Globotriaosylceramide induces oxidative stress and up-regulates cell adhesion molecule expression in Fabry disease endothelial cells

Abstract

Fabry disease, an X-linked systemic vasculopathy, is caused by a deficiency of alpha-galactosidase A resulting in globotriaosylceramide (Gb(3)) storage in cells. The pathogenic role of Gb(3) in the disease is not known. Based on previous work, we tested the hypothesis that accumulation of Gb(3) in the vascular endothelium of Fabry disease is associated with increased production of reactive oxygen species (ROS) and increased expression of cell adhesion molecules. Gb(3)-loading resulted in increased intracellular ROS production in cultured vascular endothelial cells in a dose-dependent manner. Increased Gb(3) also induced expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. Reduction of endogenous Gb(3) by treatment of the cells with an inhibitor of glycosphingolipid synthase or alpha-galactosidase A led to decreased expression of adhesion molecules. Plasma from Fabry patients significantly increased ROS generation in endothelial cells when compared with plasma from non-Fabry controls. This effect was not influenced by reduction of intracellular Gb(3). This study provided direct evidence that excess intracellular Gb(3) induces oxidative stress and up-regulates the expression of cellular adhesion molecules in vascular endothelial cells. In addition, other factors in patient's plasma may also contribute to oxidative stress in Fabry vascular endothelial cells.

Figures

Figure 1. Assessment of ROS production in primary ECs

(A) Immunostaining for Gb3 in primary ECs. Significant lysosomal accumulation of Gb3 could be detected in all 3 Fabry patients’ ECs (Pt. 1–3) but not in control’s ECs (cont). (B) Intracellular ROS generation in primary ECs was assessed by DCF fluorescence dye. There was no significant difference between the cells from Fabry patients and controls (P > 0.5, Mann-Whitney U test). The results are presented as mean of the data from six wells for each cell line. The results shown are representative of three independent experiments.

Figure 2. Effects of Gb3 on the generation of ROS in IMFE1 cells

IMFE1 cells were incubated with the medium containing Gb3/albumin complex for 3 days. Increase of intracellular Gb3 was verified by TLC (A) and immunostaining against Gb3 (B). Intracellular Gb3 was significantly increased after incubation with Gb3 (10 µmol/L) without significant changes in other GSLs. (C) Intracellular ROS generation was significantly increased in the cells loaded with exogenous Gb3 (5 and 10 µmol/L). The effect on ROS production was dependent on the concentrations of Gb3 in the media. Addition of 100 µmol/L vitamin C into the loading media significantly lowered ROS generation in the cells and abrogated the effect of Gb3 on ROS generation. The data are presented as mean ± SE (n = 6). Mann-Whitney U test was used to compare the statistical significance. Results shown are representative of three independent experiments.

Figure 3. Effects of Gb3 on CAMs expression in IMFE1 cells

IMFE1 cells were incubated in the medium containing Gb3 (10 µmol/L) for 2 days, then cells were treated with or without TNF-α (100 units/ml) for 4 hours. Total RNA was extracted and mRNA levels of CAMs were analyzed by TaqMan RT-PCR. Gb3-loading significantly increased steady state mRNA levels of all three CAMs analyzed, and increased ICAM-1 mRNA after TNF-α-stimulation. Results are obtained from three wells each and are representative of two independent experiments. The data are expressed as a ratio to untreated cells and presented as mean ± SE (n = 3), * P < 0.05, Mann-Whitney U test. Different y-axis scales are used for the absence (left) and presence (right) TNF-α treatment.

Figure 4. Effects of EtDO-P4 and α-galactosidase A (α-Gal A) treatments on CAMs expression

IMFE1 cells were treated with 1µmol/L EtDO-P4 for 10–17 days (A–C) or 0.08 IU/ml recombinant α-Gal A for 4 days (D and E). (A) Reduction of Gb3 in EtDO-P4 treated cells was verified with TLC. Graphs shown are the results of two independent experiments. (B) Quantitative data of GSLs levels obtained from the experiments shown in (A). The data are presented as mean ± SE (n = 2). (C) Effect of EtDO-P4 treatment on CAMs expression in presence and absence of TNF-α stimulation. (D) Lysosomal accumulation of Gb3 was reduced significantly by α-Gal A treatment when analyzed by immunostaining against Gb3. (E) Effect of α-Gal A treatment on ICAM-1 expression in the presence and absence of TNF-α stimulation. Different y-axis scales are used for the absence (left) and presence (right) of TNF-α treatment. Results are obtained from three wells and presented as mean ± SE (n = 3), * P < 0.05, Mann-Whitney U test.

Figure 5. Western blot analysis for CAMs expression after Gb3-loading and EtDO-P4 treatment

IMFE1 cells were incubated with 10 µmol/L Gb3 for 2 days (A) or 1 µmol/L EtDO-P4 for 17 days (B). The cells were treated with and without TNF-α (100 units/ml) for 6 hours before being harvested for Western blot analysis. β-actin was used as loading control. Results are representative of two independent experiments.

Figure 6. Effect of patients’ plasma on ROS generation in ECs

Endothelial cells with or without indicated pre-treatments were incubated with 20% plasma obtained from either Fabry patients (n = 6) or non-Fabry controls (n = 6) for 2 days, and intracellular ROS generation was assessed. (A) Effect of plasma on IMFE1 cells. (B) Effect of plasma on primary normal ECs (HMVEC). (C) IMFE1 cells were treated with 1 µM EtDO-P4 for 3 weeks to reduce endogenous Gb3. The cells then were incubated with 20% plasma in the presence of EtDO-P4 for 2 days. (D) IMFE1 cells were incubated with 0.08 IU/ml recombinant α-Gal A for 4 days to deplete endogenous Gb3. Then, the cells were incubated with 20% human plasma or FBS in the absence of α-Gal A for 2 days before DCF dye analysis. * P < 0.02, cells incubated with Fabry plasma were compared with the cells incubated with non-Fabry plasma; † P < 0.05, cells treated with α-Gal A were compared with un-treated cells (both cells were incubated with non-Fabry plasma after enzyme treatment); ‡ P < 0.03, cells treated with α-Gal A were compared with un-treated cells (both cells were incubated with FBS after treatment). Mann-Whitney U test was used to compare for statistical significance.