Retinal Pigment Epithelial Proliferation Research Articles

The T-Box Transcription Factor TBX2 Regulates Cell Proliferation in the Retinal Pigment Epithelium

ABSTRACTPurpose: Vertebrate eye development and function critically depend on the regulation of proliferation of retinal pigment epithelium (RPE) cells. Hence, a thorough analysis of the molecular parameters controlling RPE cell proliferation is crucial for our understanding of the physiology of this cell type both in health and in disease. The T-box transcription factor TBX2 is an important cell cycle regulator in development and oncogenesis, but its specific role in RPE cell proliferation is far from clear. The purpose of the present study is to investigate whether TBX2 plays any role in regulating RPE cell proliferation.Materials and methods: The expression of TBX2 in RPE cells was analyzed in wildtype mice and ARPE-19 cells by co-staining for RPE-specific markers and cell proliferation. In vitro, the role of TBX2 was studied by manipulating its levels using RNAi and analyzing the effects on DNA synthesis and cell growth and on gene expression at the RNA and protein levels.Results: Here, we find that TBX2 is expressed in RPE cells both in vivo and in vitro. Specific knockdown of TBX2 in the human RPE cell line ARPE-19 leads to an accumulation of cells at G1. This cell cycle arrest is accompanied by changes in the levels of known cell cycle regulators and, in particular, by an increase in the levels of the tumor-suppressor gene CCAAT/enhancer-binding protein delta (CEBPD). In fact, simultaneous knockdown of both TBX2 and CEBPD interferes with the reduction in cell proliferation brought about by TBX2 reduction alone.Conclusions: Our results provide novel insights into the regulatory mechanisms of cell proliferation in the RPE and may contribute to our understanding of normal RPE maintenance and its pathology in degenerative and proliferative disorders of the eye.

Inhibition of Proliferation and Epithelial Mesenchymal Transition in Retinal Pigment Epithelial Cells by Heavy Chain-Hyaluronan/Pentraxin 3

Proliferative vitreoretinopathy (PVR) is mediated by proliferation and epithelial mesenchymal transition (EMT) of retinal pigment epithelium (RPE). Because heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) purified from human amniotic membrane exerts anti-inflammatory and anti-scarring actions, we hypothesized that HC-HA/PTX3 could inhibit these PVR-related processes in vitro. In this study, we first optimized an ARPE-19 cell culture model to mimic PVR by defining cell density, growth factors, and cultivation time. Using this low cell density culture model and HA as a control, we tested effects of HC-HA/PTX3 on the cell viability (cytotoxicity), proliferation (EGF + FGF-2) and EMT (TGF-β1). Furthermore, we determined effects of HC-HA/PTX3 on cell migration (EGF + FGF-2 + TGF-β1) and collagen gel contraction (TGF-β1). We found both HA and HC-HA/PTX3 were not toxic to unstimulated RPE cells. Only HC-HA/PTX3 dose-dependently inhibited proliferation and EMT of stimulated RPE cells by down-regulating Wnt (β-catenin, LEF1) and TGF-β (Smad2/3, collagen type I, α-SMA) signaling, respectively. Additionally, HA and HC-HA/PTX3 inhibited migration but only HC-HA/PTX3 inhibited collagen gel contraction. These results suggest HC-HA/PTX3 is a non-toxic, potent inhibitor of proliferation and EMT of RPE in vitro, and HC-HA/PTX3’s ability to inhibit PVR formation warrants evaluation in an animal model.

ROCK Inhibition Promotes Attachment, Proliferation, and Wound Closure in Human Embryonic Stem Cell-Derived Retinal Pigmented Epithelium.

PurposeNonexudative (dry) age-related macular degeneration (AMD), a leading cause of blindness in the elderly, is associated with the loss of retinal pigmented epithelium (RPE) cells and the development of geographic atrophy, which are areas devoid of RPE cells and photoreceptors. One possible treatment option would be to stimulate RPE attachment and proliferation to replace dying/dysfunctional RPE and bring about wound repair. Clinical trials are underway testing injections of RPE cells derived from pluripotent stem cells to determine their safety and efficacy in treating AMD. However, the factors regulating RPE responses to AMD-associated lesions are not well understood. Here, we use cell culture to investigate the role of RhoA coiled coil kinases (ROCKs) in human embryonic stem cell–derived RPE (hESC-RPE) attachment, proliferation, and wound closure.MethodsH9 hESC were spontaneously differentiated into RPE cells. hESC-RPE cells were treated with a pan ROCK1/2 or a ROCK2 only inhibitor; attachment, and proliferation and cell size within an in vitro scratch assay were examined.ResultsPharmacological inhibition of ROCKs promoted hESC-RPE attachment and proliferation, and increased the rate of closure of in vitro wounds. ROCK inhibition decreased phosphorylation of cofilin and myosin light chain, suggesting that regulation of the cytoskeleton underlies the mechanism of action of ROCK inhibition.ConclusionsROCK inhibition promotes attachment, proliferation, and wound closure in H9 hESC-RPE cells. ROCK isoforms may have different roles in wound healing.Translational RelevanceModulation of the ROCK-cytoskeletal axis has potential in stimulating wound repair in transplanted RPE cells and attachment in cellular therapies.

Trichostatin A Inhibits Retinal Pigmented Epithelium Activation in an In Vitro Model of Proliferative Vitreoretinopathy

Purpose: Proliferative vitreoretinopathy (PVR) is a blinding disorder that develops after a retinal tear or detachment. Activation of the retinal pigmented epithelium (RPE) is implicated in PVR; however, the mechanisms leading to enhanced RPE proliferation, migration, and contraction remain largely unknown. This study utilized an in vitro model of PVR to investigate the role of acetylation in RPE activation and its contribution to the progression of this disease.Methods: ARPE-19 cells, primary cultures of porcine RPE, and induced pluripotent stem cell-derived RPE (iPS-RPE) were utilized for cellular and molecular analyses. Cells treated with transforming growth factor beta 2 (TGFβ2; 10 ng/mL) alone or in the presence of the broad-spectrum histone deacetylase (HDAC) inhibitor, trichostatin A (TSA; 0.1 μM), were assessed for contraction and migration through collagen contraction and scratch assays, respectively. Western blotting and immunofluorescence analysis were performed to assess α-smooth muscle actin (α-SMA) and β-catenin expression after TGFβ2 treatment alone or in combination with TSA.Results: TGFβ2 significantly increased RPE cell contraction in collagen matrix and this effect was inhibited in the presence of TSA (0.1 μM). In agreement with these data, immunofluorescence analysis of TSA-treated iPS-RPE wounded monolayers revealed decreased α-SMA as compared with control. Scratch assays to assess wound healing revealed TSA inhibited TGFβ2-mediated iPS-RPE cell migration.Conclusions: Our findings indicate a role of acetylation in RPE activation. Specifically, the HDAC inhibitor TSA decreased RPE cell proliferation and TGFβ2-mediated cell contraction and migration. Further investigation of pharmacological compounds that modulate acetylation may hold promise as therapeutic agents for PVR.

Purple Sweet Potato Anthocyanin Inhibits the Proliferation of Human Retinal Pigment Epithelial Cell by Blocking Cell Cycle and Inducing Apoptosis

Purple Sweet Potato Anthocyanin (PSPA), a class of naturally occurring anthocyanins derived from purple sweet potato storage roots, possesses unique color and multiple bioactivities. This study investigated the anti-proliferative effect of PSPA in human Retinal Pigment Epithelial (RPE) cells, the proliferation of which accounts for Proliferative Vitreoretinopathy (PVR). Blueberry Anthocyanin (BBA) was used as a contrast. PSPA and BBA inhibited RPE proliferation time-and dose-dependently through blocking the cell cycle in G0/G1 phase and inducing apoptosis via ROS accumulation, DNA damage and caspase 3/7 activation. Meanwhile, PSPA showed stronger potential than BBA in inhibiting RPE growth. Hence, we highlighted the importance of dietary supplementation of anthocyanins in PVR prevention and the application of PSPA in health industry.

LYTAK1, a TAK1 inhibitor, suppresses proliferation and epithelial‑mesenchymal transition in retinal pigment epithelium cells.

The proliferation of retinal pigment epithelium (RPE) cells following epithelial-mesenchymal transition (EMT) is critical in proliferative vitreoretinopathy (PVR), which results in retinal detachment and the loss of vision. The current study was conducted to examine the importance of transforming growth factor β-1 (TGF-β1)-activated kinase 1 (TAK1) inhibitor (LYTAK1) in regulating EMT and the proliferation of RPE cells. RPE cells were pre-treated with increasing concentrations of LYTAK1 prior to treatment with TGF-β1 for 24 h. The effect of LYTAK1 on RPE cell proliferation was examined using a Cell Counting kit-8 assay. The expression levels of TAK1, smooth muscle actin, fibronectin, p-Smad2, p-Smad3, nuclear factor (NF)-κB p65 and IκB kinase α were detected by western blotting. LYTAK1 suppressed the proliferation and migration of RPE cells. Additionally, LYTAK1 significantly prevented TGF-β1-induced EMT by decreasing the levels of fibronectin and α-smooth muscle actin. It was demonstrated that the effects of LYTAK1 were via the Smad signaling pathway. The present study also determined, that the underlying mechanism of the effects of LYTAK1 on EMT in RPE cells involves downregulation of the NF-κB signaling pathway. In conclusion, TAK1 transcription factor was shown to be important in TGF-β1-induced EMT in human RPE cells. Thus, the results of this study aid in elucidating the pathogenesis of human PVR. In addition, this study suggests that specific inhibition by LYTAK1 may provide a novel approach for the treatment and prevention of PVR.

Morphologic changes in the retina after selective retina therapy.

To investigate structural changes in the retina by histologic evaluation and in vivo spectral domain optical coherence tomography (SD-OCT) following selective retina therapy (SRT) controlled by optical feedback techniques (OFT). SRT was applied to 12 eyes of Dutch Belted rabbits. Retinal changes were assessed based on fundus photography, fluorescein angiography (FAG), SD-OCT, light microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM) at each of the following time points: 1h, and 1, 3, 7, 14 and 28days after SRT. BrdU (5'-bromo-2'-deoxy-uridine) incorporation assay was also conducted to evaluate potential proliferation of RPE cells. SRT lesions at1 h after SRT were ophthalmoscopically invisible. FAG showed leakage in areas corresponding to SRT lesions, and hyperfluorescence disappeared after 7days. SD-OCT showed that decreased reflectivity corresponding to RPE damage was restored to normal over time in SRT lesions. Histologic analysis revealed that the damage in SRT lesions was primarily limited to the retinal pigment epithelium (RPE) and the outer segments of the photoreceptors. SEM and TEM showed RPE cell migration by day 3after SRT, and restoration of the RPE monolayer with microvilli by 1week after SRT. At 14 and 28days, ultrastructures of the RPE, including the microvilli and tight junctions, were completely restored. The outer segments of the photoreceptors also recovered without sequelae. Interdigitation between the RPE and photoreceptors was observed. BrdU incorporation assay revealed proliferation of RPE on day 3after SRT, and peak proliferation was observed on day 7after SRT. Based on multimodal imaging and histologic assessment, our findings demonstrate that SRT with OFT could selectively target the RPE without damaging the neurosensory retina. Therefore, the use of SRT with OFT opens the door to the possibility of clinical trials of well-defined invisible and nondestructive retina therapy, especially for macular disease.

Wnt/β-Catenin Signaling Mediates Regeneration of Retinal Pigment Epithelium After Laser Photocoagulation in Mouse Eye

Laser photocoagulation of retinal pigment epithelium (RPE) is used to stimulate the regenerative processes of the RPE. However, the molecular mechanisms that control RPE proliferation and the epithelial-mesenchymal transition (EMT) during regeneration remain poorly understood. We investigated the role of Wnt/β-catenin signaling in the regeneration of mouse RPE after laser photocoagulation. C57BL/6J mice were photocoagulated unilaterally. To determine the β-catenin-dependent Wnt signal transduction in the photocoagulated RPE, the expression levels of Wnts, β-catenin, and their target genes were analyzed using real time-PCR and Western blotting. Retinal pigment epithelium proliferation and EMT were determined by 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay and by profiling expression of EMT markers, respectively, in eyes injected intravitreally with a Wnt/β-catenin signaling antagonist, Dkk-1, after laser photocoagulation. Expression of several of the 19 Wnt genes was significantly increased in laser-treated RPE. The expression levels of β-catenin signaling target genes cyclin D1, Otx2, and Mitf were higher in laser-treated RPE than in control RPE. The number of EdU-positive cells in the laser-treated area was significantly lower in the Dkk-1-injected group than in the laser-treated group or laser-treated + vehicle-injected group. There were more Otx2- and Mitf-positive cells after laser photocoagulation and markedly fewer in the Dkk-1-injected group. Otx2- and Mitf-positive cells were colocalized with EdU-positive cells. The EMT markers vimentin and α-smooth muscle actin (α-SMA) were upregulated in the laser-treated area and significantly downregulated in the Dkk-1-injected group. Laser photocoagulation activates a Wnt/β-catenin signal transduction pathway, promoting both RPE proliferation and EMT. Wnt/β-catenin signaling also upregulates the expression of Otx2 and Mitf, key transcription factors in RPE formation. Our results demonstrate an important role for Wnt/β-catenin signaling in RPE proliferation and EMT, and suggest that manipulating Wnt/β-catenin signaling may have therapeutic potential for RPE regeneration.

Advanced Clinical Imaging and Tissue-based Biomarkers of the Eye for Toxicology Studies in Minipigs.

There is increased interest to use minipigs in ocular toxicology studies due to their anatomical similarities with human eyes and as a substitute for nonhuman primates. This requires adaptation of enhanced optical coherence tomography (OCT) techniques and of ocular relevant immunohistochemistry (IHC) or in situ hybridization (ISH) markers to porcine eyes. In this study, OCT and OCT angiography (AngioOCT) were performed on adult Göttingen minipigs. To increase structural information on retinal and choroidal vasculature, OCT data were speckle denoized and choroidal blood vessels were segmented with threshold filtering. In addition, we established a set of IHC and ISH markers on Davidson's fixed paraffin-embedded minipig eyes: neurofilament-160, neuronal nuclei, calretinin, protein kinase C-α, vimentin, glial fibrillary acidic protein, glutamine synthetase, ionized calcium-binding adaptor molecule-1, rhodopsin, synaptophysin, postsynaptic density protein-95, retinal pigment epithelium (RPE)-specific protein-65, von Willebrand factor, α-smooth muscle actin, desmin, and Ki-67, thus enabling visualization of retinal neuronal and glial cells, photoreceptors, synapses, RPE, blood vessels, myocytes, macrophages, or cell proliferation. Using ISH, transcripts of vascular endothelial growth factor A, angiopoietin-2, and endothelial tyrosine kinase were visualized. This article describes for the first time in minipig eyes speckle noise-free OCT, AngioOCT, and a set of IHC/ISH markers on Davidson's fixed paraffin-embedded tissues and helps to establish the minipig for ocular toxicology and pharmacology studies.

A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells.

The integrity of the epithelium is maintained by a complex but regulated interplay of processes that allow conversion of a proliferative state into a stably differentiated state. In this study, using human embryonic stem cell (hESC) derived Retinal Pigment Epithelium (RPE) cells as a model; we have investigated the molecular mechanisms that affect attainment of the epithelial phenotype. We demonstrate that RPE undergo a Mesenchymal–Epithelial Transition in culture before acquiring an epithelial phenotype in a FOXM1 dependent manner. We show that FOXM1 directly regulates proliferation of RPE through transcriptional control of cell cycle associated genes. Additionally, FOXM1 modulates expression of the signaling ligands BMP7 and Wnt5B which act reciprocally to enable epithelialization. This data uncovers a novel effect of FOXM1 dependent activities in contributing towards epithelial fate acquisition and furthers our understanding of the molecular regulators of a cell type that is currently being evaluated as a cell therapy.

Retinal pigment epithelial cell proliferation.

The human retinal pigment epithelium forms early in development and subsequently remains dormant, undergoing minimal proliferation throughout normal life. Retinal pigment epithelium proliferation, however, can be activated in disease states or by removing retinal pigment epithelial cells into culture. We review the conditions that control retinal pigment epithelial proliferation in culture, in animal models and in human disease and interpret retinal pigment epithelium proliferation in context of the recently discovered retinal pigment epithelium stem cell that is responsible for most invitro retinal pigment epithelial proliferation. Retinal pigment epithelial proliferation-mediated wound repair that occurs in selected macular diseases is contrasted with retinal pigment epithelial proliferation-mediated fibroblastic scar formation that underlies proliferative vitreoretinopathy. We discuss the role of retinal pigment epithelial proliferation in age-related macular degeneration which is reparative in some cases and destructive in others. Macular retinal pigment epithelium wound repair and regression of choroidal neovascularization are more pronounced in younger than older patients. We discuss the possibility that the limited retinal pigment epithelial proliferation and latent wound repair in older age-related macular degeneration patients can be stimulated to promote disease regression in age-related macular degeneration.

Suppression of the proliferation of hypoxia-Induced retinal pigment epithelial cell by rapamycin through the /mTOR/HIF-1α/VEGF/ signaling.

Rapamycin, a highly specific inhibitor of mammalian target of rapamycin (mTOR), exhibits significant antitumor/antiangiogenic activity in human cancer cells. Its effect on the retinal pigment epithelial (RPE) cells was rarely investigated. This study assessed the proliferation of hypoxia-induced RPE and the inhibitory effects of rapamycin using 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and examined the expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in RPE cells with or without rapamycin under normoxic and hypoxic conditions using real-time PCR and Western blot. We found that hypoxia increased the levels of mTOR, HIF-1α, and VEGF. The suppression of HIF-1α and VEGF by rapamycin was associated with dephosphorylation of mTOR and the downstream effector ribosomal protein S6 kinase (P70S6K) and 4E-binding protein-1 (4E-BP1) of mTORC1. Rapamycin only inhibited the protein levels and did not change the mRNA expression of HIF-1α. No cytotoxicity to the RPE cells by rapamycin was caused under either normoxia or hypoxia. Our data suggest that rapamycin suppresses hypoxia-induced RPE cell proliferation through a mechanism related to the targeting of mTOR/HIF-1α/VEGF signaling. Rapamycin may potentially provide a safe and effective novel treatment for choroidal vascular disease.

PROLIFERACINĖS VITREORETINOPATIJOS PATOGENEZĖ IR GYDYMAS: AKTUALIJOS GERESNEI AKIŲ LIGŲ PRIEŽIŪRAI UŽTIKRINTI

Proliferative vitreoretinopathy (PVR) is defined as retinal pigment epithelium proliferation associated growth and contraction of avascular fibrous – cellular membranes within the vitreous body. It develops after rhegmatogenous retinal detachment, when liberated cells of retinal pigment epithelium gain access through retinal tear to vitreous body, or after serious retinal surgery, in which extensive retinopexy by lasering or cryotherapy is performed, or in cases of serious penetrating eye injuries. While retinal surgery is improving PVR gains more importance. Despite effective initial treatment pathological membranes progress on surface of the retina, that causes its traction and deterioration of visual functions. This paper reviews data from latest publications about PVR pathogenic mechanisms, risk factors, treatment and its complications, as well as prevention.

Pseudotumoral and Multiple Retinal Pigment Epithelium Proliferation in Vogt-Koyanagi-Harada Disease.

We report a case of pseudotumoral retinal pigment epithelium (RPE) proliferation in Vogt-Koyanagi-Harada (VKH) disease, in a 50-year-old female who presented with a juxtapapillary and peripheral subretinal hyperpigmented lesions in the left eye and “sunset glow fundus,” hyperpigmented striae, and multiple atrophic chorioretinal spots in the periphery. The darkly pigmented exuberant larger subretinal mass extended to the periphery with associated subretinal fibrosis. This patient demonstrated the entire clinical presentation of VKH disease, which tends to course with a chronic, bilateral, granulomatous panuveitis and exudative retinal detachment associated with poliosis, vitiligo, alopecia, and central nervous system and auditory signs. Our case is unique for the presence of exuberant, pseudotumoral RPE proliferation at the juxtapapillary region and peripheral area. Although this complication has rarely been reported, a high index of suspicion is warranted for early diagnosis and avoids unnecessary treatments of a pseudotumor.

Retinal Pigment Epithelial Cell Conditioned Medium Enhances the Yield of RPE Cells Differentiated from Human Embryonic Stem Cells

Geographic atrophy (GA) in patients with age-related macular degeneration (AMD) is characterized by the loss of retinal pigment epithelial (RPE) cells in the macular region. Human embryonic stem cells (hESC)-derived RPE have shown promise in cellular therapy for these diseases. However, the differentiation from hESC is a very lengthy process with an initially low RPE yield. The purpose of this study was to determine whether polarized hESC-RPE conditioned medium (CM) could improve the yield of RPE differentiated from hESC and to identify the mechanism of this effect. Two hESC lines, hES3 and H9, were differentiated into RPE by culturing in CM or regular medium for up to 8 weeks. We found substantially more pigmented cells in CM based on surface area quantification, and Q-RT PCR showed significantly higher expression of RPE-specific genes in the H9 and hES3 cells cultured in CM. The results indicated that CM increased the yield of RPE cells differentiated from H9 and hES3 lines via increased RPE colony formation and proliferation. These findings may be beneficial to the manufacturing process for future clinical studies.

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

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

Effects of p75 neurotrophin receptor on regulating hypoxia-induced angiogenic factors in retinal pigment epithelial cells.

Retinal pigment epithelium (RPE) exerts critical roles in the maintenance of the normal functions of the retina, whereas RPE dysfunction can induce retina neovascularization. p75 neurotrophin receptor (p75(NTR)) has been shown to play essential roles in angiogenesis. However, the function of p75(NTR) in the RPE remains unclear. In the present study, we demonstrated that p75(NTR) was highly expressed in the human choroidal neovascularization membranes. For in vitro study, RPE was exposed to hypoxia, and a knockdown of p75(NTR) was achieved via lentivirus-mediated RNA interference. The results showed that hypoxia induced the expression of p75(NTR) in the RPE, and the knockdown of p75(NTR) rescued RPE proliferation activity and inhibited apoptosis which induced by hypoxia. After the deletion of p75(NTR), RPE-secreted pro-angiogenic factors (vascular endothelial growth factor and platelet-derived growth factor), inflammatory factors [interleukin 1 beta (IL1β), IL18, and stromal cell-derived factor 1], and matrix metalloproteinases (MMPs) (MMP3 and MMP9) were down-regulated under hypoxic conditions. While the RPE secreted anti-angiogenic factors (pigment epithelium-derived factor) and angiostatin, the tissue inhibitors of metalloproteinases (TIMPs) (TIMP-1 and TIMP-3) were up-regulated after the knockdown of p75(NTR). The human umbilical vein endothelial tube formation ability can be inhibited when it is co-cultured with the supernatant extract from p75(NTR)-knockdown RPE under hypoxic induction. These results suggest that the knockdown of p75(NTR) suppressed pro-angiogenic factors which induced by hypoxia while promoting the anti-angiogenesis-related factors in the RPE. It is indicated that p75(NTR) could be a potential therapeutic target for RPE hypoxia or oxidative stress diseases.

Development of a surface to increase retinal pigment epithelial cell (ARPE-19) proliferation under reduced serum conditions

Age related macular degeneration of the eye is brought about by damage to the retinal pigment epithelium (RPE) and is a major cause of adult blindness. One potential treatment method is transplantation of RPE cells grown in vitro. Maintaining RPE cell viability and physiological function in vitro is a challenge, and this must also be achieved using materials that can be subsequently used to deliver an intact cell sheet into the eye. In this paper, plasma polymerisation has been used to develop a chemically modified surface for maintaining RPE cells in vitro. Multiwell plates modified with a plasma copolymer of allylamine and octadiene maintained RPE cell growth at a level similar to that of TCPS. However, the addition of bound glycosaminoglycans (GAGs) to the plasma polymerised surface significantly enhanced RPE proliferation. Simply adding GAG to the culture media had no positive effect. It is shown that a combination of plasma polymer and GAG is a promising method for developing suitable surfaces for cell growth and delivery, that can be applied to any substrate material.

The Effects of Vital Dyes on Retinal Pigment Epithelium Cells in Oxidative Stress

Purpose: To determine the effect of the most commonly used vital dyes in vitrectomy [trypan blue at 0.15% concentration and indocyanine green (ICG) at 0.5% concentration] on the viability of human retinal pigment epithelium (RPE) cell lines (ARPE-19) exposed to oxidative stress. Methods: ARPE-19 cells unexposed or exposed to oxidative stress (hypoxic chamber) were treated for 1 min with one of the dyes. RPE proliferation was measured by ³H-thymidine incorporation, adhesion by ability to adhere to fibronectin, and safety by annexin V staining. Results: Proliferation: The dyes affected the proliferation of RPE cells differently under non-hypoxic and hypoxic conditions (p = 0.001). In non-hypoxic conditions, there was no statistically significant difference between the proliferation of the treated (both dyes) and untreated RPE cells (p = 0.279). Under hypoxia, both dyes significantly suppressed proliferation, more so with ICG (p = 0.001). Adhesion: The dyes affected adhesion differently under non-hypoxic and hypoxic conditions (p = 0.04). In non-hypoxic conditions, both increased the adhesive properties of RPE cells to fibronectin, ICG more than trypan blue (p = 0.001). Under hypoxia, both dyes suppressed adhesion, with no statistically significant difference between treated and non-treated RPE cells. Apoptosis: Both dyes increased early apoptosis of RPE cells compared with no treatment (p = 0.001), ICG more than trypan blue. Hypoxia increased the apoptosis of both dyes compared to non-hypoxic conditions (p = 0.02). Conclusions: In hypoxic conditions, both dyes showed an inhibition of RPE adhesion to fibronectin and proliferation capacity and an increase in early apoptosis compared with non-hypoxic conditions. Apoptosis was greater in ICG-treated RPE cells than in trypan blue-treated cells.

Spectral-Domain Optical Coherence Tomographic Findings at Each Stage of Punctate Inner Choroidopathy

To describe the natural course of punctate inner choroidopathy (PIC) using spectral-domain optical coherence tomography (SD-OCT). Prospective observational case series. Forty-two consecutive patients (60 eyes) with PIC with at least 3 months of follow-up. Serial SD-OCT images were obtained from both eyes of each participant at each visit. The morphologic characteristics of each stage of PIC lesions observed by SD-OCT. Continued stage progression of PIC lesions was observed in 27 eyes (45%), among which choroidal thickness changes were observed in 8 eyes (30%). Stage I lesion showed a normal appearance or slight irregularities in the outer nuclear layer. Stage II lesion appeared as a focal elevation of the retinal pigment epithelium (RPE) with corresponding disruption of the inner and outer segments of the photoreceptor interface. Stage III lesion broke through the RPE, forming a hump-shaped chorioretinal nodule with moderate reflectivity beneath the outer plexiform layer (OPL), generally with subsequent disruption of Bruch's membrane. Nodules occasionally invaded the inner retina, causing segmental retinal phlebitis in 2 eyes. Stage IV lesion regressed in a retrograde manner with tissue loss from the photoreceptor layer and inner choroid, finally leaving a V-shaped hernia of the OPL and inner retina into the choroid. Stage V lesion gradually eliminated the photoreceptors around the lesion; this process was accompanied by RPE proliferation at multiple levels, which reduced retinal herniation. Parafoveal stage V lesions caused late occult macular atrophy in 4 eyes. Choroidal thickness increased throughout the active phase and reached a peak at stage III; this parameter then significantly decreased at stage IV and gradually reached a minimum that was lower than the initial value at stage I. Punctate inner choroidopathy is a chorioretinitis rather than an inner choroidopathy. Spectral-domain OCT characterized a 5-stage evolution of PIC lesions: choroidal infiltration, formation of sub-RPE nodules, and then chorioretinal nodules, regression, and retinal herniation.