EVIDENCE THAT NITRIC-OXIDE IS AN ENDOGENOUS ANTIANGIOGENIC MEDIATOR

1 The involvement of nitric oxide (NO) in the regulation of angiogenes is was examined in the in vivo system of the chorioallantoic membrane (CAM) of the chick embryo and in the matrigel tube formation assay. 2 Sodium nitroprusside (SNP) (0.37-28 nmol/disc), which releases NO spon taneously, caused a dose-dependent inhibition of angiogenesis in the C AM in vivo and reversed completely the angiogenic effects of alpha-thr ombin (6.7 nmol/disc) and the protein kinase C (PKC) activator 4-beta- phorbol-12-myristate-13-acetate (PMA) (0.97 nmol/disc). In addition, S NP (28 x 10(-6) M) stimulated the release of guanosine 3'-5'-cyclic mo nophosphate (cyclic GMP) from the CAM in vitro. 3 In the matrigel tube formation assay, an in vitro assay of angiogenesis, both SNP (1-3 x 1 0(-6) M) and the cell permeable cyclic GMP analogue, Br-cGMP (0.3-1.0 x 10(-3) M) reduced tube formation. 4 The inhibitors of NO synthase, N -G-monomethyl-L-arginine (L-NMMA) (3.8-102 nmol/disc) and N-G-nitro-L- arginine methylester (L-NAME) (1.3-34.2 nmol/disc) stimulated angiogen esis in the CAM in vivo, in a dose-dependent fashion, D-NMMA and D-NAM E on the other hand had no effect on angiogenesis in this system. 5 L- Arginine (10.9 nmol/disc), although it had a modest antiangiogenic eff ect by itself, was capable of abolishing the angiogenic effects of L-N MMA (34.2 nmol/disc) and of L-NAME (3.8 nmol/disc). 6 Dexamethasone, a n inhibitor of the induction of NO synthase, at 0.2-116.1 nmol/disc, s timulated angiogenesis in the CAM, whereas at 348.4-1161 nmol/disc it inhibited this process. Combination of 38.7 nmol/disc dexamethasone wi th L-NAME (9.3 nmol/disc) resulted in a potentiation of the angiogenic effect of the former. It appears therefore that both the constitutive and the inducible NO synthase may contribute to the NO-mediated inhib ition of angiogenesis. 7 Superoxide dismutase (SOD), which prevents th e destruction of NO, at 300 i.u./disc had a modest antiangiogenic effe ct in the CAM, by itself. In addition, SOD, prevented alpha-thrombin ( 6.7 nmol/disc) and PMA (0.97 nmol/disc) from stimulating angiogenesis in the CAM. 8 These results suggest that NO may be an endogenous antia ngiogenic molecule of pathophysiological importance.