saccharina latissima) inhibits hepatic inflammation and fibrosis in a mouse model of diet-induced nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH), closely associated with obesity, is a health concern worldwide. We investigated whether the consumption of U.S.-grown sugar kelp (Saccharina latissima), an edible brown alga, can prevent obesity-associated metabolic disturbances and NASH in a mouse model of diet-induced NASH. Male C57BL/6J mice were fed a low-fat diet, a high-fat/high-sucrose/high-cholesterol diet (HF), or a HF diet containing sugar kelp (HF-Kelp) for 14 weeks. HF-Kelp group showed lower body weight with increased O2 consumption, CO2 production, physical activity, and energy expenditure compared with the HF. In the liver, there were significant decreases in weight, triglycerides, total cholesterol, and steatosis with HF-Kelp. The HF-Kelp group decreased hepatic expression of a macrophage marker adhesion G protein-coupled receptor E1 (Adgre1) and an M1 macrophage marker integrin alpha x (Itgax). HF-Kelp group also exhibited decreased liver fibrosis, as evidenced by less expression of fibrogenic genes and collagen accumulation than those of HF group. In epididymal white adipose tissue (eWAT), HF-Kelp group exhibited decreases in eWAT weight and adipocyte size compared with those of the HF. HF-Kelp group showed decreased expression of collagen type VI alpha 1 chain, Adgre1, Itgax, and tumor necrosis factor α in eWAT. We demonstrated, for the first time, that the consumption of U.S-grown sugar kelp prevented the development of obesity and its associated metabolic disturbances, steatosis, inflammation, and fibrosis in the liver and eWAT of a diet-induced NASH mouse model.

Physalin B attenuates liver fibrosis via suppressing LAP2α-HDAC1-mediated deacetylation of the transcription factor GLI1 and hepatic stellate cell activation.

Liver fibrosis is one of the leading causes of morbidity and mortality worldwide but lacks any acceptable therapy. The transcription factor glioma-associated oncogene homologue 1 (GLI1) is a potentially important therapeutic target in liver fibrosis. This study investigates the anti-fibrotic activities and potential mechanisms of the phytochemical, physalin B. Two mouse models (CCl4 challenge and bile duct ligation) were used to assess antifibrotic effects of physalin B in vivo. Mouse primary hepatic stellate cells (pHSCs) and human HSC line LX-2 also served as in vitro liver fibrosis models. Liver fibrogenic genes, GLI1 and GLI1 downstream genes were examined using Western blot and quantitative real-time PCR (qRT-PCR). GLI1 acetylation and LAP2α-HDAC1 interaction were analysed by co-immunoprecipitation. In vivo, physalin B administration attenuated hepatic histopathological injury and collagen accumulation and decreased expression of fibrogenic genes. Physalin B dose-dependently suppressed fibrotic marker expression in LX-2 cells and mouse pHSCs. Mechanistic studies showed that physalin B inhibited GLI activity by non-canonical Hedgehog signalling. Physalin B blocked formation of lamina-associated polypeptide 2α (LAP2α)/histone deacetylase 1 (HDAC1) complexes, thus inhibiting HDAC1-mediated GLI1 deacetylation. Physalin B up-regulated acetylation of GLI1, down-regulated expression of GLI1 and subsequently inhibited HSC activation. Physalin B exerted potent antifibrotic effects in vitro and in vivo by disrupting LAP2α/HDAC1 complexes, increasing GLI1 acetylation and inactivating GLI1. This indicates that the phytochemical physalin B may be a potential therapeutic candidate for the treatment of liver fibrosis.

Protective effect of LNA-anti-miR-132 therapy on liver fibrosis in mice

microRNAs (miRs) are small regulatory RNAs that are frequently deregulated in liver disease. Liver fibrosis is characterized by excessive scarring caused by chronic inflammatory processes. In this study, we determined the functional role of miR-132 using a locked nucleic acid (LNA)-anti-miR approach in liver fibrosis. A significant induction in miR-132 levels was found in mice treated with CCl4 and in patients with fibrosis/cirrhosis. Inhibition of miR-132 in mice with LNA-anti-miR-132 caused decreases in CCl4-induced fibrogenesis and inflammatory phenotype. An attenuation in collagen fibers, α SMA, MCP1, IL-1β, and Cox2 was found in LNA-anti-miR-132-treated mice. CCl4 treatment increased caspase 3 activity and extracellular vesicles (EVs) in control but not in anti-miR-132-treated mice. Inhibition of miR-132 was associated with augmentation of MMP12 in the liver and Kupffer cells. In vivo and in vitro studies suggest miR-132 targets SIRT1 and inflammatory genes. Using tumor cancer genome atlas data, an increase in miR-132 was found in hepatocellular carcinoma (HCC). Increased miR-132 levels were associated with fibrogenic genes, higher tumor grade and stage, and unfavorable survival in HCC patients. Therapeutic inhibition of miR-132 might be a new approach to alleviate liver fibrosis, and treatment efficacy can be monitored by observing EV shedding.

Impacts of n-acetyl cysteine on gibberellic acid-induced hepatorenal dysfunction through modulation of pro-inflammatory cytokines, antifibrotic and antioxidant activity.

The extensive usage of gibberellic acid (GA3) in agriculture and plant growth is generally associated with enormous human and public health hazards. The present research assesses the impact of n-acetyl cysteine (NAC) on the hepatorenal injury persuaded by GA3 for this purpose, After two weeks of adaptation twenty-four rats allocated into four groups (6 rats/group) as follows: control group, supplied with saline only; n-acetyl cysteine (NAC) group, provided with 150mg/kg/bw by stomach tube (orally) dissolved in saline; Positive GA3 group, received GA3 (55mg/kg/bw) orally; Protective group received NAC (150mg/kg/bw) and GA3 (55mg/kg/bw) as in NAC and GA3 groups. Rats received their treatments for consecutive 3weeks. On day 22, rats were anesthetized, then euthanized. Blood and tissue samples were obtained for biochemical, antioxidants markers analysis, gene expression, and histopathological examination. Our results revealed significant changes in serum AST, ALT, urea, uric acid, total protein, and albumin levels with a substantial rise of MDA and NO concentration in GA3 treated rats along with a considerable decrease of the GSH and overexpression of the inflammatory hepatic and renal cytokines (IL-10, TNF-α, NOS) and fibrotic gene expression TGF-β1, and α-SMA, with boost expression of nuclear factor-kappa (NFk B). NAC co-administered with GA3 significantly normalized the kidney and liver function and the antioxidant state, besides normal histological structure of both liver and kidney tissue and downregulated expression of the pro-inflammatory cytokines as well as, fibrogenic gene expression. PRACTICAL APPLICATIONS: The current study confirmed that GA3 induced hepto-renal dysfunction that was ameliorated by NAC administration. Moreover, our findings confirmed the antioxidant capability of n-acetyl cysteine and afford robust evidence about the ameliorative effect of the n-acetyl cysteine to attenuate the hepatorenal injury induced by gibberellic acid through modulation of the antioxidant defense system fibrogenic, and pro-inflammatory cytokines expression.

Coordinated signaling of activating transcription factor 6α and inositol-requiring enzyme 1α regulates hepatic stellate cell-mediated fibrogenesis in mice.

Liver injury and the unfolded protein response (UPR) are tightly linked, but their relationship differs with cell type and injurious stimuli. UPR initiation promotes hepatic stellate cell (HSC) activation and fibrogenesis, but the underlying mechanisms are unclear. Despite the complexity and overlap downstream of UPR transducers inositol-requiring protein 1α (IRE1α), activating transcription factor 6α (ATF6α), and protein kinase RNA-like ER kinase (PERK), previous research in HSCs primarily focused on IRE1α. Here, we investigated the fibrogenic role of ATF6α or PERK in vitro and HSC-specific UPR signaling in vivo. Overexpression of ATF6α, but not the PERK effector activating transcription factor 4 (ATF4), promoted HSC activation and fibrogenic gene transcription in immortalized HSCs. Furthermore, ATF6α inhibition through Ceapin-A7, or Atf6a deletion, disrupted transforming growth factor β (TGFβ)-mediated activation of primary human hepatic stellate cells (hHSCs) or murine hepatic stellate cells (mHSCs), respectively. We investigated the fibrogenic role of ATF6α in vivo through conditional HSC-specific Atf6a deletion. Atf6aHSCΔ/Δ mice displayed reduced fibrosis and HSC activation following bile duct ligation (BDL) or carbon tetrachloride (CCl4)-induced injury. The Atf6aHSCΔ/Δ phenotype differed from HSC-specific Ire1a deletion, as Ire1aHSCΔ/Δ mice showed reduced fibrogenic gene transcription but no changes in fibrosis compared with Ire1afl/fl mice following BDL. Interestingly, ATF6α signaling increased in Ire1aΔ/Δ HSCs, whereas IRE1α signaling was upregulated in Atf6aΔ/Δ HSCs. Finally, we asked whether co-deletion of Atf6a and Ire1a additively limits fibrosis. Unexpectedly, fibrosis worsened in Atf6aHSCΔ/ΔIre1aHSCΔ/Δ mice following BDL, and Atf6aΔ/ΔIre1aΔ/Δ mHSCs showed increased fibrogenic gene transcription. ATF6α and IRE1α individually promote fibrogenic transcription in HSCs, and ATF6α drives fibrogenesis in vivo. Unexpectedly, disruption of both pathways sensitizes the liver to fibrogenesis, suggesting that fine-tuned UPR signaling is critical for regulating HSC activation and fibrogenesis.NEW & NOTEWORTHY ATF6α is a critical driver of hepatic stellate cell (HSC) activation in vitro. HSC-specific deletion of Atf6a limits fibrogenesis in vivo despite increased IRE1α signaling. Conditional deletion of Ire1α from HSCs limits fibrogenic gene transcription without impacting overall fibrosis. This could be due in part to observed upregulation of the ATF6α pathway. Dual loss of Atf6a and Ire1a from HSCs worsens fibrosis in vivo through enhanced HSC activation.

Limb-bud and Heart (LBH) mediates proliferation, fibroblast-to-myofibroblast transition and EMT-like processes in cardiac fibroblasts.

Cardiac fibrosis is an important pathological change after myocardial infarction (MI). Its progression is essential for post-MI infarct healing, during which transforming growth factor beta1 (TGF-β1) plays a critical role. Limb-bud and Heart (LBH), a newly discovered target gene of TGF-β1, was shown to promote normal cardiogenesis. αB-crystallin (CRYAB), an LBH-interacting protein, was demonstrated to be involved in TGF-β1-induced fibrosis. The roles and molecular mechanisms of LBH and CRYAB during cardiac fibrosis remain largely unexplored. In this study, we investigated the alterations of LBH and CRYAB expression in mouse cardiac tissue after MI. LBH and CRYAB were upregulated in activated cardiac fibroblasts (CFs), while in vitro TGF-β1 stimulation induced the upregulation of LBH, CRYAB, and fibrogenic genes in primary CFs of neonatal rats. The results of the ectopic expression of LBH proved that LBH accelerated CF proliferation under hypoxia, mediated the expression of CRYAB and fibrogenic genes, and promoted epithelial-mesenchymal transition (EMT)-like processes in rat CFs, while subsequent CRYAB silencing reversed the effects induced by elevated LBH expression. We also verified the protein-protein interaction (PPI) between LBH and CRYAB in fibroblasts. In summary, our work demonstrated that LBH promotes the proliferation of CFs, mediates TGF-β1-induced fibroblast-to-myofibroblast transition and EMT-like processes through CRYAB upregulation, jointly functioning in post-MI infarct healing. These findings suggest that LBH could be a promising potential target for the study of cardiac repair and cardiac fibrosis.

The KLB rs17618244 gene variant is associated with fibrosing MAFLD by promoting hepatic stellate cell activation.

BackgroundThe rs17618244 G>A β-Klotho (KLB) variant has been associated with increased risk of ballooning and inflammation in pediatric patients with metabolic associated fatty liver disease (MAFLD), by reducing KLB expression. In hepatocytes, KLB downregulation induced fat accumulation and the expression of inflammatory and lipotoxic genes. We aimed to examine firstly the impact of the KLB rs17618244 variation on liver damage in adult patients with MAFLD and secondly its effect on hepatic stellate cells (HSCs) activation.MethodsThe impact of the KLB rs17618244 variant on histological liver damage was surveyed in a retrospective cohort of 1111 adult patients with MAFLD. Subgroup analysis was performed according to the presence of obesity (BMI>35; n = 708). Immortalized HSCs (LX-2) were transfected with the KLB wild type (LX-2_KLBwt), or with the mutant one carrying the rs17618244 (LX-2_KLBmut).FindingsAt ordinal regression analysis the KLB rs17618244 variant was associated with hepatic fibrosis (OR 1.23, 95% C.I.1.004–1.51; p = 0.04), but not with steatosis, inflammation and ballooning. By stratifying patients according to the presence of obesity, the KLB A allele was further associated with lobular inflammation (OR 1.32, 95% C.I.1.02–1.72; p = 0.03) and cirrhosis (OR 2.51, 95% C.I.1.23–5.05; p = 0.01) Moreover, hepatic KLB expression correlated with that of fibrogenic genes. LX-2_KLBmut cells showed reduced KLB protein levels paralleled by an induction of pro-fibrogenic genes and enhanced proliferative rate.InterpretationThe KLB rs17618244 variant is associated with hepatic fibrosis, inflammation and cirrhosis mainly in obese patients with MAFLD and HSCs which carry this mutation are highly proliferative and acquire a myofibroblast-like phenotype.FundingRicerca Finalizzata Ministero della Salute GR-2019–12,370,172 (NP), Ricerca Corrente Fondazione IRCCS Cà Granda (PD and ALF), Ricerca Finalizzata Ministero della Salute RF-2013–02,358,319 (ALF), and Ricerca Corrente and 5 × 1000 Ministero della Salute (AA).

CDK9 inhibition improves diabetic nephropathy by reducing inflammation in the kidneys

Diabetic nephropathy (DN) is a chronic inflammatory renal disease induced by hyperglycemia. Recent studies have implicated cyclin-dependent kinase 9 (CDK9) in inflammatory responses and renal fibrosis. In this study, we explored a potential role of CDK9 in DN by using cultured mouse mesangial cell line SV40 MES-13 and streptozotocin-induced type 1 mouse model of diabetes. We inhibited CDK9 in mice and in cultured cells by a highly selective CDK9 inhibitor, LDC000067 (LDC), and evaluated inflammatory and fibrogenic outcome by mRNA and protein analyses. Our studies show that treatment of diabetic mice with LDC significantly inhibits the levels of inflammatory cytokines and fibrogenic genes in kidney specimens. These reductions were associated with improved renal function. We also found that LDC treatment suppressed MAPK-AP1 activation. We then confirmed the involvement of CDK9 in cultured SV40 MES-13 cells and showed that deficiency in CDK9 prevents glucose-induced inflammatory and fibrogenic proteins. This protection was also afforded by suppression of MAPK-AP1. Taken together, our results how that hyperglycemia activates CDK9-MAPK-AP1 axis in kidneys to induce inflammation and fibrosis, leading to renal dysfunction. Our findings also suggest that CDK9 may serve as a potential therapeutic target for DN.

PAX5 activates telomerase activity and proliferation in keloid fibroblasts by transcriptional regulation of SND1, thus promoting keloid growth in burn-injured skin.

Staphylococcal nuclease domain-containing 1 (SND1) that functioned as an oncogene in a variety of tumors was upregulated in burn-injured skin tissues, and this study aims to investigate the effect of SND1 on keloid and elucidate the underlying mechanism. Keloid fibroblasts (KFs) and normal skin fibroblasts (NFs) were isolated from the keloid tissues and adjacent normal skin tissues of keloid patients. The SND1 expression was assessed in keloid tissues and KFs with Western blot assay. Gain- and loss-of-function experiments were performed to investigate the role of SND1 in proliferation, colony formation, telomerase activity, expression of fibrogenic genes and production of pro-inflammatory factors in KFs. Chromatin immunoprecipitation (CHIP) and Dual-luciferase reporter gene assays were used to verify the interaction of Paired-box gene 5 (PAX5) on SND1 promoter. Then, a series of rescue experiments were performed to verify the effects of SND1 overexpression on PAX5 knockdown-mediated KF functions. Finally, the role of SND1 in keloid formation in vivo was validated in mice with keloid implantation. SND1 was upregulated in keloid tissues and KFs. SND1 positively regulated proliferation, colony formation, telomerase activity, production of pro-inflammatory factors and expression of fibrogenic genes. PAX5 directly bound to the SND1 promoter to transcriptionally regulate SND1 expression and positively regulated SND1-mediated KF functions via the ERK/JNK pathway. In vivo assay further demonstrated that SND1 displayed a positive effect on keloid formation. SND1 transcriptionally regulated by PAX5 promotes keloid formation through activating telomerase activity via the ERK/JNK signaling pathways, which provides a promising therapeutic target for clinical treatment of burned skin keloid.

Neoagarooligosaccharide Protects against Hepatic Fibrosis via Inhibition of TGF-β/Smad Signaling Pathway.

Hepatic fibrosis occurs when liver tissue becomes scarred from repetitive liver injury and inflammatory responses; it can progress to cirrhosis and eventually to hepatocellular carcinoma. Previously, we reported that neoagarooligosaccharides (NAOs), produced by the hydrolysis of agar by β-agarases, have hepatoprotective effects against acetaminophen overdose-induced acute liver injury. However, the effect of NAOs on chronic liver injury, including hepatic fibrosis, has not yet been elucidated. Therefore, we examined whether NAOs protect against fibrogenesis in vitro and in vivo. NAOs ameliorated PAI-1, α-SMA, CTGF and fibronectin protein expression and decreased mRNA levels of fibrogenic genes in TGF-β-treated LX-2 cells. Furthermore, downstream of TGF-β, the Smad signaling pathway was inhibited by NAOs in LX-2 cells. Treatment with NAOs diminished the severity of hepatic injury, as evidenced by reduction in serum alanine aminotransferase and aspartate aminotransferase levels, in carbon tetrachloride (CCl4)-induced liver fibrosis mouse models. Moreover, NAOs markedly blocked histopathological changes and collagen accumulation, as shown by H&E and Sirius red staining, respectively. Finally, NAOs antagonized the CCl4-induced upregulation of the protein and mRNA levels of fibrogenic genes in the liver. In conclusion, our findings suggest that NAOs may be a promising candidate for the prevention and treatment of chronic liver injury via inhibition of the TGF-β/Smad signaling pathway.

Insulin treatment improves liver histopathology and decreases expression of inflammatory and fibrogenic genes in a hyperglycemic, dyslipidemic hamster model of NAFLD

BackgroundNon-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are highly prevalent comorbidities in patients with Type 2 diabetes. While many of these patients eventually will need treatment with insulin, little is known about the effects of insulin treatment on histopathological parameters and hepatic gene expression in diabetic patients with co-existing NAFLD and NASH. To investigate this further, we evaluated the effects of insulin treatment in NASH diet-fed hamsters with streptozotocin (STZ) -induced hyperglycemia.MethodsForty male Syrian hamsters were randomized into four groups (n = 10/group) receiving either a NASH-inducing (high fat, fructose and cholesterol) or control diet (CTRL) for four weeks, after which they were treated with STZ or sham-injected and from week five treated with either vehicle (CTRL, NASH, NASH-STZ) or human insulin (NASH-STZ-HI) for four weeks by continuous s.c. infusion via osmotic minipumps.ResultsNASH-STZ hamsters displayed pronounced hyperglycemia, dyslipidemia and more severe liver pathology compared to both CTRL and NASH groups. Insulin treatment attenuated dyslipidemia in NASH-STZ-HI hamsters and liver pathology was considerably improved compared to the NASH-STZ group, with prevention/reversal of hepatic steatosis, hepatic inflammation and stellate cell activation. In addition, expression of inflammatory and fibrotic genes was decreased compared to the NASH-STZ group.ConclusionsThese results suggest that hyperglycemia is important for development of inflammation and profibrotic processes in the liver, and that insulin administration has beneficial effects on liver pathology and expression of genes related to inflammation and fibrosis in a hyperglycemic, dyslipidemic hamster model of NAFLD.

Neutrophil-to-hepatocyte communication via LDLR-dependent miR-223-enriched extracellular vesicle transfer ameliorates nonalcoholic steatohepatitis.

Neutrophil infiltration around lipotoxic hepatocytes is a hallmark of nonalcoholic steatohepatitis (NASH); however, how these 2 types of cells communicate remains obscure. We have previously demonstrated that neutrophil-specific microRNA-223 (miR-223) is elevated in hepatocytes to limit NASH progression in obese mice. Here, we demonstrated that this elevation of miR-223 in hepatocytes was due to preferential uptake of miR-223-enriched extracellular vesicles (EVs) derived from neutrophils as well other types of cells, albeit to a lesser extent. This selective uptake was dependent on the expression of low-density lipoprotein receptor (LDLR) on hepatocytes and apolipoprotein E (APOE) on neutrophil-derived EVs, which was enhanced by free fatty acids. Once internalized by hepatocytes, the EV-derived miR-223 acted to inhibit hepatic inflammatory and fibrogenic gene expression. In the absence of this LDLR- and APOE-dependent uptake of miR-223-enriched EVs, the progression of steatosis to NASH was accelerated. In contrast, augmentation of this transfer by treatment with an inhibitor of proprotein convertase subtilisin/kexin type 9, a drug used to lower blood cholesterol by upregulating LDLR, ameliorated NASH in mice. This specific role of LDLR and APOE in the selective control of miR-223-enriched EV transfer from neutrophils to hepatocytes may serve as a potential therapeutic target for NASH.

Aramchol downregulates stearoyl CoA-desaturase 1 in hepatic stellate cells to attenuate cellular fibrogenesis

SummaryBackground & AimsAramchol is a fatty acid-bile acid conjugate that reduces liver fat content and is being evaluated in a phase III clinical trial for non-alcoholic steatohepatitis (NASH). Aramchol attenuates NASH in mouse models and decreases steatosis by downregulating the fatty acid synthetic enzyme stearoyl CoA desaturase 1 (SCD1) in hepatocytes. Although hepatic stellate cells (HSCs) also store lipids as retinyl esters, the impact of Aramchol in this cell type is unknown.MethodsWe investigated the effects of Aramchol on a human HSC line (LX-2), primary human HSCs (phHSCs), and primary human hepatocytes (phHeps).ResultsIn LX-2 and phHSCs, 10 μM Aramchol significantly reduced SCD1 mRNA while inducing PPARG (PPARγ) mRNA, with parallel changes in the 2 proteins; ACTA2, COL1A1, β-PDGFR (bPDGFR) mRNAs were also significantly reduced in LX-2. Secretion of collagen 1 (Col1α1) was inhibited by 10 μM Aramchol. SCD1 knockdown in LX-2 cells phenocopied the effect of Aramchol by reducing fibrogenesis, and addition of Aramchol to these cells did not rescue fibrogenic gene expression. Conversely, in LX-2 overexpressing SCD1, Aramchol no longer suppressed fibrogenic gene expression. The drug also induced genes in LX-2 that promote cholesterol efflux and inhibited ACAT2, which catalyses cholesterol synthesis. In phHeps, Aramchol also reduced SCD1 and increased PPARG mRNA expression.ConclusionsAramchol downregulates SCD1 and elevates PPARG in HSCs, reducing COL1A1 and ACTA2 mRNAs and COL1A1 secretion. These data suggest a direct inhibitory effect of Aramchol in HSCs through SCD1 inhibition, as part of a broader impact on both fibrogenic genes as well as mediators of cholesterol homeostasis. These findings illustrate novel mechanisms of Aramchol activity, including potential antifibrotic activity in patients with NASH and fibrosis.Lay summaryIn this study, we have explored the potential activity of Aramchol, a drug currently in clinical trials for fatty liver disease, in blocking fibrosis, or scarring, by hepatic stellate cells, the principal collagen-producing (i.e. fibrogenic) cell type in liver injury. In both isolated human hepatic stellate cells and in a human hepatic stellate cell line, the drug suppresses the key fat-producing enzyme, stearoyl CoA desaturase 1 (SCD1), which leads to reduced expression of genes and proteins associated with hepatic fibrosis, while inducing the protective gene, PPARγ. The drug loses activity when SCD1 is already reduced by gene knockdown, reinforcing the idea that inhibition of SCD1 is a main mode of activity for Aramchol. These findings strengthen the rationale for testing Aramchol in patients with NASH.

Transient ACE (Angiotensin-Converting Enzyme) Inhibition Suppresses Future Fibrogenic Capacity and Heterogeneity of Cardiac Fibroblast Subpopulations.

Transient ACE (angiotensin-converting enzyme) inhibition in spontaneously hypertensive rats is known to protect against future injury-induced cardiac inflammation, fibrosis, and dysfunction; however, the mechanisms of protection have not been delineated. Here, we used single-cell RNA sequencing to test the hypothesis that transient ACE inhibitor treatment would induce a persistent shift in cardiac fibroblast subpopulations. Adult male spontaneously hypertensive rats (11 weeks old, hypertensive with cardiac hypertrophy) were treated for 2 weeks with an ACE inhibitor, enalapril (30 mg/kg per day, PO), or water (untreated spontaneously hypertensive rats) followed by a 2-week washout period (n=7/group). Cardiac fibroblasts were isolated from the left ventricle and subjected to single-cell RNA sequencing. Nine clusters of fibroblasts were identified, with 98% of cells in clusters 0 to 6. The transient treatment produced significant changes both within and across clusters. Cluster 1 depicted a highly fibrogenic gene profile, with cluster 6 serving as a gateway to cluster 1. Transient ACE inhibition depleted the gateway and expanded cluster 0, which was the least fibrogenic profile. Moreover, within cluster 1 fibroblasts, ACE inhibition reduced expression of individual fibrosis genes (eg, COL1A1, COL3A1, and FN1; all P<1×10-35). Clusters 2 to 5 reflected proliferative, moderately fibrogenic, translationally active, and less inflammatory subsets of fibroblasts, all of which exhibited attenuated fibrogenic gene expression after transient ACE inhibition. In conclusion, transient ACE inhibition shifts cardiac fibroblast subpopulations and degree of activation resulting in an overall reduced fibrogenic phenotype.

Inhibition of hyaluronan synthesis by 4-methylumbelliferone ameliorates non-alcoholic steatohepatitis in choline-deficient L-amino acid-defined diet-induced murine model.

Hyaluronan (HA) as a glycosaminoglycan can bind to cell-surface receptors, such as TLR4, to regulate inflammation, tissue injury, repair, and fibrosis. 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, is a drug used for the treatment of biliary spasms. Currently, therapeutic interventions are not available for non-alcoholic steatohepatitis (NASH). In this study, we investigated the effects of 4-MU on NASH using a choline-deficient amino acid (CDAA) diet model. CDAA diet-fed mice showed NASH characteristics, including hepatocyte injury, hepatic steatosis, inflammation, and fibrogenesis. 4-MU treatment significantly reduced hepatic lipid contents in CDAA diet-fed mice. 4-MU reversed CDAA diet-mediated inhibition of Ppara and induction of Srebf1 and Slc27a2. Analysis of serum ALT and AST levels revealed that 4-MU treatment protected against hepatocellular damage induced by CDAA diet feeding. TLR4 regulates low molecular weight-HA-induced chemokine expression in hepatocytes. In CDAA diet-fed, 4-MU-treated mice, the upregulated chemokine/cytokine expression, such as Cxcl1, Cxcl2, and Tnf was attenuated with the decrease of macrophage infiltration into the liver. Moreover, HA inhibition repressed CDAA diet-induced mRNA expression of fibrogenic genes, Notch1, and Hes1 in the liver. In conclusion, 4-MU treatment inhibited liver steatosis and steatohepatitis in a mouse model of NASH, implicating that 4-MU may have therapeutic potential for NASH.

Discovery and Biological Evaluation of N-Methyl-pyrrolo[2,3-b]pyridine-5-carboxamide Derivatives as JAK1-Selective Inhibitors.

Janus kinase 1 (JAK1) plays a key role in most cytokine-mediated inflammatory and autoimmune responses through JAK/STAT signaling; thus, JAK1 inhibition is a promising therapeutic strategy for several diseases. Analysis of the binding modes of current JAK inhibitors to JAK isoforms allowed the design of N-alkyl-substituted 1-H-pyrrolo[2,3-b] pyridine carboxamide as a JAK1-selective scaffold, and the synthesis of various methyl amide derivatives provided 4-((cis-1-(4-chlorobenzyl)-2-methylpiperidin-4-yl)amino)-N-methyl-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (31g) as a potent JAK1-selective inhibitor. In particular, the (S,S)-enantiomer of 31g (38a) exhibited excellent potency for JAK1 and selectivity over JAK2, JAK3, and TYK2. On investigating the effect of 31g on hepatic fibrosis, it was found that it reduces the proliferation and fibrogenic gene expression of TGF-β-induced hepatic stellate cells (HSCs). Specifically, 31g significantly inhibited TGF-β-induced migration of HSCs at 0.25 μM in wound-healing assays.