In thiis study,we show thai carbachol stimulates the accumulation of inositol phosphates(InsPs)in human rellnal pigment epithelium (RPE)cells and atropine blocks the carbachol-induced effect ,suggesting the existence of musearinie acelyleholine receptors in human RPE cells. In contrast,noradrenaline,serotonin, cpidermal growth factor (EGF),isoproterenol,and
NECA (5'-[N-ethyl]-carboxamido-adenosine)do not influence the basal levels of InsPs.Moreover,isoprmerenol and NECA do not affect the carhaehol elevated levels of InsPs.EGF,howcvcr,does potentiate the carhaehol stimulated elevation of InsPs in a dose-dependent manner ,suggesting an interaction between EGF and musearinie receptors in cultured human RPE cells.
（Chin J Ocul Fundus Dis,1994,10:220-222）
Replacement of diseased retinal pigment epithelium (RPE) cells with healthy RPE cells by transplantation is one option to treat several retinal degenerative diseases including age-related macular degeneration, which are caused by RPE loss and dysfunction. A cellular scaffold as a carrier for transplanted cells, may hold immense promise for facilitating cell migration and promoting the integration of RPE cells into the host environment. Scaffolds can be prepared from a variety of natural and synthetic materials. Strategies, such as surface modification and structure adjustment, can improve the biomimetic properties of the scaffolds, optimize cell attachment and cellular function following transplantation and lay a foundation of clinical application in the future.
Severely coagulated retinae by argon laser of 20 Chinese hamsters were investigated with transmission electron-microscopy. The results revealed destruction of retinal pigment epithelium-Bruch's membrane-choroid capillary complex at the coagulated foci, and leakage of fluid and blood cells through the choroidal vessels into the subretinal space. Several days after laser burn the subretinal fluid was found to subside and the RPE cells surrounding the burned lesions started to proliferate. The smaller lesions were covered by the proliferating RPE 10 days after coagulation, but poor regeneration of RPE in large necrotic areas. Neovascularization was usually associated with obvious defect of Bruch's membrane and restoration of RPE barrier was most likely impossible.
（Chin J Ocul Fundus Dis,1992,8:14-16）
Objective
To study the relationship between the expression of sonic hedgehog (Shh) and vascular endothelial growth factor (VEGF) in hypoxic human retinal pigment epithelial (hRPE) cells.
Methods
Cultured hRPE-19 cells (3rd - 6th generations) were used in this experiment. hRPE-19 cells were divided into three groups including the control group, the hypoxia experimental group (100 μmol/L CoCl2) and the inhibition group (pretreatment with 20 μmol/L cyclopamine 1 hour before hypoxia). After culturing for 4, 8, 12 and 24 hours, the mRNA level of Shh and VEGF genes in these cells were measured by fluorescence quantitative polymerase chain reaction, and the protein level of Shh and VEGF in the supernatants were measure by enzyme-linked immunosorbent assay. The relationship between the expression of Shh and VEGF was analyzed by Pearson?correlation?analysis.
Results
The control group expressed low levels of Shh and VEGF mRNA/protein. The expression of Shh and VEGF mRNA/protein in the hypoxia experimental group was significantly higher than that in the control group (F=178.364, 183.732, 77.456, 91.572; P＜0.01). The expression of Shh and VEGF mRNA in the inhibition group was significantly lower than that in the hypoxia experimental group (F=68.745, 121.834; P＜0.01). In the hypoxia experimental group, the expression of VEGF protein was positively correlated with the expression of Shh protein (r=0.942, P＜0.05); and the expression of VEGF and Shh mRNA was positively correlated (r=0.970, P＜0.01). However, there was no significant correlation in the expression of VEGF and Shh mRNA in the inhibition group (r=0.915, P＞0.05).
Conclusion
There is a positive correlation between the expression of Shh and VEGF in hypoxic hRPE cells.
ObjectiveTo observe the macular choroidal and retinal pigment epithelium (RPE) thickness in tilted disc syndrome (TDS).
MethodsThis is a descriptive study. Thirty eyes of 22 TDS patients (TDS group) and 30 eyes of 15 normal subjects (control group) were analyzed. Among TDS group, there were 8 males (11 eyes) and 14 females (19 eyes), the average age was (9.00±2.78) years old. The best corrected visual acuity (BCVA) was 0.3-1.0, and the average spherical equivalent degree was (-3.44±2.22) DS. Among the control group, there were 8 males (16 eyes) and 7 females (14 eyes), the average age was (9.33±1.11) years old. The best corrected visual acuity (BCVA)≥1.0, and the average spherical equivalent degree was (-3.18±1.13)DS. The difference of the spherical equivalent degree between two groups was not statistically significant (t=-1.648, P=0.110). Enhanced depth imaging techniques of frequency-domain optical coherence tomography was used to measure the thickness of choroid and RPE at totally 17 sites. There sites included subfoveal, 4 sites each (500, 1000, 1500 and 2000 μm from the fovea) at the horizontal (nasal/temple) and vertical (superior/inferior) directions.
ResultsThe subfoveal choroidal thickness was (235.53±51.77) μm and (273.45±60.3) μm in TDS patients and control respectively, the difference was significant(t=-2.612,P=0.011). The difference of the choroidal thickness of the other 8 horizontal sites (F=24.180) and 8 vertical sites (F=23.390) in TDS group was statistically significant (P=0.000). The TDS choroidal thickness of all horizontal sites except nasal 1000 μm site was thinner than corresponding sites of the control group (P<0.05). The TDS choroidal thickness of the subfoveal site and 4 inferior vertical sites was thinner than corresponding sites of the control group (P<0.05). The subfoveal RPE thickness was (32.56±5.00) μm and (36.58±3.60) μm in TDS patients and control respectively, the difference was significant(t=-3.567,P=0.001). The subfoveal RPE thickness was the thickest among other 16 sites in both groups, and the TDS RPE thickness of all sites was thinner than control group, the difference was statistically significant (P<0.05).
ConclusionThe choroidal and RPE thickness of TDS patient was thinner than normal subjects.
Based on the pathogenic mechanisms of age-related macular degeneration (AMD), tremendous preclinical and clinical trials have demonstrated that cell transplantation which aim to replace impaired retinal pigment epithelium (RPE) with healthy RPE cells is a promising approach to treat AMD. So far, choices of cell sources mainly are autologous RPE, iris pigment epithelium, fetal RPE, human embryonic stem cell-derived RPE and human induced pluripotent stem cell-derived RPE, and some of them are undergoing clinical researches. Grafting manners in cell-based therapies are various including RPE sheet or RPE-choroid complex transplantation, RPE cell suspension injection, and RPE sheet transplantation with scaffolds. This review is limited to cell-based therapies for RPE that damaged first in the progress of AMD and focus on recent advances in cell sources, transplantation methods, preclinical and clinical trials, and the obstacles that must be overcome.
Pachychoroid spectrum diseases includes central serous chorioretinopathy (CSC), pachychoroid pigment epitheliopathy, pachychoroid neovasculopathy, and polypoidal choroidal vasculopathy, which share common characteristics, including focal or diffused increased choroidal thickness, choroidal hyper-permeability, and dilated choroidal vessels. These diseases are likely to represent a continuum of the same pathogenic process. Similar features and association among them suggest that they may have similar etiology. It is of great clinical significance to understand the composition and typical morphological changes of pachychoroid-related diseases and to explore its possible pathogenesis.
ObjectiveTo observe the changes of eotaxin-1(CCL11), eotaxin-2(CCL24)and eotaxin-3(CCL26)in ranibizumab treated light-injured human retinal pigment epithelium (RPE) cells ARPE-19 and investigate the effects of vascular endothelial growth factor (VEGF) antagonist to the expressions of eotaxins.
MethodsCultured human RPE cells(8th-12th generations)were divided into light-injured group, ranibizumab treated group and normal control group. Cells of the three groups were exposed to the blue light at the intensity of(600±100) Lux for 12 h to establish the light injured model, while cell culture dishes of the normal control group were wrapped with double-layer foil. The cells of ranibizumab treated group were treated with VEGF-A antagonist(ranibizumab)at the final concentration of 0.125 mg/ml for 24 hours directly after the illumination. The mRNA and protein of VEGF-A, eotaxin-1, eotaxin-2, eotaxin-3, NF-κB were determined by Real time-PCR, enzyme-linked immunosorbent assay, Western blot, immunohistochemical staining at 0, 3, 6, 12, 24 hours after light damage.
ResultsThe mRNA and protein level of VEGF-A, eotaxin-1, eotaxin-2, eotaxin-3, NF-κB in the light-injuried group increased significantly compared to that in normal control group (P < 0.05). After treating with ranibizumab, the expression of eotaxin-1, eotaxin-2, eotaxin-3, NF-κB were significantly suppressed (P < 0.05).
ConclusionThe suppression of over-expression of VEGF in human RPE may down-regulate the expression of eotaxins, via the suppression of NF-κB.
Objective To observe the effect of bone forming protein 4 (BMP4) on the proliferation and migration of human retinal pigment epithelium (RPE) cells under oxidative stress, and to preliminarily explore its effect on epithelial-mesenchymal transition (EMT) of RPE cells. MethodsHuman RPE cells cultured in vitro were divided into normal group, pure 4-hydroxynonenal (HNE) group (4-HNE group), 4-HNE+NC group and 4-HNE+ small interfering BMP (siBMP4) group. The effect of 4-HNE on the proliferation of RPE cells was detected by thiazole blue colorimetry. The effects of 4-HNE and BMP4 on cell migration were determined by cell scratch test. The expression of BMP4 was detected by immunofluorescence staining, Western blot and real-time quantitative polymerase chain reaction. The transfection efficiency of siBMP4 was observed by fluorescence microscopy. Mitochondrial reactive oxygen species (MitoSOX) were detected by flow cytometry. The expression of EMT markers E-cadherin and Fibronection were detected by immunofluorescence assay. t-test was used for comparison between the two groups, and one-way analysis of variance was used for comparison between the three groups. ResultsCompared with normal group, cell proliferation and migration ability of 4-HNE group were significantly enhanced, with statistical significance (t=21.619, 24.469; P＜0.05). The expression of BMP4 in cells was significantly increased, and the difference was statistically significant (t=19.441, P＜0.05). The relative expression levels of BMP4 mRNA and protein were also significantly increased, with statistical significance (t=26.163, 37.163; P＜0.05). After transfection with siBMP4 for 24 h, the transfection efficiency of BMP4 in RPE cells was＞90%. Compared with 4-HNE group and 4-HNE+NC group, the relative expression levels of BMP4 protein (F=27.241), mRNA (F=36.943), cell mobility (F=46.723) and MitoSOX expression levels (F=39.721) in normal group and 4-HNE+siBMP4 group were significantly decreased. The differences were statistically significant (P＜0.05). The epithelial marker E-cadherin increased significantly, while the mesenchymal marker Fibronection decreased significantly, with statistical significance (F= 51.722, 45.153; P＜0.05). ConclusionsBMP4 inhibits RPE proliferation and migration under oxidative stress. BMP4 is involved in inducing EMT in RPE cells.
ObjectiveTo observe the expression of hot shock protein 47 (HSP47) in pre-retinal membrane of proliferative vitreoretinopathy (PVR) and the influence of transforming growth factor-β2 (TGF-β2) on the expression of HSP47 in retinal pigment epithelial (RPE) cell.
MethodsPre-retinal membranes were collected and observed by hematoxylin-eosin, Masson and immunohistochemical staining. Cultured ARPE-19 cells were treated with TGF-β2 at serial concentration (0, 1, 5, 10 ng/ml) and time (0, 12, 24, 48 hours), respectively. And then the mRNA and protein expressions of HSP47 and Col-Ⅰ were measured by fluorescence quantitative reverse transcription polymerase chain reaction and Western blot at the same time.
ResultsA lot of epithelial cells with pigmental particles were observed in pre-retinal membranes of PVR, much accumulated collagen protein was observed in the specimens, and HSP47 positive expression was bserved in cytoplasm and stroma of most of the epithelioid cells. Compared with 0 ng/ml group, the expressions of HSP47 mRNA in ARPE-19 were up-regulated by 1.32, 2.35, 1.85 fold, significant differences were observed in all groups (F=27.21, P<0.05); the expressions of protein were up-regulated by 2.33, 2.89, 2.60 fold, significant differences were observed in all groups (F=39.78, P<0.05). The expressions of Col-Ⅰ mRNA were up-regulated by 1.29, 1.52, 2.11 fold, significant differences were observed in all groups (F=23.45, P<0.05); the expressions of protein were up-regulated by 1.18, 1.49, 2.11 fold and significant differences were observed in all groups (F=29.10, P<0.05). Compared with 0 hour group, the expressions of HSP47 mRNA were up-regulated by 1.56, 1.84, 2.86 fold in ARPE-19 cells stimulated by 5 ng/ml TGF-β2 for 12, 24 and 48 hours, and the differences were all significant (F=31.56, P<0.05); the expressions of protein were up-regulated by 2.08, 2.37, 2.80 fold, and the differences were all significant (F=49.18, P<0.05). The expressions of Col-Ⅰ mRNA were up-regulated by 1.57, 1.86, 2.78 fold and the differences were all significant (F=54.43, P<0.05), the expressions of protein were up-regulated by 1.38, 1.59, 2.16 fold and the differences were all significant (F=42.52, P<0.05).
ConclusionTGF-β2 may play a role in the pathologic process of PVR by promoting the expression of HSP47 and then increasing the synthesis and accumulation of Col-Ⅰ.
