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Epigenetics has been recognized to play an important role in physiological and pathological processes of the human body. Accumulating evidence has indicated that epigenetic mechanisms contribute to the pathogenesis of age-related macular degeneration (AMD). Although the susceptibility related to genetic variants has been revealed by genome-wide association studies, those genetic variants may predict AMD risk only in certain human populations. Other mechanisms, particularly those involving epigenetic factors, may play an important role in the pathogenesis of AMD. Therefore, we briefly summarize the most recent reports related to such epigenetic mechanisms, including DNA methylation, histone modification, and non-coding RNA, and the interplay of genetic and epigenetic factors in the pathogenesis of AMD.

Age-related macular degeneration (AMD) is a progressive eye disease and the most common cause of blindness among the elderly. AMD is characterized by early atrophy of the choriocapillaris and retinal pigment epithelium (RPE). Although AMD is a multifactorial disease with many environmental and genetic risk factors, a hallmark of the disease is the origination of extracellular deposits, or drusen, between the RPE and Bruch membrane. Human retinal G-protein-coupled receptor (RGR) gene generates an exon-skipping splice variant of RGR-opsin (RGR-d; NP_001012740) that is a persistent component of small and large drusen. Herein, the findings show that abnormal RGR proteins, including RGR-d, are pathogenic in an animal retina with degeneration of the choriocapillaris, RPE, and photoreceptors. A frameshift truncating mutation resulted in severe retinal degeneration with a continuous band of basal deposits along the Bruch membrane. RGR-d produced less severe disease with choriocapillaris and RPE atrophy, including focal accumulation of abnormal RGR-d protein at the basal boundary of the RPE. Degeneration of the choriocapillaris was marked by a decrease in endothelial CD31 protein and choriocapillaris breakdown at the ultrastructural level. Fundus lesions with patchy depigmentation were characteristic of old RGR-d mice. RGR-d was mislocalized in cultured cells and caused a strong cell growth defect. These results uphold the notion of a potential hidden link between AMD and a high-frequency RGR allele.

We sought to characterize the angiofibrotic and apoptotic effects of vascular endothelial growth factor (VEGF)-inhibition on fibrovascular epiretinal membranes in eyes with traction retinal detachment because of proliferative diabetic retinopathy.Membranes were excised from 20 eyes of 19 patients (10 randomized to intravitreal bevacizumab, 10 controls) at vitrectomy. Membranes were stained with antibodies targeting connective tissue growth factor (CTGF) or VEGF and colabeled with antibodies directed against endothelial cells (CD31), myofibroblasts, or retinal pigment epithelium markers. Quantitative and colocalization analyses of antibody labeling were obtained through immunofluorescence confocal microscopy. Masson trichrome staining, cell counting of hematoxylin and eosin sections, and terminal dUTP nick-end labeling staining were performed.High levels of fibrosis were observed in both groups. Cell apoptosis was higher (P = 0.05) in bevacizumab-treated membranes compared with controls. The bevacizumab group had a nonsignificant reduction in colocalization in CD31-CTGF and cytokeratin-VEGF studies compared with controls. Vascular endothelial growth factor in extracted membranes was positively correlated with vitreous levels of VEGF; CTGF in extracted membranes was negatively correlated with vitreous levels of CTGF.Bevacizumab suppresses vitreous VEGF levels, but does not significantly alter VEGF or CTGF in diabetic membranes that may be explained by high baseline levels of fibrosis. Bevacizumab may cause apoptosis within fibrovascular membranes.

Background Choroidal neovascularization (CNV) is a leading cause of central vision loss complicated with age-related macular degeneration. Although intravitreal anti-VEGF therapy is widely used in wet age-related macular degeneration, optimal treatment regimens for the disease are still under investigation. EphrinB2 and EphB4 regulate angiogenesis, and interruption of EphB4/ephrinB2 has been demonstrated to inhibit angiogenesis. In the current study, we studied the effects of soluble EphB4 (sEphB4) on laser induced CNV in a rat model by intravitreous injection and the underlying mechanism. Methods Male rats (Brown-Norway) were used in the study. CNV was induced by laser and the sEphB4 was injected intravitreous after laser at days 3 and 7. The CNV lesions were evaluated by three methods: fluorescein angiography (FA) in vivo, CNV volume by confocal analysis of choroidal flat-mounts and H&E staining. The expression of fibronectin (FN), VEGFR-2, phospho-VEGFR-2 (pVEGFR-2), the double labeling of EphB4 with FN was analyzed by immunofluorescence. The interaction of FN with EphB4 and the effects of intraocular injection of sEphB4 on the inhibition of pVEGFR-2 were determined by western blot. Results The FA leakage and CNV volume were significantly inhibited by the injection of the sEphB4. Further, histology analysis showed that CNV lesion was significantly smaller in the rats with sEphB4 injection than rats with placebo application. The expressions of pVEGFR-2 and FN in the CNV lesions were reduced compared with controls. Conclusions Our study suggests that the inhibition of CNV by sEphB4 may be through suppression of VEGFR-2 phosphorylation and the expression of FN. sEphB4 may be a new potential therapeutic strategy of CNV.

Endoplasmic reticulum (ER) stress is linked to several pathological conditions including age-related macular degeneration. Excessive ER stress initiates cell death cascades which are mediated, in part, through mitochondrial dysfunction. Here, we identify αB crystallin as an important regulator of ER stress-induced cell death. Retinal pigment epithelial (RPE) cells from αB crystallin (-/-) mice, and human RPE cells transfected with αB crystallin siRNA, are more vulnerable to ER stress induced by tunicamycin. ER stress-mediated cell death is associated with increased levels of reactive oxygen species, depletion of glutathione in mitochondria, decreased superoxide dismutase activity, increased release of cytochrome c, and activation of caspases 3 and 4. The ER stress signaling inhibitors, salubrinal and 4-(2-aminoethyl) benzenesulfonyl fluoride, decrease mitochondrial damage and reduce RPE apoptosis induced by ER stress. Prolonged ER stress decreases levels of αB crystallin, thus exacerbating mitochondrial dysfunction. Overexpression of αB crystallin protects RPE cells from ER stress-induced apoptosis by attenuating increases in Bax, CHOP, mitochondrial permeability transition, and cleaved caspase 3. Thus, these data collectively demonstrate that αB crystallin provides critical protection of mitochondrial function during ER stress-induced RPE apoptosis.

Proliferative vitreoretinopathy (PVR) is a serious complication of retinal detachment and ocular trauma and its recurrence may lead to irreversible vision loss. Epithelial to mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is a critical step in the pathogenesis of PVR, which is characterized by fibrotic membrane formation and traction retinal detachment. In this study, we investigated the potential impact of resveratrol (RESV) on EMT and the fibrotic process in cultured RPE cells and further examined the preventive effect of RESV on PVR development using a rabbit model of PVR. We found that RESV induces mesenchymal to epithelial transition (MET) and inhibits transforming growth factor-β2(TGF-β2)-induced EMT of RPE cells by deacetylating SMAD4. The effect of RESV on MET was dependent on sirtuin1 activation. RESV suppressed proliferation, migration and fibronectin synthesis induced by platelet-derived growth factor-BB or TGF-β2. In vivo, RESV inhibited the progression of experimental PVR in rabbit eyes. Histological findings showed that RESV reduced fibrotic membrane formation and decreased α-SMA expression in the epiretinal membranes. These results suggest the potential use of RESV as a therapeutic agent to prevent the development of PVR by targeting EMT of RPE.

alphaB-crystallin is a chaperone belonging to the small heat shock protein family. Herein we show attenuation of intraocular angiogenesis in alphaB-crystallin knockout (alphaB-crystallin(-/-)) mice in 2 models of intraocular disease: oxygen-induced retinopathy and laser-induced choroidal neovascularization. Vascular endothelial growth factor A (VEGF-A) mRNA and hypoxia inducible factor-1alpha protein expression were induced during retinal angiogenesis, but VEGF-A protein expression remained low in alphaB-crystallin(-/-) retina versus wild-type mice, whereas VEGF-R2 expression was not affected. Both alphaB-crystallin and its phosphorylated serine59 formwere expressed, and immunoprecipitation revealed alphaB-crystallin binding to VEGF-A but not transforming growth factor-beta in cultured retinal pigment epithelial (RPE) cells. alphaB-crystallin and VEGF-A are colocalized in the endoplasmic reticulum in RPE cells under chemical hypoxia. alphaB-crystallin(-/-) RPE showed low VEGF-A secretion under serum-starved conditions compared with wild-type cells. VEGF-A is polyubiquitinated in control and alphaB-crystallin siRNA treated RPE; however, mono-tetra ubiquitinated VEGF-A increases with alphaB-crystallin knockdown. Endothelial cell apoptosis in newly formed vessels was greater in alphaB-crystallin(-/-) than wild-type mice. Proteasomal inhibition in alphaB-crystallin(-/-) mice partially restores VEGF-A secretion and angiogenic phenotype in choroidal neovascularization. Our studies indicate an important role for alphaB-crystallin as a chaperone for VEGF-A in angiogenesis and its potential as a therapeutic target.

We hypothesized that neutrophils and their secreted factors mediate breakdown of the integrity of the outer blood-retina-barrier by degrading the apical tight junctions of the retinal pigment epithelium (RPE). The effect of activated neutrophils or neutrophil cell lysate on apparent permeability of bovine RPE-Choroid explants was evaluated by measuring[H]mannitol flux in a modified Ussing chamber. The expression of matrix metalloproteinase- (MMP-) 9 in murine peritoneal neutrophils, and the effects of neutrophils on RPE tight-junction protein expression were assessed by confocal microscopy and western blot. Our results revealed that basolateral incubation of explants with neutrophils decreased occludin and ZO-1 expression at 1 and 3 hours and increased the permeability of bovine RPE-Choroid explants by >3-fold (P<.05). Similarly, basolateral incubation of explants with neutrophil lysate decreased ZO-1 expression at 1 and 3 hours (P<.05) and increased permeability of explants by 75%. Further, we found that neutrophils prominently express MMP-9 and that incubation of explants with neutrophils in the presence of anti-MMP-9 antibody inhibited the increase in permeability. These data suggest that neutrophil-derived MMP-9 may play an important role in disrupting the integrity of the outer blood-retina barrier.

Matrix metalloproteinases (MMP)-2 and -9 play an important role in the pathogenesis of choroidal neovascularization (CNV). Retinal pigment epithelial cells (RPE) are an important source of MMPs in the outer retinal environment, however little is known about the local factors that modulate MMP secretion in these cells. The purpose of this study was to determine the effects of CNV involved growth factors and the extracellular matrix molecule fibronectin on MMP-2 and -9 secretion by cultured human RPE.MMP-2 and -9 secretion was studied using gelatin zymography, Western blot, and ELISA assay of RPE culture supernatants. The effects of stimulating the cells for 36 hours with vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bGFG), tumor necrosis factor-alpha (TNF-alpha), or fibronectin (FN), all angiogenic factors found in CNV membranes, was determined.Resting RPE cells secreted MMP-2 but not MMP-9. Stimulation with TNF-alpha induced secretion of MMP-9 and increased the secretion of MMP-2. MMP-2 secretion was also increased by stimulation with FN and VEGF, but not bFGF.The results indicated that the angiogenic molecules VEGF, FN, and TNF-alpha stimulate MMP-2 and -9 secretion from RPE and thus further promote CNV.

Inflammatory mediators have been proposed to play a critical role in the pathogenesis of choroidal neovascularization, a blinding complication of age-related macular degeneration. We evaluated the expression of TNF-alpha in human choroidal neovascular membranes and found that it colocalized with cells expressing VEGF, angiopoietin (Ang)-1 and Ang2. In cultured choroidal endothelial cells we found that TNF-alpha increased Ang2 mRNA (increased transcription) and protein levels prior to those of Ang1 and VEGF. The results raise the possibility that during neovascularization, TNF-alpha may modulate endothelial plasticity and survival by sequential inactivation of Tie2 followed by activation of Tie2 and VEGF receptors.

To determine the effect of the calcium signaling modulating drug carboxyamido-triazole (CAI) on substeps of exudative age-related macular degeneration (AMD) in vitro.Zymography and ELISA determined the effect of CAI on MMP-2 production of choroidal endothelial cells (CECs) stimulated by bFGF and VEGF. The effects of CAI on attachment of retinal pigment endothelial (RPE) cells/CECs onto fibronectin, laminin, collagen IV, and migration toward fibronectin were investigated. Proliferation induced by serum and bFGF (10 microg/ml) with and without CAI (0.1-10 microM) was measured by cell counting and 3H-uptake. Viability and apoptosis of the exposed cells was assessed by an MTT and an apoptosis assay.CAI inhibited serum- and bFGF-induced proliferation, cell attachment onto fibronectin and collagen IV, but only CEC attachment onto laminin. Inhibition of MMP-2 production was observed (10 microM CAI). CAI reduced the cellular viability by apoptosis induction.CAI inhibits substeps of exudative macular degeneration and may be of value for the treatment of the disease.

Aims To determine whether hepatocyte growth factor (HGF) and connective tissue growth factor (CTGF) are expressed in human specimens of proliferative vitreoretinopathy (PVR) and to propose a model of PVR pathogenesis based upon the known activities of these growth factors. Methods Immunohistochemical methods (ABC Elite) were used to demonstrate the presence of HGF and CTGF in cryostat sections of five human PVR membranes. Results In each of the five PVR membranes, stromal cells were immunohistochemically positive for both HGF and CTGF. Based upon this information and the known actions of these growth factors, a model of PVR pathogenesis was developed. In this model, injury of the retina induces an inflammatory response that upregulates HGF expression inducing the formation of multilayered groups of migratory retinal pigment epithelial cells (RPE). These RPE, present in a provisional extracellular matrix, come in contact with vitreous containing TGF-β. The TGF-β is activated, upregulating expression of CTGF. Under the influence of TGF-β and CTGF, RPE become myofibroblastic and fibrosis ensues. Retinal traction induces further detachment continuing the cycle of retinal injury. Conclusions HGF and CTGF are expressed in PVR membranes and may play important roles in the pathogenesis of PVR. The expression and function of these growth factors should be critically examined in human PVR specimens, in in vitro cultures of RPE, and in animal models of PVR.

Choroidal neovascularization (CNV) is a blinding complication of age-related macular degeneration that manifests as the growth of immature choroidal blood vessels through Bruch’s membrane, where they can leak fluid or hemorrhage under the retina. Here, we demonstrate that the histone deacetylase inhibitor (HDACi) trichostatin A (TSA) can down-regulate the pro-angiogenic hypoxia-inducible factor-1α and vascular endothelial growth factor (VEGF), and up-regulate the anti-angiogenic and neuro-protective pigment epithelium derived factor in human retinal pigment epithelial (RPE) cells. Most strikingly, TSA markedly down-regulates the expression of VEGF receptor-2 in human vascular endothelial cells and, thus, can knock down pro-angiogenic cell signaling. Additionally, TSA suppresses CNV-associated wound healing response and RPE epithelial-mesenchymal transdifferentiation. In the laser-induced model of CNV using C57Bl/6 mice, systemic administration of TSA significantly reduces fluorescein leakage and the size of CNV lesions at post—laser days 7 and 14 as well as the immunohistochemical expression of VEGF, VEGFR2, and smooth muscle actin in CNV lesions at post-laser day 7. This report suggests that TSA, and possibly HDACi’s in general, should be further evaluated for their therapeutic potential for the treatment of CNV.

Up to 50 million people worldwide are afflicted with the devastating blinding disease age-related macular degeneration (AMD)1–3. The vast majority of patients have the currently untreatable “dry” or atrophic form of AMD, characterized by NLRP3 inflammasome-driven degeneration of the retinal pigment epithelium (RPE) supportive cell layer4,5. Blockade of the NLRP3 inflammasome is a next-generation therapeutic target in dry AMD; however, it was recently reported that inflammasome-mediated production of IL18 potentially safeguards the retina against the other, often more visually devastating form of AMD, for which dry AMD patients are at greatly increased risk of developing, known as choroidal neovascularization (CNV)6. Therefore, it is essential, prior to initiating inflammasome-targeting clinical trials, to directly and rigorously assess whether modulating IL18 or the NLRP3 inflammasome affects CNV and RPE cell health.

Hepatocyte growth factor (HGF) is normally expressed by mesenchymal cells while its receptor, c-Met, is expressed in epithelial cells. Since HGF is critically involved in epithelial-mesenchyme interactions and the retinal pigment epithelium (RPE) is present at the interface between the retina and choroid, this study was initiated to determine whether the RPE expresses or responds to HGF in vitro. Cultured adult and fetal human RPE expressed mRNA for HGF and c-Met by RT-PCR. ELISA assay demonstrated the secretion of HGF into RPE culture supernatants. Tyrosine phosphorylation of c-Met was constitutively found in 72 hour RPE cultures and could be rapidly induced in serum-starved cells by concentrated RPE supernatants. HGF was mitogenic for cultured RPE (100 ng/ml.) and stimulated their chemotaxis (maximal response at 50 ng/ml). RPE are one of only a very limited number of epithelia that express both HGF and its receptor, suggesting the possibility of an autocrine action for this growth factor.

Tumor necrosis factor-alpha (TNF) has been implicated in the pathogenesis of several retinal diseases. Soluble forms of the TNF receptors, p55 (55 kDa) and p75 (75 kDa), have recently been identified in biological fluids and may regulate TNF activity. The potential biological significance of these receptors for the human retina was examined by determining their presence in human vitreous and their release from eye cup explants in which the retina has been removed leaving an intact retinal pigment epithelium (HRPE). Normal human vitreous and conditioned medium from eye-cup HRPE explants demonstrated the presence of soluble p55 and p75. Soluble p55 was significantly more abundant than p75 in all vitreous samples (P0.03). Conditioned medium from eye-cup HRPE explants contained significantly more soluble p55 than p75 (P0.00002). Enzyme-linked immunosorbent assay showed the presence of soluble p55, and not p75, in conditioned medium from primary cultured HRPE cells. Activation of the protein kinase C pathway in these cells with the phorbol ester PMA significantly increased the release of soluble p55 (Por = 0.001); whereas, pharmacological inhibition of protein kinase C with calphostin-C significantly decreased the shedding of p55 (Por = 0.001). The results indicate that primary cultured HRPE cells shed p55 and regulate this shedding in part through the protein kinase C pathway. The presence of soluble TNF receptors within normal human vitreous and within conditioned medium from the eye-cup HRPE explant model suggests that these soluble receptors may have a biological function in the eye.

Proliferative vitreoretinopathy (PVR) is characterized by the proliferation and migration of retinal pigment epithelial (RPE) cells in the vitreous cavity. The drug hypericin, which is already in clinical use as an antidepressant, has shown promise as an antiviral and antineoplastic agent. To investigate the therapeutic potential of hypericin in PVR, we incubated RPE cells in standard medium with various serum concentrations containing 0.5 to 5 microM hypericin. In some experiments we studied the effects of hypericin in conjunction with the RPE growth stimulating cytokine tumor necrosis factor alpha (TNF-alpha). Dose-dependent inhibition of RPE cell proliferation with IC50 values of 0.7 microM and 3.3 microM in 1% and 5% serum respectively, was found. Even in conjunction with TNF-alpha, hypericin inhibited RPE proliferation with an IC50 value of 1.5 microM. The drug inhibited PKC activity in cells treated with a 2.5 microM dose by 72% after 30 min and by 100% after 180 min. Finally, hypericin induced RPE cells to undergo apoptotic cell death, as shown by the presence of DNA laddering. These results suggest that hypericin may have potential as a therapeutic drug for PVR and that its antiproliferative and apoptotic effects on RPE cells in vitro are in part mediated by PKC.

Epigenetics has become an increasingly important area of biomedical research. Increasing evidence shows that epigenetic alterations influence common pathologic responses including inflammation, ischemia, neoplasia, aging, and neurodegeneration. Importantly, epigenetic mechanisms may have a pathogenic role in many complex eye diseases such as corneal dystrophy, cataract, glaucoma, diabetic retinopathy, ocular neoplasia, uveitis, and age-related macular degeneration. The emerging emphasis on epigenetic mechanisms in studies of eye disease may provide new insights into the pathogenesis of complex eye diseases and aid in the development of novel treatments for these diseases.

Bone morphorgenetic protein (BMP)-4 has been shown to play a pivotal role in eye development; however, its role in mature retina or ocular angiogenic diseases is unclear. Activating downstream Smad signaling, BMP4 can be either pro-angiogenic or anti-angiogenic, depending on the context of cell types and associated microenvironment. In this study, we generated transgenic mice over-expressing BMP4 in retinal pigment epithelial (RPE) cells (Vmd2-Bmp4 Tg mice), and used the laser-induced choroidal neovascularization (CNV) model to study the angiogenic properties of BMP4 in adult eyes. Vmd2-Bmp4 Tg mice displayed normal retinal histology at 10 weeks of age when compared with age-matched wildtype mice. Over-expression of BMP4 in RPE in the transgenic mice was confirmed by real-time PCR and immunostaining. Elevated levels of Smad1,5 phosphorylation were found in BMP4 transgenic mice compared to wildype mice. Over-expression of BMP4 was associated with less severe CNV as characterized by fluorescein angiography, CNV volume measurement and histology. While control mice showed increased levels of vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9 after laser injury, Vmd2-Bmp4 Tg showed no increase in either VEGF or MMP-9. Further, we found that TNF-induced MMP-9 secretion in vitro was reduced by pretreatment of RPE cells with BMP4. The inhibition of MMP-9 was Smad-dependent because BMP4 failed to repress TNF-induced MMP-9 expression when Smad1,5 was silenced by siRNA. In summary, our studies identified an anti-angiogenic role for BMP4 in laser-induced CNV, mediated by direct inhibition of MMP-9 and indirect inhibition of VEGF.

Bone morphogenetic protein-4 (BMP4) may be involved in the molecular switch that determines which late form of age-related macular degeneration (AMD) an individual develops. BMP4 expression is high in retinal pigment epithelium (RPE) cells in late, dry AMD patients, while BMP4 expression is low in the wet form of the disease, characterized by choroidal neovascularization (CNV). Here, we sought to determine the mechanism by which BMP4 is down-regulated in CNV. BMP4 expression was decreased within laser-induced CNV lesions in mice at a time when tumor necrosis factor (TNF) expression was high (7 d postlaser) and was reexpressed in RPE when TNF levels declined (14 d postlaser). We found that TNF, an important angiogenic stimulus, significantly down-regulates BMP4 expression in cultured human fetal RPE cells, ARPE-19 cells, and RPE cells in murine posterior eye cup explants. We identified two specificity protein 1 (Sp1) binding sites in the BMP4 promoter that are required for basal expression of BMP4 and its down-regulation by TNF. Through c-Jun NH2-terminal kinase (JNK) activation, TNF modulates Sp1 phosphorylation, thus decreasing its affinity to the BMP4 promoter. The down-regulation of BMP4 expression by TNF in CNV and mechanisms established might be useful for defining novel targets for AMD therapy.—Xu, J., Zhu, D., He, S., Spee, C., Ryan, S. J., Hinton, D. R. Transcriptional regulation of bone morphogenetic protein 4 by tumor necrosis factor and its relationship with age-related macular degeneration.

PURPOSE. To investigate the role of fibronectin containing extra domain A (FN-EDA) in the pathogenesis of proliferative vitreoretinopathy (PVR) and the regulation of FN-EDA by transforming growth factor (TGF)- and connective tissue growth factor (CTGF) in retinal pigment epithelial (RPE) cells. METHODS. Expression of FN-EDA in normal human retinas and PVR membranes was evaluated by immunohistochemistry. The effects of TGF and CTGF on FN-EDA mRNA and protein expression in primary cultures of human RPE cells were analyzed at different time points by real-time PCR and Western blot, respectively. The interaction of CTGF with TGF2 or with its type II receptor TGFRII was examined by ELISA, immunoprecipitation, and solid-phase binding assays. RESULTS. FN-EDA was abundantly expressed in PVR membranes but absent from the RPE monolayer in normal human retinas. Treatment of RPE cells with TGF2 induced FN-EDA expression in a time- and dose-dependent manner, but CTGF alone had no effect. However, CTGF, through its N-terminal half fragment, augmented TGF2-induced expression of FN-EDA at the protein level. This effect was blocked by antibodies against TGF 2o r TGFRII. Interaction of TGF 2o r TGFRII with CTGF was dose dependent and specific. CTGF directly bound TGF2 and TGFRII at its N- and C-terminal domains, respectively. CONCLUSIONS. These findings suggest that CTGF promotes the profibrotic activities of TGF acting as a cofactor through direct protein interactions and complex regulatory mechanisms. (Invest Ophthalmol Vis Sci. 2011;52:5068‐5078) DOI:10.1167/iovs.117191

The retinal pigment epithelium plays an important role in maintaining photoreceptor cell survival and function. Normal retinal pigment epithelial (RPE) cells are quiescent without migration or proliferation.1–3 RPE cells in the stationary monolayer start to migrate in response to pathologic changes in the microenvironment associated with conditions such as proliferative vitreoretinopathy (PVR),4 age-related macular degeneration,5 and diabetic retinopathy.6 RPE cell migration is a complex biological process involving changes in cell attachment, spreading, and cytoskeletal reorganization and is regulated by cell matrix, matrix-dependent enzymes, cytokines, and growth factors.7–9 RPE cell migration is also mediated by cell membrane-associated signaling, especially receptor tyrosine kinases.10,11 Recent reports show that EphB4 and its ligand EphrinB2 mediate cell migration in a variety of cells, such as neurons, retinal vascular endothelial cells, and choroidal endothelial cells.12,13 The Ephs and Ephrins constitute the largest of the receptor tyrosine kinase families, with 14 receptors and eight ligands.14–16 This family is subdivided into EphA and EphB groups and regulates a diverse array of the cellular functions (migration, repulsion, adhesion, and vessel maturation).14–17 The interaction between the Eph receptor and the Ephrin ligand activates forward and reverse signaling through interactions with cytoplasmic signaling proteins. The nature of the downstream signaling pathways and the consequent regulation of cell migration had been demonstrated in various cell culture systems.18–22 Kertesz23 has highlighted the role of EphB4 in angiogenesis through its effects on endothelial cell migration.23 Steinle12 reported that the agonistic EphB4/Fc chimera can stimulate retinal endothelial cell migration by activation of PI3K, Src, and other signaling pathways. In a previous study, we found that sEphB4 inhibits vascular endothelial growth factor (VEGF)-induced choroidal endothelial cell migration.13 We have also demonstrated that platelet-derived growth factor-BB (PDGF) increases RPE cell migration through the activation of p42/44 mitogen-activated protein kinase (MAPK).24,25 Furthermore, PDGF has been found to be expressed in fibrotic PVR membranes and choroidal neovascular membranes, which are associated with RPE migration.26,27 Although previous studies have focused on the roles of EphB4 and EphrinB2 in endothelial cell function and cancer cells, little is known about their role in migration of ocular cells such as RPE cells. Therefore, the aim of the present study was to determine the expression of EphB4 and EphrinB2 in retinal pigment epithelium, to evaluate the effect of sEphB4 on RPE cell migration induced by PDGF, and to investigate the signaling pathways involved.

Variants in the gene for complement factor H (CFH) have been implicated as a major risk factor for the development of age-related macular degeneration (AMD). Little is known, however, about the factors regulating local expression and secretion of CFH by retinal pigment epithelial cells (RPE).Cultured human early passage RPE cells, highly differentiated, polarized human RPE cultures, and bovine RPE explants were incubated in the presence or absence of recombinant human or bovine interferon-gamma (IFN-gamma; 25 ng/ml). CFH expression in cell lysates, and secretion into culture supernatants were examined by Western blot. CHF expression and localization was analyzed by confocal microscopy. Migration assay was performed in a modified Boyden chamber with early passage human RPE cells after stimulation with recombinant CFH protein (1-100 ng/ml).CFH was expressed in the cell lysates of RPE cells, and this expression was significantly upregulated by IFN-gamma. Immunoreactivity for CFH was detected in RPE cells of bovine explants and highly differentiated human RPE monolayers, and the level of immunoreactivity increased after IFN-gamma stimulation. Confocal microscopy revealed that CFH was predominantly localized in the apical cytoplasm of polarized human RPE. Western blot confirmed that IFN-gamma increased CFH secretion into RPE supernatants. Dose-dependent RPE cell chemotactic migration was induced by CFH.IFN-gamma promotes CFH expression in the apical compartment of RPE cells and increases secretion of CFH into RPE culture supernatants. Furthermore, CFH promotes chemotactic migration of RPE. This study suggests that interactions between CFH and IFN-gamma have the potential to play a role in the pathogenesis of AMD.

Fibrosis plays an important role in the pathogenesis of several common blinding disorders, including proliferative diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, and PVR;1–4 however, much needs to be learned about the basic pathophysiology of fibrosis in the intraocular environment.1 PVR may be viewed as a prototypical example of a protracted intraocular wound healing response that occurs when traction-generating cellular membranes develop in the vitreous and on the inner or outer surfaces of the retina following rhegmatogenous retinal detachment or major ocular trauma.5–7 RPE cells play a critical role in this epiretinal membrane formation8,9 These cells proliferate and migrate from the RPE monolayer to form sheets of dedifferentiated cells within a provisional extracellular matrix (ECM) containing fibronectin and thrombospondin.9–11 The protracted wound healing response causes the cellular membrane to become progressively more paucicellular and fibrotic.11 Experimental models of PVR have been developed to evaluate intraocular proliferation;12–16 however, these models typically exhibit cellular fibrinous strands without prominent fibrotic responses. Studies evaluating the role of those factors that elicit this fibrotic response are of particular interest. Normal ocular wound healing involves a tightly coordinated series of events: recruitment and activation of inflammatory cells, release of cytokines and growth factors, activation, proliferation and migration of ocular cells, secretion of extracellular matrix, tissue remodeling, and repair.1, 17 CTGF is an important stimulant of fibrosis,18 but its role in intraocular wound healing or PVR has not been studied in detail. CTGF is a secreted, cysteine-rich, heparin-binding polypeptide growth factor,19 20 that is rapidly upregulated after stimulation with serum or transforming growth factor-β (TGF-β). Various CTGF fragments have been shown to accumulate in tissue culture or body fluids while retaining their biologic activity.20–22 CTGF functions as a downstream mediator of TGF- β action on fibroblasts; it stimulates cell proliferation and cell matrix deposition (collagen 1 and fibronectin),18,20,23 and it may induce apoptosis.24,25 In addition to its action as a growth factor, CTGF has been implicated as an adhesive substrate in fibroblasts, mediated through α6β1 integrin.26 Importantly, CTGF is coordinately expressed with TGF-β and it demonstrates increased expression in numerous fibrotic disorders, including systemic sclerosis,27,28 pulmonary, renal, and myocardial fibrosis,29–32 and atherosclerosis.33 In the present study, we examine the process by which CTGF mediates the transformation of activated RPE into a fibrotic epiretinal membrane. Our results identify CTGF as a major mediator of retinal fibrosis and potentially an effective therapeutic target.

Induction of glucose-regulated protein (GRP)-78 in the endoplasmic reticulum (ER) is a protective mechanism cells use to adapt to ER stress. We evaluated the expression of GRP-78 and its regulation by an oxidant tert-butyl hydroperoxide (tBH) in human retinal pigment epithelium (RPE) cells.We used a carboxy-H2-DCFDA staining method to detect tBH-induced accumulation of reactive oxygen species (ROS) in RPE cells, and analyzed the expression of GRP-78 in normal human fetal and adult retinas and in cultured human RPE cells by immunohistochemistry. The effects of tBH (10-100 microM) on GRP-78 and on growth arrest and DNA damage inducible genes 153 (GADD153) protein and mRNA expression were studied using Western blot and real-time polymerase chain reaction.Sections of fetal retinas were negative for GRP-78. Adult retinas showed moderate cytoplasmic GRP-78 staining in the RPE and choroid. tBH-induced ROS accumulation in RPE cells showed partial colocalization with the ER. GRP-78 and GADD153 mRNA and protein expression in cultured RPE cells were significantly upregulated by treatment with tBH.tBH increases oxidative stress, increases accumulation of ROS in the ER, and upregulates expression of GRP-78 and GADD153. This supports the connection between oxidative stress and ER stress, and suggests that GRP-78 may serve a protective role in the RPE response to oxidative stress.

We outline current in vitro and in vivo models for experimental proliferative vitreoretinopathy (PVR) and provide a detailed protocol of our standardized in vivo PVR model. PVR is the leading cause of failed surgical procedures for the correction of rhegmatogenous retinal detachment. The pathogenesis of this multifactorial condition is still not completely understood. Experimental models for PVR help us understand the factors that play a role in the pathogenesis of the disease process in a controlled manner and allow for reproducible preclinical assessment of novel therapeutic interventions. We describe a cell injection model in detail that uses homologous retinal pigment epithelial (RPE) cell cultures to induce PVR over a 2-8 week period.

Aim: To report the early vitreous involvement in a rare familial amyloidotic polyneuropathy (FAP) mutation and associated vitreous vascular endothelial growth factor (VEGF) levels. Design: Observational case series. Methods: Review of clinical, pathological, photographic, and angiographic records of two FAP siblings with severe vitreous involvement. Laboratory ELISA analysis of vitreous samples for VEGF, and DNA sequence analysis of peripheral blood for transthyretin (TTR) mutational analysis. Results: Two patients underwent 25-gauge vitrectomy in three eyes with marked improvement of visual acuity. Neovascularisation seen intraoperatively responded to endolaser. Analysis of vitrectomy samples for VEGF showed raised levels in all three specimens. Mutational analysis revealed an isolated Glu54Gly mutation in the transthyretin gene. Conclusions: Early involvement of the vitreous occurs in a rare transthyretin mutation of FAP, with increased vitreous levels of VEGF.

OBJECTIVES To assess the effect of bevacizumab injection on connective tissue growth factor (CTGF) and vascular endothelial growth factor (VEGF) in the ocular fluids of patients with diabetic traction retinal detachment, and to determine whether intraoperative and postoperative complications are decreased in eyes given adjunctive preoperative bevacizumab injection. METHODS Twenty eyes of 19 patients were randomized to receive intravitreal bevacizumab or sham injection 3 to 7 days before vitrectomy for severe proliferative diabetic retinopathy. We collected aqueous samples before injection and at the time of vitrectomy and extracted undiluted vitreous samples. RESULTS Five eyes had decreased vascularization of membranes from preinjection to the time of vitrectomy (all in the bevacizumab treatment arm). Median visual acuities were 20/400 in control eyes at baseline and postoperative month 3 (POM3) and 8/200 in the bevacizumab-treated group at baseline and 20/100 at POM3 (P= .30 between control and bevacizumab-treated groups at POM3). All retinas were attached at POM3. Vitreous levels of VEGF were significantly lower in the bevacizumab group than in the control group (P= .03). Vitreous levels of CTGF were slightly lower in the bevacizumab group compared with the control group, but this difference was not statistically significant (P= .38). Levels of CTGF in the aqueous were strongly correlated with CTGF levels in the vitreous of controls (Spearman correlation coefficient, 0.95 [P< .001]). CONCLUSIONS Intravitreal bevacizumab injection reduces vitreous levels of VEGF and produces a clinically observable alteration in diabetic fibrovascular membranes. Ocular fluid levels of CTGF are not significantly affected within the week after VEGF inhibition. Retinal reattachment rates and visual acuity are not significantly altered by preoperative intravitreal bevacizumab injection at POM3. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT01270542.

Dexamethasone (DEX) is a glucocorticoid that is widely used after vitreoretinal surgery and may have mitogenic properties. This study was initiated to determine if human cultured retinal pigment epithelium (RPE) cells express glucocorticoid receptor and proliferate in response to DEX stimulation. Glucocorticoid receptor mRNA was detected in RPE cells by means of reverse transcriptase polymerase chain reaction. To determine the effect of DEX on RPE cell proliferation in vitro, we cultured human RPE cells with various concentrations for DEX for 5 days and determined the effects on cell number and incorporation of tritiated thymidine. DEX treatment with a DEX dose of 1 microgram/ml in the presence or absence of serum resulted in a maximal 2-3 times increase in cell number. Autoradiography after thymidine incorporation revealed a greater than 2-fold increase in incorporating cells at this same dose. These results indicate that DEX causes cultured human RPE cells to proliferate and suggest that it may have a growth factor-like action.

Transplantation of normal retinal pigment epithelium (RPE) into a diseased eye holds promise for treatment of several blinding disorders. Previuos studies have involved immunosuppression and implantation of freshly isolated cells. We report here the successful transplantation of cultured human RPE cells into rabbits that were not immunosuppressed. A modified pars plana transvitreal technique was used for RPE transplantation. The cultured RPE cells, loaded with carbon as a marker, were transplanted into the denuded Bruch's membrane of albino rabbits. The animals were followed for from 1 week to 3 months. On histologic examination at 2 months, no infiltrating lymphocytes were found in the vitreous cavity or choroid, even though Bruch's membrane was damaged. At about 3 months there were some macrophages in the subretina of transplanted eyes, indicating that an immunoreaction does occur eventually. Electron microscopy of the transplanted RPE showed apical-basal polarity and gap junctions. Restored function was attested to by the presence of phagosomes and phagocytosed outer segments in the transplanted cells. Our findings suggest that there is a weak, delayed immunoreaction to human RPE cells transplanted beneath the retina of the rabbit; however, functional recovery of the transplanted cells occurs before this immune response develops.

PURPOSE The purpose of this study was to evaluate the effect of a soluble monomeric form of the EphB4 extracellular domain (sEphB4) on choroidal endothelial cell (CEC) migration and tube formation and on experimental laser-induced choroidal neovascularization (CNV). METHODS EphrinB2 and EphB4 expression in CECs was investigated by Western blot analysis and immunohistochemistry. Effects of sEphB4 (0.5-3 microg/mL) on CEC migration were evaluated with a modified Boyden chamber assay. Tube formation was assayed in CEC cultures in collagen gel. CNV was induced in rats by laser photocoagulation. The effects of intravitreal injection of sEphB4 on CNV development were evaluated at day 14 by fluorescein angiography (FA), confocal volumetric analysis of isolectin-B4 labeled flatmounts, and histologic examination of CNV membranes. RESULTS CEC cells express both EphB4 and EphrinB2, according to Western blot analysis. Immunohistochemical sections of rat eye showed immunoreactivity for both EphB4 and EphrinB2 in the choroidal endothelium. sEphB4 reduced CEC migration in response to vascular endothelial growth factor (P < 0.01). Similarly, sEphB4 inhibited CEC tube formation in a dose-dependent manner. EphB4, and to a lesser extent EphrinB2, were detected on vascular channels within laser-induced CNV membranes. Intravitreal injection of sEphB4 inhibited laser-induced CNV formation. CNV membranes showed a reduction in leakage score (P < 0.05), and membrane volumes were reduced in size (P < 0.05). Histologic analysis revealed that vascularity was reduced in sEphB4-treated membranes. CONCLUSIONS Recombinant soluble monomeric EphB4 exerts an inhibitory effect on choroidal angiogenesis in vitro and in vivo. It should be further evaluated for its potential as a novel therapy for CNV.

Purpose Hepatocyte growth factor (HGF) regulates barrier function of retinal pigment epithelial (RPE) cells. The purpose of this study was to determine whether overexpression of HGF in the RPE induces retinal detachment (RD). Methods E1/E3-deleted adenoviral vectors encoding HGF (Ad CMV.HGF), green fluorescent protein (Ad CMV.GFP), or connective tissue growth factor (AdCMV.CTGF) were injected subretinally in adult pigmented rabbits (5 x 10(4) plaque-forming units [pfu]/eye). Animals were observed for up to 28 days with fundus photography. HGF expression in the retina and vitreous was determined using immunohistochemistry, and ELISA. Histopathologic examinations were performed with light and electron microscopy. Results Control eyes injected with AdCMV.GFP showed GFP expression almost exclusively in the RPE monolayer. Eyes injected with AdCMV.HGF showed strong HGF immunopositivity in RPE cells at the injection site. Elevated HGF levels were found in the vitreous peaking at postinjection day 7, diminishing to baseline by postinjection day 28. Eyes injected with AdCMV.HGF developed chronic RD and chronic inflammation in the choroid within the time frame of HGF expression. Groups of proliferating RPE cells were seen in the subretinal space in the region of the RD, and in some cases multilayered cellular membranes developed. No RD and minimal morphologic changes were seen in the eyes injected with AdCMV.GFP or AdCMV.CTGF. Conclusions Overexpression of HGF in RPE induces chronic, serous RD with subretinal proliferation of RPE. This work provides insight into the pathogenesis of RD and suggests that HGF should be further investigated as a target for therapeutic intervention in RD.

Objective To evaluate the expression of connective tissue growth factor (CTGF) in choroidal neovascular membranes from patients with age-related macular degeneration and the effect of CTGF on choroidal endothelial cell (CEC) function. Methods Using immunohistochemical methods, we analyzed CTGF expression in 13 surgically excised choroidal neovascular membranes related to age-related macular degeneration. The expression of CTGF in retinal pigment epithelial and CEC cultures was determined by means of reverse transcriptase polymerase chain reaction and Western blot, and its regulation by vascular endothelial growth factor and transforming growth factor β was determined. The effects of CTGF on bovine CEC proliferation, attachment, migration, and tube formation were measured. Results Vascularized human choroidal neovascular membranes showed strong CTGF immunoreactivity. Double staining disclosed colocalization of CTGF with retinal pigment epithelial cells and CECs. The CTGF induced a significant increase in attachment and migration of CECs; however, it did not stimulate CEC proliferation. The CTGF protein was up-regulated in retinal pigment epithelial cells and CECs by stimulation with transforming growth factor β and vascular endothelial growth factor, respectively. Conclusions The expression of CTGF in choroidal neovascular membranes, its regulation by angiogenic growth factors, and its proangiogenic effects on CEC function suggest that CTGF may play a role in the pathogenesis of choroidal neovascularization. Clinical Relevance Multiple growth factors are involved in the pathogenesis of choroidal neovascularization in age-related macular degeneration.