Interaction of pyridinium bis-retinoid (A2E) with bilayer lipid membranes

The accumulation of lipofuscin granules within the retinal pigment epithelium (RPE) cells is correlated with the progression of age-related macular degeneration. One of the fluorophores contained in lipofiscin granules is pyridinium bis-retinoid (A2E). To test its membrane-toxic effect, the interaction of A2E with bilayer lipid membranes (BLM) was studied. The incorporation of charged A2E molecules into the membranes has been detected as a change of either zeta-potential of multilayer liposomes or boundary potential of BLM. It was shown that the presence of up to 25mol% of A2E did not destabilize the bilayers made of saturated phosphatidylcholine (PC). However, the destabilizing effect became very significant when BLM contained negatively charged lipids such as cardiolipin or phosphatidylserine. The electrical breakdown measurements revealed that the A2E-induced decrease of BLM stability was primarily associated with the growing probability of lipid pore formation. It was found from the measurements of boundary potential of BLM that exposure of A2E to light initiates its transformation into at least two products. One of them is epoxy-A2E, which, being hydrophilic, moves from the membrane into water solution. The other product is a non-identified hydrophobic substance. Illumination of A2E-containing BLM made from unsaturated PC by visible light caused the membrane damage presumably due to oxidation of these lipids by singlet oxygen generated by excited A2E molecules. However, this effect was very weak compared to the effect of known photosensitizers. The illumination of BLM with A2E also leads to the damage of gramicidin incorporated into the membrane, as was detected by measuring the conductance of channels formed by this peptide.     

Fluorescence emission and excitation spectra of fluorophores of lipofuscin granules isolated from retinal pigment epithelium of human cadaver eyes

Visible light-induced changes in fluorescence characteristics of lipofuscin granules (LG) isolated from retinal pigment epithelium of human cadaver eyes are compared with the analogous age-related changes and correlated with the content and photooxidation of LG main fluorophore, A2E. We used HPLC to examine changes of LG fluorophore composition with donor age, as well as before and after visible-light irradiation (the latter HPLC tests were also done with synthetic A2E). Visible light induces oxidation of LG fluorophores. As a result, their fluorescence characteristics change: the emission spectrum is blue-shifted by 25–40 nm. The observed age-dependent changes in the relative content of LG fluorophores and their oxidized derivatives were qualitatively similar with those caused by irradiation. To improve the accuracy of a new noninvasive diagnostic method, fundus autofluorescence imaging, it is important to know the ratio of nonoxidized and oxidized fluorophore derivatives depending on age and eye pathology.     

Lipofuscin and A2E Accumulate with Age in the Retinal Pigment Epithelium of Nrl−/− Mice†

Lipofuscin is a fluorescent material with significant phototoxic potential that accumulates with age in the retinal pigment epithelium (RPE) of the eye. It is thought to be a factor in retinal degeneration diseases. The most extensively characterized lipofuscin component, N‐retinylidene‐N‐retinylethanolamine (A2E), has been proposed to be a byproduct of reactions involving the visual pigment chromophore. To examine the impact of the visual pigment and photoreceptor cell type on lipofuscin accumulation, we analyzed the RPE from Nrl^?/? mice of various ages for lipofuscin fluorescence and A2E levels. The photoreceptor cells of the Nrl^?/? retina contain only cone‐like pigments, and produce cone‐like responses to photostimulation. The cone‐like nature of these cells was confirmed by the presence of RPE65. Lipofuscin was measured with fluorescence imaging, whereas A2E was quantified by UV/VIS absorbance spectroscopy coupled to HPLC. The identity of A2E was corroborated with tandem mass spectrometry. Lipofuscin and A2E accumulated with age, albeit to lower levels compared with wild type mice. The emission spectra of RPE lipofuscin granules from Nrl^?/? mice were similar to those from wild type mice, with λ_maxca 610 nm. These results demonstrate that cone visual pigments can contribute to the production of lipofuscin and A2E.     

Photophysical Studies of A2-E, Putative Precursor of Lipofuscin, in Human Retinal Pigment Epithelial Cells

With age, human retinal pigment epithelial cells accumulate lipofuscin that can absorb photons in the visible range leading to light-induced damage and impaired vision. A putative precursor of lipofuscin, 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E, 5E,7E- octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6-(2,6,6-trimethyl-1 - cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]-pyridinium (A2-E), has recently been isolated and characterized from aged human retinal pigment epithelial cells. We have found that A2-E inhibits the growth of human retinal pigment epithelial cells at concentrations greater than 1 microM. Time-resolved fluorescence measurements of 1 microM A2-E in solution, performed under 413 nm excitation, showed that fluorescence wave forms integrated across the spectrum (450-600 nm) were best-fitted with three decay times in the nanosecond and subnanosecond time scale: 6.6, 1.9 and 0.33 ns. Untreated retinal pigment epithelial cells were characterized by three fluorescence lifetimes: 6.3, 1.7 and 0.35 ns. In retinal pigment epithelial cells treated with 1 microM A2-E, the fluorescence decay was significantly faster, with the marked presence (approximately equal to 30%) of a fourth short lifetime (0.12 ns). These fluorescence decay times for A2-E bound to human retinal pigment epithelial cells are similar to those of lipofuscin granules isolated from aged human retinal pigment epithelial cells. This similarity supports the hypothesis that A2-E is a precursor of lipofuscin and suggests that A2-E may play a role in the overall light damage associated with age-related retinal diseases.     

Fluorophores of lipofuscin granules responsible for human eye fundus autofluorescence

Individual fluorophores and/or their groups contained in a chloroform extract of lipofuscin granules isolated from retinal pigment epithelium of human cadaver eyes were studied by HPLC. Their spectral characteristics were studied, which made it possible to evaluate the contribution of particular fluorophores and/or their groups to the general image of human eye fundus autofluorescence. Many components, being conjugates of all-trans-retinal of different nature, contribute to the total fluorescence spectrum of the chloroform extract. The fluorophore A2E is not predominant.     

Effect of UV radiation and hydrogen peroxide on the antiradical and antioxidant activities of DOPA-melanin and melanosomes from retinal pigment epithelial cells

The effect of continuous UV radiation and hydrogen peroxide on destruction and antioxidant properties of synthetic DOPA-melanin (prepared by oxidation of 3,4-dihydroxyphenylalanine (DOPA)) and melanosomes isolated from cells of the retinal pigment epithelium (RPE) was investigated. The kinetics of melanin destruction was recorded based on the accumulation of fluorescent low-molecular-weight reaction products, the antiradical activity of melanin was determined by chemiluminescence method, the concentration of free radical products was measured by electron paramagnetic resonance, and the antioxidant activity of melanins was estimated by their inhibitory effect on lipid peroxidation. It was shown that UVC—UVA irradiation (up to 5 hours) of DOPA-melanin and melanosomes of retinal pigment epithelium decreased neither the latency period of luminol chemiluminescence nor the inhibitory action of pigments on Fe²⁺- and UV-induced peroxidation of cardiolipin liposomes. However, very long UV irradiation gave rise to fluorescent destruction products, decreased the concentration of paramagnetic centers in the pigment (especially light-dependent ones), and decreased the antiradical and antioxidant activities. For example, UV irradiation of DOPA-melanin during 52 h resulted in approximately a 2-fold decrease in the concentration of paramagnetic centers and decline of antiradical and antioxidant activities. However, even with such a hard irradiation the pigment retained significant inhibitory activity against lipid peroxidation. The oxidative destruction of DOPA-melanin in the presence of hydrogen peroxide in the dark resulted in complete destruction of the polymer and loss of its protective properties. It is assumed that destruction of RPE cell melanin is caused mainly by oxidative processes.     

Comparison of A2E Cytotoxicity and Phototoxicity with all-trans-Retinal in Human Retinal Pigment Epithelial Cells†

All-trans-retinal is the precursor of A2E, a fluorophore within lipofuscin, which accumulates in human retinal pigment epithelial (hRPE) cells and contributes to age-related macular degeneration. Here we have compared the in vitro dark cytotoxicity and visible-light-mediated photoreactivity of all-trans-retinal and A2E in hRPE cells. All-trans-retinal caused distinct cytotoxicity in hRPE cells measured with cell metabolic activity (MTS) and lactate dehydrogenase release assays. Significant increases in intracellular oxidized glutathione (GSSG), extracellular GSH and GSSG levels and lipid hydroperoxide production were observed in cells incubated in the dark with 25 and 50 μm all-trans-retinal. Light modified all-trans-retinal’s harmful action and decreased extracellular glutathione and hydroperoxide levels. A2E (<25 μm ) did not affect cell metabolism or cytoplasmic membrane integrity in the dark or when irradiated. 25 μm A2E raised the intracellular GSSG level in hRPE cells to a much smaller extent than 25 μm all-trans-retinal. A2E did not induce glutathione efflux or hydroperoxide generation in the dark or after irradiation. These studies support our previous conclusions that although A2E may be harmful at high concentrations or when oxidized, its phototoxic properties are insignificant compared to those of all-trans-retinal. The endogenous production of A2E may serve as a protective mechanism to prevent damage to the retina by free all-trans-retinal.     

Complexation of polycations to anionic liposomes: composition and structure of the interfacial complexes

Poly(N-ethyl-4-vinylpyridinium bromide) (a polycation with a degree of polymerization of 1100) was adsorbed onto liposomes composed of egg lecithin with a 0.05-0.20 molar fraction (nu) of anionic headgroups provided by cardiolipin (a doubly anionic lipid). According to electrophoretic mobility data, this led to total charge neutralization of the liposomes, whereupon the liposomes adopted a positive charge as additional polymer continued to adsorb. Although the liposomes aggregated at the charge-neutralization point, they disassembled into individual liposomes after becoming positively charged. The degree of polymer adsorption was shown to reach a limit. Thus, by measuring the free polymer content in a liposome suspension, it was possible to determine the polymer concentration at which the liposome surface became saturated with polymer. Beyond this point, an electrostatic/steric barrier at the surface suppressed further adsorption. Dynamic light scattering studies of liposomes with and without adsorbed polymer allowed calculation of the polymer film thickness which ranged from 22 to 35 nm as the molar fraction of cardiolipin (nu) increased from 0.05 to 0.20. The greater the content on the anionic lipid in the bilayer, the thicker the polymer film. The maximum number of polymer molecules adsorbed onto the liposomes was estimated: 1-2 molecules for nu = 0.05; 3 molecules for nu = 0.1; 4- molecules for nu = 0.15; and 6 molecules for nu = 0.2. The polymer appears to lie on the liposome surface, rather than embedding into the bilayer, because addition of NaCl easily dislodges the polymer from the liposome into the bulk water.     

Evidence for the hydration effect at the semiconductor phospholipid-bilayer interface by TiO2 photocatalysis

The interactions of TiO2 with phospholipid bilayers found in cell membrane walls were observed to perturb the bilayer structure under UVA light irradiation. The structure changes in the phospholipid bilayers upon contact with TiO2 under light and in the dark were followed by X-ray diffraction. Hydration effects at the semiconductor-phospholipid interface played an important role in the degradation of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) bilayers taken as cell wall lipid bilayer models. Evidence is provided that the fluidity of the phospholipid bilayers plays a significant role when interacting in the dark with the TiO2 or in processes mediated by TiO2 under light irradiation.     

Probing the spatial dependence of the emission spectrum of single human retinal lipofuscin granules using near-field scanning optical microscopy

The emission spectra of single lipofuscin granules are examined using spectrally resolved confocal microscopy and near-field scanning optical microscopy (NSOM). The emission spectrum varies among the granules examined revealing that individual granules are characterized by different distributions of fluorophores. The range of spectra observed is consistent with in vivo spectra of human retinal pigment epithelium cells. NSOM measurements reveal that the shape of the spectrum does not vary with position within the emissive regions of single lipofuscin granules. These results suggest that the relative distribution of fluorophores within the emissive regions of an individual granule is homogeneous on the spatial scale approximately 150 nm.     

THE USE OF A COMPUTERIZED SPECTRORADIOMETER TO PREDICT PHYTOCHROME PHOTOEQUILIBRIA UNDER POLYCHROMATIC IRRADIATION

Abstract A method is described for predicting the effect of polychromatic irradiation upon the photo-stationary equilibrium of the plant photoreceptor phytochrome. This method follows from the rate equations for phototransformation and utilizes the in vivo action spectra for phytochrome phototransformation (Pratt and Briggs, 1966). A scanning spectroradiometer interfaced with a microcomputer is used to determine a spectral photon distribution from 360 to 800 nm. The products of the photon fluence rate and the relative quantum efficiencies at 2-nm intervals are summed over the entire visible range to yield a predicted percentage of the pigment in the Pfr form. This value was determined under eight different polychromatic light sources and was generally within 7% Pfr of the value measured in etiolated corn coleoptiles under the same light sources.     

Controlling liposomal drug release with low frequency ultrasound: mechanism and feasibility

The ability of low-frequency ultrasound (LFUS) to release encapsulated drugs from sterically stabilized liposomes in a controlled manner was demonstrated. Three liposomal formulations having identical lipid bilayer compositions and a similar size ( approximately 100 nm) but differing in their encapsulated drugs and methods of drug loading have been tested. Two of the drugs, doxorubicin and methylpredinisolone hemisuccinate, were remote loaded by transmembrane gradients (ammonium sulfate and calcium acetate, respectively). The third drug, cisplatin, was loaded passively into the liposomes. For all three formulations, a short exposure to LFUS (<3 min) released nearly 80% of the drug. The magnitude of drug release was a function of LFUS amplitude and actual exposure time, irrespective of whether irradiation was pulsed or continuous. Furthermore, no change in liposome size distribution or in the chemical properties of the lipids or of the released drugs occurred due to exposure to LFUS. Based on our results, we propose that the mechanism of release is a transient introduction of porelike defects in the liposome membrane, which occurs only during exposure to LFUS, after which the membrane reseals. This explains the observed uptake of the membrane-impermeable fluorophore pyranine from the extraliposomal medium during exposure to LFUS. The implications of these findings for clinical applications of controlled drug release from liposomes are discussed.     

PHOTOPHYSICAL STUDIES ON HUMAN RETINAL LIPOFUSCIN

Fluorescent material generated in the human retina accumulates within lipofuscin granules of the retinal pigment epithelium (RPE) during aging. Its presence has been suggested to contributed to various diseases including age-related macular degeneration. Because this material absorbs light at wave lengths as long as 550 nm, photophysical studies were performed to determine whether lipofuscin could contribute to light damage and to determine if its composition is similar to a synthetically prepared lipofuscin. Time-resolved experiments were performed to monitor (1) fluorescence decay, (2) the UV-visible absorption of longer-lived excited states and (3) the formation and decay of singlet oxygen at 1270 nm. Steady-state and time-resolved fluorescence studies indicate that human and synthetic lipofuscin have fluorophores in common. Time-resolved absorption experiments on human retinal lipofuscin and synthetic lipofuscin showed the presence of at least two transient species, one absorbing at 430 nm (lifetime caμs) and a second absorbing at 580 nm, which decays via second order kinetics. In addition, there is a third absorbing species stable to several hundred milliseconds. The transient species at 430 nm is quenched by oxygen, suggesting that it is a triplet state. Subsequent studies showed the formation of singlet oxygen, which was monitored by its phosphorescence decay at 1270 nm. These studies demonstrate that lipofuscin can act as a sensitizer for the generation of reactive oxygen species that may contribute to the age-related decline of RPE function and blue light damage.     

UV-A induced oxidative stress is more prominent in naturally pigmented aged human RPE cells compared to non-pigmented human RPE cells independent of zinc treatment

To investigate the effects of zinc supplementation on human amelanotic (ARPE-19) and native pigmented retinal pigment epithelial cells (hRPE) under normal light conditions and after ultraviolet A light exposure. hRPE cells, containing both melanin and lipofuscin granules, were prepared from human donor eyes of 60-70 year old patients. Cells of the amelanotic ARPE-19 cell line and pigmented hRPE cells were treated with zinc chloride and subjected to oxidative stress by UV-A irradiation. Intracellular H(2)O(2) formation was measured using a fluorescence oxidation assay. Additionally, apoptosis and viability assays were performed. Control cells were treated identically except for irradiation and zinc supplementation. Under normal light conditions, zinc treated hRPE cells produced less H(2)O(2) than unsupplemented hRPE cells. Viability and apoptosis events did not change. After UV-A irradiation, ARPE and hRPE cells were greatly impaired in all tests performed compared to the non-irradiated controls. No differences were found after zinc supplementation. hRPE cells showed a higher apoptosis and mortality rate than non-pigmented cells when stressed by UV-A light. ARPE cells never showed any zinc related effects. In contrast, without irradiation, zinc supplementation reduced H(2)O(2) production in pigmented hRPE cells slightly. We did not find any zinc effect in irradiated hRPE cells. After UV light exposure, pigmented cells showed a higher apoptosis and mortality than cells lacking any pigmentation. We conclude that cells with pigmentation consisting of melanin and lipofuscin granules have more prooxidative than antioxidative capacity when stressed by UV light exposure compared to cells lacking any pigmentation.     

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 alpha-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.     

Anthocyanins protect against A2E photooxidation and membrane permeabilization in retinal pigment epithelial cells

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 component of ocular lipofuscin

The presence of lipofuscin in postmitotic cells is considered a hallmark of the aging process. In the retinal pigment epithelium (RPE), lipofuscin is found as micrometer-sized spherical particles and characterized by its yellow autofluorescence when exposed to blue light. This exposure to light is also known to produce reactive oxygen intermediates (ROI), but the particular molecular constituent(s) responsible for this phototoxicity have yet to be completely identified. Resulting mostly from the autophagocytosis of intracellular organelles, the composition of lipofuscin is poorly defined but known to contain protein, lipids and several fluorophores. The subsequent identification of one of the fluorophores in lipofuscin, A2E, generated much interest and resulted in a variety of studies to understand its potential role in the phototoxicity of lipofuscin. Several modes of toxicity have been suggested through which A2E can affect the health of RPE cells. These modes include photoinduced production of ROI, which places additional oxidative stress on RPE cells, the disruption of membrane integrity through its natural role as an amphiphilic detergent and inhibition of key cellular functions. This article presents the current understanding of the photochemistry of A2E and its involvement as a phototoxic agent in RPE cells.     

Comparison of the aerobic photoreactivity of A2E with its precursor retinal

A2E (2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E, 3E,5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]pyridinium) is a blue-absorbing molecular constituent of human ocular lipofuscin and contributes to the golden-yellow emission of this pigment. Lipofuscin photoproduces toxic reactive oxygen intermediates (ROI), but the specific molecular components responsible for this phototoxicity remain unidentified. In this article the aerobic photoreactivity of A2E is quantified by comparison with its biosynthetic precursor, all-trans-retinal, and with other appropriate standards. Under blue-light exposure the efficacies for formation of cholesterol (Ch) hydroperoxides and the superoxide radical anion (O2*-) were determined using high-pressure liquid chromatography with electrochemical detection and electron spin resonance oximetry and spin trapping, respectively. Photogeneration of singlet oxygen after blue-light excitation of A2E was demonstrated unambiguously by the Ch peroxidation assay. After blue-light irradiation of A2E, O2*- were detected, but the concentration was insufficient to account for the measured production of O2*- by the solvent extract of lipofuscin granules. The collective data support the conclusion that A2E does not produce sufficient concentrations of ROI to be the primary phototoxic constituent of lipofuscin.     

A new approach to measuring the action spectrum for singlet oxygen production by human retinal lipofuscin

The human retinal pigment epithelial (RPE) layer contains a complex mixture of components called lipofuscin; this mixture forms with age and with various genetic disorders such as Stargardt's disease. Its presence may contribute to retinal deterioration via several mechanisms including photochemical processes. In the lipofuscin mixture, both type I and II mechanisms have been identified, with the latter consisting of the generation of singlet oxygen. Several components of that mixture have been identified, most notably a bis-retinoid pyridinium compound called A2E and its derivatives. Photo-oxidative studies on the compound A2E have revealed that its dominant photochemical mechanism is via free radical or type I processes. Because singlet oxygen is an important photooxidative intermediate in tissue, its generation in the RPE may contribute to retinal maculopathies. It is therefore necessary to determine which specific component(s) in the lipofuscin mixture produce singlet oxygen upon excitation with light. This was ascertained by evaluating the action spectrum for singlet oxygen production for the whole lipofuscin mixture using time-resolved spectroscopy. Singlet oxygen was generated by excitation of the sample at different wavelengths while maintaining a constant beam energy, and was directly detected by its phosphorescence decay at 1270 nm using a Ge photodiode. The action spectrum for singlet oxygen sensitization by the organic soluble portion of lipofuscin had an absorption maximum at ca 380 nm, which is to the blue of A2E (maximum at 430 nm). Compounds with a similar absorption maximum eluted in the HPLC earlier than A2E and were detected in human lipofuscin. The concentration of this component apparently increased in concentration in human RPE lipofuscin mixture as a function of age up to 90 years old.     

Light-mediated and H-bond facilitated liposomal release: the role of lipid head groups in release efficiency

Syntheses of coumarin-containing lipids and liposomal formulations incorporating these lipids are studied. The influence of the lipid head groups in enhancing the release efficiency of these liposomes under light irradiation is studied and a molecular mechanism is provided.