title: Endothelin-1 cooperates with hypoxia to induce vascular-like structures through vascular endothelial growth factor-C, -D and -A in lymphatic endothelial cells.
abstract: OBJECTIVE
  Lymphangiogenesis refers to the formation of new lymphatic vessels and is thought to constitute conduits for the tumor cells to metastasize. We previously demonstrated that endothelin (ET)-1 through its binding with ETB receptor (ET(B)R) expressed on lymphatic endothelial cells (LEC), induced cell growth and invasiveness. Since vascular endothelial growth factor (VEGF)-A/-C/-D, and hypoxia play key role in lymphatic differentiation, in this study we investigated the involvement of these growth factors and hypoxia in ET-1-induced lymphangiogenesis.
  
  
  METHODS
  Real time PCR and ELISA were used to quantify VEGF-A/-C/-D. LEC morphological differentiation was analyzed by tube formation assay on Matrigel.
  
  
  RESULTS
  Hypoxia, as well as ET-1, induced an increase in VEGF-A/-C and -D expression that was reduced in the presence of a selective ET(B)R antagonist, BQ788, and enhanced when ET-1 was administered under hypoxic conditions. We analyzed the role of hypoxia on LEC morphological differentiation, and found that hypoxia increased the formation of vascular-like structures on Matrigel and that in combination with ET-1 this effect was markedly enhanced. The use of specific antibodies neutralizing VEGF-A, or recombinant VEGFR-3/(Flt-4)/Fc that block VEGF-C/-D, inhibited the effect of ET-1 as well that of hypoxia.
  
  
  CONCLUSIONS
  These results demonstrated that ET-1 and hypoxia act, at list in part, through VEGF to induce lymphangiogenic events and that these two stimuli may cooperate to induce VEGF-A/-C/-D expression and lymphatic differentiation. These data further support the role of ET-1 as potent lymphangiogenic factor that relies on the interplay with hypoxic microenvironment and with VEGF family members.
authors.affiliation: Microbiology Section, Department of Experimental Medicine, University of Brescia, Italy.
authors.forename: Emirena
authors.initials: E
authors.lastname: Garrafa
authors.forename: Valentina
authors.initials: V
authors.lastname: Caprara
authors.forename: Valeriana
authors.initials: V
authors.lastname: Di Castro
authors.forename: Laura
authors.initials: L
authors.lastname: Rosanò
authors.forename: Anna
authors.initials: A
authors.lastname: Bagnato
authors.forename: Francesca
authors.initials: F
authors.lastname: Spinella
mesh_terms: D002454:Cell Differentiation; D015687:Cell Hypoxia; D042783:Endothelial Cells / Q000378:metabolism; D065131:Endothelin B Receptor Antagonists; D019332:Endothelin-1 / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D006801:Humans; D008198:Lymph Nodes / Q000166:cytology / Q000378:metabolism; D042583:Lymphangiogenesis / Q000502:physiology*; D009842:Oligopeptides / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D044023:Receptor, Endothelin B / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*; D042582:Vascular Endothelial Growth Factor C / Q000235:genetics / Q000378:metabolism*; D042643:Vascular Endothelial Growth Factor D / Q000235:genetics / Q000378:metabolism*
chemical_list: D065131:Endothelin B Receptor Antagonists; D019332:Endothelin-1; D009842:Oligopeptides; D010880:Piperidines; D044023:Receptor, Endothelin B; C467484:VEGFA protein, human; C467486:VEGFC protein, human; D042461:Vascular Endothelial Growth Factor A; D042582:Vascular Endothelial Growth Factor C; D042643:Vascular Endothelial Growth Factor D; C086539:BQ 788