Degradation of basement membrane by
metalloproteinases (
MMP) is a critical step in
tumor angiogenesis. To evaluate in vitro angiogenesis, several models have been employed, including bovine cornea, fenestrated rat brain,
Matrigel, and others. These models did not provide quantitative analysis of capillary formation. The current study aimed for a novel approach to in vitro assay of angiogenesis with a "wet scanning electron microscope (SEM)" to investigate suppression of
tumor angiogenesis by the
MMP inhibitor,
SI-27. The effects of noncytotoxic concentrations of
SI-27 (1-100 microM) were determined on nonmitogenic
vascular endothelial growth factor (
VEGF) (10 ng/ml)-mediated cell motility and in vitro angiogenesis of human umbilical vein endothelial cells (HUVECs). Activities of
MMP and
tissue inhibitor of metalloproteinase (TIMP) were determined by
enzyme-linked
immunosorbent assay (ELISA). Subsequently, the inhibitory effect of
SI-27 was examined on in vitro angiogenesis stimulated by supernatants of human
glioma cell lines (U87MG, U251MG, or U373MG). In vitro angiogenesis was quantitatively analyzed with a variable-pressure SEM. Cell motility and in vitro angiogenesis by HUVECs were significantly increased by
VEGF along with elevated MMP-1 and -2 activity, whereas
SI-27 significantly suppressed
VEGF-mediated in vitro angiogenesis and inactivated both MMP-1 and MMP-2, but not inhibited cell motility. The angiogenesis promoted by
glioma supernatants showed a significant reduction in the presence of
SI-27.
SI-27, a novel
MMP inhibitor, inhibited
tumor angiogenesis in vitro. It can be anticipated to prevent
tumor growth through its angiosuppressive effect. Quantitative analysis with a variable-pressure SEM is a novel approach to in vitro angiogenesis assay.