Esteban Mezey

Transforming Growth Factor-β1 Induces an Epithelial-to-Mesenchymal Transition State in Mouse Hepatocytes in Vitro

Liver fibrosis is a progressive pathologic process that involves deposition of excess extracellular matrix leading to distorted architecture and culminating in cirrhosis. The role of transforming growth factor-β (TGF-β) as a key molecule in the development and progression of hepatic fibrosis via the activation of hepatic stellate cells, among other fibroblast populations, is without controversy. We hereby show that TGF-β1 induces an epithelial-to-mesenchymal transition (EMT) state in mature hepatocytes in vitro. EMT state was marked by significant upregulation of α1(I) collagen mRNA expression and type I collagen deposition. Similar changes were found in a “normal” mouse hepatocyte cell line (AML12), thus confirming that hepatocytes are capable of EMT changes and type I collagen synthesis. We also show that in hepatocytes in the EMT state, TGF-β1 induces the snail-1 transcription factor and activates the Smad2/3 pathway. Evidence for a central role of the TGF-β1/Smad pathway is further supported by the inhibition of EMT by Smad4 silencing using small interference RNA technology. In conclusion, TGF-β1, a known pro-apoptotic cytokine in mature hepatocytes, is capable of mediating phenotypic changes and plasticity in the form of EMT, resulting in collagen deposition. Our findings support a potentially crucial role for EMT in the development and progression of hepatic fibrogenesis.

Inositol Pyrophosphates Inhibit Akt Signaling, Thereby Regulating Insulin Sensitivity and Weight Gain

The inositol pyrophosphate IP7 (5-diphosphoinositolpentakisphosphate), formed by a family of three inositol hexakisphosphate kinases (IP6Ks), modulates diverse cellular activities. We now report that IP7 is a physiologic inhibitor of Akt, a serine/threonine kinase that regulates glucose homeostasis and protein translation, respectively, via the GSK3β and mTOR pathways. Thus, Akt and mTOR signaling are dramatically augmented and GSK3β signaling reduced in skeletal muscle, white adipose tissue, and liver of mice with targeted deletion of IP6K1. IP7 affects this pathway by potently inhibiting the PDK1 phosphorylation of Akt, preventing its activation and thereby affecting insulin signaling. IP6K1 knockout mice manifest insulin sensitivity and are resistant to obesity elicited by high-fat diet or aging. Inhibition of IP6K1 may afford a therapeutic approach to obesity and diabetes.

Decreased Jejunal Uptake of Labeled Folic Acid (3H-PGA) in Alcoholic Patients: Roles of Alcohol and Nutrition

Abstract The jejunal uptake of labeled folic acid (3H-PGA) was measured in chronic alcoholic patients without definite liver disease. The intestine was perfused through a triple lumen tube with a solution containing 3H-PGA in a concentration of 25 ng per milliliter. Uptake was low in eight patients who gave a history of poor diet during the alcoholic binge that preceded admission (20.4 per cent ± 4.16 S.D.). A significantly greater value was obtained in nine abstinent subjects fed a hospital diet for two weeks (36.5 per cent ± 5.75). The subsequent administration of ethanol for two weeks to seven patients did not change the mean uptake of 3H-PGA (34.8 per cent ± 9.87). This study confirms that the absorption of folic acid is decreased in malnourished, actively drinking alcoholics. The data suggest that the functional defect is caused by poor nutrition rather than a toxic effect of ethanol on the jejunum.

A randomized placebo controlled trial of vitamin E for alcoholic hepatitis

BACKGROUND/AIMS The effect of vitamin E administration on clinical and laboratory parameters of liver function and on markers of fibrogenesis was assessed in patients with mild to moderate alcoholic hepatitis in a double blind placebo controlled randomized trial. METHODS Twenty-five patients received 1000 I.U. of vitamin E per day, while 26 patients received placebo for 3 months. The patients were followed for 1 year after entry into the trial. RESULTS Vitamin E did not result in significant greater decreases in serum aminotransferases and serum bilirubin or in greater increases in serum albumin as compared with placebo. Prothrombin time did not change, while serum creatinine remained in the normal range. Monocyte nuclear nuclear factor-kappa B binding activity decreased in patients who remained abstinent, regardless of whether they received vitamin E. As regards markers of hepatic fibrogenesis, vitamin E treatment decreased serum hyaluronic acid (P<0.05) while serum aminoterminal peptide of type III procollagen did not change in either group. Four patients in the treatment group and five in the placebo group died during the 1-year study. CONCLUSIONS Vitamin E treatment improves serum hyaluronic acid but has no beneficial effects on tests of liver function in patients with mild to moderate alcoholic hepatitis.

Intestinal Malabsorption In Folate-Deficient Alcoholics

Malnutrition with folate deficiency is frequently found among alcoholics, and could be caused in part by decreased intestinal absorption. To evaluate the relationship of nutrition and absorption in alcoholics, intestinal absorption was studied in patient volunteers placed on a folate-deficient diet with ethanol. Intestinal absorption was measured by conventional means and by the technique of triple lumen perfusion of the jejunum. In 2 patients who ingested ethanol, 200 g per day with a low folate diet, the dietary induction of folate deficiency was followed by decreased absorption of d-xylose, labeled folic acid (3H-pteroylglutamic acid), glucose, fluid, and sodium. Mild net secretion of fluid and sodium into the intestinal lumen was observed in a patient who remained sober on the low folate diet and in a patient who ingested ethanol, 300 g per day, with a regular diet. The morphology of the jejunal mucosa was not affected. These preliminary data suggest that the combination of dietary folate deficiency and prolonged ethanol intake results in intestinal malabsorption of several water-soluble substances, which may account in part for the poor nutrition often found in binge drinkers.

Human bile contains MicroRNA‐laden extracellular vesicles that can be used for cholangiocarcinoma diagnosis

Cholangiocarcinoma (CCA) presents significant diagnostic challenges, resulting in late patient diagnosis and poor survival rates. Primary sclerosing cholangitis (PSC) patients pose a particularly difficult clinical dilemma because they harbor chronic biliary strictures that are difficult to distinguish from CCA. MicroRNAs (miRs) have recently emerged as a valuable class of diagnostic markers; however, thus far, neither extracellular vesicles (EVs) nor miRs within EVs have been investigated in human bile. We aimed to comprehensively characterize human biliary EVs, including their miR content. We have established the presence of extracellular vesicles in human bile. In addition, we have demonstrated that human biliary EVs contain abundant miR species, which are stable and therefore amenable to the development of disease marker panels. Furthermore, we have characterized the protein content, size, numbers, and size distribution of human biliary EVs. Utilizing multivariate organization of combinatorial alterations (MOCA), we defined a novel biliary vesicle miR‐based panel for CCA diagnosis that demonstrated a sensitivity of 67% and specificity of 96%. Importantly, our control group contained 13 PSC patients, 16 with biliary obstruction of varying etiologies (including benign biliary stricture, papillary stenosis, choledocholithiasis, extrinsic compression from pancreatic cysts, and cholangitis), and 3 with bile leak syndromes. Clinically, these types of patients present with a biliary obstructive clinical picture that could be confused with CCA. Conclusion: These findings establish the importance of using extracellular vesicles, rather than whole bile, for developing miR‐based disease markers in bile. Finally, we report on the development of a novel bile‐based CCA diagnostic panel that is stable, reproducible, and has potential clinical utility. (Hepatology 2014;60:896–907)

Familial hemochromatosis: Characteristics of the pre-cirrhotic stage in a large kindred

Ffty asymptomatic members of a kindred with familial hemochromatosis were studied in an effort to clarify some of the physiologic abnormalities present in the pre-cirrhotic or latent stage of the disease. Using excess hepatic iron as a marker for inheritance of hemochromatosis, results of liver biopsies on 31 family members suggest an auto-somal dominant mode of inheritance with incomplete expressivity. In addition to a relationship between alcohol intake and excess liver iron, there was a strong association between the level of alcohol intake and the presence of hepatic fibrosis in those subjects with excess iron stores. Both serum iron and transferrin saturation were significantly higher in family members with iron overload than in those who were not affected. Only transferrin saturation was significantly correlated with the severity of hepatic iron deposition. Studies of glucose tolerance (OGTT, IVITT, glucose clamp studies) demonstrated a defect in carbohydrate metabolism associated with deficient insulin secretion and insulin resistance, both of which were related to the degree of hepatic iron depostion. In this kindred we have found no evidence for a contribution of inheritance to the carbohydrate intolerance of hemochromatosis. Iron overload was not related to activity of hepatic collagen proline hydroxylase or urinary excretion of peptide-bound hydroxyproline. Serum ferritin, previously thought to be a reliable marker of reticuloendothelial iron stores, was normal in 19 of 20 family members with iron overload.

MicroRNA down-regulated in human cholangiocarcinoma control cell cycle through multiple targets involved in the G1/S checkpoint

MicroRNAs (miRs) recently emerged as prominent regulators of cancer processes. In the current study we aimed at elucidating regulatory pathways and mechanisms through which miR‐494, one of the miR species found to be down‐regulated in cholangiocarcinoma (CCA), participates in cancer homeostasis. miR‐494 was identified as down‐regulated in CCA based on miR arrays. Its expression was verified with quantitative real‐time reverse‐transcription polymerase chain reaction (qRT‐PCR). To enforce miR expression, we employed both transfection methods, as well as a retroviral construct to stably overexpress miR‐494. Up‐regulation of miR‐494 in cancer cells decreased growth, consistent with a functional role. mRNA arrays of cells treated with miR‐494, followed by pathway analysis, suggested that miR‐494 impacts cell cycle regulation. Cell cycle analyses demonstrated that miR‐494 induces a significant G1/S checkpoint reinforcement. Further analyses demonstrated that miR‐494 down‐regulates multiple molecules involved in this transition checkpoint. Luciferase reporter assays demonstrated a direct interaction between miR‐494 and the 3′‐untranslated region of cyclin‐dependent kinase 6 (CDK6). Last, xenograft experiments demonstrated that miR‐494 induces a significant cancer growth retardation in vivo. Conclusion: Our findings demonstrate that miR‐494 is down‐regulated in CCA and that its up‐regulation induces cancer cell growth retardation through multiple targets involved in the G1‐S transition. These findings support the paradigm that miRs are salient cellular signaling pathway modulators, and thus represent attractive therapeutic targets. miR‐494 emerges as an important regulator of CCA growth and its further study may lead to the development of novel therapeutics. (HEPATOLOGY 2011)

Leptin enhances the effect of transforming growth factor β in increasing type I collagen formation

Hepatic fibrosis produced by carbon tetrachloride and by Schistosoma masoni is markedly decreased in leptin deficient ob/ob mice as compared to control mice. Leptin is present in activated rat stellate cells, which are the principal collagen producing cells in the liver. The purpose of this study was to identify the leptin receptor and to determine the effects of leptin on type I collagen expression in the human stellate cell line, LX-1. Leptin protein was detected in the LX-1 cells. The leptin receptor (OB-R) was demonstrated by immunofluorescent staining and confocal microscopy. However, only the short forms (Ob-R(s)), but not the long forms (Ob-R(l)), of leptin receptor mRNA expression were detected. Leptin increased alpha(1)(I) collagen mRNA and type I collagen production. Leptin did not increase TGFbeta1 mRNA or protein in the cultured LX-1 cells. Leptin, however, increased TGFbeta type II receptor mRNA and protein and augmented the effect of TGFbeta1 on collagen production. In conclusion, this study shows that the effect of leptin in increasing type I collagen production in stellate cells is mediated by actions of leptin in increasing the effectiveness of TGFbeta on fibrogenesis by means of an enhancement of the TGFbeta type II receptor.

Coordinated effects of microRNA-494 induce G2/M arrest in human cholangiocarcinoma

MicroRNA (miRs) have emerged as salient regulators in cancer homeostasis and, recently, as putative therapeutics. Cholangiocarcinomas (CCA) are aggressive cancers with survival usually measured in months. mRNA arrays followed by pathway analysis revealed that miR-494 is a major modulator of the cell cycle progression from gap 2 (G₂) to mitosis (M). We performed fluorescence activated cell sorting (FACS) as well as differential interference contrast (DIC) microscopy, and confirmed that miR-494 induces a significant arrest in G₂/M in CCA cells. Furthermore, we verified that miR-494 modulates the protein level of six genes involved in the G₂/M transition: Polo-like Kinase 1 (PLK1), pituitary tumor-transforming gene 1 (PTTG1), Cyclin B1 (CCNB1), cell-division cycle 2 (CDC2), cell-division cycle 20 (CDC20) and topoisomerase II α (TOP2A). Next, we identified direct binding of miR-494 to the open reading frame (ORF) and downregulation of PTTG1 and TOP2A. In summary, our findings suggest that miR-494 has a global regulatory role in cell cycle progression, exerted by concerted effects on multiple proteins involved in gap 1 (G₁) to synthesis (S), as described previously, as well as G₂ to M progression. Therefore, it appears that the simultaneous effects of a single miR species on multiple targets along the same canonical pathway is advantageous for the usage of miRs as therapeutics. In addition, our data suggest that miRs act within a narrow range. miR expression above the upper threshold does not appear to induce further effects, which is reassuring in terms of off-target effects of miR surrounding noncancerous tissue.