Human Pigment Epithelial Cells Research Articles

Therapeutic Extracellular Vesicles from Tonsil-Derived Mesenchymal Stem Cells for the Treatment of Retinal Degenerative Disease.

Retinal degenerative disease (RDD), one of the most common causes of blindness, is predominantly caused by the gradual death of retinal pigment epithelial cells (RPEs) and photoreceptors due to various causes. Cell-based therapies, such as stem cell implantation, have been developed for the treatment of RDD, but potential risks, including teratogenicity and immune reactions, have hampered their clinical application. Stem cell-derived extracellular vesicles (EVs) have recently emerged as a cell-free alternative therapeutic strategy; however, additional invasiveness and low yield of the stem cell extraction process is problematic. To overcome these limitations, we developed therapeutic EVs for the treatment of RDD which were extracted from tonsil-derived mesenchymal stem cells obtained from human tonsil tissue discarded as medical waste following tonsillectomy (T-MSC EVs). To verify the biocompatibility and cytoprotective effect of T-MSC EVs, we measured cell viability by co-culture with human RPE without or with toxic all-trans-retinal. To elucidate the cytoprotective mechanism of T-MSC EVs, we performed transcriptome sequencing using RNA extracted from RPEs. The in vivo protective effect of T-MSC EVs was evaluated using Pde6b gene knockout rats as an animal model of retinitis pigmentosa. T-MSC EVs showed high biocompatibility and the human pigment epithelial cells were significantly protected in the presence of T-MSC EVs from the toxic effect of all-trans-retinal. In addition, T-MSC EVs showed a dose-dependent cell death-delaying effect in real-time quantification of cell death. Transcriptome sequencing analysis revealed that the efficient ability of T-MSC EVs to regulate intracellular oxidative stress may be one of the reasons explaining their excellent cytoprotective effect. Additionally, intravitreally injected T-MSC EVs had an inhibitory effect on the destruction of the outer nuclear layer in the Pde6b gene knockout rat. Together, the results of this study indicate the preventive and therapeutic effects of T-MSC EVs during the initiation and development of retinal degeneration, which may be a beneficial alternative for the treatment of RDD.

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells using the Sleeping Beauty Transposon System.

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 µL of resuspension buffer and combined with 2 µL of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells using the Sleeping Beauty Transposon System

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 µL of resuspension buffer and combined with 2 µL of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.

Resveratrol protects human retinal pigment epithelial cells from toxicity of high glucose by up-regulation of miR-26a

Resveratrol protects human retinal pigment epithelial cells from toxicity of high glucose by up-regulation of miR-26a

Inhibitory effects of NADPH oxidase 4 inhibitor on VEGF expression in hypoxia-induced human RPE cells

Objective To observe the inhibitory effects of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) on vascular endothelial growth factor (VEGF) expression in hypoxia-induced human retinal pigment epithelial cells. Methods The cultured APRE-19 cells were divided into Avastin group and VAS2870 group, and then the Avastin group was subdivided into the normoxic control group, hypoxia control group, 0.25 mg/ml Avastin intervention group, 0.50 mg/ml Avastin intervention group and 0.75 mg/ml Avastin intervention group, and the VAS2870 group was subdivided into 1 μmol/ml VAS2870 intervention group, 3 μmol/ml VAS2870 intervention group and 5 μmol/ml VAS2870 intervention group.CoCl2 of final concentration of 300 mol/L was added to the medium to establish the cytochemical hypoxia model.The expressions of NOX4 and VEGF in human retinal pigment epithelial cells were located and evaluated by immunofluorescence staining, and relative expressing levels of NOX4 and VEGF proteins were compared by Western blot assay. Results The relative expression of NOX4 was 0.657±0.153, 1.000±0.200, 1.206±0.300, 1.260±0.200 and 1.413±0.273, and the relative expression of VEGF-A was 0.821±0.110, 1.210±0.100, 0.672±0.100, 0.340±0.120 and 0.300±0.130 in the normoxic control group, hypoxia control group, 0.25 mg/ml Avastin intervention group, 0.50 mg/ml Avastin intervention group and 0.75 mg/ml Avastin intervention group, respectively, with statistically significant differences among the groups (F=17.631, P<0.001; F=4.777, P<0.05). The relative expression of NOX4 protein in 0.75 mg/ml Avastin intervention group was significantly lower than that in normoxia control group (P<0.001). The relative expression of VEGF-A protein in the cells of the 0.25, 0.50 and 0.75 mg/ml Avastin intervention group was significantly lower than that in hypoxia control group (P<0.05). The expression of NOX4 protein in the cells was 0.970±0.120, 1.060±0.130, 0.880±0.130, 0.567±0.135 and 0.450±0.120, and the relative expression of VEGF-A protein was 0.387±0.135, 0.627±0.125, 0.370±0.140, 0.363±0.140 and 0.160±0.100 in the normoxia control group, hypoxia control group, 1 μmol/ml VAS2870 intervention group, 3 μmol/ml VAS2870 intervention group and 5 μmol/ml VAS2870 intervention group, respectively, with statistically significant differences among them (F=12.933, P<0.001; F=4.948, P<0.05). The relative expression of VEGF-A protein in the 1, 3 and 5 μmol/ml VAS2870 intervention group was significantly lower than that in hypoxia control group (P<0.05). Conclusions NOX4 inhibitor can inhibit the expression of VEGF-A protein in hypoxia-induced human RPE cells by down-regulating the NOX4 level. Key words: Hypoxia; Retinal pigment epithelium, human; Nicotinamide adenine dinucleotide phosphate oxidase 4; Vascular endothelial growth factor; Inhibitor

Inhibitory effect of Tanshinone IIA on the proliferation of human retinal pigment epithelial cells in hypoxia and its mechanism

Objective To investigate the effect of Tanshinone ⅡA on the proliferation and the signaling pathway of human retinal pigment epithelial (RPE) cells in hypoxia. Methods CoCl2 (150 μmol/L) was used to simulate hypoxic condition and the ARPE-19 cells cultured in vitro were divided into blank control group, hypoxia control group, Tanshinone ⅡA group and hypoxia-inducible factor-1α (HIF-1α) inhibitor group.The different doses of Tanshinone ⅡA were used to treat ARPE-19 for 24, 48 and 72 hours, respectively.The inhibitory rate of cell proliferation of different groups were detected by MTT after 24, 48 and 72 hours of administration cultured, and the apoptosis rate and the cell cycle distribution of cells in the hypoxia were analyzed by flow cytometry.Real-time PCR and Western blot were used to detect the expressions of mRNA and protein of HIF-1α and vascular endothelial growth factor (VEGF). Results MTT assay showed that Tanshinone ⅡA could inhibit the proliferation of ARPE-19 cells in a dose- and time-dependent manner, and the proliferation inhibitory rate gradually increased in the 1, 5 and 10 mg/L Tanshinone ⅡA groups, with significant differences between any two groups (all at P 0.05). Conclusions Tanshinone ⅡA can inhibit the proliferation of RPE cells, induce apoptosis by arresting cells at G0/G1 phase.The mechanism is related to the suppression of HIF-1α/VEGF signaling pathway. Key words: Tanshinone ⅡA; Retinal pigment epithelial cells; Hypoxia-inducible factor-1α; Vascular endothelial growth factor; Proliferation

Controlling crystalline phase of TiO2 thin films to evaluate its biocompatibility

ABSTRACTThis paper describes studies performed to understand the biocompatibility of TiO2 thin films with controlled crystalline phases using human pigment epithelium cells (ARPE-19). TiO2 films were deposited by radio frequency magnetron sputtering using different gas pressure, oxygen partial pressure and magnetron power. Thermal annealing was used to control the microstructure and crystalline phase of the resulting films. Results show that films deposited at low power (75 and 100 W) developed anatase phase and as power increases (>125 W), a mixture of rutile and anatase is observed. For films deposited at higher power (>150 W), only rutile phase was obtained. These results indicate that plasma power is a critical parameter to control TiO2 crystalline phase. Finally, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay showed that there is no significative difference in cell proliferation among TiO2 crystalline phases and indicates that the biocompatibility is mostly defined by the surface chemistry of the films.

Role of mitochondrial DNA in human retinal pigment epithelium cells apoptosis induced by blue light

Objective To research the role of mitochondrial DNA mediate the cultured human retinal pigment epithelium (hRPE) cell apoptosis induced by blue light and the relationship with time. Methods Established the blue light damage model of cultured hRPE cells in vitro with light emitting diode (LED) blue light density of (4.0±0.5)mW/cm2 adjusted by FL-1D blue light illumination meter, and the illumination time was set as 0, 0.5, 1, 2, 4, 6, 12 and 24 hours, then the cells were grouped according to the illumination time.Immunofluorescence were used to identify the cells; the expressions of caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, bax and bcl-2 were detected with Western blot.Quantitative PCR was used to detect the copy number of mitochondrial DNA and PCR was used to detect mitochondrial DNA 4977bp common deletion. Results Immunofluorescence results showed that the RPE65 protein was expressed in the cytoplasm.The expressions of bax were upregulated after illumination for 1 hour, cleaved caspase-3 were upregulated after illumination for 2 hours, caspase-3, caspase-9, cleaved caspase-9 were upregulated after illumination for 4 hours, while the expression of bcl-2 was downregulated after illuminated for 2 hours, with significant differences compared to the normal control group (all at P<0.05). The copy number of mitochondrial DNA in 0.5, 1, 2, 4, 6, 12 and 24 hours groups was downregulated, with significant differences compared to the normal control group (all at P<0.05). The expressions of 4977bp common deletion in 0.5, 1, 2, 4, 6, 12 and 24 hours groups were increased, with significant differences compared with the normal control group (all at P<0.05). Conclusions Blue light can cause cell apoptosis, especially mitochondrial apoptosis, in hRPE probably motivated by mitochondrial DNA damage. Key words: Mitochondrial DNA; Apoptosis; Oxidative stress; Human retinal pigment epithelium cell

Mediated effects of endoplasmic reticulum stress on light-induced apoptosis and inflammation of human retinal pigment epithelial cell

Background The light damage model of retinal pigment epithelium (RPE) cells is a research direction of retinal degeneration diseases, and RPE cell apoptosis induced by light damage and inflammation is an important pathologic basis of light-induced RPE cell damage.However, whether endoplasmic reticulum stress (ERS) paticipates in light-induced RPE cell damage is rarely reported. Objective This study was to explore the effects of ERS on light-induced RPE cell damage. Methods Human RPE cell line (ARPE-19) was cultured, and light damage models were created by irradiating the cells for 3-, 6-, 12- and 24-hours with white fluorescent lamp with the intensity of (2 000±500)lx for the selection of optimal irradiating time, and the cells in the normal control group were cultured in the dark environment.The cells were divided into normal control group, light exposure group and 4-phenylbutyric acid (4-PBA) pretreated+ light exposure group.The cells from 4-PBA pretreated+ light exposure group were cultued firstly with 4-PBA for 30 minutes and followed by light exposure for 12 hours.The apoptisis rate of the cells and intracellular reactive oxygen species (ROS) content were detected by flow cytometry; the concentrations of interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the cell supernatant were assyed by ELISA.The relative expressing levels of activating transcription factor 6 (ATF-6), C/enhancer binding protein homologous protein (CHOP) and caspase-12 mRNA and protein in the cells were detected by real-time quantitative PCR and Western blot, respectively. Results The cultured cells showed a long spindle shape, the border was not clear, the cytoplasm was degranulation, and the cell fragments increased.Flow cytometry showed that compared with the normal control group, the ROS content in the cells and the apoptosis rate were evidently increased with the lapse of light exposure time (F=763.00, 119.30, both at P<0.01). ELISA results showed that the concentrations of IL-1β and TNF-α in the cell supernatant were significantly higher in the light exposure 6-hour group than those in the normal control group with the peak value in the light exposure 12-hour group.Compared with the normal control group, the relative expression levels of ATF-6, CHOP and caspase-12 mRNA and protein in the cells were elevated in the light exposure group and peaked in the light exposure 12-hour group.In addition, the relative expression levels of ATF-6 mRNA, CHOP mRNA and caspase-12 mRNA in the cells were significantly reduced in 4-PBA pretreated+ light exposure group compared with the light exposure group (F=281.69, 473.88, 308.45, all at P<0.01), and their proteins were also significantly reduced (F=47.86, 57.93, 106.59, all at P<0.01). The apoptosis rate, concentrations of IL-1β and TNF-α in the cell supernatant were significantly reduced in 4-PBA pretreated+ light exposure group compared with the light exposure group (F=88.64, 245.47, 101.01, all at P<0.01). Conclusions The light exposure at (2 000±500)lx induces intracellular ROS accumulation and activates the ERS response, which results in apoptosis and inflammatory process of human RPE cells.4-PBA, a inhibitor of ERS, can suppress light-induced ERS response and therefore reduces the apoptosis rate and inhibits inflammatory process. Key words: Oxidative stress; Light/adverse effects; Endoplasmic reticulum/pathology; Pigment epithelium of eye/pathology; Cell line; Human; Apotosis; Inflammation

Protective effects of tissue factor targeting peptide on human retinal pigment epithelial cell damage induced by blue light

Background Light-induced retinal damage results in the damage of retinl pigment epithelial (RPE) cells and therefore affects the pathogenesis and development of age-related macular degeneration (AMD). Studies showed that tissue factor (TF) is overexpressed in oxidative damaged RPE cells and the choroidal neovascularization (CNV) of AMD, speculating that the suppression of TF can prevent the damage of RPE cells and inhibit CNV. Objective This study was conducted to observe the protective effects of TF targeting peptide (TF-TP), a new drug of autologous synthesis, on human RPE-cells induced by blue light. Methods Human RPE cells were isolated from donor eye and cultured.Cultured cells were divided into blank control group, model group and TF-TP treated group.Light-induced RPE cell damage model was established by exposuring the cells in the blue light of (4.0±0.5)mW/cm2 for 12 hours in the model group, and different concentrations (10, 100, 150, 200, 300 μmol/L) of TF-TP were added into the medium to pretreat the cells for 24 hours and then exposed the cells to the blue light for 12 hours in the TF-TP groups.The cell viability was determined by CCK-8 assay.The morphology and ultrastructure in the cells were observed under the inverted microscope and transmission electron microscope.The apoptosis of the cells was assayed by Hoechst staining.The expressions of TF and apoptosis-related protein bax, bcl-2 in the cells were determined by Western blot. Results CCK-8 assay showed that there was no significant difference in the cell viability among blank control group and different concentrations TF-TP groups (F=2.15, P=0.11). The cell survival rate of blank control group, model group and 150 μmol/L TF-TP group was (100.0±0.00)%, (43.79±6.55)% and (63.45±3.57)%, and the survial rate was increased in the 150 μmol/L TF-TP group compared with the model group (P=0.00), and 150 μmol/L was detemined as a optimal concentration of TF-TP.A lot of shrinkage, deformation, suspension cells were exhibited under the optical microscope, and decrease of microvilli structure, rupture of mitochondrial cristae and vacuolar degeneration of the cells were found in the model group, and the damage of the cells were evidently lightened in the 150 μmol/L TF-TP group.The apoptosis rate of the cells were (0.98±0.19)%, (9.98±0.82)% and (5.73±0.88)% in the blank group, model group and 150 μmol/L TF-TP group, respectively, with a significant difference among the groups (F=206.18, P=0.00), and the apoptosis rate of the cells in the 150 μmol/L TF-TP group was significantly lower than that in the model group (P<0.05). Compared with the blank control group, the relative expression of bax and TF was obviously increased and that of bcl-2 was decreased in the model group; while the expression of bax and TF was lower, and that of bcl-2 was higher in the 150 μmol/L TF-TP group compared with the model group (all at P<0.05). Conclusions Pretreation of TF-TP can lessen cell apoptosis and increase cell survival rate and therefore plays a protective role to blue light-induced human RPE cells possibly by inhibiting bax/bcl-2 apoptotic pathways mediated by TF. Key words: Tissue factor; Human; Retinal pigment epithelium/metabolism; Epithelial cells/ radiation effects; Light; Apoptosis

Inhibitory effects of autophagy inhibitor 3-MA on high glucose-induced proliferation of human retinal pigment epithelium cells

Background Diabetic retinopathy (DR) is one of the common complications of diabetes.Retinal pigment epithelium (RPE) cells, as important constituent cells of the retina, play important roles in the development and progression of DR. Objective This study was to investigate the suppressive effects of autophagy inhibitor 3-MA on the proliferation of human retinal pigment epithelium cells (hRPECs) following high glucose culture. Methods HRPECs were divided into control group, high-glucose group and 3-MA+ high glucose group.The cells were cultured by the DMEM/F12 with 5 mmol/L glucose in the control group, and by the DMEM/F12 with 5 mmol/L glucose in the high glucose group and by the DMEM/F12 with 10 mmol/L 3-MA (for 1 hour firstly) and 30 mmol/L glucose in the 3-MA+ high glucose group.The cells were inoculated into 24-well plate with the content of 1×105/well, and then the cells were consecutively cultured for 24 hours with DMEM/F12 containing 0.5% fetal bovine serum after achieved attached 80% confluence.The morphology and ultrastructure of the cells were examined by optics microscope and transmission electronic microscope.The proliferative rate of the cells was assayed by CCK -8 kit.The expression of autophagy-related gene microtubule associated protein light chain 3B (LC3B) in the cells was detected by Western blot and LC3B-Ⅱ/LC3B-Ⅰ value was quantitatively evaluated among the groups. Results The cells grew well with uniform size and regulatory arrangement in the control group.The cells in the high glucose group enlarged and the number of cells evidently increased.In the 3-MA+ high glucose group, the cells decreased with a disorder arrangement.Under the transmission electron microscope, the cells were normal with the round- or oval-like nucleus and normal organelles in the control group, and autolysosome could be seen in the cells in the high glucose group.In the 3-MA+ high glucose group, some autophagic bodies were found.The proliferative rate of the cells was (100.0±2.0)%, (116.9±5.2)% and (103.7±4.7)% in the control group, high glucose group and 3-MA+ high glucose group respectively, showing a significant difference among the groups (F=13.526, P=0.006). The proliferative rate was considerably raised in the high glucose group compared with the control group and 3-MA+ high glucose group (both at P 0.05). Compared with the control group, the expressing intensity of LC3B-Ⅰ protein was weakened and that of LC3-Ⅱprotein was enhanced, and the expression intensity of LC3B-Ⅰ and LC3B-Ⅱproteins in the 3-MA+ high glucose group was similar to that in the control group.The LC3-Ⅱ/LC3-Ⅰ ratio was 0.131±0.065, 2.504±0.097 and 0.274±0.007 in the control group, high glucose group and 3-MA+ high glucose group, respectively, with significant differences among the groups (F=1 694.676, P=0.000), and the LC3-Ⅱ/LC3-Ⅰ ratio was increased in the high glucose group in comparison with the control group and the 3-MA+ high glucose group (all at P 0.05). Conclusions High glucose culture of hRPECs can activate autophagy process and promote cell proliferation.3-MA, an autophagy inhibitor, suppresses the high glucose-induced growth of HRPECs by inhibiting autophagy process. Key words: Autophagy/drug effects; Glucose/pharmacology; Retinal pigment epithelium cell/drug effects; Ultrastructure; Biomarkers; Humans

Effect of exosomes derived from human umbilical cord mesenchymal stem cells on the expression of vascular endothelial growth factor A in blue light injured human retinal pigment epithelial cells

Objective To 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. Methods hUCMSC 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. Results hUCMSC 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. Conclusion hUCMSC 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. Key words: Mesenchymal stem cells; Exosomes; Retinal pigment epithelium; Vascular endothelial growth factor A; Animal experimentation

Transthyretinin repress retinal microvascular endothelial cells under high glucose and hypoxia environment

Objective To explore transthyretin (TTR) effect on retinal vascular endothelial cells (hREC) under high glucose and hypoxia environment. Methods hREC and human retinal pigment epithelial cell (hRPEC) were cultured at low-glucose (LG), high glucose (HG) and hypoxia. The glucose concentration was increased from 5.5 mmol/L up to 25 mmol/L, and hypoxia was induced by 200 μmol/L CoCl2. The cells were divided into LG group, LG-hypoxia group, HG group, HG-hypoxia group according to the different cell culture environment. The growth index was detected at 0, 4, 8, 16, 24, 36, 48, 60, 72 hours after cultured. Furthermore, hREC and hRPEC were also cultured with additional TTR (4 μmol/L), respectively. Then transwell co-culture system was employed to reveal the effects of hRPEC on the growth of hREC. Results At 72 hours after cultured, the growth index of hREC and hRPEC in LG group were increased as compared with LG-hypoxia group and HG group (hREC: F=17.098, 22.970; P<0.05. hRPEC: F=45.442, 9.011; P<0.05); the growth index of hREC and hRPEC were decreased in HG group and HG-hypoxia group (hREC: F=146.184, P<0.05; hRPEC: F=27.907, P<0.05). Additionally, hREC could be significantly repressed by added TTR during culture with high concentration of glucose (F=161.430, 24.106; P<0.05). hREC could be significantly increased by added TTR during culture with low concentration of glucose (F=200.486, 48.662; P<0.05). In co-culture process, hRPEC revealed inhibition activity against hREC under both natural and abnormal environment (LG group: F=15.711, P<0.05; LG-hypoxia group: F=45.659, P<0.05; HG group: F=7.857, P<0.05; HG-hypoxia group: F=6.348, P<0.05). Conclusion Under high glucose and hypoxia environment, the growth of hREC from neovascular could be inhibited by TTR. Key words: Retinal Vessels/cytology; Endothelial cells/physiology; Prealbumin/drug effects

TSG-6 inhibited the HCMV infection-induced expression of TNF-α and IL-6

Objective To investigate the roles of tumor necrosis factor alpha stimulus gene-6 (TSG-6) in the inflammatory responses induced by human cytomegalovirus (HCMV) infection and the possible mechanism. Methods Human lung fibroblast cells (MRC-5) and human retinal pigment epithelial cells (ARPE-19) were infected with HCMV strains. A normal cell control group and an inactivated virus infection group were set up as well. qRT-PCR analysis and Western blot assay were performed to measure the expression of TSG-6 at mRNA and protein levels in each group. The recombinant human TSG-6 protein (rhTSG-6) was administrated to the HCMV-infected MRC-5 cell cultures at the concentration of 100 ng/ml. The supernatants were harvested to measure the levels of TNF-α and IL-6 and the transcriptional activity of NF-κB by using ELISA, indirect immunofluorescence assay (IFA) and Western blot assay. Meanwhile, a normal cell control group and a virus control group were set up for comparison. Results The expression of TSG-6 at mRNA level in HCMV-infected MRC-5 cells were 19 and 376 times of that in normal cells at the time points of 72 h and 96 h after infection. The expression of TSG-6 at mRNA level in HCMV-infected ARPE-19 cells were 15 and 354 times of that in normal cells at the time points of 96 h and 120 h after infection. Results of the Western blot assay further confirmed the increased expression of TSG-6 at protein level at the corresponding time points. Compared with the virus control group, the levels of TNF-α and IL-6 in the supernatants of HCMV-treated MRC-5 cell culture were significantly decreased upon the intervention of rhTSG-6 protein (P<0.05). Moreover, TSG-6 protein could suppress the expression and nuclear translocation of NF-κB. Conclusion TSG-6 was involved in the HCMV infection-induced inflammation responses and played a role in inhibiting the expression of HCMV infection-related inflammatory factors and affecting the nuclear localization of NF-κB. Key words: Human cytomegalovirus; Tumor necrosis factor αstimulated gene 6; NF-κB

Role of Shh signal transduction pathway in vascular endothelial growth factor expression under hypoxia in cultured human retinal pigment epithelial cells

Objective To investigate the role of sonic hedgehog (Shh) signal transduction pathway in the expression of vascular endothelial growth factor (VEGF) under hypoxia in cultured human retinal pigment epithelial (hRPE) cells. Methods ARPE-19 were cultured and divided into normal ARPE-19 (Cont) and hypoxia group (100 μmol/L CoCl2 Cobalt Chloride + ARPE-19); hypoxia group was further divided into CoCl2 group, cyclopamine group (CYA) and dimethyl sulfoxide (DMSO) group. 20 μmol/L cyclopamine was added to the CYA group 1 hour before hypoxia, 1‰ DMSO was added into DMSO group at the same time. The hRPE cells were cultured under hypoxia for 4, 8, 12, 24 hours. The expression of Shh and VEGF were determined by Real-time fluorescent quantitate PCR (RT-PCR). The amount of VEGF in the hRPE-conditioned supernatant was measured using enzyme linked immunosorbent assay (ELISA) at 4, 8, 12, 24 hours, respectively. Results RT-PCR tests showed that the level of Shh and VEGF of hRPE was time dependently increased (Shh: F=45.260, P=0.001; VEGF: F=264.938, P=0.001). The level of Shh and VEGF of hRPE in the group treated with cyclopamine was decreased (P<0.01). ELISA tests showed that the amount of VEGF in hRPE supernatant was significantly increased in time-dependent manner (F=3 156.676, P=0.001), and it was down-regulated by cyclopamine under hypoxia (P<0.01). Conclusion Shh signal transduction pathway could play a role in the VEGF expression induced by hypoxia in hRPE cells. Key words: Vascular endothelial growth factors; Retinal pigment epithelium; Cells, cultured

Ameliorative effect of VIP family members on blood retinal barrier breakdown in diabetic macular edema

Diabetic macular edema (DME) is one of the main complications of diabetic retinopathy [1]. This pathology is owed to impairment of the blood-retinal barrier (BRB) [2]. Many factors, such as hypoxia, contribute to barrier dysfunction and progression of the disease. Low oxygen tension is one of the main events involved in the formation of new blood vessels that characterize the typical uncontrolled angiogenesis in proliferative stage of diabetic retinopathy. In the last decades, various studies have focused their attention on the role of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) in the pathophysiology of DME. However, the effects of these peptides in maintaining the integrity of the BRB exposed to hypoxia remains to be elucidated. In the present work we have studied, for the first time, the effect of these peptides on outer BRB integrity following hypoxic insult in an experimental model of DME. To this end, we have used the human retinal pigment epithelial cells (ARPE-19) to test the effect of both peptides on cellular permeability, transepithelial electrical resistance, tight junctions expression and hypoxia-induced apoptosis. Results have demonstrated that both PACAP and VIP are able to rescue the integrity of cell monolayer during the hypoxic event, minimizing apoptotic damages induced by low tissue oxygen tension through the activation of phosphoinositide 3 kinase /Akt and mammalian mitogen activated protein kinase/Erk kinase signaling pathways. Furthermore, these peptides modulate the expression of vascular endothelial growth factor which is one of the downstream transcription factor activated during the hypoxic process. In conclusion, we have demonstrated that PACAP and VIP are able to counteract the damage induced by hypoxia on BRB through the modulation of hypoxia inducible factors expression.

The correlation between the concentrations of VEGF and PEDF and Ca2+-PKC signaling pathways in human retinal pigment epithelial cells cultured in vitro after exposuring to blue light

To investigate the concentrations of vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), inositol triphosphate (IP3) and diacylglycerol (DAG) in human retinal pigment epithelium (RPE) cells after exposuring to blue light, and to explore the relationship with Ca2+-PKC signaling pathways, to evaluate the role of Ca2+-PKC signaling pathways of blue-light irradiation induced apoptosis in RPE cells. The fourth generation human RPE cells in vitro were exposured to blue light (2000±500 lux) for 6 hours, 24 hours prolongation of post-exposure culture. The concentrations of VEGF, PEDF, IP3 and DAG were assayed by enzyme linked immunosorbent assay (ELISA). Cells were randomly divided into 6 groups, group A (control), group B (exposure to blue light), group C (exposure to blue light+PMA), group D (exposure to blue light+Calphostin C), group E (exposure to blue light+Nifedipine), group F (exposure to blue light+Calphostin C+Nifedipine). Flow cytometry was used to detect the apoptosis rate of human RPE cells in A, B and F group. Comparing with group A (584.38±10.66), the concentration of VEGF in group B (700.70±5.88), group C (698.21±6.66) and group E (648.30±4.91) was higher, the difference was statistically significant (P=0.002, 0.002, 0.016). Comparing with group B (700.70±5.88), the concentration of VEGF in Group D (623.87±3.12) and E (648.30±4.91) was lower (P=0.001, 0.002). Comparing with group A (75.96±1.70), the concentration of PEDF in Group B (71.82±1.67) and C (72.43±0.58) was lower (P=0.004, 0.011), but the concentration of PEDF in Group D (86.31±1.35) and E (93.72±1.24) was higher (P=0.000, 0.000). Comparing with group B (71.82±1.67), the concentration of PEDF in Group D (86.31±1.35) and E (93.72±1.24) was higher (P=0.000, 0.000). Comparing with group A (7.70±0.29), the ratio of VEGF to PEDF in Group B (9.85±0.34) and Croup C (9.64±0.02) was higher (P=0.008, 0.027) Comparing with group B, The ratio of VEGF to PEDF in Group D (7.23±0.08) and E (6.92±0.06) was lower (P=0.016, 0.015). Comparing with group A (108.42±0.75, 995.47± 13.61), the concentration of IP3 and DAG in Group B (117.24±1.06, 1070.10±10.07), C (137.12±2.71, 1046.40±7.90), D (139.17±1.40, 1041.13±9.76) and E (149.61±0.77, 1273.14±10.89) was higher, the difference was statistically significant (P=0.003, 0.007, 0.000, 0.000, 0.000, 0.000, 0.000, 0.000). Comparing with group B, the concentration of IP3 in Group C, D and E was higher (P=0.011, 0.000, 0.000). Comparing with group B, the concentration of DAG in Group C and D was lower (P=0.021, 0.007). Comparing with group B, the concentration of DAG in Group E was higher (P=0.000). Comparing with group A (10.27±1.88), the apoptosis rate of RPE cells in Group B(25.07±2.66) and F(19.37±3.23) was higher, the difference was statistically significant (P=0.001, 0.009). Comparing with group B (25.07±2.66), the apoptosis rate of RPE cells in Group F (19.37±3.23) was lower (P=0.038). (1) After exposuring to blue light, the concentrations of VEGF, IP3 and DAG are increased and the ratio of VEGF to PEDF is also increased and the concentration of PEDF is decreased in human RPE cells. (2) L-Type Calcium Channels and Ca2+-PKC signaling pathways may be regulate the concentrations of VEGF, PEDF, IP3 and DAG in RPE cells after exposuring to blue light by feedback regulation. (3) The application of Calphostin C combined with Nifedipine may be restrain the apoptosis of RPE cells after exposuring to blue light.

Epithelial-mesenchymal transition of human retinal pigment epithelial cells under the high glucose condition in vitro

Background Several types of cells participate in the formation of proliferative membrane in proliferative retinopathy (PVR), and the proliferation, migration and epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells play an important role.Many studies have confirmed high blood glucose is the basic pathogenesis of diabetic retinopathy (DR).However, whether EMT could be induced in RPE cells under the high glucose condition has not been reported. Objective This study was to investigate the effects of high glucose on the migration and EMT of RPE cells in high glucose culture model in vitro. Methods Human RPE cell line D407 were cultured and passaged in DMEM/F12 medium with 10% fetal bovine serum, and 6-8 generations of cells were used in experiment.The cells were divided into 3 groups based on different glucose concentrations in medium.The glucose at the final concentration 5.5 mmol/L or 60.0 mmol/L was respectively used in the normal control group or high glucose group, and the DMEM with 5.5 mmol/L glucose and mannitol was used in the hypertonic control group.The migration rate of the cells were detected 0, 24, 48 and 72 hours after scratching by wound-scratch test.Real-time PCR was used to detect the relative expressions of zonula occludens-1 (ZO-1) and α-smooth muscle actin (α-SMA) in the cells. Results Cultured cells showed a polygon shape with the clear nucleolus and dense arrangement in the normal control group and the hypertonic control group, but the cells were larger and elongated with the lapse of culture time with the indistinct structure and loose arrangement.At 48 hours after scratching, migrating cells were seen in the scratching area, and the scratching area disappeared at 72 hours after scratching in the high glucose group, but the scratching area still was existed in the normal control group or hypertonic control group.The migrating rate of the cells was higher in the high glucose group than that in the normal control group or hypertonic control group, showing total differences among 3 groups and various time points (Fgroup =328.600, P=0.000;Ftime=773.270, P=0.000).Compared with the normal control group, the expression level of ZO-1 mRNA was significantly lower, and α-SMA mRNA level was higher 48 hours and 72 hours in the high glucose group than those in the normal control group (all at P<0.05). Conclusions High glucose induce the migration and EMT of RPE cells in vitro, which may be associated with the pathogenesis of proliferative diabetic retinopathy. Key words: Human; Retinal pigment epithelium cell; High glucose; Epithelial-mesenchymal transition; Diabetes mellitus/complication, retinopathy; Cell culture

The effect of blue light on human retinal pigment epithelium cells α1D subunit protein expression and vascular endothelial growth factor and basic fibroblast growth factor secretion in vitro

To investigate the effect of blue light on human retinal pigment epithelium (RPE) α1D subunit protein expression and its relationship with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) secretion in vitro. The fourth generation cultured human RPE cells in vitro were randomly divided into 4 groups, group A (control), group B (exposure to blue light), group C (exposure to blue light+nifedipine), group D [exposure to blue light+(-)Bay K8644]. Cells were exposed to blue light (2 000 ± 500) lx for 6 hours, and cells culture completed 24 hours later. VEGF and bFGF concentration were assayed by enzyme linked immunosorbent assay (ELISA). Real-time polymerase chain reaction was used to analysis L-type calcium channel α1D subunit mRNA expression. Western blot was used to examine the protein expression of L-type calcium channel α1D subunit. Analysis of variance was used to compare the difference of α1D subunit mRNA and protein expression, VEGF and bFGF concentration between groups. Correlation analysis was used to show the relationship between α1D subunit protein expression and concentration of VEGF and bFGF. (1) There is significant statistically difference in the population mean of VEGF and bFGF concentration in four groups (F = 99.441, 21.310, P = 0.000,0.000) . VEGF and bFGF concentration in group B (3 281.51 ± 251.73, 1 346.81 ± 62.27) and group D (3 808.01 ± 94.01, 1 485.82 ± 108.97) was higher than it was in group A (2 401.09 ± 228.07, 1 232.42 ± 65.41) , which was statistically different (P = 0.000, 0.000, 0.019, 0.000). And it was higher in group D (3 808.01 ± 94.01, 1 485.82 ± 108.97) compared with group B (3 281.51 ± 251.73, 1 346.81 ± 62.27) (P = 0.000, 0.006). While, it was lower in group C (1 927.28 ± 143.11, 1 149.39 ± 62.99) than it was in group B (3 281.51 ± 251.73, 1 346.81 ± 62.27) (P = 0.000, 0.000) . (2) The mean of mRNA expression of α1D subunit between four groups was statistically significant (F = 50.320, P = 0.000) . It was higher in group B (210 ± 23) , group D (478 ± 80) and group C (232 ± 14) than group A (100 ± 20). It was statistically significant different (P = 0.023, 0.006, 0.010). And it was higher in group D (478 ± 80) than group B (210 ± 23) and C (232 ± 14) (P = 0.032, 0.039). (3) There was statistically significant difference in the expression of L-type calcium channel α1D subunit protein in four groups (F = 1 940.363, P = 0.000) . It was significantly higher (P = 0.000,0.000) in group B (0.974 2 ± 0.014 7) and group D (0.654 9 ± 0.005 0) than group A (0.503 2 ± 0.007 5). And it was higher in group D (0.654 9 ± 0.005 0) than it was in group C (0.413 9 ± 0.008 8) (P = 0.000). (4) There was positive correlation between the L-type calcium channel α1D subunit protein expression and VEGF concentration secreted by retinal pigment epithelium cells (r = 0.674, F = 8.333, P = 0.016), but there was no correlation with bFGF concentration (r = 0.537, F = 4.061, P = 0.072). L-type calcium channel α1D subunit may be involved in blue light induced damage on human retinal pigment epithelial cells. Blue light exposure can induce the mRNA and protein expression of α1D subunit, VEGF and bFGF concentration in retinal pigment epithelium cells Increased. And there was a positive correlation between α1D subunit protein expression and the VEGF concentration.

Characteristic of ocular safety profile of triamcinolone made in China

To characterize the safety profile of triamcinolone acetonide made in China (Transton) and triamcinolone acetonide acetate (Tongyong) for their ocular application. Experimental study. In vitro cell viability assay was performed on 3 types of human ocular cells to evaluate the cytotoxicity of the simulated vitreal concentrations (from a 1:15 dilution as if injected into 1.5 ml of rabbit vitreous to 1:50 dilution as if injected into 5 ml of human vitreous) of Transton and Tongyong using MTT method.In vivo 28 guinea pigs, randomly divided into four groups, were used for evaluating either intravitreal 6 µl of the two types of triamcinolone suspension or 18 µl of their supernatant. Following the injections, the eyes were monitored by biomicroscopy, ophthalmoscopy, tonometry, electroretinography, and histology. The Dunnett's test was used to analyze in vitro cell viability. Paired sample t-test and Generalized Estimating Equations (GEE) were respectively used to compare electroretinography data and intraocular pressure between experimental eyes and the control eyes. The undiluted supernatant of Transton and Tongyong was toxic to human scleral fibroblasts when compared with their control groups (MTT values = 0.046 ± 0.036 and 0.044 ± 0.05 versus 0.367 ± 0.106 and 0.413 ± 0.128) (P < 0.01) or with the BSS group (0.368 ± 0.106 and 0.441 ± 0.137) (P < 0.01). 1:15 or greater dilution of supernatant of Transton and Tongyong did not show cytotoxicity on cultured human retinal pigment epithelium cells or Müller cell (P > 0.05), but 1:15 dilution of Tongyong supernatant showed cytotoxicity on the Müller cells (MTT value = 0.366 ± 0.062 versus 0.417 ± 0.042 for BSS) (P = 0.03). In vivo, neither intravitreal 6 µl (0.25 mg, equivalent to 4 mg in 0.1 ml for human eyes) of the suspension nor 18 µl (equivalent resultant preservative concentration to intravitreal 4 mg in 0.1 ml for rabbit eyes) of the supernatant of Transton and Tongyong showed ocular toxicity. The equivalent dose (6 µl) to 4 mg in 0.1 ml intravitreal suspension for human eye or equivalent resultant preservative concentration to 0.1 ml intravitreal suspension for rabbit eye is found safe in guinea pig eyes.