Effect of an E1⁻E4⁻ Ad vector on growth and survival of primary human endothelial cells. (A) Growth of endothelial cells in response to growth factors after exposure to an E1⁻E4⁻ vector. Subconfluent primary human endothelial cells were cultured in growth factor medium, as in Fig 1A, after exposure to E1⁻E4⁻ Adβgal (50 pfu/cell) compared with E1⁻E4⁺ Adβgal (50 pfu/cell) or no vector (“control”). Shown is the number of viable cells as assessed by trypan blue dye exclusion. (B) Survival of endothelial cells cultured in growth factor–free medium after exposure to an E1⁻E4⁻ Ad vector and E1⁻E4⁻ ORF6 Ad vector. Confluent primary human endothelial cells were cultured in growth factor–free medium after exposure to E1⁻E4⁻ Adβgal, E1⁻E4⁻ ORF6 Ad vector, E1⁻E4⁺ Adβgal, or control, as in panel A. (C) Effect of an E1⁻E4⁻ Ad vector on cellular DNA synthesis of primary endothelial cells. Cells were exposed to the E1⁻E4⁻ Adβgal vector or no vector (“control”) and cultured in growth factor medium or growth factor–free medium, with [³H]thymidine uptake quantified as in Fig 2A. The data for panels A through C represent the mean ± standard error of triplicate measurements. (D) Evaluation of the status of the cellular DNA in cultured endothelial cells after exposure to an E1⁻E4⁻ Ad vector. Cells exposed to the E1⁻E4⁻ Adβgal vector or no vector (“control”) were cultured in growth factor medium or growth factor–free medium as described in Fig 2B, and evaluated by propidium iodide staining and flow cytometry. The data for day 1 is displayed with a linear abscissa to best show the >2n peaks; the data for day 5 and day 12 is displayed with log scale to best show the <2n fragmented DNA. The <2n, 2n, and 4n DNA peaks are indicated. (E) Levels of Bcl2 and Bax protein in human endothelial cells after exposure to E1⁻E4⁻ Adβgal vector. Confluent endothelial cells were exposed to the vector or no vector (“control”) and cultured in growth factor–free medium. The analysis was identical to that in Fig 3. (F) (see page 2939) Evaluation of apoptosis in cells infected with E1⁻E4⁻Ad vector. Subconfluent endothelial cells cultured in poly-D-lysine–coated glass coverslips were infected with E1⁻E4⁻ Ad vector as described in the Fig 1legend. After vector infection, cells were maintained in growth factor medium for 10 days or in growth factor–free medium for 1 day, and apoptotic cells were identified by assessment of free DNA 3′-OH ends.