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）
ObjectiveTo observe the effect of subretinal injection of retinal pigment epithelium (RPE) cells for RPE in mice. MethodsA total of 30 postnatal day 7 C57BL/6J mice were randomly divided into normal mice group, OIR model group and OIR model cell transplanted group, 10 mice in each group. The OIR model was induced in mice of OIR model group and OIR model cell transplanted group. The RPE cells were subretinal injected into the RPE of mice in OIR model cell transplanted group. At 20 days after the injection, the RPE thickness was evaluated by fluorescence microscope. The expression of RPE65, Bestrophin and zonula occludens-1 (ZO-1) were estimated by Western blot and real-time quantitative PCR (RT-PCR). ResultsThe thickness of RPE in OIR model mice was thinner than that in normal mice; the thickness of RPE in OIR model cell transplantation mice was significantly thicker than that in the OIR model mice. The results of Western blot and RT-PCR indicated that the differences of protein (F=8.597, 18.864, 25.691) and mRNA expression (F=39.458, 11.461, 34.796) of RPE65, Bestrophin, ZO-1 were statistically significant between OIR model group and OIR model cell transplanted group (P < 0.05). ConclusionsSubretinal injection of RPE cells can promote RPE thickening. RPE65 and Bestrophin protein relative expression levels increased, ZO-1 protein relative expression levels reduced; mRNA expression levels of RPE65, Bestrophin and ZO-1 genes increased.
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.
The effects of various receptor agonists/antagonists on the accumulation of inositol phosphates(InsPs) in cultured rat retinal pigment epitheium (RPE) cells were analysed. The results showed that serotonin and the 5-HT2 agonists such as alpha;-methyl-serotonin, quipazine, and DOI (1-[2.5-dimethoxy-4-iodopheny1]-2-aminopropane) all stimulated InsPs accumulation in rat RPE cells. The serotonin-induced stimulation of InsPs was effectively blocked by ketanserin, a 5-HT2 antagonist, but also attenuated by the active phorbol ester PMA (4ft-phorbol 12-myristate 13-acetate), a potent protein kinase C activator.The data presented provide clear evidence for the presence of 5-HT2 receptors coupled to phosphoinositide metabolism on cultured rat RPE cells. （Chin J Ocul Fundus Dis,1994,10:80-83）
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.
ObjectiveTo study how CD73 is shed from the retinal pigment epithelium (RPE) surface.MethodsCD73 shedding was induced by treating RPE with lipopolysaccharides (LPS) and TNF-α. After Phospholipase C (PLC) or pan matrix metalloproteinase (MMP) inhibitors were added, surface amount of CD73 was evaluated by flow cytometry (FACS). Then selective inhibitors or their corresponding siRNAs of MMP-2 and MMP-9 were applied to the treatments of RPE; and their effects on induced CD73 shedding were evaluated by FACS. By site directed mutagenesis, mutations were introduced to Lys547-Phe548 coding sites of CD73 cDNA, which was cloned in a pcDNA mammalian expression vector. Both wt-CD73 and mutated-CD73 were over expressed in CD73-/- RPE and their induced shedding was compared.ResultsLPS and TNF-α induced CD73 shedding from RPE was completely blocked by the addition of pan MMP inhibitor but not PLC inhibitor. Selective MPP-9, but not MMP-2, inhibitor or its siRNA blocked CD73 shedding. In CD73-/- RPE induced CD73 shedding was happened to overexpressed wt-CD73 but not Lys547-Phe548 sites mutant CD73.ConclusionMMP-9 is responsible for shedding CD73 from RPE through hydrolyzing its Lys547 -Phe548 sites.
ObjectiveTo observe the effect of TGF-β receptor inhibitor Compound C on the directed differentiation of human embryonic stem cells (hESC) into retinal pigment epithelial (RPE) cells. MethodsH1 hESC were divided into control group and experimental group. When the hESC reached over confluence, the medium was changed to knockout serum replacement medium without bFGF to induce RPE differentiation. The experimental group was supplemented with 1 μmol/L TGF-β receptor inhibitor Compound C at the first six days of induction. Real-time PCR was carried out to examine the expression of paired-box gene 6 (PAX6), microphthalmia-associated transcription factor (MITF), cellular retinaldehyde blinding protein (CRALBP), and RPE65 in both groups at the 1, 3, 5 weeks of the induction process. hESC-derived RPE (hESC-RPE) cells were isolated mechanically and purified. Real-time PCR, Western blot and immunofluorescence were used to characterize the purified hESC-RPE cells. ResultsPigmented colonies were observed in experimental group at the 4 weeks of the induction process, while no pigmented colony could be detected in the control group. All the purified pigmented cells from experimental group showed polygons morphology. Experimental group showed significantly higher expression of RPE marker genes PAX6, MITF, CRALBP and RPE65 than the control group(P<0.05). Compared with the hESC and ARPE-19 cells line, purified hESC-RPE cells showed much higher expression of PAX6, MITF, CRALBP and RPE65(P<0.05).High expression level of PAX6 and RPE65 proteins were observed in hESC-RPE cells. Immunofluorescence verified the expression of PAX6 and ZO-1 in hESC-RPE cells. ConclusionTGF-β receptor inhibitor Compound C significantly improved the differentiation efficiency of hESC into RPE.
Objective To investigate the impact of photodynamic therapy (PDT) with verteporfin on the expression of pigment epithelial derivative factor (PEDF) mRNA and vascular endothelial growth factor (VEGF) mRNA in adult retinal pigment epithelial (RPE) cells in vitro. Methods The changes of cellular viability before and after PDT were assessed by methyl thiazolyl tetrazolum (MTT) colorimetric assay. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was conducted to detect the expression of PEDF and VEGF mRNA in RPE cells before and after PDT. Results PDT caused the death of RPE cells. The cellular mortality was positively correlated with the power of photocoagulation and the concentration of verteporfin. Conclusion PDT could downregulate the expression of PEDF and VEGF mRNA in adult RPE cells in vitro, which may relate to the cure or relapse of subfoveal choroidal neovascular membrane after PDT. (Chin J Ocul Fundus Dis, 2006, 22: 256-260)
ObjectiveTo evaluate the functional and anatomical outcomes of autologous single retinal pigment epithelium (RPE) transplantation for severe obsolete submacular hemorrhage (SMH) in late age-related macular degeneration (AMD). MethodsA retrospective clinical study. From January 2012 to December 2015, 11 patients with AMD (11 eyes) with obsolete SMH who were diagnosed and treated by pars plana vitrectomy (PPV) combined with autologous RPE transplantation at the Department of Ophthalmology, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine were included. Among them, there were 9 eyes in 9 males and 2 eyes in 2 females. All the eyes underwent the examinations of best corrected visual acuity (BCVA) and optical coherence tomography; 4 eyes underwent macular fixation function (MAIA) at the same time. The BCVA examination was carried out using the international standard visual acuity chart, which was converted into logarithm of the minimum angle of resolution (logMAR) visual acuity during statistics. All eyes were treated with PPV combined with autologous single-layer RPE transplantation or autologous RPE-choroidal full-thickness transplantation, and were divided into S group and C group, with 5 and 6 eyes respectively. The differences of age (t=－0.363), gender composition ratio (χ2=0.549), course and thickness of SMH (t=0.118, 0.231), average times of anti-vascular endothelial growth factor drug treatments (t=0.129), times of PPV (t=－0.452) between the two groups were not statistically significant (P>0.05). The follow-up period was 6-40 months after the operation, and the BCVA, MAIA, graft status and complications of the eyes after the operation were observed. The comparison of continuous variables between groups was performed by independent-sample t test; the comparison of categorical variables was performed by χ2 test. ResultsAt the last follow-up, the average logMAR BCVA of the eyes in group S and C were 1.62±0.34 and 1.03±0.20, respectively; group C was better than group S, however, the difference was not statistically significant (t=1.532, P=0.160). There were 4 eyes (80%, 4/5) and 6 eyes (100%, 6/6) in S group and C group with BCVA better than preoperative, the difference was no statistical significance (χ2=0.677, P=0.895). There were 2 (40%, 2/5) and 3 (50%, 3/6) eyes with logMAR BCVA better than 1.0 in S group and C group, and the difference was not statistically significant (χ2=0.572, P=0.423). After the operation, 6 eyes of grafts were in good condition and 5 eyes were in poor condition; the BCVA of grafts in good condition was significantly higher than that of poor condition, the difference was statistically significant (t=4.894, P=0.001). Among the 4 eyes that underwent MAIA examination, 2 eyes were unstable and diffusely fixed on the graft; the fixation point was located at the normal retina adjacent to the graft area in 2 eyes. Secondary subretinal hemorrhage occurred in 3 eyes after the operation; the intraocular pressure was high in 1 eye after the operation. During the follow-up period, no intraocular infection, secondary retinal detachment, recurrent choroidal neovascularization or low intraocular pressure occurred in all eyes. ConclusionsBoth autologous single-layer RPE transplantation and autologous RPE-choroidal full-thickness transplantation can help stabilize or even improve the visual function of eyes with severe SMH secondary to advanced AMD. The visual acuity after surgery is closely related to the state of the graft.
ObjectiveTo observe the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells (hUCMSC) on the expression of vascular endothelial growth factor (VEGF) A in blue light injured human retinal pigment epithelial (RPE) cells. MethodshUCMSC were cultured with exo-free fetal bovine serum for 48 hours, and then the supernatants were collected to isolate and purify exosomes by gradient ultracentrifugation method. Transmission electron microscopy was used to identify the morphology of exosomes. Surface specific maker protein CD63 and CD90 were detected via Western blot. Cultured ARPE-19 cells were divided into normal control group, blue light injured group and hUCMSC exosomes treated group. Cells were exposed to the blue light at the intensity of (2000±500) Lux for 12 hours to establish the light injured models. The cells of hUCMSC exosomes treated group were treated by different concentrations of exosomes for 8, 16, 24 hours. The mRNA and protein of VEGF-A were determined by real time-polymerase chain reaction and Western blot. Immunofluorescence assay were used to detect the expression levels of VEGF-A. ResultshUCMSC exosomes were successfully isolated, they exhibited round or oval shape and their diameter ranged from 50 to 100 nm with membrane structure through electron microscope. hUCMSC exosomes expressed the common surface marker protein CD63 and the surface marker protein CD90 of hUCMSC. The protein and mRNA level of VEGF A in the blue light injured group increased significantly compared to that in normal control group (t=-16.553, -19.456; P < 0.05). After treating with low, middle and high concentration of hUCMSC exosomes for 8, 16 and 24 hours, the protein and mRNA level of VEGF A of injured RPE were significantly decreased (P < 0.05). With the treated time and concentration of hUCMSC exosomes improved, the protein and mRNA level of VEGF A of injured RPE gradually decreased (P < 0.05). Immunofluorescence assay showed the protein level of VEGF-A of injured RPE gradually decreased with the same concentration of hUCMSC exosomes treated over time. ConclusionhUCMSC exosomes can effectively down-regulate the mRNA and protein level of VEGF-A in blue light injured RPE, the effect depends on the concentration and treated time of hUCMSC exosomes.