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High glucose increases lysyl oxidase expression and activity in retinal endothelial cells: mechanism for compromised extracellular matrix barrier function.

Chronopoulos A, Tang A, Beglova E, Trackman PC, Roy S - Diabetes (2010)

Bottom Line: In cells grown in HG medium, LOX activity and cell monolayer permeability was significantly increased, as were LOX and proLOX immunostaining.Small interfering RNA- or BAPN-induced-specific blockage of LOX expression or activity, respectively, reduced cell monolayer permeability.HG-induced increased LOX expression and activity compromises barrier functional integrity, a prominent lesion of diabetic retinopathy.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine and Ophthalmology, Boston University School of Medicine, Boston, Massachusetts, USA.

ABSTRACT

Objective: In diabetes, retinal vascular basement membrane (BM) undergoes significant thickening and compromises vessel function including increased vascular permeability, a prominent lesion of early diabetic retinopathy. In this study we determined whether altered expression and activity of lysyl oxidase (LOX), a cross-linking enzyme, may compromise vascular basement membrane functional integrity under high-glucose (HG) conditions.

Research design and methods: Rat retinal endothelial cells (RRECs) grown in normal (5 mmol/l) or HG (30 mmol/l glucose) medium for 7 days were assessed for expression of LOX and proLOX by Western blot analysis and LOX enzyme activity. To determine whether HG alters cellular distribution patterns of LOX and proLOX, immunostaining with respective antibodies was performed. Similarly, cells grown in normal or HG medium were subjected to both LOX inhibition with β-aminopropionitrile (BAPN) and by small interfering RNA knockdown, and respectively examined for cell monolayer permeability. Additionally, retinas of streptozotocin (STZ)-induced diabetic rats were analyzed to determine if diabetes altered LOX expression.

Results: Western blot analysis revealed significantly increased LOX and proLOX expression in cells grown in HG medium compared with those grown in normal medium. The increased LOX level was strikingly similar to LOX upegulation in the diabetic retinas. In cells grown in HG medium, LOX activity and cell monolayer permeability was significantly increased, as were LOX and proLOX immunostaining. Small interfering RNA- or BAPN-induced-specific blockage of LOX expression or activity, respectively, reduced cell monolayer permeability.

Conclusions: HG-induced increased LOX expression and activity compromises barrier functional integrity, a prominent lesion of diabetic retinopathy.

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Related in: MedlinePlus

Western blot analysis of LOX protein levels in RRECs grown in normal (N) or HG media. Graph shows that the LOX protein level was significantly upregulated in cells grown in HG medium compared with cells grown in normal (N) medium. Cells exposed to mannitol for 7 days exhibited no change in LOX expression. Data are presented as mean ± SD (*P < 0.005; n = 9).
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Figure 1: Western blot analysis of LOX protein levels in RRECs grown in normal (N) or HG media. Graph shows that the LOX protein level was significantly upregulated in cells grown in HG medium compared with cells grown in normal (N) medium. Cells exposed to mannitol for 7 days exhibited no change in LOX expression. Data are presented as mean ± SD (*P < 0.005; n = 9).

Mentions: Western blot analysis indicated that RRECs grown in HG medium for 7 days exhibited significant increase in the expression of LOX and proLOX protein levels compared with those of RRECs grown in normal medium (136 ± 18% of control, P < 0.005, n = 9; 157 ± 32% of control, P < 0.0005, n = 9, respectively). Exposure of the cells to 30 mmol/l mannitol for 7 days had no effect on LOX expression. The β-actin protein expression used as an internal control confirmed equal protein loading for all groups (Figs. 1 and 2).


High glucose increases lysyl oxidase expression and activity in retinal endothelial cells: mechanism for compromised extracellular matrix barrier function.

Chronopoulos A, Tang A, Beglova E, Trackman PC, Roy S - Diabetes (2010)

Western blot analysis of LOX protein levels in RRECs grown in normal (N) or HG media. Graph shows that the LOX protein level was significantly upregulated in cells grown in HG medium compared with cells grown in normal (N) medium. Cells exposed to mannitol for 7 days exhibited no change in LOX expression. Data are presented as mean ± SD (*P < 0.005; n = 9).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2992778&req=5

Figure 1: Western blot analysis of LOX protein levels in RRECs grown in normal (N) or HG media. Graph shows that the LOX protein level was significantly upregulated in cells grown in HG medium compared with cells grown in normal (N) medium. Cells exposed to mannitol for 7 days exhibited no change in LOX expression. Data are presented as mean ± SD (*P < 0.005; n = 9).
Mentions: Western blot analysis indicated that RRECs grown in HG medium for 7 days exhibited significant increase in the expression of LOX and proLOX protein levels compared with those of RRECs grown in normal medium (136 ± 18% of control, P < 0.005, n = 9; 157 ± 32% of control, P < 0.0005, n = 9, respectively). Exposure of the cells to 30 mmol/l mannitol for 7 days had no effect on LOX expression. The β-actin protein expression used as an internal control confirmed equal protein loading for all groups (Figs. 1 and 2).

Bottom Line: In cells grown in HG medium, LOX activity and cell monolayer permeability was significantly increased, as were LOX and proLOX immunostaining.Small interfering RNA- or BAPN-induced-specific blockage of LOX expression or activity, respectively, reduced cell monolayer permeability.HG-induced increased LOX expression and activity compromises barrier functional integrity, a prominent lesion of diabetic retinopathy.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine and Ophthalmology, Boston University School of Medicine, Boston, Massachusetts, USA.

ABSTRACT

Objective: In diabetes, retinal vascular basement membrane (BM) undergoes significant thickening and compromises vessel function including increased vascular permeability, a prominent lesion of early diabetic retinopathy. In this study we determined whether altered expression and activity of lysyl oxidase (LOX), a cross-linking enzyme, may compromise vascular basement membrane functional integrity under high-glucose (HG) conditions.

Research design and methods: Rat retinal endothelial cells (RRECs) grown in normal (5 mmol/l) or HG (30 mmol/l glucose) medium for 7 days were assessed for expression of LOX and proLOX by Western blot analysis and LOX enzyme activity. To determine whether HG alters cellular distribution patterns of LOX and proLOX, immunostaining with respective antibodies was performed. Similarly, cells grown in normal or HG medium were subjected to both LOX inhibition with β-aminopropionitrile (BAPN) and by small interfering RNA knockdown, and respectively examined for cell monolayer permeability. Additionally, retinas of streptozotocin (STZ)-induced diabetic rats were analyzed to determine if diabetes altered LOX expression.

Results: Western blot analysis revealed significantly increased LOX and proLOX expression in cells grown in HG medium compared with those grown in normal medium. The increased LOX level was strikingly similar to LOX upegulation in the diabetic retinas. In cells grown in HG medium, LOX activity and cell monolayer permeability was significantly increased, as were LOX and proLOX immunostaining. Small interfering RNA- or BAPN-induced-specific blockage of LOX expression or activity, respectively, reduced cell monolayer permeability.

Conclusions: HG-induced increased LOX expression and activity compromises barrier functional integrity, a prominent lesion of diabetic retinopathy.

Show MeSH
Related in: MedlinePlus