Xanthine derivative KMUP-1 ameliorates retinopathy.

Retinal neovascularization (RNV) and cell apoptosis observed in retinopathy are the most common cause of vision loss worldwide. Increasing vascular endothelial growth factor (VEGF), which was driven by hypoxia or inflammation, would result in RNV. This study investigated the anti-inflammatory and anti-apoptotic xanthine-based derivative KMUP-1 on hypoxia-induced conditions in vitro and in vivo. In the oxygen-induced retinopathy animal model, KMUP-1 mitigated vaso-obliteration and neovascularization. In the cell model of hypoxic endothelium cultured at 1% O2, KMUP-1 inhibited endothelial migration and tube formation and had no cytotoxic effect on cell growth. Upregulation of pro-angiogenic factors, HIF-1α and VEGF, and pro-inflammatory cytokines, IL-1ß and TNF-α, expression in the retinal-derived endothelial cells, RF/6 A cells, upon hypoxia stimulation, was suppressed by KMUP-1 treatment. RF/6 A cells treated with KMUP-1 showed a reduction of PI3K/Akt, ERK, and RhoA/ROCKs signaling pathways and induction of protective pathways such as eNOS and soluble guanylyl cyclase at 1% O2. Furthermore, KMUP-1 decreased the expression of VEGF, ICAM-1, TNF-α, and IL-1ß and increased the BCL-2/BAX ratio in the oxygen-induced retinopathy mouse retina samples. In conclusion, the results of this study suggest that KMUP-1 has potential therapeutic value in retinopathy due to its triple effects on anti-angiogenesis, anti-inflammation, and anti-apoptosis in hypoxic endothelium.

LncRNA TUG1 Promotes Apoptosis, Invasion, and Angiogenesis of Retinal Endothelial Cells in Retinopathy of Prematurity via MiR-145-5p.

Purpose: Retinopathy of prematurity (ROP) is a common retinal vascular disease in premature neonates. In recent years, there is increasing evidence that the long non-coding RNA taurine upregulated gene 1 (TUG1) plays a regulatory role in vascular diseases, suggesting a potential role for TUG1 in vascular endothelial cells. We hypothesized that TUG1 may be associated with ROP. Our aim, therefore, was to explore the biological functions of TUG1 in aberrant retinal development. Methods: We used the mouse oxygen-induced retinopathy (OIR) model to simulate the pathological changes of retinal in ROP. Quantitative real-time polymerase chain reaction was used to detect the expression of TUG1, miR-145-5p and cellular communication network factor 1 (CCN1). Human retinal endothelial cells (HRECs) were treated with CoCl2 to mimic hypoxia conditions. Cellular functional changes were observed after transfection with RNA interference (RNAi)-TUG1 and miR-145-5p mimics. The apoptosis of HRECs was detected by flow cytometry, the migration ability was detected by wound healing and transwell migration assays, and the ability of angiogenesis was detected by tube formation assay. The potential binding sites between TUG1, miR-145-5p, and CCN1 were verified by dual-luciferase reporter assays. The degree of retinopathy was evaluated by staining retinal sections with hematoxylin and eosin, and the expression of CCN1, HIF-1α, VEGF, caspase-3, Bcl-2, IL-1ß, and TNF-α protein was analyzed by Western blotting and immunohistochemistry. Results: In the retina tissue of OIR mice, TUG1, miR-145-5p, and CCN1 were differentially expressed. Knocking down TUG1 attenuated apoptosis, migration, and angiogenesis induced by hypoxia on HRECs, as did miR-145-5p overexpression. Results from reporter assays indicate direct interactions between TUG1, miR-145-5p, and CCN1. Intravitreal injection of miR-145-5p mimics reduced the degree of retinopathy. Conclusion: TUG1 acts as a molecular sponge of miR-145-5p to regulate CCN1 expression and thus regulate the development of retinal neovascularization. This regulatory mechanism may provide a new theoretical basis for the prevention and treatment of ROP.

In-Depth Molecular Characterization of Neovascular Membranes Suggests a Role for Hyalocyte-to-Myofibroblast Transdifferentiation in Proliferative Diabetic Retinopathy.

Background: Retinal neovascularization (RNV) membranes can lead to a tractional retinal detachment, the primary reason for severe vision loss in end-stage disease proliferative diabetic retinopathy (PDR). The aim of this study was to characterize the molecular, cellular and immunological features of RNV in order to unravel potential novel drug treatments for PDR. Methods: A total of 43 patients undergoing vitrectomy for PDR, macular pucker or macular hole (control patients) were included in this study. The surgically removed RNV and epiretinal membranes were analyzed by RNA sequencing, single-cell based Imaging Mass Cytometry and conventional immunohistochemistry. Immune cells of the vitreous body, also known as hyalocytes, were isolated from patients with PDR by flow cytometry, cultivated and characterized by immunohistochemistry. A bioinformatical drug repurposing approach was applied in order to identify novel potential drug options for end-stage diabetic retinopathy disease. Results: The in-depth transcriptional and single-cell protein analysis of diabetic RNV tissue samples revealed an accumulation of endothelial cells, macrophages and myofibroblasts as well as an abundance of secreted ECM proteins such as SPARC, FN1 and several types of collagen in RNV tissue. The immunohistochemical staining of cultivated vitreal hyalocytes from patients with PDR showed that hyalocytes express α-SMA (alpha-smooth muscle actin), a classic myofibroblast marker. According to our drug repurposing analysis, imatinib emerged as a potential immunomodulatory drug option for future treatment of PDR. Conclusion: This study delivers the first in-depth transcriptional and single-cell proteomic characterization of RNV tissue samples. Our data suggest an important role of hyalocyte-to-myofibroblast transdifferentiation in the pathogenesis of diabetic vitreoretinal disease and their modulation as a novel possible clinical approach.

Effect of Probenecid on Endothelial Cell Growth Rate and Retinal Angiogenesis in an Oxygen-Induced Retinopathy Model.

Objectives: Probenecid is an anion transport inhibitor, which, according to the connectivity map (CMap; a biological application database), interferes with hypoxia-induced gene expression changes in retinal vascular endothelial cells (ECs). Here, we investigated the influence of probenecid on retinal EC cytotoxicity and retinal neovascularization in a murine oxygen-induced retinopathy (OIR) model. Methods: The retinal EC growth rate in the presence of hypoxia-mimicking concentrations of cobalt chloride (CoCl2) was determined using the thiazolyl blue tetrazolium bromide (MTT) assay and proliferating cell nuclear antigen (PCNA) expression. In OIR rats, probenecid was administered by intraperitoneal injection (i.p.) from postnatal day (P) 1 to P7. The concentrations of vitreous humor vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF)-1α, and placental growth factor (PlGF) were determined by using the ELISA kit at P21. The amount of newly formed vascular lumen was evaluated by histopathological examination. Retinopathy and neovascularization were assessed by scoring isolectin B4 fluorescein-stained retinal flat mounts. Western blots for liver tissue HIF-1α and hepcidin (HAMP) were performed. Results: In vitro, probenecid led to the recession of the hypoxia-induced EC growth rate. In vivo, compared to the OIR retina, the upregulation of VEGF, HIF-1α, and PlGF in phase II retinopathy of prematurity (ROP) was inhibited by probenecid administration. Moreover, probenecid ameliorated neovascularization and resulted in significantly reduced relative leakage fluorescence signal intensity in fluorescein-stained retinal flat mounts (p < 0.05). Probenecid alleviated the liver overactivation of HAMP and downregulation of HIF-1α in OIR rats. Conclusions: This is the first demonstration that implies that probenecid might be a protective compound against retinal angiogenesis in OIR. These changes are accompanied with decreased hyperoxia-mediated hepcidin overproduction. Although the relevance of the results to ROP needs further research, these findings may help establish potential pharmacological targets based on the CMap database.