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.
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 clinical characterisitics of choroidal excavation in the macula. MethodsA total of 22 patients (22 eyes) with choroidal excavation diagnosed by spectral domain high definition optical coherence tomography (HD-OCT) were enrolled in this study. The patients included 12 males (54.50%) and 57 females (45.50%). The age was ranged from 21 to 82 years old, with an average of (41.44±13.17) years. All the patients were affected unilaterally, including 9 right eyes and 13 left eyes. The corrected vision, slit lamp microscope with preset lens, fundus photography, HD-OCT and fluorescence fundus angiography (FFA)were measured for all patients. The clinical characterisitics and concomitant diseases were observed. Seventeen eyes were followed for a period between 3 to 12 months. The lesions change were evaluated by HD-OCT. ResultsThere were 18 eyes (81.8%) with symptoms of micropsia and metamorphopsia, 4 eyes (18.2%) without symptoms. The corrected vision was ranged from 0.3 to 1.2, 12 eyes (54.54%) with moderate or high myopia. Fundus examination presents yellowish-white exudation in 12 eyes (54.54%), yellowish-white exudation accompanied with hemorrhage in 9 eyes (40.91%), grayish yellow reflex halo in 1 eye (4.55%). HD-OCT showed that the retinal pigment epithelium (RPE) layer was involved in the excavation, and the photoreceptor outer segment and pigment junction (OPR) layer was disappeared in all eyes. The external limiting membrane and the photoreceptor inner segment/outer segment junction layer were preserved in 13 eyes (59.09%) and disappeared in 9 eyes (40.91%). There were 10 eyes (18.18%) with a single lesion, 4 eyes (18.18%) with idiopathic choroidal neovascularization, 4 eyes (18.18%) with punctate inner choroidopathy, 1 eye (4.55%) with polypoidal choroidal vasculopathy, 1 eye (4.55%) with macular preretinal menbrance, 1 eye (4.55%) with central serous chorioretinopathy. FFA showed hypofluorescence in early phase, hyperfluorescence in late phase, without obvious leakage. There was no noticeable changes in size and morphological changes in the follow-up period. ConclusionsChoroidal excavation in the macula occurs mostly in middle-aged people with myopia. It can be associated with many fundus diseases. The excavation is located in RPE layer, and OPR layer disappeared. Choroidal excavation in the macula develops slowly.
ObjectiveTo study the role of Rac1 in the epithelial-mesenchymal transition (EMT) process of retinal pigment epithelial cells (RPE) induced by transforming growth factorβ(TGF-β). MethodsHuman ARPE-19 cells were divided into 4 groups including control group, TGF-βgroup, TGF-β+NSC23766 group, NSC23766 group. NSC23766 was added to medium 2 hours before TGF-βtreatment to block the Rac1 receptors.α-smooth muscle actin (α-SMA) expression was measured by immunofluorescence and Western blot. Cell scratch assay, invasion assay and gel contraction experiments were used to measure cell migration, invasion, cell contraction. ResultsThe expression ofα-SM A was higher in TGF-βgroup, compared with the control group, TGF-β+NSC23766 group (F=825.314, P < 0.05). Cell scratch assay showed that the cellular gap was less in GF-βgroup, compared with the control group, TGF-β+NSC23766 group, NSC23766 group (F=177.351, P < 0.05). Cell invasion assay showed that, the number of cells pass through the fiber membrane was the same in TGF-βgroup and other 3 groups (F=0.371, P=0.055). Gel contraction assay showed that TGF-βcan promote the cellular contraction, compare to the control group, TGF-β+NSC23766 group, NSC23766 group, the difference was statistically significant (F=40.473, P < 0.05). ConclusionRac1 play a role in TGF-β-induced behavioral changes of RPE cells; NSC23766 inhibit RPE cellular behavior change by regulating Rac1 activation.
ObjectiveTo observe the effect of complement receptor 1 (CR1) on barrier of cultured human retinal epithelial cells (hRPE) under complement-activated oxidative stress. MethodsThe third to fifth passage of hRPE cultured on Transwell insert were used to establish a stable hRPE monolayer barrier. The hRPE monolayer barrier was exposed to 500 μmol/L ten-butyl hydroperoxide and 10% normal human serum to establish the hRPE monolayer barrier model of complement-activated oxidative stress in vitro. hRPE monolayer barriers under complement-activated oxidative stress were divided into two groups including model group and CR1 treatment (1 μg/ml) group. Model group and CR1 treatment group were treated with 1 μl phosphate buffer solution (PBS) or CR1 for 4 hours. Normal hRPE monolayer barrier were used as control in transepithelial resistance (TER) measurement experiment. TER was measured to evaluate the barrier function of hRPE. The hRPE-secreted vascular endothelial growth factor (VEGF) and chemokine (C-C Motif) Ligand 2 (CCL2), together with complement bioactive fragments (C3a, C5a) and membrane-attack complex (MAC) in the supernatant were detected by enzyme-linked immune sorbent assay. ResultsStable hRPE monolayer barrier was established 3 weeks after hRPE seeded on Transwell insert. Complement-activated oxidative stress resulted in a sharp decrease of TER to 54.51% compared with normal hRPE barrier. CR1 treatment could significantly improve TER of barrier under complement-activated oxidative stress to 63.48% compared with normal hRPE barrier(t=21.60, P < 0.05). Compared with model group, CR1 treatment could significantly decrease the concentration of VEGF and CCL2 by 11.48% and 23.47% secreted by hRPE under complement-activated oxidative stress (t=3.26, 2.43; P < 0.05). Compared with model group, CR1 treatment could also decreased the concentration of C3a, C5a and MAC by 24.00%, 27.87%, 22.44%.The difference were statistically significant (t=9.86, 2.63, 6.94; P < 0.05). ConclusionsCR1 could protect the barrier function of hRPE cells against complement-activated oxidative stress. The underlying mechanism may involve inhibiting complement activation and down-regulating the expression of VEGF and CCL2.
RCBTB1 gene associated hereditary retinopathy is an extremely rare inherited retinal disease (IRD) discovered recently. The mutation of RCBTB1 gene can lead to a variety of IRD clinical phenotypes, such as early retinitis pigmentosa and delayed chorioretinal atrophy. The hereditary mode of RCBTB1 gene associated retinopathy is autosomal recessive. RCBTB1 gene plays an important role in maintaining mitochondrial function and anti-oxidative stress defense mechanism of retinal pigment epithelium cells. In the future, it is necessary to further determine whether there is a genotypic and phenotypic correlation in the age of onset of RCBTB1 gene associated retinopathy or multi-organ involvement, and evaluate the safety and efficacy of adeno-associated virus-mediated RCBTB1 gene replacement therapy in animal models, to explore the feasibility of gene replacement therapy and stem cell therapy.
ObjectiveTo observe the effect of exosomes secreted by retinal pigment epithelial (RPE) cells which damaged by blue light to Nod-like receptor protein (NLRP3).MethodsCultured ARPE-19 cells were divided into 2 groups; one group of RPE cells were exposed to blue light irradiation for 6 hours, the other group was cultured in routine environment. Total exosomes were extracted from the two groups by differential ultracentrifugation in low-temperature, and examined by transmission electron microscope to identify their forms. The exosomes were then incubated with normal ARPE-19 cells. The expression level of CD63, interleukin (IL)-1β, IL-18 and caspase-1 on the exosome surface were measured by Western blotting. The expressions of NLRP3 mRNA in RPE cells were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR).ResultsBlue light damaged the cellular morphology. Transmission electron microscopy showed that the exosomes were 50-200nm in diameter and like double-concave disks. Blue light damaged cell-derived exosomes had significantly higher expression of IL-1β (t=18.04), IL-18 (t=12.55) and caspase-1 (t=14.70) than the control group (P＜0.001). ARPE-19 cells cultured with blue light damaged cell-derived exosomes also had significantly higher expression of IL-1β (t=18.59), IL-18 (t=23.95) and caspase-1 (t=35.27) than control exosomes (P＜0.001). RT-PCR showed that the relative expression of NLRP3 mRNA of PRE cells in experimental group and control group were 1.000±0.069 and 0.2±0.01, respectively, the difference was significant (t=12.20, P＜0.001).ConclusionThe expression IL-1β, IL-18 and caspase-1 and NLRP3 mRNA were upregulated by exosomes secreted by blue light damaged-RPE cells.
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.
ObjectiveTo observe the efficacy of intravitreal injection of aflibercept (IVA) in the treatment of exudative age-related macular degeneration (wAMD) combined with RPE detachment (PED).MethodsA retrospective case study. From June 2018 to June 2019, 32 eyes (overall group) of 27 wAMD patients with PED were included in the study. All eyes were treated with IVA. The initial loading dose was 2.0 mg, which was injected once a month for 2 consecutive months and and then use a PRN regimen after evaluation. According to the maximum height of PED (PEDH) 2 months after treatment, the overall group was divided into the response group and the partial response group, with 20 (62.50%) and 12 (37.50%) eyes respectively. The response group: PEDH decreased by ≥25% compared with before treatment. The partial response: PEDH decreased by ＜25%. The macular fovea was scanned with the 3D-OCT 2000 instrument from Topcon (Japan). PEDH, PED area (PEDA), PED volume (PEDV), and macular foveal retinal thickness (CMT) were measured. There was no significant difference in BCVA, CMT, PEDH, PEDA, and PEDV of the eyes in the response group and the partial response group (t=－0.791, －0.488, －0.900, －1.130, －0.400; P=0.435, 0.630, 0.380, 0.270, 0.690). The changes of BCVA, PEDH, PEDA, PEDV, CMT in each group were observed before treatment and 1, 2, 4, and 6 months after treatment. The comparison of BCVA and PED-related indicators and CMT before and after treatment were performed by repeated measures analysis of variance.ResultsCompared with before treatment, the BCVA, CMT, PEDH, PEDA and PEDV of the eyes in the overall group, the response group, and the partial response group were obviously improved after treatment. Among them, there were statistically significant differences in all indicators of the overall group and the response group (FBCVA=5.871, 3.798; P=0.001, 0.019. FCMT=24.526, 14.109; P=0.000, 0.001. FPEDH=12.569, 12.091; P=0.000, 0.000. FPEDA=7.534, 6.286; P=0.000, 0.000. FPEDV=5.139, 4.104; P=0.004, 0.014); there was no statistically significant difference in PED-related indicators in the partial response group (FPEDH=3.210, P=0.054; FPEDA=1.913, P=0.183; FPEDV=3.500, P=0.051), the difference between BCVA and CMT was statistically significant (FBCVA=3.033, P=0.027; FCMT=11.140, P=0.001). Two months after treatment, the eye number of PEDH reduction rate ＜25%, 25%-＜50%, 50%-＜75%, and ≥75% were 12 (37.50%), 8 (25.00%), 9 (28.13%), and 3 (9.38%) in the overall group, respectively. And PED in one eye (3.13%) was completely eliminated. Six months after treatment, the proportion was 13 (40.23%), 5 (15.63%), 7 (21.88%) and 7 (21.88%), respectively, among which 4 eyes (12.50%) with PED were completely resolved.ConclusionsAflibercept treatment of wAMD combined with PED can restore its anatomical indicators and improve visual function of patients in a short time; the efficacy of PED in the PRN stage is related to the efficacy of the loading dose stage.
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.