Vasohibin as an endothelium-derived negative feedback regulator of angiogenesis
J. Clin. Invest. Kazuhide Watanabe, et al. 114:898 doi:10.1172/JCI21152 [Go to this article.]

Figure 6
Vasohibin suppresses tumor growth and tumor angiogenesis. (A) The synthesis of vasohibin protein in LLC cells. Cell extracts were prepared from mock or vasohibin transfectants (Vh-bulk) for Western blotting. Clone 16 and clone 19 were vasohibin-producing clones. (B) Effect of vasohibin on the proliferation of LLC cells in vitro. Proliferation of mock transfectants, vasohibin transfectants, clone 16, and clone 19 was determined. (C) Effect of secreted vasohibin from LLC cells on the migration of HUVECs. Mock or vasohibin transfectants were plated on the lower compartment of a modified Boyden chamber and the migration of HUVECs toward the lower chamber of the Transwell insert was analyzed. Values are expressed as mean ± SD of 4 samples. (D) Effect of vasohibin gene transfection on the growth of LLC cells in vivo. BDF1 mice were inoculated intradermally with LLC cells. Tumor volume was determined consecutively. (E) Effect of vasohibin gene transfection on tumor angiogenesis. Paraffin sections were prepared from tumors for the immunostaining of CD31; sections obtained on day 8 after inoculation are shown. Visualization with a DAKO LSAB+/HRP kit is shown at left, and that with streptavidin-Cy3 conjugate on the right. Yellow lines trace vascular lumens. Scale bars: 50 μm. (F) Quantitative analysis of tumor vascular area. Total vascular area per field was determined using NIH Image and compared. Values are expressed as mean ± SD of 6 random fields. *P < 0.05; **P < 0.01.