Peripheral Retinal Pigment Epithelium Research Articles

Age-related reduction in the functional properties of adult stem cells located in the peripheral region of human retinal pigment epithelium.

Adult stem cells (SCs) with self-renewal and multilineage potential have been reported upon culturing human retinal pigment epithelial (RPE) cells. The current study aimed to identify the location of SCs in human RPE and to elucidate the age-related changes. Peripheral, equatorial, and central RPE cells from donors of three age groups were analyzed for their sphere-forming, clonal, and label-retaining cell properties. Furthermore, native human RPE flatmounts were immunostained for SC and proliferating cell markers. Cells with higher sphere-forming and clonal ability were identified only in young donors (<30 years) and were restricted to the periphery. Upon culturing, cells from peripheral and equatorial regions had the label-retaining cell (LRC) property. With aging, the LRCs were restricted to the periphery and were reduced. In young donors, Ki67 + proliferating cells were not observed in native RPE. However, such cells were observed in the peripheral RPE of older donors correlating with the need for regeneration. The native RPE cells were negative for SC marker expression. The above findings highlighted the presence of SCs with the ability to proliferate in the peripheral RPE and a reduction in these functional properties of SCs with aging.

Multimodal imaging study and clinical management in Coats' disease

Aims/Purpose: Coats' disease is an idiopathic ocular condition characterized by retinal telangiectasia, aneurysms, and exudation, frequently affects unilateral eyes of young males. Rare pathology of difficult diagnosis and clinical management. Follow‐up by multimodal imaging of clinical improvement after correct treatment in Coats' Disease.Methods: Morales Meseguer University Hospital, Murcia, Spain. Observational study using multimodal imaging of clinical cases in patients diagnosed with Coats' disease. Retrospective follow‐up during several visits between 2021–2023. Images taken with optical coherence tomography (OCT), fluorescein angiography (FA) and autofluorescence.Results: Presentation two clinical cases. A 56‐year‐old female and a 28‐year‐old male diagnosed with Coats disease in right eye. Best corrected visual acuity (BVAC) of 0.6 and 0.5, respectively. Female had macular oedema (ME) with hard exudates in temporal arcade territory with areas of venous and arterial sheathing, macroaneurysm in inferior temporal arcade territory. Hypertrophy of the peripheral retinal pigment epithelium. OCT shows EM, subretinal fluid (SRF) and hyperreflective dots in middle layers. Male with a history of vitrectomy and phacoemulsification after regmatogenous and tractional retinal detachment secondary to the disease, presents pseudophakia, angular synechiae and iridotomies, vascular malformations, inferior haemorrhages, zone of temporal and nasal inferior exudation with FSR area, inferior tear pigmented with laser. OCT shows ME with FSR. Both cases, FA proves blood fluids levels and saccular aneurysms of arterial arterioles and venules. We note an improvement BVAC and EM with resolution of FSR on multimodal imaging study after intravitreal and focal laser treatment.Conclusions: Multimodal imaging in the follow‐up of Coats' disease is essential to assess correct treatment. Treatment with focal laser and intravitreal therapy is safe and effective in control of Coats disease.

Location of Retinal Pigment Epithelial Cells in the Eye Is Critical to Their Function

The eye is an extension of the brain and a highly sophisticated sensory organ. The intricacy of the eye is well appreciated on examination of the exquisite posterior pole, composed of numerous interconnected cell and tissue types. These types broadly include the multicellular neural retina; a single layer of epithelial cells providing support to the retina, called the retinal pigment epithelium; a multilaminar extracellular matrix called the Bruch membrane; and the choroid, a thin, trilaminar connective tissue adjacent to the Bruch membrane, composed of a dense web of vessels, pigments, and immune cells. 1 Choudhary M. Malek G. Potential therapeutic targets for age-related macular degeneration: the nuclear option. Prog Retin Eye Res. 2022; 101130 PubMed Google Scholar The posterior pole can be partitioned into two regions, the macula and the periphery, adding another tier of complexity to the eye. The anatomic macula, when evaluated histologically, is an oval-shaped region approximately 5 mm in diameter, centrally located in the posterior pole of eye. Though covering approximately 4% of the retina, it is densely composed of cone photoreceptors, facilitating high-resolution color vision. The rest of the retina is rich in rod photoreceptors, involved in side, or peripheral, and night vision. Other regional differences include, but are not limited to, variances in the structural architecture of the retinal layers, cellular distribution, and blood flow. 2 Johnson M. Dabholkar A. Huang J.D. Presley J.B. Chimento M.F. Curcio C.A. Comparison of morphology of human macular and peripheral Bruch's membrane in older eyes. Curr Eye Res. 2007; 32: 791-799 Crossref PubMed Scopus (35) Google Scholar , 3 Quinn N. Csincsik L. Flynn E. Curcio C.A. Kiss S. Sadda S.R. Hogg R. Peto T. Lengyel I. The clinical relevance of visualising the peripheral retina. Prog Retin Eye Res. 2019; 68: 83-109 Crossref PubMed Scopus (70) Google Scholar , 4 Sabeti F. Lane J. Rohan E.M.F. Rai B.B. Essex R.W. McKone E. Maddess T. Correlation of central versus peripheral macular structure-function with acuity in age-related macular degeneration. Transl Vis Sci Technol. 2021; 10: 10 Crossref PubMed Scopus (9) Google Scholar Thus, it may come as no surprise that human retinal diseases demonstrate regional selectivity, in which a cell or tissue type is particularly vulnerable in one area while in adjacent zones may remain mostly unaffected. In this issue of the American Journal of Pathology, Mullin et al 5 Mullin N.K. Voigt A.P. Boese E.A. Liu X. Stone E.M. Tucker B.A. Mullins R.F. Transcriptomic and chromatin accessibility analysis of the human macular and peripheral retinal pigment epithelium at the single-cell level. Am J Pathol. 2023; Abstract Full Text Full Text PDF PubMed Google Scholar explore several essential questions in this realm: Is there region-associated heterogeneity in retinal pigment epithelial cells, whose dysfunction is central to the development of several vision-debilitating diseases?; What are the differences in the expression profiles of retinal pigment epithelial cells in the macula versus the periphery?; and Could regional differences in the levels of expression in retinal pigment epithelial cells be linked to susceptibility to disease? Transcriptomic and Chromatin Accessibility Analysis of the Human Macular and Peripheral Retinal Pigment Epithelium at the Single-Cell LevelThe American Journal of PathologyPreviewSome human retinal diseases are characterized by pathology that is restricted to specific cell types and to specific regions of the eye. Several disease entities either selectively affect or spare the macula, the retina region at the center of the posterior pole. Photoreceptor cells in the macula are involved in high-acuity vision and require metabolic support from non-neuron cell types. Some macular diseases involve the retinal pigment epithelium (RPE), an epithelial cell layer with several metabolic-support functions essential for the overlying photoreceptors. Full-Text PDF

SIRT6 overexpression in the nucleus protects mouse retinal pigment epithelium from oxidative stress.

Retinal pigment epithelium (RPE) is essential for the survival of retinal photoreceptors. To study retinal degeneration, sodium iodate (NaIO3) has been used to cause oxidative stress-induced RPE death followed by photoreceptor degeneration. However, analyses of RPE damage itself are still limited. Here, we characterized NaIO3-induced RPE damage, which was divided into three regions: periphery with normal-shaped RPE, transitional zone with elongated cells, and center with severely damaged or lost RPE. Elongated cells in the transitional zone exhibited molecular characteristics of epithelial-mesenchymal transition. Central RPE was more susceptible to stresses than peripheral RPE. Under stresses, SIRT6, an NAD+-dependent protein deacylase, rapidly translocated from the nucleus to the cytoplasm and colocalized with stress granule factor G3BP1, leading to nuclear SIRT6 depletion. To overcome this SIRT6 depletion, SIRT6 overexpression was induced in the nucleus in transgenic mice, which protected RPE from NaIO3 and partially preserved catalase expression. These results demonstrate topological differences of mouse RPE and warrant further exploring SIRT6 as a potential target for protecting RPE from oxidative stress-induced damage.

Disturbances of Ocular Circulation in Color Doppler Imaging, Retinal Changes and Electrophysiological Tests with Neuro-Ophthalmological Clinical Symptoms in the Course of CADASIL Syndrome—A Case Report

The authors present a new paper examining the disturbances in ocular circulation and electrophysiological changes in the presence of neuro-ophthalmic manifestations in a patient with cerebral autosomal dominant arteriopathy with subcortical infracts and leucoencephalopathy (CADASIL). Symptoms reported by the patient included: transient vision loss (TVL), migraines, diplopia, bilateral peripheral visual field loss and convergence insufficiency. CADASIL was confirmed by the presence of NOTCH3 gene mutation (p.Cys212Gly), the presence of granular osmiophilic material (GOM) in cutaneous vessels in an immunohistochemistry test (IHC) and bilateral focal vasogenic lesions in the white matter of the cerebral hemisphere, with micro-focal infarct in the left external capsule on a magnetic resonance imaging test (MRI). Color Doppler imaging (CDI) confirmed decreased blood flow and increased vascular resistance in the retinal and posterior ciliary arteries, with reduced P50 wave amplitude on a pattern electroretinogram (PERG). An eye fundus examination and fluorescein angiography (FA) revealed the constriction of retinal vessels and a peripheral retinal pigment epithelium (RPE) atrophy with focal drusen. The authors suggest that the cause of TVL may be changes in the hemodynamics of the retinochoroid vessels associated with the narrowing of small vessels and the presence of druses in the retina-which is supported by a decrease in the amplitude of the P50 wave in PERG, changes in OCT correlating simultaneously with changes in MRI imaging and other neurological symptoms.

Transcriptomic and Chromatin Accessibility Analysis of the Human Macular and Peripheral Retinal Pigment Epithelium at the Single-Cell Level

Some human retinal diseases are characterized by pathology that is restricted to specific cell types and to specific regions of the eye. Several disease entities either selectively affect or spare the macula, the retina region at the center of the posterior pole. Photoreceptor cells in the macula are involved in high-acuity vision and require metabolic support from non-neuronal cell types. Some macular diseases involve the retinal pigment epithelium (RPE), an epithelial cell layer with several metabolic-support functions essential for the overlying photoreceptors. In the current study, the ways in which RPE confers region-specific disease susceptibility were determined by examining heterogeneity within RPE tissue from human donors. RPE nuclei from the macular and peripheral retina were profiled using joint single-nucleus RNA and ATAC sequencing. The expression of several genes differed between macular and peripheral RPE. Region-specific ATAC peaks were found, suggesting regulatory elements used exclusively by macular or peripheral RPE. Across anatomic regions, subpopulations of RPE were identified that appeared to have differential levels of expression of visual cycle genes. Finally, loci associated with age-related macular degeneration were examined for a better understanding of RPE-specific disease phenotypes. These findings showed variations in the regulation of gene expression in the human RPE by region and subpopulation, and provide a source for a better understanding of the molecular basis of macular disease.

Clinical Characterization of Autosomal Dominant and Autosomal Recessive PROM1 Mutation With a Report of Novel Mutation.

This study aims to provide clinical characterization of PROM1 mutation with a report of novel mutation. This study is a retrospective case series of six patients from a single institution with multimodal imaging, electroretinography, and genetic testing. Six patients aged 12 to 47 years were identified. Patients with autosomal recessive (AR) variants showed more severe panretinal dystrophy with symmetrical macular involvement and peripheral retinal pigment epithelium atrophy. The autosomal dominant (AD) variants, on the other hand, showed milder macular involvement with bull's eye maculopathy phenotype with minimal peripheral involvement. Among patients with AR variants, a younger patient with aberrant splicing showed a milder phenotype compared with patients with a nonsense mutation and an additional ABCA4 mutation. The authors describe patients with PROM1 retinopathy inherited AD and AR inherited patterns. Novel mutations of c.1909C>T and c.2050C>T were identified, leading to truncation of the protein at sequence p.Gln637* and p.Arg684*, respectively. [Ophthalmic Surg Lasers Imaging Retina 2022;53:422-428.].

A case of bilateral pachychoroid disease: polypoidal choroidal vasculopathy in one eye and peripheral exudative hemorrhagic chorioretinopathy in contralateral eye

BackgroundWe report a case of bilateral pachychoroid disease manifesting polypoidal choroidal vasculopathy (PCV) with punctate hyperfluorescent spot (PHS) in one eye, and peripheral exudative hemorrhagic choroidal retinopathy (PEHCR) with central serous chorioretinopathy (CSC) and PHS in the contralateral eye.Case presentation: A 51-year-old healthy woman presented with complaint of blurred vision in her right eye. Corrected visual acuity was 20/20 in the right and 24/20 in the left eye. Fundus examination was normal in the left eye. In the right eye, fundus finding of an orange-red nodular lesion and optical coherence tomography (OCT) finding of polypoidal lesions led to a diagnosis of PCV. Four aflibercept intravitreal injections were performed in her right eye. After treatment, indocyanine green angiography (ICGA) confirmed residual polypoidal lesions with branching vascular networks and PHS with choroidal vascular hyperpermeability. OCT showed PHS associated with small sharp-peaked retinal pigment epithelium (RPE) elevation in peripheral fundus and small RPE elevation in posterior fundus. Based on the above findings, PCV with PHS was finally diagnosed in the right eye. Posttreatment corrected visual acuity in the right eye was 20/20. She presented again 32 months later, with complaint of vision loss in her left eye. Left corrected visual acuity was 20/20, and fundus examination showed mild vitreous hemorrhage. Vitrectomy was performed. In temporal midperipheral fundus, fluorescein angiography revealed CSC, and OCT showed pachychoroid. ICGA depicted abnormal choroidal networks and PHS in peripheral fundus. Furthermore, polypoidal lesions were confirmed by OCT. Based on the above findings, PEHCR and CSC with PHS was finally diagnosed in the left eye. Postoperative corrected visual acuity in the left eye was 20/20, and aflibercept intravitreal injection was performed for prevention of recurrence of vitreous hemorrhage.ConclusionsThis is the first case report of PCV with PHS in one eye, and PEHCR with CSC and PHS in the contralateral eye. This case suggests that PCV, PEHCR, and CSC may be linked pathologies of pachychoroid spectrum disease.

Development of Cell Delivery System for the Subretinal Space Using Biodegradable Nanosheets

Age-related macular degeneration (AMD) is a major ophthalmic disease that causes visual impairment and blindness, particularly in elderly people. The developing choroidal vessels invade Bruch’s membrane and disrupt the monolayer of retinal pigment epithelial (RPE) cells. Subsequently, degeneration of photoreceptors occurs. Hence, subretinal transplantation of RPE cells to the site of degeneration would offer an ideal treatment providing the transplanted cells could generate a monolayer, reconstruct the RPE–photoreceptor interface, and inhibit further development of choroidal vessels. Although transplantation of autologous peripheral RPE cells has been tested by injection of cell suspensions using a syringe needle, limited visual improvement result due to the low viability of the injected cells, and their restricted distribution and integration into the subretinal tissue.Recently, we investigated the use of biodegradable polymeric nanosheets as cell scaffolds [1,2]. Because this system enabled the easy manipulation of the cell/nanosheet constructs with a syringe needle, it raises the possibility of transplanting cell sheets into a narrow space, such as the subretinal space, in a minimally-invasive manner. So far, poly(lactic-co-glycolic acid) (PLGA) was used as the material for the nanosheets. However, this biodegradable polymer is relatively inflammatory because it produces lactic and glycolic acids as degradation intermediates and these acid dissociation constants are about 3.8. In this study, we fabricated biodegradable nanosheets using polycaprolactone (PCL) and collagen as low inflammatory materials and investigated a minimally invasive and effective method for cell transplantation into the subretinal space (Fig.1(a)).Micropatterned nanosheets consisting of PCL, collagen and Nile red were prepared by a combination of spin-coating and micro-contact printing technique (Fig.1(b)). The diameter and thickness of the nanosheets used for the following experiments were 200 μm and 200 nm, respectively. The cell/nanosheet constructs were prepared by culturing rat retinal pigment epithelial (RPE-J) cells on the micropatterned nanosheets in a monolayer. A hand-made glass capillary needle was as thick as a 27G medical needle, and the tip angle was 30 degrees. Also, it was coated with 2-methayloxyethyl phosphorylcholine (MPC) to prevent cells from attaching the inner surface of the needle. The cell/nanosheet constructs and a sodium hyaluronate solution with the medium were loaded into the capillary needle. Subsequently, the cell/nanosheet was injected into the subretinal space through the sclera of rat. Several days after implantation, the rats were euthanized, and the eyeballs were enucleated.The micropatterned nanosheet was shown in Fig.1(c). The nanosheets had sufficient strength and flexibility to be handled in water. The shape or diameter of the nanosheets could be varied by adjusting the microstructure of the PDMS stamp. Also, the thickness of the nanosheets could be controlled by changing the concentration of the solution. As shown in Fig.1b, the cells formed a monolayer on the nanosheet of PCL/collagen. Finally, we attempted to deliver the RPE-J cell/nanosheet constructs to the subretinal space of a rat eye in vivo. The hand-made glass capillary needle shape allowed the needle insertion along the retina through the sclera. The fluorescence of PCL/collagen nanosheet and cells could be observed in the eyeballs under excitation light. Also, as shown in Fig.1(e), it was confirmed that the cell/nanosheet construct was successively delivered into the subretinal space.We fabricated biodegradable nanosheets using PCL and collagen as low inflammatory materials. Also, we developed a minimally invasive and effective method for cell transplantation into the subretinal space using capillary needles and confirmed that cell/nanosheet was successfully delivered. This method holds great promise for transplantation of monolayer cells and for the development of local cell delivery systems. In order to further improve this method, we plan to evaluate the inflammatory properties of the nanosheets and the efficacy of the delivered cells.

Regeneration of the zebrafish retinal pigment epithelium after widespread genetic ablation.

The retinal pigment epithelium (RPE) is a specialized monolayer of pigmented cells within the eye that is critical for maintaining visual system function. Diseases affecting the RPE have dire consequences for vision, and the most prevalent of these is atrophic (dry) age-related macular degeneration (AMD), which is thought to result from RPE dysfunction and degeneration. An intriguing possibility for treating RPE degenerative diseases like atrophic AMD is the stimulation of endogenous RPE regeneration; however, very little is known about the mechanisms driving successful RPE regeneration in vivo. Here, we developed a zebrafish transgenic model (rpe65a:nfsB-eGFP) that enabled ablation of large swathes of mature RPE. RPE ablation resulted in rapid RPE degeneration, as well as degeneration of Bruch’s membrane and underlying photoreceptors. Using this model, we demonstrate for the first time that zebrafish are capable of regenerating a functional RPE monolayer after RPE ablation. Regenerated RPE cells first appear at the periphery of the RPE, and regeneration proceeds in a peripheral-to-central fashion. RPE ablation elicits a robust proliferative response in the remaining RPE. Subsequently, proliferative cells move into the injury site and differentiate into RPE. BrdU incorporation assays demonstrate that the regenerated RPE is likely derived from remaining peripheral RPE cells. Pharmacological disruption using IWR-1, a Wnt signaling antagonist, significantly reduces cell proliferation in the RPE and impairs overall RPE recovery. These data demonstrate that the zebrafish RPE possesses a robust capacity for regeneration and highlight a potential mechanism through which endogenous RPE regenerate in vivo.

Topographic protein profiling of the age-related proteome in the retinal pigment epithelium of Callithrix jacchus with respect to macular degeneration

In the retinal pigment epithelium (RPE) several factors within the macular compared to peripheral regions cause differences in physiological aging. The molecular mechanisms during aging in the context of topography are not well known. The proteome of RPE of different aged macular-bearing primates Callithrix jacchus was thus analysed with ion mobility mass spectrometry. Macular and periphery of neonate RPE were well differentiated from aged tissues as demonstrated by principal component analysis. This finding was mainly due to proteins involved in major developmental processes and the visual cycle. The distinction of adult from senile tissue and macular from periphery was more subtle. The hypotheses of inflammation increasing with age was supported. High expression levels of proteins related to oxidative stress (e.g., cathepsin B) and chaperones (e.g., HSP90) were detected in aged RPE as confirmed by Western blot and immunohistochemical analysis. Decreased levels of proteins participating in angiostatic properties (e.g., thrombospondin 1) and the integrity of tissue basement membranes with age (e.g., nidogen 1) were in agreement with neovascularization. This study presents targets for further investigations of the mechanisms of the aging process with the aim to elucidate predictive factors for the conversion of physiological aging into pathological conditions. SignificanceThe current study characterized the different protein profiles of the retinal pigment epithelium (RPE) of the macula-bearing, non-human primate Callithrix jacchus during life-time. In addition, the subproteomes of macular and peripheral RPE were investigated. Differently expressed proteins described developmental processes in neonate tissue and destructive mechanisms in aged samples. Insights into the physiological aging process of the RPE and its conversion into pathophysiological conditions were gained. They assist in designing therapeutical approaches to counteract age-related diseases of the retina.

Clinical application of ultra-wide field fundus autofluorescence imaging

Ultra-wide field fundus autofluorescence (FAF) imaging is a new noninvasive technique with an imaging range of about 200 °. It can detect peripheral retinal lesions that cannot be found in previous FAFs and more objectively reflect intracellular content and distribution of lipofuscin in the retinal pigment epithelium (RPE) and RPE cell metabolic status. The ultra-wide field FAF can find the abnormal autofluorescence (AF) in the peripheral retina of the eyes of age-related macular degeneration (AMD), and different AF manifestations may have an impact on the diagnosis and treatment of the different AMD subtypes. It is helpful to evaluate subretinal fluid in the eyes of central serous choroidal retinopathy and can accurately detect the changes in the outer retina of the eyes without subretinal fluid. It can help to determine the type of uveitis and fully display the evolution of the disease. It can also assess the peripheral photoreceptor cell layer and RPE in patients with retinal dystrophy and retinitis pigmentosa, and comprehensively evaluate their retinal function and monitor the progress of disease. It can also assist in the evaluation of the short-term efficacy and RPE cell function after the scleral buckling surgery for patients with rhegmatogenous retinal detachment. In the future, ultra-wide field FAF may change the knowledge and intervention strategy of ocular fundus diseases and promote the clinical and scientific research in this field. Key words: Retinal diseases/diagnosis; Choroid diseases/diagnosis; Macular degeneration/diagnosis; Diagnostic techniques, ophthalmological; Fluorescence; Review

RETINAL DETACHMENT ASSOCIATED WITH PERIPHERAL TEAR OF THE RETINAL PIGMENT EPITHELIUM IN PACHYCHOROID SPECTRUM DISEASE

To describe a rare case of an exudative retinal detachment associated with a peripheral tear of the retinal pigment epithelium in a young myopic pachychoroid eye. Clinical case report and literature review. Imaging was obtained with color fundus photography, spectral domain optical coherence tomography, fluorescein angiography, and fluorescein angiography. A 33-year-old white man presented with an exudative choroidopathy resulting in an exudative neurosensory detachment, associated with peripheral retinal pigment epithelium tears. Spectral domain optical coherence tomography demonstrated a pachychoroid (628 μm) with subfoveal fluid. Despite a secondary pars-plana vitrectomy with silicone oil and later, Densiron-68 (Labtician Ophthalmics Inc) endotamponade, there was an area of persistent subretinal fluid inferiorly. A previously placed scleral buckle was removed, and scleral window surgery performed, with complete resolution of fluid and restoration of vision. Choroidal thickness improved to 333 μm postoperatively. Peripheral retinal pigment epithelium tear-associated retinal detachment can be seen in patients with pachychoroid spectrum disease. Scleral window surgery may be considered in the management of these retinal detachments.

Mislocalisation of BEST1 in iPSC-derived retinal pigment epithelial cells from a family with autosomal dominant vitreoretinochoroidopathy (ADVIRC).

Autosomal dominant vitreoretinochoroidopathy (ADVIRC) is a rare, early-onset retinal dystrophy characterised by distinct bands of circumferential pigmentary degeneration in the peripheral retina and developmental eye defects. ADVIRC is caused by mutations in the Bestrophin1 (BEST1) gene, which encodes a transmembrane protein thought to function as an ion channel in the basolateral membrane of retinal pigment epithelial (RPE) cells. Previous studies suggest that the distinct ADVIRC phenotype results from alternative splicing of BEST1 pre-mRNA. Here, we have used induced pluripotent stem cell (iPSC) technology to investigate the effects of an ADVIRC associated BEST1 mutation (c.704T > C, p.V235A) in patient-derived iPSC-RPE. We found no evidence of alternate splicing of the BEST1 transcript in ADVIRC iPSC-RPE, however in patient-derived iPSC-RPE, BEST1 was expressed at the basolateral membrane and the apical membrane. During human eye development we show that BEST1 is expressed more abundantly in peripheral RPE compared to central RPE and is also expressed in cells of the developing retina. These results suggest that higher levels of mislocalised BEST1 expression in the periphery, from an early developmental stage, could provide a mechanism that leads to the distinct clinical phenotype observed in ADVIRC patients.

Proliferation and Migration of Peripheral Retinal Pigment Epithelial Cells Are Associated with the Upregulation of Wingless-Related Integration and Bone Morphogenetic Protein Signaling in Dark Agouti Rats

Objective: The aim of this study was to investigate the possible migration of proliferating peripheral retinal pigment epithelial (RPE) cells and their association with differential gene expressions. Materials and Methods: The RPE layer was obtained from the inner aspect of the eyeball of dark agouti rats (12-13 weeks old) and was mounted on glass slides. The peripheral RPE cell proliferation was evaluated using bromodeoxyuridine immunohistochemistry (n = 10). The cell migration was examined using the Dil tracer technique (n = 40) at the end of weeks 6, 10, 14 and 18. Affymetrix microarray analysis was used to investigate differential gene expressions in peripheral and central RPE cells, which was authenticated by RT-PCR using 4 RPE-specific genes (n = 10). Results: In this study, peripheral RPE cells divided and appeared in clusters, but equatorial and central RPE cells rarely divided. The peripheral RPE cells migrated to the central RPE region in a time-dependent manner up to the end of week 14, but not later. The microarray analysis showed the expression of 9,645 out of a total of 35,220 genes studied. Among the 9,645 genes, 573 were differentially expressed (438 were upregulated and 135 were downregulated) in peripheral RPE cells as compared to central RPE cells. Of these 573 genes, 56 were associated with signaling pathways related to the regulation of cell proliferation, including Pax6, TGFβ, BMP and Wnt, and 404 were associated with pathways of cell migration. Conclusions: In this study, peripheral RPE cells divided and migrated to the central region. This process was associated with differential gene expressions in these cells.

Prohibitin as the Molecular Binding Switch in the Retinal Pigment Epithelium.

Previously, our molecular binding study showed that prohibitin interacts with phospholipids, including phosphatidylinositide and cardiolipin. Under stress conditions, prohibitin interacts with cardiolipin as a retrograde response to activate mitochondrial proliferation. The lipid-binding switch mechanism of prohibitin with phosphatidylinositol-3,4,5-triphosphate and cardiolipin may suggest the role of prohibitin effects on energy metabolism and age-related diseases. The current study examined the region-specific expressions of prohibitin with respect to the retina and retinal pigment epithelium (RPE) in age-related macular degeneration (AMD). A detailed understanding of prohibitin binding with lipids, nucleotides, and proteins shown in the current study may suggest how molecular interactions control apoptosis and how we can intervene against the apoptotic pathway in AMD. Our data imply that decreased prohibitin in the peripheral RPE is a significant step leading to mitochondrial dysfunction that may promote AMD progression.

Investigating mitochondria as a target for treating age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness among older adults in the developed world. Although the pathological mechanisms have not been definitively elucidated, evidence suggests a key role for mitochondrial (mt) dysfunction. The current study used our unique collection of human retinal samples graded for the donor's stage of AMD to address fundamental questions about mtDNA damage in the retina. To evaluate the distribution of mtDNA damage in the diseased retina, damage in the retinal pigment epithelium (RPE) and neural retina from individual donors were compared. To directly test a long-held belief that the macula is selectively damaged with AMD, RPE mtDNA damage was measured in the macula and peripheral sections from individual donors. Small segments of the entire mt genome were examined to determine whether specific regions are preferentially damaged. Our results show that mtDNA damage is limited to the RPE, equivalent mtDNA damage is found in the macular and peripheral RPE, and sites of damage are localized to regions of the mt genome that may impact mt function. These results provide a scientific basis for targeting the RPE mitochondria with therapies that protect and enhance mt function as a strategy for combating AMD.

Selective Impairment of a Subset of Ran-GTP-binding Domains of Ran-binding Protein 2 (Ranbp2) Suffices to Recapitulate the Degeneration of the Retinal Pigment Epithelium (RPE) Triggered by Ranbp2 Ablation

Retinal pigment epithelium (RPE) degeneration underpins diseases triggered by disparate genetic lesions, noxious insults, or both. The pleiotropic Ranbp2 controls the expression of intrinsic and extrinsic pathological stressors impinging on cellular viability. However, the physiological targets and mechanisms controlled by Ranbp2 in tissue homeostasis, such as RPE, are ill defined. We show that mice, RPE-cre::Ranbp2(-/-), with selective Ranbp2 ablation in RPE develop pigmentary changes, syncytia, hypoplasia, age-dependent centrifugal and non-apoptotic degeneration of the RPE, and secondary leakage of choriocapillaris. These manifestations are accompanied by the development of F-actin clouds, metalloproteinase-11 activation, deregulation of expression or subcellular localization of critical RPE proteins, atrophic cell extrusions into the subretinal space, and compensatory proliferation of peripheral RPE. To gain mechanistic insights into what Ranbp2 activities are vital to the RPE, we performed genetic complementation analyses of transgenic lines of bacterial artificial chromosomes of Ranbp2 harboring loss of function of selective Ranbp2 domains expressed in a Ranbp2(-/-) background. Among the transgenic lines produced, only Tg(RBD2/3*-HA)::RPE-cre::Ranbp2(-/-)-expressing mutations, which selectively impair binding of RBD2/3 (Ran-binding domains 2 and 3) of Ranbp2 to Ran-GTP, recapitulate RPE degeneration, as observed with RPE-cre::Ranbp2(-/-). By contrast, Tg(RBD2/3*-HA) expression rescues the degeneration of cone photoreceptors lacking Ranbp2. The RPE of RPE-cre::Ranbp2(-/-) and Tg(RBD2/3*-HA)::RPE-cre::Ranbp2(-/-) share proteostatic deregulation of Ran GTPase, serotransferrin, and γ-tubulin and suppression of light-evoked electrophysiological responses. These studies unravel selective roles of Ranbp2 and its RBD2 and RBD3 in RPE survival and functions. We posit that the control of Ran GTPase by Ranbp2 emerges as a novel therapeutic target in diseases promoting RPE degeneration.

Enhanced retinal pigment epithelium regeneration after injury in MRL/MpJ mice

Regenerative medicine holds the promise of restoring cells and tissues that are destroyed in human disease, including degenerative eye disorders. However, development of this approach in the eye has been limited by a lack of animal models that show robust regeneration of ocular tissue. Here, we test whether MRL/MpJ mice, which exhibit enhanced wound healing, can efficiently regenerate the retinal pigment epithelium (RPE) after an injury that mimics the loss of this tissue in age-related macular degeneration. The RPE of MRL/MpJ and control AKR/J mice was injured by retro-orbital injection of sodium iodate at 20 mg/kg body weight, which titration studies indicated was optimal for highlighting strain differences in the response to injury. Five days after sodium iodate injection at this dose, electroretinography of both strains revealed equivalent retinal responses that were significantly reduced compared to untreated mice. At one and two months post-injection, retinal responses were restored in MRL/MpJ but not AKR/J mice. Bright field and fluorescence microscopy of eyecup cryosections indicated an initial central loss of RPE cells and RPE65 immunostaining in MRL/MpJ and AKR/J mice, with preservation of peripheral RPE. Phalloidin staining of posterior eye whole mounts confirmed this pattern of RPE loss, and revealed a transition region characterized by RPE cell shedding and restructuring in both strains, suggesting a similar initial response to injury. At one month post-injection, central RPE cells, RPE65 immunostaining and phalloidin staining were restored in MRL/MpJ but not AKR/J mice. BrdU incorporation was observed throughout the RPE of MRL/MpJ but not AKR/J mice after one month of administration following sodium iodate treatment, consistent with RPE proliferation. These findings provide evidence for a dramatic regeneration of the RPE after injury in MRL/MpJ mice that supports full recovery of retinal function, which has not been observed previously in mammalian eyes. This model should prove useful for understanding molecular mechanisms that underlie regeneration, and for identifying factors that promote RPE regeneration in age-related macular degeneration and related diseases.

Human retinal pigment epithelial SPARC expression and age: an immunohistochemical study.

The aim of this study was to investigate the temporal and spatial expression of the matricellular protein SPARC (Secreted Protein, Acidic and Rich in Cysteine; also known as osteonectin) in human retinal pigment epithelial (RPE) cells, and compare the results with Bruch's membrane thickness, employing immuno-histochemistry. Eyes from 36 human donors, 16 being <or=65 and 20 >65 years old, were included in the study. Intensity of SPARC immunoreactivity was evaluated using Aequitas Image Analysis software with two-way analysis of variance (ANOVA). Bruch's membrane thickness was assessed by profile analysis and the association between RPE SPARC immunoreactivity and Bruch's membrane thickness was investigated by fitting a linear mixed effects model to the data set. Intensity of SPARC immunostaining in RPE cells was significantly lower in older donors (p<0.05 and p<0.001 for posterior and peripheral RPE cells, respectively). The anatomical localisation of the RPE cells also affected the intensity of SPARC staining, which was lower in posterior compared to peripheral cells (p<0.01). No correlation was observed between SPARC immunoreactivity in RPE cells and the thickness of the underlying Bruch's membrane, in either posterior or peripheral regions. Our results suggest that RPE cell SPARC levels decline with age, a change that may play a role in the pathogenesis of age-related diseases such as age-related macular degeneration.