Lysosomal oxidation of LDL alters lysosomal pH, induces senescence, and increases secretion of pro-inflammatory cytokines in human macrophages

Abstract

We have shown that aggregated LDL is internalized by macrophages and oxidized in lysosomes by redox-active iron. We have now investigated to determine whether the lysosomal oxidation of LDL impairs lysosomal function and whether a lysosomotropic antioxidant can prevent these alterations. LDL aggregated by SMase (SMase-LDL) caused increased lysosomal lipid peroxidation in human monocyte-derived macrophages or THP-1 macrophage-like cells, as shown by a fluorescent probe, Foam-LPO. The pH of the lysosomes was increased considerably by lysosomal LDL oxidation as shown by LysoSensor Yellow/Blue and LysoTracker Red. SMase-LDL induced senescence-like properties in the cells as shown by β-galactosidase staining and levels of p53 and p21. Inflammation plays a key role in atherosclerosis. SMase-LDL treatment increased the lipopolysaccharide-induced secretion of TNF-α, IL-6, and MCP-1. The lysosomotropic antioxidant, cysteamine, inhibited all of the above changes. Targeting lysosomes with antioxidants, such as cysteamine, to prevent the intralysosomal oxidation of LDL might be a novel therapy for atherosclerosis.

Keywords: antioxidants; atherosclerosis; lipid peroxidation; low density lipoprotein; lysosomes.

Fig. 1.

Lipid peroxidation and ROS in human macrophages. THP-1 macrophages or HMDMs were treated with no LDL, native LDL, or SMase-LDL (both at 200 μg protein/ml LDL protein) in the presence or absence of 5, 10, or 25 μM cysteamine for 24 h. The cells were then incubated with either 2 μM Foam-LPO for 15 min or 10 μM DHE for 30 min, harvested, and assayed by flow cytometry. A: MFI of of Foam-LPO in red channel of healthy THP-1 macrophages, native-LDL-treated THP-1 macrophages, and SMase-LDL-treated THP-1 macrophages. B: MFI of Foam-LPO in red channel of SMase-LDL-treated THP-1 macrophages in the presence or absence of cysteamine (Cys) (10 and 25 μM). C, D: Lipid peroxidation calculated from the ratio between the MFI of the green channel (FL1) and red channel (FL3) in THP-1 macrophages (C) and HMDMs (D). E, F: Overall ROS production in HMDM control, native LDL-treated, and SMase-LDL-treated cells. G: The effect of cysteamine (25 μM) on SMase-LDL ROS production. H: Analysis of MFI of ROS generation. ***P < 0.001, **P < 0.01 compared with untreated cells, ^###P < 0.001, ^##P < 0.01, ^#P < 0.05; ANOVA followed by Tukey’s test; n = 3–6 independent experiments.

Fig. 2.

Effect of SMase-LDL and cysteamine (Cys) on LysoTracker Red accumulation by macrophages. THP-1 macrophages or HMDMs (1 × 10⁶) were cultured in 12-well tissue culture plates in RPMI medium (containing 10% v/v FCS) alone or containing native LDL or SMase-LDL with or without cysteamine (10 or 25 μM) for 72 h. All LDL concentrations were 100 μg protein/ml. After 72 h, cells were treated with 500 nM LysoTracker Red for 30 min and then assayed by flow cytometry. The MFI peak of LysoTracker Red in the red channel was then measured. A: MFI in red channel of healthy THP-1 macrophages, native-LDL-treated macrophages, and SMase-LDL-treated macrophages. B: MFI in red channel of SMase-LDL treated THP-1 macrophages in the presence or absence of cysteamine (10 and 25 μM). C: Data expressed as percentage loss of MFI of LysoTracker Red in the red channel compared with untreated control macrophages in THP-1 macrophages. D: Data expressed as percentage loss of MFI of LysoTracker Red in the red channel compared with untreated macrophages in HMDMs. **P < 0.01, ***P < 0.001 compared with untreated cells; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared with SMase-LDL-treated cells; ANOVA followed by Tukey’s test; n = 4 independent experiments.

Fig. 3.

Effect of lysosomal oxidation of SMase-LDL on the pH of lysosomes in THP-1 macrophages. THP-1 macrophages (A) or HMDMs (D) were cultured in a black 96-well microplate at 1 × 10⁵ per well in RPMI medium (containing 10% v/v FCS) with no LDL, native LDL, or SMase-LDL (both at 100 μg protein/ml) with or without cysteamine (Cyst/Cys) (10 or 25 μM) for 72 h. The cells were then treated with 5 μM LysoSensor Yellow/Blue for 30 min at 37°C. The samples were then read in a FLUOstar Optima fluorometer, with excitation at 355 nm. The ratio of emission at 440 nm and 535 nm was then calculated for each sample and the pH values determined from a standard plot. B, E: The effect of cysteamine on control THP-1 macrophages (B) and HMDMs (E). C: The effect of cysteamine on native LDL-treated THP-1 cells. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with SMase-LDL-treated macrophages; ANOVA followed by Tukey’s test of at least four independent experiments.

Fig. 4.

Effect of lysosomal oxidation of LDL on senescence in human macrophages. HMDMs were cultured in 12-well tissue culture plates at 3,000 cells per well in RPMI medium (containing 10% v/v lipoprotein-deficient serum) containing either no LDL (A), native LDL (B), SMase-LDL alone (C), or SMase-LDL (D) (all at 100 μg protein/ml) with 10 μM cysteamine (Cys) for 72 h. The cells were then stained to identify any senescent cells by a lysosomal β-galactosidase activity assay and p53 and p21 expression. E, F: The percentage of senescent cells in HMDMs (E) and THP-1 cells (F), which had been treated in the same way. The images shown are representative of three independent experiments. G, H: The MFI for p53 (G) and p21 (H) expression in HMDMs. I, J: A comparison of p53 (I) and p21 (J) MFI under various treatment conditions. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control cells; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 for the indicated comparison; ANOVA followed by Tukey’s test of at least three independent experiments.

Fig. 5.

Effect of SMase-LDL on cytokine expression in macrophages. THP-1 macrophages or HMDMs were incubated in fresh RPMI-1640 medium (containing 10% v/v FBS), alone or with native LDL or SMase-LDL (both at 50 μg protein/ml) for either 12 or 24 h, and the medium was assayed for various pro-inflammatory cytokines. Some of the wells were preincubated with cysteamine (Cys) (10 or 25 μM) for 24 h prior to SMase-LDL treatment. After SMase-LDL treatment, the cells were washed with PBS and then stimulated with LPS (10 ng/ml) for 4 h at 37°C, and the medium was collected and assayed for cytokine levels. *P < 0.05, **P < 0. 01, and ***P < 0.001 compared with the control cells; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 for the indicated comparison. The data shown are from at least three independent experiments and were analyzed by one-way ANOVA followed by Tukey’s posttest.

Fig. 6.

Effect of cysteamine on LDL oxidation catalyzed by iron at pH 4.5. SMase-LDL (50 μg protein/ml) in NaCl/sodium acetate buffer (pH 4.5) was incubated with 5 μM FeSO₄ in the presence or absence of cysteamine (25 μM) at 37°C in capped quartz cuvettes. Oxidation was monitored by measuring the change in attenuance at 234 nm (A) or loss of LDL-tryptophan fluorescence against appropriate reference cuvettes (C). This is a representative example of three independent experiments. B: Time taken to reach an attenuance of 0.1 during the oxidation with iron. D: Decrease in LDL-tryptophan fluorescence after 150 min of oxidation. ***P < 0.001; t-test; n = 3 independent experiments.