Artificial senescence of bovine retinal pigment epithelial cells induced by near-ultraviolet in vitro

Abstract

RPE cells irradiated by near-ultraviolet (NUV) were characterized at cellular, biochemical and molecular levels in order to determine whether light-induced RPE changes contribute to the senescence of RPE cells in vitro. Biochemical and molecular parameters of cellular senescence were studied by using both bovine RPE cells at confluence repeatedly irradiated by NUV (peaking at 365 nm) and RPE cells at different levels of population doubling (PDL). After repeated NUV irradiation, RPE proliferation was markedly suppressed. In parallel, the BrdU index significantly reduced to a minimum level, similar to RPE cells undergoing multiple population doublings. NUV irradiation resulted in a decrease in cellular alkali-soluble melanin and an increase in lipofuscin-like fluorophores. The lipofuscin-like fluorophores, isolated from RPE cells exposing repeated NUV irradiation, represented a gradual hyperchromic change and red-shift, reaching the wavelength maxima (560–572 nm), at excitation wavelength of 365 nm, a typical range of ‘age pigment’. These phenomena were substantially eliminated in oxygen-free conditions. Both the NUV-irradiated RPE cells and RPE cells at 20 Pd expressed 4 to 8-fold and 2 to 4-fold less PEDF and TIMP-3 genes, respectively. As result of experiments using chronic photochemical treatment, RPE cells represented several characteristics of cellular senescence. In addition to alterations of the melanin/lipofuscin system, DNA synthesis was greatly suppressed in NUV-irradiated RPE cells, indicating replicative senescence. The phenomena of downregulation of the possible senescence markers imply that photochemical reactions of RPE cells accelerate the process of RPE senescence.