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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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Cryosections of normal and diabetic rat retinas immunostained with anti-LOX primary antibody and rhodamine-conjugated secondary antibody. Blood vessel in the diabetic retina shows increased LOX immunostaining compared with those of the normal retina.
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Figure 5: Cryosections of normal and diabetic rat retinas immunostained with anti-LOX primary antibody and rhodamine-conjugated secondary antibody. Blood vessel in the diabetic retina shows increased LOX immunostaining compared with those of the normal retina.

Mentions: Blood glucose levels measured routinely and at the time of death confirmed the presence of hyperglycemia in these rats compared with control nondiabetic rats (326 ± 16 vs. 104 ± 12, P < 0.01). After 3 weeks of diabetes, retinal LOX protein levels were significantly increased (125 ± 4.8% of control, P < 0.05) compared with those in control nondiabetic rats (n = 5) (Fig. 4). The β-actin protein expression was used as an internal control for protein loading and was similar in all groups. The immunohistochemical analysis of the retinal sections suggests that the perivascular tissue as well as the basement membrane of the retinal capillaries in the diabetic retinas show increased LOX immunostaining compared with blood vessels in the normal retinas (Fig. 5).


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)

Cryosections of normal and diabetic rat retinas immunostained with anti-LOX primary antibody and rhodamine-conjugated secondary antibody. Blood vessel in the diabetic retina shows increased LOX immunostaining compared with those of the normal retina.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Cryosections of normal and diabetic rat retinas immunostained with anti-LOX primary antibody and rhodamine-conjugated secondary antibody. Blood vessel in the diabetic retina shows increased LOX immunostaining compared with those of the normal retina.
Mentions: Blood glucose levels measured routinely and at the time of death confirmed the presence of hyperglycemia in these rats compared with control nondiabetic rats (326 ± 16 vs. 104 ± 12, P < 0.01). After 3 weeks of diabetes, retinal LOX protein levels were significantly increased (125 ± 4.8% of control, P < 0.05) compared with those in control nondiabetic rats (n = 5) (Fig. 4). The β-actin protein expression was used as an internal control for protein loading and was similar in all groups. The immunohistochemical analysis of the retinal sections suggests that the perivascular tissue as well as the basement membrane of the retinal capillaries in the diabetic retinas show increased LOX immunostaining compared with blood vessels in the normal retinas (Fig. 5).

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