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Andreas Pollreisz, Taras Afonyushkin, Olga Oskolkova, Florian Gruber, Valery Bochkov, Ursula Schmidt-Erfurth; Mechanisms of VEGF upregulation in human RPE cells: Role of oxidized phospholipids and the ATF4 arm of the unfolded protein response. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6103.
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© ARVO (1962-2015); The Authors (2016-present)
The aim of this study was to elucidate the role of oxidized phospholipids (OxPLs) and UVA-1 light as proangiogenic mediators in human retinal pigment epithelial (RPE) cells.
Quantification of OxPLs in the vitreous and in UVA-1 irradiated primary human RPE cells was performed by reversed-phase liquid chromatography coupled to mass spectrometry. UVA-1 irradiation of RPE cells was performed with a Sellamed-3000 UVA-1 device. RPE cells were transfected with small interfering RNA targeting ATF4. mRNA levels of proangiogenic genes in RPE cells were determined by real-time quantitative PCR and VEGF-A concentrations measured in culture medium by ELISA.
The presence of OxPLs in the human eye was documented by analyzing lipid extracts from human vitreous by mass spectrometry. Multiple species of oxidized phosphatidylcholines (OxPCs) were detected, including biologically active fragmented species POVPC, PGPC, PONPC and PAzPC. UVA-1 irradiation of RPE cells led to substantial accumulation of these and many other molecular species of OxPCs. In vitro oxidized OxPCs and other classes of OxPLs upregulated the expression of VEGF in RPE cells, which critically depended on the ATF4 arm of the unfolded protein response (UPR).
Our data prove that different species of OxPLs are present in the human vitreous and generated in RPE cells upon irradiation with UVA-1 light. These oxidized products are capable of inducing changes in gene expression of VEGF, relevant to angiogenesis via the ATF4 arm of UPR. These results suggest new mechanistic insights into the known association between light exposure and risk of AMD development.
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