Ocular proteomic and transcriptomic changes with aging in a rabbit model of lensectomy with intraocular lens insertion.

Children that undergo intraocular surgery have an exaggerated postoperative response compared to adults that can result in significant postoperative challenges and reduced post-operative visual acuity. Rabbits were used as an animal model for investigating aging differences, treatment options, and surgical techniques for anterior chamber surgical interventions due to similarities in anterior chamber size and decreasing postoperative response with age. In our study, juvenile and adult rabbits underwent lensectomy with intraocular lens (IOL) insertion to determine how ocular RNA transcripts and proteins change with age. Rabbits underwent lensectomy with IOL insertion, and aqueous humor (AH) was collected immediately prior to surgery and at the peak of the postoperative response on post-operative day 3. Proteins related to coagulation and inflammation were assessed using targeted mass spectrometry. In addition, the cornea and iris/ciliary body tissues were dissected, and transcripts analyzed using RNA sequencing. While clinically, juvenile rabbits have greater fibrin formation following intraocular surgery compared to older rabbits, this change does not appear to be related to relative abundance levels of coagulation and inflammatory proteins in the AH. Gene transcript levels from a variety of immune response and inflammatory pathways reflected significant increases when comparing operated to unoperated ocular tissues, indicating the significant impact that surgery has on each ocular structure. This work further advances our understanding of how the rabbit eye proteomic and transcriptomic changes in response to surgery with aging, as we seek to ultimately identify the mechanisms for the exaggerated postoperative responses after pediatric intraocular surgery.

The Inhibitory Effect of Blue Light on Phagocytic Activity by ARPE-19 Cells.

Chronic exposure of the retina to short wavelength visible light is a risk factor in pathogenesis of age-related macular degeneration. The proper functioning and survival of photoreceptors depends on efficient phagocytosis of photoreceptor outer segments (POS) by retinal pigment epithelium. The purpose of this study was to analyze the phagocytic activity of blue light-treated ARPE-19 cells, and to examine whether the observed effects could be related to altered levels of POS phagocytosis receptor proteins and/or to oxidation of cellular proteins and lipids. POS phagocytosis was measured by flow cytometry. Phagocytosis receptor proteins αv and ß5 integrin subunits and Mer tyrosine kinase (MerTK) were quantified by western blotting. The intact functional heterodimer αvß5 was quantified by immunoprecipitation followed by immunoblotting. Cellular protein and lipid hydroperoxides were analyzed by coumarin boronic acid probe and iodometric assay, respectively. Cell irradiation induced reversible inhibition of specific phagocytosis and transient reductions in phagocytosis receptor proteins. Full recovery of functional heterodimer was apparent. Significant photooxidation of cellular proteins and lipids was observed. The results indicate that transient inhibition of specific phagocytosis by blue light could be related to the reduction in phagocytosis receptor proteins. Such changes may arise from oxidative modifications of cell phagocytic machinery components.

Tissue Plasminogen Activator Effects on Fibrin Volume and the Ocular Proteome in a Juvenile Rabbit Model of Lensectomy.

Purpose: To investigate the use of tissue plasminogen activator (tPA) and its effects on the ocular proteome as a therapeutic intervention for postoperative inflammation and fibrin formation following intraocular lens (IOL) insertion in a juvenile rabbit model. Methods: Twenty-six rabbits, 6 to 7 weeks old, underwent lensectomy with IOL insertion. Following examination on day 3, 100 µL of either 25 µg of recombinant rabbit tPA or balanced salt solution (control) was injected into the anterior chamber. On postoperative day 4, rabbits underwent examination, and eyes were harvested and fixed for 9.4-Tesla magnetic resonance imaging (MRI). Three masked observers quantified fibrin scar volume using Horos Project software. Aqueous humor (AH) was collected immediately prior to surgery and on postoperative days 3 and 4. Proteins related to coagulation and inflammation were assessed in AH samples using targeted mass spectrometry via parallel reaction monitoring. Results: tPA significantly reduced the volume of fibrin 24 hours following administration compared with control eyes (0.560 mm3 vs. 3.29 mm3; P < 0.0001). Despite the reduced fibrin scar, proteins related to the coagulation and complement cascade were not significantly different following tPA injection. Conclusions: tPA may be a safe candidate for reduction of postoperative fibrin scarring after intraocular surgery. MRI can provide a quantitative value for fibrin volume changes. Translational Relevance: tPA is a candidate to treat ocular fibrin scarring. MRI can quantify the efficacy of treatments in future dose-response studies. Targeted mass spectrometry can provide critical data necessary to help decipher the effect on the abundance of targeted proteins following pharmacological intervention.

Quantitative proteomic analysis of aqueous humor after rabbit lensectomy reveals differences in coagulation and immunomodulatory proteins.

Compared to adults, children experience increased postoperative scarring and inflammation following intraocular surgery. While the underlying causes of the exaggerated immune response in children are not understood, proteins play key roles in postoperative scarring and wound healing processes. To identify and quantify proteins associated with the robust postoperative immune response, this study applied quantitative proteomics approaches to a juvenile rabbit model of lensectomy with intraocular lens (IOL) insertion. Twenty-six 6-7 week-old New Zealand white rabbits underwent unilateral portions of lensectomy with IOL insertion including: anterior chamber paracentesis, corneal incision with wound suture, lensectomy only, and lensectomy with IOL insertion. Aqueous humor was collected immediately prior and three days after each procedure. Semi-quantitative protein discovery was achieved by label-free quantitation using data dependent and data independent acquisition modes. Based on the discovery results, targeted quantitation by parallel reaction monitoring of 3 proteins of interest, fibrinogen-beta chain, transforming growth factor beta-2, and retinol binding protein 3, was used to confirm the observed quantitative trends. Total protein concentration levels increased with each progressive surgical step of lensectomy with IOL insertion. Proteins related to the complement and coagulation cascades were found to increase in relative abundance, while proteins related to ocular immunosuppression decreased in abundance following surgery. These data provide insights into the postoperative response by providing the first surgical step-wise views of the AH proteome before and after surgery. Overall, this work provides the foundation for future investigations targeting specific proteins for therapeutic interventions aimed at minimizing postoperative complications after pediatric intraocular surgery.

Sustained Anti-Vascular Endothelial Growth Factor Activity of Aflibercept (Eylea) After Storage in Polycarbonate Syringes Used for Intravitreal Injection: A Pathway to Safety and Efficiency.

Purpose: Aflibercept (Eylea™, Regeneron) is supplied in single-use glass vials along with 1 cc polycarbonate syringes. We sought to determine if storage of aflibercept for sustained periods within these syringes would result in loss of antivascular endothelial growth factor (anti-VEGF) activity. Methods: Aflibercept samples were drawn from commercially available glass vials into manufacturer-supplied 1-mL syringes and stored at 4°C. Anti-VEGF activity was assessed using enzyme-linked immunosorbent assays at the following storage durations: 0, 4, 9, 14, and 28 days. Frozen samples stored at -20°C for 28 and 56 days were also assayed. Also, a subset of aflibercept samples was stored and then diluted to 1:10 and progressively smaller concentrations and the assays repeated. Aggregation of aflibercept was tested using a dynamic light scattering assay. Results: There were no statistical differences in anti-VEGF activity among aflibercept samples of 1:1 or 1:10 dilution stored at either 4°C or -20°C at any of the storage intervals (P > 0.05). We also observed persistence of robust anti-VEGF activity for up to 14 days when diluted poststorage to 1:16,000, a concentration that would be expected after >7 vitreous half-lives within the eye (estimated at >50 days). No evidence of drug aggregation in specimens stored for 14 days was observed. Conclusions: Our findings support feasibility of prefilling and storage of aflibercept within manufacturer-supplied polycarbonate syringes for as long as 14 days before use under pharmacy-based sterile conditions, facilitating greater safety and efficiency in many clinics delivering anti-VEGF therapy.

Zeaxanthin and α-tocopherol reduce the inhibitory effects of photodynamic stress on phagocytosis by ARPE-19 cells.

Zeaxanthin and α-tocopherol have been previously shown to efficiently protect liposomal membrane lipids against photosensitized peroxidation, and to protect cultured RPE cells against photodynamic killing. Here the protective action of combined zeaxanthin and α-tocopherol was analyzed in ARPE-19 cells subjected to photodynamic (PD) stress mediated by rose Bengal (RB) or merocyanine-540 (MC-540) at sub-lethal levels. Stress-induced cytotoxicity was analyzed by the MTT assay. The peroxidation of membrane lipids was determined by HPLC-EC (Hg) measurements of cholesterol hydroperoxides using cholesterol as a mechanistic reporter molecule. The specific phagocytosis of FITC-labeled photoreceptor outer segments (POS) isolated from bovine retinas was measured by flow cytometry, and the levels of phagocytosis receptor proteins αv integrin subunit, ß5 integrin subunit and MerTK were quantified by Western blot analysis. Cytotoxicity measures confirmed that PD stress levels used for phagocytosis analysis were sub-lethal and that antioxidant supplementation protected against higher, lethal PD doses. Sub-lethal PD stress mediated by both photosensitizers induced the accumulation of 5α-OOH and 7α/ß-OOH cholesterol hydroperoxides and the addition of the antioxidants substantially inhibited their accumulation. Antioxidant delivery prior to PD stress also reduced the inhibitory effect of stress on POS phagocytosis and partially reduced the stress-induced diminution of phagocytosis receptor proteins. The use of a novel model system where oxidative stress was induced at sub-lethal levels enable observations that would not be detectable using lethal stress models. Moreover, novel observations about the protective effects of zeaxanthin and α-tocopherol on photodynamic damage to ARPE-19 cell membranes and against reductions in the abundance of receptor proteins involved in POS phagocytosis, a process essential for photoreceptor survival, supports the importance of the antioxidants in protecting of the retina against photooxidative injury.

Photic injury to cultured RPE varies among individual cells in proportion to their endogenous lipofuscin content as modulated by their melanosome content.

PURPOSE: We determined whether photic stress differentially impairs organelle motility of RPE lipofuscin and melanin granules, whether lethal photic stress kills cells in proportion to lipofuscin abundance, and whether killing is modulated by melanosome content. METHODS: Motility of endogenous lipofuscin and melanosome granules within the same human RPE cells in primary culture was quantified by real-time imaging during sublethal blue light irradiation. Cell death during lethal irradiation was quantified by dynamic imaging of the onset of nuclear propidium iodide fluorescence. Analyzed were individual cells containing different amounts of autofluorescent lipofuscin, or similar amounts of lipofuscin and a varying content of phagocytized porcine melanosomes, or phagocytized black latex beads (control for light absorbance). RESULTS: Lipofuscin granules and melanosomes showed motility slowing with mild irradiation, but slowing was greater for lipofuscin. On lethal irradiation, cell death was earlier in cells with higher lipofuscin content, but delayed by the copresence of melanosomes. Delayed death did not occur with black beads, suggesting that melanosome protection was due to properties of the biological granule, not simple screening. CONCLUSIONS: Greater organelle motility slowing of the more photoreactive lipofuscin granule compared to melanosomes suggests that lipofuscin mediates mild photic injury within RPE cells. With lethal light stress endogenous lipofuscin mediates killing, but the effect is cell autonomous and modulated by coincident melanosome content. Developing methods to quantify the frequency of individual cells with combined high lipofuscin and low melanosome content may have value for predicting the photic stress susceptibility of the RPE monolayer in situ.

Photosensitized oxidative stress to ARPE-19 cells decreases protein receptors that mediate photoreceptor outer segment phagocytosis.

PURPOSE: To determine whether previously shown photodynamic (PD)-induced inhibition of specific photoreceptor outer segment (POS) phagocytosis by ARPE-19 cells is associated with reductions in receptor proteins mediating POS phagocytosis, and if PD treatment with merocyanine-540 (MC-540) produces additional effects leading to its inhibition of nonspecific phagocytosis. METHODS: ARPE-19 cells preloaded with MC-540 or rose bengal (RB) were sublethally irradiated with green light. Phagocytosis of POS was measured by flow cytometry and POS receptor proteins (Mer tyrosine kinase receptor [MerTK] and integrin subunits αv and ß5) and ß-actin were quantified by Western blotting at 0.5 and 24 hours after irradiation, with comparison to samples from nonsensitized control cultures. The intact integrin heterodimer αvß5 was quantified by immunoprecipitation followed by blotting. The distribution of N-cadherin, ZO-1, and F-actin was visualized by fluorescence microscopy. RESULTS: Mild PD stress mediated by both photosensitizers that elicits no significant morphologic changes produces transient and recoverable reductions in MerTK. The individual αv and ß5 integrin subunits are also reduced but only partially recover. However, there is sufficient recovery to support full recovery of the functional heterodimer. Light stress mediated by MC-540 also reduced levels of actin, which is known to participate in the internalization of particles regardless of type. CONCLUSIONS: After PD treatment POS receptor protein abundance and phagocytosis show a coincident in time reduction then recovery suggesting that diminution in receptor proteins contributes to the phagocytic defect. The additional inhibition of nonspecific phagocytosis by MC-540-mediated stress may result from more widespread effects on cytosolic proteins. The data imply that phagocytosis receptors in RPE cells are sensitive to oxidative modification, raising the possibility that chronic oxidative stress in situ may reduce the efficiency of the RPE's role in photoreceptor turnover, thereby contributing to retinal degenerations.

Photoaging of human retinal pigment epithelium is accompanied by oxidative modifications of its eumelanin.

Although photodegradation of the retinal pigment epithelium (RPE) melanin may contribute to the etiology of age-related macular degeneration, the molecular mechanisms of this phenomenon and the structural changes of the modified melanin remain unknown. Recently, we found that the ratio of pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) to pyrrole-2,3,5-tricarboxylic acid (PTCA) is a marker for the heat-induced cross-linking of eumelanin. In this study, we examined UVA-induced changes in synthetic eumelanins to confirm the usefulness of the PTeCA/PTCA ratio as an indicator of photo-oxidation and compared changes in various melanin markers and their ratios in human melanocytes exposed to UVA, in isolated bovine RPE melanosomes exposed to strong blue light and in human RPE cells from donors of various ages. The results indicate that the PTeCA/PTCA ratio is a sensitive marker for the oxidation of eumelanin exposed to UVA or blue light and that eumelanin and pheomelanin in human RPE cells undergo extensive structural modifications due to the life-long exposure to blue light.

Oxidative stress increases HO-1 expression in ARPE-19 cells, but melanosomes suppress the increase when light is the stressor.

PURPOSE: Phagocytized melanosomes in ARPE-19 cells were previously shown to decrease susceptibility to oxidative stress induced by hydrogen peroxide treatment and increase stress due to light irradiation relative to cells containing control black latex beads. Here we asked whether differential expression of antioxidant enzymes in cells containing pigment granules could explain the outcomes. METHODS: ARPE-19 cells were loaded by phagocytosis with porcine RPE melanosomes or black latex beads (control particles). Heme oxygenase-1 (HO-1), HO-2, glutathione peroxidase (GPx), and catalase were quantified by Western blot analysis before and after treatment with sublethal hydrogen peroxide or blue light (400-450 nm). The stress was confirmed as sublethal by cell survival analysis using real-time quantification of propidium iodide fluorescence. RESULTS: Phagocytosis itself produced transient changes in protein levels of some antioxidant enzymes, but steady-state levels (7 days after phagocytosis) did not differ in cells containing melanosomes versus beads. Sublethal stress, induced by either hydrogen peroxide or light, had no effect on catalase or HO-2 in either particle-free or particle-loaded cells. In contrast, HO-1 protein was upregulated by treatment with both hydrogen peroxide and light. Particle content did not affect the HO-1 increase induced by hydrogen peroxide, but the increase induced by blue light irradiation was partially blocked in cells containing black beads and blocked even more in cells containing melanosomes. CONCLUSIONS: The results do not implicate differential antioxidant enzyme levels in stress protection by melanosomes against hydrogen peroxide, but they suggest a multifaceted role for melanosomes in regulating light stress susceptibility in RPE cells.

Melanosome-iron interactions within retinal pigment epithelium-derived cells.

Melanosomes were recently shown to protect ARPE-19 cells, a human retinal pigment epithelium (RPE) cell line, against oxidative stress induced by hydrogen peroxide. One postulated mechanism of antioxidant action of melanin is its ability to bind metal ions. The aim here was to determine whether melanosomes are competent to bind iron within living cells, exhibiting a property previously shown only in model systems. The outcomes indicate retention of prebound iron and accumulation of iron by granules after iron delivery to cells via the culture medium, as determined by both colorimetric and electron spin resonance analyses for bound-to-melanosome iron. Manipulation of iron content did not affect the pigment's ability to protect cells against H(2) O(2) , but the function of pigment granules within RPE cells should be extended beyond a role in light irradiation to include participation in iron homeostasis.

Dynamic analyses reveal cytoprotection by RPE melanosomes against non-photic stress.

PURPOSE: Isolated melanosomes are known to have antioxidant properties but whether the granules perform an antioxidant function within cells is unclear. The aim of this study was to determine whether retinal pigment epithelium (RPE) melanosomes are competent to protect cultured cells against non-photic oxidative stress induced by treatment with H(2)O(2). METHODS: Porcine melanosomes, either untreated or irradiated with visible light to simulate age-related melanin photobleaching, were introduced by phagocytosis into ARPE-19 cells. Cells were treated with H(2)O(2) using two delivery methods: as a pulse, or by continuous generation following addition of glucose oxidase to the medium. Cell survival in melanosome-containing cells was compared to survival in cells containing phagocytosed control latex beads using two real-time cell death assays. RESULTS: Following H(2)O(2) delivery by either method, greater resistance to critical concentrations of H(2)O(2) was seen for cells containing melanosomes than for cells containing beads. Melanosome-mediated protection manifested as a delay in the time of onset of cell death and a slower rate of cell death over time. Photobleaching diminished the stress resistance conferred by the pigment granules. Individual cells in co-cultures were differentially sensitive to oxidative stress depending upon their particle content. Additional features of the time course of the cell death response were revealed by the dynamic analyses conducted over hours post oxidant treatment. CONCLUSIONS: The results show, for the first time, that melanosomes perform a cytoprotective function within cultured cells by acting as an antioxidant. The outcomes imply that melanosomes perform functions within RPE cells aside from those related to light irradiation, and also suggest that susceptibility to ubiquitous pro-oxidizing agents like H(2)O(2) is partly determined by discrete features of individual RPE cells such as their granule content.

Intravitreally injected human immunoglobulin attenuates the effects of Staphylococcus aureus culture supernatant in a rabbit model of toxin-mediated endophthalmitis.

OBJECTIVE: To determine whether human immunoglobulin attenuates the toxic effects of Staphylococcus aureus culture supernatant in a rabbit model of endophthalmitis. METHODS: Immunoglobulin binding to products of S aureus strain RN4220 was tested by Western blot analysis using known toxins (beta-hemolysin and toxic shock syndrome toxin-1) and a concentrated culture supernatant containing S aureus exotoxins (pooled toxin). To induce endophthalmitis, pooled toxin was injected into the rabbit vitreous. For immunoglobulin treatment, immunoglobulin and pooled toxin were either mixed and injected simultaneously or immunoglobulin was injected immediately after or 6 hours after pooled toxin injection. Severity of endophthalmitis was graded according to a 9-day course with clinical examination (slitlamp biomicroscopy or indirect ophthalmoscopy) and evaluation of histologic sections. RESULTS: The toxic effects of pooled toxin were markedly reduced when immunoglobulin was mixed with pooled toxin and injected simultaneously. Delayed injection of immunoglobulin diminished its ability to reduce toxicity. Clinical and histologic signs of toxicity were partially attenuated when immunoglobulin was injected immediately after pooled toxin, but only minimal clinically detectable reductions in toxicity were observed when immunoglobulin injection was delayed for 6 hours. CONCLUSION: Pooled human immunoglobulin can attenuate the toxic intravitreal effects of a concentrated culture supernatant containing S aureus exotoxins. Clinical Relevance Immunoglobulin may represent a novel adjuvant in the treatment of bacterial endophthalmitis. To optimize the potential therapeutic benefit, maximizing the mixture of immunoglobulin with bacterial products and early intervention are likely to be important.

Antioxidants and ocular cell type differences in cytoprotection from formic acid toxicity in vitro.

Retinal photoreceptors and retinal pigment epithelial (RPE) cells are among the cell types that are sensitive to poisoning with methanol and its toxic metabolite formic acid. When exposed to formic acid in vitro, cultured cell lines from photoreceptors (661W) and the RPE (ARPE-19) were previously shown to accumulate similar levels of formate, but cytotoxic effects are greater in 661W cells. Here catalase and glutathione were analyzed in the two retinal cell lines to determine whether differences in these antioxidant systems contributed to cell-type-specific differences in cytotoxicity. Cells were exposed to formic acid (pH 6.8) in the culture medium in the presence or absence of a catalase activity inhibitor, 3-amino-1,2,4-triazole (AT), or a glutathione synthesis inhibitor, buthionine L-sulfoximine (BSO). Catalase protein, catalase enzyme activity, glutathione, glutathione peroxidase activity, cellular ATP, and cytotoxicity were analyzed. Compared to ARPE-19, 661W cells show lower antioxidant levels: 50% less glutathione, glutathione peroxidase and catalase protein, and 90% less catalase enzyme activity. In both cell types, formic acid treatment produced decreases in glutathione and glutathione peroxidase, and glutathione synthesis inhibition with BSO produced greater ATP depletion and cytotoxicity than formic acid treatment alone. In contrast, formate exposure produced decreases in catalase protein and activity in 661W cells, but increases in activity in ARPE-19. Treatment with the catalase inhibitor AT increased the formate sensitivity only of the ARPE-19 cells. ARPE-19 cells, therefore, may be less susceptible to formate toxicity due to higher levels of antioxidants, especially catalase, which increases on formate treatment and which has a significant cytoprotective effect for the RPE cell line.

Age-related changes in the photoreactivity of retinal lipofuscin granules: role of chloroform-insoluble components.

PURPOSE: Lipofuscin accumulates in human retinal pigment epithelium (RPE) cells with age and may be the main factor responsible for the increasing susceptibility of RPE to photo-oxidation with age. As the composition, absorption, and fluorescence of lipofuscin undergo age-related changes, the purpose of this study was to determine whether photoreactivity of lipofuscin granules also changes with the donor age. METHODS: To determine whether the photoreactivity of lipofuscin itself is age related, lipofuscin granules were isolated from human RPE and pooled into age groups. Photoreactivity was assessed by measuring action spectra of photo-induced oxygen uptake and photogeneration of reactive oxygen species. Separation of chloroform-soluble (ChS) and -insoluble (ChNS) components by Folch's extraction was used to determine the factors responsible for the age-related increase in lipofuscin photoreactivity. RESULTS: The observed rates of photo-induced oxygen uptake and photo-induced accumulation of superoxide-derived spin adducts indicated that when normalized to equal numbers of lipofuscin granules, aerobic photoreactivity of lipofuscin increased with age. Both ChS and ChNS mediated photogeneration of singlet oxygen, superoxide radical anion, and photo-oxidation of added lipids and proteins. Although both ChS and ChNS exhibited substantial photoreactivities, neither exhibited significant age-related changes when normalized to equal dry mass. In contrast, ChNS contents in lipofuscin granules significantly increased with aging. CONCLUSIONS: Aerobic photoreactivity of RPE lipofuscin substantially increases with aging. This effect may be ascribed to the increased content of insoluble components.

Formate, the toxic metabolite of methanol, in cultured ocular cells.

Methanol has neurotoxic actions on the human retina due to its metabolite, formic acid, which is a mitochondrial toxin. In methanol poisoned animals, morphologic changes were seen both in retinal photoreceptors and in cells of the underlying retinal pigment epithelium (RPE). Here the effects of formate exposure on the two retinal cell types were analyzed in more detail in vitro using photoreceptor (661W) and RPE (ARPE-19) cell lines. Cells were exposed for time courses from minutes to days to sodium formate at pH 7.4 or to formic acid at pH 6.8, to simulate the metabolic acidosis that accompanies methanol poisoning. Formate accumulation, cellular ATP, cytotoxicity (lactate dehydrogenase (LDH) release) and cell phenotype were analyzed. Formate accumulated with a similar biphasic pattern in both cell types, and to similar levels whether delivered as sodium formate or as formic acid. ATP changes with sodium formate treatment differed between cell types with only 661W cells showing a rapid (within minutes), transient ATP increase. The subsequent ATP decrease was earlier in 661W cells (6 h) than the ATP decrease in ARPE-19 cells (24 h), and although both cell types showed evidence of cytotoxicity, the effects were greater for 661W cells. Both cell types showed enhanced morphologic and biochemical changes with formic acid treatment including earlier and/or greater effects on ATP depletion and cytotoxicity; again effects were more pronounced in 661W cells. Formate therefore is toxic for both cell lines, with 661W cells exhibiting greater sensitivity. Medium pH also appears to play a significant role in formate toxicity in vitro.

Spectroscopic and morphological studies of human retinal lipofuscin granules.

The emission properties of ocular lipofuscin granules isolated from human retinal pigment epithelial cells are examined by using steady-state fluorescence spectroscopy and spectrally resolved confocal microscopy. The shape of the emission spectrum of a thick sample of lipofuscin granules dried on glass varies with excitation energy. The polarization of this emission is wavelength-dependent, exhibiting significant polarization near the excitation wavelength and becoming mostly depolarized over the majority of the emission spectrum. These results show that the yellow-emitting fluorophores [e.g., 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)] are excited as a result of energy transfer within the granules and therefore are not the dominant blue-absorbing chromophores within lipofuscin granules. Atomic force microscopy images show lipofuscin granules to be an aggregated structure. Bulk and in vivo emission measurements must therefore take into account the effect of Raleigh scattering. When corrected for scattering, the emission spectrum of a thick lipofuscin deposit or intracellular lipofuscin resembles that for A2E. The sum of the emission spectra of a collection of individual granules also resembles the emission spectrum of A2E, but the spectrum of individual granules varies significantly. This result suggests that the agreement between the emission spectra of lipofuscin and A2E is fortuitous, and the collective data indicate the presence of several blue-absorbing chromophores in lipofuscin and show A2E is not the dominant yellow-emitting fluorophore in many of the granules studied.

Loss of melanin from human RPE with aging: possible role of melanin photooxidation.

The pigment melanin, which is believed to play a photoprotective role, was quantified here in human RPE cells from donors of different age. Electron spin resonance (ESR) spectroscopy was shown to provide a quantitative measure of melanin and was used as a non-destructive measure of melanin content. Results indicated an age-related melanin loss in RPE cells, with melanin content diminishing 2.5-fold between the first and the ninth decade of life. To determine whether photo-oxidation may contribute to age-related changes in RPE melanin, RPE in human eyecups, isolated human and bovine RPE cells, purified melanin granules, or synthetic dopa melanin were irradiated with various wavelengths and intensities of visible light. Samples were analysed for changes in melanin content by ESR spectroscopy, and by absorption and emission spectrophotometry. The concentration of hydrogen peroxide was measured in some samples, and some human eyecups were examined by transmission electron microscopy. Irradiation of RPE in eyecups with intense visible light was found to produce a time-dependent photobleaching of melanosomes that was accompanied by the formation of hydrogen peroxide. Photobleaching of isolated RPE melanosomes and synthetic dopa melanin resulted in enhanced melanin fluorescence, as previously shown for melanin from aged donors by others, and significantly reduced ESR signal intensity, resembling the changes in melanin with aging observed here. We conclude that the content of melanin in RPE cells undergoes an age-related change to which photo-oxidation may contribute. This observation raises the question of whether age-related changes in melanin reduce the photoprotective role of the pigment in aging RPE cells.

Photoreactivity of aged human RPE melanosomes: a comparison with lipofuscin.

PURPOSE: To determine whether aging is accompanied by changes in aerobic photoreactivity of retinal pigment epithelial (RPE) melanosomes isolated from human donors of different ages, and to compare the photoreactivity of aged melanosomes with that of RPE lipofuscin. METHODS: Human RPE pigment granules were isolated from RPE cells pooled into groups according to the age of the donors. Photoreactivity was determined by blue-light-induced oxygen uptake and photogeneration of reactive oxygen species. Short-lived radical intermediates were detected by spin-trapping, hydrogen peroxide by an oxidase electrode, singlet oxygen by cholesterol assay, and lipid hydroperoxides by iodometric assay. RESULTS: Blue-light photoexcitation of melanosomes resulted in age-related increases in both oxygen uptake and the accumulation of superoxide anion spin adducts. The efficiencies of these processes, however, were still significantly lower than that induced by photoexcited lipofuscin. During irradiation of melanosomes, a substantial amount of oxygen was converted into hydrogen peroxide, whereas for lipofuscin, hydrogen peroxide accounted for not more than 3% of oxygen consumed. In contrast to lipofuscin, photoexcited melanosomes did not substantially increase the rate of oxidative reactions in the presence of polyunsaturated lipids or albumin. However, oxygen uptake was significantly elevated in the presence of ascorbate. Thus, the rate of photo-induced oxygen uptake in samples containing both ascorbate and melanosomes approached that observed in lipofuscin samples. CONCLUSIONS: Blue-light-induced photoreactivity of melanosomes increases with age, perhaps providing a source of reactive oxygen species and leading to depletion of vital cellular reductants, which, together with lipofuscin, may contribute to cellular dysfunction.