Figure 2

Pressure-induced endothelial activation promotes reactive oxygen species (ROS) production and arginase II via NFĸB-dependent pathways. (A-D) Cultured HUVECs were pressurised (0, 60 or 120 mmHg) for 1 h in a flow-through chamber and the % change in intensity of 2′-7′ dichlorodihydrofluorescein diacetate (A-B; DCFHDADA; 5 μM; n = 5–7) and peroxyfluor-6 acetoxymethyl ester (C-D; PF6-AM; 5 μM; n = 6–7) assessed. Propidium iodide (PI; 5 μM) was also visualized to assess cell death. (E) Quantification of leukocyte adhesion (at 10 min) during high pressure (120 mmHg) coupled with either NADPH oxidase inhibitor (apocynin; 3 μM), xanthine oxidase inhibitor (allopurinol; 100 μM), arginase inhibitor (BEC, [S-(2-boroethyl)-L-cysteine], angiotensin receptor 1 antagonist (candesartan; 100 nM), mitochondrial ROS inhibitor (cyclosporine A; 2 μM), 100 μM), cholesterol chelant (methyl-β-cyclodextrin, MβCD; 10 mM) or the cytochrome P450 inhibitor (miconazole; 30 μM) (n = 4–5). (F and G) Real-time PCR of pressurised carotid arteries (0, 80 and 120 mmHg) was performed to determine the gene expression of arginase I (ArgI; n = 3–4) and arginase II (ArgII; n = 4–7) isoforms. (H-J) HUVECs were seeded onto glass slides, unpressurised (0 mmHg) or pressurised to 120 mmHg and fluorescently probed and imaged in the absence and presence of the NADPH oxidase inhibitor, apocynin (3 µM) or the arginase inhibitor BEC (100 µM) to determine the nuclear (I) and cytosolic (J) expression of NFĸB (nuclear factor κB, n = 5–11). (K) CCL2 and (L) ICAM1 gene expression in HUVECs pressurised to 120 mmHg was assessed in the presence and absence of the NFκB inhibitor, SN50 (20 µM), which prevents translocation of cytosolic NFκB to the nucleus (n = 5–6). All results are expressed as mean ± SEM. Data were analysed with a Student’s t-test (E, K, and L), a one-way (F-G and I-J), or a two-way ANOVA (B, D) with Bonferroni post hoc test where *p < 0.05, **p < 0.01 and ***p < 0.001.