ABSTRACT
Title
New tools for the study of cGMP dependent functions: validation in VEGF induced permeability and angiogenesis
Authors
L. Morbidelli1,2, M. Monti2, R. Solito2, F. Finetti1, A. Papapetropoulos3 and M. Ziche1
1Department of Biotechnology, Section of Pharmacology, University of Siena, Siena, and 2Noxamet srl, via A. Moro 2 Siena, Italy; and 3Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, Patras, Greece.
1Department of Biotechnology, Section of Pharmacology, University of Siena, Siena, and 2Noxamet srl, via A. Moro 2 Siena, Italy; and 3Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, Patras, Greece.
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) and cGMP-dependent protein kinase I (PKG-I) inhibition on VEGF responses in vitro and in vivo.
Treatment of endothelial cells (EC) with VEGF stimulated eNOS phosphorylation and cGMP accumulation; pre-treatment 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 and the peptides DT-2 or DT3 (selective PKG-I inhibitors) blocked the mitogenic effects of VEGF. In addition, cells in which sGC and PKG-I were inhibited exhibited no migration and sprouting in response to VEGF. By studying the mechanisms through which NS-2028 and DT2/DT3 inhibited EC functions, we found that p38 and VASP were involved. To test whether sGC also mediated the angiogenic effects of VEGF in vivo, we used the rabbit cornea assay. Animals receiving NS-2028 orally and DT3 locally displayed a reduced neovascular response to VEGF. As increased vascular permeability occurs prior to new blood vessel formation, we determined the effect of NS-2028 and DT3 in vascular leakage. Using a modified Miles assay we observed that NS-2028 and DT3 attenuated VEGF–induced permeability.
Overall, we provide evidence that sGC and PKG-I mediates the angiogenic and permeability-promoting activities of VEGF, indicating the significance of sGC and PKG-I as a downstream effector of VEGF-triggered responses.
1: Morbidelli L, et al . Am J Physiol Regul Integr Comp Physiol. 2010;298(3):R824-32.
2: Koika V, et al. Vascul Pharmacol. 2010;53(5-6):215-22.
Treatment of endothelial cells (EC) with VEGF stimulated eNOS phosphorylation and cGMP accumulation; pre-treatment 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 and the peptides DT-2 or DT3 (selective PKG-I inhibitors) blocked the mitogenic effects of VEGF. In addition, cells in which sGC and PKG-I were inhibited exhibited no migration and sprouting in response to VEGF. By studying the mechanisms through which NS-2028 and DT2/DT3 inhibited EC functions, we found that p38 and VASP were involved. To test whether sGC also mediated the angiogenic effects of VEGF in vivo, we used the rabbit cornea assay. Animals receiving NS-2028 orally and DT3 locally displayed a reduced neovascular response to VEGF. As increased vascular permeability occurs prior to new blood vessel formation, we determined the effect of NS-2028 and DT3 in vascular leakage. Using a modified Miles assay we observed that NS-2028 and DT3 attenuated VEGF–induced permeability.
Overall, we provide evidence that sGC and PKG-I mediates the angiogenic and permeability-promoting activities of VEGF, indicating the significance of sGC and PKG-I as a downstream effector of VEGF-triggered responses.
1: Morbidelli L, et al . Am J Physiol Regul Integr Comp Physiol. 2010;298(3):R824-32.
2: Koika V, et al. Vascul Pharmacol. 2010;53(5-6):215-22.