Fuxiang Ye

Plasma-activated medium suppresses choroidal neovascularization in mice: a new therapeutic concept for age-related macular degeneration

Choroidal neovascularization (CNV) is the main pathogenesis of age-related macular degeneration (AMD), which leads to severe vision loss in many aged patients in most advanced country. CNV compromises vision via hemorrhage and retinal detachment on account of pathological neovascularization penetrating the retina. Plasma medicine represents the medical application of ionized gas “plasma” that is typically studied in the field of physical science. Here we examined the therapeutic ability of plasma-activated medium (PAM) to suppress CNV. The effect of PAM on vascularization was assessed on the basis of human retinal endothelial cell (HREC) tube formation. In mice, laser photocoagulation was performed to induce CNV (laser-CNV), followed by intravitreal injection of PAM. N-Acetylcysteine was used to examine the role of reactive oxygen species in PAM-induced CNV suppression. Fundus imaging, retinal histology examination, and electroretinography (ERG) were also performed to evaluate PAM-induced retinal toxicity. Interestingly, HREC tube formation and laser-CNV were both reduced by treatment with PAM. N-acetylcysteine only partly neutralized the PAM-induced reduction in laser-CNV. In addition, PAM injection had no effect on regular retinal vessels, nor did it show retinal toxicity in vivo. Our findings indicate the potential of PAM as a novel therapeutic agent for suppressing CNV.

Interleukin-18 Induces Retinal Pigment Epithelium Degeneration in Mice

PURPOSE To examine the effectiveness of interleukin-18 (IL-18) on choroidal neovascularization (CNV) and retinal pigment epithelium (RPE) in humans and mice. METHODS Serum IL-18 levels in patients with wet and dry AMD who were older than 50 years were measured and compared with those of age-matched controls. In mice, laser photocoagulation was performed in the retina to induce experimental CNV, and CNV volume was measured in eyes injected with recombinant IL-18 (rIL-18) and IL-18 neutralizing antibody (nIL-18Ab) compared with those injected with control. Tube formation assay was performed on human retinal endothelial cells (HREC) with rIL-18 administration in vitro. After subretinal injection of rIL-18, fundus change in the injected eyes was evaluated; active caspase-3 level was measured in the RPE/choroid complex, and tight junction integrity in RPE was visualized by zonula occludens-1 (ZO-1) staining. RESULTS Serum IL-18 levels in dry AMD patients were higher than those in control. Mouse rIL-18 or nIL-18Ab did not induce significant change in CNV volume compared with controls or change tube formation in HREC. Subretinal injection of rIL-18 induced retinal degeneration in the mice fundus; ZO-1 staining showed considerably disturbed RPE structure, and active caspase-3 expression was significantly higher after rIL-18 induction. CONCLUSIONS Interleukin-18 did not show a pro- or antiangiogenic effect on mouse laser-induced CNVs (laser-CNVs), whereas it directly induced RPE cell apoptosis in the mouse eye. Our results suggested that IL-18 is associated with dry AMD, but not with wet AMD.

Histamine H4 receptor as a new therapeutic target for choroidal neovascularization in age-related macular degeneration

The present treatment for choroidal neovascularization (CNV) associated with age‐related macular degeneration (AMD) is not sufficient. Hence, we examined the therapeutic efficacy of reducing histamine H4 receptor expression on CNV in mice.

Malondialdehyde induces autophagy dysfunction and VEGF secretion in the retinal pigment epithelium in age-related macular degeneration.

Age-related macular degeneration (AMD) is a major cause of blindness in developed countries and is closely related to oxidative stress, which leads to lipid peroxidation. Malondialdehyde (MDA) is a major byproduct of polyunsaturated fatty acid (PUFA) peroxidation. Increased levels of MDA have been reported in eyes of AMD patients. However, little is known about the direct relationship between MDA and AMD. Here we show the biological importance of MDA in AMD pathogenesis. We first confirmed that MDA levels were significantly increased in eyes of AMD patients. In ARPE-19 cells, a human retinal pigment epithelial cell line, MDA treatment induced vascular endothelial growth factor (VEGF) expression alternation, cell junction disruption, and autophagy dysfunction that was also observed in eyes of AMD patients. The MDA-induced VEGF increase was inhibited by autophagy-lysosomal inhibitors. Intravitreal MDA injection in mice increased laser-induced choroidal neovascularization (laser-CNV) volumes. In a mouse model fed a high-linoleic acid diet for 3 months, we found a significant increase in MDA levels, autophagic activity, and laser-CNV volumes. Our study revealed an important role of MDA, which acts not only as a marker but also as a causative factor of AMD pathogenesis-related autophagy dysfunction. Furthermore, higher dietary intake of linoleic acid promoted CNV progression in mice with increased MDA levels.

Nuclear Factor (Erythroid-Derived)-Related Factor 2-Associated Retinal Pigment Epithelial Cell Protection under Blue Light-Induced Oxidative Stress

Purpose. It is a matter of increasing concern that exposure to light-emitting diodes (LED), particularly blue light (BL), damages retinal cells. This study aimed to investigate the retinal pigment epithelium (RPE) damage caused by BL and to elucidate the role of nuclear factor (erythroid-derived)-related factor 2 (Nrf2) in the pathogenesis of BL-induced RPE damage. Methods. ARPE-19, a human RPE cell line, and mouse primary RPE cells from wild-type and Nrf2 knockout (Nrf2 −/−) mice were cultured under blue LED exposure (intermediate wavelength, 450 nm). Cell death rate and reactive oxygen species (ROS) generation were measured. TUNEL staining was performed to detect apoptosis. Real-time polymerase chain reaction was performed on NRF2 mRNA, and western blotting was performed to detect Nrf2 proteins in the nucleus or cytoplasm of RPE cells. Results. BL exposure increased cell death rate and ROS generation in ARPE-19 cells in a time-dependent manner; cell death was caused by apoptosis. Moreover, BL exposure induced NRF2 mRNA upregulation and Nrf2 nuclear translocation in RPE. Cell death rate was significantly higher in RPE cells from Nrf2 −/− mice than from wild-type mice. Conclusions. The Nrf2 pathway plays an important role in protecting RPE cells against BL-induced oxidative stress.

Role of Caveolin-1 for Blocking the Epithelial-Mesenchymal Transition in Proliferative Vitreoretinopathy.

Purpose Proliferative vitreoretinopathy (PVR) is one of the most severe ocular diseases. Fibrotic changes in retinal cells are considered to be involved in the pathogenesis of PVR. Epithelial-mesenchymal transition (EMT) of RPE cells is one of the main concepts in the pathogenesis of fibrovascular membranes (FVMs) in PVR. In this study, we examined the expression of Caveolin-1 in human FVMs from patients with PVR. We also examined the role of Caveolin-1 in the pathogenesis of PVR. Methods Western blotting, real-time PCR, and immunohistochemistry were performed with human FVMs and mouse eyes with PVR. Cell migration assays were performed to evaluate the involvement of Caveolin-1 in EMT using primary human and mouse RPE cells. Results Caveolin-1 was expressed in human FVMs and upregulated in the mouse eyes with PVR. The alpha-smooth muscle actin (αSMA) expression and migration ability were increased in RPE cells with knockout or knockdown of Caveolin-1, whereas zonula occludens-1 (ZO-1) immunohistochemistry showed reduced morphology and expression of ZO-1. In addition, migration assays showed that Caveolin-1 reduction increased RPE cell migration abilities. Conclusions These results indicated that Caveolin-1 in RPE cells prevents PVR by blocking EMT.

Expression of Vascular Endothelial Growth Factor by Retinal Pigment Epithelial Cells Induced by Amyloid-β Is Depressed by an Endoplasmic Reticulum Stress Inhibitor

Purpose: Amyloid-β (Aβ) is a 36- to 43-amino-acid peptide that is a constituent of drusen, and it has been demonstrated to upregulate vascular endothelial growth factor (VEGF) expression by retinal pigment epithelial (RPE) cells. This study aimed to determine whether 4-phenylbutyl phosphonylacetate (PBA), a known endoplasmic reticulum (ER) stress inhibitor, can reduce Aβ-induced expression of VEGF in RPE cells. Methods: Aβ was added to the medium of regularly cultured or polarized ARPE-19 cells, a human RPE cell line, with or without PBA. The levels of VEGF and ER stress markers, namely GRP78/Bip, cleaved caspases 4 and 12 and GADD153/C-EBP homologous protein, were determined by enzyme-linked immunoassay, immunocytochemistry and Western blotting. Results: Exposure of ARPE-19 cells to Aβ induced GRP78/Bip expression and activated caspases 4 and 12; however, their expression was decreased by simultaneous exposure to PBA. Aβ increased the expression of VEGF both in regularly cultured and polarized ARPE-19 cells, but it was suppressed by PBA. PBA did not cause RPE cell apoptosis. Conclusion: Aβ has been suggested to be involved in the development of age-related macular degeneration; therefore, our findings suggest that drugs that target ER stress should be considered for the treatment of age-related macular degeneration.

Nutritional Supplementation Inhibits the Increase in Serum Malondialdehyde in Patients with Wet Age-Related Macular Degeneration

Purpose. To compare serum levels of malondialdehyde (MDA) in patients with wet age-related macular degeneration (wAMD), patients with dry AMD (dAMD), and patients without AMD and to evaluate the efficacy of nutritional supplementation for treating elevated serum MDA in patients with wAMD. Methods. MDA levels were measured in sera from 20 patients with wAMD, 20 with dAMD, and 24 without AMD. Patients with wAMD were randomized to receive or not receive nutritional supplementation (10 patients in each group), and MDA levels were measured after 3 months of treatment. Results. MDA levels in patients with wAMD were significantly greater compared with patients without AMD. In eyes with wAMD, there was a significant correlation between MDA levels and choroidal neovascularization lesion area. Serum MDA levels decreased in most patients that received supplementation and significantly increased in those who did not. Conclusion. Baseline serum MDA levels were elevated in patients with wAMD, and MDA levels were directly correlated with choroidal neovascularization lesion area. In addition, nutritional supplementation appeared to exert a protective effect against oxidative stress in patients with wAMD.

Increased Ocular Levels of MicroRNA-148a in Cases of Retinal Detachment Promote Epithelial–Mesenchymal Transition

PURPOSE The purpose of this study was to determine microRNA expression in vitreous and subretinal fluid (SRF) samples from patients with retinal detachment (RD). The pathological importance of the identified microRNA transcript levels was analyzed in vitro. METHODS Vitreous fluid was collected from 10 patients with macular hole (MH), vitreomacular traction syndrome (VMTS), or foveoschisis and from 11 patients with RD. Subretinal fluid was collected from 7 patients with RD. Of these, blood serum was collected in 4 patients. MicroRNA microarray profiling was performed to identify microRNA transcripts that were present in vitreous fluid, and more redundantly detected in SRF, of patients with RD, but not detected in control eyes. Western blotting and scratch assays were performed in ARPE-19 cells and primary human RPE cell lines transfected with microRNA to elucidate the effect of identified microRNA transcripts on epithelial-mesenchymal transition (EMT). RESULTS MicroRNA microarray profiling revealed that hsa-miR-148a-3p was the most redundantly detected transcript in SRF and vitreous fluid from patients with RD, but not those with the other diseases. Expression levels of hsa-miR-148a-3p were higher in SRF samples than in blood serum samples in 3 out of 4 patients. Following hsa-miR-148a-3p mimic transfection, ARPE-19 and human RPE cells demonstrated increased expression of α-smooth muscle actin by Western blotting and increased migration ability during scratch assays. CONCLUSIONS The results of the present study indicate that hsa-miR-148a-3p was specifically detected in RD and promotes EMT in RPE.

Correlation between miR-148 Expression in Vitreous and Severity of Rhegmatogenous Retinal Detachment.

Purpose. We had earlier reported positive hsa-miR-148a-3p expression in eyes with rhegmatogenous retinal detachment (RRD) and its involvement in the epithelial-mesenchymal transition of retinal pigment epithelium in vitro. Here we investigated the association of hsa-miR-148a-3p expression levels in the vitreous fluid of patients with RRD with severity of RRD. Methods. The hsa-miR-148a-3p expression levels in the vitreous fluid, range (degree) of retinal detachment (RD), and pixels of retinal break were measured in 27 eyes with RRD. The association of hsa-miR-148a-3p expression levels with other factors was evaluated by multiple regression analysis. Results. The hsa-miR-148a-3p expression levels, time from onset of RRD to vitrectomy, range of RD, and pixels of retinal breaks were 23.68 ± 43.00, 12.07 ± 15.36 days, 155.85 ± 86.67 degrees, and 37000 ± 67100 pixels, respectively. Five eyes with RRD had vitreous hemorrhage preoperatively. The hsa-miR-148a-3p expression levels were significantly associated with pixels of retinal breaks (β = 0.699) and the time from onset of RRD to vitrectomy (β = 0.358) but not with the range of RD or presence of vitreous hemorrhage. Conclusion. The hsa-miR-148a-3p expression levels in the vitreous fluid were significantly associated with the size of retinal break and disease duration.