Figure 1.
Anion-exchange chromatography of low-density lipoprotein (LDL) from a hypercholesterolemic patient.
Chromatograms showing the L5 percentage before treatment (left), after 3 months of atorvastatin treatment (10 mg/day; center), and 3 months after the discontinuation of treatment (right) in hypercholesterolemic patient (baseline plasma LDL-cholesterol level, 226 mg/dL). Treatment with atorvastatin for 3 months reduced the level of L5, and discontinuation of treatment for 3 months resulted in the recurrence of elevated L5.
Table 1.
Characteristics of Patients with Hypercholesterolemia and Healthy Control Subjects Before Atorvastatin Treatment.
Table 2.
Characteristics of Patients with Hypercholesterolemia Before and After Atorvastatin Treatment.
Figure 2.
Analysis of C-reactive protein (CRP) expression by using Western blot and enzyme-linked immunosorbent assay (ELISA).
(A, B) C-reactive protein expression in human aortic endothelial cells (HAECs) treated with L5 from 5 hypercholesterolemic patients (patients labeled as A–E). L5 increased endothelial CRP expression by up to 2.5-fold more than did L1. (C) L5 treatment (10–100 µg/mL) increased endothelial CRP expression in a dose-dependent manner. (D) ELISA analysis of CRP in the conditioned culture medium (CCM) of HAECs treated with phosphate-buffered saline (PBS), L1, or L5. For all experiments, n = 3, and bars shown in graphs represent standard deviation. *P<0.05, **P<0.01, ***P<0.001 vs. PBS or untreated control.
Figure 3.
Detection of total reactive oxygen species (ROS) in human aortic endothelial cells (HAECs).
(A) L5 (50 µg/mL) from patients increased ROS production (green) in HAECs after 20 minutes (top and middle row) and after 24 hours (bottom row). Compared to the phosphate-buffered saline (PBS) control, L1 treatment had no effect. Staining with 4′,6-diamidino-2-phenylindole (DAPI) (blue) indicated HAEC apoptosis 24 hours after L5 (50 µg/mL) exposure. The pretreatment of cells with TS92 attenuated the L5-induced ROS increase after 20 minutes (middle row). (B) Blocking the production of ROS by adding ROS inhibitor N-acetyl cysteine (NAC) attenuated the L5-induced increase in CRP levels. For all experiments, n = 3, and bars shown in graphs represent standard deviation. *P<0.05, **P<0.01 vs. untreated control.
Figure 4.
Effects of lectin-like oxidized LDL receptor-1 (LOX-1) inhibition in human aortic endothelial cells (HAECs).
(A) HAECs were pretreated with LOX-1 neutralizing antibody TS92 before the introduction of L1 or L5 into cell culture. (B) HAECs were pretreated with TS92 before the introduction of recombinant human CRP (5 or 50 µg/mL) into cell culture. For all experiments, n = 3, and bars shown in graphs represent standard deviation. *P<0.05 vs. untreated control.
Figure 5.
Time-course analysis of L1 and L5 internalization by human aortic endothelial cells (HAECs) and L5-induced C-reactive protein (CRP) expression.
(A) Results of fluorescence microscopy showing that DiI-L1 and DiI-L5 (each red) were internalized by HAECs at different time points. The nuclei of HAECs were co-stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). (B) Western blot showing that L5 augmented endothelial CRP expression in a time-dependent manner. Time point 0 represents the phosphate-buffered saline (PBS) control. *P<0.05 vs. PBS-treated control. (C) L5 deprivation study showed that internalized L5 continued to induce CRP and lectin-like oxidized receptor-1 (LOX-1) expression 24 hours after replacing L5 conditioned culture media with fresh EGM2 media. L5 exposure times are shown. HAECs were incubated with recombinant CRP for 2 hours as a positive control. Time point 0 represents the PBS control. For all experiments, n = 3, and bars in graphs represent standard deviation. †P<0.05 vs. PBS-treated CRP control, *P<0.05 vs. PBS-treated LOX-1 control.