Identification of interferon-beta-stimulated genes that inhibit angiogenesis in vitro.

Interferons (IFNs) have proven antitumor activity against a variety of human malignancies, which may result, at least in part, from inhibition of angiogenesis. The objective of this study was to identify IFN-stimulated genes (ISGs) that played a role in mediation of angiogenic inhibition. IFN-beta was a more potent antiangiogenic agent compared to IFN-alpha2b (80% versus 20%, respectively) and suggests that IFNs inhibited angiogenesis by preventing endothelial cell differentiation, and not by direct antiproliferative effects. To identify ISGs that were key inhibitors of angiogenesis, we utilized an in vitro fibrin gel angiogenic assay which closely recapitulated the in vivo processes of angiogenesis. DNA microarray analysis of IFN-beta-treated endothelial cells in the fibrin gel assay identified 11 ISGs that were induced >10-fold during angiogenesis inhibition. Recombinant IP-10 inhibited angiogenesis in a dose-dependent fashion, but was a less effective inhibitor compared to IFN-beta, suggesting that additional ISGs are involved in inhibiting angiogenesis. ISG20 was upregulated by microarray analysis, but did not inhibit angiogenesis when overexpressed in human umbilical vein endothelial cells (HUVECs). However, a dominant negative mutant of ISG20 inhibited angiogenesis by 43%. Results suggest that IFN-induced angiogenic inhibition was likely mediated by multiple ISGs; our novel finding is that decreased exonuclease activity in HUVECs associated with expression of the ISG20 ExoII mutant inhibited angiogenesis.

IFN-alpha1,8 inhibits tumor-induced angiogenesis in murine angiosarcomas.

Interferon-alpha (IFN-alpha) has proved effective in the treatment of hemangiomas, hemangioblastomas, and Kaposi's sarcoma. To investigate the ability of IFNs to inhibit angiosarcoma, we used two transformed murine endothelial cell lines that form angiosarcomas in vivo. SVR and MS1-VEGF cell lines express oncogenic H-ras or vascular endothelial growth factor (VEGF), respectively. IFN-alpha1,8, which is active against murine and human cells, inhibited SVR and MS1-VEGF proliferation in vitro by 40% at 10(3) U/mL (p = 0.028). In vivo, IFN-alpha1,8 inhibited SVR tumor volume by 71% (p = 0.047) and MS1-VEGF volume by 79% (p = 0.003). Tumor-induced angiogenesis was decreased in SVR tumors by 52% (p = 0.005) and in MS1-VEGF tumors by 58% (p = 0.001). Sera from IFN-alpha1,8-treated mice bearing either SVR or MS1-VEGF tumors demonstrated a 5-fold increase in IP-10/CXCL10 (p = 0.001), an IFN-induced antiangiogenic protein. Both recombinant IP-10 and IFN-alpha1,8 inhibited human umbilical vein endothelial cell (HUVEC) vessel formation in the fibrin gel assay, a three-dimensional culture model of angiogenesis, by 56% at 25 ng/mL and 50% at 1.2 ng/mL, respectively (p < 0.001). An IP-10 blocking antibody restored vessel formation to 80% of untreated controls (p = 0.001). Given the magnitude of the in vivo response, these data suggested that the antitumor effects of IFN-alpha1,8 were likely mediated through angiogenesis inhibition rather than solely by direct inhibition of tumor cell proliferation.