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.

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.

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.

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.

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.

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.

Expression of molecular equivalent of hypothalamic-pituitary-adrenal axis in adult retinal pigment epithelium.

We have investigated expression of molecular elements of the hypothalamic-pituitary-adrenal (HPA) axis in the human retinal pigment epithelium (RPE) cells. The presence of corticotropin-releasing factor (CRF); urocortins I, II and III; CRF receptor type 1 (CRFR1); POMC and prohormone convertases 1 and 2 (PC1 and PC2) mRNAs were shown by RT-PCR; the protein products were detected by ELISA, western blot or immunocytochemical methods in an ARPE-19 cell line derived from an adult human donor. CRFR2 was below the level of detectability. The CRFR1 was functional as evidenced by CRF stimulation of cAMP and inositol triphosphate production as well as by ligand induction of transcriptional activity of inducible cis-elements cAMP responsive element (CRE), activator protein 1 responsive element (AP-1) and POMC promoter) in ARPE-19 using luciferase reporter assay. Immunoreactivities representative of CRF, pre-urocortin, CRFR1 receptor and ACTH were also detected in mouse retina by in situ immunocytochemistry. Finally, using RT-PCR, we detected expression of genes encoding four key enzymes participating in steroids synthesis (CYP11A1, CYP11B1, CYP17 and CYP21A2) and showed transformation of progesterone into cortisol-immunoreactivity in cultured ARPE-19 cells. Therefore, we suggest that ocular tissue expresses CRF-driven signalling system that follows organisational structure of the HPA axis.

Restoration of vision in RPE65-deficient Briard dogs using an AAV serotype 4 vector that specifically targets the retinal pigmented epithelium.

Previous studies have tested gene replacement therapy in RPE65-deficient dogs using recombinant adeno-associated virus 2/2 (rAAV2/2), -2/1 or -2/5 mediated delivery of the RPE65 gene. They all documented restoration of dark- and light-adapted electroretinography responses and improved psychophysical outcomes. Use of a specific RPE65 promoter and a rAAV vector that targets transgene expression specifically to the RPE may, however, provide a safer setting for the long-term therapeutic expression of RPE65. Subretinal injection of rAAV2 pseudotyped with serotype 4 (rAAV2/4) specifically targets the RPE. The purpose of our study was to evaluate a rAAV2/4 vector carrying a human RPE65cDNA driven by a human RPE65 promoter, for the ability to restore vision in RPE65-/- purebred Briard dogs and to assess the safety of gene transfer with respect to retinal morphology and function. rAAV2/4 and rAAV2/2 vectors containing similar human RPE65 promoter and cDNA cassettes were generated and administered subretinally in eight affected dogs, ages 8-30 months (n = 6 with rAAV2/4, n = 2 with rAAV2/2). Although fluorescein angiography and optical coherence tomography examinations displayed retinal abnormalities in treated retinas, electrophysiological analysis demonstrated that restoration of rod and cone photoreceptor function started as soon as 15 days post-injection, reaching maximal function at 3 months post-injection, and remaining stable thereafter in all animals treated at 8-11 months of age. As assessed by the ability of these animals to avoid obstacles in both dim and normal light, functional vision was restored in the treated eye, whereas the untreated contralateral eye served as an internal control. The dog treated at a later age (30 months) did not recover retinal function or vision, suggesting that there might be a therapeutic window for the successful treatment of RPE65-/- dogs by gene replacement therapy.

Shroom2 (APXL) regulates melanosome biogenesis and localization in the retinal pigment epithelium.

Shroom family proteins have been implicated in the control of the actin cytoskeleton, but so far only a single family member has been studied in the context of developing embryos. Here, we show that the Shroom-family protein, Shroom2 (previously known as APXL) is both necessary and sufficient to govern the localization of pigment granules at the apical surface of epithelial cells. In Xenopus embryos that lack Shroom2 function, we observed defects in pigmentation of the eye that stem from failure of melanosomes to mature and to associate with the apical cell surface. Ectopic expression of Shroom2 in naïve epithelial cells facilitates apical pigment accumulation, and this activity specifically requires the Rab27a GTPase. Most interestingly, we find that Shroom2, like Shroom3 (previously called Shroom), is sufficient to induce a dramatic apical accumulation of the microtubule-nucleating protein gamma-tubulin at the apical surfaces of naïve epithelial cells. Together, our data identify Shroom2 as a central regulator of RPE pigmentation, and suggest that, despite their diverse biological roles, Shroom family proteins share a common activity. Finally, because the locus encoding human SHROOM2 lies within the critical region for two distinct forms of ocular albinism, it is possible that SHROOM2 mutations may be a contributing factor in these human visual system disorders.

Glutamate acts at NMDA receptors on fresh bovine and on cultured human retinal pigment epithelial cells to trigger release of ATP.

The photoreceptors lie between the inner retina and the retinal pigment epithelium (RPE). The release of glutamate by the phototoreceptors can signal changes in light levels to inner retinal neurons, but the role of glutamate in communicating with the RPE is unknown. Since RPE cells are known to release ATP, we asked whether glutamate could trigger ATP release from RPE cells and whether this altered cell signalling. Stimulation of the apical face of fresh bovine RPE eyecups with 100 mum NMDA increased ATP levels more than threefold, indicating that both receptors for NMDA and release of ATP occurred across the apical membrane of fresh RPE cells. NMDA increased ATP levels bathing cultured human ARPE-19 cells more than twofold, with NMDA receptor inhibitors MK-801 and d-AP5 preventing this release. Blocking the glycine site of the NMDA receptor with 5,7-dichlorokynurenic acid prevented ATP release from ARPE-19 cells. Release was also blocked by channel blocker NPPB and Ca(2+) chelator BAPTA, but not by cystic fibrosis transmembrane conductance regulator (CFTR) blocker glibenclamide or vesicular release inhibitor brefeldin A. Glutamate produced a dose-dependent release of ATP from ARPE-19 cells that was substantially inhibited by MK-801. NMDA triggered a rise in cell Ca(2+) that was blocked by MK-801, by the ATPase apyrase, by the P2Y(1) receptor antagonist MRS2179 and by depletion of intracellular Ca(2+) stores with thapsigargin. These results suggest that glutamate stimulates NMDA receptors on the apical membrane of RPE cells to release ATP. This secondary release can amplify the glutaminergic signal by increasing Ca(2+) inside RPE cells, and might activate Ca(2+)-dependent conductances. The interplay between glutaminergic and purinergic systems may thus be important for light-dependent interactions between photoreceptors and the RPE.

Overexpression of FasL in retinal pigment epithelial cells reduces choroidal neovascularization.

Choroidal neovascularization (CNV) is responsible for the severe visual loss in age-related macular degeneration. CNV formation is considered to be due to an imbalance between pro- and antiangiogenic factors that lead to neovascular growth from the choriocapillaris into the subretinal space. To define whether FasL overexpression in retinal pigment epithelial cells (RPE) can inhibit choroidal neovascularization through Fas-FasL-mediated apoptosis, we examined the role of this pathway in a mouse model of laser-induced choroidal neovascularization. FasL was expressed in the retinal pigment epithelium of transgenic mice. Polymerase chain reaction (PCR), immunoblot, and immunohistochemistry confirmed that the transgene FasL was specifically expressed in RPE. The established laser model was used to induce choroidal neovascularization (CNV) in wild-type (WT) and transgenic mice. CNV formation was compared with respect to fluorescein angiographic leakage (at days 0 and 14 after laser injury) and histological appearance. The lesions were assessed on RPE-choroidal flatmounts after CD31-labeling and with confocal microscopy after perfusion with rhodamine-labeled concanavalin A (Con A). Apoptosis was quantified by TUNEL positivity and caspase activation. FasL mRNA and protein were highly expressed in the RPE of the transgenic mice before and after laser photocoagulation. In contrast, FasL was only weakly expressed in the RPE layer of WT C57BL/6J mice. While ruptures of Bruch's membrane and CNV formation were observed histologically two weeks after laser photocoagulation in transgenic as well as control eyes, the shape and size of CNV lesions were reduced in the transgenic mice. The area of leakage was decreased by 70% in FasL transgenic mice compared with WT mice (P<0.005). The number of TUNEL-positive cells was greater in FasL-overexpressing mice and correlated with the expression of activated caspases. Th expression of other antiangiogenic factors such as PEDF remained unchanged. The specific overexpression of FasL in RPE layer reduced CNV formation in our laser model. Our results strongly point to the FasL-Fas pathway as a potential therapeutic target in controlling pathological choroidal neovascularization.

Differential expression of retinal pigment epithelium (RPE) IP-10 and interleukin-8.

Interferon-gamma induced protein of 10 kDa (IP-10) is a C-X-C chemokine that attracts T lymphocytes and inhibits angiogenesis. In this study, we investigated the expression of IP-10 by human retinal pigment epithelial cells (HRPE) and compared IP-10 expression to that of interleukin-8 (IL-8), which is a leukocytic chemoattractant and pro-angiogenic factor. Cultured HRPE cells were incubated with either IL-1 beta (0.2-20 ng/ml) or tumor necrosis factor (TNF)-alpha (0.2-20 ng/ml) alone or in combination with interferon-gamma (IFN-gamma) (1000 U/ml). HRPE cells were also incubated with: (1) media conditioned by activated human T lymphocytes (CM), or (2) the same CM treated with neutralizing antibodies to IL-1, TNF, and/or IFN-gamma. IL-8 and IP-10 protein levels were measured by ELISA and mRNA levels by Northern blot analysis of HRPE cells. HRPE cells produced very high levels of IP-10 in response to either IL-1 beta/IFN-gamma, TNF-alpha/IFN-gamma or CD3-activated T-lymphocyte CM. The levels of IP-10 were at least tenfold higher (p<.001) than IL-8 measured in the same samples. Neutralizing antibodies to TNF and IFN-gamma, but not to IL-1, abrogated the ability of the CD3-activated T lymphocytes CM to induce HRPE IP-10 (p<.001). HRPE cells produce differential levels of IP-10 and IL-8 in response to various combinations of recombinant and T-lymphocyte-secreted pro-inflammatory cytokines. This may be important in evolving inflammatory and angiogenic ocular responses.

Expression of endostatin in human choroidal neovascular membranes secondary to age-related macular degeneration.

Endostatin is an endogenous angiogenesis inhibitor which requires E-selectin for its antiangiogenic activity. The aim of this study was to investigate the expression of endostatin in human choroidal neovascular membranes (CNV) secondary to age-related macular degeneration (AMD) with regard to vascularization and proliferative activity. An interventional case series of 36 patients who underwent removal of CNV were retrospectively investigated. Thirty-six CNV were analyzed by light microscopic immunohistochemistry for the expression of CD34 (endothelial cells, EC), CD105 (activated EC), Ki-67 (cell proliferation), Cytokeratin 18 (epithelial cells), VEGF (vascular endothelial growth factor), E-selectin and endostatin. Donor eyes (n=7) including one with AMD were used as controls. Endostatin immunoreactivity was present in choroidal vessels of five as well as in the retinal pigment epithelium (RPE)-Bruch's membrane complex of two donor eyes without AMD. In one eye with AMD, endostatin was detected in RPE, Bruch's membrane and choroidal vessels. Ninety-two percent (33/36) of CNV disclosed endostatin staining. RPE-Bruch's membrane complex, choroidal vessels and stroma were positive in 50% (18/36), 72% (26/36), and 78% (28/36) of the membranes, respectively. Both control eyes and CNV expressed all the investigated markers except E-selectin being positive only in membranes. Endostatin, an endogenous angiogenesis inhibitor, is expressed in CNV and its therapeutic up-regulation may be a new strategy in the treatment of neovascular AMD.

Proteomic analysis of mature melanosomes from the retinal pigmented epithelium.

The protein content of melanosomes in the retinal pigment epithelium (RPE) was analyzed by mass spectrometry. More than 100 proteins were found to be common to two out of three variations of sample preparation. Some proteins normally associated with other organelles were detected. Several lysosomal enzymes were detected, with the presence of cathepsin D confirmed by immunoelectron microscopy, thus supporting the previously suggested notion that melanosomes may contribute to the degradation of ingested photoreceptor outer segment disks.

Fas-ligand is stored in secretory lysosomes of ocular barrier epithelia and released with microvesicles.

Previously we described the release of hr44 from the ciliary epithelium to coincide with the loss of the late endosomal/lysosomal marker protein CD63 in mildly inflamed rat eyes. We showed that both proteins are released with microvesicles into the supernatant of cultured retinal pigmented epithelial cells (ARPE-19). Here we wish to determine whether there is a concomitant loss of fas-ligand (FasL) in vivo and whether ocular epithelial cells have secretory lysosomes similar to T cells, from where FasL and hr44 could derive. FasL plays an important role in immunity, immune cell homeostasis and in the maintenance of immune privilege in the eye. However the mode of release of FasL from ocular epithelial cells or its activity in the eye is not fully understood. In normal rat eyes, FasL was detected in the epithelia of the iris and ciliary body and in the anterior region of the retinal pigmented epithelium. FasL is expressed constitutively and is associated with vesicular structures in the normal ciliary epithelium but is not detectable in the ciliary epithelium of inflamed eyes. In contrast, the posterior RPE, which under normal conditions is negative for FasL and hr44 showed strong staining for both molecules in areas adjacent to sub-retinal inflammatory infiltrates. Immunofluorescence and Western blot analysis indicated that cultured ARPE-19 cells express both the soluble and membrane form of FasL. The intracellular concentration of FasL was significantly increased in cells grown in presence of interferon (INF)-gamma. The microvesicles released by cultured ARPE-19 cells and previously shown to be positive for hr44 and CD63 are also positive for membrane FasL. Expression of a recombinant fluorescent construct of FasL together with immuno-staining for CD63 demonstrated that FasL localises to the endocytic compartment of ARPE-19 cells and of melanoma cells (positive control). In cells with lysosomes devoid of specialised secretory functions (e g. HeLa cells) recombinant FasL localised to the cell membrane, demonstrating that RPE cells have secretory lysosomes. We suggest that ocular epithelial cells release soluble FasL and the membrane form of FasL with vesicles. Both forms may contribute in different ways to the effectiveness of the ocular immune response and immune privilege.

Pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in aged human choroid and eyes with age-related macular degeneration.

The purpose of this study was to examine the localization and relative levels of vascular endothelial growth factor (VEGF; an angiogenic factor) and pigment epithelium-derived factor (PEDF; an antiangiogenic factor) in aged human choroid and to determine if the localization or their relative levels changed in age-related macular degeneration (AMD). Ocular tissues were obtained from eight aged control donors (age range, 75-86 years; mean age, 79.8 years) with no evidence or history of chorioretinal disease and from 12 donors diagnosed with AMD (age range, 61-105 years; mean age, 83.9 years). Tissues were cryopreserved and streptavidin alkaline phosphatase immunohistochemistry was performed with rabbit polyclonal anti-human VEGF and rabbit polyclonal anti-human PEDF antibodies. Binding of the antibodies was blocked by preincubation of the antibody with an excess of recombinant human PEDF or VEGF peptide. Choroidal blood vessels were identified with mouse anti-human CD-34 antibody in adjacent tissue sections. Three independent observers graded the immunohistochemical reaction product. The most prominent sites of VEGF and PEDF localization in aged control choroid were RPE-Bruch's membrane-choriocapillaris complex including RPE basal lamina, intercapillary septa, and choroidal stroma. There was no significant difference in immunostaining intensity and localization of VEGF and PEDF in aged control choroids. The most intense VEGF immunoreactivity was observed in leukocytes within blood vessels. AMD choroid had a similar pattern and intensity of VEGF immunostaining to that observed in aged controls. However, PEDF immunoreactivity was significantly lower in RPE cells (p=0.0073), RPE basal lamina (p=0.0141), Bruch's membrane (p<0.0001), and choroidal stroma (p=0.0161) of AMD choroids. The most intense PEDF immunoreactivity was observed in disciform scars. Drusen and basal laminar deposits (BLDs) were positive for VEGF and PEDF. In aged control subjects, VEGF and PEDF immunostaining was the most intense in RPE-Bruch's membrane-choriocapillaris complex. In AMD, PEDF was significantly lower in RPE cells, RPE basal lamina, Bruch's membrane and choroidal stroma. These data suggest that a critical balance exists between PEDF and VEGF, and PEDF may counteract the angiogenic potential of VEGF. The decrease in PEDF may disrupt the balance and be permissive for the formation of choroidal neovascularization (CNV) in AMD.

Experimental autoimmune uveitis induced by immunization with retinal pigment epithelium-specific 65-kDa protein peptides.

PURPOSE: To investigate the pathogenic potential and sites of retinal pigment epithelium-specific 65-kDa protein (RPE65) for inducing experimental autoimmune uveitis (EAU) in Lewis rats. METHODS: Twenty-six peptides were chemically synthesized based on the amino acid sequences of human RPE65. These peptides spanned the entire RPE65 sequence. Each peptide was injected into a footpad and the peritoneum of Lewis rats. The eyes were examined by slit-lamp biomicroscopy, and the findings were correlated with the histological findings. The serum antibody titer and lymphocyte reactivity against each peptide was also determined by enzyme-liked immunosorbent assay (ELISA) and lymphocyte proliferation assay, respectively. RESULTS: Active immunization of rats resulted in the induction of EAU with 14 (3 severe and 11 mild) of the 26 peptides. The clinical course of the EAU was similar to that induced by the injection of retinal antigens such as S-antigen or inter-photoreceptor retinoid binding protein (IRBP). However, the histopathologic changes differed from the EAU induced by these retinal antigens. The inflammation was induced mainly from the retinal pigment epithelium (RPE) and the choroid, while the retina was relatively well-preserved except for some granulomatous changes adjacent to the RPE. CONCLUSIONS: Active immunization with peptides making up RPE65 will induce EAU. RPE65 has multiple EAU-inducing sites for Lewis rats.

Vascular endothelial growth factor expression and secretion by retinal pigment epithelial cells in high glucose and hypoxia is protein kinase C-dependent.

Retinal pigment epithelial (RPE) cells express vascular endothelial growth factor (VEGF) in response to high glucose or hypoxia. We hypothesised that VEGF expression and secretion by RPE cells in high glucose and hypoxia are regulated by protein kinase C (PKC). Primary cultured RPE cells from Sprague-Dawley rats were growth-arrested for 48 hr in 0.5% FBS in 5.6 or 30 mm D-glucose. Cells were exposed to hypoxic conditions (<1% O(2), 5% CO(2)) for the last 15-18 hr of growth-arrest. PKC -alpha, -beta(1), -delta, -epsilon, and -zeta were expressed by RPE cells and exposure to high glucose for 48 hr had no effect on expression as demonstrated by Western immunoblotting. High glucose, hypoxia or VEGF stimulated translocation of a number of the PKC isozymes to the membrane or particulate fractions implying activation. In response to high glucose or acute phorbol myristate acetate (PMA) stimulation, VEGF mRNA analysed by RT-PCR was increased. Intracellular VEGF protein identified by immunoblotting and confocal immunofluorescence imaging was significantly increased by high glucose, hypoxia or acute PMA stimulation. Calphostin C or a specific inhibitor of PKC-zeta prevented high glucose-stimulated VEGF expression in high glucose. VEGF secretion, as measured by ELISA in the culture medium, was enhanced in hypoxia but not in high glucose. Following exposure of RPE cells to PMA for 24 hr, PKC-delta was significantly down regulated, whereas PKC-alpha, -beta, -epsilon and -zeta remained unchanged. Secretion of VEGF in normal or high glucose, or hypoxia was significantly reduced following treatment with PMA for 24 hr but not with the PKC-zeta inhibitor. We conclude that in high glucose and hypoxia PKC isozymes are activated and are necessary for VEGF expression. Secretion of VEGF is enhanced in hypoxia and appears to be regulated by PKC-delta. RPE cells may contribute to the pathogenesis of retinopathy caused by high glucose and hypoxia through the expression and secretion of VEGF that are regulated by PKC isozymes.

Inhibition of p53 by lentiviral mediated shRNA abrogates G1 arrest and apoptosis in retinal pigmented epithelial cell line.

We silenced p53 gene expression in ARPE-19, a human retinal pigmented epithelial cell line using RNA interference. The effect of silencing the p53 gene in proliferating ARPE-19 cells was studied. Four short hairpin RNAs (shRNAs) targeting different regions of human p53 mRNA were delivered individually into ARPE-19 cells using lentiviral vector to produce stable cell lines. p53 mRNA and protein levels were reduced to varying extents in the four shRNA-transduced ARPE-19 cell lines. The cell line that showed greatest reduction (85-90%) of p53 expression showed decreased p21 promoter activation after DNA damage with camptothecin, etoposide and MMS. Whereas treatment of wild type ARPE-19 cells with camptothecin resulted in apoptosis, silencing p53 expression increased their survival. Cell cycle analyses indicated that irradiation resulted in a G(1) arrest in ARPE-19 cells, and that the arrest was significantly reduced in p53-silenced cells. Thus, p53 plays a central role in the response of ARPE-19 cells to DNA damaging agents that act via different mechanisms. Additionally, ARPE-19 cells with reduced p53 expression behave similar to tumor cell lines with mutated or non-functional p53. The present data demonstrate the utility of lentiviral vectors to create stable isogenic cell lines with reduced expression of a specific gene, thereby permitting the study of the function of a gene, the pathways controlled by it, and the effect of therapeutics on a cell with altered genetic makeup in a pair-wise fashion.

Mechanisms for the induction of HNE- MDA- and AGE-adducts, RAGE and VEGF in retinal pigment epithelial cells.

Pathological features of age-related macular degeneration such as the formation of extracellular deposits and neovascularization are frequently viewed as outcomes of compromising processes within retinal pigment epithelial cells, but the initiating circumstances are poorly understood. Here we tested the hypothesis that photooxidation events initiated by A2E, a blue light-excitable aging fluorophore of the retinal pigment epithelium, can set the stage for altered cellular signaling and changes in the expression of genes that can impact the extracellular milieu. Proteins modified by lipid peroxidation products (4-hydroxynonenal; malondialdhyde) and advanced glycation end products were detected at sites of blue light irradiation both in association with the cultured A2E-laden retinal pigment epithelial cells and within the fibronectin substrate on which the cells were grown. RAGE, the cell surface receptor that transduces the effects of advanced glycation end products, was also upregulated, and RAGE expression co-localized with the deposition of advanced glycation end products. Blue light triggered alterations in gene expression was also evidenced by elevations in both transcripts and protein for vascular endothelial growth factor, a potent angiogenic and permeability-enhancing factor. These findings indicate that cell associated and extracellular modification of proteins by lipid peroxidation products and advanced glycation end products together with increased expression of RAGE and vascular endothelial growth factor may be induced consequent to blue light illumination of A2E-burdened retinal pigment epithelial cells. Thus, photooxidative events that are not an immediate threat to retinal pigment epithelial cell viability may nevertheless elicit sustained perturbation that could ultimately alter neighboring tissues and impact retinal pigment epithelial cell function.