The role of retinal pigment epithelium in the early stage of development of subretinal neovascularization

In order to evaluate the role of retinal pigment epithelium (RPE) in the early stage of experimental subretinal neovascularization, we severely damaged RPE cells before the development of subretinal neovascularization. Three adult rhesus monkeys were used in this study. One mol/l l-ornithine hydrochloride saline solution was injected intravitreously at 2 weeks before intense krypton laser photocoagulation on the retina at the posterior pole, and clinical and histopathological course were studied at 1 to 28 days after photocoagulation. As a result, no evidence of new vessel formation could be observed clinically throughout the entire course. Histopathologically, at 3 days after photocoagulation, slight proliferation of RPE cells was identified by electron microscopy at the margin of the laser lesions, and budding of vascular endothelial cells derived from choroidal microvessels was observed in the choroid. However, no newly-formed vessels extended into the subretinal space at 7 days or more after photocoagulation. These results confirmed our hypothesis that proliferated RPE cells had inductive effect on the growth of endothelial cells in the early stage of development of subretinal neovascularization.     

The role of retinal pigment epithelium in the involution of subretinal neovascularization

The extravascular milieu around laser-induced experimental subretinal neovascularization (SRN) was studied during the evolution of the neovascular membrane from its early leaky stage to its late involuted stage. When the first signs of visible leakage appeared on angiography, newly formed vessels were spread in the subretinal space around the break in Bruch's membrane, fluid was accumulating in the subretinal space, and retinal pigment epithelial (RPE) cells were proliferating in a papillary pattern around the newly formed vessels; the RPE proliferation began with the undamaged cells at the edges of the laser injury. With further maturation, the RPE continued to envelope the subretinal vessels. This RPE proliferation was associated with the disappearance of fluid between the enveloped vessels and the sensory retina, and the gradual cessation of fluorescein leakage during angiography. At the end of the involution process, when the neovascular membrane no longer demonstrated any leakage, the subretinal vessels were found to be tightly enveloped by RPE cells, and no fluid separated them from the sensory retina. The authors' results suggest that involution of the neovascular membrane with maturation, as demonstrated by the cessation of visible fluorescein leakage, is the result of RPE proliferation that tightly envelopes the newly formed vessels and probably resorbs the previously accumulated subretinal fluid, as well as preventing its further accumulation in the subretinal space.     

A role of the retinal pigment epithelium in the involution of subretinal neovascularization]

We clarified a role of the retinal pigment epithelium (RPE) in the regression of experimentally induced subretinal neovascularization (SRN) in monkey. Eight eyes of 5 rhesus monkeys were used in this study. Two weeks after intense krypton laser photocoagulation to the posterior pole of the fundus, 0.5M l-ornithine hydrochloride solution 0.03 ml was injected intravitreously for the purpose of selective RPE damage. After ornithine injection, SRN continued without any evidence of spontaneous regression over 8 weeks following photocoagulation. Histopathologically, SRN developed with wide lumen in the subretinal space accompanied with serous detachment of the sensory retina, and new vessels were not enveloped completely by the proliferating RPE cells. We already showed that experimentally induced subretinal neovascularizations naturally regress spontaneously by envelopment of RPE cells 5 to 8 weeks after photocoagulation. Our results suggested that SRN persist actively without regression due to incomplete enclosure by RPE by selective damage of RPE at the active stage of SRN. We have confirmed that the RPE cells played an important role at the involution stage of SRN.     

Persistent subretinal indocyanine green induces retinal pigment epithelium atrophy

PURPOSE: To describe a case of a patient with macular hole with subretinal indocyanine green (ICG) during vitrectomy. DESIGN: Interventional case report. METHODS: A 66-year-old woman with macular hole underwent a vitrectomy with ICG. RESULTS: After application of ICG into the vitreous, ICG was introduced in the subretinal space. Indocyanine green was found to be present for more than 6 months. Retinal pigment epithelium atrophy appeared at the site of the lesion. CONCLUSIONS: Although ICG may be a useful tool for distinguishing the internal limiting membrane and other tissues careful application is required to prevent side effects.     

Resorption of subretinal fluid by transepithelial flow of the retinal pigment epithelium

The efficiency of flow through the retinal pigment epithelium (RPE) was calculated by studying the postoperative course of the subretinal fluid after nondrainage surgery of rhegmatogenous retinal detachment. In ten eyes of ten patients the preoperative volume of the subretinal fluid, area of the RPE participating in resorption of the fluid, elapsed time until resorption of the fluid, and efficiency of the flow were studied using B-scan ultrasonography. The flow through the RPE was 261 l/cm² per day and could drain more than 50% of the vitreous volume to the choroidal circulation. These data coincide well with early reattachment of neural retina in nondrainage surgery. No statistically significant difference in this flow was seen between young and old patients.     

Observations on the retinal pigment epithelium and retinal macrophages in experimental retinal detachment.

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.     

Functional and morphological analysis of the subretinal injection of retinal pigment epithelium cells

Replacement of retinal pigment epithelium (RPE) cells by transplantation is a potential treatment for some retinal degenerations. Here, we used a combination of invasive and noninvasive methods to characterize the structural and functional consequences of subretinal injection of RPE cells. Pigmented cells from primary cultures were injected into albino mice. Recovery was monitored over 8 weeks by fundus imaging, spectral domain optical coherence tomography (sdOCT), histology, and electroretinography (ERG). sdOCT showed that retinal reattachment was nearly complete by 1 week. ERG response amplitudes were reduced after injection, with cone-mediated function then recovering better than rod function. Photoreceptor cell loss was evident by sdOCT and histology, near the site of injection, and is likely to have been the main cause of incomplete recovery. With microscopy, injected cells were identified by the presence of apical melanosomes. They either established contact with Bruch's membrane, and thus became part of the RPE monolayer, or were located on the apical surface of the host's cells, resulting in apposition of the basal surface of the injected cell with the apical surface of the host cell and the formation of a series of desmosomal junctions. RPE cell density was not increased, indicating that the incorporation of an injected cell into the RPE monolayer was concomitant with the loss of a host cell. The transplanted and remaining host cells contained large vacuoles of ingested debris as well as lipofuscin-like granules, suggesting that they had scavenged the excess injected and host cells, and were stressed by the high digestive load. Therefore, although significant functional and structural recovery was observed, the consequences of this digestive stress may be a concern for longer-term health, especially where RPE cell transplantation is used to treat diseases that include lipofuscin accumulation as part of their pathology.     

Impacts of hypoxia-inducible factor-1 knockout in the retinal pigment epithelium on choroidal neovascularization

Purpose. Hypoxia-inducible factor (HIF)-1 is a key oxygen sensor and is believed to play an important role in neovascularization (NV). The purpose of this study is to determine the role of retinal pigment epithelium (RPE)-derived HIF-1α on ocular NV. Methods. Conditional HIF-1α knockout (KO) mice were generated by crossing transgenic mice expressing Cre in the RPE with HIF-1α floxed mice, confirmed by immunohistochemistry, Western blot analysis, and fundus fluorescein angiography. The mice were used for the oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) models. Results. HIF-1α levels were significantly decreased in the RPE layer of ocular sections and in primary RPE cells from the HIF-1α KO mice. Under normal conditions, the HIF-1α KO mice exhibited no apparent abnormalities in retinal histology or visual function as shown by light microscopy and electroretinogram recording, respectively. The HIF-1α KO mice with OIR showed no significant difference from the wild-type (WT) mice in retinal levels of HIF-1α and VEGF as well as in the number of preretinal neovascular cells. In the laser-induced CNV model, however, the disruption of HIF-1α in the RPE attenuated the over expression of VEGF and the intercellular adhesion molecule 1 (ICAM-1), and reduced vascular leakage and CNV area. Conclusions. RPE-derived HIF-1α plays a key role in CNV, but not in ischemia-induced retinal NV.     

Effect of cytokeratin 17 on retinal pigment epithelium degeneration and choroidal neovascularization

AIM: To study the effects of cytokeratin 17 (CK17) on sodium iodate (NaIO3) induced rat retinal pigment epithelium (RPE) degeneration, laser induced rat choroidal neovascularization (CNV), and oxidative stress of human retinal pigment epithelium cells (ARPE-19) and human umbilical vein endothelial cell (HUVEC). METHODS: Thirty 8-week-old male Brown Norway rats were randomly divided into 3 groups, 10 rats in control group treated with solvent alone; 10 rats in NaIO3 group treated with solvent and 35 mg/kg NaIO3 injection through hypoglossal vein and 10 rats in CK17+NaIO3 group treated with 1% CK17 eye drop 3 times a day for 1wk before and 4wk after NaIO3 injection. RPE function was measured with c-wave of electroretinogram (ERG). Another 20 rats were randomly divided into 2 groups. Of them 10 rats in CK17 group were anesthetized to receive Nd:YAG laser and given 1% CK17 eye drop before same as above; 10 rats in control were received Nd:YAG and treated with solvent. The development of choroidal neovascularization (CNV) was determined by fundus fluorescein angiography (FFA) performed on 4wk after laser. Methylthiazoly tetrazolium (MTT) assay was used to study effect of CK17 on various oxidants induced injury in ARPE-19 and HUVEC in vitro. RESULTS: Four weeks after NaIO3 injection, the c-wave amplitude of ERG was 0.393±0.02 V in the control group, 0.184±0.018 V in NaIO3 group and 0.3±0.01 V in CK17+NaIO3 group. There was a significant reversal of the c-wave by CK17 as compared to NaIO3 group (P<0.01). Four weeks after laser, the size of the CNV lesion was 2.57±0.27 mm2 in control group and 1.64±0.08 mm2 in CK17 group. The lesion size significantly diminished in CK17 group (P<0.01). The in vitro results showed CK17 also reversed the various oxidants induced injuries in ARPE-19 at the dose of 100 μg/mL and enhanced the injury in HUVECs at different concentrations. CONCLUSION: CK17 can significantly protect RPE from NaIO3 induced degeneration in vivo and in vitro and also could reverse the various oxidants induced injuries in vitro. It inhibits the development of CNV in rat model, interfered with vascular endothelial cell proliferation in vitro.     

Activated macrophages in experimental subretinal neovascularization

Laser-induced subretinal neovascularization (SRN) in monkey retinas was investigated by immunohistochemical techniques to identify the presence and location of activated macrophages. Retinal lesions were examined 3, 8 and 14 days after intensive argon laser treatment, and the distribution of interleukin-1 (IL-1)-containing cells in the lesions was determined by the orthogonal reconstruction of serial sections. Macrophages were present in the subretinal space of day 3 and day 8 lesions. These IL-1-containing cells were distributed about the area of rupture of Bruch's membrane and were quite common in lesions taken 3 days following laser treatment. While still apparent, the number was decreased at 8 days, and none were found 14 days after laser treatment. The temporal and spatial distribution of IL-1-staining macrophages paralleled the development of SRN, suggesting a relationship between the presence of activated macrophages and the initiation of neovascularization in this model.     

Selective neovascularization of the retinal pigment epithelium in rat photoreceptor degeneration in vivo.

Photoreceptor cell degeneration in rodents from a variety of causes results in neovascularization of the retinal pigment epithelium as a late stage phenomenon. Even though the vessels within the pigment epithelium arise from the retinal circulation, they can manifest the choroidal endothelial cell phenotype of fenestrated endothelial cells. In order to study the detailed cellular events which result in incorporation of retinal vessels within the retinal pigment epithelium, a morphological and morphometric analysis of the RPE and vasculature was performed in rats. Urethane, given subcutaneously to newborn rats, results in a photoreceptor degeneration but does not affect the RPE, choroid or inner retinal layers. Retinas were studied from rats of 8 to 24 weeks of age, the time period when vascularization of the RPE occurs. Loss of retinal vessels is first seen at 12 weeks, primarily in substantial dropout of vessel profiles in the outer plexiform layer (OPL) vessel bed. There is a gradient of loss from the OPL bed to the nerve fiber layer (NFL) bed and from the central to peripheral region. Total vessel density of the experimental retinas is greater than controls at 8 and 12 weeks. This occurs because there is marked loss of retinal thickness, due to photoreceptor degeneration, without a comparable loss of vessel profiles. The total retinal vessel density decreases from 8 to 20 weeks, and appears to stabilize at 20 and 24 weeks. Analysis of the separate vessel beds shows that this apparent stabilization is due to continued loss of vessels within the sensory retina, and increased presence of vascular profiles within the RPE. Total absence of the photoreceptor cell is necessary for incorporation of vessels within the RPE. Since new vessel profiles develop in the RPE but not the adjacent sensory retina, we speculate that the RPE may stimulate neovascularization of the RPE. A model of the cellular events leading to RPE neovascularization is proposed.     

Inhibition of experimental choroidal neovascularization by overexpression of tissue inhibitor of metalloproteinases-3 in retinal pigment epithelium cells

PURPOSE: To evaluate the feasibility of introducing exogenous tissue inhibitor of metalloproteinases-3 gene into the rat retinal pigment epithelium using hemagglutinating virus of Japan liposomes and to assess the effect of tissue inhibitor of metalloproteinases-3 overexpression in retinal pigment epithelium cells on the formation of experimental choroidal neovascularization. METHODS: Hemagglutinating virus of Japan liposomes containing hemagglutin epitope-tagged tissue inhibitor of metalloproteinases-3 gene were injected into the subretinal space in rat eyes. Localization of oligonucleotides was evaluated by fluorescence microscopy. Exogenous tissue inhibitor of metalloproteinases-3 mRNA expression was assessed by reverse transcribed polymerase chain reaction. Exogenous tissue inhibitor of metalloproteinases-3 protein expression was visualized by immunostaining with monoclonal antibody 12CA5 against the hemagglutin epitope. Three days after transfection of tissue inhibitor of metalloproteinases-3 gene into retinal pigment epithelium cells, intense laser photocoagulation was performed and the incidence of choroidal neovascularization was assessed by fluorescein fundus angiography. RESULTS: Exogenous tissue inhibitor of metalloproteinases-3 mRNA expression in the choroid and retina was detected on day 3. The efficiency of tissue inhibitor of metalloproteinases-3 gene transfection into retinal pigment epithelium cells was greatest on day 7 and decreased gradually thereafter. The incidence of choroidal neovascularization in tissue inhibitor of metalloproteinases-3 gene-transfected eyes was markedly decreased compared with controls. CONCLUSIONS: This study shows that tissue inhibitor of metalloproteinases-3 gene can be transferred into rat retinal pigment epithelium using the hemagglutinating virus of Japan-liposome method and that tissue inhibitor of metalloproteinases-3 gene overexpression can inhibit development of experimental choroidal neovascularization. This method may represent a future treatment modality for human macular degeneration associated with choroidal neovascularization.     

Presumed combined hamartoma of the retina and retinal pigment epithelium with preretinal neovascularization

PURPOSE: To describe a case of presumed combined hamartoma of the retina and retinal pigment epithelium associated with preretinal neovascularization. DESIGN: Observational case report. METHODS: We report clinical and angiographic findings of a 26-year-old woman. RESULTS: The patient presented with mild vitreous hemorrhage and slowly decreasing vision in the right eye. A combined hamartoma of the midperipheral retina and retinal pigment epithelium with an epiretinal membrane causing traction to the macula was found. Fluorescein angiography showed areas of capillary nonperfusion and a large preretinal neovascularization peripheral to the hamartoma. CONCLUSIONS: A combined hamartoma may be associated with retinal capillary nonperfusion and preretinal neovascularization, suggesting that significant retinal ischemia can occur with a combined hamartoma.