Antiangiogenic effects of S-nitrosocaptopril crystals as a nitric oxide donor

Abstract

Angiogenesis is the formation of new capillaries from preexisting vessels by migration and proliferation of endothelial cells, which produce a cellular signaling messenger, nitric oxide (NO). The purpose of the present study was to examine the effects of exogenous NO donors on angiogenesis by using a novel crystalline NO donor, S-nitrosocaptopril. The characteristic X-ray diffraction pattern of S-nitrosocaptopril was demonstrated for the first time. On primary capillary endothelial cells pretreated with vascular endothelium growth factor (VEGF), S-nitrosocaptopril (1–500 μM), but not captopril, produced a dose-dependent inhibition of endothelial proliferation. On chick embryos of entire living eggs, gelatin sponges adsorbed with VEGF were implanted on the embryo chorioallantoic membrane to promote vascular growth activity within the sponges. Addition of S-nitrosocaptopril crystals (0.1 mg) to the gelatin sponges markedly reduced vascular density around the sponges, whereas captopril did not inhibit neovascularization. The vascular hemoglobin content surrounding each of the gelatin sponges was determined as a confirmatory test. S-nitrosocaptopril, but not captopril, significantly decreased the hemoglobin content of the embryo tissues immediately surrounding the gelatin sponges. In conclusion, S-nitrosocaptopril exerts an inhibitory effect on angiogenesis. This newly discovered function of S-nitrosocaptopril appears to be governed by distinct structural NO moiety.

Introduction

Major discoveries in the past have proven that the vascular endothelium is more than a lining for blood vessels. The endothelial cells locally produce endothelium-derived relaxing factor (EDRF), which is now recognized as nitric oxide (NO) (Furchgott, 1988). These normally quiescent cells can rapidly proliferate with a turnover time of 5 days during angiogenesis (Folkman, 1995), which is a highly regulated phenomenon and is under the control of angiogenic stimulators and inhibitors. Unregulated angiogenesis is seen under pathological conditions of tumor growth, diabetic retinopathy, and psoriasis. Angiogenic growth factors, such as vascular endothelium growth factor (VEGF), act as autocrine or paracrine growth factors to induce angiogenesis (Ferrara and Davis-Smith, 1997). VEGF has been shown to induce EDRF-dependent vasorelaxation in a dose-dependent fashion (Ku et al., 1993).

NO has been implicated as a modulator of blood flow, motility, electrolyte and water transport, and in the function of endothelial cells, platelets, mast cells, and macrophages. Although the physiological and pathophysiological functions of NO have been extensively studied, its effects on angiogenic signal transduction pathways are poorly understood and a little information is available concerning the indirect activity of NO on angiogenic models Pipili-Synetos et al., 1994, Pipili-Synetos et al., 1995. An interesting development in NO donors has been the realization that NO can exert both beneficial and deleterious effects in different tissues and under different conditions.

S-nitrosothiols can elicit many of the various physiological regulatory functions attributed to EDRF/ NO (Myers et al., 1990), including vascular and nonvascular smooth muscle relaxation Jia and Furchgott, 1993, Jia and Stamler, 1999, inhibition of platelet aggregation (Mathews and Kerr, 1993) and inflammation (Granger and Kubes, 1996), as well as neurotransmission (Kendrick et al., 1997). It has been well known that S-nitrosothiols decompose to yield NO and the corresponding disulfide RSSR (Singh et al., 1996), the latter could then be reduced by either an intracellular spontaneous recovery mechanism (Clancy et al., 1994), or a general protein disulfide reductase (Nikitovic and Holmgrent, 1996). Therefore, a S-nitrosothiol composed of an NO group and another pharmacological active component would have considerable theoretical and practical significance.

S-nitrosocaptopril (1-[(2S)-3-nitrosomercapto-2-methyl-1-oxopropyl]-l-Proline), an exemplary S-nitrosothiol, exhibits many NO-like activities such as direct relaxation of blood vessel both in vivo and in vitro Jia and Blantz, 1998, Lin et al., 1998, and the inhibition of platelet aggregation Loscalzo et al., 1989, Amano et al., 1994. S-nitrosocaptopril nonenzymetically releases NO in a manner similar to most of S-nitrosothiols (Jia et al., 1999). There have been many efforts to make a preparation of S-nitrosocaptopril in a powder form, however, the molecular entity is reported to be too unstable to isolate as pure crystals (Loscalzo et al., 1989). This made it impossible to extensively explore the mechanism of actions of the compound or its therapeutic implication. Despite of earlier hundreds of failure in purifying and crystallizing the compound, we have most recently synthesized the red flake crystals with a good yield. The novel crystals provide us with opportunities to explore pilot information regarding the overall function of NO in angiogenesis. Additionally, owing to the inadequacy of biochemical quantitative analyses of amounts of blood vessels in tissues, we extended our previous technique of hemoglobin measurement (Jia et al., 1996) to quantify the vascular existence in the chick embryo chorioallantoic model of entire eggs. The effects of S-nitrosocaptopril on the angiogenic models were examined in comparison with the corresponding parent compound captopril.