Grafting of ARPE-19 and Schwann cells to the subretinal space in RCS rats

PURPOSE: To study the distribution of the human retinal pigment epithelium (hRPE) cell line ARPE-19 and human Schwann (hSC) cells grafted to the subretinal space of the Royal College of Surgeon (RCS) rat and the relation of graft cell distribution to photoreceptor rescue. METHODS: Cell suspensions of both donor types were injected into the subretinal space of 3-week-old dystrophic RCS rats through a transscleral approach, human fibroblast and medium were used as control grafts. All animals were maintained on oral cyclosporine. At 1, 2, 4, 6, 15, 28, and 36 weeks after grafting, animals were killed. Human cell-specific markers were used to localize donor cells. RESULTS: Both donor cell types, as revealed by antibodies survived for a substantial time. Their distribution was very different: hRPE cells formed a large clump early on and, with time, spread along the host RPE in a layer one to two cells deep, whereas hSCs formed many smaller clumps, mainly in the subretinal space. Both cells rescued photoreceptors beyond the area of donor cell distribution. The number of surviving cells declined with time. CONCLUSIONS: Both hRPE and hSC grafts can survive and rescue photoreceptors for a substantial time after grafting. The number of both donor cell types declined with time, which could be an immune-related problem and/or due to other factors intrinsic to the host RCS retina. The fact that rescue occurred beyond the area of donor cell distribution suggests that diffusible factors are involved, raising the possibility that the two cell types function in a similar manner to rescue photoreceptors.     

Contribution of rod and cone pathways to the dark-adapted electroretinogram (ERG) b-wave following retinal degeneration in RCS rats

Although the RCS rat is widely used as a model of progressive photoreceptor loss, it is unclear how the relative rod and cone functions change with age. Rod and cone b-waves were isolated using a double flash ERG paradigm. In contrast to cones, rods never reached normal functional maturity levels, and the ERG b-wave changed from being predominantly rod-driven to being purely cone-driven by age 74 days, at which point, b-waves were progressively replaced by negative STR-like (scotopic threshold response) waves that persisted up to age 180 days. A double flash commonly abolished this wave and unveiled a b-wave.     

Measuring the cone contribution to the ERG b-wave to assess function and predict anatomical rescue in RCS rats

Subretinal injections of human retinal pigment epithelial (RPE) cells early in the course of retinal degeneration in Royal College of Surgeons (RCS) rats can rescue photoreceptors. Fourteen injected animals were studied using a double flash electroretinogram (ERG): 10 were examined longitudinally and four terminally with immunohistochemistry. The proportion of cone contribution to the ERG b-wave rather than the absolute size of isolated cone response proved to be a reliable indicator of function over time and a predictor of the proportion of cones identified anatomically in the area of optimal photoreceptor rescue.     

Time course of deterioration of rod and cone function in RCS rat and the effects of subretinal cell grafting: a light- and dark-adaptation study

To examine how rod and cone function are differentially affected during retinal degeneration, and after subretinal cell grafting, we obtained light- and dark-adaptation curves by recording threshold multiunit responses from the superior colliculus of anesthetized rats. Unoperated RCS dystrophic and non-dystrophic rats were used and the effects of subretinal grafting in dystrophic rats of cells known to limit photoreceptor degeneration were examined. The adaptation curves showed that rod function was severely compromised in unoperated dystrophic RCS rats at low luminance levels, even as early as 21 days of age and that cone thresholds became gradually elevated over time. While cell transplantation preserved both rod and cone photoreceptors, rod function did not recover, although further deterioration of cone threshold responses was prevented. This raises concern that measures of outer nuclear layer thickness may not in themselves be an accurate measure of visual capabilities and efficacy of a restoration strategy.     

Local cone and rod system function in progressive cone dystrophy

PURPOSE: To compare the patterns of local cone and rod system impairment in patients with progressive cone dystrophy (CD) using psychophysical and electrophysiological techniques. METHODS: Local cone system function was assessed by measuring cone system thresholds (visual fields) and cone-mediated multifocal electroretinograms (mfERGs). Rod system function was assessed by measuring rod system thresholds (visual fields) and rod-mediated mfERGs. The results in a group of eight patients with CD were compared with those in an age-similar control group. RESULTS: All the patients had abnormal cone system visual field thresholds and cone-mediated mfERGs. Cone system psychophysical thresholds were elevated for targets presented within the central 10 degrees, but were within normal limits for targets at peripheral locations. Cone-mediated mfERG measures of amplitude scale and time scale were abnormal for most of the hexagons tested. Most of the rod-mediated psychophysical thresholds and mfERGs were within normal limits. Rod system losses tended to be patchy and scattered throughout the area tested. CONCLUSIONS: There was poor correspondence among local measures of cone and rod system losses in these patients with CD. The results suggest that the spatial pattern of cone system losses in this disease differs from the spatial pattern of rod system losses.     

Temporal and spatial characteristics of cone degeneration in RCS rats

Purpose  

The temporal and spatial characteristics of cone degeneration in the Royal College of Surgeons (RCS) rat were studied to provide information for treatment strategies of retinitis pigmentosa.     

Role of HGF/c-Met in serum-starved ARPE-19 cells

PURPOSE: Hepatocyte growth factor (HGF) and its receptor (HGFR/c-Met) regulate motility, mitogenesis, and morphogenesis in a cell type-dependent fashion. We report the role of HGF and c-Met on stress-induced ARPE-19 human retinal pigment epithelial (RPE) cells in this study. METHODS: The cells were cultured either with or without serum. Southern and Western blot analyses were done to determine the expression patterns of HGF/c-Met in serum-starved ARPE-19 cells. The cell proliferation pattern in serum-starved condition was analyzed using MTS assay. Inhibition level of cell proliferation was analyzed using a neutralizing monoclonal antibody against c-Met (2 microg/ml). RESULTS: Abnormal cell proliferation and scattering of ARPE-19 cells was observed under serum starvation. HGF/c-Met were expressed in serum-starved ARPE-19 cells. ARPE-19 cell proliferation was also enhanced with recombinant HGF treatment. Neutralization against c-Met inhibited the proliferation of serum-deprived ARPE-19 by 64.5% (n=9, S.D. 5.5%). Serum starvation appears to induce epithelial-mesenchymal transition of ARPE-19 cells, resulting in scatter, and the expression of alpha-smooth muscle actin (alpha-SMA), a marker for fibrosis. CONCLUSIONS: In conclusion, c-Met induced under non-physiologic conditions has significant effects on the activation of RPE cells.     

RAGE ligand upregulation of VEGF secretion in ARPE-19 cells

PURPOSE: The importance of VEGF in stimulating neovascular age-related macular degeneration (AMD) is well-recognized, but the initiating factors that induce local upregulation of VEGF remain unclear. The current study was conducted to test the hypothesis that activation of RAGE (receptor for advanced glycation end products [AGEs]) by its ligands, including AGEs, amyloid-beta peptide (Abeta), and S100B/calgranulins, some of which are known components of drusen and Bruch's membrane deposits, modulate secretion of VEGF by retinal pigment epithelial (RPE) cells. METHODS: ARPE-19 cells were used for all experiments. The cells were transfected with constructs encoding a signal transduction mutant of human RAGE to assess the RAGE-dependence of intracellular signaling. VEGF secretion and gene expression were assessed by ELISA and quantitative real-time PCR. SDS-PAGE and size exclusion chromatography were performed to analyze the structural changes of S100B after oxidation of its thiol groups under denaturing and nondenaturing conditions, respectively. NF-kappaB activation was assessed via electrophoretic mobility shift assay (EMSA). The impact of the NF-kappaB inhibition was assessed by using parthenolide. RESULTS: ARPE-19 cells basally secreted VEGF under normal cell culture conditions. Immobilized ligands of RAGE increased VEGF secretion in a RAGE-dependent manner. In contrast, soluble AGE-BSA, fresh Abeta, and S100B were less effective in increasing VEGF secretion. Studies with Abeta demonstrated that oligomeric and surface-immobilized forms of Abeta, but not soluble monomeric forms of Abeta, were effective upregulators of VEGF secretion via RAGE. Oxidation of S100B's thiol groups resulted in the formation of oligomers that displayed distinct RAGE biological activity compared with the simple dimeric form. RAGE-mediated upregulation of VEGF secretion by ARPE-19 cells was largely dependent on NF-kappaB, as indicated by studies with parthenolide. CONCLUSIONS: Immobilized or oligomerized ligands for RAGE induce RPE cells to increase VEGF secretion. NF-kappaB plays a central role in RAGE-dependent RPE secretion of VEGF. In AMD, activation of the RAGE axis in RPE cells may contribute to upregulation of VEGF, potentially inciting or propagating neovascular macular disease.     

A functional profile of gene expression in ARPE-19 cells

Background  

Retinal pigment epithelium cells play an important role in the pathogenesis of age related macular degeneration. Their morphological, molecular and functional phenotype changes in response to various stresses. Functional profiling of genes can provide useful information about the physiological state of cells and how this state changes in response to disease or treatment. In this study, we have constructed a functional profile of the genes expressed by the ARPE-19 cell line of retinal pigment epithelium.     

Development of ERG responses: The ISCEV rod, maximal and cone responses in normal subjects

PURPOSE: Summarize ISCEV ERG responses from normal infants and children. METHODS: The amplitudes and implicit times of the ISCEV rod, maximal dark-adapted and cone responses from a total of 409 normal infants (n = 128), children and adult controls were compiled. The subjects, aged 1 week to 52 years, were divided into seven age groups, including four in infancy (< 52 weeks). The response parameters for each age group were summarized as percentiles. RESULTS: In each ISCEV condition, the youngest infants (1-5 weeks) had significantly smaller amplitudes and longer implicit times than adults. Amplitude increased and implicit time decreased systematically with age. CONCLUSIONS: The developmental changes in ERG responses are significant. The medians and ranges herein provide provisional norms against which the ERG responses from pediatric patients can be compared.     

Preservation of photoreceptors in dystrophic RCS rats following allo- and xenotransplantation of IPE cells

Purpose  To examine whether iris pigment epithelial (IPE) cells transplanted into the subretinal space of Royal College of Surgeons (RCS) rats have the ability to rescue photoreceptors. Methods  Rat IPE (rIPE) or human IPE (hIPE) cells were transplanted subretinally in 23-day-old RCS rats. Sham injection and transplantation of ARPE-19 cells served as controls. After 12 weeks, eyes were evaluated for photoreceptor survival by morphometric analysis and electron microscopy. Results  Morphometric analysis showed photoreceptor rescue in all transplanted and sham-injected animals (number of photoreceptors/300 μm retina±sd: rIPE 41.67 ± 28; hIPE 29.50 ± 16; ARPE-19 36.12 ± 21; sham 16.56 ± 6) compared to age-matched, control rats (number of photoreceptors/300 μm retina±sd: 9.71 ± 4). Photoreceptor rescue was prominent in IPE cell-transplanted rats and was significantly greater than sham-injected eyes (p = 0.02 for rIPE and p = 0.04 for hIPE). Conclusion  Since IPE cells transplanted into the subretinal space have the ability to rescue photoreceptors from degeneration in the RCS rat without any harmful effects, IPE cells may represent an ideal cell to genetically modify and thus carry essential genetic information for the repair of defects in the subretinal space.     

LWS cone effects on rod threshold and saturation in achromats with residual cone function

Rod saturation on flashed and steady red backgrounds was investigated in normals and three achromats, two of whom were found to have some residual cone function. LWS cones selectively reduce the background level at which rod saturation occurs and elevate rod thresholds at flashed background levels well below saturation. Both of these LWS cone actions are also present in eyes with greatly reduced LWS cone function. In normal eyes LWS cones also elevate rod thresholds on steady backgrounds. We thus conclude that LWS cones influence rods through different mechanisms under transient (flashed) and steady-state background stimulation and that the increase in rod visual sensitivity observed during prolonged presentation of a background is due to a time-dependent reduction of LWS cone influence on rods. Finally, the finding that rod-cone interactions of the same magnitude found in normals can be seen in individuals where the cones' ability to mediate vision is severely reduced suggests the rod saturation paradigm as a sensitive technique for revealing residual LWS cone function.     

Assessment of local cone on- and off-pathway function using multifocal ERG technique

There is evidence that recording the photopic ON- and OFF-responses with long-duration stimuli is useful for determining the contribution of the cone ON- and OFF-pathways to the primate photopic electroretinogram (ERG). In this study, the optimal conditions for recording multifocal ON-OFF responses are described, and the technique is applied to normal subjects and two patients with unusual retinal diseases. The results from the normal subjects demonstrated that there were topographical variations of the photopic ERG waveform: when responses were normalized to the ON-response (b-wave) amplitude, the OFF-response (d-wave) amplitude increased with increasing eccentricity. The changes in the waveform in two patients suggested relatively greater defects of the hyperpolarizing or depolarizing bipolar cells. We conclude that the multifocal ERG technique with long-duration stimuli can be a useful tool to assess the function of local cone ON- and OFF-pathways in normal and diseased retinas. This revised version was published online in July 2006 with corrections to the Cover Date.     

Local cone and rod system function in patients with retinitis pigmentosa

PURPOSE. To compare local cone and rod system function in patients with retinitis pigmentosa (RP) using electrophysiological and psychophysical techniques. METHODS. Cone-mediated multifocal electroretinograms (M-ERGs), cone system threshold visual fields, rod-mediated M-ERGs, and rod system threshold visual fields were measured in seven patients with RP. RESULTS. All the patients had normal cone system visual field thresholds and normal cone-mediated M-ERG implicit times within the central 5 degrees. Both cone-mediated responses were abnormal at some peripheral retinal locations. There were significant correlations among cone system amplitude, timing, and visual field loss. All the patients had some retinal areas where the rod-mediated M-ERG amplitudes were not measurable. In areas where they were measurable, these rod-mediated M-ERG responses were often within normal limits for amplitude and timing. In contrast to the cone system data, there were no significant relationships between rod-mediated M-ERG measures and rod system threshold elevations. The cone and rod system psychophysical thresholds showed regional correspondence; the amplitude-scale and time-scale measures of the M-ERG did not. CONCLUSIONS. The results indicate that there was better local correspondence between psychophysical and electrophysiological measures in the cone system than in the rod system in patients with RP. In addition, the psychophysical measures of cone and rod system function showed better correspondence than did the electrophysiological measures.     

金雀异黄素对缺氧诱导的ARPE-19细胞中VEGF表达的影响

目的研究长期缺氧对体外培养的人ARPE-19细胞血管内皮生长因子(vascular endotheial growth factor,VEGF)表达的诱导作用,以及金雀异黄素(genistein,Gen)对其表达的影响和机理。方法使用半定量RT-PCR和酶联免疫吸附试验(ELISA)检测ARPE-19细胞中VEGFmRNA和蛋白的表达,分析不同浓度Gen以及PD98059、TyrphostinA25对ARPE-19细胞缺氧24h诱导的VEGF表达的影响。结果(1)正常对照组有少量VEGF表达,缺氧24h可明显提高ARPE-19细胞VEGFmRNA和蛋白的表达,分别为(9.566±1.528)倍和(2.636±0.499)倍;(2)Gen(50μmol·L-1,100μmol·L-1,200μmol·L-1)与缺氧组对比,分别抑制缺氧组VEGFmRNA表达的55.03%、78.72%、90.69%,蛋白的表达量为(189.60±20.20)ng·L-1(117.70±21.97)ng·L-1、(49.70±13.17)ng·L-1,抑制作用呈浓度依赖性;(3)PD98059、TyrphostinA25也可抑制缺氧诱导的VEGFmRNA表达的67.24%和56.25%,蛋白表达量为(86.33±12.47)ng·L-1、(98.08±2.42)ng·L-1。结论Gen可抑制长期缺氧诱导的ARPE-19细胞VEGF表达,并可能是通过抑制p42/p44MAPK途径和HIF-1途径共同抑制VEGF的表达,提示Gen对视网膜新生血管的形成具有预防和潜在的治疗价值。     

The local cone and rod system function in early age-related macular degeneration

To compare cone and rod system function in patients with early age- related macular degeneration (ARMD) and control group using multifocal electroretinogram (MERG) and perimetry, to investigate whether there is rod system dysfunction in the central retina in ARMD. Cone-mediated MERG, photopic sensitivity, rod-mediated MERG, and scotopic sensitivity in 16 eyes of control subjects and 24 eyes of early dry-form ARMD were measured with VERIS Science ^TM 4.0 and Octopus 101 perimetry. The latencies and average response densities of the summed responses and five ring retinal regions, average sensitivity of all locus and eight ring retinal regions in control eyes were compared with those in ARMD. Mean scotopic and photopic sensitivity of ARMD patients were significantly lower than that of normal controls. Sotopic sensitivity reduced more than photopic sensitivity and the greatest deficit was 2.5–5.0°. The amplitudes of N₁ and P₁ wave in one ring (5.0°) of rod MERG were significantly lower of ARMD patients than that of normal subjects. Our results suggest that rod function decreased and the parafoveal rod cells were predominantly damaged in ARMD. The rod function testing in macula may be a useful tool to diagnose and measure the fundus dysfunction of ARMD.