title: Glial cell-induced endothelial morphogenesis is inhibited by interfering with extracellular signal-regulated kinase signaling.
abstract: OBJECTIVE
  Tumor vasculature provides the infrastructure by which malignant tissue can be nourished; therefore, targeting angiogenesis may be an effective means of treating cancer. We showed previously that SNB19 glioblastoma cells modulate bovine retinal endothelial cells in cocultures to form capillary-like network structures, that matrix metalloproteinase-9 (MMP-9) expression is critical for endothelial morphogenesis, and that MMP-9 expression in glioblastoma cells is regulated by extracellular signal-regulated kinase-1 (ERK-1). In the present study, we investigated whether interfering with the activation of this mitogen-activated protein (MAP) kinase would repress MMP-9 synthesis and inhibit capillary formation.
  
  
  METHODS
  Cocultures of bovine retinal endothelial and SNB19 cells were analyzed for MMP-9 secretion, and phospho- and total ERK levels. These cocultures were treated with PD98059, a specific inhibitor of MAP/ERK kinase 1, or transfected with dominant-negative ERK-1 mutant containing expression vector. Alterations in capillary-like structure formation, and actin cytoskeleton and secretion of vascular endothelial growth factor (VEGF), MMP-9, and tissue inhibitor of metalloproteinase-1 were determined by immunofluorescence, gelatin zymography, and Western blotting.
  
  
  RESULTS
  We found that inhibition of the ERK-1/2 pathway with PD98059 abrogated glial cell-mediated capillary formation by the endothelial cells and reduced the levels of MMP-9 in the coculture. Strikingly, the abrogation of MAP kinase signaling by a dominant-negative ERK-1 mutant inhibited glial-induced capillary network formation by reducing VEGF levels and MMP-9 activity and increasing the levels of tissue inhibitor of metalloproteinase-1. Inhibition of ERK activity also disrupted the formation of the actin cytoskeleton, a prerequisite for endothelial cell migration.
  
  
  CONCLUSIONS
  The mechanism underlying activation of ERK is involved in reorganization of the actin cytoskeleton, and induction of VEGF and MMP-9, thereby stimulating endothelial cell morphogenesis. These studies clearly provide experimental evidence that ERK inhibition diminishes glial-induced endothelial-cell morphogenesis; therefore, interfering with ERK signaling may be a viable approach to target angiogenesis.
authors.affiliation: Program of Cancer Biology, University of Illinois College of Medicine at Peoria, Peoria, Illinois 61656, USA.
authors.forename: Nirmala
authors.initials: N
authors.lastname: Chandrasekar
authors.forename: Sanjeeva
authors.initials: S
authors.lastname: Mohanam
authors.forename: S Sajani
authors.initials: SS
authors.lastname: Lakka
authors.forename: Dzung H
authors.initials: DH
authors.lastname: Dinh
authors.forename: William C
authors.initials: WC
authors.lastname: Olivero
authors.forename: Meena
authors.initials: M
authors.lastname: Gujrati
authors.forename: Jasti S
authors.initials: JS
authors.lastname: Rao
mesh_terms: D000199:Actins / Q000737:chemistry; D000818:Animals; D002417:Cattle; D002450:Cell Communication; D042783:Endothelial Cells / Q000502:physiology*; D005786:Gene Expression Regulation; D020780:Matrix Metalloproteinase 9 / Q000235:genetics; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D018919:Neovascularization, Physiologic; D009457:Neuroglia / Q000502:physiology*; D010766:Phosphorylation; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000096:biosynthesis; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics
chemical_list: D000199:Actins; D019715:Tissue Inhibitor of Metalloproteinase-1; D042461:Vascular Endothelial Growth Factor A; D020928:Mitogen-Activated Protein Kinases; D020780:Matrix Metalloproteinase 9