Effect of VEGF/VEGFR autocrine loop on ECs.

(A) EC survival in serum-free conditions or in the presence of different concentrations of GA is shown. ECs were cultured in serum-free RPMI and left untreated or treated with GA, for a 72-hour period. Apoptotic cells were determined by annexin V staining, as described in “Materials and methods.” Results are shown as a percentage of apoptotic (annexin V⁺) cells. Each determination was done in triplicate. (B) Infection of ECs with adeno-VEGF induces Hsp90 expression. Total protein extracts were obtained from noninfected (control), adeno-null–infected, or adeno-VEGF–infected ECs, after 12 hours of culture in serum-free conditions. Results show Hsp90 levels, as determined by Western blotting. (C) ECs infected with adeno-null or adeno-VEGF are resistant to serum-free and GA-induced apoptosis. Noninfected, adeno-null–infected or adeno-VEGF–infected ECs were cultured for 72 hours in serum-free conditions, or with increasing concentrations of GA (0.02, 0.2, and 2 μM). Apoptotic cells (as determined by annexin V staining) were quantified after 72 hours. Results are shown as a percentage of apoptotic cells, and each determination was done in triplicate. Adeno-VEGF–infected cells survived significantly longer in serum-free conditions or after treatment with GA (**P < .05).