J Korean Med Sci.  2004 Feb;19(1):95-100. 10.3346/jkms.2004.19.1.95.

Methylglyoxal Induces Apoptosis Mediated by Reactive Oxygen Species in Bovine Retinal Pericytes

• Kim J
• Son JW
• Lee JA
• Oh YS
• Shinn SH

• KMID: 1785701
• DOI: http://doi.org/10.3346/jkms.2004.19.1.95

Abstract

One of the histopathologic hallmarks of early diabetic retinopathy is the loss of pericytes. Evidences suggest that the pericyte loss in vivo is mediated by apoptosis. However, the underlying cause of pericyte apoptosis is not fully understood. This study investigated the influence of methylglyoxal (MGO), a reactive -dicarbonyl compound of glucose metabolism, on apoptotic cell death in bovine retinal pericytes. Analysis of internucleosomal DNA fragmentation by ELISA showed that MGO (200 to 800 micrometer) induced apoptosis in a concentration-dependent manner. Intracellular reactive oxygen species were generated earlier and the antioxidant, N-acetyl cysteine, inhibited the MGO-induced apoptosis. NF-kB activation and increased caspase- 3 activity were detected. Apoptosis was also inhibited by the caspase-3 inhibitor, Z-DEVD-fmk, or the NF- kB inhibitor, pyrrolidine dithiocarbamate. These data suggest that elevated MGO levels observed in diabetes may cause apoptosis in bovine retinal pericytes through an oxidative stress mechanism and suggests that the nuclear activation of NF-kB are involved in the apoptotic process.

Keyword

Retina; Pericytes; Methylglyoxal; Pyruvaldehyde; Apoptosis; Reactive Oxygen Species; NF-kappaB

MeSH Terms

Acetylcysteine/pharmacology
Animals
*Apoptosis
Caspases/metabolism
Cattle
Cell Death
Cell Survival
DNA Fragmentation
Dose-Response Relationship, Drug
Enzyme-Linked Immunosorbent Assay
Flow Cytometry
Glucose/metabolism
NF-kappa B/metabolism
Nucleosomes/metabolism
Oxidative Stress
Pericytes/*drug effects
Pyruvaldehyde/*pharmacology
*Reactive Oxygen Species
Retina/cytology/*drug effects

Figure

• 

  Fig. 1 Dose-dependent cytotoxic effects of MGO in retinal pericytes. Cytotoxicity was measured by MTT assay after 6 hr. Data are means±SD of triplicate experiments. *p<0.05, **p<0.01.

• 

  Fig. 2 (A) Effect of MGO on intracellular nucleosome enrichment. Cells were treated with 200, 400, 600, 800 µM MGO for 6 hr and the nucleosome concentration within the cell and in the cell culture supernatant was measured by ELISA. (B) Caspase-3 activity in retinal pericytes. Data are means±SD of triplicate experiments. *p<0.05, **p<0.01 compared to the value of the control.

• 

  Fig. 3 Measurement of intracellular ROS production by flow cytometry using DCF-DA. (A) Cells incubated with or without MGO (control, purplish; 400 µM, green; 800 µM, red) for 2 hr. (B) Cells co-treated with 2 mM NAC were incubated with or without MGO for 2 hr. (control, purplish; 400 µM+NAC, green; 800 µM+NAC, red). Data are representative results from three separate experiments.

• 

  Fig. 4 Subcellular localization of NF-κB p65 subunits. (A, B) In control cells, NF-κB is located in the cytoplasm. (C, D) In cells treated 800 µM MGO for 6 hr, NF-κB is translocated into the nuclei (red). Magnification, (A, C) ×100; (B, D)×400.

• 

  Fig. 5 Effects of MGO on NF-κB binding. Pericytes were treated with MGO (800 µM) for 6 hr. Nuclear extracts from the treated and untreated control cells were isolated and used in an EMSA with 32P-labeled NF-κB oligonucleotide as a probe. The arrow indicates the NF-κB binding complex. Competitor, 100-fold molar excess of unlabeled NF-κB probe. Data are representative results from three separate experiments.

• 

  Fig. 6 Inhibitory effects of NAC, PDTC, and Z-DEVD-fmk on MGO-induced pericyte apoptosis. Apoptosis was measured by ELISA after 400 or 800 µM MGO treatment for 6 hr. Data are means±SD of triplicate experiments. *p<0.05, **p<0.01 vs without NAC or inhibitor.

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