αvβ3/αvβ5-Integrin blockade induces apoptosis in adherent endothelial cells, independent of anoikis. (A-B) HBMECs (106 cells/10-cm dish) were allowed to spread on vitronectin. After 2 hours, RGDfV (25 μg/mL) was added and cells were incubated for additional 12 to 72 hours. Apoptosis (cells with a sub-G0/G1 DNA content) was assessed by flow cytometry of permeabilized and propidium iodide–stained cells (combined floating and adherent cells); n = 3; most error bars are hidden by the symbols. Panel B depicts representative flow cytometry tracings of HBMECs incubated with 25 μg/mL RADfV (top) or RGDfV (bottom) for 48 hours, from a separate experiment. (C) HBMECs (106/10-cm plate) were seeded on Petri dishes coated with vitronectin (VN, left column) or PLL (middle and right columns) and incubated overnight. The VN plates and one set of PLL plates were also blocked with 1% BSA (VN and PLL/BSA; left and right columns), and one set of PLL plates was left unblocked (PLL; middle column). RGDfV (bottom row; 25 μg/mL) or the control peptide, RADfV (top row), was added for 2 hours. Cells were photographed (Olympus DP11-N digital camera) using an inverted Olympus Phase Contrast ULWCD 0.30 microscope. Original magnification, × 400. (D) HBMECs (106 cells/10-cm dish) were seeded on vitronectin- or PLL-coated plates that were blocked with BSA (VN and PLL/BSA), or on unblocked PLL-coated plates (PLL), as in Figure 1C. Cells were treated with vehicle control (-; DMSO, □), RADfV (A; ▨), or RGDfV (G; ▪; 25 μg/mL) for 24 hours. Apoptosis was assessed as in panels A-B, in triplicate samples for each condition. P < .001 between vehicle control– or RADfV-treated cells and the RGDfV-treated cells on vitronectin or on unblocked PLL. The difference was not significant on PLL/BSA (unpaired t tests, n = 3). (E) HBMECs (3 × 105 cells/well in 2-well chamber slides) were seeded and cultured for 30 hours without serum. Slides were fixed and stained for vitronectin, fibronectin, and tenascin as described in “Materials and methods.” Control slides that were stained with rabbit or mouse immunoglobulin G (IgG), but not the primary antibodies, showed only the blue nuclear counterstain (data not shown). Photographs were taken using an Olympus DP10 digital camera, on an Olympus BX40 microscope. Original magnification, × 400.
