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. 2021 Jan:133:104098.
doi: 10.1016/j.mvr.2020.104098. Epub 2020 Oct 17.

Advanced glycation end-products disrupt brain microvascular endothelial cell barrier: The role of mitochondria and oxidative stress

Anthony Dobi  1 , Sarah Rosanaly  1 , Anne Devin  2 , Pascal Baret  1 , Olivier Meilhac  3 , G Jean Harry  4 , Christian Lefebvre d'Hellencourt  5 , Philippe Rondeau  6
Affiliations

Advanced glycation end-products disrupt brain microvascular endothelial cell barrier: The role of mitochondria and oxidative stress

Anthony Dobi et al. Microvasc Res. 2021 Jan.

Abstract

During diabetes mellitus, advanced glycation end-products (AGEs) are major contributors to the development of alterations in cerebral capillaries, leading to the disruption of the blood-brain barrier (BBB). Consequently, this is often associated with an amplified oxidative stress response in microvascular endothelial cells. As a model to mimic brain microvasculature, the bEnd.3 endothelial cell line was used to investigate cell barrier function. Cells were exposed to native bovine serum albumin (BSA) or modified BSA (BSA-AGEs). In the presence or absence of the antioxidant compound, N-acetyl-cysteine, cell permeability was assessed by FITC-dextran exclusion, intracellular free radical formation was monitored with H2DCF-DA probe, and mitochondrial respiratory and redox parameters were analyzed. We report that, in the absence of alterations in cell viability, BSA-AGEs contribute to an increase in endothelial cell barrier permeability and a marked and prolonged oxidative stress response. Decreased mitochondrial oxygen consumption was associated with these alterations and may contribute to reactive oxygen species production. These results suggest the need for further research to explore therapeutic interventions to restore mitochondrial functionality in microvascular endothelial cells to improve brain homeostasis in pathological complications associated with glycation.

Keywords: Advanced glycation end-products; Diabetes; Endothelial dysfunction; Mitochondria; Oxidative stress.

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Conflict of interest statement

Conflict of interests

The authors declare that there are no conflicts of interests

Figures

Fig 1:
Fig 1:. BSA-AGEs increase endothelial barrier permeability.
A) Cell viability was determined by crystal violet cell adherence assay after treatments with BSA or BSA-AGEs at 50 μM for 24h. Bars represent the mean ± SD cell number in each condition as a percentage of control (n = 4). Permeability was assayed on bEnd.3 cells using B) 4 kDa FITC-dextran (FD4) and B) 70 kDa FITC-dextran (FD70) before and after treatments with BSA or BSA-AGEs at 50 μM for 24 h. Mannitol diluted in DMEM (1.4 M) was used as positive control. The intensity of FITC fluorescence was measured in each basolateral chamber 10 min and 20 min after the addition of FD4 or FD70 in the apical chamber. Bars represent the mean ± SD of the variations of dextran quantities (μg) having crossed cell barrier (n = 4–5). *Effect of BSA-AGEs or Mannitol (vs. BSA) **p < 0.01, ***p < 0.001 determined by Tukey’s post hoc analysis following a significant two way ANOVA (for B and C).
Fig. 2:
Fig. 2:. BSA-AGEs induce an increase in bEnd.3 oxidative stress associated with endothelial barrier permeability which could be partially reduced by N-acetyl cysteine.
ROS levels were determined with H2DCF-DA probe in bEnd.3 cells treated with BSA or BSA-AGEs at 50 μM A) for 1h or 24h, and B) for 1h in the presence or absence of 0.5 mM N-acetyl cysteine (NAC). Bars represent the mean ± SD of 2’,7’-dichlorodihydrofluorescein (DCF) fluorescence as a percentage of vehicle (n = 3–4). C) Permeability was assayed on bEnd.3 cells using 4 KDa FITC-Dextran (FD4) before and after treatments with BSA or BSA-AGEs (50 μM) for 24h, in the presence or absence of 0.5 mM NAC. The intensity of fluorescence from FITC was measured in each basolateral chamber 10 min after the addition of FD4 in the apical chamber. Bars represent the mean ± SD of the variations of dextran quantities having crossed cell barrier (n = 4–5). D) SOD2 activity was determined in lysates from cells previously treated with BSA or BSA-AGEs (50 mM) for 24 h. Bars represent the mean ± SD of the enzyme activity (expressed as % of international catalytic unit in regard to vehicle condition (n = 3). *Effect of BSA-AGEs (vs. BSA). *p < 0.05, **p < 0.01, ***p < 0.001. #Effect of NAC (vs. BSA-AGEs). #p < 0.05 determined by Tukey’s post hoc analysis following a significant two way ANOVA (for A, B and C) or by a Student t-test (for D).
Fig. 3:
Fig. 3:. BSA-AGEs alter mitochondrial respiratory parameters of bEnd.3 cells.
Oxygen consumption by bEnd.3 cells was recorded 24h after treatments with BSA or BSA-AGEs (50 μM) for 24h. A-C: measurement by OROBOROS Oxygraph-2k; A) Spontaneous mitochondrial respiration. B) state 4 respiration (in the presence of oligomycin) and C) maximal respiration (in the presence of DNP). D-F: measurement by Seahorse XF Analyzer; D) Spontaneous mitochondrial respiration. E) state 4 respiration (in the presence of oligomycin) and F) maximal respiration (in the presence of DNP),. Bars represent the mean ± SD of oxygen consumption in percentage versus vehicle condition (n = 6–8). *Effect of BSA-AGEs (vs. BSA). *p < 0.05 determined by a Student t-test.
Fig. 4:
Fig. 4:. DNP modulates ROS production in BSA-AGEs-treated cells.
A) ROS levels were determined with H2DCF-DA probe in bEnd.3 cells treated with BSA or BSA-AGEs at 50 μM for 24h, and then co-incubated with or without (w/o) DNP (0.25 to 100 μM) for an additional 45 min. B) ROS levels were measured in cells treated with BSA or BSA-AGEs at 50 μM for 24h, and then co-incubated with or without DNP at 0.25 μM for an additional 45 min. Bars represent the mean ± SD of 2’,7’-dichlorodihydrofluorescein (DCF) fluorescence normalized in percentage (n = 3–4). *Effect of BSA-AGEs (vs. BSA). **p < 0.01. #Effect of DNP (vs. BSA-AGEs). #p < 0.05, ##p < 0.01 determined by Tukey’s post hoc analysis following a significant one way ANOVA (for A) or a two way ANOVA (for B).
Fig. 5:
Fig. 5:. BSA-AGEs induced bEnd.3 oxidative stress was rather due to reduced mitochondrial respiration.
Oxygen consumption by bEnd.3 cells was recorded 24h after treatments with BSA or BSA-AGEs at 50 μM for 24h, in the presence or absence of 0.5 mM NAC. A) Spontaneous mitochondrial respiration, B) state 4 respiration and C) maximal respiration were measured in each condition using OROBOROS Oxygraph-2k. D) Biomarker of mitochondrial content, citrate synthase activity was determined in lysates from bEnd.3 cells 24h after treatment. Bars represent the mean ± SD of the studied parameter in percentage versus vehicle condition (n = 6–8). E) SOD2 activity was determined in lysates from cells treated with BSA or BSA-AGEs (50 mM) for 24 h, in the presence or absence of 0.5 mM NAC. Bars represent the mean ± SD of the enzyme activity in percentage versus vehicle condition (n = 3). *Effect of BSA-AGEs (vs. BSA). *p < 0.05. #Effect of NAC (vs. BSA-AGEs). #p < 0.05 determined by Tukey’s post hoc analysis following a two way ANOVA (for A, B, C and E).
Fig. 6:
Fig. 6:
Proposed model for BSA-AGEs-induced an increase in endothelial cell barrier permeability related to oxidative stress and mitochondrial respiration.
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