Low-Luminance Blue Light-Enhanced Phototoxicity in A2E-Laden RPE Cell Cultures and Rats.

N-retinylidene-N-retinylethanolamine (A2E) and other bisretinoids are components of lipofuscin and accumulate in retinal pigment epithelial (RPE) cells-these adducts are recognized in the pathogenesis of retinal degeneration. Further, blue light-emitting diode (LED) light (BLL)-induced retinal toxicity plays an important role in retinal degeneration. Here, we demonstrate that low-luminance BLL enhances phototoxicity in A2E-laden RPE cells and rats. RPE cells were subjected to synthetic A2E, and the effects of BLL on activation of apoptotic biomarkers were examined by measuring the levels of cleaved caspase-3. BLL modulates the protein expression of zonula-occludens 1 (ZO-1) and paracellular permeability in A2E-laden RPE cells. Early inflammatory and angiogenic genes were also screened after short-term BLL exposure. In this study, we developed a rat model for A2E treatment with or without BLL exposure for 21 days. BLL exposure caused fundus damage, decreased total retinal thickness, and caused neuron transduction injury in the retina, which were consistent with the in vitro data. We suggest that the synergistic effects of BLL and A2E accumulation in the retina increase the risk of retinal degeneration. These outcomes help elucidate the associations between BLL/A2E and angiogenic/apoptotic mechanisms, as well as furthering therapeutic strategies.

A novel bisretinoid of retina is an adduct on glycerophosphoethanolamine.

PURPOSE: Fluorescent bisretinoid compounds accumulate in retinal pigment epithelial (RPE) cells as a consequence of two processes: random reactions of vitamin A aldehyde in photoreceptor cell outer segments, and phagocytosis of discarded photoreceptor outer segment discs by RPE. The formation of bisretinoid is accentuated in some forms of retinal degeneration. The detection of a novel bisretinoid fluorophore that is a conjugate of all-trans-retinal and glycerophosphoethanolamine is reported. METHODS: Human RPE/choroid, eyes harvested from Abca4 (ATP-binding cassette transporter 4) null mutant mice, and biosynthetic reaction mixtures were analyzed by ultra performance liquid chromatography coupled to mass spectrometry and by nuclear magnetic resonance spectra and spectrofluorometry. RESULTS: A fluorescent compound in mouse eyes and in human RPE/choroid corresponded to the product of the reaction between all-trans-retinal and glycerophosphoethanolamine (A2-GPE), as determined on the basis of molecular weight (m/z 746), absorbance (approximately 338,443 nm), and retention time. Nuclear magnetic resonance spectra were consistent with a pyridinium molecule with a glycerophosphate moiety. The emission maximum of A2-GPE was approximately 610 nm. A2-GPE accumulated with age in mouse eyes and was more abundant in Abca4(-/-) mice, a model of recessive Stargardt disease. CONCLUSIONS: To date, several bisretinoids of RPE lipofuscin have been isolated and characterized, and for all of these, formation involves the membrane phospholipid phosphatidylethanolamine. Conversely, the bisretinoid A2-GPE is detected as sn-glycero-3-phosphoethanolamine (GPE) derivatized by two all-trans-retinal. The pathways by which A2-GPE may form under conditions of increased availability of all-trans-retinal, for instance in the Abca4(-/-) mouse, are discussed.

Novel lipofuscin bisretinoids prominent in human retina and in a model of recessive Stargardt disease.

Bisretinoid adducts accumulate as lipofuscin in retinal pigment epithelial (RPE) cells of the eye and are implicated in the pathology of inherited and age-related macular degeneration. Characterization of the bisretinoids A2E and the all-trans-retinal dimer series has shown that these pigments form from reactions in photoreceptor cell outer segments that involve all-trans-retinal, the product of photoisomerization of the visual chromophore 11-cis-retinal. Here we have identified two related but previously unknown RPE lipofuscin compounds. By high performance liquid chromatography-electrospray ionization-tandem mass spectrometry, we determined that the first of these compounds is a phosphatidyl-dihydropyridine bisretinoid; to indicate this structure and its formation from two vitamin A-aldehyde (A2), we will refer to it as A2-dihydropyridine-phosphatidylethanolamine (A2-DHP-PE). The second pigment, A2-dihydropyridine-ethanolamine, forms from phosphate hydrolysis of A2-DHP-PE. The structure of A2-DHP-PE was corroborated by Fourier transform infrared spectroscopy, and density functional theory confirmed the presence of a dihydropyridine ring. This lipofuscin pigment is a fluorescent compound with absorbance maxima at approximately 490 and 330 nm, and it was identified in human, mouse, and bovine eyes. We found that A2-DHP-PE forms in reaction mixtures of all-trans-retinal and phosphatidylethanolamine, and in mouse eyecups we observed an age-related accumulation. As compared with wild-type mice, A2-DHP-PE is more abundant in mice with a null mutation in Abca4 (ATP-binding cassette transporter 4), the gene causative for recessive Stargardt macular degeneration. Efforts to clarify the composition of RPE lipofuscin are important because these compounds are targets of gene-based and drug therapies that aim to alleviate ABCA4-related retinal disease.

Lipofuscin and lipofuscin-like substances.

Lipofuscin is defined as being a yellowish brown, lipid-rich, heterogeneous, cytoplasmic granular pigment emitting an intense yellow autofluorescence when excited with ultraviolet light, which accumulates in various tissues of animals during their aging. It is believed that the pigments are derived from the reaction of some of reactive secondary products including malonaldehyde, formed during membranous lipid peroxidation, with amino groups of phospholipids and proteins, etc., and that these formations are accompanied by alteration of the membrane structure and inactivation of the enzymes. The fluorescence measurement of the pigments is widely used as a parameter of lipid peroxidation in vivo as well as in vitro. However, their origin, chemical structure, biological significance or fate has not as yet been fully elucidated. This article introduces and discusses the recent studies on these problems.

Organic solvent soluble lipofuscin pigments and glutathione peroxidase in weanling and old rats.

Tissues from weanling and 19-month-old Sprague Dawley rats were spectrophotofluorometrically analyzed for organic solvent soluble lipofuscin pigments (OLP) and assayed for glutathione peroxidase (GSH-Px) activity. Significantly lower levels of OLP and higher levels of GSH-Px activity were found in the kidney, heart and testis at 19 months of age than were found in weanling rats. No age-related differences in OLP were observed in the brain, lung or liver. The results appeared to indicate an inverse relationship between OLP and GSH-Px activity in some tissues, but the magnitude of the changes observed coupled with conflicting observations in the liver suggested that GSH-Px activity and OLP concentrations were not directly related.