MEK5-ERK5 Axis Promotes Self-renewal and Tumorigenicity of Glioma Stem Cells.

Glioma stem cells (GSC) promote the malignancy of glioblastoma (GBM), the most lethal brain tumor. ERK5 belongs to the MAPK family. Here, we demonstrated that MAPK kinase 5 (MEK5)-ERK5-STAT3 pathway plays an essential role in maintaining GSC stemness and tumorigenicity by integrating genetic and pharmacologic manipulation and RNA sequencing analysis of clinical specimens. ERK5 was highly expressed and activated in GSCs. ERK5 silencing by short hairpin RNA in GSCs suppressed the self-renewal potential and GBM malignant growth concomitant with downregulation of STAT3 phosphorylation. Conversely, the activation of the MEK5-ERK5 pathway by introducing ERK5 or MEK5 resulted in increased GSC stemness. The introduction of STAT3 counteracted the GSC phenotypes by ERK5 silencing. Moreover, ERK5 expression and signaling are associated with poor prognosis in patients with GBM with high stem cell properties. Finally, pharmacologic inhibition of ERK5 significantly inhibited GSC self-renewal and GBM growth. Collectively, these findings uncover a crucial role of the MEK5-ERK5-STAT3 pathway in maintaining GSC phenotypes and GBM malignant growth, thereby providing a potential target for GSC-directed therapy. Significance: In this study, we demonstrated that MEK5-ERK5-STAT3 axis plays a critical role in maintaining stemness and tumorigenicity in GSCs by using genetic, pharmacologic, and bioinformatics tools, identifying the MEK5-ERK5-STAT3 axis as a potential target for GSC-directed therapy.

Separation of Glycoproteins Based on Sugar Chains Using Novel Stationary Phases Modified with Poly(ethylene glycol)-Conjugated Boronic-Acid Derivatives.

Boronic acid (BA) reversibly complexes with the diol structure. BA derivatives separate glycoproteins based on the differences in the sugar chains. Separation typically occurs under basic conditions, which does not guarantee the structural stability of glycoproteins. Here, 5-boronopicolinic acid (BPA) is used to prepare silica-gel based columns with poly(ethylene glycol) (PEG) as a linker to suppress nonselective adsorption and poly(ethylene imine) (PEI) as a scaffold to increase the BPA moiety density. High-performance liquid chromatography (HPLC) using only aqueous buffer solutions without organic solvents demonstrates the selective retention ability of the BPA columns for glycoproteins. BPA interacts with the diols in the sugar chains but not the proteins. In an evaluation for N-glycans, the BPA columns show a higher retention ability toward high mannose type and a lower affinity to N-acetylneuraminic acid (Neu5Ac). Finally, a pair of glycoproteins, fetuin and asialofetuin, are selectively separated due to the presence of Neu5Ac on the nonreducing end.

Erk5 in Bone Marrow Mesenchymal Stem Cells Regulates Bone Homeostasis by Preventing Osteogenesis in Adulthood.

Extracellular signal-regulated kinase 5 (Erk5) belongs to the mitogen-activated protein kinase (MAPK) family. Previously, we demonstrated that Erk5 directly phosphorylates Smad-specific E3 ubiquitin protein ligase 2 (Smurf2) at Thr249 (Smurf2Thr249) to activate its E3 ubiquitin ligase activity. Although we have clarified the importance of Erk5 in embryonic mesenchymal stem cells (MSCs) on skeletogenesis, its role in adult bone marrow (BM)-MSCs on bone homeostasis remains unknown. Leptin receptor-positive (LepR+) BM-MSCs represent a major source of bone in adult bone marrow and are critical regulators of postnatal bone homeostasis. Here, we identified Erk5 in BM-MSCs as an important regulator of bone homeostasis in adulthood. Bone marrow tissue was progressively osteosclerotic in mice lacking Erk5 in LepR+ BM-MSCs with age, accompanied by increased bone formation and normal bone resorption in vivo. Erk5 deficiency increased the osteogenic differentiation of BM-MSCs along with a higher expression of Runx2 and Osterix, essential transcription factors for osteogenic differentiation, without affecting their stemness in vitro. Erk5 deficiency decreased Smurf2Thr249 phosphorylation and subsequently increased Smad1/5/8-dependent signaling in BM-MSCs. The genetic introduction of the Smurf2T249E mutant (a phosphomimetic mutant) suppressed the osteosclerotic phenotype in Erk5-deficient mice. These findings suggest that the Erk5-Smurf2Thr249 axis in BM-MSCs plays a critical role in the maintenance of proper bone homeostasis by preventing excessive osteogenesis in adult bone marrow.

Selective Recovery of Estrogenic Endocrine Disruptors from 48 Environmental Samples Using a Substrate for Activity-Specific Concentration.

hER-MIP is a molecularly imprinted polymer (MIP) that has been shown to selectively collect human estrogen receptor (hER) binding active substances. However, environmental samples contain various chemicals depending on the location and regional differences, and the hER binding activity depends on the sample type. Thus, the general applicability of hER-MIP to actual environmental samples must be elucidated. In this study, 48 environmental samples were collected and screened with hER-MIP, and a yeast assay was performed to evaluate the adsorption characteristics of the samples according to the adsorption and elution fractions. The results showed that hER-MIP collects hER binding active substances almost selectively but does not collect constitutive androstane receptor (CAR) binding active substances selectively. CAR binding activity was detected in the adsorbed fraction because several hER binding active substances also demonstrate CAR binding activity.

LPIAT1/MBOAT7 depletion increases triglyceride synthesis fueled by high phosphatidylinositol turnover.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is a common prelude to cirrhosis and hepatocellular carcinoma. The genetic rs641738 C>T variant in the lysophosphatidylinositol acyltransferase 1 (LPIAT1)/membrane bound O-acyltransferase domain-containing 7, which incorporates arachidonic acid into phosphatidylinositol (PI), is associated with the entire spectrum of NAFLD. In this study, we investigated the mechanism underlying this association in mice and cultured human hepatocytes. DESIGN: We generated the hepatocyte-specific Lpiat1 knockout mice to investigate the function of Lpiat1 in vivo. We also depleted LPIAT1 in cultured human hepatic cells using CRISPR-Cas9 systems or siRNA. The effect of LPIAT1-depletion on liver fibrosis was examined in mice fed high fat diet and in liver spheroids. Lipid species were measured using liquid chromatography-electrospray ionisation mass spectrometry. Lipid metabolism was analysed using radiolabeled glycerol or fatty acids. RESULTS: The hepatocyte-specific Lpiat1 knockout mice developed hepatic steatosis spontaneously, and hepatic fibrosis on high fat diet feeding. Depletion of LPIAT1 in cultured hepatic cells and in spheroids caused triglyceride accumulation and collagen deposition. The increase in hepatocyte fat content was due to a higher triglyceride synthesis fueled by a non-canonical pathway. Indeed, reduction in the PI acyl chain remodelling caused a high PI turnover, by stimulating at the same time PI synthesis and breakdown. The degradation of PI was mediated by a phospholipase C, which produces diacylglycerol, a precursor of triglyceride. CONCLUSION: We found a novel pathway fueling triglyceride synthesis in hepatocytes, by a direct metabolic flow of PI into triglycerides. Our findings provide an insight into the pathogenesis and therapeutics of NAFLD.

Isolated Pancreatic Sarcoidosis Diagnosed by Endoscopic Ultrasound-guided Fine-needle Aspiration.

We herein report a 52-year-old man with multiple hypoechoic lesions in the body and tail of the pancreas detected during a screening ultrasound. Computed tomography (CT) showed no lesions other than those in the pancreas and peripheral lymph nodes. Contrast-enhanced CT identified hypovascular tumors in the pancreas. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) demonstrated partial fibrosis and noncaseating granulomas with Langhans giant cells. To our knowledge, this is the first report of isolated pancreatic sarcoidosis diagnosed by EUS-FNA. Although pancreatic sarcoidosis is very rare, clinicians should be aware of this possibility in patients presenting with multiple hypovascular pancreatic tumors.

Association of cholesterol uptake capacity, a novel indicator for HDL functionality, and coronary plaque properties: An optical coherence tomography-based observational study.

BACKGROUND: Cholesterol efflux from atherosclerotic lesion is a key function of high-density lipoprotein (HDL). Recently, we established a simple, high-throughput, cell-free assay to evaluate the capacity of HDL to accept additional cholesterol, which is herein referred to as "cholesterol uptake capacity (CUC)". OBJECTIVE: To clarify the cross-sectional relationship between CUC and coronary plaque properties. METHODS: We enrolled 135 patients to measure CUC and assess the morphological features of angiographic stenosis by optical coherence tomography (OCT). We estimated the extent of the lipid-rich plaque by multiplying the mean lipid arc by lipid length (lipid index). The extent of the OCT-detected macrophage accumulation in the target plaque was semi-quantitatively estimated using a grading system. RESULTS: Lipid-rich plaque lesions were identified in 125 patients (92.6%). CUC was inversely associated with the lipid index (R = -0.348, P < 0.0001). In addition, CUC was also inversely associated with macrophage score (R = -0.327, P < 0.0001). Conversely, neither circulating levels of HDL cholesterol nor apoA1 showed a similar relationship. CONCLUSIONS: We demonstrated that CUC was inversely related to lipid-rich plaque burden and the extent of macrophage accumulation, suggesting that CUC could be useful for cardiovascular risk stratification.

A pancreatic mucinous cystic neoplasm undergoing intriguing morphological changes over time and associated with recurrent pancreatitis: A case report.

RATIONALE: Mucinous cystic neoplasms (MCNs) are pancreatic mucin-producing cystic lesions with a distinctive ovarian-type stroma. The diagnosis is generally easy in typical cases; however, differential diagnosis is difficult in others such as in the case we report herein. PATIENT CONCERNS: A 27-year-old woman with sudden onset of epigastric pain was referred to our hospital for suspected acute pancreatitis. Contrast-enhanced computed tomography revealed a 25-mm cystic lesion in the pancreas and a low density area with delayed enhancement at the right upper side of the cystic lesion. DIAGNOSES: During its clinical course, the cystic lesion underwent various morphological changes. Eventually, it presented typical findings of MCNs, and could be accurately diagnosed. INTERVENTIONS: Laparoscopic distal pancreatectomy was performed on the patient by preserving the spleen. OUTCOMES: The patient revealed no symptoms till 1 year after the operation. LESSONS: This case of MCN with intriguing short-term morphological changes was associated with recurrent pancreatitis. A combination of imaging modalities is essential for accurate diagnosis of MCNs, and follow-up with serial imaging might be useful for certain unusual lesions.

Exogenous Administration of Low-Dose Lipopolysaccharide Potentiates Liver Fibrosis in a Choline-Deficient l-Amino-Acid-Defined Diet-Induced Murine Steatohepatitis Model.

Various rodent models have been proposed for basic research; however, the pathogenesis of human nonalcoholic steatohepatitis (NASH) is difficult to closely mimic. Lipopolysaccharide (LPS) has been reported to play a pivotal role in fibrosis development during NASH progression via activation of toll-like receptor 4 (TLR4) signaling. This study aimed to clarify the impact of low-dose LPS challenge on NASH pathological progression and to establish a novel murine NASH model. C57BL/6J mice were fed a choline-deficient l-amino-acid-defined (CDAA) diet to induce NASH, and low-dose LPS (0.5 mg/kg) was intraperitoneally injected thrice a week. CDAA-fed mice showed hepatic CD14 overexpression, and low-dose LPS challenge enhanced TLR4/NF-κB signaling activation in the liver of CDAA-fed mice. LPS challenge potentiated CDAA-diet-mediated insulin resistance, hepatic steatosis with upregulated lipogenic genes, and F4/80-positive macrophage infiltration with increased proinflammatory cytokines. It is noteworthy that LPS administration extensively boosted pericellular fibrosis with the activation of hepatic stellate cells in CDAA-fed mice. Exogenous LPS administration exacerbated pericellular fibrosis in CDAA-mediated steatohepatitis in mice. These findings suggest a key role for LPS/TLR4 signaling in NASH progression, and the authors therefore propose this as a suitable model to mimic human NASH.

Magnetic Field Stimuli-Sensitive Drug Release Using a Magnetic Thermal Seed Coated with Thermal-Responsive Molecularly Imprinted Polymer.

A new stimulus-responsive drug delivery system using Fe3O4 nanoparticles coated with molecularly imprinted polymer (MIP) is reported. Magnetic thermal seeds (MTS) with their size controlled between 10 and 20 nm that could generate heat under an alternate current (AC) magnetic field were modified with a thermal-responsive MIP by grafting polymerization for effective release of an anticancer drug, methotrexate (MTX). The MIP-coated MTS showed the superparamagnetic property as well as the selective adsorption ability toward MTX, and 80% of MXT adsorbed on the MIP-coated MTS was stimulus released at 60 °C by cleaving hydrogen bonding in the recognition sites. Finally, the MTX release from the MTX-loaded MIP-coated MTS under an AC magnetic field within 10 min was successfully demonstrated.

Efficient extraction of estrogen receptor-active compounds from environmental surface water via a receptor-mimic adsorbent, a hydrophilic PEG-based molecularly imprinted polymer.

We report an efficient screening procedure for the selective detection of compounds that are actively bound to estrogen receptor (ER) from environmental water samples using a receptor-mimic adsorbent prepared by a molecularly imprinted polymer (MIP). To mimic the recognition ability of ER, we improved the typical MIP preparation procedure using a hydrophilic matrix with a polyethylene glycol (PEG)-based crosslinker and a hydrophobic monomer to imitate the hydrophobic pocket of ER. An optimized MIP prepared with methacrylic acid as an additional functional monomer and estriol (E3), an analogue of 17ß-estradiol (E2), exhibited highly selective adsorption for ER-active compounds such as E2 and E3, with significant suppression of non-specific hydrophobic adsorption. The prepared MIP was then applied to the screening of ER-active compounds in sewage samples. The fraction concentrated by the MIP was evaluated by in vitro bioassay using the yeast two-hybrid (Y2H) method and liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOFMS). Compared to an authentic adsorbent, styrene-divinylbenzene (SDB)-based resin, the fraction concentrated by the MIP had 120% ER activity in the Y2H assay, and only 25% peak volume was detected in LC-Q-TOFMS. Furthermore, a few ER-active compounds were identified only from the fraction concentrated by the MIP, although they could not be determined in the fraction concentrated by the SDB-based resin due to ion suppression along with high levels of hydrophobic compounds. These results indicated that the newly developed MIP effectively captured ER-active compounds and while allowing most non-ER-active compounds to pass through.

Glycogenic Hepatopathy in Type 1 Diabetes Mellitus.

Glycogenic hepatopathy (GH) is a rare complication of poorly controlled type 1 diabetes mellitus (T1DM), and is characterized by elevated liver enzymes, hepatomegaly, and glycogen accumulation. We herein present the case of a 23-year-old man with poorly controlled T1DM who had liver dysfunction. Imaging studies showed severe hepatomegaly and fatty liver. The examination of a liver biopsy specimen revealed fatty droplets, ballooning, inflammation, and mild fibrosis. Subsequent periodic acid-Schiff (PAS) staining after diastase digestion confirmed GH. In this case, the improvement of hyperglycemia, not HbA1c, resulted in the improvement of the patient's liver function. This is the first report on the use of continuous glucose monitoring in patients with GH to show that continuous hyperglycemia may worsen GH.

Platelet hyperaggregability is associated with decreased ADAMTS13 activity and enhanced endotoxemia in patients with acute cholangitis.

AIM: Insufficient ADAMTS13 activity (ADAMTS13:AC) leads to increased levels of unusually large von Willebrand factor (VWF) multimers and causes microcirculatory disturbance and multiple organ failure (MOF). Endotoxin (Et) triggers the activation of coagulation and cytokine cascades, leading to MOF in severe inflammatory response syndrome. Here, we investigated the potential role of endotoxemia-related ADAMTS13 in acute cholangitis. METHODS: Twenty-four patients with acute cholangitis, including 7 with severe acute cholangitis, were recruited in this study. The levels of ADAMTS13:AC, VWF antigen (VWF:Ag), interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α in each patient were determined by enzyme-linked immunosorbent assay, whereas Et levels were determined by Et activity assay (EAA) analysis. RESULTS: The ADAMTS13:AC and VWF:Ag levels were significantly lower and higher, respectively, in patients with acute cholangitis than in controls. The EAA levels were higher in patients with acute cholangitis than in controls, and were inversely correlated with that of ADAMTS13:AC. Patients with severe acute cholangitis had significantly lower ADAMTS13:AC and higher VWF:Ag levels than those with mild to moderate cholangitis. Notably, ADMTS13:AC was directly correlated with platelet counts and inversely correlated with IL-6 levels, and the VWF:Ag/ADAMTS13:AC ratio was directly correlated with IL-8 and TNF-α levels. CONCLUSIONS: Imbalance of ADAMTS13:AC and VWF:Ag levels might be associated with severe acute cholangitis, reflecting platelet hyperaggregability. Severe acute cholangitis has severe pathophysiological features and is complicated by endotoxemia and MOF. Notably, this is the first report indicating an association between the levels of ADAMTS13:AC and VWF:Ag and those of EAA and cytokines in acute cholangitis.

Aortic Valve Replacement for the Management of Heyde Syndrome: A Case Report.

Heyde syndrome describes the triad of aortic stenosis, acquired coagulopathy, and anemia due to bleeding from intestinal angiodysplasia. An 87-year-old man with iron deficiency anemia due to melena was admitted to our hospital. On examination, a systolic murmur was heard and echocardiography confirmed the presence of aortic stenosis. Esophagogastroduodenoscopy and colonoscopy were unremarkable. Capsule endoscopy and double balloon endoscopy revealed angiodysplasia throughout the small intestine. Laboratory investigations were significant for reduced plasma levels of high molecular weight von Willebrand factor multimers. On the basis of these findings, the patient was diagnosed with Heyde syndrome. The patient required frequent blood transfusions because of the intestinal bleeding, and underwent bioprosthetic aortic valve replacement. Twenty months after the operation, the gastrointestinal bleeding resolved and the patient no longer required blood transfusions. This is the first case report to describe an improvement in bleeding from angiodysplasia, one year after aortic valve replacement. It demonstrates the effective treatment of Heyde syndrome with aortic valve replacement, and highlights the importance of considering this differential diagnosis when evaluating patients presenting with repeated episodes of gastrointestinal bleeding and a concurrent systolic murmur.

A Patient with Hepatocellular Carcinoma with Isolated Right Atrial Metastases.

Hepatocellular carcinoma (HCC) with isolated right atrial metastasis is extremely rare; most cases are considered inoperable. We herein report the case of a 74-year-old man with HCC with isolated right atrial metastases without hepatic vein invasion; the right atrial lesion was resected because of the risk of heart failure and sudden death. Postoperatively, he underwent transcatheter arterial chemoembolization and radiofrequency ablation for intrahepatic HCC. He recovered completely, with a long-term survival of 36 months. This is the first report of an HCC patient with isolated right atrial metastases without hepatic vein invasion. Tumorectomy for solitary atrial metastasis is effective for HCC patients.

Predisposing factors for hepatocellular carcinoma recurrence following initial remission after transcatheter arterial chemoembolization.

Hepatocellular carcinoma (HCC) is prone to recurrence following curative treatment. The purpose of the present study was to identify the predisposing factors of HCC recurrence following complete remission achieved by transarterial chemoembolization (TACE). A retrospective cohort study of 70 consecutive patients with HCC who underwent TACE as the initial treatment was conducted. The patients were divided into two groups according to their 1-year disease-free survival (DFS) status; the early recurrence group (ER group; n=32), with HCC recurring within 1 year of initial TACE; and the non-early recurrence group (NER group; n=38), who did not experience recurrence within 1 year. The parameters identified as significantly associated with DFS time on univariate analysis were aspartate aminotransferase (AST), alanine aminotransferase and α-fetoprotein levels, as well as the tumor number (P=0.003, P=0.027, P=0.002 and P=0.005, respectively). Multivariate analysis revealed that AST levels and tumor number were significantly associated with a shorter DFS period (P=0.009 and P=0.038, respectively). The Mantel-Haenszel test revealed a significant trend of decreasing DFS with increasing tumor number. Among the patients with HCC in the ER group, locoregional recurrence occurred more frequently in those who received TACE alone compared with those treated with TACE combined with radiofrequency ablation treatment. In summary, multinodularity of HCC is the most potent predictive factor for the recurrence of HCC within 1 year of initial TACE.

Tunable separations based on a molecular size effect for biomolecules by poly(ethylene glycol) gel-based capillary electrophoresis.

We report novel capillary gel electrophoresis (CGE) with poly(ethylene glycol) (PEG)-based hydrogels for the effective separations of biomolecules containing sugars and DNAs based on a molecular size effect. The gel capillaries were prepared in a fused silica capillary modified with 3-(trimethoxysilyl)propylmethacrylate using a variety of the PEG-based hydrogels. After the fundamental evaluations in CGE regarding the separation based on the molecular size effect depending on the crosslinking density, the optimized capillary provided the efficient separation of glucose ladder (G1 to G20). In addition, another capillary showed the successful separation of DNA ladder in the range of 10-1100 base pair, which is superior to an authentic acrylamide-based gel capillary. For both glucose and DNA ladders, the separation ranges against the molecular size were simply controllable by alteration of the concentration and/or units of ethylene oxide in the PEG-based crosslinker. Finally, we demonstrated the separations of real samples, which included sugars carved out from monoclonal antibodies, mAbs, and then the efficient separations based on the molecular size effect were achieved.

Therapeutic strategies for alcoholic liver disease: Focusing on inflammation and fibrosis (Review).

Excessive alcohol consumption is the most common cause of liver disease in the world. Chronic alcohol abuse leads to liver damage, liver inflammation, fibrosis and hepatocellular carcinoma. Inflammatory cytokines, such as tumor necrosis factor-α and interferon-γ, induce liver injury, which leads to the develo-pment of alcoholic liver disease (ALD). Hepatoprotective cytokines, such as interleukin (IL)-6 and IL-10, are also associated with ALD. IL-6 improves ALD via the activation of STAT3 and the subsequent induction of a variety of hepatoprotective genes in hepatocytes. Alcohol consumption promotes liver inflammation by incre-asing the translocation of gut-derived endotoxins to the portal circulation and by activating Kupffer cells through the lipopolysaccharide/Toll-like receptor 4 pathways. Oxidative stress and microflora products are also associated with ALD. Hepatic stellate cells play an important role in angiogenesis and liver fibrosis. Anti-angiogenic therapy has been found to be effective in the prevention of fibrosis. This suggests that blocking angiogenesis could be a promising therapeutic option for patients with advanced fibrosis. This review discusses the main pathways associated with liver inflammation and liver fibrosis as well as new therapeutic strategies.

Severe Aplastic Anemia following Parvovirus B19-Associated Acute Hepatitis.

Human parvovirus (HPV) B19 is linked to a variety of clinical manifestations, such as erythema infectiosum, nonimmune hydrops fetalis, and transient aplastic anemia. Although a few cases have shown HPVB19 infection as a possible causative agent for hepatitis-associated aplastic anemia (HAAA) in immunocompetent patients, most reported cases of HAAA following transient hepatitis did not have delayed remission. Here we report a rare case of severe aplastic anemia following acute hepatitis with prolonged jaundice due to HPVB19 infection in a previously healthy young male. Clinical laboratory examination assessed marked liver injury and jaundice as well as peripheral pancytopenia, and bone marrow biopsy revealed severe hypoplasia and fatty replacement. HPVB19 infection was diagnosed by enzyme immunoassay with high titer of anti-HPVB19 immunoglobulin M antibodies. Immunosuppressive therapy was initiated 2 months after the onset of acute hepatitis when liver injury and jaundice were improved. Cyclosporine provided partial remission after 2 months of medication without bone marrow transplantation. Our case suggests that HPVB19 should be considered as a hepatotropic virus and a cause of acquired aplastic anemia, including HAAA.

Combined treatment with dipeptidyl peptidase-4 inhibitor (sitagliptin) and angiotensin-II type 1 receptor blocker (losartan) suppresses progression in a non-diabetic rat model of steatohepatitis.

AIM: Dipeptidyl peptidase-4 (DPP4) inhibitors (DPP4-I) are oral glucose-lowering drugs for type 2 diabetes mellitus. Previously, we reported that DPP4-I (sitagliptin) exerted suppressive effects on experimental liver fibrosis in rats. Blockade of the renin-angiotensin system by angiotensin-II type 1 receptor blocker (losartan), commonly used in the management of hypertension, has been shown to significantly alleviate hepatic fibrogenesis and carcinogenesis. We aimed to elucidate the effects and possible mechanisms of a sitagliptin + losartan combination on the progression of non-diabetic non-alcoholic steatohepatitis (NASH) in a rat model. METHODS: To induce NASH, Fischer 344 rats were fed a choline-deficient L-amino acid-defined diet for 12 weeks. We elucidated the chemopreventive effects of sitagliptin + losartan, especially in conjunction with hepatic stellate cell (HSC) activation, angiogenesis, and oxidative stress, all known to play important roles in the progression of NASH. RESULTS: Sitagliptin + losartan suppressed choline-deficient L-amino acid-defined diet-induced hepatic fibrogenesis and carcinogenesis. The combination treatment exerted a greater inhibitory effect than monotherapy. These inhibitory effects occurred almost concurrently with the suppression of HSC activation, neovascularization, and oxidative stress. In vitro studies showed that sitagliptin + losartan inhibited angiotensin II-induced proliferation and expression of transforming growth factor-ß1 and α1 (I)-procollagen mRNA of activated HSC and in vitro angiogenesis, in parallel with the suppression observed in in vivo studies. CONCLUSIONS: The widely and safely used sitagliptin + losartan combination treatment in clinical practice could be an effective strategy against NASH.