Effects of organic solvents on two retinal pigment epithelial lipofuscin fluorophores, A2E and all-trans-retinal dimer.

Gene and drug therapies are being developed to alleviate vision loss in patients with Stargardt's disease and age-related macular degeneration (AMD). To evaluate the therapeutic effects of these treatments, organic solvents are routinely used to extract and quantify bisretinoid lipofuscin constituents, such as N-retinylidene-N-retinyl-ethanolamine (A2E) and all-trans-retinal dimer (ATR-dimer). By high-performance liquid chromatography (HPLC), we found that A2E and ATR-dimer were both altered by tetrahydrofuran (THF) and chloroform, but were stable in dimethyl sulfoxide (DMSO) or methanol (MeOH). In addition, cyclohexane and ethanol (EtOH) did not alter ATR-dimer, whereas an alteration of A2E occurred in EtOH. On the basis of these findings, we designed processes II-IV, generated by modifications of process I, a routine method to measure bisretinoid compounds in vivo. Extra amounts of either ATR-dimer or A2E in mouse eyecups were released by processes II-IV versus process I. Efforts to clarify the effects of organic solvents on lipofuscin pigments are important because such studies can guide the handling of these fluorophores in related experiments.

Crystal structure of the globular domain of C1QTNF5: Implications for late-onset retinal macular degeneration.

Autosomal dominant late-onset retinal macular degeneration (L-ORMD) is caused by a single S163R mutation in the C1q and tumor necrosis factor-related protein 5 (C1QTNF5) gene. The C1QTNF5 gene encodes a secreted and membrane-associated protein involved in adhesion of retinal pigmented epithelial cells (RPE) to Bruch's membrane. The crystal structure of the trimeric globular domain of human C1QTNF5 at 1.34Å resolution reveals unique features of this novel C1q family member. It lacks a Ca²âº-binding site, displays a remarkable non-uniform distribution of surface electrostatic potentials and possesses a unique sequence (F181F182G183G184W185P186) that forms a hydrophobic plateau surrounded by Lys and Arg residues with a solvent cavity underneath. S163 forms a hydrogen bond with F182 in a hydrophobic area extending to the hydrophobic plateau. The pathogenic mutation S163R disrupts this hydrogen bonding and positively charges these hydrophobic areas. Thus, our analysis provides insights into the structural basis of the L-ORMD disease mechanism.

Association between various types of obesity and macular pigment optical density.

PURPOSE: To elucidate the association between macular pigment optical density (MPOD) and various types of obesity in the South-Indian population. PATIENTS AND METHODS: In total, 300 eyes of 161 healthy volunteers of South-Indian origin were studied. MPOD was measured psychophysically at 0.25°, 0.50°, 1.00°, and 1.75° eccentricities from fovea. Anthropometric measurements included waist circumference (WC) and waist-to-hip ratio (WHR) and body mass index (BMI). Using the WHO Expert Consultation guidelines, obesity was defined based on BMI alone (BMI ≥ 23 kg/m(2)), based on WC alone (WC ≥ 90 cm for men and ≥ 80 cm for women), and based on WHR alone (≥ 0.90 for men and ≥ 0.85 for women). Isolated generalized obesity was defined as increased BMI and normal WC. Isolated abdominal obesity was defined as increased WC and normal BMI. Combined obesity was defined as increased BMI and increased WC. RESULTS: Mean MPOD at all eccentricities was not significantly different between men and women. Mean MPOD values did not significantly differ in various types of obesity, when compared with the normal subjects. On subgroup analysis, in age group ≥ 60 years, mean MPOD values were significantly higher in subjects with obesity based on BMI (0.61 vs 0.41, P=0.036), obesity based on WHR (0.67 vs 0.41, P=0.007), and isolated generalized obesity (0.66 vs 0.41, P=0.045) in comparison with normal subjects at 0.25° eccentricity. CONCLUSION: We found lack of an association between MPOD and obesity in the South-Indian population. A similar finding was also noted on age group- and gender-wise analyses.

Comparison of tight junction protein expression in the ciliary epithelia of mouse, rabbit, cat and human eyes.

Tight junctions in the nonpigmented epithelium (NPE) of the ciliary processes and the iris vascular endothelium form the ocular blood aqueous barrier that prevents leakage of proteins, immune cells and non-immune cells of blood into the anterior chamber. We attempted to determine whether ultrastructural differences in tight junctions reported in earlier studies are reflected in the expression pattern of tight junction proteins (TJP) and whether the TJP in mice, rabbits and cats resemble those of humans. For immunohistochemistry, 10 µm thick cryosections were rehydrated in PBS and fixed in 50 mM ammonium chloride at room temperature. After rinses in PBS, the sections were incubated twice in 0.1% Triton X-100, 10% goat serum, specific primary antibody or in PBS. After rinses in PBS, the sections were incubated in FITC-conjugated secondary antibody. After rinses in PBS, the sections were mounted with Vectashield mounting medium with propidium iodide, examined and photographed using a confocal microscope. The expression patterns of TJP in ocular ciliary epithelium of human, rabbit, cat and mouse were similar. Occludin immunoreactivity was observed as a sharp line along the junction between pigmented epithelium (PE) and NPE, and along the apico-lateral surfaces of NPE. Very light staining of the ciliary stroma was observed in cat and mouse. Claudin-1 was expressed along the entire boundaries of NPE and was more distinct between PE and NPE in rabbit. The ciliary stroma showed faint staining in cat and mouse. ZO-1 showed staining between PE and NPE, and at the adjacent membrane. Moderate staining was seen in PE in cat and mouse, which suggests that claudin-1, occludin and ZO-1 are expressed along the junction between PE and NPE, and the apico-lateral border of NPE. Lack of major difference in the expression patterns among the different species is important for validating the use of rabbit, mouse and cat in studies of intraocular inflammation in humans.

Specific role of lymphatic marker podoplanin in retinal pigment epithelial cells.

Podoplanin is a small transmembrane glycoprotein widely known to be a marker for lymphatic endothelial cells. In this study, we identify a novel localization of podoplanin in the retinal pigment epithelium (RPE), a cellular monolayer critically involved in the visual process. Using a small interfering RNA (siRNA)-mediated gene silencing approach, we have also demonstrated, for the first time, that podoplanin depletion in human RPE cells leads to a marked reduction of cell aggregates and tight junctions. Additionally, the podoplanin-depleted cells also exhibit a significantly lower rate of proliferation. These data together indicate that podoplanin plays a crucial role in RPE cell functions. Further investigation on this factor may reveal novel mechanisms and therapeutic strategies for RPE-related eye diseases, such as proliferative retinopathy and age-related macular degeneration.

The value of measurement of macular carotenoid pigment optical densities and distributions in age-related macular degeneration and other retinal disorders.

There is increasing recognition that the optical and antioxidant properties of the xanthophyll carotenoids lutein and zeaxanthin play an important role in maintaining the health and function of the human macula. In this review article, we assess the value of non-invasive quantification of macular pigment levels and distributions to identify individuals potentially at risk for visual disability or catastrophic vision loss from age-related macular degeneration, and we consider the strengths and weaknesses of the diverse measurement methods currently available.

Pigmentos maculares / Macular pigments

A luteína e a zeaxantina são pigmentos amarelos que se localizam na mácula. Devido à sua localização, diminuem e filtram a quantidade de luz principalmente azul que chega aos fotorreceptores, atuam como antioxidantes e podem melhorar a qualidade visual. Esta é uma revisão do seu mecanismo de incorporação, ação, possíveis aplicações e conhecimento científico a respeito.

Lutein and Zeaxanthin are yellow pigments located at the macula. Because of your location macular pigments decrease and filter the amount of blue light that reach photoreceptors, protect the outer retina from oxidative stress and may improve the vision quality. This is a review regarding incorporation mechanism, function and knowledge update.

[Specification of reflectometric measurement complex to evaluate optic density of macular pigments and concentration of phototoxic chemicals in retina].

Suggestion is to specify reflectometric measurement complex based on digital multisensor imaginery fundus-camera, in order to evaluate optic density of macular pigments and concentration of phototoxic chemicals in human retina. The authors presented a review of role played by macular pigments (zeaxanthine and lutein) in human eye viability, analyzed yellow spot as a protective light filter against harmful effects of short-wave light, increasing optic image quality in human eye and responsible for colour vision. Role of evaluating the individual density of macular pigments was stressed as a forecasting efficient criterion of occupational selection in operators performing visual tasks of detection, distance and dimensions measurement for remote objects, monitoring the changeable circumstances.

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.

[Macular pigments]. / Pigmentos maculares.

Lutein and Zeaxanthin are yellow pigments located at the macula. Because of your location macular pigments decrease and filter the amount of blue light that reach photoreceptors, protect the outer retina from oxidative stress and may improve the vision quality. This is a review regarding incorporation mechanism, function and knowledge update.

Sub-retinal drusenoid deposits in human retina: organization and composition.

We demonstrate histologically sub-retinal drusenoid debris in three aged human eyes, two of them affected by age-related maculopathy. By postmortem fundus examination, the lesions were drusen-like, i.e., they were pale spots apparently at the level of the retinal pigment epithelium (RPE). Light and electron microscopy revealed aggregations of membranous debris, the principal constituent of soft drusen, in the sub-retinal space. Immunohistochemistry and confocal microscopy confirmed the presence of molecules typically associated with drusen (positive for unesterified cholesterol, apoE, complement factor H, and vitronectin) without evidence for molecules associated with photoreceptors (lectin-binding disaccharide bridges and opsins), Müller cells (glial fibrillary acid protein and cellular retinal binding protein, CRALPB), or RPE (CRALPB). The fact that a drusenoid material, sharing some markers with conventional drusen, can occur on opposite faces of the RPE, suggests deranged polarity of normally highly vectorial processes for basolateral secretion from RPE, and that overproduction of secreted materials and direction of secretion are independently specified processes. In the future, drusenoid sub-retinal debris might be more frequently revealed by emerging high-resolution imaging techniques.

VEGF-induced choroidal damage in a murine model of retinal neovascularisation.

BACKGROUND/AIMS: Photoreceptor-specific upregulation of vascular endothelial growth factor (VEGF) in a transgenic mouse model (Kimba) of retinal neovascularisation induces retinal vascular damage which appears similar to that in diabetic retinopathy. Here we have determined whether the choroidal vasculature is also affected in Kimba. METHODS: Kimba mice were assessed with fundus fluorescein angiography for mild, moderate or severe retinal vascular leakage prior to preparation of choroidal corrosion casts for quantitative analysis using scanning electron microscopy. VEGF was located immunohistochemically. RESULTS: Choroidal abnormalities included microaneurysms, constriction, shrinkage and dropout in the capillaries and tortuosity and loops in the arteries and veins which were similar to those observed in corrosion casts of the human choroid in diabetes. Similar to human diabetes, choroidal neovascularisation was not observed. The severity of choroidal damage correlated with the extent of retinal vascular leakage. In addition to the expected presence of VEGF in photoreceptors, VEGF was also detected in the pigment epithelium and choroid in the transgenic mice. CONCLUSION: We show that elevated retinal VEGF levels trigger pathophysiological changes in the choroid. We suggest that therapies to prevent vascular damage in diabetes must target both the retinal and choroidal vasculatures.

Retinal pigment epithelium lipofuscin proteomics.

Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein ( approximately 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E(2) adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.

Cadmium accumulation in the human retina: effects of age, gender, and cellular toxicity.

Tobacco smoking and aging are among the few factors linked to age-related macular degeneration (AMD), a major cause of blindness in the elderly. Recent studies indicate that cadmium (Cd), an environmental toxic trace metal, is approximately four-fold higher in the retinas of smokers compared to non-smokers. In this study, we determined the effects of age and gender on Cd accumulation in human retinal tissues, specifically the neural retina, retinal pigment epithelium (RPE), and choroid. Cadmium levels in cultured RPE cells or retinal tissues isolated from frozen donor eyes were measured using inductively coupled plasma mass spectrometry (ICP-MS) and graphite furnace atomic absorption spectrophotometry (GF-AAS). Cadmium uptake in cultured human RPE cells (ARPE-19) was also assessed using GF-AAS. Toxic effects of cadmium were determined from cell loss (measured as a decrease in cell density) and lactate dehydrogenase release (an indicator of membrane disruption). In "young" eyes (< 55 years) Cd was highest in the retinal pigment epithelium and lowest in the neural retina. Cd was higher in all tissues in aged eyes (>or=55 years) and was significantly higher in the neural retina and RPE in older females. Cultured RPE cells exposed to Cd showed altered cell morphology, decreased cell survival, elevated ROS levels and concentration-dependent disruption of membrane integrity. We conclude that cadmium is accumulated differently in the neural retinal and RPE of older men and women. The deleterious effects of Cd on RPE cells indicate that this environmental toxin is a potentially important factor in age-related retinal disease.

Semenogelins in the human retina: Differences in distribution and content between AMD and normal donor tissues.

The two cellular targets of interest in age-related macular degeneration (AMD) are the photoreceptors and the RPE. However, the mechanisms involved in AMD pathology are not yet fully understood. In the present report, we extend our previous studies on semenogelin proteins (Sgs) in normal human retina and compare these with the distribution in retinas from AMD donor eyes. Semenogelins I (SgI) and II (SgII) are the major structural protein components of semen coagulum, but have been recently found in non-genital tissues as well. Cryo and paraffin sections of human retina were processed for both immunofluorescence and DAB reaction with a specific antibody. The presence of SgI was analyzed in retina and RPE total lysates and SgI was detected by western blot in human retina and RPE. The intensity of immunoreactivity was significantly reduced in the AMD eyes. SgI is expressed in the normal human retina and in the retina of AMD donor eyes, where localization was detected in the photoreceptors and in a few ganglion cells. We find the distribution of SgI in the AMD retinas substantially lower than observed in normal retina. SgI localization to photoreceptors and the RPE suggests a possible function related to the ability of these cells to sequester zinc.

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.

Microscopic characterization of rat retinal progenitor cells.

A progenitor cell line was developed from a postnatal day 2 (P2) rat retina to study the effects of secreted proteins of the retinal pigment epithelium (RPE) on isolated retinal progenitor cells and markers for immature and differentiated retinal cells. Progenitor cells were cloned from a P2 explant grown in secreted proteins of cultured RPE cells. A cell line was cloned from a single progenitor cell. During the period of RPE-secreted protein stimulation the cells were transformed with the psi AE1A virus. Progenitor cells formed extensive processes when grown in serum and proliferated from the explant when grown in secreted proteins of RPE cells as demonstrated by bromodeoxyuridine (BrdU). All progenitor cells at early and late passages including a cloned cell line (D4) expressed Pax6, a transcription factor essential for eye development, which was verified by Western blotting. All cells expressed nestin, an early neuroepithelial cell marker. These two traits showed the immature character of these rat retinal progenitor cells. All cells expressed the intermediate filament protein vimentin, an intermediate filament protein. Interestingly, most progenitor cells grown in serum expressed the mature cell markers opsin, but few cells expressed glial fibrillary acidic protein (GFAP). The progenitor cells responded to proteins secreted by cultured RPE cells by forming large clusters, while cells grown in retinoic acid formed long thin processes that extended from a round cell body. These progenitor cells, following treatment with secreted proteins of the RPE, will be tested for their therapeutic effect in diseased rat retinas.

Hypoxia-inducible factor expression in human RPE cells.

BACKGROUND: Hypoxia-inducible factor (HIF) is a common transcription factor for many angiogenic proteins. Retinal pigment epithelial (RPE) cells are an important source of angiogenic factors in the retina. The expression of HIF, its regulation by proline hydroxylase (PHD) enzymes, and its downstream regulation of angiogenic factors like vascular endothelial growth factor (VEGF) and erythropoietin (EPO) was studied in RPE cells in order to determine some of the molecular mechanisms underlying ischaemic retinal disease. METHODS: ARPE-19 cells were cultured for various times under hypoxic conditions. Cellular HIF and PHD isoforms were analysed and quantified using western blot and densitometry. VEGF and EPO secreted into the media were assayed using enzyme-linked immunosorbent assay (ELISA). Messenger RNA (mRNA) was quantified using real-time quantitative reverse transcriptase polymerase chain reaction (qPCR). RNA interference was achieved using siRNA techniques. RESULTS: HIF-1 alpha was readily produced by ARPE-19 cells under hypoxia, but HIF-2 alpha and HIF-3 alpha could not be detected even after HIF-1 alpha silencing. HIF-1 alpha protein levels showed an increasing trend for the first 24 h while HIF-1 alpha mRNA levels fluctuated during this time. After 36 h HIF-1 alpha protein levels declined to baseline levels, a change that was coincident with a rise in both PHD2 and PHD3. Silencing HIF-1 alpha significantly decreased VEGF secretion. Significant production of EPO could not be detected at the protein or mRNA level. CONCLUSIONS: HIF-1 alpha appears to be the main isoform of HIF functioning in ARPE-19 cells. Under hypoxia, HIF-1 alpha levels are likely self-regulated by a feedback loop that involves both transcriptional and post-translational mechanisms. VEGF production by human RPE cells is regulated by HIF-1 alpha. EPO was not produced in significant amounts by RPE cells under hypoxic conditions, suggesting that other cells and/or transcription factors in the retina are responsible for its production.

Distribution and composition of esterified and unesterified cholesterol in extra-macular drusen.

More details about the distribution of esterified and unesterified cholesterol (EC, UC), abundant druse components, would inform models of druse biogenesis and new technologies for ocular imaging. From donors with grossly normal maculas (n=10, 66-86years), whose eyes were preserved in paraformaldehyde within 6h of death, extra-macular drusen encased with retinal pigment epithelium (RPE) were isolated manually. Cryosections of pelleted drusen, stained with filipin for UC and EC, were used to investigate filipin staining patterns within single drusen (n=193) and to quantify fluorescence (n=146). From lipid extracts of other drusen/RPE and RPE samples, total cholesterol (TC) and UC were determined by enzymatic fluorimetry. Drusen contained cores, basally located regions that were intensely bright when stained for UC or deeply dark when stained for EC; many were surrounded by concentric lamellae. Within the same cores, the EC-poor regions were significantly smaller (13.0mum) than UC-rich regions (17.1mum). Drusen with highly fluorescent EC-rich shells lacked UC-rich shells. Small spots representing lakes were visible only in drusen stained for EC. Some drusen had small, refractive spherical inclusions lacking both UC and EC. Of drusen examined, 32% had a UC-rich core, 35% had an EC-poor core, 31% had an EC-rich shell, 25% had EC-rich lakes, and 4-5% had UC-, EC-poor inclusions. Shells and cores occurred in significantly non-overlapping druse populations. The percentage of TC that was esterified ranged from 32-66% for drusen/RPE and 5-21% for RPE. The disposition of cholesterol in cores may reflect the activity of invading cellular process. The greater size of UC-rich cores relative to EC-poor cores may reflect a declining gradient of enzymatic activity with increased radial distance from the putative invaders. The relative sizes of sub-domains defined by cholesterol composition are compared to sub-domains detected in drusen by in vivo imaging methods.