FIG. 6.

Ad-mediated gene expression in cytokine-treated HUVEC. Confluent HUVEC were cultured for 48 h in the absence or presence of TNF-α, IFN-γ, or a combination of the two cytokines. The cultures were subsequently infected with Adβgal, an Ad expressing the β-galactosidase marker transgene (10¹¹ particles/ml for 10 min). After 24 h, the β-galactosidase activity was quantified and expressed relative to the total protein. β-Galactosidase data in all graphs were plotted as percentages of the expression level in untreated HUVEC. For all graphs, data points represent the means ± standard errors of 12 to 20 observations. (A) Synergism of cytokines. The data compare Ad-mediated gene transfer to untreated HUVEC or HUVEC treated with TNF-α (20 ng/ml), IFN-γ (10 ng/ml), or a combination of the two cytokines for 48 h prior to infection with Adβgal. (B) Cytokine dose response. Confluent HUVEC were cultured with various doses of TNF-α (0.06 to 2,000 ng/ml) or IFN-γ (0.03 to 1,000 ng/ml) for 48 h prior to infection with Adβgal. Synergistic effects of the cytokine treatments were investigated by culturing cells with various doses of IFN-γ or TNF-α and constant levels of IFN-γ (20 ng/ml) or TNF-α (20 ng/ml), respectively, for 48 h prior to infection with Adβgal. (C) Time dependence. The cytokine effect is shown as a function of the time of cytokine treatment. Cultures of confluent HUVEC were grown in the absence or presence of TNF-α (20 ng/ml) and IFN-γ (10 ng/ml) for 0 to 24 h and then infected with an Adβgal vector. (D) Ad vector dose response. Confluent HUVEC were cultured and treated with TNF-α and IFN-γ or were left untreated. Untreated and cytokine-treated cells were then infected with graded doses (10¹¹ to 10⁸ particles/ml for 10 min) of Adβgal. β-Galactosidase expression in cytokine-treated HUVEC was significantly less than that in untreated HUVEC at each Ad concentration (P < 0.03 for comparisons at all Ad doses).