Retinal Pigment Epithelial Cell Monolayer Research Articles

Anaphylatoxin C5a receptor signaling induces mitochondrial fusion and sensitizes retinal pigment epithelial cells to oxidative stress

Mitochondrial dynamics is a morphological balance between fragmented and elongated shapes, reflecting mitochondrial metabolic status, cellular damage, and mitochondrial dysfunction. The anaphylatoxin C5a derived from complement component 5 cleavage, enhances cellular responses involved in pathological stimulation, innate immune responses, and host defense. However, the specific response of C5a and its receptor, C5a receptor (C5aR), in mitochondria is unclear. Here, we tested whether the C5a/C5aR signaling axis affects mitochondrial morphology in human-derived retinal pigment epithelial cell monolayers (ARPE-19). C5aR activation with the C5a polypeptide induced mitochondrial elongation. In contrast, oxidatively stressed cells (H2O2) responded to C5a with an enhancement of mitochondrial fragmentation and an increase in the number of pyknotic nuclei. C5a/C5aR signaling increased the expression of mitochondrial fusion-related protein, mitofusin-1 (MFN1) and − 2 (MFN2), as well as enhanced optic atrophy-1 (Opa1) cleavage, which are required for mitochondrial fusion events, whereas the mitochondrial fission protein, dynamin-related protein-1 (Drp1), and mitogen-activated protein kinase (MAPK)-dependent extracellular signal-regulated protein kinase (Erk1/2) phosphorylation were not affected. Moreover, C5aR activation increased the frequency of endoplasmic reticulum (ER)-mitochondria contacts. Finally, oxidative stress induced in a single cell within an RPE monolayer (488 nm blue laser spot stimulation) induced a bystander effect of mitochondrial fragmentation in adjacent surrounding cells only in C5a-treated monolayers. These results suggest that C5a/C5aR signaling produced an intermediate state, characterized by increased mitochondrial fusion and ER-mitochondrial contacts, that sensitizes cells to oxidative stress, leading to mitochondrial fragmentation and cell death.

Dabigatran and Wet AMD, Results From Retinal Pigment Epithelial Cell Monolayers, the Mouse Model of Choroidal Neovascularization, and Patients From the Medicare Data Base.

BackgroundAge-related macular degeneration (AMD), the leading cause of irreversible blindness in elderly Caucasian populations, includes destruction of the blood-retina barrier (BRB) generated by the retinal pigment epithelium-Bruch’s membrane complex (RPE/BrM), and complement activation. Thrombin is likely to get access to those structures upon BRB integrity loss. Here we investigate the potential role of thrombin in AMD by analyzing effects of the thrombin inhibitor dabigatran.Material and MethodsMarketScan data for patients aged ≥65 years on Medicare was used to identify association between AMD and dabigatran use. ARPE-19 cells grown as mature monolayers were analyzed for thrombin effects on barrier function (transepithelial resistance; TER) and downstream signaling (complement activation, expression of connective tissue growth factor (CTGF), and secretion of vascular endothelial growth factor (VEGF)). Laser-induced choroidal neovascularization (CNV) in mouse is used to test the identified downstream signaling.ResultsRisk of new wet AMD diagnosis was reduced in dabigatran users. In RPE monolayers, thrombin reduced TER, generated unique complement C3 and C5 cleavage products, led to C3d/MAC deposition on cell surfaces, and increased CTGF expression via PAR1-receptor activation and VEGF secretion. CNV lesion repair was accelerated by dabigatran, and molecular readouts suggest that downstream effects of thrombin include CTGF and VEGF, but not the complement system.ConclusionsThis study provides evidence of association between dabigatran use and reduced exudative AMD diagnosis. Based on the cell- and animal-based studies, we suggest that thrombin modulates wound healing and CTGF and VEGF expression, making dabigatran a potential novel treatment option in AMD.

Bruch's-Mimetic Nanofibrous Membranes Functionalized with the Integrin-Binding Peptides as a Promising Approach for Human Retinal Pigment Epithelium Cell Transplantation.

Background: This study aimed to develop an ultrathin nanofibrous membrane able to, firstly, mimic the natural fibrous architecture of human Bruch’s membrane (BM) and, secondly, promote survival of retinal pigment epithelial (RPE) cells after surface functionalization of fibrous membranes. Methods: Integrin-binding peptides (IBPs) that specifically interact with appropriate adhesion receptors on RPEs were immobilized on Bruch’s-mimetic membranes to promote coverage of RPEs. Surface morphologies, Fourier-transform infrared spectroscopy spectra, contact angle analysis, Alamar Blue assay, live/dead assay, immunofluorescence staining, and scanning electron microscopy were used to evaluate the outcome. Results: Results showed that coated membranes maintained the original morphology of nanofibers. After coating with IBPs, the water contact angle of the membrane surfaces varied from 92.38 ± 0.67 degrees to 20.16 ± 0.81 degrees. RPE cells seeded on IBP-coated membranes showed the highest viability at all time points (Day 1, p < 0.05; Day 3, p < 0.01; Days 7 and 14, p < 0.001). The proliferation rate of RPE cells on uncoated poly(ε-caprolactone) (PCL) membranes was significantly lower than that of IBP-coated membranes (p < 0.001). SEM images showed a well-organized hexa/polygonal monolayer of RPE cells on IBP-coated membranes. RPE cells proliferated rapidly, contacted, and became confluent. RPE cells formed a tight adhesion with nanofibers under high-magnification SEM. Our findings confirmed that the IBP-coated PCL membrane improved the attachment, proliferation, and viability of RPE cells. In addition, in this study, we used serum-free culture for RPE cells and short IBPs without immunogenicity to prevent graft rejection and immunogenicity during transplantation. Conclusions: These results indicated that the biomimic BM-IBP-RPE nanofibrous graft might be a new, practicable approach to increase the success rate of RPE cell transplantation.

Corneal Application of R9-SOCS1-KIR Peptide Alleviates Endotoxin-Induced Uveitis.

PurposeUveitis is an ocular inflammation that can affect individuals of all ages and is a major cause of blindness. We have tested the therapeutic efficacy of a cell penetrating peptide from the kinase inhibitory region of suppressor of cytokine signaling 1, denoted as R9-SOCS1-KIR.MethodsWe stimulated J774A.1 cells with lipopolysaccharide (LPS) in the presence of R9-SOCS1-KIR or its inactive control peptide. Effect on inflammatory pathways was followed by the nuclear translocation of nuclear factor κB p65 subunit and phosphorylated-p38. Synthesis of inflammatory markers induced by LPS was tested by reverse transcriptase polymerase chain reaction, Western blot analysis, and ELISA of cell supernatants. We monitored effects on the barrier properties of a differentiated ARPE-19 monolayer treated with LPS. We treated C57BL/6 mice topically with either R9-SOCS1-KIR or vehicle and injected their eyes intravitreally with LPS. Eyes were analyzed by fundoscopy, fluorescein angiography, optical coherence tomography, histology, Western blotting, multiplex enzyme-linked immunosorbent assay, and flow cytometry.ResultsTreatment with R9-SOCS1-KIR resulted in suppression of signaling through nuclear factor κB and p-p38 pathways. R9-SOCS1-KIR suppressed the expression of inflammatory genes, the secretion of inflammatory makers such as nitric oxide, and IL-1β induced by LPS. Increased permeability of retinal pigment epithelial cell monolayers was prevented. Corneal administration of R9-SOCS1-KIR blocked the acute inflammation observed in LPS-injected mouse eyes.ConclusionsTreatment with R9-SOCS1-KIR alleviated the inflammatory responses in cell culture. Topical delivery of this peptide on mouse eyes protected against LPS-induced damage.Translational RelevanceTopical delivery of R9-SOCS1-KIR peptide allows the patient to self-administer the drug, while preventing any systemic effects on unrelated organs.

Mechanisms of extracellular vesicle uptake in stressed retinal pigment epithelial cell monolayers

PurposeExtracellular vesicles (EVs) can mediate long-distance communication in polarized RPE monolayers. Specifically, EVs from oxidatively stressed donor cells (stress EVs) rapidly reduced barrier function (transepithelial resistance, TER) in naïve recipient monolayers, when compared to control EVs. This effect on TER was dependent on dynamin-mediated EV uptake, which occurred rapidly with EVs from oxidatively stressed donor cells. Here, we further determined molecular mechanisms involved in uptake of EVs by naïve RPE cells. MethodsRPE cells were grown as monolayers in media supplemented with 1% FBS followed by transfer to FBS-free media. Cultures were used to collect control or stress EVs upon treatment with H2O2, others served as naïve recipient cells. In recipient monolayers, TER was used to monitor EV-uptake-based activity, live-cell imaging confirmed uptake. EV surface proteins were quantified by protein chemistry. ResultsClathrin-independent, lipid raft-mediated internalization was excluded as an uptake mechanism. Known ligand-receptor interactions involved in clathrin-dependent endocytosis include integrins and proteoglycans. Desialylated glycans and integrin-receptors on recipient cells were necessary for EV uptake and subsequent reduction of TER in recipient cells. Protein quantifications confirmed elevated levels of ligands and neuraminidase on stress EVs. However, control EVs could confer activity in the TER assay if exogenous neuraminidase or additional ligand was provided. ConclusionsIn summary, while EVs from both stressed cells and control contain cargo to communicate stress messages to naive RPE cells, stress EVs contain surface ligands that confer rapid uptake by recipient cells. We propose that EVs potentially contribute to RPE dysfunction in aging and disease.

Molecular Basis of The Retinal Pigment Epithelial Changes That Characterize The Ocular Lesion in Toxoplasmosis.

When a person becomes infected with Toxoplasma gondii, ocular toxoplasmosis is the most common clinical presentation. The medical literature describes retinitis with surrounding hyperpigmentation secondary to proliferative changes in the retinal pigment epithelium, which is sufficiently characteristic that investigation often is not needed to make the diagnosis. We aimed to establish the frequency of “typical” ocular toxoplasmosis and delineate its molecular basis. Among 263 patients presenting consecutively with ocular toxoplasmosis to Ribeirão Preto General Hospital in Brazil, where T. gondii infection is endemic, 94.2% of 345 eyes had retinal hyperpigmentation. In ARPE-19 and primary human retinal pigment epithelial cell monolayers exposed to minimal numbers of T. gondii tachyzoites, the proliferation marker–KI-67–was increased in uninfected cells, which also were rendered more susceptible to infection. RT-qPCR and ELISA detected increased expression of vascular endothelial growth factor A (VEGF) and insulin-like growth factor (IGF)1, and decreased expression of thrombospondin (TSP)1 by infected cells. Blockade of VEGF and IGF1—or supplementation of TSP1—reversed the proliferation phenotype in uninfected cells. Our findings confirm that hyperpigmentation is a characteristic feature of retinitis in ocular toxoplasmosis, and demonstrate that T. gondii-infected human retinal pigment epithelial cells secrete VEGF and IGF1, and reduce production of TSP1, to promote proliferation of adjacent uninfected cells and create this disease-specific appearance.

Characterization of a tissue-engineered choroid

The choroid of the eye is a vascularized and pigmented connective tissue lying between the retina and the sclera. Increasing evidence demonstrates that, beyond supplying nutrients to the outer retina, the different choroidal cells contribute to the retina’s homeostasis, especially by paracrine signaling. However, the precise role of each cell type is currently unclear. Here, we developed a choroidal substitute using the self-assembly approach of tissue engineering. Retinal pigment epithelial (RPE) cells, as well as choroidal stromal fibroblasts, vascular endothelial cells and melanocytes, were isolated from human eye bank donor eyes. Fibroblasts were cultured in a medium containing serum and ascorbic acid. After six weeks, cells formed sheets of extracellular matrix (ECM), which were stacked to produce a tissue-engineered choroidal stroma (TECS). These stromal substitutes were then characterized and compared to the native choroid. Their ECM composition (collagens and proteoglycans) and biomechanical properties (ultimate tensile strength, strain and elasticity) were similar. Furthermore, RPE cells, human umbilical vein endothelial cells and choroidal melanocytes successfully repopulated the stromas. Physiological structures were established, such as a confluent monolayer of RPE cells, vascular-like structures and a pigmentation of the stroma. Our TECS thus recaptured the biophysical environment of the native choroid, and can serve as study models to understand the normal interactions between the RPE and choroidal cells, as well as their reciprocal exchanges with the ECM. This will consequently pave the way to derive accurate insight in the pathophysiological mechanisms of diseases affecting the choroid. Statement of significanceThe choroid is traditionally known for supplying blood to the avascular outer retina. There has been a renewed attention directed towards the choroid partly due to its implication in the development of age-related macular degeneration (AMD), the leading cause of blindness in industrialized countries. Since AMD involves the dysfunction of the choroid/retinal pigment epithelium (RPE) complex, a three-dimensional (3D) model of RPE comprising the choroid layer is warranted. We used human choroidal cells to engineer a choroidal substitute. Our approach takes advantage of the ability of cells to recreate their own environment, without exogenous materials. Our model could help to better understand the role of each choroidal cell type as well as to advance the development of new therapeutics for AMD.

Extracellular vesicle-mediated long-range communication in stressed retinal pigment epithelial cell monolayers

Retinal pigment epithelium (RPE) alterations in age-related macular degeneration occur in patches, potentially involving long-distance communication between damaged and healthy areas. Communication along the epithelium might be mediated by extracellular vesicles (EVs). To test this hypothesis, EVs were collected from supernatants of polarized ARPE-19 and primary porcine RPE monolayers for functional and biochemical assays. EVs from oxidatively stressed donor cells reduced barrier function in recipient RPE monolayers when compared to control EVs. The effect on barrier function was dependent on EV uptake, which occurred rapidly with EVs from oxidatively stressed donor cells. Mass spectrometry-based proteomic analysis of EVs identified HDAC6, which is known to reduce tight junction stability. Activity assays confirmed the presence of HDAC6 in EVs, and EV transfer assays using HDAC6 inhibitors confirmed its effect in monolayers. These findings demonstrate that EVs can communicate stress messages to healthy RPE cells, potentially contributing to RPE dysfunction.

Nanofibrous carrier for transplantation of retinal pigment epithelial cells

SummaryThe most common retinal degenerative diseases such as age‐related macular degeneration (AMD) and retinitis pigmentosa belong to serious eye diseases that cause dominant number of cases of impaired vision or even legal blindness. Because there is no treatment method to stop progression of these diseases or even treat them, it is supposable that cell therapy based on replacement of retinal pigment epithelial (RPE) cells can serve as an effective treatment for AMD and other degenerative diseases. The study was aimed at development of a new delivery technique for transplantation of RPE cells using two complementary delivery devices: a membraneous cell carrier and an injector as trans‐scleral implantation instrument. The design of the suggested nanofibrous carrier with a peripheral frame was optimized to provide a minimal diffusion barrier for the flow of nutrients in the subretinal space and at the same time to resist surgical handling. An implantation instrument together with the cell carrier ensured a safe transfer of an organized RPE cell monolayer into the subretinal space. Different designs of the carrier seeded with cultured RPE cells and the implantation instrument was assessed in both ex vivo and in vivo animal experiments.

The formation of a functional retinal pigment epithelium occurs on porous polytetrafluoroethylene substrates independently of the surface chemistry

Subretinal transplantation of functioning retinal pigment epithelial (RPE) cells may have the potential to preserve or restore vision in patients affected by blinding diseases such as age-related macular degeneration (AMD). One of the critical steps in achieving this is the ability to grow a functioning retinal pigment epithelium, which may need a substrate on which to grow and to aid transplantation. Tailoring the physical and chemical properties of the substrate should help the engineered tissue to function in the long term. The purpose of the study was to determine whether a functioning monolayer of RPE cells could be produced on expanded polytetrafluoroethylene substrates modified by either an ammonia plasma treatment or an n-Heptylamine coating, and whether the difference in surface chemistries altered the extracellular matrix the cells produced. Primary human RPE cells were able to form a functional, cobblestone monolayer on both substrates, but the formation of an extracellular matrix to exhibit a network structure took months, whereas on non-porous substrates with the same surface chemistry, a similar appearance was observed after a few weeks. This study suggests that the surface chemistry of these materials may not be the most critical factor in the development of growth of a functional monolayer of RPE cells as long as the cells can attach and proliferate on the surface. This has important implications in the design of strategies to optimise the clinical outcomes of subretinal transplant procedures.Graphical

Potential Retinal Benefits of Dietary Polyphenols Based on Their Permeability across the Blood-Retinal Barrier.

Whether all dietary polyphenols nourish the eyes via oral supplementation is controversial. Given that passage of dietary polyphenols across the blood-retina barrier (BRB) is the precondition for polyphenols to exhibit ocular benefits, the BRB permeability of polyphenols was assessed in this study. Being common dietary polyphenols in fruits and vegetables, nonanthocyanin flavonoids, anthocyanins, and phenolic acids were investigated. BRB was simulated in vitro by using a differentiated retinal pigment epithelial cell monolayer cultivated on a Transwell culture system. Penetration rate was calculated by quantitatively analyzing the polyphenols in basolateral media. The BRB permeability of different polyphenols obviously (p < 0.05) differed, as follows: phenolic acids > nonanthocyanin flavonoids > anthocyanins. Glycosylation and methylation improved the BRB permeability of nonanthocyanin flavonoids and anthocyanins. However, instability and carbonylation at the C-4 position severely suppressed the BRB permeability of anthocyanins and nonanthocyanin flavonoids. Moreover, a new metabolite was discovered during penetration of anthocyanins into the BRB. However, hydrophilic phenolic acids exhibited better BRB permeability than hydrophobic ones. Data demonstrate that BRB permeability of polyphenols was determined based on structural characteristics, hydrophilicity, stability, and metabolic changes.

Fundus autofluorescence imaging in dry AMD: 2014 Jules Gonin lecture of the Retina Research Foundation

Fundus autofluorescence (FAF) imaging allows for topographic mapping of intrisnic fluorophores in the retinal pigment epithelial cell monolayer, as well as mapping of other fluorophores that may occur with disease in the outer retina and the sub-neurosensory space. FAF imaging provides information not obtainable with other imaging modalities. Near-infrared fundus autofluorescence images can also be obtained in vivo, and may be largely melanin-derived. FAF imaging has been shown to be useful in a wide spectrum of macular and retinal diseases. The scope of applications now includes identification of diseased RPE in macular/retinal diseases, elucidating pathophysiological mechanisms, identification of early disease stages, refined phenotyping, identification of prognostic markers for disease progression, monitoring disease progression in the context of both natural history and interventional therapeutic studies, and objective assessment of luteal pigment distribution and density as well as RPE melanin distribution. Here, we review the use of FAF imaging in various phenotypic manifestations of dry AMD.

Polarized Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cell Monolayers Have Higher Resistance to Oxidative Stress-Induced Cell Death Than Nonpolarized Cultures

Oxidative stress-mediated injury to the retinal pigment epithelium (RPE) is a major factor involved in the pathogenesis of age-related macular degeneration (AMD), the leading cause of blindness in the elderly. Human embryonic stem cell (hESC)-derived RPE cells are currently being evaluated for their potential for cell therapy in AMD patients through subretinal injection of cells in suspension and subretinal placement as a polarized monolayer. To gain an understanding of how transplanted RPE cells will respond to the highly oxidatively stressed environment of an AMD patient eye, we compared the survival of polarized and nonpolarized RPE cultures following oxidative stress treatment. Polarized, nonpolarized/confluent, nonpolarized/subconfluent hESC-RPE cells were treated with H2O2. Terminal deoxynucleotidyl transferase dUTP nick end labeling stains revealed the highest amount of cell death in subconfluent hESC-RPE cells and little cell death in polarized hESC-RPE cells with H2O2 treatment. There were higher levels of proapoptotic factors (phosphorylated p38, phosphorylated c-Jun NH2-terminal kinase, Bax, and cleaved caspase 3 fragments) in treated nonpolarized RPE-particularly subconfluent cells-relative to polarized cells. On the other hand, polarized RPE cells had constitutively higher levels of cell survival and antiapoptotic signaling factors such as p-Akt and Bcl-2, as well as antioxidants superoxide dismutase 1 and catalase relative to nonpolarized cells, that possibly contributed to polarized cells' higher tolerance to oxidative stress compared with nonpolarized RPE cells. Subconfluent cells were particularly sensitive to oxidative stress-induced apoptosis. These results suggest that implantation of polarized hESC-RPE monolayers for treating AMD patients with geographic atrophy should have better survival than injections of hESC-RPE cells in suspension.

Fundus Autofluorescence in Geographic Atrophy: A Review

Fundus autofluorescence is a noninvasive imaging technology that provides information on the distribution of lipofuscin within the retinal pigment epithelial cell monolayer. Progressive accumulation of lipofuscin within retinal pigment epithelial cells is involved in the pathogenesis of geographic atrophy in age-related macular degeneration. This review contains an introduction to fundus autofluorescence, review of currently available imaging methods, and discussion of the role of autofluorescence imaging in geographic atrophy progression. The recent classification of geographic atrophy phenotypes by the Fundus Autofluorescence in Age-related Macular Degeneration Study (FAM) and the association of phenotype and atrophy progression are also summarized.

Fundus Autofluorescence and Progression of Age-related Macular Degeneration

Fundus autofluorescence imaging is an imaging method that provides additional information compared to conventional imaging techniques. It permits to topographically map lipofuscin distribution of the retinal pigment epithelial cell monolayer. Excessive accumulation of lipofuscin granules in the lysosomal compartment of retinal pigment epithelium cells represents a common downstream pathogenetic pathway in various hereditary and complex retinal diseases including age-related macular degeneration (AMD). This comprehensive review contains an introduction in fundus autofluorescence imaging, including basic considerations, the origin of the signal, different imaging methods, and a brief overview of fundus autofluorescence findings in normal subjects. Furthermore, it summarizes cross-sectional and longitudinal fundus autofluorescence findings in patients with AMD, addresses the pathophysiological significance of increased fundus autofluorescence, and characterizes different fundus autofluorescence phenotypes as well as fundus autofluorescence alterations with disease progression.

Polyurethanes as potential substrates for sub-retinal retinal pigment epithelial cell transplantation

Transplantation of cultured retinal pigment epithelial (RPE) cells under the failing macular is a potential treatment for age related macular degeneration. An important step in the development of this procedure is the identification of a suitable membrane on which to grow and transplant the cells. This paper evaluates the potential of using polyurethanes in this application since they possess several of the required properties, such as, flexibility, robustness, biostability and good biocompatiblilty although their hydrophobicity can limit cell adhesion. Three commercially available polyether urethanes (Pellethane, Tecoflex and Zytar) were evaluated in terms of their wettability using dynamic contact angle analysis and their ability to support a monolayer of functioning RPE cells (ARPE-19) . Furthermore Pellethane and Tecoflex were treated with a simple air plasma treatment and analysed as above. In the "as received condition" only a few RPE cells attached to the Pellethane and Tecoflex and remained clumped. RPE cells grew to confluence on the Zytar substrate by 7 days without further surface modification. Air gas plasma treatment of both Pellethane and Tecoflex increased the wettability of the surfaces and this resulted in the growth of a monolayer of well-spread RPE cells on both materials. Morphologically these cells grew with a normal 'cobblestone' phenotype. These results demonstrate the potential of these polyurethanes for this application.

Aquaporin-1 channels in human retinal pigment epithelium: role in transepithelial water movement.

Aquaporin (AQP) is a hexahelical integral membrane protein that functions as a constitutive channel for water and regulated channel for cations in fluid transporting tissues, including many in the eye. Although AQP1 has been cloned from a cDNA library prepared from cultures of retinal pigment epithelial (RPE) cells isolated from human fetal tissue, three separate studies failed with various immunochemical techniques to detect AQP1 protein in adult human or rat RPE preparations. The purpose of this study was to examine specifically the expression and distribution of AQP1 in adult human RPE in situ by using alternative methodologies and model systems and to determine the contribution of AQP1 to water movement across cultured RPE cells isolated from human cadaveric and fetal eyes. AQP1 in human RPE in situ was determined after biotinylation of proteins on cell surfaces and streptavidin chromatography, followed by immunoblot analyses. AQP1 distribution in a polarized in vitro RPE model was determined with indirect immunofluorescence confocal microscopy. The role of channel-mediated transport of water across RPE cell monolayers on filters was assessed by osmotic challenge assay. Expression levels of AQP1 were controlled with an adenovirus expression system and monitored by immunoblot analyses. AQP1 protein was detected in human RPE in situ and in cultures of human adult and fetal RPE cells. In functional assays, AQP1 facilitated water movement across RPE monolayers in an expression-dependent manner in two complementary model systems. The expression of AQP1 by RPE in vivo probably contributes to the efficient transepithelial water transport across RPE, maintains retinal attachment, and prevents subretinal edema.

Ultrastructural analysis of hydraulic and abrasive retinal pigment epithelial cell debridements

Differential changes in Bruch's membrane, choriocapillaris, retinal pigment epithelium, retina, and tapetum after hydraulic or abrasive debridement of the retinal pigment epithelium in the cat area centralis were documented by fluorescein angiography, histology, and transmission electron microscopy at 1-hour, 1-day, 3-day, 1-week, or 4-week time points. Abrasive debridement is associated with abnormal fluorescein angiography and incomplete ingrowth of retinal pigment epithelial cells. Transmission electron microscopy shows that abrasive debridement inflicts more long-lasting ultrastructural damage to Bruch's membrane, the choriocapillaris, tapetum, and retina than does hydraulic debridement. Because the retinal pigment epithelium can resurface abrasively debrided Bruch's membrane that is disorganized, split, reduplicated, or missing, we cannot correlate the ultrastructural appearance of Bruch's membrane with the likelihood of complete resurfacing of the debrided area. Primary choriocapillary or retinal damage in abrasive debridements may contribute to the poor outcome. Regions of retinal degeneration with no underlying retinal pigment epithelial cell monolayer were significantly larger in abrasive debridements at the 4-week than at the 1-week time point. Reduced resurfacing at the later time point suggests that not all cells resurfacing abrasively debrided areas survived over the longer term. This finding may mean that retinal pigment epithelial cells are not able to resurface completely and permanently areas showing geographic atrophy of the choriocapillaris.

Transplantation of allogenic anterior lens capsule to the subretinal space in pigs.

To investigate the consequences of transplantation of a new basement membrane to the subretinal space (SRS) as a substitution of Bruch's membrane. Porcine anterior lens capsules (ALC) were transplanted to the subretinal space of 20 eyes from 19 young Danish landrace pigs. All pigs underwent a three port localized pars plana vitrectomy. Seventeen eyes received naked ALC. In three experiments the ALC was embedded in gelatine, in order to prevent curling of the ALC. The observation period varied between zero and 49 days. The pigs were examined by ophthalmoscopy and fundus photography. Histopathological examination of enucleated eyes was performed at the end of the experiment. ALCs transplanted to the subretinal space were well-tolerated and caused no inflammation when Bruch's membrane was left undamaged. After 11 days host RPE and glial cells started to cover the ALC in a competitive fashion. When Bruch's membrane was damaged, ingrowths of choroidal vessels and fibroblasts was prominent. The use of gelatine to flatten the ALC did not prevent curling, and gelatine caused pronounced inflammation. It is possible to transplant porcine ALC to the SRS of the pig. ALCs are well-tolerated in the SRS and are covered with well-differentiated monolayers of host RPE-cells, if Bruch's membrane is left intact.

Migration of retinal pigment epithelial cells in vitro modulated by monocyte chemotactic protein-1: enhancement and inhibition.

The migration of retinal pigment epithelial (RPE) cells is an initial step in the development of proliferative vitreoretinopathy (PVR). This in vitro study was carried out to investigate the effects of monocyte chemotactic protein-1 (MCP-1) on the migration and proliferation of RPE cells. We used an in vitro wound healing model in which a small area of a confluent monolayer of human RPE (HRPE) cells was denuded with a razor blade. The cultures were subsequently incubated with MCP-1, IL-1beta, TNF-alpha, or combinations thereof. Neutralizing IgG1 of antihuman MCP-1, dexamethasone (DEX) or daunorubicin were also added to the cultures to test their inhibitory effects on migration of RPE cells. HRPE migration was measured as the number of cells that entered the denuded area. The effect of MCP-1 on proliferation of HRPE cells was examined by MTT assay. MCP-1 stimulated HRPE cell migration in a dose-dependent manner. IL-1beta or TNF-alpha slightly stimulated HRPE cell migration, but adding anti-MCP- IgG1 significantly reduced this effect. MCP-1-induced migration could be inhibited by DEX but not by daunorubicin. MCP-1 did not show a significant effect on HRPE cell proliferation. MCP-1 stimulates HRPE cell migration, suggesting that this chemokine regulates the development of PVR at the initial stage. The migration of HRPE cells induced by IL-1beta and TNF-alpha may be associated with the MCP-1 that HRPE cells secretes under the stimulation of these two cytokines. The knowledge that MCP-1-induced migration of HRPE cells is inhibited by DEX may be useful in devising an effective treatment for PVR.