mRNA transfer by EPC-derived MVs. HMECs (5 × 10⁴ cells/well) were incubated for 24 hours in DMEM plus 5% BSA with 10 μg/mL MVs derived from GFP-EPC, and the synthesis of GFP protein was evaluated by confocal microscopy (Zeiss LSM 5 Pascal confocal Laser scanning microscope equipped with an Helium/Neon 543 mm laser, an Argon 450-530 mm laser, and an EC planar NEOFluar 63 × 1.4 oil DIC objective lens; acquisition software, Zeiss LSMS version 3.2). (A) Representative micrograph showing MVs labeled with the PKH26 red fluorescent dye. (B) Representative micrograph showing that the green fluorescence of GFP was not detectable by confocal microscopy in MVs derived from GFP-EPC. (C) Representative micrograph showing the uptake of PKH26-labeled MVs by HMECs after a 30-minute incubation at 37°C. (D-G) The detection is shown of green fluorescence in HMECs incubated with MVs derived from GFP-EPC after 30 minutes (D), 6 hours (E), 24 hours (F), or 72 hours (G) at 37°C. (H) Treatment with RNase abrogated the expression of GFP by HMECs incubated for 24 hours at 37°C with MVs derived from GFP-EPCs despite that labeled MVs were internalized by HMECs (I). Three experiments were performed with similar results. Scale bar, 10 μm. (J-M) Effect of MV mRNA extracts on in vitro angiogenesis assay. HMECs (5 × 10⁴ cells/well) were plated on growth factor–reduced Matrigel in DMEM plus 0.25% BSA and were challenged with vehicle alone or were stimulated with 10 μg/mL MVs or with 6μg Lipofectamine 2000 (Invitrogen) alone (Lf) or Lf plus 3 μg MV mRNA extracts, 3 μg mRNA alone, and 1 U/mL Rnase plus Lf plus MV mRNA extracts for 6 hours at 37°C. (J) Data show the mean (± 1 SD) of total length of capillary-like structures expressed as arbitrary units by the computer analysis system in 5 different files at ×20 magnification in duplicated wells of 4 different experiments. Analysis of variance with Newmann-Keuls multicomparison test was performed; *P < .05 treatments versus vehicle; §P < .05 mRNA alone or Lf plus mRNA plus RNase versus Lf plus mRNA extracts. (K-M) Representative micrographs showing the capillary-like structure formation on Matrigel by HMECs unstimulated (K) and stimulated with either (L) Lf + mRNA extracted from MVs or (M) with Lf + mRNA + RNase (Scale bar, 20 μm). The images were obtained by Nikon inverted microscope coupled with Casti Microimage analysis system as described in “In vitro angiogenesis.”