Skip to main content
. 2022 Jul 25;11(15):2288. doi: 10.3390/cells11152288

Figure 5.

Figure 5

Interaction of endothelial cells with NE or isolated neutrophils stimulated monocyte transendothelial migration via activation of the PAR2 pathway. (A) Confluent hECs grown on collagen-coated transwell inserts (8 μm pore size) were treated with NE (20 nM) or PAR2 agonist (PAR2-AP, 7.5 μM) in the presence or absence of NE inhibitor (NEI, 1 μM), PAR2 inhibitor (PAR2 Inh, 1 μM), ROCK inhibitor (ROCK Inh, 5 μM), and MLCK inhibitor (MLCK Inh, 5 μM) for 16 h. hECs were washed with normal media before adding BCECF-AM labeled THP-1 cells (1 × 105) for 6 h. (B) Confluent hECs on transwell inserts were treated with freshly isolated neutrophils (1 × 106 cells/well) for 16 h in the presence or absence of NE inhibitor (NEI, 1 μM) or PAR2 inhibitor (I191, 1 μM). hEC monolayer on the top chamber was washed with cell media to remove neutrophils. Then, BCECF-AM labeled THP-1 cells (1 × 105 cells/well) were used to assess transmigration for 2 h. Representative images show fluorescence-labeled THP-1 cells transmigrated through the hEC monolayer were captured (A,B). For quantification, monocytes were counted in five random microscopic fields per well. The values are expressed as mean ± SD of three independent experiments. Top panel: ** p < 0.01 vs. vehicle control group, # p < 0.05 vs. NE group; lower panel: ** p < 0.01 vs. vehicle control group, # p < 0.05 vs. neutrophil (Neut) group.