Accumulation of argpyrimidine, a methylglyoxal-derived advanced glycation end product, increases apoptosis of lens epithelial cells both in vitro and in vivo

Abstract

The formation of advanced glycation end products (AGEs) has been considered to be a potential causative factor of injury to lens epithelial cells (LECs). Damage of LECs is believed to contribute to cataract formation. The purpose of this study was to investigate the cytotoxic effect of AGEs on LECs both in vitro and in vivo. We examined the accumulation of argpyrimidine, a methylglyoxal-derived AGE, and the expression of apoptosis-related molecules including nuclear factor- kappaB (NF-κB), Bax, and Bcl-2 in the human LEC line HLE-B3 and in cataractous lenses of Zucker diabetic fatty (ZDF) rats, an animal model of type 2 diabetes. In cataractous lenses from twenty-oneweek- old ZDF rats, LEC apoptosis was markedly increased, and the accumulation of argpyrimidine as well as subsequent activation of NF-κB in LECs were significantly enhanced. The ratio of Bax to Bcl-2 protein levels was also increased. In addition, the accumulation of argpyrimidine triggered apoptosis in methylglyoxal- treated HLE-B3 cells. However, the presence of pyridoxamine (an AGEs inhibitor) and pyrrolidine dithiocarbamate (a NF-κB inhibitor) prevented apoptosis in HLE-B3 cells through the inhibition of argpyrimidine formation and the blockage of NF-κB nuclear translocalization, respectively. These results suggest that the cellular accumulation of argpyrimidine in LECs is NF-κB-dependent and pro-apoptotic.

Figure 1

Argpyrimidine formation and apoptosis in LECs. (A) Grade of cataract formation in the normal ZL rat (▪) and ZDF rat (•). (B) Western blot analysis of argpyrimidine. (C) Double staining for argpyrimidine and TUNEL-positive apoptotic cells. The lens sections from the normal ZL rats and ZDF rats are stained with argpyrimidine (AP, red), TUNEL (green) and DAPI (blue). Almost all TUNEL-positive cells coincide with argpyrimidine-positive cells. The scale bar = 50 µm. All data are expressed as means ± SE, n = 8. The asterisk (^*) indicates a value of P < 0.01 vs. normal ZL rats.

Figure 2

NF-κB activation and expression of Bax and Bcl-2 in LECs. (A) Immunofluorescence staining of NF-κB (a, d), Bax (b, e) and Bcl-2 (c, f). Representative photomicrographs of lenses from the normal ZL rat (a-c) and ZDF rat (d-f). Positive signals (green) for activated NF-κB are mainly detected in the nucleus of diabetic LECs. The lens epithelium of ZDF rats shows strong immunoreactivity for Bax. However, Bcl-2 immunoreactivity does not differ between normal and diabetic rats. The scale bar = 50 µm. (B) Analysis of NF-κB DNA binding activity by an ELISA-based assay. (C) Expression of Bax and Bcl-2 protein by western blotting and the ratio of Bax to Bcl-2 protein expression levels. Values in the bar graphs represent means ± SE, n = 8. The asterisk (^*) indicates a value of P < 0.01 vs. normal ZL rats.

Figure 3

Argpyrimidine formation and subcellular localization of the NF-κB p65 subunit in HLE-B3 cells. (A) Immunofluorescence staining of argpyrimidine. (B) Fluorescent counterstaining of nuclei with DAPI. (C) Subcellular localization of the NF-κB p65 subunit. (D) Merge of the signals for NF-κB p65 subunits and DAPI. HLE-B3 cells are incubated with the indicated concentration of MGO in the presence or absence of PDTC for 24 h. In control cells, NF-κB is located in the cytoplasm. In cells treated with MGO for 24 h, argpyrimidine (AP) formation is induced by MGO and NF-κB is translocated into the nuclei. However, both PM and PDTC inhibit NF-κB nuclear translocation.

Figure 4

Effects of PDTC on apoptosis and expression of Bax and Bcl-2 in HLE-B3 cells. (A) TUNEL staining. (B) CCK-8 assay. (C) Expression of Bax and Bcl-2 protein by western blotting and the ratio of Bax to Bcl-2 protein expression level. HLE-B3 cells are incubated with the indicated concentration of MGO in the presence or absence of PDTC for 24 h. PDTC inhibits apoptosis of HLE-B3 cells in a dose-dependent manner. Values in the bar graphs represent means ± SE, n = 4. The asterisk (^*) indicates a value of P < 0.01 vs. the control group; the pound sign (^#) indicates a value of P < 0.01 vs. the MGO-treated group.