Limits...
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.

Show MeSH

Related in: MedlinePlus

Biochemical dysfunction in HMEC-1 endothelial cells in hyperglycemic culture and reversal by sulforaphane in vitro: ROS formation. A and B: Assessment of cellular ROS formation. H(L−), 5 mmol/l d(+)-glucose with 25 mmol/l l(−)-glucose; AN(nrf2), transfection with siRNA for nrf2 knockdown; AN(TK), transfection with siRNA for transketolase knockdown; and AN(scr), transfection with scrambled sequence siRNA. Data are means ± SD (n = 3). * and ○Significance with respect to normoglycemic (N) and hyperglycemic (H) control, respectively, with 1, 2, and 3 symbols representing P < 0.05, 0.01, and 0.001, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2551693&req=5

f4: Biochemical dysfunction in HMEC-1 endothelial cells in hyperglycemic culture and reversal by sulforaphane in vitro: ROS formation. A and B: Assessment of cellular ROS formation. H(L−), 5 mmol/l d(+)-glucose with 25 mmol/l l(−)-glucose; AN(nrf2), transfection with siRNA for nrf2 knockdown; AN(TK), transfection with siRNA for transketolase knockdown; and AN(scr), transfection with scrambled sequence siRNA. Data are means ± SD (n = 3). * and ○Significance with respect to normoglycemic (N) and hyperglycemic (H) control, respectively, with 1, 2, and 3 symbols representing P < 0.05, 0.01, and 0.001, respectively.

Mentions: To examine the effect of ARE-linked gene expression on biochemical dysfunction in hyperglycemia, the cellular production of ROS was quantified. Hyperglycemic culture of endothelial cells produced a threefold increased formation of ROS (Fig. 4A). This was not induced by the addition of 25 mmol/l l-glucose (which does not permeate into endothelial cells) to the normoglycemic control. Incubation of endothelial cells with sulforaphane reversed the increase in ROS by 73%, suggesting that activation of ARE-linked gene expression prevented increased ROS formation. Increased ROS formation by HMEC-1 cells in hyperglycemic cultures was prevented by incubation for 60 min with mitochondrial inhibitors (ROS formation [percentage of normoglycemic control]: 10 μmol/l p-trifluoromethoxycarbonylcyanide phenylhydrazone, 94 ± 3%; 5 μmol/l rotenone, 104 ± 3%; and 2 μmol/l myxothiazole, 88 ± 3%). This suggests that dysfunction of mitochondria was a primary source of the increased ROS and electron flux, although complexes I and III contributed to this effect. Incubation of HMEC-1 cells with these inhibitors for 24 h decreased cell viability by 40–50% and masked the effect of rotenone.


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)

Biochemical dysfunction in HMEC-1 endothelial cells in hyperglycemic culture and reversal by sulforaphane in vitro: ROS formation. A and B: Assessment of cellular ROS formation. H(L−), 5 mmol/l d(+)-glucose with 25 mmol/l l(−)-glucose; AN(nrf2), transfection with siRNA for nrf2 knockdown; AN(TK), transfection with siRNA for transketolase knockdown; and AN(scr), transfection with scrambled sequence siRNA. Data are means ± SD (n = 3). * and ○Significance with respect to normoglycemic (N) and hyperglycemic (H) control, respectively, with 1, 2, and 3 symbols representing P < 0.05, 0.01, and 0.001, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Biochemical dysfunction in HMEC-1 endothelial cells in hyperglycemic culture and reversal by sulforaphane in vitro: ROS formation. A and B: Assessment of cellular ROS formation. H(L−), 5 mmol/l d(+)-glucose with 25 mmol/l l(−)-glucose; AN(nrf2), transfection with siRNA for nrf2 knockdown; AN(TK), transfection with siRNA for transketolase knockdown; and AN(scr), transfection with scrambled sequence siRNA. Data are means ± SD (n = 3). * and ○Significance with respect to normoglycemic (N) and hyperglycemic (H) control, respectively, with 1, 2, and 3 symbols representing P < 0.05, 0.01, and 0.001, respectively.
Mentions: To examine the effect of ARE-linked gene expression on biochemical dysfunction in hyperglycemia, the cellular production of ROS was quantified. Hyperglycemic culture of endothelial cells produced a threefold increased formation of ROS (Fig. 4A). This was not induced by the addition of 25 mmol/l l-glucose (which does not permeate into endothelial cells) to the normoglycemic control. Incubation of endothelial cells with sulforaphane reversed the increase in ROS by 73%, suggesting that activation of ARE-linked gene expression prevented increased ROS formation. Increased ROS formation by HMEC-1 cells in hyperglycemic cultures was prevented by incubation for 60 min with mitochondrial inhibitors (ROS formation [percentage of normoglycemic control]: 10 μmol/l p-trifluoromethoxycarbonylcyanide phenylhydrazone, 94 ± 3%; 5 μmol/l rotenone, 104 ± 3%; and 2 μmol/l myxothiazole, 88 ± 3%). This suggests that dysfunction of mitochondria was a primary source of the increased ROS and electron flux, although complexes I and III contributed to this effect. Incubation of HMEC-1 cells with these inhibitors for 24 h decreased cell viability by 40–50% and masked the effect of rotenone.

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