title: The soluble guanylyl cyclase inhibitor NS-2028 reduces vascular endothelial growth factor-induced angiogenesis and permeability.
abstract: Nitric oxide (NO) is known to promote vascular endothelial growth factor (VEGF)-stimulated permeability and angiogenesis. However, effector molecules that operate downstream of NO in this pathway remain poorly characterized. Herein, we determined the effect of soluble guanylyl cyclase (sGC) inhibition on VEGF responses in vitro and in vivo. Treatment of endothelial cells (EC) with VEGF stimulated eNOS phosphorylation and cGMP accumulation; pretreatment with the sGC inhibitor 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS-2028) blunted cGMP levels without affecting VEGF-receptor phosphorylation. Incubation of cells with NS-2028 blocked the mitogenic effects of VEGF. In addition, cells in which sGC was inhibited exhibited no migration and sprouting in response to VEGF. To study the mechanisms through which NS-2028 inhibits EC migration, we determined the effects of alterations in cGMP levels on p38 MAPK. Initially, we observed that inhibition of sGC attenuated VEGF-stimulated activation of p38. In contrast, the addition of 8-Br-cGMP to EC stimulated p38 phosphorylation. The addition of cGMP elevating agents (BAY 41-2272, DETA NO and YC-1) enhanced EC migration. To test whether sGC also mediated the angiogenic effects of VEGF in vivo, we used the rabbit cornea assay. Animals receiving NS-2028 orally displayed a reduced angiogenic response to VEGF. As increased vascular permeability occurs prior to new blood vessel formation, we determined the effect of NS-2028 in vascular leakage. Using a modified Miles assay, we observed that NS-2028 attenuated VEGF-induced permeability. Overall, we provide evidence that sGC mediates the angiogenic and permeability-promoting activities of VEGF, indicating the significance of sGC as a downstream effector of VEGF-triggered responses.
authors.affiliation: Department of Molecular Biology, Section of Pharmacology, University of Siena, Siena, Italy.
authors.forename: Lucia
authors.initials: L
authors.lastname: Morbidelli
authors.forename: Anastasia
authors.initials: A
authors.lastname: Pyriochou
authors.forename: Sandra
authors.initials: S
authors.lastname: Filippi
authors.forename: Ioannis
authors.initials: I
authors.lastname: Vasileiadis
authors.forename: Charis
authors.initials: C
authors.lastname: Roussos
authors.forename: Zongmin
authors.initials: Z
authors.lastname: Zhou
authors.forename: Heleni
authors.initials: H
authors.lastname: Loutrari
authors.forename: Johannes
authors.initials: J
authors.lastname: Waltenberger
authors.forename: Anne
authors.initials: A
authors.lastname: Stössel
authors.forename: Athanassios
authors.initials: A
authors.lastname: Giannis
authors.forename: Marina
authors.initials: M
authors.lastname: Ziche
authors.forename: Andreas
authors.initials: A
authors.lastname: Papapetropoulos
mesh_terms: D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000502:physiology; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003315:Cornea / Q000098:blood supply; D004347:Drug Interactions; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology*; D010069:Oxadiazoles / Q000494:pharmacology*; D010078:Oxazines / Q000494:pharmacology*; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000071756:Soluble Guanylyl Cyclase; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology; D014664:Vasodilation / Q000187:drug effects / Q000502:physiology; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; C111257:NS 2028; D010069:Oxadiazoles; D010078:Oxazines; D018160:Receptors, Cytoplasmic and Nuclear; D042461:Vascular Endothelial Growth Factor A; D016222:Fibroblast Growth Factor 2; D048051:p38 Mitogen-Activated Protein Kinases; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase