Small Interfering RNA Targeted to ASPP2 Promotes Progression of Experimental Proliferative Vitreoretinopathy.

Background. Epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) is vital in proliferative vitreoretinopathy (PVR) development. Apoptosis-stimulating proteins of p53 (ASPP2) have recently been reported to participate in EMT. However, the role of ASPP2 in PVR pathogenesis has not been identified. Methods. Immunohistochemistry was used to investigate the expression of ASPP2 in epiretinal membranes of PVR patients. ARPE-19 cells were transfected with ASPP2-siRNA, followed with measurement of cell cytotoxicity, proliferation, and migration ability. EMT markers and related inflammatory and fibrosis cytokines were measured by western blot or flow cytometry. Additionally, PVR rat models were induced by intravitreal injection of ARPE-19 cells transfected with ASPP2-siRNA and evaluated accordingly. Results. Immunofluorescence analysis revealed less intense expression of ASPP2 in PVR membranes. ASPP2 knockdown facilitated the proliferation and migration of RPE cells and enhanced the expression of mesenchymal markers such as alpha smooth muscle actin, fibronectin, and ZEB1. Meanwhile, ASPP2-siRNA increased EMT-related and inflammatory cytokines, including TGF-ß, CTGF, VEGF, TNF-α, and interleukins. PVR severities were more pronounced in the rat models with ASPP2-siRNA treatment. Conclusions. ASPP2 knockdown promoted EMT of ARPE-19 cells in vitro and exacerbated the progression of experimental PVR in vivo, possibly via inflammatory and fibrosis cytokines.

Identification of proteins that interact with alpha A-crystallin using a human proteome microarray.

PURPOSE: To identify proteins interacting with alpha A-crystallin (CRYAA) and to investigate the potential role that these protein interactions play in the function of CRYAA using a human proteome (HuProt) microarray. METHODS: The active full-length CRYAA protein corresponding to amino acids 1-173 of CRYAA was recombined. A HuProt microarray composed of 17,225 human full-length proteins with N-terminal glutathione S-transferase (GST) tags was used to identify protein-protein interactions. The probes were considered detectable when the signal to noise ratio (SNR) was over 1.2. The identified proteins were subjected to subsequent bioinformatics analysis using the DAVID database. RESULTS: The HuProt microarray results showed that the signals of 343 proteins were higher in the recombinant CRYAA group than in the control group. The SNR of 127 proteins was ≥ 1.2. The SNR of the following eight proteins was > 3.0: hematopoietic cell-specific Lyn substrate 1 (HCLS1), Kelch domain-containing 6 (KLHDC6), sarcoglycan delta (SGCD), KIAA1706 protein (KIAA1706), RNA guanylyltransferase and 5'-phosphatase (RNGTT), chromosome 10 open reading frame 57 (C10orf57), chromosome 9 open reading frame 52 (C9orf52), and plasminogen activator, urokinase receptor (PLAUR). The bioinformatics analysis revealed 127 proteins associated with phosphoproteins, alternative splicing, acetylation, DNA binding, the nuclear lumen, ribonucleotide binding, the cell cycle, WD40 repeats, protein transport, transcription factor activity, GTP binding, and cellular response to stress. Functional annotation clustering showed that they belong to cell cycle, organelle or nuclear lumen, protein transport, and DNA binding and repair clusters. CRYAA interacted with these proteins to maintain their solubility and decrease the accumulation of denatured target proteins. The protein-protein interactions may help CRYAA carry out multifaceted functions. CONCLUSIONS: One-hundred and twenty-seven of 17,225 human full-length proteins were identified that interact with CRYAA. The advent of microarray analysis enables a better understanding of the functions of CRYAA as a molecular chaperone.

Polyethylene glycol-modified pigment epithelial-derived factor: new prospects for treatment of retinal neovascularization.

Pathological retinal neovascularization and choroidal neovascularization are major causes of vision loss in a variety of clinical conditions, such as retinopathy of prematurity, age-related macular degeneration, and diabetic retinopathy. Pigment epithelial-derived factor (PEDF) has been found to be the most potent natural, endogenous inhibitor of neovascularization, but its application is restricted because of its instability and short half-life. Polyethylene glycol (PEG) has been used as a drug carrier to slow clearance rate for decades. The present study investigated PEGylated-PEDF for the first time and evaluated its long-term effects on preventing angiogenesis in vitro and in vivo. PEG showed lower cytotoxicity to human umbilical vein endothelial cells (HUVECs). In vitro, PEGylated-PEDF inhibited HUVEC proliferation, migration, tube formation, and vascular endothelium growth factor secretion and induced HUVEC apoptosis in a dose-dependent manner, and it showed a statistically significant difference compared with the PEDF treatment group. In vivo, PEGylated-PEDF had a long-lasting effect in both plasma and retinal concentrations. In an oxygen-induced retinopathy model, one intravitreous injection of PEGylated-PEDF after mouse pups were moved into room air resulted in a significant difference in the inhibition of retinal neovascularization, which decreased the nonperfusion area, compared with the PEDF-treated group. Our present study demonstrated for the first time the long-term inhibitory effects of PEGylated-PEDF on the prevention of neovascularization in vitro and in vivo. These data suggest that PEGylated-PEDF could offer an innovative therapeutic strategy for preventing retinal neovascularization.

An Insertion Variant in CRH Confers an Increased Risk of Central Serous Chorioretinopathy.

Purpose: To identify a novel corticotropin-releasing hormone (CRH) gene variant relevant in patients with central serous chorioretinopathy (CSC). Methods: We performed a genetic study of CSC in families and sporadic cases with controls. Using whole-exome sequencing and linkage analysis, we identified a heterozygous insertion variant, Gln52insPro, in the CRH gene that cosegregated in two Chinese families with CSC. This variant was evaluated among an additional 1307 patients with CSC and 1438 ethnicity-matched control individuals from three independent Chinese cohorts. Results: The CRH variant was strongly associated with CSC in these cohorts of Chinese patients (Pmeta = 1.24 × 10-11; odds ratio, 3.01; 95% confidence interval, 2.15-4.21). The risk variant Gln52insPro decreased CRH gene expression. Conclusions: Our results implicate the hypothalamic-pituitary-adrenal stress response system in the pathogenesis of CSC and provide a novel rationale for therapeutic intervention.

Genetic analysis and clinical features of X-linked retinoschisis in Chinese patients.

Many mutations in the retinoschisis (RS1) gene have been identified, but there are limited clinical data relating to the different genotypes. This study investigated the genotype, clinical phenotype and therapies for X-linked juvenile retinoschisis (XLRS) patients in China to evaluate the effects of gene mutations and therapies on the prognosis of the disease. Thirty patients were recruited in the study. Genetic examination identified 8 novel RS1 gene mutations. Twenty-four patients were identified as missense mutation, which was the most common gene mutation in XLRS patients. Amino acids 102 and 209 were the most common mutation areas, accounting for a total 35.7% of all patients. Mutations affecting amino acid 102 were associated with poor results on the flash electroretinogram (ERG). Sixteen patients had various complications. Anti-vascular endothelial growth factor (VEGF) drugs were given to four patients with hemorrhage or other complications, and serious adverse events did not occur. Our outcome demonstrates that missense mutation was the leading cause of XLRS and more than half of the patients with this missense had various complications. Anti-VEGF drugs may be an effective and safe way to prevent deterioration of XLRS with certain complications. There is wide genotypic and phenotypic variability in Chinese patients with XLRS.

X-Linked Retinoschisis in Juveniles: Follow-Up by Optical Coherence Tomography.

Purpose. To explore the structural progression of X-linked retinoschisis (XLRS) in patients by using spectral-domain optical coherence tomography (SD-OCT). Design. Retrospective, observational study. Methods. Patients who were diagnosed with XLRS by genetic testing underwent comprehensive ophthalmological examinations from December 2014 to October 2016. Each eye was measured by SD-OCT using the same clinical protocol. A correlation between best-corrected visual acuity (VA) and SD-OCT measurements was observed. Results. Six patients demonstrated retinoschisis (12 eyes) and typical foveal cyst-like cavities (10 eyes) on SD-OCT images with a mean logMAR VA of 0.48. The median age was 7.5 years at the initial visit. Their foveal retinal thickness (516.9 µm) and choroid thickness (351.4 µm) decreased at a rate of 38.1 and 7.5 µm, respectively, at the 10.5-month follow-up visit; however, there were no significant differences (P = 0.622 and P = 0.406, resp.). There was no significant correlation between VA, the foveal retinal thickness, and subfoveal choroid thickness. Conclusions. SD-OCT images for XLRS patients during the juvenile period revealed no significant changes in the fundus structure, including the foveal retinal thickness and choroid thickness within one-year follow-up. There was a lack of correlation between VA, foveal retinal thickness, and subfoveal choroid thickness.

Whole-exome sequencing implicates UBE3D in age-related macular degeneration in East Asian populations.

Age-related macular degeneration (AMD) is a leading cause of irreversible central blindness among the elderly worldwide. We use exome sequencing to analyse nonsynonymous single-nucleotide variants (SNVs) across the whole genome of 216 neovascular AMD cases and 1,553 controls. As a follow-up validation, we evaluate 3,772 neovascular AMD cases and 6,942 controls from five independent cohorts in the East Asian population. Here we show strong evidence of an association at a novel, missense SNV, rs7739323, which is located in the ubiquitin protein ligase E3D (UBE3D) gene (Pmeta=1.46 × 10(-9), odds ratio (OR)=0.74, 95% confidence interval (CI): 0.63-0.88). Furthermore, ablation of the UBE3D protein lead to an abnormal amount of pigment granules deposited in retinal pigment epithelium microvilli area and an abnormal response on electroretinography (ERG) in UBE3D(+/-) heterozygous mice. Our findings indicate that the ubiquitin-proteasome system may play a role in the pathogenesis of neovascular AMD.

Placental growth factor expression is reversed by antivascular endothelial growth factor therapy under hypoxic conditions.

BACKGROUND: Clinical trials have revealed that the antivascular endothelial growth factor (VEGF) therapies are effective in retinopathy of prematurity (ROP). But the low level of VEGF was necessary as a survival signal in healthy conditions, and endogenous placental growth factor (PIGF) is redundant for development. The purpose of this study was to elucidate the PIGF expression under hypoxia as well as the influence of anti-VEGF therapy on PIGF. METHODS: CoCl2-induced hypoxic human umbilical vein endothelial cells (HUVECs) were used for an in vitro study, and oxygen-induced retinopathy (OIR) mice models were used for an in vivo study. The expression patterns of PIGF under hypoxic conditions and the influence of anti-VEGF therapy on PIGF were evaluated by quantitative reverse transcription-polymerase chain reaction (RTPCR). The retinal avascular areas and neovascularization (NV) areas of anti-VEGF, anti-PIGF and combination treatments were calculated. Retina PIGF concentration was evaluated by ELISA after treatment. The vasoactive effects of exogenous PIGF on HUVECs were investigated by proliferation and migration studies. RESULTS: PIGF mRNA expression was reduced by hypoxia in OIR mice, in HUVECs under hypoxia and anti-VEGF treatment. However, PIGF expression was reversed by anti-VEGF therapy in the OIR model and in HUVECs under hypoxia. Exogenous PIGF significantly inhibited HUVECs proliferation and migration under normal conditions, but it stimulated cell proliferation and migration under hypoxia. Anti-PIGF treatment was effective for neovascular tufts in OIR mice (P<0.05). CONCLUSION: The finding that PIGF expression is iatrogenically up-regulated by anti-VEGF therapy provides a consideration to combine it with anti-PIGF therapy.

Antiangiogenesis effects of endostatin in retinal neovascularization.

PURPOSE: Pathological retinal angiogenesis is a major cause of vision loss. Endostatin is a natural antiangiogenesis antitumor protein that is widely used in cancer studies. In this study, we investigated the efficacy and potential mechanisms of endostatin for the prevention of retinal neovascularization both in vitro and in vivo. METHODS: Human umbilical vein endothelial cells (HUVECs) were used for the in vitro studies. HUVECs were incubated with endostatin or the vascular endothelial growth factor (VEGF) and endostatin for different time points. Cell proliferation, migration, cell cycling, and tube formation studies were carried out using a Cell Counting Kit-8 assay, a Transwell assay, flow cytometry, and a Matrigel assay, respectively. Enzyme-Linked Immunosorbent Assay (ELISA) was used to study VEGF and pigment epithelial-derived factor (PEDF) protein secretion from the HUVECs at different time points. A murine oxygen-induced retinopathy (OIR) model was used for the in vivo studies. Seven-day-old C57BL/6J pups (p7) were exposed to 75% oxygen for 5 days. On p12, the animals were returned to a normal atmosphere and were immediately injected intravitreously with 1.5 µL of a 5 mg/mL endostatin solution. At p18, the mice were perfused with fluorescein-dextran-FITC, and their retinas were flat mounted to measure the nonperfused area. Retinal VEGF and PEDF levels were also measured by ELISA Kits in the OIR mice at p18. RESULTS: In vitro, endostatin inhibited HUVEC proliferation in a dose-dependent manner and also inhibited HUVEC proliferation in a VEGF-containing medium. Additionally, endostatin can inhibit migration, tube formation, and VEGF secretion in HUVECs, while also inducing apoptosis in HUVECs at several time points. These effects were statistically significant when compared to the control group (P<0.05). In vivo, a single intravitreous injection of endostatin reduced the retinal nonperfused area from 30% in the control group to 23% in the treatment group (P<0.0001). Intravitrous injection of endostatin reduced VEGF levels in retinas, while it increased PEDF levels. CONCLUSIONS: Endostatin showed convincing inhibitory effects on angiogenesis both in vitro and in vivo. The inhibitory effects may be, at least partly, resulted from the restoration of the PEDF/VEGF ratio. These data suggest that endostatin could offer an innovative pharmaceutical strategy for the prevention of retinal neovascularization.