Licarin A as a Novel Drug for Inflammatory Eye Diseases.

Purpose: This study investigated the safety and therapeutic efficacy of licarin A (LCA) in the treatment of intraocular inflammation. Methods:In vitro safety of LCA in retinal pigmented epithelial cells (ARPE-19) and human embryonic stem cell derived-retinal pigmented epithelial cells (hES-RPE) was evaluated using CellTiter-Blue® kit. The chorioallantoic membrane (CAM) assay was used to investigate LCA safety and antiangiogenic activity. In vivo safety of intravitreal LCA was accomplished by clinical examination (including assessment of intraocular pressure), electroretinography (ERG), and histopathology. Uveitis was induced in rats by subcutaneous and intravitreal injection of bacillus Calmette-Guérin (BCG) antigen of Mycobacterium bovis. Intraocular inflammation was graded by slit-lamp and fundus examination, ERG, and histopathology. Results: LCA was safe to cells and to the CAM at concentration below 12.0 µM. LCA significantly reduced the percentage of blood vessels in the CAM. Retinal safety and anti-inflammatory efficacy of intravitreal injection of LCA 6.0 µM were confirmed through clinical, functional, and histopathological evaluation. Significant reduction of inflammatory cytokines (tumor necrosis factor-α and interleukin-6) was also found, when compared to untreated animals. Conclusion: The results suggest that LCA is a potential new drug for the treatment of inflammatory eye disease.

Combined Transplantation With Human Mesenchymal Stem Cells Improves Retinal Rescue Effect of Human Fetal RPE Cells in Retinal Degeneration Mouse Model.

Purpose: We verified whether fetal RPE (fRPE) cells and mesenchymal stem cells (MSCs) cotransplantation can combine the features of these two cell types and alleviate retinal degeneration in a retinal degenerative disease mouse model. Methods: Tail vein injection of sodium iodate (NaIO3) was conducted to establish the retinal degenerative disease mouse model. MSCs and fRPE cells were transplanted either separately or combined in the subretinal space of retinal degenerative disease animals. ERG, optical coherence tomography, histologic, and immunofluorescence analyses were performed. Furthermore, the expression level of Crx, rhodopsin, Iba1, F4/80, Caspase 3, nerve growth factor, and brain-derived neurotrophic factor were assessed to investigate the mechanisms involved in cell transplantation effects. Results: Cotransplantation of fRPE and MSC cells promoted significant improvements in ERG results and in the survival rate of transplanted cells. In addition, MSC and fRPE cell cotransplantation resulted in an increase in the thickness of the total retina, as well as in the outer and inner nuclear layers. Combined transplantation also upregulated the expression level of Crx and rhodopsin and downregulated caspase 3 expression, highlighting its better photoreceptor rescue effect in relation to the single cell type transplantation. Finally, combined transplantation suppressed the expression of Iba1 and F4/80 factors while increasing the endogenous expression of nerve growth factor and brain-derived nerve growth factor neurotrophic factors. These data suggest that MSC and fRPE cell cotransplantation is able to suppress immunoreactions and promote neurotrophic factor excretion. Conclusions: Combined transplantation of MSCs and fRPE cells results in a better retinal rescue effect than single cell type transplantation in NaIO3-induced retinopathy.

Optic vesicle morphogenesis requires primary cilia.

Arl13b is a gene known to regulate ciliogenesis. Functional alterations in this gene's activity have been associated with Joubert syndrome. We found that in Arl13 null mouse embryos the orientation of the optic cup is inverted, such that the lens is abnormally surrounded by an inverted optic cup whose retina pigmented epithelium is oddly facing the surface ectoderm. Loss of Arl13b leads to the disruption of optic vesicle's patterning and expansion of ventral fates. We show that this phenotype is consequence of miss-regulation of Sonic hedgehog (Shh) signaling and demonstrate that the Arl13b-/- eye phenotype can be rescued by deletion of Gli2, a downstream effector of the Shh pathway. This work identified an unexpected role of primary cilia during the morphogenetic movements required for the formation of the eye.

The retinal pigmented epithelium - from basic developmental biology research to translational approaches.

The development of the eye has been a topic of extensive investigation, from the early studies on tissue induction to more recent breakthroughs in resolving the mechanism regulating progenitor patterning and their gradual and coordinated differentiation into diverse tissue types that function together throughout life. Among the ocular tissue types, the retinal pigmented epithelium (RPE) is at the forefront of developmental biology and stem cell research. The growing interest in this lineage stems from its importance for photoreceptor function as well as from its requirement during embryogenesis for the development of the photoreceptors and the choroid. Indeed mutations in RPE genes and epigenetic changes that occur during aging are the cause of monogenic as well as multifactorial retinal diseases. Importantly, the RPE is readily generated from stem cells, and these stem cell-derived RPE cells are currently being tested in clinical trials for transplantation in cases of retinal dystrophies; they also constitute an important model to study developmental processes in vitro. This review summarizes recent advances in our understanding of RPE development and its requirement for the development of photoreceptors and choroidal vasculature. We discuss the contribution of basic findings to therapeutic applications and the future challenges in uncovering developmental processes and mimicking them ex vivo to further advance research and therapy of retinal disorders.

Complement component C3aR constitutes a novel regulator for chick eye morphogenesis.

Complement components have been implicated in a wide variety of functions including neurogenesis, proliferation, cell migration, differentiation, cancer, and more recently early development and regeneration. Following our initial observations indicating that C3a/C3aR signaling induces chick retina regeneration, we analyzed its role in chick eye morphogenesis. During eye development, the optic vesicle (OV) invaginates to generate a bilayer optic cup (OC) that gives rise to the retinal pigmented epithelium (RPE) and neural retina. We show by immunofluorescence staining that C3 and the receptor for C3a (the cleaved and active form of C3), C3aR, are present in chick embryos during eye morphogenesis in the OV and OC. Interestingly, C3aR is mainly localized in the nuclear compartment at the OC stage. Loss of function studies at the OV stage using morpholinos or a blocking antibody targeting the C3aR (anti-C3aR Ab), causes eye defects such as microphthalmia and defects in the ventral portion of the eye that result in coloboma. Such defects were not observed when C3aR was disrupted at the OC stage. Histological analysis demonstrated that microphthalmic eyes were unable to generate a normal optic stalk or a closed OC. The dorsal/ventral patterning defects were accompanied by an expansion of the ventral markers Pax2, cVax and retinoic acid synthesizing enzyme raldh-3 (aldh1a3) domains, an absence of the dorsal expression of Tbx5 and raldh-1 (aldh1a1) and a re-specification of the ventral RPE to neuroepithelium. In addition, the eyes showed overall decreased expression of Gli1 and a change in distribution of nuclear ß-catenin, suggesting that Shh and Wnt pathways have been affected. Finally, we observed prominent cell death along with a decrease in proliferating cells, indicating that both processes contribute to the microphthalmic phenotype. Together our results show that C3aR is necessary for the proper morphogenesis of the OC. This is the first report implicating C3aR in eye development, revealing an unsuspected hitherto regulator for proper chick eye morphogenesis.

Differential behavioral outcomes following neonatal versus fetal human retinal pigment epithelial cell striatal implants in parkinsonian rats.

Following the failure of a Phase II clinical study evaluating human retinal pigment epithelial (hRPE) cell implants as a potential treatment option for Parkinson's disease, speculation has centered on implant function and survival as possible contributors to the therapeutic outcomes. We recently reported that neonatal hRPE cells, similar to hRPE cells used in the Phase II clinical study, produced short-lived in vitro and limited in vivo trophic factors, which supports that assumption. We hypothesize that the switch from fetal to neonatal hRPE cells, between the Phase I and the Phase II clinical trial may be partly responsible for the later negative outcomes. To investigate this hypothesis, we used two neonatal hRPE cell lots, prepared in a similar manner to neonatal hRPE cells used in the Phase II clinical study, and compared them to previously evaluated fetal hRPE cells for behavioral changes following unilateral striatal implantation in 6-hydroxydopamine-lesioned rats. The results showed that only fetal, not neonatal, hRPE cell implants, were able to improve behavioral outcomes following striatal implantation in the lesioned rats. These data suggest that fetal hRPE cells may be preferential to neonatal hRPE cells in restoring behavioral deficits.

Reductive carboxylation is a major metabolic pathway in the retinal pigment epithelium.

The retinal pigment epithelium (RPE) is a monolayer of pigmented cells that requires an active metabolism to maintain outer retinal homeostasis and compensate for oxidative stress. Using 13C metabolic flux analysis in human RPE cells, we found that RPE has an exceptionally high capacity for reductive carboxylation, a metabolic pathway that has recently garnered significant interest because of its role in cancer cell survival. The capacity for reductive carboxylation in RPE exceeds that of all other cells tested, including retina, neural tissue, glial cells, and a cancer cell line. Loss of reductive carboxylation disrupts redox balance and increases RPE sensitivity to oxidative damage, suggesting that deficiencies of reductive carboxylation may contribute to RPE cell death. Supporting reductive carboxylation by supplementation with an NAD+ precursor or its substrate α-ketoglutarate or treatment with a poly(ADP ribose) polymerase inhibitor protects reductive carboxylation and RPE viability from excessive oxidative stress. The ability of these treatments to rescue RPE could be the basis for an effective strategy to treat blinding diseases caused by RPE dysfunction.

Reduced Metabolic Capacity in Aged Primary Retinal Pigment Epithelium (RPE) is Correlated with Increased Susceptibility to Oxidative Stress.

One of the affected tissues in age-related macular degeneration (AMD) is the retinal pigment epithelium (RPE), a tissue that consists of terminally differentiated cells and that accumulates damage over time. In all tissues, mitochondria (mt), which play an essential role in both cell health (energy) and death (initiator of apoptosis), undergo an aging process through the accumulation of mtDNA damage, changes in mitochondrial dynamics, a reduction in biogenesis, and mitophagy, leading to an overall reduction in mitochondrial energy production and other non-energy-related functions. Here we have compared energy metabolism in primary human RPE cells isolated from aborted fetus or aged donor eyes and grown as stable monolayers. H2O2 treatment resulted in the generation of reactive oxygen species and superoxide, an effect that was significantly augmented by age. Mitochondrial metabolism, as analyzed by Seahorse respirometry, revealed reduced mitochondrial oxygen consumption (ATP production) at baseline and a complete loss of reserve capacity in aged cells. Likewise, glycolysis was blunted in aged cells. Taken together, these studies showed that RPE cells derived from aged donor eyes are more susceptible to oxidative stress, and exhibit a loss in mitochondrial respiratory reserve capacity and a reduction in glycolysis. These data suggest that while old cells may have sufficient energy at rest, they cannot mount a stress response requiring additional ATP and reducing agents. In summary, these data support the hypothesis that mitochondria or energy metabolism is a valid target for therapy in AMD.

The Contribution of Melanoregulin to Microtubule-Associated Protein 1 Light Chain 3 (LC3) Associated Phagocytosis in Retinal Pigment Epithelium.

A main requisite in the phagocytosis of ingested material is a coordinated series of maturation steps which lead to the degradation of ingested cargo. Photoreceptor outer segment (POS) renewal involves phagocytosis of the distal disk membranes by the retinal pigment epithelium (RPE). Previously, we identified melanoregulin (MREG) as an intracellular cargo-sorting protein required for the degradation of POS disks. Here, we provide evidence that MREG-dependent processing links both autophagic and phagocytic processes in LC3-associated phagocytosis (LAP). Ingested POS phagosomes are associated with endogenous LC3 and MREG. The LC3 association with POSs exhibited properties of LAP; it was independent of rapamycin pretreatment, but dependent on Atg5. Loss of MREG resulted in a decrease in the extent of LC3-POS association. Studies using DQ-BSA suggest that loss of MREG does not compromise the association and fusion of LC3-positive phagosomes with lysosomes. Furthermore, the mechanism of MREG action is likely through a protein complex that includes LC3, as determined by colocalization and immunoprecipitation in both RPE cells and macrophages. We posit that MREG participates in coordinating the association of phagosomes with LC3 for content degradation with the loss of MREG leading to phagosome accumulation.

Multiple requirements of the focal dermal hypoplasia gene porcupine during ocular morphogenesis.

Wnt glycoproteins control key processes during development and disease by activating various downstream pathways. Wnt secretion requires post-translational modification mediated by the O-acyltransferase encoded by the Drosophila porcupine homolog gene (PORCN). In humans, PORCN mutations cause focal dermal hypoplasia (FDH, or Goltz syndrome), an X-linked dominant multisystem birth defect that is frequently accompanied by ocular abnormalities such as coloboma, microphthalmia, or even anophthalmia. Although genetic ablation of Porcn in mouse has provided insight into the etiology of defects caused by ectomesodermal dysplasia in FDH, the requirement for Porcn and the actual Wnt ligands during eye development have been unknown. In this study, Porcn hemizygosity occasionally caused ocular defects reminiscent of FDH. Conditional inactivation of Porcn in periocular mesenchyme led to defects in mid- and hindbrain and in craniofacial development, but was insufficient to cause ocular abnormalities. However, a combination of conditional Porcn depletion in optic vesicle neuroectoderm, lens, and neural crest-derived periocular mesenchyme induced severe eye abnormalities with high penetrance. In particular, we observed coloboma, transdifferentiation of the dorsal and ventral retinal pigment epithelium, defective optic cup periphery, and closure defects of the eyelid, as well as defective corneal morphogenesis. Thus, Porcn is required in both extraocular and neuroectodermal tissues to regulate distinct Wnt-dependent processes during morphogenesis of the posterior and anterior segments of the eye.

Iron upregulates melanogenesis in cultured retinal pigment epithelial cells.

The purpose of our studies was to examine the relationship between iron and melanogenesis in retinal pigment epithelial cells, as prior observations had suggested that iron may promote melanogenesis. This relationship has potential clinical importance, as both iron overload and hyperpigmentation are associated with age-related macular degeneration (AMD). Human fetal retinal pigment epithelial cells and ARPE-19 cells were treated with iron in the form of ferric ammonium citrate, after which quantitative RT-PCR and electron microscopy were performed. Melanogenesis genes tyrosinase, tyrosinase-related protein 1, Hermansky-Pudlak Syndrome 3, premelanosome protein and dopachrome tautomerase were upregulated, as was the melanogenesis-controlling transcription factor, microphthalmia-associated transcription factor (MITF). Iron-treated cells had increased pigmentation and melanosome number. Multiple transcription factors upstream of MITF were upregulated by iron.

Canonical Wnt/ß-catenin signalling is essential for optic cup formation.

A multitude of signalling pathways are involved in the process of forming an eye. Here we demonstrate that ß-catenin is essential for eye development as inactivation of ß-catenin prior to cellular specification in the optic vesicle caused anophthalmia in mice. By achieving this early and tissue-specific ß-catenin inactivation we find that retinal pigment epithelium (RPE) commitment was blocked and eye development was arrested prior to optic cup formation due to a loss of canonical Wnt signalling in the dorsal optic vesicle. Thus, these results show that Wnt/ß-catenin signalling is required earlier and play a more central role in eye development than previous studies have indicated. In our genetic model system a few RPE cells could escape ß-catenin inactivation leading to the formation of a small optic rudiment. The optic rudiment contained several neural retinal cell classes surrounded by an RPE. Unlike the RPE cells, the neural retinal cells could be ß-catenin-negative revealing that differentiation of the neural retinal cell classes is ß-catenin-independent. Moreover, although dorsoventral patterning is initiated in the mutant optic vesicle, the neural retinal cells in the optic rudiment displayed almost exclusively ventral identity. Thus, ß-catenin is required for optic cup formation, commitment to RPE cells and maintenance of dorsal identity of the retina.

Functional analysis of serially expanded human iPS cell-derived RPE cultures.

PURPOSE: To determine the effects of serial expansion on the cellular, molecular, and functional properties of human iPS cell (hiPSC)-derived RPE cultures. METHODS: Fibroblasts obtained from four individuals were reprogrammed into hiPSCs and differentiated to RPE cells using previously described methods. Patches of deeply pigmented hiPSC-RPE were dissected, dissociated, and grown in culture until they re-formed pigmented monolayers. Subsequent passages were obtained by repeated dissociation, expansion, and maturation of RPE into pigmented monolayers. Gene and protein expression profiles and morphological and functional characteristics of hiPSC-RPE at different passages were compared with each other and to human fetal RPE (hfRPE). RESULTS: RPE from all four hiPSC lines could be expanded more than 1000-fold when serially passaged as pigmented monolayer cultures. Importantly, expansion of hiPSC-RPE monolayers over the first three passages (P1-P3) resulted in decreased expression of pluripotency and neuroretinal markers and maintenance of characteristic morphological features and gene and protein expression profiles. Furthermore, P1 to P3 hiPSC-RPE monolayers reliably demonstrated functional tight junctions, G-protein-coupled receptor-mediated calcium transients, phagocytosis and degradation of photoreceptor outer segments, and polarized secretion of biomolecules. In contrast, P4 hiPSC-RPE cells failed to form monolayers and possessed altered morphological and functional characteristics and gene expression levels. CONCLUSIONS: Highly differentiated, pigmented hiPSC-RPE monolayers can undergo limited serial expansion while retaining key cytological and functional attributes. However, passaging hiPSC-RPE cultures beyond senescence leads to loss of such features. Our findings support limited, controlled passaging of patient-specific hiPSC-RPE to procure cells needed for in vitro disease modeling, drug screening, and cellular transplantation.

Vax1/2 genes counteract Mitf-induced respecification of the retinal pigment epithelium.

During vertebrate eye development, the transcription factor MITF acts to promote the development of the retinal pigment epithelium (RPE). In embryos with Mitf mutations, the future RPE hyperproliferates and is respecified as retinal tissue but only in a small portion of the dorsal RPE. Using a series of genetic crosses, we show that this spatial restriction of RPE respecification is brought about by persistent expression of the anti-retinogenic ventral homeodomain gene Vax2 in the dorso-proximal and both Vax1 and Vax2 in the ventral RPE. We further show that dorso-proximal RPE respecification in Vax2/Mitf double mutants and dorso-proximal and ventral RPE respecification in Vax1/2/Mitf triple mutants result from increased FGF/MAP kinase signaling. In none of the mutants, however, does the distal RPE show signs of hyperproliferation or respecification, likely due to local JAGGED1/NOTCH signaling. Expression studies and optic vesicle culture experiments also suggest a role for NOTCH signaling within the mutant dorsal RPE domains, where ectopic JAGGED1 expression may partially counteract the effects of FGF/ERK1/2 signaling on RPE respecification. The results indicate the presence of complex interplays between distinct transcription factors and signaling molecules during eye development and show how RPE phenotypes associated with mutations in one gene are modulated by expression changes in other genes.

Matrix metalloproteinase activity creates pro-angiogenic environment in primary human retinal pigment epithelial cells exposed to complement.

PURPOSE: Mechanistic studies have shown that inflammation, complement activation, extracellular matrix (ECM) turnover, growth factor imbalance, and oxidative stress are fundamental components of age-related macular degeneration (AMD). Matrix metalloproteinases (MMPs) mediate ECM turnover but also process various bioactive molecules. Here, we tested whether complement attack on RPE monolayers changes MMP secretion and activation, thereby altering the availability of growth factors in the extracellular space. METHODS: Human embryonic RPE monolayers with stable transepithelial resistance (TER) were established. Complement activation was induced with H2O2 and normal human serum. MMP-2/9, vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) protein, and mRNA levels were analyzed by Western blotting, ELISA, and real-time PCR; activity of MMP-2/9 by gelatin zymography. RESULTS: Complement activation resulted in a loss of TER, which required transient membrane attack complex formation, activation of the alternative pathway, and VEGF secretion and signaling. Despite the generation of reactive oxygen species, cellular integrity or intracellular adenosine triphosphate (ATP) levels were unaffected. However, expression of MMP-2/9 and their protease activity was elevated. Inhibition of MMP-2/9 activity increased PEDF and decreased VEGF levels in the apical and basal supernatants but had no effect on their expression levels. VEGF levels in the supernatant correlated with the level TER reduction. CONCLUSIONS: These studies suggest that complement activation, by altering the expression and activation of MMPs, has the ability to generate a proangiogenic environment by altering the balance between VEGF and PEDF. Our findings link reported results that have been associated with AMD pathogenesis; oxidative stress; complement activation; VEGF/PEDF ratio; and MMP activity.

The orchestration of mammalian tissue morphogenesis through a series of coherent feed-forward loops.

Tissue morphogenesis requires intricate temporal and spatial control of gene expression that is executed through specific gene regulatory networks (GRNs). GRNs are comprised from individual subcircuits of different levels of complexity. An important question is to elucidate the mutual relationship between those genes encoding DNA-binding factors that trigger the subcircuit with those that play major "later" roles during terminal differentiation via expression of specific genes that constitute the phenotype of individual tissues. The ocular lens is a classical model system to study tissue morphogenesis. Pax6 is essential for both lens placode formation and subsequent stages of lens morphogenesis, whereas c-Maf controls terminal differentiation of lens fibers, including regulation of crystallins, key lens structural proteins required for its transparency and refraction. Here, we show that Pax6 directly regulates c-Maf expression during lens development. A 1.3-kb c-Maf promoter with a 1.6-kb upstream enhancer (CR1) recapitulated the endogenous c-Maf expression pattern in lens and retinal pigmented epithelium. ChIP assays revealed binding of Pax6 and c-Maf to multiple regions of the c-Maf locus in lens chromatin. To predict functional Pax6-binding sites, nine novel variants of Pax6 DNA-binding motifs were identified and characterized. Two of these motifs predicted a pair of Pax6-binding sites in the CR1. Mutagenesis of these Pax6-binding sites inactivated transgenic expression in the lens but not in retinal pigmented epithelium. These data establish a novel regulatory role for Pax6 during lens development, link together the Pax6/c-Maf/crystallin regulatory network, and suggest a novel type of GRN subcircuit that controls a major part of embryonic lens development.

Eye morphogenesis and patterning of the optic vesicle.

Organogenesis of the eye is a multistep process that starts with the formation of optic vesicles followed by invagination of the distal domain of the vesicles and the overlying lens placode resulting in morphogenesis of the optic cup. The late optic vesicle becomes patterned into distinct ocular tissues: the neural retina, retinal pigment epithelium (RPE), and optic stalk. Multiple congenital eye disorders, including anophthalmia or microphthalmia, aniridia, coloboma, and retinal dysplasia, stem from disruptions in embryonic eye development. Thus, it is critical to understand the mechanisms that lead to initial specification and differentiation of ocular tissues. An accumulating number of studies demonstrate that a complex interplay between inductive signals provided by tissue-tissue interactions and cell-intrinsic factors is critical to ensuring proper specification of ocular tissues as well as maintenance of RPE cell fate. While several of the extrinsic and intrinsic determinants have been identified, we are just at the beginning in understanding how these signals are integrated. In addition, we know very little about the actual output of these interactions. In this chapter, we provide an update of the mechanisms controlling the early steps of eye development in vertebrates, with emphasis on optic vesicle evagination, specification of neural retina and RPE at the optic vesicle stage, the process of invagination during morphogenesis of the optic cup, and maintenance of the RPE cell fate.

Neurogenin1 effectively reprograms cultured chick retinal pigment epithelial cells to differentiate toward photoreceptors.

Photoreceptors are highly specialized sensory neurons in the retina, and their degeneration results in blindness. Replacement with developing photoreceptor cells promises to be an effective therapy, but it requires a supply of new photoreceptors, because the neural retina in human eyes lacks regeneration capability. We report efficient generation of differentiating, photoreceptor-like neurons from chick retinal pigment epithelial (RPE) cells propagated in culture through reprogramming with neurogenin1 (ngn1). In reprogrammed culture, a large number of the cells (85.0% +/- 5.9%) began to differentiate toward photoreceptors. Reprogrammed cells expressed transcription factors that set in motion photoreceptor differentiation, including Crx, Nr2E3, NeuroD, and RXRgamma, and phototransduction pathway components, including transducin, cGMP-gated channel, and red opsin of cone photoreceptors (equivalent to rhodopsin of rod photoreceptors). They developed inner segments rich in mitochondria. Furthermore, they responded to light by decreasing their cellular free calcium (Ca(2+)) levels and responded to 9-cis-retinal by increasing their Ca(2+) levels after photobleaching, hallmarks of photoreceptor physiology. The high efficiency and the advanced photoreceptor differentiation indicate ngn1 as a gene of choice to reprogram RPE progeny cells to differentiate into photoreceptor neurons in future cell replacement studies.

Soluble EphB4 inhibition of PDGF-induced RPE migration in vitro.

PURPOSE: EphB4 receptor (EphB4) and its ligand (EphrinB2) play an important role in the regulation of cell adhesion, growth, and migration. The purpose of this study was to determine the effects of EphB4 blockade by soluble EphB4 (sEphB4) on retinal pigment epithelial (RPE) cell migration and proliferation, induced by platelet-derived growth factor-BB (PDGF), and to establish its relevance to proliferative vitreoretinopathy (PVR). METHODS: The expression of EphB4 and EphrinB2 in early-passage human RPE cells and in human PVR membranes was evaluated by confocal microscopy. The effect of sEphB4 (0.1-3 microg/mL) on PDGF (20 ng/mL)-induced RPE migration and proliferation was evaluated using a modified Boyden chamber assay and an MTT assay, respectively. Attachment to basement membrane matrix and fibronectin was assayed by MTT. Phosphorylation of FAK and p42/44 mitogen-activated protein kinase (MAPK) in retinal pigment epithelium was determined by Western blot analysis after exposure to sEphB4. The effect of sEphB4 on the phosphorylation of EphB4/EphrinB2 was demonstrated with the use of immunoprecipitation assays. RESULTS: EphrinB2 and EphB4 were expressed on human RPE cells in vitro and in cells within human PVR membranes. sEphB4 blocked EphB4 and EphrinB2 phosphorylation in RPE cells in vitro. sEphB4 reduced RPE migration in response to PDGF stimulation (P < 0.01). Similarly, sEphB4 inhibited RPE attachment and proliferation in a dose-dependent manner (P < 0.05). PDGF-induced phosphorylation of FAK and MAPK was inhibited by sEphB4. CONCLUSIONS: EphB4 and EphrinB2 are expressed in RPE cells and PVR membranes. sEphB4 inhibits PDGF-induced RPE cell attachment, proliferation, and migration. This effect may result from the inhibition of FAK and MAPK phosphorylation.