Eva Pipili-Synetos

Evidence that nitric oxide is an endogenous antiangiogenic mediator.

1 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. 2 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. 3 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. 4 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. 5 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). 6 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. 7 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. 8 These results suggest that NO may be an endogenous antiangiogenic molecule of pathophysiological importance.

Inhibition of angiogenesis, tumour growth and metastasis by the NO‐releasing vasodilators, isosorbide mononitrate and dinitrate

1 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 2 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 3 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 4 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 5 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.

Evidence that platelets promote tube formation by endothelial cells on matrigel

1 The involvement of platelets in neovascularization was investigated in the matrigel tube formation assay, an in vitro model of angiogenesis. 2 Platelets promoted the formation of capillary‐like structures (expressed as relative tube area) number‐ and time‐dependently. Relative tube area increased from 0.98±0.02 (n = 8) in the presence of 6.25×104, to 3.21±0.12 (n = 8) in the presence of 106 platelets/well compared to 0.54±0.04 (n = 8) in their absence. This increase was unaffected by acetyl salicylic acid (ASA), apyrase, and hirudin. Photographs from representative experiments, showed that platelets adhered along the differentiating endothelium. 3 Addition of α‐thrombin (0.1–1 i.u. ml−1), the nitric oxide (NO) donor sodium nitroprusside (SNP; 1–100 μm) or the NO synthase inhibitor, l‐NG‐arginine‐methylester (l‐NAME, 30–300 μm) to the assay, had no effect on tube formation compared to that seen with platelets alone. 4 Neuraminidase (0.01 i.u./107 platelets), which strips sialic acid residues from membrane glycoproteins, abolished the promoting effect of platelets on tube formation. The relative tube area in the presence of neuraminidase‐treated platelets was 0.81±0.03 (n = 8), in the presence of untreated platelets 1.69±0.09, P < 0.001 (n = 8) and in the absence of platelets, 0.80±0.04 (n = 8). The tetrapeptide Arg‐Gly‐Asp‐Ser (RGDS; 20–200 μm) which inhibits von Willebrand factor, fibrinogen and fibronectin‐mediated adhesion, had no effect on the promoting effect of platelets on tube formation. 5 These results indicate that platelets promote angiogenesis in vitro. This effect is largely independent from activation by α‐thrombin, is not modified by manipulating NO and prostaglandin metabolism and proceeds possibly through adhesion of the platelets to the differentiating endothelium.

Nitric oxide is involved in the regulation of angiogenesis

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.

Nitric oxide synthase expression, enzyme activity and NO production during angiogenesis in the chick chorioallantoic membrane

In order to elucidate further the role of nitric oxide (NO) as an endogenous antiangiogenic mediator, mRNA expression of inducible nitric oxide synthase (iNOS), enzyme activity and production of NO were determined in the chick chorioallantoic membrane (CAM), an in vivo model of angiogenesis. In this model, maximum angiogenesis is reached between days 9–12 of chick embryo development. After that period, vascular density remains constant. Inducible NO synthase (iNOS) mRNA expression, determined by reverse transcriptase polymerase chain reaction (RT–PCR), increased from the 8th day reaching a maximum (70% increase) at days 10–11. NO synthase activity, determined as citrulline formation in the presence of calcium, also increased from day 8 reaching a maximum around day 10 (100% increase). Similar results were obtained in the absence of calcium suggesting that the NOS determined was the inducible form. Nitric oxide production, determined as nitrites, increased from day 8 reaching a maximum around day 10 (64% increase) and remaining stable at day 13. Finally, the bacterial lipopolysaccharide LPS (which activates transcriptionally iNOS), inhibited dose dependently angiogenesis in the CAM. These results in connection with previous findings from this laboratory, showing that NO inhibits angiogenesis in the CAM, suggest that increases in iNOS expression, enzyme activity and NO production closely parallel the progression of angiogenesis in the CAM, thus providing an endogenous brake to control this process.

Protein kinase C involvement in the regulation of angiogenesis.

Using the chick chorioallantoic membrane as a model system for the study of angiogenesis, we have shown that promoters of protein kinase C (PKC) such as 4-beta-phorbol-12-myristate-13-acetate (4-beta-PMA) and 1,2-dioctanoyl-sn-glycerol (DiC8) stimulated angiogenesis. This effect was specific since 4-alpha-PMA and 1,2-dioleoyl-sn-glycerol, which either do not activate or cannot reach PKC, were devoid of angiogenic activity. Furthermore, Ro 31-8220, a specific inhibitor of PKC, suppressed both basal and 4-beta-PMA- or DiC8-induced angiogenesis. Similar results were obtained with the commonly used inhibitor of PKC, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine and with tricyclodecan-9-yl-xanthogenate, an antitumor agent which has been suggested to be an inhibitor of PKC. Activation of PKC may be, therefore, an important signalling pathway in the initiation and control of the angiogenic response.

Involvement of nitric oxide in the inhibition of angiogenesis by interleukin‐2

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.

Leukotrienes C4 and D4 promote angiogenesis via a receptor-mediated interaction

The involvement of leukotrienes in angiogenesis was investigated in the in vivo chick chorioallantoic membrane system. In this system leukotrienes C4 and D4 promoted angiogenesis in a dose-dependent manner. Leukotriene B4 was ineffective. The potent and selective peptidyl leukotriene receptor antagonist, SK&F 104353-Z2, abolished the angiogenic effects of leukotrienes C4 and D4 and reduced unstimulated angiogenesis. These results indicate that leukotrienes C4 and D4 promote angiogenesis in the chick chorioallantoic membrane via a receptor-mediated interaction.

INOSITOL PHOSPHATES FORMED IN RAT AORTA AFTER ALPHA-1-ADRENOCEPTOR STIMULATION ARE INHIBITED BY FORSKOLIN

Rat aortic smooth muscle rings without endothelial cells were subjected to alpha 1-adrenoceptor stimulation. We measured the contractile state of the smooth muscle cells and the formation of inositol phosphates (InsPs) on receptor stimulation. Using different extracellular calcium-containing solutions (2.5 mM, 0.1 mM and Ca(2+)-free) enabled us to discriminate three contractile phases after noradrenaline (10(-5) M) stimulation: an initial fast contraction (15 s) and a fast and slow component of the sustained contraction, which was established 10 min after stimulation. Under normal calcium conditions in the presence of 10 mM LiCl the formation of Ins(1,4,5)P3 was increased predominantly after stimulation, while the formation of Ins(1,3,4)P3, Ins(1,3,4,6)P4, Ins(1,3,4,5)P4, Ins(3,4,5,6)P4 and InsP5/InsP6 was also stimulated. The cAMP-inducing agent forskolin (0.5 microM) induced a relaxation of the basal tone and increased the level of the InsP4 isomers. The noradrenaline-induced contractile responses as well as the formation of InsP fractions mentioned were inhibited by forskolin. Further an increase in the formation of phosphatidylinositol bisphosphate was observed. It is concluded that in rat aorta InsPs and in particular Ins(1,4,5)P3 is involved in the different contractile phases caused by alpha 1-adrenoceptor stimulation. The relaxation induced by forskolin under these circumstances could be explained by an interaction of forskolin, most likely via the formation of cAMP, with InsPs formation at the level of phospholipase C activation.

Inhibition of TRAP-induced angiogenesis by the tripeptide Phe-Pro-Arg, a thrombin-receptor-derived peptide analogue

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 (TRAP1–14, S42FLLRNPNDKYEPF55) 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-receptor-derived 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.