Inhibition of Angiogenesis by Notch Pathway Modulation

Briefly, growth factor-depleted Matrigel was thawed overnight at 40 °C and mixed to homogeneity. Culture plates (48-well) were coated with 0.1 ml of Matrigel and allowed to gelatinize at 37 °C for 30 min. HUVECs or HUVECS and HTR8 cells (2.5 × 10⁴ each), labeled with cell tracker Red CMTPX and Green CMFDA (Molecular Probes), respectively, were cultured on Matrigel in the presence of 10% normal pregnancy serum (NPS) containing A-1254 (10 μg/ml). The concentration of A-1254 was selected from a dose response studies described earlier11 (link). The endothelial cell tube formation was monitored and recorded after 12–14 hrs of incubation using the florescence microscopy at 4 x magnification (Nikon Eclipse TS 100 coupled with CCD camera) as described11 (link)33 (link). To evaluate the significance of the Notch/Dll pathway in A-1254-induced inhibition of angiogenesis, A-1254-induced disruption of three-dimensional tube formation by HUVECs was assessed in the presence or absence of different doses of γ-secretase inhibitor, L1790 (0, 1, 5, 10, 25 μM) (Sigma-Aldrich).