Effect of sCD40L on the expression of VEGF by human endothelial cells.

HUVECs were cultured until confluence and were treated with sCD40L (Bristol Myers Squibb) as detailed below. RNA was harvested from HUVECs, and the expression of VEGF was analyzed by RPA. Unless indicated, sCD40L was used as a culture supernatant in all assays at a concentration found to have optimal bioactivity (equal to a dilution of 1:5) in positive controls. (A) Temporal expression of VEGF in resting HUVECs or HUVECs stimulated with sCD40L. The expression of VEGF is markedly induced 1 hour following treatment with sCD40L and peaks in expression between 2 and 5 hours following activation. (B) Effect of increasing concentrations of sCD40L (represented by dilutions of culture supernatant) on VEGF expression after 5 hours. (C) HUVECs cultured in the absence or presence of sCD40L and a blocking anti-CD40 antibody to determine the specificity of sCD40L for CD40-dependent VEGF expression. In these studies, identical effects of sCD40L on the induction of VEGF were obtained when purified recombinant sCD40L (Ancell) was used at a concentration of 1 to 10 μg/mL. The expression of VEGF in a renal carcinoma cell line served as a positive control (Control), and the expression of L32 and GAPDH served as internal housekeeping gene controls. For panels A and B, the expression of IL-1 is also illustrated. All autoradiographs are representative of 3 experiments with similar results.