Physalin A Restores Redox and Mitochondrial Homeostasis to Protect against Diabetic Retinopathy.

Objective To investigate the effects of leech percolate on cell division cycle of culturedhuman retinal pigment epithelium (hRPE)cells induced by thrombin.Exploring mechanism of leech percolateon preventing and curing PVR.Methods (1)Human retinal pigment epithelium(hRPE)cells were cultured invitro with digest culture method. (2)The selected concentrations of leech percolate (128mg/ml,64mg/ml,32mg/ml,16mg/ml and 8mg/ml)were based on the previous experiments.With or without 0.5NIHU/mlthrombin, hRPE cells were treated with leech percolate by various concentrations for 24hrs, and the effects ofcell division cycle of leech percolate were studied with Flow Cytometer (FCM). (3)With or without0.5NIHU/ml thrombin, hRPE cells were treated with 64mg/ml leech percolate at different times(6hr, 12hr,24hrand 48hr), and the effects of cell division cycle of leech percolate were treated with FCM.Results (1)Therewas a general tendency that leech percolate of the selected concentrations arrested hRPE cells on G0/G1 phaseand 64mg/mi leech percolate inhibited 77.9% hRPE cells on G0/G1 phase and made hRPE cells on S phaseless especially. (2)During the period of 6hrs and 48hrs the longer the treat time of 64mg/ml leech percolate, themore the RPE cells arrested on G0/G1 phase.Conclusion The results approve leech percolate can inhibitproliferation of human RPE cells cultured in vitro.Its mechanism may be that leech percolate could act on celldivision cycle and arrest RPE cells on G0/G1 phase to make proliferation power of hRPE cells lower. Key words: Human retinal pigment epithelium cells; Leech percolate; Thrombin; Flow Cytometer; Cell division cycle

Aims/hypothesisDiabetic macular edema represents the main cause of visual loss in diabetic retinopathy. Besides inner blood retinal barrier breakdown, the role of the outer blood retinal barrier breakdown has been poorly analyzed. We characterized the structural and molecular alterations of the outer blood retinal barrier during the time course of diabetes, focusing on PKCζ, a critical protein for tight junction assembly, known to be overactivated by hyperglycemia.MethodsStudies were conducted on a type2 diabetes Goto-Kakizaki rat model. PKCζ level and subcellular localization were assessed by immunoblotting and immunohistochemistry. Cell death was detected by TUNEL assays. PKCζ level on specific layers was assessed by laser microdissection followed by Western blotting. The functional role of PKCζ was then evaluated in vivo, using intraocular administration of its specific inhibitor.ResultsPKCζ was localized in tight junction protein complexes of the retinal pigment epithelium and in photoreceptors inner segments. Strikingly, in outer segment PKCζ staining was restricted to cone photoreceptors. Short-term hyperglycemia induced activation and delocalization of PKCζ from both retinal pigment epithelium junctions and cone outer segment. Outer blood retinal barrier disruption and photoreceptor cone degeneration characterized long-term hyperglycemia. In vivo, reduction of PKCζ overactivation using a specific inhibitor, restored its tight-junction localization and not only improved the outer blood retinal barrier, but also reduced photoreceptor cell-death.ConclusionsIn the retina, hyperglycemia induced overactivation of PKCζ is associated with outer blood retinal barrier breakdown and photoreceptor degeneration. In vivo, short-term inhibition of PKCζ restores the outer barrier structure and reduces photoreceptor cell death, identifying PKCζ as a potential target for early and underestimated diabetes-induced retinal pathology.

To investigate relationship of the expression of MERTK gene and the activity of protein kinase C (PKC) in the phagocytic process of human retinal pigment epithelial (hRPE) cells. Cultured hRPE cells were incubated with rod outer segments (ROS) suspension (containing ROS 1x10(10)/L) at 37 degrees C, then cells were rinsed at different times to terminate the phagocytosis. The kinetic of phagocytosis was measured by double-fluorescent labeling. The activity of PKC and the expression level of MERTK gene were measured by counting gamma-32P radio-activity with liquid scintillation and RT-PCR respectively. Change of MERTK gene expression was measured after hRPE was treated cells with stimulator or inhibitor of PKC. Statistical analysis was performed by SPSS 13.0 software. The phagocytic assay showed that the quality of bound and ingested ROS by hRPE cells increased. The quality of ingested ROS by hRPE cells at 24 hours was (2.85+/-0.11)x10(6), which reached maximum contrast with control (0.00+/-0.00)x10(6) (t=47.64, P<0.05). The activity of PKC (both in cytoplasm and on membrane) decreased during all the incubation periods compared with control [cytoplasm: (329.63+/-14.26) nmolxg(-1)xmin(-1)and on membrane: (467.67+/-68.87) nmolxg(-1)xmin(-1)], and reached the minimum at 24 h[cytoplasm: (151.13+/-17.67) nmolxg(-1)xmin(-1) and on membrane: (152.45+/-64.83) nmolxg(-1)xmin(-1); cytoplasm t=89.66 and membrane t=10.31, P<0.05]. The level of MERTK mRNA increased in pulse-chase and long-time incubation test. The gray level for 90 min was 1.8853+/-0.0077, contrasted with control 0.7246+/-0.0062, F=16,060.2167 and P<0.05. The gray level for 24 h was 0.5946+/-0.0082, contrasted with control 0.3343+/-0.0064, F=919.8421 and P<0.05. When up-regulating the activity of PKC in hRPE cells, the level of MERTK mRNA was decreased in the proceeding incubating with ROS contrasted with control (pulse-chase group F=17,142.2331, long time group F=1886.4614; P<0.05). After down-regulating the activity of PKC in hRPE cells, the level of MERTK mRNA waved between 4.4670+/-0.0092 and 5.7034+/-0.0095 in the first 30 min of incubating with ROS, which lower than control 0.9117+/-0.0021 (F=199,012.9138, P<0.05). The lower activity of PKC and the higher expression MERTK gene are very important for sustaining phagocytic process of ROS by hRPE cells. MERTK gene and PKC both as up-stream regulators are negative-correlated in the phagocytic process of hRPE.

To observe the internalization of A2E by human retinal pigmented epithelial (hRPE) cells and study whether the lipofuscin fluorophore A2E (N-retinylidene-N-retinylethanolamine) participates in blue light-induced damage to hRPE cells. A mixture of all-trans-retinal and ethanolamine was used to produce A2E in one step. A2E granules were delivered to medium of cultured hRPE cells for internalization. Confluent cultures were subsequently exposed to 450 nm (blue) light for 20 minutes with or without A2E (25, 50, 100 micromol/L). The light intensity was 70 mW/mm(2). Phototoxicity was quantified at 12, 24, 36, and 48 h after exposure by CCK-8 of viable cells. Apoptosis of cells was detected by Hoechst 33342 DNA staining and flow cytometry. The reaction of all-trans-retinal (100 mg) and ethanolamine (9.5 mg) produced 53.8 mg A2E in one step. When A2E was delivered to hRPE cells in culture, it accumulated intracellularly. Internalized A2E presented as autofluorescent granules having a perinuclear distribution. As shown by CCK-8 analysis, the A2E-fed hRPE cell viability reduced with duration after 450 nm light exposure. Conversely, blue light-exposed hRPE cells that did not contain A2E showed less loss of cell viability. The percentage of hRPE cell apoptosis with 25 micromol/L A2E 12, 24, 36 and 48 h after blue light exposure was (12.11 +/- 2.32)%, (31.21 +/- 3.72)%, (64.23 +/- 3.53)% and (58.71 +/- 3.48)% respectively. Conversely, the apoptosis was less than 5% in other hRPE cells. A2E is essential to blue light-induced hRPE cell damage. Only blue light exposure and without A2E lead to little cell injury. hRPE cells in old people which contain much lipofuscin are sensitive to blue light injury.

Diabetic macular edema (DME) is a common complication leading to a central vision loss in patients with diabetes. In this eye pathology, the hyperglycaemic/hypoxic microenvironment of pigmented epithelium is responsible for outer blood retinal barrier integrity changes. More recently, we have shown that a small peptide derived from the activity-dependent neuroprotective protein (ADNP), known as NAP, counteracts damages occurring during progression of diabetic retinopathy by modulating HIFs/VEGF pathway. Here, we have investigated for the first time the role of this peptide on outer blood retinal barrier (BRB) integrity exposed to hyperglycaemic/hypoxic insult mimicking a model in vitro of DME. To characterize NAP role on disease's pathogenesis, we have analyzed its effect on HIFs/VEGF system in human retinal pigmented epithelial cells, ARPE-19, grown in high glucose and low oxygen tension. The results have shown that NAP prevents outer BRB breakdown by reducing HIF1α/HIF2α, VEGF/VEGFRs, and increasing HIF3α expression, moreover it is able to reduce the percentage of apoptotic cells by modulating the expression of two death related genes, BAX and Bcl2. Further investigations are needed to determine the possible use of NAP in DME treatment.

Visual perception begins with the absorption of a photon by an opsin pigment, inducing isomerization of its 11-cis-retinaldehyde chromophore. After a brief period of activation, the resulting all-trans-retinaldehyde dissociates from the opsin apoprotein rendering it insensitive to light. Restoring light sensitivity to apo-opsin requires thermal re-isomerization of all-trans-retinaldehyde to 11-cis-retinaldehyde via an enzyme pathway called the visual cycle in retinal pigment epithelial (RPE) cells. Vertebrates can see over a 10(8)-fold range of background illumination. This implies that the visual cycle can regenerate a visual chromophore over a similarly broad range. However, nothing is known about how the visual cycle is regulated. Here we show that RPE cells, functionally or physically separated from photoreceptors, respond to light by mobilizing all-trans-retinyl esters. These retinyl esters are substrates for the retinoid isomerase and hence critical for regenerating visual chromophore. We show in knock-out mice and by RNA interference in human RPE cells that this mobilization is mediated by a protein called "RPE-retinal G protein receptor" (RGR) opsin. These data establish that RPE cells are intrinsically sensitive to light. Finally, we show that in the dark, RGR-opsin inhibits lecithin:retinol acyltransferase and all-trans-retinyl ester hydrolase in vitro and that this inhibition is released upon exposure to light. The results of this study suggest that RGR-opsin mediates light-dependent translocation of all-trans-retinyl esters from a storage pool in lipid droplets to an "isomerase pool" in membranes of the endoplasmic reticulum. This translocation permits insoluble all-trans-retinyl esters to be utilized as substrate for the synthesis of a new visual chromophore.

The aim of the current study was to explore the potential of TREK-TRAAK two-pore domain potassium (K2P) channels in protecting human retinal pigment epithelium (hRPE) cells against oxidative stress. hRPE cells were obtained from donors, and then cell identification and detection of the expression levels of TREK-TRAAK K2P channels in hRPE cells were conducted. Subsequently, tert-butyl hydroperoxide (t-BH) was used to induce oxidative stress in hRPE cells. Docosahexaenoic acid (DHA) was used to stimulate and fluoxetine was used to inhibit the TREK-TRAAK K2P channels. The survival rates of hRPE cells under oxidative stress were examined using flow cytometry. Apoptosis-associated factors, including Bax, Bcl-2, cleaved-caspase-3, αB-crystallin and their mRNAs, were examined using immunofluorescence, western blot and reverse transcription-polymerase chain reaction analyses. Variations in the cytoarchitecture were observed by immunofluorescence and electron microscopy. The cells examined in the present study were identified as hRPE cells. All members in the TREK-TRAAK K2P channel family (including TREK-1, TREK-2 and TRAAK) were found to be expressed in hRPE cells. Stimulation of TREK-TRAAK K2P channels increased the survival rates of hRPE cells under oxidative stress and the levels of intracellular protective factors, such as Bcl-2 and αB-crystallin. By contrast, inhibition of these channels decreased the cell survival rates and increased apoptosis enhancing factors, such as Bax and cleaved-caspase-3. Further examination of the cytoarchitecture revealed that TREK-TRAAK K2P channels protected the integrity of the hRPE cell structure against oxidative stress. In conclusion, the present study suggested that the activated TREK-TRAAK K2P channels serve a role in protecting hRPE cells against the oxidative stress induced by t-BH, which indicated that these K2P channels are potential novel targets in retinal protection and provided a new direction for research and therapy in retinal degeneration diseases.

BackgroundLow-level red light (LLRL) irradiation may inhibit myopia occurrence and progression. Understanding how LLRL inhibits fibrosis in human retinal pigment epithelial (hRPE) cells is critical to inhibiting myopia progression and developing novel therapeutic strategies. Here, we explored the effects of LLRL on hRPE cells in a myopia-simulating hypoxic microenvironment and elucidated the mechanisms through which it inhibits scleral remodeling.MethodsWe first used the MTT assay to analyze hRPE cell proliferation under hypoxic conditions after LLRL irradiation at varying frequencies over different durations. RNA sequencing was used to screen for key signaling molecules leading to hRPE cell fibrosis. Western blotting, reverse transcription quantitative polymerase chain reaction, and immunofluorescence assay were used to detect the role of ubiquitin binding enzyme E2 C (UBE2C) in hRPE cell fibrosis under LLRL irradiation.ResultsLLRL was noted to regulate the extracellular matrix, inhibiting fibrosis in hypoxic hRPE cells. Moreover, supernatant of LLRL-treated hypoxic hRPE cells inhibited scleral remodeling in human scleral fibroblasts. Mechanistically, LLRL inhibited cell fibrosis by regulating UBE2C activation of the AKT/mTOR pathway.ConclusionIn a hypoxic environment, LLRL irradiation can prevent fibroblast transformation in hRPE cells, indicating its potential in scleral remodeling inhibition. Our results revealed the molecular mechanism through which red light controls myopia and provide evidence for further basic and clinical research.Graphical abstract

目的 探讨以腺病毒为载体对体外培养的人视网膜色素上皮(human retinal pigment epithelial,hRPE)细胞进行人内皮抑素(human endostatin,hES)感染,获得高效表达hES转基因细胞的可行性.方法 常规培养hRPE细胞,传代至第3～第5代后,应用携带有绿色荧光蛋白(green fluores-cent protein,GFP)的hES重组腺病毒按感染复数30进行感染.培养24 h后,在荧光显微镜下计数培养的hRPE细胞的GFP表达阳性率;用免疫组织化学法观察hES在已感染hRPE细胞中的表达情况;提取hRPE细胞总RNA,采用RT-PCR技术对产物行琼脂糖凝胶电泳;用Western blot法检测感染hRPE细胞培养上清液中hES的表达水平.结果 hES重组腺病毒感染后,hRPE细胞形态正常.感染后24 h,GFP即呈100%表达,弥漫整个胞质;免疫组织化学法观察可见,hRPE细胞浆内呈棕黄色的阳性反应,而在感染空载腺病毒的对照组中细胞胞浆无染色;RT-PCR反应可见,细胞裂解产物中有hES阳性条带;Western blot法检测结果 表明,培养上清液中有hES蛋白的表达.结论 hES重组腺病毒载体能有效感染体外培养的hRPE细胞,并稳定表达hES蛋白。

Matrigel and Activin A promote cell-cell contact and anti-apoptotic activity in cultured human retinal pigment epithelium cells

To explore the effect of hypoxia inducible factor-1 alpha (HIF-1 alpha) gene on the expression of vascular endothelial growth factor (VEGF) in human retinal pigment epithelial (hRPE) cells under hypoxia conditions by using small hairpin loop RNA (shRNA) to silence HIF-1 alpha. CoCl(2) (150 micromol/L) was used to simulate the hypoxia environment for hRPE cells. After choosing a target site of HIF-1 alpha mRNA, shRNA was designed and synthesized by this target site. hRPE cells were transfected by this shRNA in vitro. Then, these cells were cultured under hypoxia conditions (150 micromol/L CoCl(2)). The mRNA expression of HIF-1 alpha and VEGF was measured by semi-quantitative reverse transcription PCR (RT-PCR). The protein level of HIF-1 alpha and VEGF was studied by western blot analysis. After hRPE cells were transfected by HIF-1 alpha-specific shRNA, RT-PCR showed that the expression of HIF-1 alpha mRNA was inhibited by 77.1%, and western blot analysis showed that the level of HIF-1 alpha protein was significantly decreased in hRPE cells under hypoxia conditions. Moreover, the expression of VEGF mRNA was inhibited by 27.8% and the level of VEGF protein was also significantly decreased in transfected hRPE cells under hypoxia conditions. Under hypoxia conditions, HIF-1 alpha-specific shRNA effectively keeps HIF-1 alpha gene silenced, and consequently down-regulates VEGF expression against hypoxia. These results suggest that HIF-1 alpha is one of the most important cytokines for retinal neovascularization.

Ocular toxoplasmosis is mediated by monocytes infected with Toxoplasma gondii that are disseminated to target organs. Although infected monocytes can easily access to outer blood-retinal barrier due to leaky choroidal vasculatures, not much is known about the effect of T. gondii-infected monocytes on outer blood-retinal barrier. We prepared human monocytes, THP-1, infected with T. gondii and human retinal pigment epithelial cells, ARPE-19, grown on transwells as an in vitro model of outer blood-retinal barrier. Exposure to infected monocytes resulted in disruption of tight junction protein, ZO-1, and decrease in transepithelial electrical resistance of retinal pigment epithelium. Supernatants alone separated from infected monocytes also decreased transepithelial electrical resistance and disrupted tight junction protein. Further investigation revealed that the supernatants could activate focal adhesion kinase (FAK) signaling in retinal pigment epithelium and the disruption was attenuated by FAK inhibitor. The disrupted barrier was partly restored by blocking CXCL8, a FAK activating factor secreted by infected monocytes. In this study, we demonstrated that monocytes infected with T. gondii can disrupt outer blood-retinal barrier, which is mediated by paracrinely activated FAK signaling. FAK signaling can be a target of therapeutic approach to prevent negative influence of infected monocytes on outer blood-retinal barrier.

To investigate the expressions of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) of cultured human retinal pigment epithelial (hRPE) cells under mechanical stress in vitro, so as to mimic and understand the role of these 2 inflammatory cytokines in the early stage of development of primary retinal detachment. Ferric oxide beads coated with collagen were added to the culture plate containing wall-attaching hRPE cells and then the cells were incubated and washed to remove the unbound beads. A magnet was put to the culture plate to provide vertical magnetic force. Cytochalasin D (CD) was added to the culture fluid to inhibit the phagocytizing, secreting, and moving functions of the hRPE. Before the experiment and 15 min, 0.5 h, 1 h, 4 h, and 8 h the beginning of experiment, the MCP-1 mRNA and IL-8 mRNA in the hRPE cells were measured with reverse transcriptase polymerse chain reaction (RT-PCR), and the MCP-1 and IL-8 protein expression in the supernatants was tested with enzyme-linked immunosorbent assay (ELISA) kits. MCP-1 and IL-8 mRNA were expressed at very low levels in the RPE cells and supernatant not exposed to magnetic force. After exposure to magnetic force, both MCP-1 and IL-8 mRNA demonstrated "double peaks" expression. Their first peak levels appeared within 0.5 h, being 3.30 ng/L +/- 0.12 ng/L and 1.88 ng/L +/- 0.08 ng/L respectively. The first peaks of MCP-1 and IL-8 protein expression were within 1 h, being 552.05 ng/L +/- 7.64 ng/Land 236.67 ng/L +/- 14.30 ng/L respectively, and the second peaks appeared about 4 hours later. After cytochalasin D pretreated, the expression and secretion of both MCP-1 and IL-8 of the hRPE cells were significantly decreased to the levels of 2.36 ng/L +/- 0.27 ng/L and 1.64 ng/L +/- 0.08 ng/L, and 353.80 ng/L +/- 16.68 ng/L and 101.86 ng/L +/- 15.92 ng/L respectively. Mechanical stress induces the RPE cells to express MCP-1 and IL-8, and this effect was inhibited in part by pretreatment of CD, indicating that the cytoskeleton may be involved in the effect and that these two inflammatory cytokines take a part in the early stage of development of primary retinal detachment.

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

Distinct CD40L receptors mediate inflammasome activation and secretion of IL-1β and MCP-1 in cultured human retinal pigment epithelial cells