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.
Retinitis pigmentosa (RP) is a genetic disorder of photoreceptor cell apoptosis and retinal pigment epithelium (RPE) cell atrophy caused by gene mutation. The clinical manifestations are night blindness, peripheral visual field loss and progressive vision loss. RPE cell apoptosis plays an important role in the progression of RP, and exogenous implantation of RPE cells as an alternative therapy has shown certain efficacy in animal experiments and clinical trials. With the diversification of cell sources, the update of surgical techniques and the continuous emergence of biological materials, more possibilities and hopes are provided for cell therapy. To further promote the development of this field in the future, it is still necessary to strengthen the cooperation between medicine, bioengineering and other disciplines in the future to jointly promote the innovation and development of therapeutic methods. It is believed that RPE cell transplantation therapy will show a brighter prospect in the future
Objective To investigate the effects of exosomes from cultured human retinal pigment epithelium (ARPE-19) cells affected by oxidative stress on the proliferation and expression of vascular endothelial growth factor-A (VEGF-A) and Akt of ARPE-19 cells. Methods Culture ARPE-19 cells. The concentration of 2.5 μmol/L rotenone was selected to simulate oxidative stress and isolated ARPE-19-exosome. Exosomes were isolated by ExoQuick exosome precipitation solution. Transmission electron microscopy was used to identify the morphology of exosomes. Western blot was used to detect exosomes’ surface-specific maker protein CD63. ARPE-19 cells affected by oxidative stress were cultured with exosome as experimental group, normal ARPE-19 cells were cultured with exosome as control group. The cell proliferation was examined by methyl thiazolyl tetrazolium assay. Western blot and immunofluorescence assay were used to detect the expression levels of VEGF-A and Akt protein. Real-time quantitative polymerase chain reaction (RT-PCR) was used to detect the levels of VEGF-A mRNA and Akt mRNA. Results The diameter of normal ARPE-19-exosomes ranged from 50 to 150 nm. The isolated exosomes expressed CD63. AREP-19 cells were cultured with ARPE-19 (affected by rotenone)-exosome, the cell viability in experimental group was significantly reduced than in the control group. Green fluorescence was observed in the cytoplasm under fluorescence microscope. Compared with the control group, VEGF-A was up-regulated expressed and Akt was down-regulated expressed. Western blot results showed that, VEGF-A protein expression in the experimental group were higher than the control group. Akt protein expression in the experimental group were less than the control group. The difference was statically significant (t=3.822, 6.527;P＜0.05). RT-PCR results showed that VEGF-A mRNA expression levels was higher in the experimental group than the control group. Akt mRNA expression levels was lower in the experimental group than the control group. The difference was statically significant (t=8.805, −7.823;P＜0.05). Conclusions Exosomes from ARPE-19 cells affected by oxidative stress inhibit the proliferation of normal ARPE-19 cells, increase the expression of VEGF-A and reduce the expression of Akt.
ObjectiveTo assess the occurrence of CNV in patients presenting with flat irregular pigment epithelial detachments (FIPED). MethodsForty-five patients (49 eyes) with FIPED on OCT were enrolled in this retrospective study. There were 25 males (28 eyes) and 20 females (21 eyes). The mean age was 61.022±9.292 years. FFA, ICGA, spectral domain OCT and OCT angiography (OCTA) were performed in all patients during the same period. The FIPED was defined as an irregular elevation of the RPE allowing distinct visualization of Bruch’s membrane on OCT B-scan. The abnormal vascular signals from the deep retinal layer to the choroid layer on OCTA was defined as CNV. The CNV was classified into a type 1 CNV and a type 2 CNV according to the OCT characteristics. The CNV was classified into a typical and occult CNV according to the characteristics of the FFA image. Of all 49 eyes, fundus angiography revealed 18 eyes (36.7%) with CNV, and 31 eyes (63.3%) with no characteristic signs of CNV. FFA examination found that CNV in 8 eyes (classic CNV in 1 eyes, occult CNV in 7 eyes), which confirmed by OCT were type 1 CNV; transmitted fluorescence in 41 eyes. ICGA examination showed that CNV-like hyperfluorescence spots in 18 eyes, suspicious hyperfluorescence spots in late stage in 20 eyes, and choroidal high permeability in 11 eyes, respectively; and 18 CNV eyes were confirmed to be type 1 CNV by OCT. To compare the detection of CNV by OCTA and fundus angiography. ResultsOf the 49 eyes with FIPED, OCTA detected 36 eyes (73.5%) of type 1 CNV, and full or partial strong reflex signals were seen in FIPED; 13 eyes (26.5%) were not associated with CNV, and some strong reflection signals were found in FIPED in 9 eyes, 4 eyes with weak reflection signal. The FFA was examined for 1, 7 eyes of the classic and occult CNV, which confirmed to be type 1 CNV by OCTA. Among the 18 eyes with CNV which detected by ICGA, OCTA also found type 1 CNV. Among the 20 eyes with ICGA’s late suspicious strong fluorescent spots, OCTA showed 17 eyes of type 1 CNV; in 11 eyes with high choroidal permeability, OCTA showed type 1 CNV in 1 eye. Among the 36 eyes with CNV which detected by OCT, there were SRD in 32 eyes, no SRD in 2 eyes and retinal interlamellar cavities in 2 eyes. ConclusionOCTA can detect 73.5% of FIPED eyes with CNV. Compared with traditional fundus angiography, OCTA has a higher detection rate of CNV under FIPED. The FIPED of the internal strong reflection signal has a certain diagnostic value for the type 1 CNV.
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 expressions of miR-183 and retinal dehydrogenase 11 (RDH11) in exosomes derived from bone marrow mesenchymal stem cells (BMSC), and to preliminarily explore their targeting relationship and their effects on retinal pigment epithelial (RPE) cells. MethodsBMSC from C57BL/6 (C57) mice were isolated and cultured, and BMSC-derived exosomes were identified. BMSC were divided into blank group, simulation blank control group (mimic-NC group), miR-183 simulation group (miR-183-mimic group). C57 mice and retinal degeneration 10 (rd10) mouse RPE cells were cultured with reference to literature methods. RPE cells from rd10 mice were transfected with BMSC exosomes and co-cultured and divided into control group, exosome group, mimic-NC-exosome group (mimic-NC-exo group), miR-183-mimic-exosome group (miR-183-mimic-exo group). The relative expression levels of miR-183, RDH11 mRNA and protein in C57 mice, rd10 mice and RPE cells in each group were detected by real-time quantitative polymerase chain reaction and western blotting. The targeting relationship between miR-183 and RDH11 was analyzed by bioinformatics website and dual luciferase reporter. Cell counting kit 8 was used to detect the effect of miR-183 on BMSC exosomes on RPE cell proliferation; in situ labeling end labeling method was used to detect RPE cells apoptosis. One-way ANOVA was used to compare multiple groups. ResultsCompared with C57 mouse RPE cells, the relative expression of miR-183 in rd10 mouse RPE cells was down-regulated, and the relative expression of RDH11 mRNA was up-regulated, and the differences were statistically significant (t=5.230, 8.548; P=0.006, 0.001). Compared with the blank group and the mimic-NC group, the relative expression of miR-183 mRNA in the exosomes of the miR-183-mimics group was significantly increased (F=60.130, P＜0.05). After 24 h of co-culture, exosomes entered RPE cells. Compared with the mimic-NC-exo group, the relative expression of miR-183 mRNA in RPE cells in the miR-183-mimic-exo group was significantly increased, the proliferation ability was enhanced (t=7.311, P=0.002), and the number of apoptotic cells was decreased (F=10.949, P=0.012), and the differences were statistically significant (t=4.571, P=0.002). Bioinformatics website and dual-luciferase report confirmed that miR-183 has a targeting relationship with RDH11. Compared with the mimic-NC group, the relative expression of RDH11 mRNA and protein in the exosomes of the miR-183-mimic group was decreased, and the difference was statistically significant (t=5.361, 6.591; P=0.006, 0.003). After co-culture, compared with the control group, there was no significant difference in the relative expression of RDH11 mRNA and protein in RPE cells in the exosome group (t=0.169, 1.134; P=0.874, 0.320); The relative expressions of RDH11 mRNA and protein in RPE cells in -183-mimic-exo group were decreased, and the difference was statistically significant (t=5.554, 5.546; P=0.005, 0.005). ConclusionUp-regulation of BMSC-derived exosomal miR-183 promote the proliferation of RPE cells in vitro by targeting the expression of RDH11 and reduce the number of apoptosis.
ObjectiveTo observe confocal scanning laser ophthalmoscope (cSLO) based retinal imaging and color fundus camera in pigment epithelial detachment (PED) of polypoidal choroidal vasculopathy (PCV).MethodsPED of 30 patients (32 eyes) were recruited from June 2016 to June 2017 in the Beijing Tongren Hospital who were detected in high-definition OCT (HD-OCT) and diagnosed as PCV by FFA and ICGA. There were 16 males (17 eyes) and 14 females (15 eyes); aged from 50-83 years, with the mean age of 66.59 years. The photographs of ocular fundus including color fundus camera, cSLO imaging, HD-OCT, FFA and ICGA were analyzed. Multimodal imaging results were regarded as gold standard. Sensitivity and specificity were calculated in serous and hemorrhagic PED diagnosis using color fundus camera and cSLO imaging. The positive number of PED was used to compare between two modes fundus imaging by using χ2 test.ResultsTwenty serous PED eyes, 3 hemorrhagic PED eyes and 9 serous/hemorrhagic PED eyes were determined using multimodal imaging. The sensitivity and specificity of color fundus camera were 45% and 100% in detecting serous PED and 100% and 91% in detecting hemorrhagic PED. The sensitivity and specificity of cSLO imaging were 83% and 100% in detecting serous PED and 50% and 86% in detecting hemorrhagic PED. The positive number of serous PED in cSLO imaging was significantly higher than color fundus camera (χ2=7.752, P=0.011). The positive number of hemorrhagic PED in cSLO imaging shows no obvious difference compared with color fundus camera (χ2=1.164, P=0.419).ConclusionThe sensitivity and positive number of detecting serous PED with PCV in cSLO fundus imaging were higher than the color fundus camera technology.
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.