Young P. Jang

Complement activation by photooxidation products of A2E, a lipofuscin constituent of the retinal pigment epithelium

Recent studies have implicated local inflammation and activation of complement amongst the processes involved in the pathogenesis of age-related macular degeneration (AMD). Several lines of investigation also indicate that bis-retinoid pigments, such as A2E, that accumulate as lipofuscin in retinal pigment epithelial (RPE) cells, contribute to the disease process. In an investigation of a potential trigger for complement activation in AMD, we explored the notion that the complex mixture of products resulting from photooxidation of A2E might include a range of fragments that could be recognized by the complement system as “foreign” and that could serve to activate the complement system, leading to low-grade inflammation. To this end, we established an in vitro assay by using human serum as a source of complement, and we measured products of C3 activation by enzyme immunoassay. Accordingly, we found that the C3 split products inactivated C3b (iC3b) and C3a were elevated in serum, overlying ARPE-19 cells that had accumulated A2E and were irradiated to induce A2E photooxidation. Precoating of microtiter plates with two species of oxidized A2E, peroxy-A2E, and furano-A2E, followed by incubation with serum, also activated complement. We suggest that products of the photooxidation of bis-retinoid lipofuscin pigments in RPE cells could serve as a trigger for the complement system, a trigger than would predispose the macula to disease and that, over time, could contribute to chronic inflammation. These findings link four factors that have been posited as being associated with AMD: inflammation, oxidative damage, drusen, and RPE lipofuscin.

A2E-epoxides Damage DNA in Retinal Pigment Epithelial Cells VITAMIN E AND OTHER ANTIOXIDANTS INHIBIT A2E-EPOXIDE FORMATION

The autofluorescent pigments that accumulate in retinal pigment epithelial cells with aging and in some retinal disorders have been implicated in the etiology of macular degeneration. The major constituent is the fluorophore A2E, a pyridinium bisretinoid. Light-exposed A2E-laden retinal pigment epithelium exhibits a propensity for apoptosis with light in the blue region of the spectrum being most damaging. Efforts to understand the events precipitating the death of the cells have revealed that during irradiation (430 nm), A2E self-generates singlet oxygen with the singlet oxygen in turn reacting with A2E to generate epoxides at carbon-carbon double bonds. Here we demonstrate that A2E-epoxides, independent of singlet oxygen, exhibit reactivity toward DNA with oxidative base changes being at least one of these lesions. Mass spectrometry revealed that the antioxidants vitamins E and C, butylated hydroxytoluene, resveratrol, a trolox analogue (PNU-83836-E), and bilberry extract reduce A2E-epoxidation, whereas single cell gel electrophoresis and cell viability studies revealed a corresponding reduction in the incidence of DNA damage and cell death. Vitamin E, a lipophilic antioxidant, produced a more pronounced decrease in A2E-epoxidation than vitamin C, and treatment with both vitamins simultaneously did not confer additional benefit. Studies in which singlet oxygen was generated by endoperoxide in the presence of A2E revealed that vitamin E, butylated hydroxytoluene, resveratrol, the trolox analogue, and bilberry reduced A2E-epoxidation by quenching singlet oxygen. Conversely, vitamin C and ginkgolide B were not efficient quenchers of singlet oxygen under these conditions.

A2E, a byproduct of the visual cycle

A substantial portion of the lipofuscin that accumulates with age and in some retinal disorders in retinal pigment epithelial (RPE) cells, forms as a consequence of light-related vitamin A recycling. Major constituents of RPE lipofuscin are the di-retinal conjugate A2E and its photoisomers. That the accretion of A2E has consequences for the cell, with the adverse effects of A2E being attributable to its amphiphilic structure and its photoreactivity, is consistent with evidence of an association between atrophic age-related macular degeneration (AMD) and excessive lipofuscin accumulation.

The all-trans-retinal dimer series of lipofuscin pigments in retinal pigment epithelial cells in a recessive Stargardt disease model

The bis-retinoid pigments that accumulate in retinal pigment epithelial cells as lipofuscin are associated with inherited and age-related retinal disease. In addition to A2E and related cis isomers, we previously showed that condensation of two molecules of all-trans-retinal leads to the formation of a protonated Schiff base conjugate, all-trans-retinal dimer-phosphatidylethanolamine. Here we report the characterization of the related pigments, all-trans-retinal dimer-ethanolamine and unconjugated all-trans-retinal dimer, in human and mouse retinal pigment epithelium. In eyecups of Abcr−/− mice, a model of recessive Stargardt macular degeneration, all-trans-retinal dimer-phosphatidylethanolamine was increased relative to wild type and was more abundant than A2E. Total pigment of the all-trans-retinal dimer series (sum of all-trans-retinal dimer-phosphatidylethanolamine, all-trans-retinal dimer-ethanolamine, and all-trans-retinal dimer) increased with age in Abcr−/− mice and was modulated by amino acid variants in Rpe65. In in vitro assays, enzyme-mediated hydrolysis of all-trans-retinal dimer-phosphatidylethanolamine generated all-trans-retinal dimer-ethanolamine, and protonation/deprotonation of the Schiff base nitrogen of all-trans-retinal dimer-ethanolamine was pH-dependent. Unconjugated all-trans-retinal dimer was a more efficient generator of singlet oxygen than A2E, and the all-trans-retinal dimer series was more reactive with singlet oxygen than was A2E. By analyzing chromatographic properties and UV-visible spectra together with mass spectrometry, mono- and bis-oxygenated all-trans-retinal dimer photoproducts were detected in Abcr−/− mice. The latter findings are significant to an understanding of the adverse effects of retinal pigment epithelial cell lipofuscin.

Characterization of peroxy-A2E and furan-A2E photooxidation products and detection in human and mouse retinal pigment epithelial cell lipofuscin.

The nondegradable pigments that accumulate in retinal pigment epithelial (RPE) cells as lipofuscin constituents are considered to be responsible for the loss of RPE cells in recessive Stargardt disease, a blindness macular disorder of juvenile onset. This autofluorescent material may also contribute to the etiology of age-related macular degeneration. The best characterized of these fluorophores is A2E, a compound consisting of two retinoid-derived side arms extending from a pyridinium ring. Evidence indicates that photochemical mechanisms initiated by excitation from the blue region of the spectrum may contribute to the adverse effects of A2E accumulation, with the A2E photooxidation products being damaging intermediates. By studying the oxidation products (oxo-A2E) generated using oxidizing agents that add one or two oxygens at a time, together with structural analysis by heteronuclear single quantum correlation-NMR spectroscopy, we demonstrated that the oxygen-containing moieties generated within photooxidized A2E include a 5,8-monofuranoid and a cyclic 5,8-monoperoxide. We have shown that the oxidation sites can be assigned to the shorter arm of A2E, to the longer arm, or to both arms by analyzing changes in the UV-visible spectrum of A2E, and we have observed a preference for oxidation on the shorter arm. By liquid chromatography-mass spectrometry, we have also detected both monofuran-A2E and monoperoxy-A2E in aged human RPE and in eye cups of Abca4/Abcr–/– mice, a model of Stargardt disease. Because the cytotoxicity of endoperoxide moieties is well known, the production of endoperoxide-containing oxo-A2E may account, at least in part, for cellular damage ensuing from A2E photooxidation.

Anthocyanins Protect Against A2E Photooxidation and Membrane Permeabilization in Retinal Pigment Epithelial Cells

Abstract The pyridinium bisretinoid A2E, an autofluorescent pigment that accumulates in retinal pigment epithelial cells with age and in some retinal disorders, can mediate a detergent-like perturbation of cell membranes and light-induced damage to the cell. The photodynamic events initiated by the sensitization of A2E include the generation of singlet oxygen and the oxidation of A2E at carbon–carbon double bonds. To assess the ability of plant-derived anthocyanins to modulate adverse effects of A2E accumulation on retinal pigment epithelium (RPE) cells, these flavylium salts were isolated from extracts of bilberry. Nine anthocyanin fractions reflecting monoglycosides of delphinidin, cyanidin, petunidin and malvidin were obtained and all were shown to suppress the photooxidation of A2E at least in part by quenching singlet oxygen. The anthocyanins tested exhibited antioxidant activity of variable efficiency. The structural characteristics relevant to this variability likely included the ability to form a stable quinonoidal anhydro base at neutral pH, a conjugated diene structure in the C (pyrane) ring, the presence of hydroxyl groups on the B (benzene) ring and the relative hydrophobicity conferred by the arrangement of substituents on the B ring. Cells that had taken up anthocyanins also exhibited a resistance to the membrane permeabilization that occurs as a result of the detergent-like action of A2E.

A2E, a fluorophore of RPE lipofuscin: can it cause RPE degeneration?

In atrophic age-related macular degeneration (AMD) and Stargardt disease, the death of retinal pigment epithelial (RPE) cell death leads to photoreceptor cell degeneration and visual impairment. Nevertheless, the cause of RPE atrophy is poorly understood. One factor that may place RPE cells at risk is the accumulation of critical levels of lipofuscin. Indeed, several lines of evidence indicate that the excessive accumulation of lipofuscin by RPE cells is significant in terms of the etiology of AMD. Firstly, histological analyses of human donor eyes (Wing et al., 1978; Weiter et al., 1986), in addition to fundus spectrophotometry (Delori et al., 1995a; Delori et al., 2001), and confocal ophthalmoscopy (von Ruckmann et al., 1997), have shown that RPE cells overlying the macula, with the exception of RPE in the cone-rich fovea, exhibit the most pronounced age-related accumulation of fluorescent material. Lipofuscin levels in RPE cells are also topographically correlated with histopathological indicators of AMD (Feeney-Burns et al., 1984; Dorey et al., 1989) and with the loss of photoreceptor cells in aged eyes (Dorey et al., 1989). Interestingly, increased fundus autofluorescence at the borders of geographic atrophy is considered to represent an enhanced accumulation of RPE lipofuscin and to implicate the latter in the disease process (Holz et al., 1999; Holz et al., 2001). While the amassing of lipofuscin by RPE is a feature of aging, excessive accretion also occurs in Stargardt disease, some forms of retinitis pigmentosa and cone-rod dystrophy (Weingeist et al., 1982; Rabb et al., 1986; Lopez et al., 1990; Delori et al., 1995b; Kennedy et al., 1995).

A2E, A Fluorophore of RPE Lipofuscin, Can Destabilize Membrane

Studies of Stargardt disease suggest a role for RPE lipofuscin in the RPE cell atrophy that characterizes macular degeneration. The best known constituent of RPE lipofuscin is the pyridinium bisretinoid, A2E (Eldred and Lasky, 1993; Parish et al., 1998). Amongst the properties of A2E that may be damaging to the RPE cell is its ability to destabilize cell membranes (Sparrow et al., 1999). A hydrophilic head group combined with a pair of hydrophobic side-arms are the structural correlates of this behavior. This amphiphilic structure accounts for the tendency of A2E to aggregate (Sakai et al., 1996; De and Sakmar, 2002), a behavior first recognized in deuterated chloroform (CDCl3), the broadening of the 1H NMR signal indicating that the protonated pyridinium moieties of A2E were closely packed within the interior of micelles while the hydrophobic chains contacted the solvent. Further evidence of the detergent-like behaviour of A2E has been revealed in experiments demonstrating the ability of A2E to induce concentration-dependent membrane leakage (Sparrow et al., 1999). In studies employing unilamellar vesicles, it has also been shown that A2E, at critical micellar concentrations, can solubilize membranes (De and Sakmar, 2002).

Photooxidation of RPE lipofuscin bisretinoids enhances fluorescence intensity.

Light-related cycling of chemically reactive vitamin A aldehyde leads to the formation autofluorescent bis-retinoid pigments that accumulate as lipofuscin in retinal pigment epithelial cells. The amassing of these diretinoid compounds is implicated in the pathogenesis of age-related macular degeneration and in some inherited forms of retinal degeneration. For all of these fluorophores, extended conjugation systems confer absorbance maxima in the visible spectrum. We report that an increase in fluorescence emission can accompany photoxidation of the bisretinoids A2E and all-trans-retinal dimer. These findings are relevant to the quantification of RPE lipofuscin based on inference from fluorescence intensity.

OT-674 Suppresses Photooxidative Processes Initiated by an RPE Lipofuscin Fluorophore

The pathological processes involved in age‐related macular degeneration (AMD) include retinal pigment epithelial (RPE) cell degeneration; oxidative mechanisms likely contribute to the demise of these cells. Indeed, RPE cells may be particularly susceptible to photooxidative mechanisms since they accumulate retinoid‐derived photoreactive compounds that constitute the lipofuscin of the cell. Thus we undertook to test the capacity of OT‐674, the reduction product (Tempol‐H) of the nitroxide Tempol, to suppress photooxidative processes initiated by the RPE lipofuscin fluorophore A2E. Accordingly, when ARPE‐19 cells that had accumulated A2E were irradiated at 430 nm, pretreatment with OT‐674 (0.01–10 mm) was found to confer a resistance to cell death. Monitoring by quantitative HPLC also showed that OT‐674 reduced A2E photooxidation in a cell‐free system. Moreover, when presented with a singlet oxygen generator, OT‐674 served as a quencher of singlet oxygen that was more effective than Trolox and α‐tocopherol. We conclude that OT‐674 is a potent antioxidant that suppresses photooxidative processes generated in cultured RPE cells by the lipofuscin fluorophore A2E. As oxidative damage to RPE cells is considered to be a risk factor for AMD, antioxidant therapy with OT‐674 may serve a protective role.