Function of CD40 ligation for VEGF-induced angiogenesis.
HUVECs were cultured in 24-well tissue culture plates in 5% FBS for 48 hours in the absence or presence of sCD40L or VEGF. HUVECs were seeded at 5 × 104 cells per well in 0.75 mL medium, and cell numbers were counted daily by means of a calibrated grid (100 × magnification). Treatment of HUVECs with VEGF resulted in enhanced survival of HUVECs and promoted growth (as described13) and served as positive control. Treatment of HUVECs with heat-inactivated sCD40L supernatant served as negative control. (A) Representative photomicrographs of HUVEC cultures after 48 hours in medium alone, VEGF (5 ng/mL), and sCD40L (Bristol Myers Squibb). (B) Relative increase in number of HUVECs compared with control (medium alone) after 48 hours of culture in the presence of sCD40L or VEGF, in the absence or presence of a neutralizing anti-VEGF antibody. The median values of 4 independent experiments performed in duplicate cultures are illustrated. There is enhanced growth of HUVECs following treatment with sCD40L, and this effect is abrogated by addition of a neutralizing anti-VEGF antibody into cell cultures. Controls for anti-VEGF were cultures of VEGF in the absence or presence of antibody. Additional negative controls (not shown) were cultures in the presence of murine sCD40L (Bristol Myers Squibb) generated in the same manner as the human sCD40L used in these experiments.
