title: Sequential Functions of CPEB1 and CPEB4 Regulate Pathologic Expression of Vascular Endothelial Growth Factor and Angiogenesis in Chronic Liver Disease.
abstract: OBJECTIVE
  Vascular endothelial growth factor (VEGF) regulates angiogenesis, yet therapeutic strategies to disrupt VEGF signaling can interfere with physiologic angiogenesis. In a search for ways to inhibit pathologic production or activities of VEGF without affecting its normal production or functions, we investigated the post-transcriptional regulation of VEGF by the cytoplasmic polyadenylation element-binding proteins CPEB1 and CPEB4 during development of portal hypertension and liver disease.
  
  
  METHODS
  We obtained transjugular liver biopsies from patients with hepatitis C virus-associated cirrhosis or liver tissues removed during transplantation; healthy human liver tissue was obtained from a commercial source (control). We also performed experiments with male Sprague-Dawley rats and CPEB-deficient mice (C57BL6 or mixed C57BL6/129 background) and their wild-type littermates. Secondary biliary cirrhosis was induced in rats by bile duct ligation, and portal hypertension was induced by partial portal vein ligation. Liver and mesenteric tissues were collected and analyzed in angiogenesis, reverse transcription polymerase chain reaction, polyA tail, 3' rapid amplification of complementary DNA ends, Southern blot, immunoblot, histologic, immunohistochemical, immunofluorescence, and confocal microscopy assays. CPEB was knocked down with small interfering RNAs in H5V endothelial cells, and translation of luciferase reporters constructs was assessed.
  
  
  RESULTS
  Activation of CPEB1 promoted alternative nuclear processing within noncoding 3'-untranslated regions of VEGF and CPEB4 messenger RNAs in H5V cells, resulting in deletion of translation repressor elements. The subsequent overexpression of CPEB4 promoted cytoplasmic polyadenylation of VEGF messenger RNA, increasing its translation; the high levels of VEGF produced by these cells led to their formation of tubular structures in Matrigel assays. We observed increased levels of CPEB1 and CPEB4 in cirrhotic liver tissues from patients, compared with control tissue, as well as in livers and mesenteries of rats and mice with cirrhosis or/and portal hypertension. Mice with knockdown of CPEB1 or CPEB4 did not overexpress VEGF or have signs of mesenteric neovascularization, and developed less-severe forms of portal hypertension after portal vein ligation.
  
  
  CONCLUSIONS
  We identified a mechanism of VEGF overexpression in liver and mesentery that promotes pathologic, but not physiologic, angiogenesis, via sequential and nonredundant functions of CPEB1 and CPEB4. Regulation of CPEB4 by CPEB1 and the CPEB4 autoamplification loop induces pathologic angiogenesis. Strategies to block the activities of CPEBs might be developed to treat chronic liver and other angiogenesis-dependent diseases.
authors.affiliation: Program of Molecular Medicine, Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain.
authors.forename: Vittorio
authors.initials: V
authors.lastname: Calderone
authors.affiliation: Program of Liver, Digestive System and Metabolism, IDIBAPS Biomedical Research Institute, CIBERehd, University of Barcelona, Barcelona, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Gallego
authors.affiliation: Program of Molecular Medicine, Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain.
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Fernandez-Miranda
authors.affiliation: Program of Liver, Digestive System and Metabolism, IDIBAPS Biomedical Research Institute, CIBERehd, University of Barcelona, Barcelona, Spain.
authors.forename: Ester
authors.initials: E
authors.lastname: Garcia-Pras
authors.affiliation: Program of Molecular Medicine, Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Maillo
authors.affiliation: Program of Liver, Digestive System and Metabolism, IDIBAPS Biomedical Research Institute, CIBERehd, University of Barcelona, Barcelona, Spain.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Berzigotti
authors.affiliation: Program of Liver, Digestive System and Metabolism, IDIBAPS Biomedical Research Institute, CIBERehd, University of Barcelona, Barcelona, Spain.
authors.forename: Marc
authors.initials: M
authors.lastname: Mejias
authors.affiliation: Program of Molecular Medicine, Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain.
authors.forename: Felice-Alessio
authors.initials: FA
authors.lastname: Bava
authors.affiliation: Program of Cancer and Human Molecular Genetics, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Angulo-Urarte
authors.affiliation: Program of Cancer and Human Molecular Genetics, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain.
authors.forename: Mariona
authors.initials: M
authors.lastname: Graupera
authors.affiliation: Program of Cancer, Hospital del Mar Research Institute (IMIM), Barcelona, Spain.
authors.forename: Pilar
authors.initials: P
authors.lastname: Navarro
authors.affiliation: Program of Liver, Digestive System and Metabolism, IDIBAPS Biomedical Research Institute, CIBERehd, University of Barcelona, Barcelona, Spain.
authors.forename: Jaime
authors.initials: J
authors.lastname: Bosch
authors.affiliation: Program of Liver, Digestive System and Metabolism, IDIBAPS Biomedical Research Institute, CIBERehd, University of Barcelona, Barcelona, Spain. Electronic address: mercefernandez@ub.edu.
authors.forename: Mercedes
authors.initials: M
authors.lastname: Fernandez
authors.affiliation: Program of Molecular Medicine, Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain; Institucio Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain. Electronic address: raul.mendez@irbbarcelona.org.
authors.forename: Raul
authors.initials: R
authors.lastname: Mendez
mesh_terms: D020413:3' Untranslated Regions; D000328:Adult; D000818:Animals; D016022:Case-Control Studies; D002460:Cell Line; D002908:Chronic Disease; D004195:Disease Models, Animal; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D006975:Hypertension, Portal / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008103:Liver Cirrhosis / Q000378:metabolism* / Q000473:pathology / Q000821:virology; D008105:Liver Cirrhosis, Biliary / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008297:Male; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D008875:Middle Aged; D009389:Neovascularization, Pathologic; D026723:Polyadenylation; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013997:Time Factors; D014157:Transcription Factors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D039221:mRNA Cleavage and Polyadenylation Factors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
chemical_list: D020413:3' Untranslated Regions; C490386:CPEB1 protein, human; C539243:CPEB1 protein, rat; C554939:CPEB4 protein, human; C554943:CPEB4 protein, rat; C490387:Cpeb1 protein, mouse; C477604:Cpeb4 protein, mouse; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014157:Transcription Factors; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D039221:mRNA Cleavage and Polyadenylation Factors; C467480:vascular endothelial growth factor A, mouse; C467498:vascular endothelial growth factor A, rat
keywords: Alternative RNA Processing; CPEB; Cytoplasmic Polyadenylation; Portal Hypertension