Atherogenesis May Involve the Prooxidant and Proinflammatory Effects of Ferryl Hemoglobin
Figure 6
FerrylHb disrupts endothelial monolayer and induces leukocyte adhesion in HUVECs. (a) Confluent HUVECs grown in 6-well plates were exposed to Hb and ferrylHb at a dose of 0–20 μg/mL overnight. Images are 100X, taken with an inverted microscope (Carl Zeiss 426126), and analyzed by ImageJ software. (b) Confluent HUVECs grown in hanging cell culture inserts were treated with heme, Hb, metHb, and ferrylHb (10 μmol/L heme groups each) for 12 hours. Fluorescein (1 μmol/L) was added into the apical filter compartment and was detected in the lower compartment after a 60-minute incubation. Endothelial permeability is expressed as fold increase over nontreated cells. (c) Confluent HUVECs were treated with heme, Hb, metHb, and ferrylHb (10 μmol/L heme) for 12 hours. Monocytes were labeled and added to HUVECs (105 cells/well) for 30 minutes at 37°C. Cells were stained with TRITC-conjugated phalloidin and with Hoechst. Images are 400x. Results are shown as mean ± S.D. () from one representative experiment of three. Images are representative of 3 independent experiments.
