Although recent research showed that advanced glycation endproduct (AGE) and hydroquinone (HQ) are related to the pathogenesis of age-related macular degeneration (AMD), the mechanism how AGE and HQ induce or accelerate AMD remains elusive. In the present study, we examined the effects of AGE and HQ on changes of human retinal pigment epithelial (RPE) cell numbers and found that the viable cell numbers were markedly reduced by HQ by apoptosis and that AGE prevented the decreases of HQ-treated cell numbers by increased replicative DNA synthesis of RPE cells without changing apoptosis. Real-time RT-PCR revealed that vascular endothelial growth factor (VEGF)-A mRNA was increased by HQ treatment and the addition of HQ+AGE resulted in a further increment. The increase of VEGF secretion was confirmed by ELISA, and inhibition of VEGF signaling by chemical inhibitors and small interfering RNA decreased the HQ+AGE-induced increases in RPE cell numbers. The deletion analysis demonstrated that -102 to -43 region was essential for the VEGF-A promoter activation. Site-directed mutaions of specificity protein 1 (SP1) binding sequences in the VEGF-A promoter and RNA interference of SP1 revealed that SP1 is an essential transcription factor for VEGF-A expression. These results indicate that HQ induces RPE cell apoptosis, leading to dry AMD, and suggest that AGE stimulation in addition to HQ enhances VEGF-A transcription via the AGE-receptor for AGE pathway in HQ-damaged cells. As a result, the secreted VEGF acts as an autocrine/paracrine growth factor for RPE and/or adjacent vascular cells, causing wet AMD.
Keywords: AGE, advanced glycation endproduct; AMD, age-related macular degeneration; Advanced glycation endproduct(s); Age-related macular degeneration; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; HQ, hydroquinone; Hydroquinone; IdU, 5ʹ-Indo-2ʹ-deoxyuridine; RAGE, receptor for advanced glycation endproduct; RPE, retinal pigment epithelial; RT-PCR, reverse transcription polymerase chain reaction;; Retinal pigment epithelial cells; SP1, specificity protein 1; SR, scavenger receptor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; VEGF, vascular endothelial growth factor; Vascular endothelial growth factor; WST-8, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt; siRNA, small interfering RNA.