Altered lipoproteins in patients with systemic lupus erythematosus are associated with augmented oxidative stress: a potential role in atherosclerosis

Fig. 1. Comparison of serum lipid profile between SLE patients and heathy controls. a Fasting levels of total cholesterol, triglyceride (TG), HDL, and LDL were compared between SLE patients (n = 35) and HCs (n = 15). SLE patients had higher levels of TG and lower levels of HDL than HCs. b The serum activity of CETP was significantly higher in SLE patients than in HCs. c Equivalent amounts of HDL2, HDL3, and LDL pooled from SLE (n = 19) patients and HCs (n = 8) were subjected to SDS-PAGE (6% gels for HDL2 and HDL3 and 15% gels for LDL). HDL2 and HDL3 ran as a single band which was identified as apoA-I on Western blot (WB). The intensity of the band of apoA-I was weaker in SLE samples than in HC samples (left and middle panels). The proteins derived from SLE-LDL ran as multiple fragments (right panel, arrow), whereas HC-LDL ran as a single band. The numbers under the bands represent the relative band intensity whereas the intensity of SLE bands was set as 1.00). Results are representative of three independent experiments. Data are expressed as the mean and SEM. CETP cholesterol ester transfer protein, M markers, HC healthy control, SLE systemic lupus erythematosus

Fig. 2. Increased oxidation of lipoproteins from systemic lupus erythematosus (SLE) patients. a HDL2, HDL3, and LDL were isolated from SLE patients (n = 19) and healthy controls (HCs) (n = 8), and their oxidation status (i.e., MDA levels) was measured. All lipoprotein fractions from SLE patients showed significantly higher levels of oxidation than those from HCs. b LDL was incubated in the presence of 5 μM CuSO4, and the formation of conjugated dienes over time was measured as a marker of de novo oxidation. LDL from SLE patients was significantly more susceptible to oxidation than that from HCs. c Serum paraoxonase activity was significantly lower in SLE samples than in HC samples. Data are expressed as the mean and SEM. HC healthy controls, MDA malondialdehyde, SLE systemic lupus erythematosus

Fig. 3. Monocytes show increased uptake of LDL from SLE patients. a and b THP-1 cells were incubated with LDL isolated from SLE patients (n = 19) and HCs (n = 8). THP-1 cells phagocytosed significantly more LDL from SLE patients and showed greater foam cell formation (NBD-positive area: 2501 ± 401.2 AUs vs. 524.1 ± 59.9 AUs, respectively; p < 0.001). c Human fibroblast cells were incubated with LDL from SLE patients or HCs, and β-galactosidase activity (a surrogate marker for cellular senescence) was measured. β-galactosidase activity was higher in fibroblasts treated with SLE-LDL (blue) than in those treated with HC-LDL. d SLE-LDL induced significantly higher β-galactosidase activity than HC-LDL (β-gal positive area: 70.9 AUs vs. 16.3 AUs, respectively; p < 0.001). Representative images (×400 magnification) from at least three independent experiments are shown. Data are expressed as the mean and SEM. AU arbitrary units, gal galactosidase, HC healthy control, MDA malondialdehyde, NBD 22-(N-7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino-23, 24-bisnor-5-cholen-3-ol, SLE systemic lupus erythematosus

Fig. 4. LDL from SLE patients induces oxidative stress in vivo. a Zebrafish embryos were injected with LDL from SLE patients (n = 19) or HCs (n = 8), and oxidation of dihydroethidium (DHE, bright red; a surrogate marker for reactive oxygen species production) was observed under a fluorescence microscope. The embryos injected with SLE-LDL showed increased fluorescence compared with those treated with PBS or HC-LDL. b SLE-LDL induced significantly more oxidation than HC-LDL. c The embryos injected with oxLDL alone showed strong fluorescence, which was reduced upon co-injection of HDL3 from HCs or SLE patients. d HDL3 from SLE patients induced significantly more DHE oxidation than that from HCs. e Injection of SLE-LDL reduced the embryo survival to a greater extent than HC-LDL or PBS. f The embryos treated with purified oxLDL showed a markedly reduced survival rate after 48 hours. HDL3 from SLE patients and HCs increased the survival of embryos treated with oxLDL. HC-HDL3 improved the survival to a greater extent than SLE-HLD3. Data are expressed as the mean and SEM (* p < 0.001). AU arbitrary units, DHE dihydroethidium, HC healthy control, MDA malondialdehyde, SLE systemic lupus…