Soluble Guanylyl Cyclase Inhibition Reduces Vascular Endothelial Growth Factor-Induced Angiogenesis and Permeability

Anastasia Pyriochou1, Lucia Morbidelli2, Athanasios Giannis3, Marina Ziche2, Andreas Papapetropoulos1. 1University of Patras, Patra, Greece, 2University of Siena, Siena, Italy, 3University of Leipzig, Leipzig, German.

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen that also enhances vascular permeability (Ferrara et al., 2003). Although nitric oxide (NO) has been shown to contribute to VEGF signalling (Papapetropoulos et al., 1997; Ziche et al., 1997) and to promote permeability and neovascularization (Fukumura et al., 2001; Murohara et al., 1998), the downstream effector molecules have poorly been characterized. We sought to determine whether inhibition of soluble guanylyl cyclase (sGC), the intracellular target of NO, by NS-2028 (4H-8-Bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one), altered the actions of VEGF on the vasculature. Statistical comparisons between groups were performed using ANOVA post hoc test or Student’s t test as appropriate. Data are expressed as mean±s.e.m.; *P<0.05 indicates significance vs vehicle or VEGF-treated groups. Treatment of human umbilical vein endothelial cells (HUVEC) with NS-2028 (10 μM;) reduced VEGF (20 ng/ml)-induced cGMP accumulation by 90 ± 16 % (n=4; P<0.05), abolished VEGF (20 ng/ml)-stimulated cell motility (n=5; P<0.05) and inhibited cell growth by 25 ± 8 % (n=3; P<0.05). NS-2028 (10 μM) also inhibited VEGF (20 ng/ml)-stimulated activation of p38 MAPK (n=3; P<0.05), essential in VEGF-induced endothelial cell migration. Furthermore in a rat cultured aortic ring assay system VEGF (50 ng/ml) caused a 3-fold increase in capillary sprouting that was completely inhibited by NS-2028 (10 μM, n=4; P<0.05). Rabbits receiving NS-2028 orally (1 g/l water) displayed a reduced angiogenic response to VEGF (300 ng / pellet) in the eye corneal assay (n=5 animals/group; P<0.05). As increased vascular permeability precedes new blood vessel formation in many cases, we determined the effect of NS-2028 in VEGF-simulated vascular leakage. Using a modified Miles assay in mice we observed that NS-2028 (10 mg/kg, i.p.) attenuated VEGF (300 ng / intradermal injection)–induced permeability (n=6; P<0.05). Overall, we have used NS-2028 to provide evidence that sGC mediates the angiogenic and permeability promoting activities of VEGF in vitro and in vivo, implicating sGC as a down-stream effector of VEGF-triggered responses.

Ferrara N. et al., (2003) Nat. Med. 9, 669-676.
Fukumura D. et al., (2001) PNAS 98, 2604-2609.
Murohara T. et al., (1998) Circulation 97, 99-107.
Papapetropoulos A. et al., (1997) J. Clin. Invest. 100, 3131-3139.
Ziche M. et al., (1997) J. Clin. Invest. 99, 2625-2634.

Experiments have been performed in accordance with the guidelines of the European Economic Community for animal care and welfare (EEC Law No. 86/609).