Ligation of CD40 results in enhanced VEGF promoter activity.

(A) HUVECs were first incubated for 24 hours in 0.5% FBS containing EBS. Subsequently, a 2.6-kb full-length VEGF promoter-luciferase construct (1 μg DNA per milliliter) was introduced into HUVECs by lipofection as detailed in “Materials and methods.” HUVECs were then cultured for 24 hours in the absence or presence of sCD40L (Bristol Myers Squibb) in duplicate wells for each condition. Luciferase activity was analyzed with the use of equal amounts of total cellular protein for each sample. Illustrated is the luciferase activity in lysates in transfected but unstimulated HUVECs and in HUVECs following stimulation with sCD40L. Mock-transfected HUVECs served as negative control. Luciferase activity was consistently induced (approximately 4-fold) in sCD40L-treated cells compared with untreated cells. Representative of 3 independent experiments performed in duplicate cultures. (B) The expression of VEGF mRNA in the same HUVECs used in panel A, either untreated or following treatment with sCD40L for 5 and 24 hours. The induced expression of VEGF mRNA was similar (approximately 4-fold) to the induced luciferase activity illustrated in panel A.