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  • Excessive retinol intake exacerbates choroidal neovascularization through upregulated vascular endothelial growth factor in retinal pigment epithelium in mice.

Excessive retinol intake exacerbates choroidal neovascularization through upregulated vascular endothelial growth factor in retinal pigment epithelium in mice.

Experimental eye research (2015-01-13)
Xue Tan, Hidenori Takahashi, Junko Nishida, Aya Aoki, Tatsuya Inoue, Yasuo Yanagi
ABSTRACT

As a part of the visual cycle, all-trans-retinol (all-trans-ROL), the major form of vitamin A in circulating blood, is transported to the retinal pigment epithelium (RPE). All-trans-ROL is essential for normal retina function. However, recent researches have shown that excessive retinol intake can cause increase of all-trans-retinal. This can lead to the accumulation of lipofuscin, which is important in the pathogenesis of retina degeneration disease, such as dry type age-related macular degeneration (AMD). Since there are few reports regarding the involvement of all-trans-ROL in exudative AMD, we investigated the effects of all-trans-ROL in vitro and in vivo. We evaluated vascular endothelial growth factor (VEGF) expression in ARPE-19 cells and THP-1 cells after all-trans-ROL treatment using ELISA and real-time RT-PCR. In-vitro tube formation assay was performed with HUVEC cells using the conditioned medium (CM) obtained from ARPE-19 cells treated with all-trans-ROL. Transcriptional activity of retinoic acid receptor (RAR) was evaluated using luciferase assay. In mice, VEGF expressions were investigated in the retina and RPE/choroid after three weeks of excessive oral retinol intake. Laser-induced choroidal neovascularization (CNV) models were evaluated after they were fed with various doses of retinol. VEGF mRNA expression and VEGF production were significantly increased in all-trans-ROL treated ARPE-19 cells, which were inhibited by an RAR antagonist LE540. In contrast, there were no significant changes in VEGF production in THP-1 cells. Transcriptional activity of RAR was upregulated by all-trans-ROL treatment in ARPE-19 cells. The CM, obtained from ARPE-19 cells treated with all-trans-ROL, induced more capillary-like tube formation than cells treated with control vehicles. In vivo, the high retinol diet group has increased VEGF expression in the RPE/choroid and larger lesion size was induced. Our results suggest that all-trans-ROL is a pro-angiogenic factor. Excessive retinoid intake may be a potential risk factor for exudative AMD.

MATERIALS
Product Number
Brand
Product Description

Supelco
Streptomycin solution, ~1 mg/mL in 1 mM EDTA, analytical standard
Sigma-Aldrich
Streptomycin sulfate salt, powder
Sigma-Aldrich
Xylazine, ≥99%
Sigma-Aldrich
Retinol, synthetic, ≥95% (HPLC), crystalline
Sigma-Aldrich
Streptomycin sulfate salt, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Streptomycin sulfate salt, powder, BioXtra, suitable for mouse embryo cell culture
Sigma-Aldrich
Retinol, ≥95.0% (HPLC), ~2700 U/mg
Sigma-Aldrich
Retinol, BioXtra, ≥97.5% (HPLC), ~3100 U/mg
Streptomycin sulfate, European Pharmacopoeia (EP) Reference Standard