Novel approach to analysis of in vitro tumor angiogenesis with a variable-pressure scanning electron microscope: suppression by matrix metalloproteinase inhibitor SI-27.
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.
Yoshida
D
D
Department of Neurosurgery, Nippon Medical School, Tokyo, Japan. dyoshida@nms.ac.jp
Noha
M
M
Watanabe
K
K
Sugisaki
Y
Y
Teramoto
A
A
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D002633:Chemotaxis / Q000187:drug effects; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects; D006801:Humans; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000032:analysis / Q000502:physiology; D008854:Microscopy, Electron / Q000379:methods*; D015394:Molecular Structure; D009363:Neoplasm Proteins / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000502:physiology; D009389:Neovascularization, Pathologic / Q000473:pathology*; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000494:pharmacology; D019108:Tight Junctions / Q000187:drug effects / Q000648:ultrastructure; D019714:Tissue Inhibitor of Metalloproteinases / Q000032:analysis; D014407:Tumor Cells, Cultured / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis / Q000494:pharmacology
D020533:Angiogenesis Inhibitors; D017077:Culture Media, Conditioned; D061965:Matrix Metalloproteinase Inhibitors; D009363:Neoplasm Proteins; D009842:Oligopeptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; C421267:SI 27; D019714:Tissue Inhibitor of Metalloproteinases; D042461:Vascular Endothelial Growth Factor A; D020782:Matrix Metalloproteinases