Fig. 3

OxHDL increases LOX-1 expression at the cellular plasma membrane through the ROS/NOX-2 pathway. Endothelial ROS were determined by means of the ROS-sensitive dye, DCF (a), Amplex red (f), and DHE/HPLC (k), in HAEC and HUVEC exposed to oxHDL (50 μg/ml) and HDL (50  μg/ml) for 0, 6, 12, 18, and 24 h (N = 6). Endothelial ROS were determined by means of the ROS-sensitive dye, DCF (b), Amplex red (g) and DHE/HPLC (l), in HAEC and HUVEC exposed to vehicle, oxHDL (50 μg/ml) and HDL (50 μg/ml) in the absence (–) or presence (+) of κ-carrageenan (250 μg/ml) for 6 h (N = 6). Endothelial ROS were determined by means of the ROS-sensitive dye, DCF (c), Amplex red (h), and DHE/HPLC (m), in HAEC and HUVEC exposed to vehicle and oxHDL (50 μg/ml) in the absence or presence of the NAD(P)H oxidase inhibitors DPI (10 μM) and Apocynin (10 mM) for 0, 6, 12, 18, and 24 h (N = 6). Endothelial ROS were determined by means of the ROS-sensitive dye, DCF (d and e), Amplex red (i and j) and DHE/HPLC (n and o), in HAEC and HUVEC exposed to vehicle and oxHDL (50 μg/ml) in the absence or presence of transfection with a siRNA against NOX-1 (siNOX1), NOX-2 (siNOX2), and NOX-4 (siNOX4) for 0, 6, 12, 18, and 24 h. Data are expressed normalized to 0 time (N = 6). Statistical differences for HAEC and HUVEC samples were assessed by a one-way analysis of variance (ANOVA) (Kruskal–Wallis) followed by Dunn’s post hoc test. *P < 0.05. Graph bars show the mean ± SD. LOX-1 expression was measured in the membrane-rich fraction from ECs exposed to HDL (50 μg/ml) and oxHDL (50 μg/ml) for 24 h in the absence (–) or presence (+) of NAC (5 mM) (p and q), GSH (1 mM) (r–s), DPI (10 μM) (t and u), and Apocynin (10 mM) (v–x) Data are expressed normalized to vehicle condition (N = 3). Statistical differences were assessed by a two-way analysis of variance (ANOVA) followed by Tukey post hoc test. **P < 0.01. Graph bars show the mean ± SD