Korean J Ophthalmol.  2012 Apr;26(2):123-131. 10.3341/kjo.2012.26.2.123.

Effect of Advanced Glycation End Products on Oxidative Stress and Senescence of Trabecular Meshwork Cells

• Park CH
• Kim JW

• KMID: 1364866
• DOI: http://doi.org/10.3341/kjo.2012.26.2.123

Abstract

PURPOSE
To investigate the effect of advanced glycation end products (AGE) on oxidative stress and cellular senescence in cultured human trabecular meshwork cells (HTMC).
METHODS
Primarily cultured HTMC were exposed to 0, 10, 50, 100, 200 microg/mL of glycated bovine serum albumin (G-BSA) for 5 days. Also co-exposed were L-arginine, sepiapterin, and antioxidant N-acetylcysteine (NAC). Cellular survival and production of nitric oxide (NO), superoxide, and reactive oxygen species were assessed by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assay, Griess assay, cytochrome c assay, and dichlorofluorescin diacetate assay, respectively. Senescence-associated beta-galactosidase staining was performed to quantify the degree of cellular senescence.
RESULTS
G-BSA decreased cellular survival, NO production, and increased superoxide production significantly in a dose-dependent manner. The effects of G-BSA were abolished with co-exposure of L-arginine, sepiapterin, and NAC. G-BSA enhanced cellular senescence accompanied by increased production of reactive oxygen species. G-BSA-induced cellular senescence was suppressed by application of L-arginine, sepiapterin, and NAC.
CONCLUSIONS
AGE enhances cellular senescence of HTMC accompanied with increased oxidative stress. AGE-induced oxidative stress and cellular senescence could be delayed by application of anti-oxidants.

Keyword

Advanced glycation end products; Aging; Antioxidants; Oxidative stress; Trabecular meshwork cells

MeSH Terms

Acetylcysteine/metabolism
Apoptosis/drug effects/physiology
Arginine/metabolism
Cell Aging/drug effects/*physiology
Cell Survival/drug effects/physiology
Cells, Cultured
Glycosylation End Products, Advanced/metabolism/*toxicity
Humans
Nitric Oxide/metabolism
Oxidative Stress/*physiology
Pterins/metabolism
Reactive Oxygen Species/metabolism
Serum Albumin, Bovine/metabolism/toxicity
Trabecular Meshwork/drug effects/*metabolism/*pathology

Figure

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  Fig. 1 Effect of advanced glycation end products (AGE) on the survival of trabecular meshwork cells. Exposure to 100 µg/mL AGE significantly decreased cellular survival (*p < 0.05).

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  Fig. 2 Effect of advanced glycation end products (AGE) on the survival of trabecular meshwork cells. The decreased cellular survival caused by AGE was abolished by exposure to L-arginine, sepiapterin, and N-acetylcysteine (NAC).

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  Fig. 3 Effect of advanced glycation end products (AGE) on the production of nitric oxide in trabecular meshwork cells. AGE significantly decreased nitric oxide production in a dose-dependent manner (*p < 0.05).

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  Fig. 4 L-arginine, sepiapterin, and N-acetylcysteine (NAC) abolished advanced glycation end products (AGE)'s inhibitory effects on the production of nitric oxide in trabecular meshwork cells exposed to AGE concentrations of 50 µg/mL and 100 µg/mL compared to non-exposed controls (*p < 0.05).

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  Fig. 5 Effect of advanced glycation end products (AGE) on the production of superoxide in trabecular meshwork cells. AGE significantly increased superoxide production in a dose-dependent manner (*p < 0.05).

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  Fig. 6 L-arginine, sepiapterin, and N-acetylcysteine (NAC) significantly decreased advanced glycation end products (AGE)-induced superoxide production compared to non-exposed trabecular meshwork cells (*,**p < 0.05).

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  Fig. 7 Effect of advanced glycation end products (AGE) on the generation of reactive oxygen species in trabecular meshwork cells. AGE significantly increased the production of reactive oxygen species in a dose-dependent manner (*p < 0.05).

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  Fig. 8 The advanced glycation end products (AGE)-induced increase in the generation of reactive oxygen species was abolished by antioxidant N-acetylcysteine (NAC), but not by L-arginine or sepiapterin, compared to non-exposed trabecular meshwork cells (*,**p < 0.05).

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  Fig. 9 Effect of advanced glycation end products (AGE) on the induction trabecular meshwork cell apoptosis assessed using flow cytometry and Annexin V/propidium iodide staining. AGE increased cellular apoptosis in a dose-dependent manner compared to non-exposed controls (*p < 0.05).

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  Fig. 10 Senescence-associated b-galactosidase staining of trabecular meshwork cells after exposure to advanced glycation end products (AGE). AGE (100 µg/mL) increased the number of senescent cells, which stained blue, (B) compared to non-exposed controls (A). Magnification, ×100.

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  Fig. 11 Effect of advanced glycation end products (AGE) on the senescence of trabecular meshwork cells. AGE significantly increased the percentage of senescence-associated β-galactosidase (SA-β-gal) (+) cells in a dose-dependent manner (*p < 0.05).

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  Fig. 12 L-arginine, sepiapterin, and N-acetylcysteine (NAC) significantly decreased the advanced glycation end products (AGE)-induced senescence of trabecular meshwork cells compared to controls exposed to 50 µg/mL (*p < 0.05) or 100 µg/mL (**p < 0.05) AGE. SA-β-gal = senescence-associated β-galactosidase.

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