Adaptive induction of NF-E2-related factor-2-driven antioxidant genes in endothelial cells in response to hyperglycemia

Abstract

Hyperglycemia in diabetes mellitus promotes oxidative stress in endothelial cells, which contributes to development of cardiovascular diseases. Nuclear factor erythroid 2-related factor-2 (Nrf2) is a transcription factor activated by oxidative stress that regulates expression of numerous reactive oxygen species (ROS) detoxifying and antioxidant genes. This study was designed to elucidate the homeostatic role of adaptive induction of Nrf2-driven free radical detoxification mechanisms in endothelial protection under diabetic conditions. Using a Nrf2/antioxidant response element (ARE)-driven luciferase reporter gene assay we found that in a cultured coronary arterial endothelial cell model hyperglycemia (10-30 mmol/l glucose) significantly increases transcriptional activity of Nrf2 and upregulates the expression of the Nrf2 target genes NQO1, GCLC, and HMOX1. These effects of high glucose were significantly attenuated by small interfering RNA (siRNA) downregulation of Nrf2 or overexpression of Keap-1, which inactivates Nrf2. High-glucose-induced upregulation of NQO1, GCLC, and HMOX1 was also prevented by pretreatment with polyethylene glycol (PEG)-catalase or N-acetylcysteine, whereas administration of H(2)O(2) mimicked the effect of high glucose. To test the effects of metabolic stress in vivo, Nrf2(+/+) and Nrf2(-/-) mice were fed a high-fat diet (HFD). HFD elicited significant increases in mRNA expression of Gclc and Hmox1 in aortas of Nrf2(+/+) mice, but not Nrf2(-/-) mice, compared with respective standard diet-fed control mice. Additionally, HFD-induced increases in vascular ROS levels were significantly greater in Nrf2(-/-) than Nrf2(+/+) mice. HFD-induced endothelial dysfunction was more severe in Nrf2(-/-) mice, as shown by the significantly diminished acetylcholine-induced relaxation of aorta of these animals compared with HFD-fed Nrf2(+/+) mice. Our results suggest that adaptive activation of the Nrf2/ARE pathway confers endothelial protection under diabetic conditions.

Fig. 1.

A: reporter gene assay showing the effects of increasing concentrations of glucose on nuclear factor erythroid 2-related factor-2 (Nrf2)/antioxidant response element (ARE) reporter activity in cultured primary human coronary arterial endothelial cells (CAECs). Cells were transiently cotransfected with ARE-driven firefly luciferase and cytomegalovirus (CMV)-driven Renilla luciferase constructs followed by glucose treatment. Cells were then lysed and subjected to luciferase activity assay. After normalization relative luciferase activity was obtained from 4–6 independent transfections. Data are means ± SE. *P < 0.05. B: effect of glucose on mRNA expression of Nqo1, Gclc, and Hmox1 in cultured primary CAECs. Data are means ± SE (n = 5 in each group). The effects of glucose were significant (P < 0.05) for each target. C: effects of small interfering RNA (siRNA) downregulation of Nrf2 (siNrf2) or overexpression of Keap-1 on high glucose (HG, 30 mmol/l)-induced mRNA expression of Nqo1, Gclc, and Hmox1 in cultured primary CAECs. Data are means ± SE (n = 5 in each group). *P < 0.05 vs. control; #P < 0.05 vs. HG only. D: representative Western blots showing protein expression of NQO1 in CAECs treated with HG, with or without siNrf2 treatment (scr, scrambled siRNA control). β-Actin was used as a loading control. Numbers are normalized densitometric values (see methods). Experiments were repeated 3 times with similar results.

Fig. 2.

A: representative fluorescent images showing stronger MitoSOX staining (red fluorescence) in high glucose-treated cultured primary human CAECs compared with untreated controls. Original magnification ×10. Blue fluorescence: nuclear counterstaining with Hoechst 33258. B: in CAECs high glucose (HG, 30 mmol/l) induces mitochondrial oxidative stress, as shown by the significant increases in the mean fluorescence intensity of oxidized MitoSOX (flow cytometry data). Overexpression of Keap-1 significantly increases HG-induced mitochondrial O₂^·− production. Data are means ± SE (n = 6 in each group). *P < 0.05 vs. baseline; #P < 0.05 vs. HG only.

Fig. 3.

A: effect of pretreatment with polyethylene glycol (PEG)-catalase (1,000 U/ml) and N-acetylcysteine (NAC, 25 mmol/l) on high glucose (HG, 30 mmol/l, for 24 h)-induced changes in mRNA expression of Nqo1, Gclc, and Hmox1 in cultured primary human CAECs. Data are means ± SE (n = 5 in each group). *P < 0.05 vs. control; #P < 0.05 vs. HG only. B: effect of siRNA knockdown of Nrf2 (siNrf2) on H₂O₂ (10⁻⁵ mol/l, for 24 h)-induced changes in mRNA expression of Nqo1, Gclc, and Hmox1 in cultured primary human CAECs. Data are means ± SE (n = 5 in each group). *P < 0.05 vs. control; #P < 0.05 vs. H₂O₂ only. C: effects of siNrf2 and overexpression of Keap-1 on sulforaphane (SFN, 2.5 μmol/l, for 24 h)-induced changes in mRNA expression of Nqo1, Gclc, and Hmox1 in cultured primary human CAECs. Data are means ± SE (n = 5 in each group). *P < 0.05 vs. control; #P < 0.05 vs. SFN only.

Fig. 4.

In primary human CAECs high glucose (HG) significantly increased apoptotic cell death as shown by increased terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) positivity (assessed by flow cytometry, see methods; P < 0.05 vs. control). siRNA knockdown of Nrf2 (siNrf2) significantly augmented HG-induced endothelial apoptosis. Data are mean ± SE (n = 6 for each group). *P < 0.05 vs. untreated; #P < 0.05 vs. HG only.

Fig. 5.

A: reporter gene assay showing that in primary human CAECs high glucose (HG) significantly increased NF-κB activity. Cells were transiently cotransfected with NF-κB-driven firefly luciferase and CMV-driven Renilla luciferase constructs followed by HG treatment. Cells were then lysed and subjected to luciferase activity assay. After normalization relative luciferase activity was assessed from 4–6 independent transfections. siRNA knockdown of Nrf2 (siNrf2) significantly augmented HG-induced endothelial NF-κB activation. Data are means ± SE (n = 5 in each group). *P < 0.05 vs. untreated; #P < 0.05 vs. HG only. B: in cultured endothelial cells HG significantly increased mRNA expression of ICAM-1. Knockdown of Nrf2 significantly augmented HG-induced ICAM-1 mRNA expression. Data are means ± SE (n = 5 in each group). *P < 0.05 vs. untreated; #P < 0.05 vs. HG only.

Fig. 6.

A: expression of Gclc and Hmox1 mRNA in aortas isolated from Nrf2^+/+ mice and Nrf2^−/− mice fed a standard diet (SD) or a high-fat diet (HFD). Data are means ± SE (n = 5 or 6 in each group). *P < 0.05 vs. SD; #P < 0.05 vs. Nrf2^+/+. B: analysis of protein expression of NQO1, GPX, and GCLC in aortic segments from Nrf2^+/+ mice and Nrf2^−/− mice fed SD or HFD. Shown are spliced bands from Western blot experiments, organized according to the experimental groups. Numbers are normalized densitometric values for the respective bands (see methods). The molecular masses of the bands recognized by the antibodies directed against NQO1, GPX, and GCLC are approximately 31, 22, and 73 kDa, respectively.

Fig. 7.

A: superoxide and peroxide production in segments of aortas isolated from Nrf2^+/+ mice and Nrf2^−/− mice fed SD or HFD, as measured by the dihydroethidium (DHE, left) and dichlorofluorescein [5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (H₂DCFDA), right] fluorescence methods. Data are means ± SE (n = 5 or 6 in each group). AU, arbitrary units. *P < 0.05 vs. SD; #P < 0.05 vs. Nrf2^+/+. B: relaxation to acetylcholine in aortic segments isolated from Nrf2^+/+ mice and Nrf2^−/− mice fed SD or HFD. Data are means ± SE (n = 5 or 6 for each data point). *P < 0.05 vs. Nrf2^+/+.

Fig. 8.

Expression of ICAM-1 (A) and TNFα (B) mRNA in aortas isolated from Nrf2^+/+ mice and Nrf2^−/− mice fed SD or HFD. Data are means ± SE (n = 5 or 6 in each group). *P < 0.05 vs. SD; #P < 0.05 vs. Nrf2^+/+.