Latent Antithrombin inhibiert die Angiogenese im A-Mel-3-Fortner Melanom durch verzögerte Ausreifung der neugebildeten Kapillaren

Abstract

Previous in vitro and in vivo studies have indicated potent antiangiogenic and antitumor activity of latent antithrombin. With the use of intravital fluorescence microscopy, the aim of the present study was to further elucidate the inhibitory action of latent antithrombin on the different steps of angiogenesis, i.e. (i) capillary sprouting, (ii) capillary maturation or (iii) network formation, in melanoma tumor growth. For this purpose, a dorsal skinfold chamber was prepared in 27 Syrian golden hamsters, and 2×105 syngenic A-Mel-3-Fortner melanoma cells were implanted onto the skin muscle surface. The process of tumor angiogenesis was analyzed and quantified at days 3, 5, 7 and 10 after implantation using intravital fluorescence microscopy and computer-assisted image analysis. The analysis included the determination of the size of the microvascular network (in relation to the size of tumor area), microvessel density (defined as the length of red blood cell perfused microvessels in relation to the size of tumor area) as well as capillary diameters, red blood cell velocity and blood perfusion. Animals daily received latent (n = 8) or native (n = 11) antithrombin in a dosage of 25 mg/kg. Nontreated animals (n = 8) served as controls (mean ± SEM; ANOVA, unpaired Student’s t-test). During initial angiogenesis, latent and native antithrombin-treated tumors showed comparable capillary sprouting, but significantly larger capillary diameters compared to controls (day 5: 9.0 ± 0.5 μm and 9.3 ± 0.5 μm versus 7.4 ± 0.4 μm; p < 0.05), indicating a delay in capillary maturation. In contrast, neither latent nor native antithrombin affected microvessel density, which increased from ∼125cm/cm2 to ∼260cm/cm2 between day 3 and day 10. This observation reflects the lack of effect on capillary sprouting. However, analysis of the overall size of the microvascular network indicated a significant reduction at day 10 in latent (89 ± 10 mm2/mm2) and native antithrombin-treated tumors (96 ± 6 mm2/mm2) compared to untreated controls (134 ± 12 mm2/mm2). In conclusion, latent antithrombin does not inhibit capillary sprouting per se, but reduces overall tumor angiogenesis by delaying the maturation of the newly formed capillaries. The comparable effectiveness of native versus latent antithrombin is probably due to the fact that native antithrombin contains substantial amounts of spontaneously formed latent antithrombin.