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Activation of NF-E2-related factor-2 reverses biochemical dysfunction of endothelial cells induced by hyperglycemia linked to vascular disease.

Xue M, Qian Q, Adaikalakoteswari A, Rabbani N, Babaei-Jadidi R, Thornalley PJ - Diabetes (2008)

Bottom Line: The effects of sulforaphane on multiple pathways of biochemical dysfunction, increased reactive oxygen species (ROS) formation, hexosamine pathway, protein kinase C (PKC) pathway, and increased formation of methylglyoxal were assessed.Activation of nrf2 by sulforaphane induced nuclear translocation of nrf2 and increased ARE-linked gene expression, for example, three- to fivefold increased expression of transketolase and glutathione reductase.This also abolished the counteracting effect of sulforaphane, suggesting mediation by nrf2 and related increase of transketolase expression.

View Article: PubMed Central - PubMed

Affiliation: Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, University Hospital, Coventry, UK.

ABSTRACT

Objective: Sulforaphane is an activator of transcription factor NF-E2-related factor-2 (nrf2) that regulates gene expression through the promoter antioxidant response element (ARE). Nrf2 regulates the transcription of a battery of protective and metabolic enzymes. The aim of this study was to assess whether activation of nrf2 by sulforaphane in human microvascular endothelial cells prevents metabolic dysfunction in hyperglycemia.

Research design and methods: Human microvascular HMEC-1 endothelial cells were incubated in low and high glucose concentrations (5 and 30 mmol/l, respectively), and activation of nrf2 was assessed by nuclear translocation. The effects of sulforaphane on multiple pathways of biochemical dysfunction, increased reactive oxygen species (ROS) formation, hexosamine pathway, protein kinase C (PKC) pathway, and increased formation of methylglyoxal were assessed.

Results: Activation of nrf2 by sulforaphane induced nuclear translocation of nrf2 and increased ARE-linked gene expression, for example, three- to fivefold increased expression of transketolase and glutathione reductase. Hyperglycemia increased the formation of ROS-an effect linked to mitochondrial dysfunction and prevented by sulforaphane. ROS formation was increased further by knockdown of nrf2 and transketolase expression. This also abolished the counteracting effect of sulforaphane, suggesting mediation by nrf2 and related increase of transketolase expression. Sulforaphane also prevented hyperglycemia-induced activation of the hexosamine and PKC pathways and prevented increased cellular accumulation and excretion of the glycating agent methylglyoxal.

Conclusions: We conclude that activation of nrf2 may prevent biochemical dysfunction and related functional responses of endothelial cells induced by hyperglycemia in which increased expression of transketolase has a pivotal role.

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Nuclear translocation of nrf2 in human HMEC-1 endothelial cells in vitro activated by sulforaphane. Nuclear fraction (A) and cytosolic fraction (B) immunoblotting for nrf2 (98-kDa band). Densitometric intensity ratios are means ± SD (n = 3). N, 5 mmol/l glucose; H, 30 mmol/l glucose; N+SFN, 5 mmol/l glucose + 4 μmol/l sulforaphane; and H+SFN, 30 mmol/l glucose + 4 μmol/l sulforaphane. *P < 0.05, **P < 0.01, and ***P < 0.001 with respect to N; ○P < 0.05 and ○○P < 0.01 with respect to H.
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f2: Nuclear translocation of nrf2 in human HMEC-1 endothelial cells in vitro activated by sulforaphane. Nuclear fraction (A) and cytosolic fraction (B) immunoblotting for nrf2 (98-kDa band). Densitometric intensity ratios are means ± SD (n = 3). N, 5 mmol/l glucose; H, 30 mmol/l glucose; N+SFN, 5 mmol/l glucose + 4 μmol/l sulforaphane; and H+SFN, 30 mmol/l glucose + 4 μmol/l sulforaphane. *P < 0.05, **P < 0.01, and ***P < 0.001 with respect to N; ○P < 0.05 and ○○P < 0.01 with respect to H.

Mentions: We investigated the activation status of nrf2 in human microvascular endothelial cells by assessing nuclear translocation of human nrf2 by immunoblotting in cytosolic and nuclear fractions and confocal microscopy of nrf2-GFP fusion protein. HMEC-1 endothelial cells incubated in model hyperglycemia (30 mmol/l glucose) showed no significant nuclear translocation of nrf2 with respect to normoglycemic control (5 mmol/l glucose) after incubation for 6 h. Addition of 4 μmol/l sulforaphane gave a twofold increase in nuclear nrf2 in both normoglycemic and hyperglycemic cultures. In the normoglycemic culture, the concentration of nrf2 in the cytosol was decreased concomitantly; whereas in the hyperglycemic culture, the concentration of nrf2 in the cytosol was increased. This suggests that the double insult of hyperglycemia and sulforaphane increased the cellular content of nrf2 protein (Fig. 2A and B). This concentration of sulforaphane did not induce significant cytotoxicity in HMEC-1 cells in incubations for up to 48 h, as assessed in previous studies (31). Transketolase activity of HMEC-1 cells was determined spectrophotometrically, as we have previously described (9).


Activation of NF-E2-related factor-2 reverses biochemical dysfunction of endothelial cells induced by hyperglycemia linked to vascular disease.

Xue M, Qian Q, Adaikalakoteswari A, Rabbani N, Babaei-Jadidi R, Thornalley PJ - Diabetes (2008)

Nuclear translocation of nrf2 in human HMEC-1 endothelial cells in vitro activated by sulforaphane. Nuclear fraction (A) and cytosolic fraction (B) immunoblotting for nrf2 (98-kDa band). Densitometric intensity ratios are means ± SD (n = 3). N, 5 mmol/l glucose; H, 30 mmol/l glucose; N+SFN, 5 mmol/l glucose + 4 μmol/l sulforaphane; and H+SFN, 30 mmol/l glucose + 4 μmol/l sulforaphane. *P < 0.05, **P < 0.01, and ***P < 0.001 with respect to N; ○P < 0.05 and ○○P < 0.01 with respect to H.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2551693&req=5

f2: Nuclear translocation of nrf2 in human HMEC-1 endothelial cells in vitro activated by sulforaphane. Nuclear fraction (A) and cytosolic fraction (B) immunoblotting for nrf2 (98-kDa band). Densitometric intensity ratios are means ± SD (n = 3). N, 5 mmol/l glucose; H, 30 mmol/l glucose; N+SFN, 5 mmol/l glucose + 4 μmol/l sulforaphane; and H+SFN, 30 mmol/l glucose + 4 μmol/l sulforaphane. *P < 0.05, **P < 0.01, and ***P < 0.001 with respect to N; ○P < 0.05 and ○○P < 0.01 with respect to H.
Mentions: We investigated the activation status of nrf2 in human microvascular endothelial cells by assessing nuclear translocation of human nrf2 by immunoblotting in cytosolic and nuclear fractions and confocal microscopy of nrf2-GFP fusion protein. HMEC-1 endothelial cells incubated in model hyperglycemia (30 mmol/l glucose) showed no significant nuclear translocation of nrf2 with respect to normoglycemic control (5 mmol/l glucose) after incubation for 6 h. Addition of 4 μmol/l sulforaphane gave a twofold increase in nuclear nrf2 in both normoglycemic and hyperglycemic cultures. In the normoglycemic culture, the concentration of nrf2 in the cytosol was decreased concomitantly; whereas in the hyperglycemic culture, the concentration of nrf2 in the cytosol was increased. This suggests that the double insult of hyperglycemia and sulforaphane increased the cellular content of nrf2 protein (Fig. 2A and B). This concentration of sulforaphane did not induce significant cytotoxicity in HMEC-1 cells in incubations for up to 48 h, as assessed in previous studies (31). Transketolase activity of HMEC-1 cells was determined spectrophotometrically, as we have previously described (9).

Bottom Line: The effects of sulforaphane on multiple pathways of biochemical dysfunction, increased reactive oxygen species (ROS) formation, hexosamine pathway, protein kinase C (PKC) pathway, and increased formation of methylglyoxal were assessed.Activation of nrf2 by sulforaphane induced nuclear translocation of nrf2 and increased ARE-linked gene expression, for example, three- to fivefold increased expression of transketolase and glutathione reductase.This also abolished the counteracting effect of sulforaphane, suggesting mediation by nrf2 and related increase of transketolase expression.

View Article: PubMed Central - PubMed

Affiliation: Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, University Hospital, Coventry, UK.

ABSTRACT

Objective: Sulforaphane is an activator of transcription factor NF-E2-related factor-2 (nrf2) that regulates gene expression through the promoter antioxidant response element (ARE). Nrf2 regulates the transcription of a battery of protective and metabolic enzymes. The aim of this study was to assess whether activation of nrf2 by sulforaphane in human microvascular endothelial cells prevents metabolic dysfunction in hyperglycemia.

Research design and methods: Human microvascular HMEC-1 endothelial cells were incubated in low and high glucose concentrations (5 and 30 mmol/l, respectively), and activation of nrf2 was assessed by nuclear translocation. The effects of sulforaphane on multiple pathways of biochemical dysfunction, increased reactive oxygen species (ROS) formation, hexosamine pathway, protein kinase C (PKC) pathway, and increased formation of methylglyoxal were assessed.

Results: Activation of nrf2 by sulforaphane induced nuclear translocation of nrf2 and increased ARE-linked gene expression, for example, three- to fivefold increased expression of transketolase and glutathione reductase. Hyperglycemia increased the formation of ROS-an effect linked to mitochondrial dysfunction and prevented by sulforaphane. ROS formation was increased further by knockdown of nrf2 and transketolase expression. This also abolished the counteracting effect of sulforaphane, suggesting mediation by nrf2 and related increase of transketolase expression. Sulforaphane also prevented hyperglycemia-induced activation of the hexosamine and PKC pathways and prevented increased cellular accumulation and excretion of the glycating agent methylglyoxal.

Conclusions: We conclude that activation of nrf2 may prevent biochemical dysfunction and related functional responses of endothelial cells induced by hyperglycemia in which increased expression of transketolase has a pivotal role.

Show MeSH
Related in: MedlinePlus