title: Angiogenic actions of angiopoietin-1 require endothelium-derived nitric oxide.
abstract: Angiopoietin1 (Ang1) is a novel angiogenic factor with important actions on endothelial cell (EC) differentiation and vascular maturation. Ang1 has been shown to prevent EC apoptosis through activation of PI3-kinase/Akt, a pathway that is also known to activate endothelium nitric oxide synthase (eNOS). Therefore, we hypothesized that the angiogenic effects of Ang1 would also be dependent on the PI3-kinase/Akt pathway, possibly mediated by increased eNOS activity and NO release. Treatment of human umbilical vein endothelial cells with recombinant Ang1* (300 ng/ml) for 15 minutes resulted in PI3-kinase-dependent Akt phosphorylation, comparable to that observed with vascular endothelial growth factor (VEGF) (50 ng/ml), and increased NO production in a PI3-kinase/Akt-dependent manner. Capillary-like tube formation induced by Ang1* in fibrin matrix at 24 hours (differentiation index, DI: 13.74 +/- 0.76 versus control 1.71 +/- 0.31) was abolished in the presence of the selective PI3-kinase inhibitor, LY294002 (50 micro mol/L) (DI: 0.31 +/- 0.31, P < 0.01) or the NOS inhibitor, L-NAME (3 mmol/L) (DI: 4.10 +/- 0.59, P < 0.01). In subcutaneous Matrigel implants in vivo, addition of recombinant Ang1* or wild-type Ang1 from conditioned media of COS-1 cells transfected with a pFLAG Ang1 expression vector, induced significant neovascularization to a degree similar to VEGF. Finally, angiogenesis in vivo in response to both Ang1 and VEGF was significantly reduced in eNOS-deficient compared with wild-type mice. In summary, our results demonstrate for the first time that endothelial-derived NO is required for Ang1-induced angiogenesis, and that the PI3-kinase signaling mediates the activation of eNOS and NO release in response to Ang1.
authors.affiliation: Terrence Donnelly Heart Center and Division of Cardiology, St. Michael's Hospital, Toronto, Ontario, Canada.
authors.forename: Saeid
authors.initials: S
authors.lastname: Babaei
authors.forename: Krystyna
authors.initials: K
authors.lastname: Teichert-Kuliszewska
authors.forename: Qiuwang
authors.initials: Q
authors.lastname: Zhang
authors.forename: Nina
authors.initials: N
authors.lastname: Jones
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Dumont
authors.forename: Duncan J
authors.initials: DJ
authors.lastname: Stewart
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D043925:Angiogenesis Inducing Agents / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D042683:Angiopoietin-1; D000818:Animals; D019556:COS Cells; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009025:Morpholines / Q000494:pharmacology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D018919:Neovascularization, Physiologic / Q000187:drug effects; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D042787:Receptor, TIE-2; D014162:Transfection; D000077191:Wortmannin
chemical_list: C467630:ANGPT1 protein, human; D000730:Androstadienes; D043925:Angiogenesis Inducing Agents; D042683:Angiopoietin-1; C467628:Angpt1 protein, mouse; D002867:Chromones; D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D009569:Nitric Oxide; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D019001:Nitric Oxide Synthase; D020794:Receptor Protein-Tyrosine Kinases; D042787:Receptor, TIE-2; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019331:NG-Nitroarginine Methyl Ester; D000077191:Wortmannin