Apoptotic EC-conditioned media contain MFG-E8 and reprogram macrophages.

<p><b>A</b> Percentage of cells with increased chromatin condensation and cell membrane permeabilization (as evaluated with HO and PI staining) in HUVEC exposed to normal medium with growth factors without serum (normal serum-starved, NSS) or serum starvation (SS) for 1 h to 4 h (*<i>p</i><0.001 versus Normal, <i>n</i> = 3). Example of HO/PI staining on serum starved HUVEC for 4 h showing chromatin condensation, right panel. <b>B</b> MEC and HUVEC were serum-starved for 1 h to 4 h. Supernatants (upper panels) and cells (lower panels) were harvested. Immunoblotting of MEC protein extracts showed that MFG-E8 levels decreased over time in parallel with increased active caspase-3 levels (lower left panel). HUVEC also exhibited reduced intracellular MFG-E8 levels over time (lower right panel). MFG-E8 levels increased over time in serum-starved conditioned medium (SSC) from EC (upper panels). β-Actin and Ponceau red staining were loading controls. Representative of 3 experiments. <b>C</b> Percentage of cells with increased chromatin condensation and cell membrane permeabilization (as evaluated with HO and PI staining) in HUVEC exposed to MMC 0.01 mg/mL or vehicle in normal medium and serum starvation (as positive control) for 15 h (left panel), *<i>p</i><0.0001 versus vehicle, <i>n</i> = 3. Immunoblot for hMFG-E8 in supernatant of EC treated with MMC (right panel). Ponceau red staining is shown as loading control. Representative of 2 experiments. <b>D</b> Immunoblot for hMFG-E8 in supernatants conditioned by necrotic HUVEC (3 freeze-thaw cycles) and serum-starved HUVEC as positive control. Ponceau red staining included as loading control. Representative of 2 experiments. <b>E</b> Immunoblot for mMFG-E8 from total medium conditioned by apoptotic EC (Total SSC), supernatant after removal of apoptotic blebs by centrifugation at 50 000 g (SSC without (W/O) blebs) and apoptotic blebs (Blebs) purified from total SSC by centrifugation, supernatant obtained from the supernatant after 50 000 g and 200 000 g centrifugation (SSC W/O exo.) and exosome-like nanovesicle fraction pelleted after the 200 000 g centrifugation (Exo.). Proteins from equal initial volumes were precipitated by TCA. Ponceau red staining is shown as loading control of samples. Representative of 2 experiments. <b>F</b> MEC were serum-starved for 4 h, the SSC were harvested, centrifuged to remove apoptotic cells. Murine macrophages were exposed to SSC or serum starvation (SS) for 24 h. ELISA were performed for TGF-β₁, VEGF, IL-10, (left panel) MCP-1 and MIP-2 (right panel), *<i>p</i><0.05, representative of <i>n</i> = 14, 12, 4, 7 and 9 separate experiments respectively.