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Hiroki Tsujinaka, Asako Itaya-Hironaka, Akiyo Yamauchi, Sumiyo Sakuramoto-Tsuchida, Takanori Fujimura, Ryogo Shobatake, Tadanobu Yoshikawa, Naonori Masuda, Mitsunobu Ohtsuki, Shin Takasawa, Nahoko Ogata; Human retinal pigment epithelial cell proliferation by the combined addition of hydroquinone and advanced glycation end-products via up-regulation of VEGF gene.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5796.
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© ARVO (1962-2015); The Authors (2016-present)
To explore the molecular pathophysiology that might explain the association between cigarette smoke, aging/diabetes, and age-related macular degeneration (AMD) by examining the effects of hydroquinone (HQ), a toxic compound in cigarette smoke-related tar, and/or advanced glycation endproducts (AGE), compounds produced during the aging and under conditions of hyperglycemia, on human retinal pigment epithelial (hRPE) cells.
ARPE-19 and h1RPE7, hRPE cells, were treated with HQ (20 µM), AGEs (300 µg/ml), and HQ+AGE for 12 h. After the treatment, viable cell numbers, apoptosis, and DNA synthesis were measured by WST-8 cleavage, TUNEL method, and 5’-indo-2’-deoxyuridine incorporation, respectively. VEGF expression was evaluated by real-time RT-PCR and ELISA. Deleted and mutated human VEGF promoters were inserted uperstream of a luciferase gene in pGL4 and transcriptional activity was measured after HQ+AGE stimulation. To identify the key factor for HQ+AGE-stimulated VEGF expression, knockdown experiments using small interfering RNA (siRNA) were performed.
The viable cells were reduced by HQ treatment (P<0.0001). The addition of HQ+AGE partially prevented the loss of viable cells (P<0.002). HQ+AGE did not prevent the HQ-induced apoptosis. DNA synthesis was increased by the addition of HQ+AGE (P=0.0001), suggesting that AGE stimulated cell proliferation of HQ-damaged cells. Real-time RT-PCR revealed that VEGF mRNA was increased by HQ+AGE. Inhibitors for VEGF/VEGF receptor as well as siVEGF decreased the HQ+AGE-induced increases in hRPE cell numbers (P<0.05). Introduction of siRNA for receptor for AGE (RAGE) attenuated the HQ+AGE-induced increase in hRPE cells (P=0.0175). Deletion analysis of VEGF promoter revealed that GC boxes in the -102~-43 region were essential for the VEGF promoter activation. Site-directed mutations of the GC boxes in the VEGF promoter and RNA interference for SP1 revealed that SP1 is the key transcription factor for VEGF expression in HQ+AGE-stimulated hRPE cells.
HQ induces hRPE cell apoptosis and could lead to dry AMD. AGE stimulation enhances VEGF transcription via the AGE-RAGE pathway in HQ-damaged cells. As a result, the secreted VEGF acts as an autocrine/paracrine growth factor for hRPE and/or adjacent vascular cells, causing wet AMD.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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