N-Acetylcysteine protects against cobalt chloride-induced endothelial dysfunction by enhancing glucose-6-phosphate dehydrogenase activity

Abstract

Hypoxia-induced endothelial dysfunction is known to be involved in the pathogenesis of several vascular diseases. However, it remains unclear whether the pentose phosphate pathway (PPP) is involved in regulating the response of endothelial cells to hypoxia. Here, we established an in vitro model by treating EA.hy926 (a hybrid human umbilical vein cell line) with cobalt chloride (CoCl₂ ; a chemical mimic that stabilizes HIF-1α, thereby leading to the development of hypoxia), and used this to investigate the involvement of PPP by examining expression of its key enzyme, glucose-6-phosphate dehydrogenase (G6PD). We report that CoCl₂ induces the accumulation of HIF-1α, leading to endothelial cell dysfunction characterized by reduced cell viability, proliferation, tube formation, and activation of cytokine production, accompanied with a significant decrease in G6PD expression and activity. The addition of 6-aminonicotinamide (6-AN) to inhibit PPP directly causes endothelial dysfunction. Additionally, N-Acetylcysteine (NAC), a precursor of glutathione, was further evaluated for its protective effects; NAC displayed a protective effect against CoCl₂ -induced cell damage by enhancing G6PD activity, and this was abrogated by 6-AN. The effects of CoCl₂ and the involvement of G6PD in endothelial dysfunction have been confirmed in primary human aortic endothelial cells. In summary, G6PD was identified as a novel target of CoCl₂ -induced damage, which highlighted the involvement of PPP in regulating the response of endothelial cell CoCl₂ . Treatment with NAC may be a potential strategy to treat hypoxia or ischemia, which are widely observed in vascular diseases.

Keywords: N-Acetylcysteine; endothelial cells; glucose-6-phosphate dehydrogenase; hypoxia; pentose phosphate pathway.

Fig. 1

Treatment with CoCl₂ caused endothelial dysfunction. (A,B) EA.hy926 endothelial cells treated with CoCl₂ (400 μm) showed accumulation of HIF‐1α (120 kD) and lactate production; (C) cell viability was measured with CCK‐8 assay; (D,E) EdU assay was applied to evaluate the proliferation ability of endothelial cells upon CoCl₂ treatment for 48 h at 400 μm. Scale bars = 200 μm; (F,G) Matrigel assay was applied to evaluate the tube formation capacity of endothelial cells upon 400 μm CoCl₂ treatment for 48 h (right panel) compared to control group (left panel). Scale bars = 200 μm; (H–K) Real‐time PCR was applied to measure gene expression of VEGF, IL‐1β, IL‐6, ICAM‐1 in EA.hy926 cells stimulated with 400 μm CoCl₂ for 48 h. Data are expressed as mean ± SEM of independent experiments (n ≥ 3). Student's t‐test or One‐way ANOVA test followed by Tukey's multiple comparison test was used. *P < 0.05, **P < 0.01, and ***P < 0.001 vs control group. [Colour figure can be viewed at wileyonlinelibrary.com]

Fig. 2

Treatment with CoCl₂ inhibited G6PD expression and activity. (A,B) Treatment of EA.hy926 cells with CoCl₂ at 400 μm time‐dependently inhibited G6PD (58 kD) expression; (C,D) G6PD activity, and products were inhibited by CoCl₂ (400 μm) treatment for 48 h as determined by G6PD activity Assay kit and NADP/NADPH Assay kit; (E) Low oxygen (1%) stimulation for 24 and 48 h inhibited G6PD expression in EA.hy926 cell line, which was comparable to CoCl₂ treatment at 400 μm. Data are expressed as mean ± SEM of independent experiments (n ≥ 3). Student's t‐test was used. *P < 0.05 vs control group.

Fig. 3

Pentose phosphate pathway is involved in endothelial dysfunction. (A,B) Addition of 6‐AN to EA.hy 926 cells time‐ and dose‐dependently decreased cells viability according to CCK‐8 assay; (C) Addition of 6‐AN at 50 μm for 48 h inhibited G6PD activity as measured by G6PD activity assay kit; (D,E) Addition of 6‐AN at 50 μm for 24 or 48 h inhibited cell proliferation according to EdU assay. Scale bars = 200 μm; (F and G) Addition of 6‐AN at 50 μm for 48 h inhibited tube formation. Scale bars = 200 μm; (H–K) Treatment with 6‐AN at 50 μm for 48 h induced activation of VEGF, IL‐1β, IL‐6, ICAM‐1, respectively. Data are expressed as mean ± SEM of independent experiments (n ≥ 3). Student's t‐test or One‐way ANOVA test followed by Tukey's multiple comparison test was used. *P < 0.05 vs control group. [Colour figure can be viewed at wileyonlinelibrary.com]

Fig. 4

NAC ameliorated CoCl₂ induced endothelial cell dysfunction. (A–E) Cell viability, proliferation, and tube formation capacity were measured by CCK‐8 assay, EdU assay, and matrigel tube formation in EA.hy926 cells stimulated with 400 μm CoCl₂ for 48 h in the presence or absence of NAC (10 mm) pretreatment for 1 h, respectively. Scale bars = 200 μm; (F–I) Real‐time PCR was applied to measure gene expression of VEGF, IL‐1β, IL‐6, and ICAM‐1 in EA.hy926 cells stimulated with 400 μm CoCl₂ for 48 h pretreated with or without NAC (10 mm) for 1 h; (J,K) Pretreatment with NAC at 10 mm for 1 h did not rescue decreased G6PD protein level caused by 400 μm CoCl₂ for 48 h; (L) Reduced G6PD activity by treatment of 400 μm CoCl₂ for 48 h was efficiently rescued by pretreatment with NAC at 10 mm for 1 h. Data are expressed as mean ± SEM of independent experiments (n ≥ 3). Student's t‐test or One‐way ANOVA test followed by Tukey's multiple comparison test was used. *P < 0.05 vs control group; #P < 0.05 vs CoCl₂ group. [Colour figure can be viewed at wileyonlinelibrary.com]

Fig. 5

6‐AN abrogated the protective effect of NAC on endothelial cell damage induced by CoCl₂. EA.hy926 endothelial cells were treated with CoCl₂ in the presence or absence of NAC (10 mm) or 6‐AN (50 μm) pretreatment for 1 h. Cell viability, proliferation, tube formation capacity, and cytokine expression were measured by CCK‐8 assay(A), EdU assay (B,C), matrigel assay (D,E), and Real‐time PCR (F,I) in EA.hy926 cells stimulated with 400 μm CoCl₂ for 48 h, respectively. Scale bars = 200 μm. Data are expressed as mean ± SEM of independent experiments (n ≥ 3). Student's t‐test or One‐way ANOVA test followed by Tukey's multiple comparison test was used. *P < 0.05 vs control group, #P < 0.05 vs CoCl₂ group, &P < 0.05 vs NAC + CoCl₂ group. [Colour figure can be viewed at wileyonlinelibrary.com]

Fig. 6

Synthetic phenotypic change of VSMCs induced by conditional medium from CoCl₂‐treated endothelial cells is ameliorated by NAC which is G6PD dependent. mRNA level of IL‐6 (A), iNOS (B), and MMP9 (C) was evaluated in A7r5 rat aortic VSMC cell line treated with conditional medium (CM, black bars) from HAECs treated by CoCl₂ in the presence or absence of NAC or 6‐AN for 48 h, or in fresh medium (control, gray bars) supplemented with CoCl₂ (400 μm), NAC (10 mm), or 6‐AN (50 μm) for 24 h. Data are expressed as mean ± SEM of independent experiments (n = 3). One‐way ANOVA test followed by Tukey's multiple comparison test was used. *P < 0.05 vs control group, #P < 0.05 vs CoCl₂ group, &P < 0.05 vs NAC + CoCl₂ group. [Colour figure can be viewed at wileyonlinelibrary.com]