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As the retinal pigment epithelium (RPE) ages, a number of structural changes occur, including loss of melanin granules, increase in the density of residual bodies, accumulation of lipofuscin, accumulation of basal deposits on or within Bruch’s membrane, formation of drusen (between the basal lamina of the RPE and the inner collagenous layer of Bruch’s membrane), thickening of Bruch’s membrane, microvilli atrophy and disorganization of the basal infoldings. Although these changes are well known, the basic mechanisms involved in them are frequently poorly understood. These age-related changes progress slowly and vary in severity in different individuals. These changes are also found in age-related macular degeneration (AMD), a late onset disease that severely impacts the RPE, but they are much more pronounced than during normal aging. However, the changes in AMD lead to severe loss of vision. Given the many supporting functions which the RPE serves for the retina, it is important to decipher the age-related changes in this epithelium in order to understand age-related changes in vision.

Purpose

A cardinal pathological feature of age-related macular degeneration (AMD) is the deposition of extracellular material between the retinal pigment epithelium (RPE) and Bruch's membrane, pathologically described as sub-RPE deposits. Both the presence and local organization of these deposits contribute to the clinical manifestations of AMD, including localized deposits clinically recognized as drusen. The biogenesis of sub-RPE deposits remains elusive. This work explores the pathological processes of sub-RPE deposit formation.

Methods

Matrigel was injected to the subretinal space of rats to create an amorphous deposit. Tissue sections were examined by light or confocal microscopy.

Results

In the presence of the subretinal deposit of Matrigel, RPE cells leave Bruch's membrane to migrate toward photoreceptors and then form a new layer between the deposit and photoreceptors, resulting in RPE translocation. The new RPE layer displaces the deposit to the sub-RPE location and therefore it becomes a sub-RPE deposit. The RPE mobilization requires the presence of photoreceptors. Bruch's membrane devoid of RPE attachment becomes vulnerable to invasion by new blood vessels from the choroid.

Conclusions

Our work supports a novel model of sub-RPE deposit formation in which excessive material first accumulates in the subretinal space, disrupting the physical contact between RPE cells and photoreceptors. To restore the contact, RPE cells migrate toward photoreceptors and form a new layer. The subretinal material is consequently displaced to the sub-RPE location and becomes sub-RPE deposit. Our data also provide evidence that the presence of sub-RPE deposit is sufficient to induce choroidal neovascularization to penetrate Bruch's membrane.

PURPOSE

To describe the features of intraretinal retinal pigment epithelium (RPE) migration documented on a prototype spectral domain high speed, ultrahigh resolution optical coherence tomography (OCT) device in a group of patients with early to intermediate dry age-related macular degeneration (AMD). To correlate intraretinal RPE migration on OCT to RPE pigment clumping on fundus photographs.

DESIGN

Retrospective, non-comparative, non-interventional case series.

PARTICIPANTS

Fifty-five eyes of 44 patients seen at the New England Eye Center between December 2007 and June 2008 with early to intermediate dry AMD.

METHODS

3D OCT scan sets from all patients were analyzed for presence of intraretinal RPE migration, defined as small discreet hyper-reflective and highly-backscattering lesions within the neurosensory retina. Fundus photographs were also analyzed to determine the presence of RPE pigment clumping, defined as black-colored, often spiculated areas of pigment clumping within the macula. OCT en face images were correlated with fundus photographs to demonstrate correspondence of intraretinal RPE migration on OCT and RPE clumping on fundus photo.

MAIN OUTCOME MEASURES

Drusen, Dry AMD, intraretinal RPE migration, RPE pigment clumping.

RESULTS

54.5% of eyes (61.4% of patients) demonstrated intraretinal RPE migration on OCT scans. 56.4% of the fundus photographs demonstrated RPE pigment clumping. All eyes with intraretinal RPE migration on OCT had corresponding RPE pigment clumping on fundus photographs. RPE pigment migrated most frequently into the outer nuclear layer (66.7% of eyes) and less frequently into more anterior retinal layers. Intraretinal RPE migration mainly occurred above areas of drusen (73.3% of eyes).

CONCLUSIONS

The appearance of intraretinal RPE migration on OCT is a common occurrence in early to intermediate dry AMD, occurring in 54.5% of eyes or 61.4% of patients. The area of intraretinal RPE migration on OCT always correlated to areas of pigment clumping on fundus photography. Conversely, all but one eye with RPE pigment clumping on fundus photography also had areas of intraretinal RPE migration on OCT. The high incidence of intraretinal RPE migration observed above areas of drusen suggests that drusen may play physical and catalytic roles in facilitating intraretinal RPE migration in dry AMD patients.

Summary

Vertebrate retinal pigment epithelium (RPE) cells are derived from the multipotent optic neuroepithelium, develop in close proximity to the retina, and are indispensible for eye organogenesis and vision. Recent advances in our understanding of RPE development provide evidence for how critical signaling factors operating in dorso-ventral and distal-proximal gradients interact with key transcription factors to specify three distinct domains in the budding optic neuroepithelium: the distal future retina, the proximal future optic stalk/optic nerve, and the dorsal future RPE. Concomitantly with domain specification, the eye primordium progresses from a vesicle to a cup, RPE pigmentation extends towards the ventral side, and the future ciliary body and iris form from the margin zone between RPE and retina. While much has been learned about the molecular networks controlling RPE cell specification, key questions concerning the cell proliferative parameters in RPE and the subsequent morphogenetic events still need to be addressed in greater detail.

Purpose

The vertebrate retina develops from the center to the periphery. In amphibians and fish the retinal margin continues to proliferate throughout life, resulting in retinal expansion. This does not happen in mammals. However, some mammalian peripheral retinal pigment epithelial (RPE) cells continue to divide, perhaps as a vestige of this mechanism. The RPE cells are adjacent to the ciliary margin, a known stem cell source. Here we test the hypothesis that peripheral RPE is fundamentally different from central RPE by challenging different regions with microscopic laser burns and charting differential responses in terms of levels of proliferation and the regions over which this proliferation occurs.

Methods

Microscopic RPE lesions were undertaken in rats at different eccentricities and the tissue stained for proliferative markers Ki67 and bromodeoxyuridine (BrdU) and the remodeling metalloproteinase marker 2 (MMP2).

Results

All lesions produced local RPE proliferation and tissue remodeling. Significantly more mitosis resulted from peripheral than central lesions. Unexpectedly, single lesions also resulted in RPE cells proliferating across the entire retina. Their number did not increase linearly with lesion number, indicating that they may be a specific population. All lesions repaired and formed apparently normal relations with the neural retina. Repaired RPE was albino.

Conclusions

These results highlight regional RPE differences, revealing an enhanced peripheral repair capacity. Further, all lesions have a marked impact on both local and distant RPE cells, demonstrating a pan retinal signaling mechanism triggering proliferation across the tissue plane. The RPE cells may represent a distinct population as their number did not increase with multiple lesions. The fact that repairing cells were hypopigmented is of interest because reduced pigment is associated with enhanced proliferative capacities in the developing neural retina.

We report a case of intraocular mycosis fungoides in a 48-year-old man. The patient presented with decreased visual acuity, white subretinal lesions, and vitritis. Post-mortem histopathology revealed malignant T cell infiltrates consistent with mycosis fungoides in the retina, vitreous, and between the retinal pigment epithelium (RPE) and Bruch's membrane Focal atrophy of the RPE, along with the sub-RPE infiltrates, correlated with the clinically visible fundus lesions.

Images

After experimental retinal detachment in rabbits macrophages are a prominent feature in the subretinal space or within the retina. Two sources for these macrophages are identified. The retinal pigment epithelium (RPE) may undergo metaplasia and actively 'bud'; the evolving macrophage is then formed by a vitreal protrusion of the cytoplasm of an RPE cell which is 'nipped off' by lateral protrusions from adjacent cells. In addition, in regions of RPE proliferation, blood-borne cells were found in Bruch's membrane and among the mass of proliferated RPE cells, suggesting that blood-borne cells may pass from the choroidal circulation through Bruch's membrane and the RPE layer.

Images

Background

Drusen are extracellular lesions characteristic of aging and age-related maculopathy, a major retinal disease of the elderly. We determined the relative proportions of lipids and proteins in drusen capped with retinal pigment epithelium (RPE) and in RPE isolated from non-macular regions of 36 human retinas with grossly normal maculas obtained <6 hr after death.

Methodology/Principal Findings

Druse pellets were examined by light and electron microscopy. Component proteins were extracted using novel methods for preserved tissues, separated, subjected to tryptic digestion and LC-MS(MS)2 analysis using an ion trap mass spectrometer, and identified with reference to databases. Lipid classes were separated using thin layer chromatography and quantified by densitometry. Major druse components were esterified cholesterol (EC), phosphatidylcholine (PC), and protein (37.5±13.7, 36.9±12.9, and 43.0±11.5 ng/druse, respectively). Lipid-containing particles (median diameter, 77 nm) occupied 37–44% of druse volume. Major proteins include vitronectin, complement component 9, apoE, and clusterin, previously seen in drusen, and ATP synthase subunit β, scavenger receptor B2, and retinol dehydrogenase 5, previously seen in RPE. Drusen and RPE had similar protein profiles, with higher intensities and greater variability in drusen. C8, part of the complement membrane attack complex, was localized in drusen by immunofluorescence.

Conclusions/Significance

At least 40% of druse content is comprised by lipids dominated by EC and PC, 2 components that are potentially accounted for by just one pathway, the secretion of lipoproteins by RPE. Manipulating genes encoding apolipoprotein pathways would be a fruitful approach to producing drusen with high EC content in laboratory animals. Therapies that directly mitigate drusen should prepare for the substantial volume of neutral lipids. The catalog of major druse proteins is nearing completion.

Purpose

To investigate the capacity of mature retinal pigment epithelium (RPE) cells to enter the cell cycle in vivo using a range of RPE-specific and proliferative specific markers in both pigmented and albino rats.

Methods

Whole-mounted retinas of both Dark Agouti and albino rats were immunolabeled with cell cycle markers Ki67 or PCNA and double labeled with RPE cell marker RPE65 or CRALBP. The number and distribution of these cells was mapped. An additional group of Dark Agouti rats were given repeated intraperitoneal injections of Bromodeoxyuridine (BrdU )for 20 days and then sacrificed 30 days later. The retinas were then processed for BrdU detection and Otx, a RPE cell-specific marker. For comparison, human RPE tissue from a postmortem donor was also labeled for Ki67.

Results

In both pigmentation phenotypes, a subpopulation of mature RPE cells in the periphery were positive for both cell cycle markers. These cells were negative for Caspase 3, hence were not apoptotic. Ki67-positive cells were also seen in human RPE. Further, many cells positive for BrdU were identified in similar retinal regions, confirming that RPE cells not only enter the cell cycle but also divide, albeit at a slow cell cycle rate. There was a ten fold increase in the number of RPE cells positive for cell cycle markers in albino (approximately 200 cells) compared to pigmented rats (approximately 20 cells).

Conclusions

Peripheral RPE cells in rats have the capacity to enter the cell cycle and complete cellular division.

A pathological study was performed on the necropsy eyes of a 59-year old-woman who had suffered for nine years from multifocal posterior uveitis. The disease had been controlled by steroid therapy with good preservation of visual function. Extensive investigation did not reveal the aetiology. On macroscopic examination numerous focal lesions with various degrees of pigmentation were observed scattered across the fundi. These lesions were studied by light and electron microscopy and immunohistochemistry. There was ongoing chorioretinal inflammation in the foci, producing destruction of Bruch's membrane, the retinal pigment epithelium (RPE), and the outer retina. The focal scars showed migration of RPE and glial cells and neovascularisation. Capillary and venule endothelial cells were swollen at the inflammatory sites. Attempts to establish a cause for this condition were unsuccessful.

Images

Human retinal pigment epithelial (RPE) cells synthesize an extraneous splice isoform of retinal G protein-coupled receptor (RGR). In this study, we analyzed the exon-skipping variant of RGR (RGR-d) that is found in extracellular deposits. RPE-choroid tissue sections were prepared from postmortem human eyes from donors of various ages. RGR-d was analyzed in drusen and Bruch's membrane by immunohistochemical localization. Extracellular RGR-d is present in most drusen, including hard, soft, confluent and early-stage. Initial drusen formation is known to be preferentially associated with the intercapillary regions of Bruch's membrane. We corroborated this significant association of drusen, including early-stage drusen, with the intercapillary regions. The distribution of extracellular RGR-d in Bruch's membrane differs in old and young donors. In older persons, nodes of concentrated RGR-d accumulate at intercapillary loci, predominantly at the lateral edges of the capillaries of the choriocapillaris. RGR-d loci at the lateral capillary wall appear numerous in old, but not young, donors. Intensely immunostained RGR-d loci can be found at the base of early-stage drusen mounds in the older donors and may precede the formation of these drusen.

Age-related macular degeneration (AMD) represents the major cause of vision loss in industrialized nations. Laminar deposits in Bruch's membrane (BM) are among the first prominent histopathologic features, along with drusen formation, and have been found to contain oxidized lipids. Increases in concentrations of oxidized LDL (oxLDL) in plasma are observed with age and high fat high (HFHC) cholesterol diet. CD36 is the principal receptor implicated in uptake of oxLDL, and is expressed in the retinal pigment epithelium (RPE). We determined if CD36 participates in oxLDL uptake in RPE and correspondingly in clearance of sub-retinal deposits. Uptake of oxLDL by RPE in vitro and in vivo was CD36-dependent. CD36 deficiency in mice resulted in age-associated accumulation of oxLDL and sub-retinal BM thickening, despite fed a regular diet. Conversely, treatment of HFHC-fed ApoE null mice with a CD36 agonist, EP80317 (300 μg/kg/day), markedly diminished thickening of BM, and partially preserved (in part) photoreceptor function. In conclusion, our data uncover a new role for CD36 in the clearance of oxidized lipids from BM and in the prevention of age-dependent sub-retinal laminar deposits.

Background

Intensive light exposure and beta-amyloid (Aβ) aggregates have been known as a risk factor for macular degeneration and an important component in the pathologic drusen structure involved in this disorder, respectively. However, it is unknown whether Aβ deposition mediates or exacerbates light exposure-induced pathogenesis of macular degeneration. Several studies including the one from us already showed accumulation of Aβ deposits in the retina in Alzheimer's transgenic mice. Using histopathological analysis combined with electroretinographic functional assessment, we investigated the effects of cyclic intensive light exposure (CILE) on the architecture of retina and related function in the APPswe/PS1bigenic mouse.

Results

Histopathological analysis has found significant loss of outer nuclear layer/photoreceptor outer segment and outer plexiform layer along with abnormal hypo- and hyper-pigmentation in the retinal pigment epithelium (RPE), remarkable choroidal neovascularization (CNV), and exaggerated neuroinflammatory responses in the outer retina of APPswe/PS1 bigenic mice following cyclic intensive light exposure (CILE), whereas controls remained little change contrasted with age-matched non-transgenic littermates. CILE-induced degenerative changes in RPE are further confirmed by transmission electron microcopy and manifest as formation of basal laminar deposits, irregular thickening of Bruch's membrane (BrM), deposition of outer collagenous layer (OCL) in the subretinal space, and vacuolation in the RPE. Immunofluorescence microscopy reveals drusenoid Aβ deposits in RPE as well as neovessels attached which are associated with disruption of RPE integrity and provoked neuroinflammatory response as indicated by markedly increased retinal infiltration of microglia. Moreover, both immunohistochemistry and Western blots detect an induction of vascular endothelial growth factor (VEGF) in RPE, which corroborates increased CNV in the outer retina in the bigenic mice challenged by CILE.

Conclusions

Our findings demonstrate that degenerative changes in the outer retina in the APPswe/PS1 bigenic mouse induced by CILE are consistent with these in AMD. These results suggest that an Alzheimer's transgenic animal model with accumulation of Aβ deposits might be an alternative animal model for AMD, if combined with other confounding factors such as intensive light exposure for AMD.

This study showed that mouse retina and retinal pigment epithelial (RPE) cells express the heme transporters FLVCR, BCRP, and PCFT. FLVCR is localized to the apical membrane, and BCRP and PCFT are localized to the basolateral membrane in RPE cells. Hemochromatosis, a genetic disease with iron overload, is associated with upregulation of FLVCR and PCFT, but with downregulation of BCRP in retina and RPE.

Purpose.

FLVCR, BCRP, and PCFT/HCP-1 represent the three heme transporters identified thus far in mammalian cells, but there is very little known about their expression and regulation in the retina. In this study, the expression of these transporters in mouse retina and retinal pigment epithelium (RPE) and their regulation in the iron-overload disease hemochromatosis were examined.

Methods.

The expression of FLVCR, BCRP, and PCFT in mouse retina and primary mouse RPE cells was studied by RT-PCR and immunofluorescence. Polarized localization of the transporters in RPE was studied by co-localization using a specific marker of the RPE apical membrane. Uptake of heme in primary RPE cells was determined using zinc-mesoporphyrin, a fluorescent heme analogue. The regulation of heme transporters by iron overload was studied in two genetic models of hemochromatosis (HFE-null mouse and HJV-null mouse) and in two nongenetic models of iron overload (cytomegalovirus infection and treatment with ferric ammonium citrate).

Results.

All three heme transporters were expressed in the retina and RPE. In the RPE, the expression of FLVCR was restricted to the apical membrane, and the expression of BCRP and PCFT was restricted to the basolateral membrane. In all cases of iron overload, the expression of FLVCR and PCFT was upregulated and that of BCRP was downregulated.

Conclusions.

Hemochromatosis is associated not only with excessive accumulation of free iron in the retina and RPE but also with excessive accumulation of heme. Since heme is toxic at high levels, as is free iron, heme-induced oxidative damage may also play a role in hemochromatosis-associated retinal pathology.

Drusen are extracellular deposits that lie beneath the retinal pigment epithelium (RPE) and are the earliest signs of age-related macular degeneration (AMD). Recent proteome analysis demonstrated that amyloid β (Aβ) deposition was specific to drusen from eyes with AMD. To work toward a molecular understanding of the development of AMD from drusen, we investigated the effect of Aβ on cultured human RPE cells as well as ocular findings in neprilysin gene–disrupted mice, which leads to an increased deposition Aβ. The results showed that Aβ treatment induced a marked increase in VEGF as well as a marked decrease in pigment epithelium-derived factor (PEDF). Conditioned media from Aβ-exposed RPE cells caused a dramatic increase in tubular formation by human umbilical vein endothelial cells. Light microscopy of senescent neprilysin gene–disrupted mice showed an increased number of degenerated RPE cells with vacuoles. Electron microscopy revealed basal laminar and linear deposits beneath the RPE layer, but we did not observe choroidal neovascularization (CNV). The present study demonstrates that Aβ accumulation affects the balance between VEGF and PEDF in the RPE, and an accumulation of Aβ reproduces features characteristic of human AMD, such as RPE atrophy and basal deposit formation. Some other factors, such as breakdown of integrity of Bruch membrane, might be necessary to induce CNV of AMD.

In this study, the authors describe the signaling pathways of cytokine induction by 7-ketocholesterol. This chronic inflammatory pathway may be of importance to the RPE and choriocapillaris, which are in direct contact with age-related oxidized lipoprotein deposits.

Purpose.

7-Ketocholesterol (7KCh) accumulates in oxidized lipoprotein deposits and is known to be involved in macrophage foam cell formation and atherosclerosis. 7-KCh is present in the primate retina and is associated with oxidized lipoprotein deposits located in the choriocapillaris, Bruch's membrane, and retinal pigment epithelium (RPE). 7-KCh can also be formed in the retina as a consequence of light-induced iron release. The purpose of this study was to examine the signaling pathways involved in the 7KCh-mediated inflammatory response focusing on three cytokines, VEGF, IL-6, and IL-8

Methods.

ARPE-19 cells were treated with 7KCh solubilized in hydroxypropyl-β-cyclodextrin. Cytokines were quantified by qRT-PCR (mRNA) and ELISA (protein) using commercially available products. NFκB activation was determined by IκBα mRNA induction

Results.

Treatment of ARPE-19 cells with 15 μM 7KCh markedly induced the expression of VEGF, IL-6, and IL-8. No increase in NOX-4 expression or ROS formation was detected. 7KCh induced the phosphorylation of ERK1/2 and p38MAPK, and inhibitors to these kinases markedly reduced the cytokine expression but did not affect the IκBα mRNA expression. By contrast, inhibition of PI3K and PKCζ significantly decreased the cytokine and IκBα mRNA expression. Inhibition of the IκB kinase complex essentially ablated all cytokine induction

Conclusions.

7KCh induces cytokines via three kinase signaling pathways, AKT-PKCζ-NFκB, p38 MAPK, and ERK. The MAPK/ERK pathways seem to preferentially enhance cytokine induction downstream from NFκB activation. The results of this study suggest that 7KCh activates these pathways through interactions in the plasma membrane, but the mechanism(s) remains unknown.

Three middle-aged male patients are described with a peculiar patterned dystrophy of the macula. The basic lesions are discrete yellow plaques typically confined to the macular area and radiating from the fovea. They appear to be located at the level of the retinal pigment epithelium (RPE). With the passage of time some of the yellow plaques altered in extent and configuration, and atrophic changes appeared or extended. Visual acuity and electrophysiological tests are either normal or only moderately affected. The lesions appear to be distinct from the patterned dystrophies of the retina already described and from other conditions characterised by yellow or white deposits at the level of the RPE.

Images

Aim: To evaluate the morphology and visual function of the macula in eyes with adult onset vitelliform macular dystrophy (AVMD).

Methods: 12 eyes of six patients with AVMD were examined by ophthalmoscopy, scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), and multifocal electroretinography (mfERGs). The macular lesions were bilateral in all patients and varied from the typical vitelliform (five eyes), faded vitelliform changes with retinal pigment epithelium (RPE) atrophy (five eyes), and a normal fovea associated with small flecks around the macula (two eyes).

Results: SLO demonstrated small abnormal bright spots in the deep retina throughout the posterior retina in all cases. OCT showed a highly reflective fusiform thickened layer at the level of the RPE and choriocapillaris in patients with a submacular yellow vitelliform lesion. A well circumscribed, optically clear space was observed beneath the retinal layer in the macular lesions with RPE atrophy. The mfERGs were significantly reduced not only in the macular area but also in the outermost ring (20–30°) of the mfERGs.

Conclusions: The submacular materials that accumulate within the RPE or subepithelial layers reported in previous histopathological studies of vitelliform lesions can be detected by OCT. In the macular lesions with RPE atrophy, the material may have disappeared leaving a subretinal or subepithelial optical clear space. These SLO and mfERG observations suggest that the morphological and functional abnormalities may not be localised just in the macular area but may be present throughout the posterior pole in eyes with AVMD.

Scanning electron microscopic observation of Bruch's membrane was performed after removal of retinal pigment epithelium (RPE) with the osmium tetroxide treatment. Eight human eyes from subjects at various ages (from newborn to 77 years old) were examined in order to investigate aging changes in Bruch's membrane. The collagen fibres of the inner collagenous zone in young eyes formed a tightly interwoven membrane, and the meshes were regular and fine. In old eyes the meshes were irregular and coarse, and deposits were observed. Deposits were embedded in the collagen fibres of the inner collagenous zone, or attached to the surface on the inner-collagenous-zone side of the basement membrane of RPE.

Images

Aims: To report the histopathology of two specimens of polypoidal choroidal vasculopathy (PCV) obtained from two eyes of Japanese patients.

Methods: Specimens were obtained under direct visualisation during macular translocation surgery with 360 degree retinotomy. The clinical findings were correlated with the light microscopic findings of the two specimens.

Results: One specimen from a 77 year old man was the central portion of the lesion that lay under the sensory retina on the retinal pigment epithelium (RPE). The specimen was made up mainly of fibrous tissue with small, thin walled vessels. Indocyanine green angiography after surgery revealed that active leaking polypoidal element remained under the RPE. Another specimen obtained from a 62 year old man was made up of a fibrovascular membrane situated within Bruch's membrane. The part of this specimen inferior to the foveal region included a collection of dilated, thin walled blood vessels without pericytes, surrounded by macrophages that stained positive for CD68. The dilated vessels appeared to be correlated with the orange coloured polyps observed by ophthalmoscopy, the polypoidal structure seen in indocyanine green angiograms, and the pyramidal elevation with intermediate reflectivity by optical coherence tomography.

Conclusion: Polypoidal structures are located within Bruch's space. They are composed of clusters of dilated, thin walled blood vessels surrounded by macrophages and fibrin material. The positive immunohistochemical staining for vascular endothelial growth factor in the RPE and the vascular endothelial cells suggests that this fibrovascular complex is a subretinal choroidal neovascularisation.

Aim: Selective retinal pigment epithelium (RPE) laser treatment is a new technique which selectively damages the RPE while sparing the neural retina. One difficulty is the inability to visualise the laser lesions. The aim of the study was to investigate whether fundus autofluorescence (AF) is changed because of the RPE damage, and thus might be used for treatment control. Additionally, the clinical course of patients with various macular diseases was evaluated.

Methods: 26 patients with macular diseases (diabetic maculopathy (DMP), soft drusen maculopathy (AMD), and central serous retinopathy (CSR)) were treated and followed up for at least 6 months. Treatment was performed with a train of repetitive short laser pulses (800 ns) of a frequency doubled Nd:YAG laser (parameters: 532 nm, 50 and 500 pulses at 100 and 500 Hz, retinal spot diameter 200 μm, pulse energies 75–175 μJ). AF was excited by 488 nm and detected by a barrier filter at 500 nm (HRA, Heidelberg Engineering, Germany). Patients were examined by ophthalmoscopy, fluorescein angiography, and autofluorescence measurements at various times after treatment (10 minutes, 1 hour, 1 and 6 weeks, 3, 6, and 12 months).

Results: Fluorescein angiography showed leakage from the irradiated areas for about 1 week after treatment. None of the laser lesions was ophthalmoscopically visible during treatment. Identification of the lesions was possible by AF imaging showing an intensity decay in the irradiated area in 22 out of 26 patients, predominantly in patients with CSR and AMD. Lesions could be identified 10 minutes after treatment as hypoautofluorescent spots, which were more pronounced 1 hour later. During follow up the laser spots became hyperautofluorescent. In patients with DMP some AF images were less helpful because of diffuse oedema and larger retinal thickness. In these cases ICG angiography was able to confirm therapeutic success very well. Most of the patients have had benefit from the treatment, with best results obtained for CSR patients.

Conclusion: Imaging of non-visible selective RPE laser effects can be achieved by AF measurements predominantly in patients without retinal oedema. Therefore, AF may replace invasive fluorescein angiography in many cases to verify therapeutic laser success. Selective laser treatment has the potential to improve the prognosis of macular diseases without the risk of laser scotomas.

For the past 10 years, number of evidence has shown that activation of complement cascade has been associated with age-related macular degeneration (AMD). The genome wide association study in American population with dominantly dry-type AMD has revealed strong association with single nucleotide polymorphism (SNP) of complement genes. Protein composition of drusen, a deposit observed in sub-retinal space between Bruch’s membrane and retinal pigment epithelial (RPE), contains active complement molecules in human and monkey. These evidences have leaded us to consider the possibility of suppressing complement cascade in the retina to delay or reverse the onset of AMD. To test is hypothesis we used the C3 inhibitor Compstatin on primate model with early-onset macular degeneration which develop drusen in less than 2 years after birth. Our preliminary result showed drusen disappearance after 6 months of intravitreal injection.

Summary

Compared with neural crest-derived melanocytes, retinal pigment epithelium (RPE) cells in the back of the eye are pigment cells of a different kind. They are a part of the brain, form an epithelial monolayer, respond to distinct extracellular signals, and provide functions that far exceed those of a light-absorbing screen. For instance, they control nutrient and metabolite flow to and from the retina, replenish 11-cis-retinal by re-isomerizing all-trans-retinal generated during photoconversion, phagocytose daily a portion of the photoreceptors’ outer segments, and secrete cytokines that locally control the innate and adaptive immune systems. Not surprisingly, RPE cell damage is a major cause of human blindness worldwide, with age-related macular degeneration a prevalent example. RPE replacement therapies using RPE cells generated from embryonic or induced pluripotent stem cells provide a novel approach to a rational treatment of such forms of blindness. In fact, RPE-like cells can be obtained relatively easily when stem cells are subjected to a two-step induction protocol, a first step that leads to a neuroectodermal fate and a second to RPE differentiation. Here, we discuss the characteristics of such cells, propose criteria they should fulfill in order to be considered authentic RPE cells, and point out the challenges one faces when using such cells in attempts to restore vision.

In the classic retinoid cycle, 11-cis retinol is synthesized in the retinal pigment epithelium (RPE) by two enzymes: Isomerase I (RPE65) and lecithin:retinol acyltransferase (LRAT). The purpose of this study is to provide experimental evidence for two active isomerases in the cone-dominated chicken eye: an LRAT-dependent Isomerase I in the RPE and an ARAT (acyl CoA:retinol acyltransferase)-dependent isomerase (Isomerase II) in the retina. First, we show that whole chicken retina in vitro, removed from the RPE/choroid and sclera, produces 11-cis retinoids upon light exposure, indicating the existence of RPE-independent isomerase (Isomerase II) activity in the retina. RT-PCR studies show high levels of RPE65 expression in the RPE, low levels in the retina, and none in primary Müller cell cultures, indicating the presence of Isomerase I in the RPE and a minimal amount in the retina. Activities of the RPE and retina isomerases were then measured by enzyme assays with specific enzyme inhibitors. 2,2′-Bipyridine, a known Isomerase I inhibitor, and N-ethyl-maleimide (NEM), a known LRAT inhibitor, significantly reduced Isomerase I activity but not Isomerase II activity. Progesterone, a known ARAT inhibitor, completely blocked Isomerase II activity but not Isomerase I activity. Thus the present study reports novel results to distinguish the biochemical properties of Isomerase I from Isomerase II, as well a difference in their locations in the chicken eye. Based on these differences, the cone-dominated chicken eye must contain two retinoid cycles: a classic visual cycle for retinoid exchange between the RPE and the retina supported by Isomerase I in the RPE, and an additional visual cycle for retinoid processing in the retina supported by Isomerase II.

Background

Age-related macular degeneration (AMD) is a leading cause of blindness that affects the central region of the retinal pigmented epithelium (RPE), choroid, and neural retina. Initially characterized by an accumulation of sub-RPE deposits, AMD leads to progressive retinal degeneration, and in advanced cases, irreversible vision loss. Although genetic analysis, animal models, and cell culture systems have yielded important insights into AMD, the molecular pathways underlying AMD's onset and progression remain poorly delineated. We sought to better understand the molecular underpinnings of this devastating disease by performing the first comparative transcriptome analysis of AMD and normal human donor eyes.

Methods

RPE-choroid and retina tissue samples were obtained from a common cohort of 31 normal, 26 AMD, and 11 potential pre-AMD human donor eyes. Transcriptome profiles were generated for macular and extramacular regions, and statistical and bioinformatic methods were employed to identify disease-associated gene signatures and functionally enriched protein association networks. Selected genes of high significance were validated using an independent donor cohort.

Results

We identified over 50 annotated genes enriched in cell-mediated immune responses that are globally over-expressed in RPE-choroid AMD phenotypes. Using a machine learning model and a second donor cohort, we show that the top 20 global genes are predictive of AMD clinical diagnosis. We also discovered functionally enriched gene sets in the RPE-choroid that delineate the advanced AMD phenotypes, neovascular AMD and geographic atrophy. Moreover, we identified a graded increase of transcript levels in the retina related to wound response, complement cascade, and neurogenesis that strongly correlates with decreased levels of phototransduction transcripts and increased AMD severity. Based on our findings, we assembled protein-protein interactomes that highlight functional networks likely to be involved in AMD pathogenesis.

Conclusions

We discovered new global biomarkers and gene expression signatures of AMD. These results are consistent with a model whereby cell-based inflammatory responses represent a central feature of AMD etiology, and depending on genetics, environment, or stochastic factors, may give rise to the advanced AMD phenotypes characterized by angiogenesis and/or cell death. Genes regulating these immunological activities, along with numerous other genes identified here, represent promising new targets for AMD-directed therapeutics and diagnostics.

Please see related commentary: http://www.biomedcentral.com/1741-7015/10/21/abstract