title: VEGF-induced retinal angiogenic signaling is critically dependent on Ca²⁺ signaling by Ca²⁺/calmodulin-dependent protein kinase II.
abstract: OBJECTIVE
  The authors conducted an in vitro investigation of the role of Ca(2+)-dependent signaling in vascular endothelial growth factor (VEGF)-induced angiogenesis in the retina.
  
  
  METHODS
  Bovine retinal endothelial cells (BRECs) were stimulated with VEGF in the presence or absence of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM; intracellular Ca(2+) chelator), U73122 (phospholipase C (PLC) inhibitor), xestospongin C (Xe-C), and 2-aminoethoxydiphenyl borate (2APB) (inhibitors of inositol-1,4,5 triphosphate (IP(3)) signaling). Intracellular Ca(2+) concentration ([Ca(2+)](i)) was estimated using fura-2 Ca(2+) microfluorometry, Akt phosphorylation quantified by Western blot analysis, and angiogenic responses assessed using cell migration, proliferation, tubulogenesis, and sprout formation assays. The effects of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 were also evaluated on VEGF-induced Akt signaling and angiogenic activity.
  
  
  RESULTS
  Stimulation of BRECs with 25 ng/mL VEGF induced a biphasic increase in [Ca(2+)](i), with an initial transient peak followed by a sustained plateau phase. VEGF-induced [Ca(2+)](i) increases were almost completely abolished by pretreating the cells with BAPTA-AM, U73122, Xe-C, or 2APB. These agents also inhibited VEGF-induced phosphorylation of Akt, cell migration, proliferation, tubulogenesis, and sprouting angiogenesis. KN93 was similarly effective at blocking the VEGF-induced activation of Akt and angiogenic responses.
  
  
  CONCLUSIONS
  VEGF increases [Ca(2+)](i) in BRECs through activation of the PLC-IP(3) signal transduction pathway. VEGF-induced phosphorylation of the proangiogenic protein Akt is critically dependent on this increase in [Ca(2+)](i) and the subsequent activation of CaMKII. Pharmacologic inhibition of Ca(2+)-mediated signaling in retinal endothelial cells blocks VEGF-induced angiogenic responses. These results suggest that the PLC/IP(3)/Ca(2+)/CaMKII signaling pathway may be a rational target for the treatment of angiogenesis-related disorders of the eye.
authors.affiliation: Centre for Vision and Vascular Science, School of Medicine, Dentistry and Biomedical Sciences, The Queen's University of Belfast, Institute of Clinical Sciences, The Royal Victoria Hospital, Belfast, Northern Ireland.
authors.forename: Elayappan
authors.initials: E
authors.lastname: Banumathi
authors.forename: Anna
authors.initials: A
authors.lastname: O'Connor
authors.forename: Sangiliyandi
authors.initials: S
authors.lastname: Gurunathan
authors.forename: David A
authors.initials: DA
authors.lastname: Simpson
authors.forename: J Graham
authors.initials: JG
authors.lastname: McGeown
authors.forename: Tim M
authors.initials: TM
authors.lastname: Curtis
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001896:Boron Compounds / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002417:Cattle; D002465:Cell Movement / Q000502:physiology; D049109:Cell Proliferation; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D004963:Estrenes / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000037:antagonists & inhibitors / Q000378:metabolism; D047028:Macrocyclic Compounds / Q000494:pharmacology; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology*; D010080:Oxazoles / Q000494:pharmacology; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism; D011760:Pyrrolidinones / Q000494:pharmacology; D012171:Retinal Vessels / Q000166:cytology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*
chemical_list: D001896:Boron Compounds; D004791:Enzyme Inhibitors; D004963:Estrenes; D047028:Macrocyclic Compounds; D010080:Oxazoles; D011760:Pyrrolidinones; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C511704:xestospongin C; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D015544:Inositol 1,4,5-Trisphosphate; C109986:2-aminoethoxydiphenyl borate; D051057:Proto-Oncogene Proteins c-akt; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D010738:Type C Phospholipases; D002118:Calcium