To investigate the effect of an intravitreally administered CCR2 antagonist, INCB3344, on a mouse model of choroidal neovascularization (CNV).
CNV was induced by laser photocoagulation on Day 0 in wild type mice. INCB3344 or vehicle was administered intravitreally immediately after laser application. On Day 14, CNV areas were measured on retinal pigment epithelium (RPE)-choroid flat mounts and histopathologic examination was performed on 7 µm-thick sections. Macrophage infiltration was evaluated by immunohistochemistry on RPE-choroid flat mounts and quantified by flow cytometry on Day 3. Expression of vascular endothelial growth factor (VEGF) protein in RPE-choroid tissue was examined by immunohistochemistry and ELISA, VEGF mRNA in sorted macrophages in RPE-choroid tissue was examine by real-time PCR and expression of phosphorylated extracellular signal-regulated kinase (p-ERK 1/2) in RPE-choroid tissue was measured by Western blot analysis on Day 3. We also evaluated the efficacy of intravitreal INCB3344 to spontaneous CNV detected in Cu, Zn-superoxide dismutase (SOD1) deficient mice. Changes in CNV size were assessed between pre- and 1week post-INCB3344 or vehicle administration in fundus photography and fluorescence angiography (FA).
The mean CNV area in INCB3344-treated mice decreased by 42.4% compared with the vehicle-treated control mice (p<0.001). INCB3344 treatment significantly inhibited macrophage infiltration into the laser-irradiated area (p<0.001), and suppressed the expression of VEGF protein (p = 0.012), VEGF mRNA in infiltrating macrophages (p<0.001) and the phosphorylation of ERK1/2 (p<0.001). The area of spontaneous CNV in Sod1−/− mice regressed by 70.35% in INCB3344-treated animals while no change was detected in vehicle-treated control mice (p<0.001).
INCB3344 both inhibits newly forming CNV and regresses established CNV. Controlling inflammation by suppressing macrophage infiltration and angiogenic ability via the CCR-2/MCP-1 signal may be a useful therapeutic strategy for treating CNV associated with age-related macular degeneration.
In the classic retinoid cycle, 11-cis retinol is synthesized in the retinal pigment epithelium (RPE) by two enzymes: Isomerase I (RPE65) and lecithin:retinol acyltransferase (LRAT). The purpose of this study is to provide experimental evidence for two active isomerases in the cone-dominated chicken eye: an LRAT-dependent Isomerase I in the RPE and an ARAT (acyl CoA:retinol acyltransferase)-dependent isomerase (Isomerase II) in the retina. First, we show that whole chicken retina in vitro, removed from the RPE/choroid and sclera, produces 11-cis retinoids upon light exposure, indicating the existence of RPE-independent isomerase (Isomerase II) activity in the retina. RT-PCR studies show high levels of RPE65 expression in the RPE, low levels in the retina, and none in primary Müller cell cultures, indicating the presence of Isomerase I in the RPE and a minimal amount in the retina. Activities of the RPE and retina isomerases were then measured by enzyme assays with specific enzyme inhibitors. 2,2′-Bipyridine, a known Isomerase I inhibitor, and N-ethyl-maleimide (NEM), a known LRAT inhibitor, significantly reduced Isomerase I activity but not Isomerase II activity. Progesterone, a known ARAT inhibitor, completely blocked Isomerase II activity but not Isomerase I activity. Thus the present study reports novel results to distinguish the biochemical properties of Isomerase I from Isomerase II, as well a difference in their locations in the chicken eye. Based on these differences, the cone-dominated chicken eye must contain two retinoid cycles: a classic visual cycle for retinoid exchange between the RPE and the retina supported by Isomerase I in the RPE, and an additional visual cycle for retinoid processing in the retina supported by Isomerase II.
Retinal pigment epithelial (RPE) cell dysfunction plays a central role in various retinal degenerative diseases, but knowledge is limited regarding the pathways responsible for adult RPE stress responses in vivo. RPE mitochondrial dysfunction has been implicated in the pathogenesis of several forms of retinal degeneration. Here we have shown that postnatal ablation of RPE mitochondrial oxidative phosphorylation in mice triggers gradual epithelium dedifferentiation, typified by reduction of RPE-characteristic proteins and cellular hypertrophy. The electrical response of the retina to light decreased and photoreceptors eventually degenerated. Abnormal RPE cell behavior was associated with increased glycolysis and activation of, and dependence upon, the hepatocyte growth factor/met proto-oncogene pathway. RPE dedifferentiation and hypertrophy arose through stimulation of the AKT/mammalian target of rapamycin (AKT/mTOR) pathway. Administration of an oxidant to wild-type mice also caused RPE dedifferentiation and mTOR activation. Importantly, treatment with the mTOR inhibitor rapamycin blunted key aspects of dedifferentiation and preserved photoreceptor function for both insults. These results reveal an in vivo response of the mature RPE to diverse stressors that prolongs RPE cell survival at the expense of epithelial attributes and photoreceptor function. Our findings provide a rationale for mTOR pathway inhibition as a therapeutic strategy for retinal degenerative diseases involving RPE stress.
To investigate the capacity of mature retinal pigment epithelium (RPE) cells to enter the cell cycle in vivo using a range of RPE-specific and proliferative specific markers in both pigmented and albino rats.
Whole-mounted retinas of both Dark Agouti and albino rats were immunolabeled with cell cycle markers Ki67 or PCNA and double labeled with RPE cell marker RPE65 or CRALBP. The number and distribution of these cells was mapped. An additional group of Dark Agouti rats were given repeated intraperitoneal injections of Bromodeoxyuridine (BrdU )for 20 days and then sacrificed 30 days later. The retinas were then processed for BrdU detection and Otx, a RPE cell-specific marker. For comparison, human RPE tissue from a postmortem donor was also labeled for Ki67.
In both pigmentation phenotypes, a subpopulation of mature RPE cells in the periphery were positive for both cell cycle markers. These cells were negative for Caspase 3, hence were not apoptotic. Ki67-positive cells were also seen in human RPE. Further, many cells positive for BrdU were identified in similar retinal regions, confirming that RPE cells not only enter the cell cycle but also divide, albeit at a slow cell cycle rate. There was a ten fold increase in the number of RPE cells positive for cell cycle markers in albino (approximately 200 cells) compared to pigmented rats (approximately 20 cells).
Peripheral RPE cells in rats have the capacity to enter the cell cycle and complete cellular division.
This study showed that mouse retina and retinal pigment epithelial (RPE) cells express the heme transporters FLVCR, BCRP, and PCFT. FLVCR is localized to the apical membrane, and BCRP and PCFT are localized to the basolateral membrane in RPE cells. Hemochromatosis, a genetic disease with iron overload, is associated with upregulation of FLVCR and PCFT, but with downregulation of BCRP in retina and RPE.
FLVCR, BCRP, and PCFT/HCP-1 represent the three heme transporters identified thus far in mammalian cells, but there is very little known about their expression and regulation in the retina. In this study, the expression of these transporters in mouse retina and retinal pigment epithelium (RPE) and their regulation in the iron-overload disease hemochromatosis were examined.
The expression of FLVCR, BCRP, and PCFT in mouse retina and primary mouse RPE cells was studied by RT-PCR and immunofluorescence. Polarized localization of the transporters in RPE was studied by co-localization using a specific marker of the RPE apical membrane. Uptake of heme in primary RPE cells was determined using zinc-mesoporphyrin, a fluorescent heme analogue. The regulation of heme transporters by iron overload was studied in two genetic models of hemochromatosis (HFE-null mouse and HJV-null mouse) and in two nongenetic models of iron overload (cytomegalovirus infection and treatment with ferric ammonium citrate).
All three heme transporters were expressed in the retina and RPE. In the RPE, the expression of FLVCR was restricted to the apical membrane, and the expression of BCRP and PCFT was restricted to the basolateral membrane. In all cases of iron overload, the expression of FLVCR and PCFT was upregulated and that of BCRP was downregulated.
Hemochromatosis is associated not only with excessive accumulation of free iron in the retina and RPE but also with excessive accumulation of heme. Since heme is toxic at high levels, as is free iron, heme-induced oxidative damage may also play a role in hemochromatosis-associated retinal pathology.
AIMS—To investigate the distribution, persistence, and stability of fluorescently labelled phosphorothioate oligonucleotides (PS-ODNs) in normal and laser photocoagulated retina following intravitreal injection in the rat.
METHODS—Fluorescently labelled PS-ODNs were injected intravitreally into pigmented eyes at doses of 0.5-10.0 nmol in 2.0 µl solution. The dynamics of PS-ODNs was evaluated by fluorescent microscopy of cryosections and flat mounted retinal pigment epithelium (RPE)-choroid-sclera. Genescan analysis was used to assess the integrity of PS-ODNs in the retina after injection. The dynamics of PS-ODNs was also evaluated in the retina following krypton laser photocoagulation with a protocol producing choroidal neovascularisation (CNV).
RESULTS—Following intravitreal injection the PS-ODNs demonstrated dose and time dependent distribution and persistence in the retina, where they accessed all neural layers. However, they preferentially accumulated in the RPE layer, demonstrated as bright granules in the cytoplasm of the cells. Injections of 5.0 and 7.5 nmol of PS-ODNs exhibited strong fluorescence in the retina for 6 weeks after injection. Genescan analysis demonstrated that the PS-ODNs remained almost completely intact for at least 12 weeks. Following laser treatment, the PS-ODNs were concentrated in the regions of laser photocoagulation and retained high intensity for at least 8 weeks after injection, particularly localised to macrophages, RPE, and the local choroidal tissue.
CONCLUSIONS—These results indicate that PS-ODNs are stable and accessible to most neural layers of the retina, and they preferentially accumulate in the RPE layer following intravitreal injection. The successful delivery of PS-ODNs into normal and laser photocoagulated retina suggests that PS-ODNs may have potential in the development of therapy for attenuating retinal degenerations and CNV.
Within the vertebrate eye, the retinal pigment epithelium (RPE) juxtaposes with the retina, but how the RPE plays a role in retinal morphogenesis remains elusive. It has been shown that the loss of function of the polarity proteins, such as Nagie oko (Nok), disrupts RPE integrity and retinal lamination. However, it is unclear whether or not such defects are caused in a tissue-autonomous fashion. Here, by taking advantage of the nok mutation, we have generated a transgenic model to restore the Nok function in the RPE, but not in the retina. With this model, we show that Nok is required for RPE integrity in a tissue-autonomous manner. However, proper retinal epithelial polarity does not require retinal expression of Nok prior to embryonic photoreceptor genesis; rather, it requires a Nok-mediated intact RPE. Interestingly, sporadic wildtype RPE donor cells are not sufficient to maintain proper retinal polarity. We further show that RPE-mediated retinal epithelial polarity underlies proper patterning of retinal ganglion cells and the cells of the inner nuclear layer. Nevertheless, during embryonic photoreceptor genesis, an intact RPE is not sufficient to maintain retinal epithelial polarity and retinal cellular pattern formation. Our results show that the subcellular architecture and cellular pattern formation of a tissue may be regulated by neighboring tissues through tissue-tissue interactions.
RPE; retina; cellular pattern formation; Nok; polarity; transgenesis
Cell cycle progression is governed by the coordinated activities of kinases, phosphatases, and the ubiquitin system. The entire complement of ubiquitin pathway components that mediate this process in retinal pigment epithelial (RPE) cells remains to be identified. This study was undertaken to determine whether the human ubiquitin-conjugating enzyme, UBE2E3, is essential for RPE cell proliferation.
UBE2E3 expression and localization in telomerase-immortalized, human RPE cells was determined with a UBE2E3-specific antibody. The necessity for UBE2E3 in RPE proliferation was determined using small interfering (si)RNA to target the expression of the enzyme. Cell counts and immunolabeling for the proliferation marker Ki-67 and the cyclin-dependent kinase inhibitor p27Kip1 were performed to assess the consequences of UBE2E3 depletion. A mouse strain harboring a disrupted allele of UbcM2 (the mouse counterpart of UBE2E3) with the coding sequence for β-galactosidase was used to track the developmental expression of the enzyme in murine RPE cells.
UBE2E3 localized in the nucleus of the immortalized RPE cells. Depletion of the enzyme by siRNA resulted in a cell-cycle exit accompanied by a loss of Ki-67, an increase in p27Kip1, and a doubling in cell area. Rescue experiments confirmed the specificity of the RNA interference. In vivo, UbcM2 was transcriptionally downregulated during RPE development in the mouse.
UBE2E3 is essential for the proliferation of RPE-1 cells and is downregulated during RPE layer maturation in the developing mouse eye. These findings indicate that UBE2E3 is a major enzyme in modulating the balance between RPE cell proliferation and differentiation.
The present study examines the effects of Cd81-null mutation on the development of the retinal pigment epithelium (RPE), specifically cell size and number of cells with multiple nuclei. The outlines of RPE in retinal flat mounts were stained with rhodamine-labeled phalloidin and RPE nuclei with Hoechst stain. The RPE layer was sampled to define the number of cells, the size of the RPE cells and the number of nuclei within the cells. The Cd81-null mutation caused an increase in the number of cells within the RPE layer. The cells were smaller than those in the wild type mice. Furthermore there was an increase in the number of mono-nucleated cells. In the posterior portion of the eye there was a significant increase in the number of multi-nucleated cells. The data indicate that CD81 plays a significant role in the final stages of RPE development, controlling cell number and overall developmental pattern.
TAPA; Growth regulation; Retina; Tetraspanin
Vascular endothelial growth factor (VEGF-A or VEGF) is a potent growth factor for the development of retinal and choroidal vasculatures. To define the temporal requirement of the retinal pigmented epithelium (RPE)-derived VEGF in choroidal vascular development, we generated conditional VEGF knockout mice using an inducible Cre/lox system. The loss of the RPE-derived VEGF was confirmed with immunoblotting and immunohistochemistry. Retinal function and structure were assessed with electroretinography and histology, respectively. Choroidal vascular density was analyzed with computer-assisted semi-quantitative assay using fluorescently labeled choroidal flat-mounts. Induction of RPE-specific VEGF disruption at embryonic day 10 (E10) or E13 for two days caused regulatable decreases in choroidal vascular density, photoreceptor function, and photoreceptor outer nuclear layer thickness. The loss of the RPE-produced VEGF after E15 did not cause detectable defects in choroidal vasculatures and photoreceptor function and morphology. These results suggest that the RPE-derived VEGF plays a critical role in choroidal vascular development during organogenesis before embryonic day 15.
choroid; RPE; VEGF; inducible; Cre/lox
MCT3, a specific marker of differentiated RPE, is downregulated after wounding. This report demonstrates for the first time a role for cell-cell contacts in restoring MCT3 expression after injury.
MCT3 is a proton-coupled monocarboxylate transporter preferentially expressed in the basolateral membrane of the retinal pigment epithelium (RPE) and has been shown to play an important role in regulating pH and lactate concentrations in the outer retina. Decreased expression of MCT3 in response to trauma or disease could contribute to pathologic changes in the retina. The present study followed the expression of MCT3 after wounding and re-epithelialization of chick RPE explant and human fetal (hf) RPE cultures.
Immunofluorescence microscopy and immunoblotting were performed to determine changes in MCT expression after scratch wounding and re-epithelialization of chick RPE/choroid explant cultures and hfRPE cell monolayers.
MCT3 expression and basolateral polarity were maintained in chick RPE/choroid explant cultures and hfRPE monolayers. Wounding resulted in loss of MCT3 and the upregulation of MCT4 expression in migrating cells at the edge of the wound. On re-epithelialization, MCT3 was detected in chick and hfRPE cells when cells became hexagonally packed and pigmented. However, in hfRPE cells, MCT4 was consistently expressed throughout the epithelial monolayer. RPE cells at the edges of chick explants and hfRPE cultures with a free edge expressed MCT4 but not MCT3.
Wounding of RPE monolayers resulted in dedifferentiation of the cells at the edge of the wound, as evidenced by a loss of MCT3 and increased MCT4 expression. Collectively, these findings suggest that both cell-cell and cell-substrate interactions are essential in directing and maintaining differentiation of the RPE and expression of MCT3.
Transplants of the retina are among the new strategies being used in the treatment of genetic and degenerative macular diseases. Moreover, various cell cultures are being tested to treat retinal disorders.
Literature dated from 2004 to 2011 was comprehensively examined via Medline and PubMed searches for the following terms: auto-, homo-, heterologous transplantation, retina, stem cells, cultivated cells.
Tissue and cell therapy of retinal diseases are reviewed, including full-thickness retina/retinal pigment epithelium (RPE)/choroid graft; full and partial thickness RPE/choroid complex grafts; RPE/Bruch membrane complex graft; and RPE, iris pigment epithelium and stem cell grafts. Recommendations for transplants, as well as the benefits and weaknesses of specific techniques in retina transplants, are discussed.
Auto- and allogenic transplants of a full or partial thickness retina/RPE/Bruch membrane/choroid complex represent an alternative treatment offered to patients with some macular diseases. Stem cell transplantation to reconstruct and regenerate the macula requires further biomolecular and animal research studies.
eye ball; transplantation; autologous; homologous; heterologous; retina; stem cells
To investigate the effects of curcumin on the development of experimental choroidal neovascularization (CNV) with underlying cellular and molecular mechanisms.
C57BL/6N mice were pretreated with intraperitoneal injections of curcumin daily for 3 days prior to laser-induced CNV, and the drug treatments were continued until the end of the study. The CNV area was analyzed by fluorescein-labeled dextran angiography of retinal pigment epithelium (RPE)-choroid flat mounts on day 7 and 14, and CNV leakage was evaluated by fluorescein angiography (FA) on day 14 after laser photocoagulation. The infiltration of F4/80 positive macrophages and GR-1 positive granulocytes were evaluated by immunohistochemistry on RPE-choroid flat mounts on day 3. Their expression in RPE-choroid complex was quantified by real-time PCR (F4/80) and Western blotting (GR-1) on day 3. RPE-choroid levels of vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, and intercellular adhesion molecule (ICAM)-1 were examined by ELISA on day 3. Double immunostaining of F4/80 and VEGF was performed on cryo-sections of CNV lesions on day 3. The expression of nuclear factor (NF)-κB and hypoxia-inducible factor (HIF)−1α in the RPE-choroid was determined by Western blotting.
Curcumin-treated mice had significantly less CNV area (P<0.05) and CNV leakage (P<0.001) than vehicle-treated mice. Curcumin treatment led to significant inhibition of F4/80 positive macrophages (P<0.05) and GR-1 positive granulocytes infiltration (P<0.05). VEGF mainly expressed in F4/80 positive macrophages in laser injury sites, which was suppressed by curcumin treatment (P<0.01). Curcumin inhibited the RPE-choroid levels of TNF-α (P<0.05), MCP-1 (P<0.05) and ICAM-1 (P<0.05), and suppressed the activation of NF-κB in nuclear extracts (P<0.05) and the activation of HIF−1α (P<0.05).
Curcumin treatment led to the suppression of CNV development together with inflammatory and angiogenic processes including NF-κB and HIF−1α activation, the up-regulation of inflammatory and angiogenic cytokines, and infiltrating macrophages and granulocytes. This provides molecular and cellular evidence of the validity of curcumin supplementation as a therapeutic strategy for the suppression of age-related macular degeneration (AMD)-associated CNV.
Hemochromatosis is an iron overload disorder with age-dependent oxidative stress and dysfunction in a variety of tissues. Mutations in HFE are responsible for most cases with hemochromatosis. We recently demonstrated that Hfe is expressed exclusively in the basal membrane of retinal pigment epithelium (RPE). Here we used Hfe−/− mice to examine ferritin levels (an indirect readout for iron levels) and morphological changes in retina. We found increased ferritin accumulation in retina in 18-month-old but not in 2-month-old mice with considerable morphological damage compared to age-matched controls. The retinal phenotype included hypertrophy and hyperplasia of RPE. RPE cells isolated from Hfe−/− mice exhibited a hyperproliferative phenotype. We also compared the gene expression profile between wild type and Hfe−/− RPE cells by microarray analysis. These studies showed that many cell cycle-related genes were differentially regulated in Hfe−/− RPE cells. One of the genes upregulated in Hfe−/− RPE cells was Slc7a11 that codes for the ‘transporter proper’ xCT in the heterodimeric cystine/glutamate exchanger (xCT/4F2hc). This transporter plays a critical role in cellular glutathione status and cell cycle progression. We confirmed the microarrray data by monitoring xCT mRNA levels by RT-PCR and also by measuring transport function. We also found increased levels of glutathione and the transcription factor/cell cycle promoter AP1 in Hfe−/− RPE cells. Wild type mouse RPE cells and human RPE cell lines, when loaded with iron by exposure to ferric ammonium citrate, showed increased expression and activity of xCT, reproducing the biochemical phenotype observed with Hfe−/− RPE cells.
hemochromatosis; retinal pigment epithelium; cell proliferation; cystine/glutamate exchanger; glutathione; transcription factor AP1
The authors show that RPE cells induce MDSC differentiation, which could be another mechanism by which RPE cells regulate immune responses in the retina.
To test whether retinal pigment epithelial (RPE) cells are able to induce myeloid-derived suppressor cell (MDSC) differentiation from bone marrow (BM) progenitors.
BM cells were cocultured with or without RPE cells in the presence of GM-CSF and IL-4. Numbers of resultant MDSCs were assessed by flow cytometry after 6 days of incubation. The ability of the RPE cell–induced MDSCs to inhibit T cells was evaluated by a CFSE-based T-cell proliferation assay. To explore the mechanism by which RPE cells induce MDSC differentiation, PD-L1–deficient RPE cells and blocking antibodies against TGF-β, CTLA-2α, and IL-6 were used. RPE cell-induced MDSCs were adoptively transferred into mice immunized with interphotoreceptor retinoid-binding protein in complete Freund's adjuvant to test their efficacy in suppressing autoreactive T-cell responses in experimental autoimmune uveitis (EAU).
RPE cells induced the differentiation of MDSCs. These RPE cell–induced MDSCs significantly inhibited T-cell proliferation in a dose-dependent manner. PD-L1–deficient RPE cells induced MDSC differentiation as efficiently as wild-type RPE cells, and neutralizing TGF-β or CTLA-2α did not alter the numbers of induced MDSCs. However, blocking IL-6 reduced the efficacy of RPE cell–induced MDSC differentiation. Finally, adoptive transfer of RPE cell–induced MDSCs suppressed IRBP-specific T-cell responses that led to EAU.
RPE cells induce the differentiation of MDSCs from bone marrow progenitors. Both cell surface molecules and soluble factors are important in inducing MDSC differentiation. PD-L1, TGF-β, and CTLA-2α were not measurably involved in RPE cell–induced MDSC differentiation, whereas IL-6 was important in the process. The induction of MDSCs could be another mechanism by which RPE cells control immune reactions in the retina, and RPE cell–induced MDSCs should be further investigated as a potential approach to therapy for autoimmune posterior uveitis.
The primary cilium, an organelle that transduces extracellular signals important for development and tissue homeostasis, is typically assembled upon cell cycle exit and disassembled upon cell cycle re-entry. Cilium assembly is thought to be suppressed in cycling cells, however the extent of suppression is not clear. For example, primary cilia are present in certain proliferating cells during development, and a period of reciliation has been reported to occur in late G1 in murine 3T3 cells released from serum starvation-induced quiescence. Human retinal pigmented epithelial (hTERT-RPE1; herein, RPE1) cells are commonly used to investigate pathways regulating cilium disassembly, however the ciliary disassembly profile of these cells remains uncertain. A period of reciliation has not been observed. Here, we analyse the ciliary disassembly profile of RPE1 cells by immunofluorescence microscopy. The results suggest a profile similar to 3T3 cells, including a period of reciliation in late G1 and a second wave of deciliation in S phase. We present evidence that arresting cells in early S phase with hydroxyurea or excess thymidine prevents the second wave of deciliation, and that deciliation is initiated shortly after release from a thymidine block, consistent with coupling to DNA replication. These findings support the often overlooked notion that cilium formation can occur in late G1, and suggest that RPE1 cells could serve as a model system for studying the molecular pathways that direct this process, in addition to those that stimulate cilium disassembly. We also present immunofluorescence data indicating that cyclin B1 localises to primary cilia.
•Ciliary disassembly profile of human RPE1 cells was analysed by immunofluorescence.•After release from serum-starvation, RPE1 cells re-assemble cilia in late G1 phase.•Cilium disassembly is not essential for S phase entry, but is coupled to DNA replication.•Cyclin B1 localises to primary cilia.
Primary cilia; Cyclin B1; CDK1; Aurora A; DNA replication; Cilium disassembly; AurA, Aurora kinase A; BrdU, bromodeoxyuridine; CDK, cyclin-dependent kinase; DAPI, 4’,6-diamidino-2-phenylindole; FBS, fetal bovine serum; HU, hydroxyurea; Mim, mimosine; siRNA, short interfering RNA; SS, serum-starved; Thy, thymidine
The retinal pigment epithelium (RPE) plays an essential role in maintaining the health of the retina. The RPE is also the site of pathologic processes in a wide variety of retinal disorders including monogenic retinal dystrophies, age-related macular degeneration, and retinal detachment. Despite intense interest in the RPE, little is known about its molecular response to ocular damage or disease. We have conducted a comprehensive analysis of changes in transcript abundance (the “genomic response”) in the murine RPE following light damage. Several dozen transcripts, many related to cell-cell signaling, show significant increases in abundance in response to bright light; transcripts encoding visual cycle proteins show a decrease in abundance. Similar changes are induced by retinal detachment. Environmental and genetic perturbations that modulate the RPE response to bright light suggest that this response is controlled by the retina. In contrast to the response to bright light, the RPE response to retinal detachment over-rides these modulatory affects.
retinal pigment epithelium; transcription; micro-array; mouse; ocular disease; light damage; retinal detachment
Age-related macular degeneration (AMD) is a leading cause of blindness that
affects the central region of the retinal pigmented epithelium (RPE), choroid, and
neural retina. Initially characterized by an accumulation of sub-RPE deposits, AMD
leads to progressive retinal degeneration, and in advanced cases, irreversible
vision loss. Although genetic analysis, animal models, and cell culture systems
have yielded important insights into AMD, the molecular pathways underlying AMD's
onset and progression remain poorly delineated. We sought to better understand the
molecular underpinnings of this devastating disease by performing the first
comparative transcriptome analysis of AMD and normal human donor eyes.
RPE-choroid and retina tissue samples were obtained from a common cohort of 31
normal, 26 AMD, and 11 potential pre-AMD human donor eyes. Transcriptome profiles
were generated for macular and extramacular regions, and statistical and
bioinformatic methods were employed to identify disease-associated gene signatures
and functionally enriched protein association networks. Selected genes of high
significance were validated using an independent donor cohort.
We identified over 50 annotated genes enriched in cell-mediated immune responses
that are globally over-expressed in RPE-choroid AMD phenotypes. Using a machine
learning model and a second donor cohort, we show that the top 20 global genes are
predictive of AMD clinical diagnosis. We also discovered functionally enriched
gene sets in the RPE-choroid that delineate the advanced AMD phenotypes,
neovascular AMD and geographic atrophy. Moreover, we identified a graded increase
of transcript levels in the retina related to wound response, complement cascade,
and neurogenesis that strongly correlates with decreased levels of
phototransduction transcripts and increased AMD severity. Based on our findings,
we assembled protein-protein interactomes that highlight functional networks
likely to be involved in AMD pathogenesis.
We discovered new global biomarkers and gene expression signatures of AMD. These
results are consistent with a model whereby cell-based inflammatory responses
represent a central feature of AMD etiology, and depending on genetics,
environment, or stochastic factors, may give rise to the advanced AMD phenotypes
characterized by angiogenesis and/or cell death. Genes regulating these
immunological activities, along with numerous other genes identified here,
represent promising new targets for AMD-directed therapeutics and diagnostics.
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The pterygium is a growth onto the cornea of fibrovascular tissue that is continuous with the conjunctiva, whereas the mechanisms of cell proliferation in pterygium epithelium are unknown.
To analyse the histopathology and the expression of cell cycle‐related molecules in pterygium tissues.
Seven pterygia were surgically removed using the bare‐sclera procedure, and three normal bulbar conjunctivas were also obtained. Formalin‐fixed, paraffin‐wax‐embedded tissues were analysed by immunohistochemistry with anti‐p27(KIP1), cyclin D1 and Ki‐67 antibodies.
Conjunctival epithelium consisted of several layers of round cells with a few goblet cells. Nuclear immunoreactivity for p27(KIP1) was noted in many normal epithelial cells, where cyclin D1 and Ki‐67‐positive nuclei were intermingled. A variety of goblet cells were located in the superficial layer of the pterygium head as well as those of the body epithelia. Several pterygium epithelial cells were p27(KIP1) positive, whereas nuclear immunoreactivity for cyclin D1 and Ki‐67 was detected in many epithelial cells. By contrast, immunoreactivity for p27(KIP1), cyclin D1 and Ki‐67 was hardly detected in the pterygium stroma.
It is suggested that pterygium growth and development are associated with the proliferation of epithelium, which is possibly involved in the expression of cell cycle‐related molecules.
Age-related macular degeneration (AMD) is the major cause of non-preventable blindness. Severe forms of AMD involve breaching of the retinal pigment epithelial (RPE) barrier by underlying choroidal endothelial cells (CECs), followed by migration into, and subsequent neovascularization of the neurosensory retina. However, little is known about the interactions between RPE and CECs and the signaling events leading to CEC transmigration. While soluble chemotactic factors secreted from RPE can contribute to inappropriate CEC transmigration, other unidentified stimuli may play an additional role. Using a coculture model that maintains the natural structural orientation of CECs to the basal aspect of RPE, we show that “contact” with RPE and/or RPE extracellular matrix increases CEC transmigration of the RPE barrier. From a biochemical standpoint, contact between CECs and RPE results in an increase in the activity of the GTPase Rac1 within the CECs; this increase is dependent on upstream activation of PI 3-K and Akt1. To confirm a link between these signaling molecules and increased CEC transmigration, we performed transmigration assays while inhibiting both PI 3-K and Rac1 activity, and observed that both decreased CEC transmigration. We hypothesize that contact between CECs and RPE stimulates a signaling pathway involving PI 3-K, Akt1, and Rac1 that facilitates CEC transmigration across the RPE barrier, an important step in the development of neovascular AMD.
Mutations in the MYO7A gene cause a deaf-blindness disorder, known as Usher syndrome 1B. In the retina, the majority of MYO7A is in the retinal pigmented epithelium (RPE), where many of the reactions of the visual retinoid cycle take place. We have observed that the retinas of Myo7a-mutant mice are resistant to acute light damage. In exploring the basis of this resistance, we found that Myo7a-mutant mice have lower levels of RPE65, the RPE isomerase that has a key role in the retinoid cycle. We show for the first time that RPE65 normally undergoes a light-dependent translocation to become more concentrated in the central region of the RPE cells. This translocation requires MYO7A, so that, in Myo7a-mutant mice, RPE65 is partly mislocalized in the light. RPE65 is degraded more quickly in Myo7a-mutant mice, perhaps due to its mislocalization, providing a plausible explanation for its lower levels. Following a 50–60% photobleach, Myo7a-mutant retinas exhibited increased all-trans-retinyl ester levels during the initial stages of dark recovery, consistent with a deficiency in RPE65 activity. Lastly, MYO7A and RPE65 were co-immunoprecipitated from RPE cell lysate by antibodies against either of the proteins, and the two proteins were partly colocalized, suggesting a direct or indirect interaction. Together, the results support a role for MYO7A in the translocation of RPE65, illustrating the involvement of a molecular motor in the spatiotemporal organization of the retinoid cycle in vision.
The results of this study suggest that COX-2 modulates VEGF expression in choroidal neovascularization and implicates a potential therapeutic role for nonsteroidal anti-inflammatory drugs.
To assess the degree of laser-induced choroidal neovascular membrane formation in wild-type (WT) and COX-2 null mice and to measure vascular endothelial growth factor (VEGF), interleukin (IL)-1β, and tumor necrosis factor (TNF)-α levels in the retina and choroid.
Four laser burns were placed in each eye of WT and COX-2 null mice to induce choroidal neovascularization. Fluorescein angiography (FA) was performed at 14 days, and retinal pigment epithelium-choroid-sclera (choroidal) flat mounts were prepared. The retina and choroid were isolated from WT and COX-2 null mice at 24, 72, and 168 hours after laser photocoagulation and from unlasered eyes and were tested for VEGF, IL-1β, and TNF-α.
COX-2 null mice demonstrated 58% (P = 0.001) and 48% (P = 0.001) reductions in CNV formation on FA and choroidal flat mounts, respectively, compared with WT mice. For unlasered mice, mean VEGF concentrations in the retina and choroid were 1.2 ± 0.42 pg/mg protein for WT but only 0.42 ± 0.2 pg/mg protein for COX-2 null mice (P < 0.05). After laser photocoagulation, WT mice showed significantly greater VEGF and IL-β expression in the retina and choroid by 168 hours (P < 0.05) and 72 hours (P < 0.05), respectively, compared with COX-2 null mice.
COX-2 null mice exhibited significantly less choroidal neovascular membrane formation associated with reduced expression of VEGF. The results of this study suggest that COX-2 modulates VEGF expression in CNV and implicates a potential therapeutic role for nonsteroidal anti-inflammatory drugs.
After experimental retinal detachment in rabbits macrophages are a prominent feature in the subretinal space or within the retina. Two sources for these macrophages are identified. The retinal pigment epithelium (RPE) may undergo metaplasia and actively 'bud'; the evolving macrophage is then formed by a vitreal protrusion of the cytoplasm of an RPE cell which is 'nipped off' by lateral protrusions from adjacent cells. In addition, in regions of RPE proliferation, blood-borne cells were found in Bruch's membrane and among the mass of proliferated RPE cells, suggesting that blood-borne cells may pass from the choroidal circulation through Bruch's membrane and the RPE layer.
β-Secretase (BACE1) is a major drug target for combating Alzheimer's disease (AD). Here we show that BACE1−/− mice develop significant retinal pathology including retinal thinning, apoptosis, reduced retinal vascular density and an increase in the age pigment, lipofuscin. BACE1 expression is highest in the neural retina while BACE2 was greatest in the retinal pigment epithelium (RPE)/choroid. Pigment epithelial-derived factor, a known regulator of γ-secretase, inhibits vascular endothelial growth factor (VEGF)-induced in vitro and in vivo angiogenesis and this is abolished by BACE1 inhibition. Moreover, intravitreal administration of BACE1 inhibitor or BACE1 small interfering RNA (siRNA) increases choroidal neovascularization in mice. BACE1 induces ectodomain shedding of vascular endothelial growth factor receptor 1 (VEGFR1) which is a prerequisite for γ-secretase release of a 100 kDa intracellular domain. The increase in lipofuscin following BACE1 inhibition and RNAI knockdown is associated with lysosomal perturbations. Taken together, our data show that BACE1 plays a critical role in retinal homeostasis and that the use of BACE inhibitors for AD should be viewed with extreme caution as they could lead to retinal pathology and exacerbate conditions such as age-related macular degeneration.
angiogenesis; β-secretase; lipofuscin; retina; retinal pigment epithelium
A primary function of cadherins is to regulate cell adhesion. Here, we demonstrate a broader function of cadherins in the differentiation of specialized epithelial cell phenotypes. In situ, the rat retinal pigment epithelium (RPE) forms cell-cell contacts within its monolayer, and at the apical membrane with the neural retina; Na+, K(+)-ATPase and the membrane cytoskeleton are restricted to the apical membrane. In vitro, RPE cells (RPE-J cell line) express an endogenous cadherin, form adherens junctions and a tight monolayer, but Na+,K(+)-ATPase is localized to both apical and basal-lateral membranes. Expression of E- cadherin in RPE-J cells results in restriction and accumulation of both Na+,K(+)-ATPase and the membrane cytoskeleton at the lateral membrane; these changes correlate with the synthesis of a different ankyrin isoform. In contrast to both RPE in situ and RPE-J cells that do not form desmosomes, E-cadherin expression in RPE-J cells induces accumulation of desmoglein mRNA, and assembly of desmosome-keratin complexes at cell-cell contacts. These results demonstrate that cadherins directly affect epithelial cell phenotype by remodeling the distributions of constitutively expressed proteins and by induced accumulation of specific proteins, which together lead to the generation of structurally and functionally distinct epithelial cell types.