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. 2016 Jan 27;36(2):274–284. doi: 10.1161/ATVBAHA.115.306827

Figure 3.

Figure 3.

Proteolysis of apolipoprotein A-I (apoA-I) impairs its ability to attenuate nuclear factor-κB (NF-κB) activation to bind to human coronary artery endothelial cells (HCAECs) and to prevent transmigration of THP-1 monocytes across HCAEC monolayer. A, HCAECs were incubated for 16 h with untreated or chymase-treated apoA-I and then activated with tumor necrosis factor-α (TNF-α) for 15 min. Nonactivated cells (control) and TNF-α-activated cells preincubated in only medium (buffer) acted as a reference. Nuclear NF-κB p65 activity was determined by measuring nuclear translocation of the NF-κB p65 subunit. The basal activity of the nonactivated HCAECs (control) was set as 1. Data represent the means±SD from triplicate wells and are representative of 2 independent experiments. *P<0.05. B, ApoA-I was labeled with 125I and then treated with chymase as described in Figure 1B. HCAECs were incubated with the indicated concentrations of 125I-labeled untreated or chymase-treated apoA-I in the absence or presence of a 40-fold excess of untreated apoA-I for 2 h at 4°C. High-affinity binding of 125I-apoA-I to HCAECs was calculated by subtracting the values of the nonspecific binding from the total binding. A representative pair of binding curves is shown in the top panel. Data (mean±SD) from 4 independent experiments from duplicate wells are expressed as percentages of untreated apoA-I (bottom). *P<0.01. C, Confluent HCAEC monolayers grown on a transwell insert were first incubated with untreated or chymase-treated apoA-I added either to the apical or basolateral compartment, then activated with TNF-α for 5 h, and finally incubated with fluorescently labeled THP-1 cells added to the apical compartment. Migration across the endothelial monolayer of the cells was quantified by measuring the fluorescence signal of the transmigrated cells. Migration in the absence of any additions was designated as 100% (control). Data shown represent the means±SD from 3 independent experiments performed in triplicate wells. *P<0.05; #P<0.05 (untreated apoA-I vs buffer).