FIGURE 5.
Reverse-mode NCX is required for thrombin-induced angiogenesis. HUVECs in Opti-MEM-I® medium containing 2% v/v FCS, 0.1% w/v BSA with or without thrombin (0.5 units/ml) were seeded in 24-well plates precoated with growth factor-reduced MatrigelTM in the presence of KB-R7943, SN-6, SEA0400, or vehicle. Images were obtained after 18 h. A, representative images at ×4 magnification are shown for key conditions. Tubule length (B) or branching points per field of view (C) from five fields of view at ×10 magnification were quantified. Mean tubule length of unstimulated controls (C) was set at 1. Bars represent means ± S.E. (n = 3 in duplicate). *, p < 0.05 versus the thrombin-stimulated control. D, representative images of HUVECs treated with the indicated concentrations of reverse-mode NCX inhibitors as in A without thrombin stimulation. E, quantification of tubule length for the cells in D was done as in B. F, images of HUVECs transfected with control (C) or NCX1-targeting (NCX1) siRNA with or without thrombin (Thr; 0.5 units/ml) and seeded on MatrigelTM as above for 6 h. G, quantification of tubule length from n = 4 in duplicate experiments was carried out as in B. H, HUVECs in Opti-MEM-I® containing 0.1% w/v BSA, 1 μg/ml hydrocortisone, and the indicated concentrations of KB-R7943, SN-6, SEA0400, or vehicle were seeded in 96-well plates precoated with collagen-I, with or without thrombin (0.5 units/ml). Cell numbers were determined by alkaline phosphatase assay after 48 h. The absorbance of the no-thrombin control was set to 1. Bars represent means ± S.E. (n = 3 in triplicate). *, p < 0.05 versus the thrombin-stimulated control. I, effect of KB-R7943 (10 μm), SN-6 (10 μm), or SEA0400 (1 μm) on HUVEC proliferation/viability in the absence of thrombin was assessed as in H. *, p < 0.05 versus the control.