The involvement of nitric oxide (NO) in the regulation of angiogenesis was examined in the in vivo system of the chorioallantoic membrane (CAM) of the chick embryo and in the matrigel tube formation assay. Sodium nitroprusside (SNP) (0.37–28 nmol/disc), which releases NO spontaneously, caused a dose‐dependent inhibition of angiogenesis in the CAM in vivo and reversed completely the angiogenic effects of α‐thrombin (6.7 nmol/disc) and the protein kinase C (PKC) activator 4‐β‐phorbol‐12‐myristate‐13‐acetate (PMA) (0.97 nmol/disc). In addition, SNP (28 × 10−6 m) stimulated the release of guanosine 3′‐5′‐cyclic monophosphate (cyclic GMP) from the CAM in vitro. In the matrigel tube formation assay, an in vitro assay of angiogenesis, both SNP (1–3 × 10−6 m) and the cell permeable cyclic GMP analogue, Br‐cGMP (0.3–1.0 × 10−3 m) reduced tube formation. The inhibitors of NO synthase, NG‐monomethyl‐l‐arginine (l‐NMMA) (3.8–102 nmol/disc) and NG‐nitro‐l‐arginine methylester (l‐NAME) (1.3–34.2 nmol/disc) stimulated angiogenesis in the CAM in vivo, in a dose‐dependent fashion. d‐NMMA and d‐NAME on the other hand had no effect on angiogenesis in this system. l‐Arginine (10.9 nmol/disc), although it had a modest antiangiogenic effect by itself, was capable of abolishing the angiogenic effects of l‐NMMA (34.2 nmol/disc) and of l‐NAME (3.8 nmol/disc). Dexamethasone, an inhibitor of the induction of NO synthase, at 0.2–116.1 nmol/disc, stimulated angiogenesis in the CAM, whereas at 348.4–1161 nmol/disc it inhibited this process. Combination of 38.7 nmol/disc dexamethasone with 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 inhibition of angiogenesis. Superoxide dismutase (SOD), which prevents the destruction of NO, at 300 i.u./disc had a modest antiangiogenic effect in the CAM, by itself. In addition, SOD, prevented α‐thrombin (6.7 nmol/disc) and PMA (0.97 nmol/disc) from stimulating angiogenesis in the CAM. These results suggest that NO may be an endogenous antiangiogenic molecule of pathophysiological importance.

The effect of the nitric oxide (NO)‐producing nitrovasodilators isosorbide mononitrate (ISMN) and isosorbide dinitrate (ISDN) were assessed on (a) the in vivo model of angiogenesis of the chick chorioallantoic membrane (CAM) and (b) on the growth and metastatic properties of the Lewis Lung carcinoma (LLC) in mice Isosorbide 5‐mononitrate (ISMN) and isosorbide dinitrate (ISDN), inhibited angiogenesis in the CAM dose‐dependently. ISMN was more potent in inhibiting this process. Both compounds were capable of completely reversing the angiogenic effect of α‐thrombin. These effects of ISMN and ISDN on angiogenesis were comparable to those previously observed with sodium nitroprusside which generates NO non‐enzymatically Mice, implanted intramuscularly with LLC, received daily i.p. injections of ISMN for 14 days resulting in a significant decrease in the size of the primary tumour and a reduction in the number and size of metastatic foci in the lungs. ISDN had a similar but less pronounced effect than that observed with ISMN Addition of ISMN or ISDN to cultures of bovine, rabbit and human endothelial cells and to cultures of LLC cells had no effect on their growth characteristics These results indicate that ISMN and ISDN inhibit angiogenesis and tumour growth and metastasis in an animal tumour model. The possibility should therefore be considered that these nitrovasodilators which are widely used therapeutically and have well characterized pharmacological profiles, may also possess antitumour properties in the clinic.

The in vivo model of the chick embryo chorioallantoic membrane (CAM) was used to study the involvement of nitric oxide (NO) in angiogenesis. The nitrovasodilator sodium nitroprusside (NaNP) and the amino acid, L-arginine, inhibited angiogenesis, assessed as both collagenous protein biosynthesis and vascular density. NG -monomethyl-L-arginine (L-NMMA), an NO synthase inhibitor, increased both collagenous protein biosynthesis and vascular density, indicating that this agent promotes angiogenesis. These results suggest that NO may participate in the regulation of angiogenesis. Manipulation of NO synthesis therefore, may prove to be another approach for controlling angioproliferative diseases.

Interleukin‐2 (IL‐2), an immunoregulatory cytokine possessing antitumour activity, is an inducer of nitric oxide (NO) synthesis in mice and man. In this study, the possibility that IL‐2 possesses antiangiogenic properties that account for its antitumour effects in vivo was examined. IL‐2 caused a dose‐dependent inhibition of angiogenesis in the chick embryo chorioallantoic membrane (CAM). This inhibition was completely reversed by the NO synthase inhibitor NG‐nitro‐L‐arginine methylester (L‐NAME). Furthermore, IL‐2 was capable of stimulating NO synthase activity in the CAM in vitro and this effect was suppressed by L‐NAME. Addition of IL‐2 to human umbilical vein endothelial cells (HUVECs) in culture, had no effect on their growth characteristics. These results suggest that IL‐2 may be an important antiangiogenic molecule causing its effect via nitric oxide synthesis. The antiangiogenic activity of IL‐2 may be, at least in part, responsible for its antitumour properties. British Journal of Pharmacology (1997) 122, 793–795; doi:

We have recently shown that thrombin promotes angiogenesis by a mechanism independent of fibrin formation. In the present paper, we investigated the effect of the thrombin-receptor-activating tetradecapeptide (TRAPH4, S42FLLRNPNDKYEPFss ) for its effects on angiogenesis in the chick chorioallantoic membrane (CAM) system of angiogenesis. A dose-dependent promotion of angiogenesis is evident with TRAP. In contrast, a thrombin-receptorderived tripeptide analogue H-Phe-Pro-Arg-OH (FPR), which was designed based on the S42FLLR46 sequence, caused an inhibition of angiogenesis in the CAM, and when it was combined with TRAP it caused a complete reversal of the angiogenesis-promoting effect of TRAP. These results indicate that the proteolytic exposure of the receptor N-terminal tetradecapeptide by thrombin can activate the post-thrombotic events related to angiogenesis. These events can be modulated by constrained peptide analogues such as FPR.

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