Martinez-Anso, E. (Eduardo)

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    Blockade of Wnt signaling inhibits angiogenesis and tumor growth in hepatocellular carcinoma
    (American association for cancer research, 2009) Shan, J. (Juanjuan); Kawa, M. (Milosz); Qian, C. (Cheng); Dong, A. (Aiwen); Hu, J. (Jie); Prieto, J. (Jesús); Fernandez-Ruiz, V. (Verónica); Martinez-Anso, E. (Eduardo)
    Aberrant activation of Wnt signaling plays an important role in hepatocarcinogenesis. In addition to direct effects on tumor cells, Wnt signaling might be involved in the organization of tumor microenvironment. In this study, we have explored whether Wnt signaling blockade by exogenous expression of Wnt antagonists could inhibit tumor angiogenesis and control tumor growth. Human Wnt inhibitory factor 1 (WIF1) and secreted frizzled-related protein 1 (sFRP1) were each fused with Fc fragment of human IgG1 to construct fusion proteins WIF1-Fc and sFRP1-Fc. The recombinant adenoviral vectors carrying WIF1-Fc and sFRP1-Fc driven by cytomegalovirus promoter were constructed. Ad-WIF1-Fc or Ad-sFRP1-Fc induced the expression and correct conformation of WIF1-Fc and sFRP1-Fc fusion proteins. These molecules caused down-regulation of E2F1, cyclin D1, and c-myc and promoted cell apoptosis in hepatocellular carcinoma cells. Treatment of established hepatocellular carcinoma tumors with Ad-WIF1-Fc and/or Ad-sFRP1-Fc resulted in significant inhibition of tumor growth and prolonged animal survival. The antineoplastic effect was associated with increased apoptosis of tumor cells, reduced microvessel density, and decreased expression of vascular endothelial growth factor and stromal cell-derived factor-1. Tube formation and migration of human microvascular endothelial cells and mouse endothelial progenitor cells (EPC) were significantly inhibited by both WIF1-Fc and sFRP1-Fc. In addition, these molecules blocked EPC differentiation and caused EPC apoptosis. Our data indicate that Wnt antagonists WIF1-Fc and sFRP1-Fc inhibit Wnt signaling and exert potent antitumor activity by increasing the apoptosis rate in tumor cells and by impairing tumor vascularization.
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    Interplay among cardiotrophin-1, prostaglandins, and vascular endothelial growth factor in rat liver regeneration
    (Wiley Blackwell, 2005) Beraza, N. (Naiara); Prieto, J. (Jesús); Bustos, M. (Matilde); Iñiguez, M. (María); Marques, J.M. (Juan Martín); Martinez-Anso, E. (Eduardo)
    Prostaglandins are hepatoprotective molecules generated in liver regeneration by the rapid induction of cyclooxygenase-2 (COX-2). Cardiotrophin-1 (CT-1) and vascular endothelial growth factor (VEGF) are other hepatoprotective mediators upregulated at 24 hours after partial hepatectomy. The interactions among these molecules during liver regeneration have not yet been defined. Here we show that rats subjected to partial hepatectomy treated with NS-398, a specific COX-2 inhibitor, exhibited cell cycle arrest, increased hepatocyte apoptosis, persistent extracellular signal-regulated kinase (ERK) 1/2 activation, and increased interleukin-6 production. These changes were associated with downregulation of CT-1 and COX-1 and altered pattern of VEGF expression. Administration of an adenovirus encoding CT-1 to NS-398-treated rats restituted normal levels of COX-1, prostaglandins, and VEGF in the liver after partial hepatectomy and restored normal liver regeneration. Furthermore, the stimulation of isolated rat hepatocytes with CT-1 increased COX-1, COX-2, and VEGF messenger RNAs and prostaglandin synthesis. Conversely, the addition of prostaglandin E1 to the culture increased CT-1 and VEGF production. In conclusion, COX-2 activation and production of prostaglandins soon after partial hepatectomy are essential requirements for hepatocyte proliferation and for the correct induction of both CT-1 and VEGF. CT-1 can restore liver regeneration after COX-2 inhibition by increasing VEGF, COX-1 expression, and prostaglandin synthesis.
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    Decreased cardiotrophin-1 levels are associated with a lower risk of developing the metabolic syndrome in overweight/obese children after a weight loss program
    (Elsevier, 2013) Martinez, J.A. (José Alfredo); Moreno-Aliaga, M. J. (María Jesús); Azcona-San-Julian, M.C. (María Cristina); Garcia-Calzon, S. (Sonia); Rendo-Urteaga, T. (Tara); Bustos, M. (Matilde); Chueca, M. (María); Oyarzabal, M. (M.); Martinez-Anso, E. (Eduardo); Marti-del-Moral, A. (Amelia)
    Objective: Cardiotrophin-1 (CT-1) shares some similarities with other cytokines, and participates in the control of energy metabolism. Higher circulating levels are observed in obese humans, but little information is gathered in weight loss (WL) programs. Therefore, we aimed to investigate the association of serum CT-1 levels with metabolic variables and the risk of developing metabolic syndrome (MetS) after a WL program in overweight/obese children. Subjects and Methods: Forty-four overweight/obese children (mean age 11.5 yr; 50% males) undergoing a 10-week WL program were enrolled. Subjects were dichotomized at the median of Body Mass Index-Standard Deviation Score (BMI-SDS) change, as high and low responders after intervention. Results: CT-1 levels were significantly reduced (-48 fmol/mL, p=0.043) in the high responder group after the WL program. They had significantly lower body weight (-3.7 kg, p<0.001), body fat mass (-8%, p<0.001), BMI-SDS (-0.78, p<0.001) and waist circumference (-5.4 cm, p<0.001), and a significant improvement in lipid and glucose profiles (p<0.05). Interestingly, decreased CT-1 levels significantly predicted changes in total cholesterol (41%) and LDL-cholesterol (28%). Moreover, in our participants the lower the CT-1 levels, the higher the reduction in MetS risk components, after the 10- week intervention, (p-ANCOVA=0.040, p-trend=0.024). Conclusion: We showed, for the first time, a reduction in serum CT-1 levels after a WL program and this decrease in CT-1 was strongly associated with a reduction in cholesterol levels and in MetS risk factors in overweight/obese children. Our findings may suggest that CT-1 could be an indirect marker for the diagnosis of MetS in this population.
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    In situ localization of anion exchanger-2 in the human kidney
    (Springer Verlag, 2000) Alava, E. (Enrique) de; Garcia-Corchon, C. (C.); Medina, J.F. (Juan Francisco); Raquel; Prieto, J. (Jesús); Martinez-Anso, E. (Eduardo); Castillo, J. (José E.)
    Na+-independent anion exchangers (AE) are a family of membrane carriers that mediate the electroneutral exchange of Cl- for HCO3- ions across plasma membranes. They are involved in intracellular pH and cell volume regulation as well as in transepithelial acid-base transport. While anion exchanger-1 (AE1) has been localized previously in the human kidney, thus far there has been no definite report on anion exchanger-2 (AE2) in this human tissue. Accordingly, immunohistochemistry was carried out on surgical specimens of the human kidney (fixed in formalin and embedded in paraffin), using a specific AE2 monoclonal antibody. Strong immunostaining was observed at the basolateral membrane of cells of thick ascending limbs and distal convoluted tubules, colocalizing with the basal membranous labyrinth of cellular interdigitations, typical of these segments. In fact, AE2 staining was attenuated at the macula densa, where basal infoldings are scarce. Additionally, in situ hybridization experiments on formalin-fixed tissue demonstrated the presence of AE2 mRNA in the same segments of the distal nephron. On the other hand, control immunohistochemistry with a monoclonal antibody against AE1 gave the expected immunoreactivity at the basal pole of the type A intercalated cells of connecting tubules and cortical collecting ducts, and in erythrocytes. Our results indicate that, depending on the nephron segment and corresponding cell types, AE1 and AE2 proteins are differentially involved in the Na+-independent exchange of Cl- for HCO3- at the basolateral membrane of polarized kidney epithelial cells.
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    Treatment of murine fulminant hepatitis with genetically engineered endothelial progenitor cells
    (Elsevier, 2011) Berasain, C. (Carmen); D'Avola, D. (Delia); Kawa, M. (Milosz); Qian, C. (Cheng); Sangro, B. (Bruno); Iñarrairaegui, M. (Mercedes); Prieto, J. (Jesús); Herrero, J.I. (José Ignacio); Quiroga, J. (Jorge); Schmitz, V. (Volker); Iñiguez, M. (María); Fernandez-Ruiz, V. (Verónica); Martinez-Anso, E. (Eduardo)
    BACKGROUND & AIMS: Cell therapy has been used to attenuate liver injury. Here we evaluated whether genetic engineering of either bone marrow-derived mononuclear cells (MNC) or endothelial progenitor cells (EPC) many enhance their hepatoprotective properties. METHODS: Mice with ConA-induced hepatitis or with lethal fulminant hepatitis resulting from administration of an adenovirus encoding CD40L (AdCD40L) received an intra-splenic injection of saline or 2 × 10(6) unmodified MNC or EPC or the same cells transduced ex vivo with an adenovirus expressing luciferase (MNCLUC and EPCLUC) or encoding the hepatoprotective cytokine cardiotrophin-1 (CT-1) (MNCCT-1 and EPCCT-1). We analyzed the extent of liver damage, the intensity of inflammatory reaction, and animal survival. RESULTS: Luciferase immunohistochemistry showed that after injection into the spleen, the engineered cells migrated efficiently to the damaged liver. In mice with ConA hepatitis EPCCT-1, but not other forms of cell therapy, significantly decreased serum transaminases and induced more intense histological improvement than other treatments. This superior therapeutic effect was associated with upregulation of cytoprotective molecules including IGF-I and EGF, lower expression of proinflammatory cytokines, IL-1b and TNFα, and decreased granzyme B levels. In AdCD40L-induced lethal fulminant hepatitis, EPCCT-1 also exceeded other cell therapies in attenuating the expression of proinflammatory mediators and hepatic injury enabling 35.7% survival while mortality was 100% in the other treatment groups. CONCLUSIONS: Genetic engineering of EPC to overexpress CT-1 enhances the hepatoprotective properties of EPC and constitutes a therapy that deserves consideration for acute liver failure. Copyright © 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
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    Presence of leptin receptors in rat small intestine and leptin effect on sugar absorption
    (Elsevier, 1998) Martinez, J.A. (José Alfredo); Urdaneta, E. (Elena); Lostao, M.P. (María Pilar); Barber, A. (Ana); Martinez-Anso, E. (Eduardo)
    Leptin is involved in food intake and thermogenesis regulation. Since leptin receptor expression has been found in several tissues including small intestine, a possible role of leptin in sugar absorption by the intestine was investigated. Leptin inhibited D-galactose uptake by rat small intestinal rings 33% after 5 min of incubation. The inhibition increased to 56% after 30 min. However, neither at 5 min nor at 30 min did leptin prevent intracellular galactose accumulation. This leptin effect was accompanied by a decrease of the active sugar transport apparent Vmax (20 vs. 4.8 micromol/g wet weight 5 min) and apparent Km (15.8 vs. 5.3 mM) without any change in the phlorizin-resistant component. On the other hand, immunohistochemical experiments using anti-leptin monoclonal antibodies recognized leptin receptors in the plasma membrane of immune cells located in the lamina propria. These results indicate for the first time that leptin has a rapid inhibitory effect on sugar absorption and demonstrate the presence of leptin receptors in the intestinal mucosa.
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    Effect of a beta3-adrenergic agonist on liver glucokinase gene expression in alloxan-diabetic rats
    (Springer Verlag, 1999) Martinez, J.A. (José Alfredo); Milagro-Yoldi, F.I. (Fermín Ignacio); Gomez-Ambrosi, J. (Javier); Martinez-Anso, E. (Eduardo)
    Beta-adrenergic agonists have been shown to elicit powerful hypoglycemic effects when administered to different animal models of diabetes. However, the intimate mechanism involved in this process remains unclear. In this context, treatment of alloxan-induced diabetic rats with the beta3-adrenergic agonist Trecadrine (1 mg x kg(-1) x day(-1), s. c.) for four days, normalized glycemia with no changes in plasma insulin levels. Liver glucokinase, a key enzyme in the regulation of glucose storage in hepatocytes, whose gene expression is significantly decreased in alloxan-diabetic rodents, showed a recovery in its mRNA levels after Trecadrine administration. These data suggest that beta3-adrenergic agonists enhance glucose storage in liver, probably through a non-insulin dependent mechanism of action.
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    Immunohistochemical detection of chloride/bicarbonate anion exchangers in human liver
    (Wiley Blackwell, 1994) Medina, J.F. (Juan Francisco); Diez-Martinez, J. (Javier); Prieto, J. (Jesús); Martinez-Anso, E. (Eduardo); Castillo, J. (José E.)
    Sodium-independent Cl-/HCO3- exchange activity has been observed in isolated rat hepatocytes and intrahepatic bile duct epithelial cells, where it is involved in intracellular pH regulation and, possibly, biliary bicarbonate secretion. Monoclonal antibodies to the membrane domain of human chloride/bicarbonate anion exchanger proteins, AE1 and AE2, were prepared so that we might determine by immunohistochemical methods the presence and location of these antiporters in the human liver. To obtain the antibody against AE1, we immunized mice with injections of washed human erythrocytes. The selected monoclonal antibody was found to be specific for the 17-kD proteolytic membrane fragment of AE1 protein. The antibody to AE2 was produced with a 14-mer synthetic peptide, whose sequence corresponds specifically to amino acid residues 871 to 884 in the deduced primary structure of human kidney AE2 protein. When the monoclonal antibody to AE2 peptide was employed for the immunohistochemical study of liver specimens (by both immunofluorescence and immunoperoxidase), a clearly defined staining was present at the canalicular membrane of hepatocytes, as well as the luminal side of the membrane of bile duct epithelial cells from small and medium-sized bile ducts. No staining was observed in the liver parenchyma with the monoclonal antibody to AE1, which instead strongly decorated the erythrocytes in liver blood vessels. We conclude that AE2 immunoreactivity is present in human liver, where it localizes very specifically to the membrane regions, which appear most probably involved in the transport of bicarbonate to bile (i.e., the canalicular membrane of hepatocytes and the apical side of epithelial cells of small and medium bile ducts).
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    Decreased anion exchanger 2 immunoreactivity in the liver of patients with primary biliary cirrhosis
    (Wiley Blackwell, 1997) Medina, J.F. (Juan Francisco); Prieto, J. (Jesús); Vazquez, J.J. (Jesús Jaime); Martinez-Anso, E. (Eduardo)
    Chloride-bicarbonate anion exchanger 2 (AE2) is expressed in a variety of tissues, including the liver and salivary glands, where it may participate in the generation of hydroionic fluxes into secretions. We have previously reported decreased hepatic levels of AE2 messenger RNA in patients with primary biliary cirrhosis (PBC), a cholestatic condition frequently associated with pluriglandular exocrine failure. Here we investigated the expression of AE2 protein in the liver of PBC patients. Using a monoclonal antibody against an AE2 peptide, immunohistochemistry was performed on liver biopsy specimens from subjects with normal liver (n = 7), patients with PBC (n = 13), and patients with cirrhosis or cholestasis other than PBC (n = 17 and 11, respectively). Immunostaining was graded from 0 to 7, according to its intensity and distribution. AE2 immunoreactivity was observed in normal livers, as previously reported, and in many pathological liver biopsy specimens, being mainly restricted to canaliculi and the luminal membrane of terminal and interlobular bile ducts. Canalicular and ductular scores were significantly reduced in the PBC group compared with each control group (normal liver and cirrhosis or cholestasis other than PBC), whereas no differences in immunoreactivity scores were observed among control groups. When four patients with primary sclerosing cholangitis (PSC) were analyzed, they also differed from those with PBC. These results suggest that PBC is characterized by diminished expression of AE2 in the liver. Reduced levels of this transporter protein might be involved in the pathogenesis of cholestasis in PBC.
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    Innate functions of immunoglobulin M lessen liver gene transfer with helper-dependent adenovirus
    (2014) Dubrot, J. (Juan); Morales-Kastresana, A. (Aizea); Unzu, C. (Carmen); Serrano-Mendioroz, I. (Irantzu); Azpilicueta, A. (Arantza); Ochoa, M.C. (María Carmen); Melero, I. (Ignacio); Fontanellas-Romá, A. (Antonio); Sampedro, A. (Ana); Martinez-Anso, E. (Eduardo)
    The immune system poses obstacles to viral vectors, even in the first administration to preimmunized hosts. We have observed that the livers of B cell-deficient mice were more effectively transduced by a helper-dependent adenovirus serotype-5 (HDA) vector than those of WT mice. This effect was T-cell independent as shown in athymic mice. Passive transfer of the serum from adenovirus-naïve WT to Rag1KO mice resulted in a reduction in gene transfer that was traced to IgM purified from serum of adenovirus-naïve mice. To ascribe the gene transfer inhibition activity to either adenoviral antigen-specific or antigen-unspecific functions of IgM, we used a monoclonal IgM antibody of unrelated specificity. Both the polyclonal and the irrelevant monoclonal IgM inhibited gene transfer by the HDA vector to either cultured hepatocellular carcinoma cells or to the liver of mice in vivo. Adsorption of polyclonal or monoclonal IgMs to viral capsids was revealed by ELISAs on adenovirus-coated plates. These observations indicate the existence of an inborn IgM mechanism deployed against a prevalent virus to reduce early post-infection viremia. In conclusion, innate IgM binding to adenovirus serotype-5 capsids restrains gene-transfer and offers a mechanism to be targeted for optimization of vector dosage in gene therapy with HDA vectors.