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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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A: Graph shows the effect of LOX siRNA on LOX protein level in RRECs. In cells grown in HG medium and transfected with LOX siRNA, the LOX protein expression was significantly decreased compared with that in cells grown in HG medium and transfected with scrambled siRNA. Data are expressed as mean ± SD (*P < 0.005, **P < 0.05). B: Effect of reduced LOX activity on cell monolayer permeability and effect of reduced LOX expression on cell monolayer permeability. The permeability of FITC-conjugated dextran molecules was significantly decreased to near normal level in cells grown in HG medium after incubation with BAPN compared with RRECs grown in HG. Data are expressed as mean ± SD (*P < 0.05, n = 6; **P < 0.05; n = 6). The permeability of FITC-conjugated dextran molecules was also significantly decreased to near normal level in cells grown in HG medium after transfection with LOX siRNA compared with untransfected RRECs grown in HG. Data are expressed as mean ± SD (***P < 0.005; n = 3). N, normal.
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Figure 7: A: Graph shows the effect of LOX siRNA on LOX protein level in RRECs. In cells grown in HG medium and transfected with LOX siRNA, the LOX protein expression was significantly decreased compared with that in cells grown in HG medium and transfected with scrambled siRNA. Data are expressed as mean ± SD (*P < 0.005, **P < 0.05). B: Effect of reduced LOX activity on cell monolayer permeability and effect of reduced LOX expression on cell monolayer permeability. The permeability of FITC-conjugated dextran molecules was significantly decreased to near normal level in cells grown in HG medium after incubation with BAPN compared with RRECs grown in HG. Data are expressed as mean ± SD (*P < 0.05, n = 6; **P < 0.05; n = 6). The permeability of FITC-conjugated dextran molecules was also significantly decreased to near normal level in cells grown in HG medium after transfection with LOX siRNA compared with untransfected RRECs grown in HG. Data are expressed as mean ± SD (***P < 0.005; n = 3). N, normal.

Mentions: We examined the effect of LOX siRNA in cells grown under HG conditions. In cells grown in HG medium and transfected with LOX siRNA, the LOX expression was significantly reduced compared with that of cells grown in HG medium and transfected with scrambled siRNA (88 ± 8% of control vs. 119 ± 16% of control, P < 0.05, n = 3) (Fig. 7A). When RRECs grown in HG medium were transfected with the LOX siRNA, the permeability of the cell monolayer significantly decreased compared with that of the HG cells transfected with scrambled siRNA (185 ± 35%, P < 0.005; 333 ± 14% of control, P < 0.005; n = 3) (Fig. 7B). Since the LOX siRNA that we used was specifically targeted against the LOX transcript, the data presented here demonstrate that LOX overexpression is involved in increased permeability.


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)

A: Graph shows the effect of LOX siRNA on LOX protein level in RRECs. In cells grown in HG medium and transfected with LOX siRNA, the LOX protein expression was significantly decreased compared with that in cells grown in HG medium and transfected with scrambled siRNA. Data are expressed as mean ± SD (*P < 0.005, **P < 0.05). B: Effect of reduced LOX activity on cell monolayer permeability and effect of reduced LOX expression on cell monolayer permeability. The permeability of FITC-conjugated dextran molecules was significantly decreased to near normal level in cells grown in HG medium after incubation with BAPN compared with RRECs grown in HG. Data are expressed as mean ± SD (*P < 0.05, n = 6; **P < 0.05; n = 6). The permeability of FITC-conjugated dextran molecules was also significantly decreased to near normal level in cells grown in HG medium after transfection with LOX siRNA compared with untransfected RRECs grown in HG. Data are expressed as mean ± SD (***P < 0.005; n = 3). N, normal.
© Copyright Policy - creative-commons
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Figure 7: A: Graph shows the effect of LOX siRNA on LOX protein level in RRECs. In cells grown in HG medium and transfected with LOX siRNA, the LOX protein expression was significantly decreased compared with that in cells grown in HG medium and transfected with scrambled siRNA. Data are expressed as mean ± SD (*P < 0.005, **P < 0.05). B: Effect of reduced LOX activity on cell monolayer permeability and effect of reduced LOX expression on cell monolayer permeability. The permeability of FITC-conjugated dextran molecules was significantly decreased to near normal level in cells grown in HG medium after incubation with BAPN compared with RRECs grown in HG. Data are expressed as mean ± SD (*P < 0.05, n = 6; **P < 0.05; n = 6). The permeability of FITC-conjugated dextran molecules was also significantly decreased to near normal level in cells grown in HG medium after transfection with LOX siRNA compared with untransfected RRECs grown in HG. Data are expressed as mean ± SD (***P < 0.005; n = 3). N, normal.
Mentions: We examined the effect of LOX siRNA in cells grown under HG conditions. In cells grown in HG medium and transfected with LOX siRNA, the LOX expression was significantly reduced compared with that of cells grown in HG medium and transfected with scrambled siRNA (88 ± 8% of control vs. 119 ± 16% of control, P < 0.05, n = 3) (Fig. 7A). When RRECs grown in HG medium were transfected with the LOX siRNA, the permeability of the cell monolayer significantly decreased compared with that of the HG cells transfected with scrambled siRNA (185 ± 35%, P < 0.005; 333 ± 14% of control, P < 0.005; n = 3) (Fig. 7B). Since the LOX siRNA that we used was specifically targeted against the LOX transcript, the data presented here demonstrate that LOX overexpression is involved in increased permeability.

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