Exosomes mediate an epithelial‐mesenchymal transition cascade in retinal pigment epithelial cells: Implications for proliferative vitreoretinopathy

Exosomes have recently emerged as a pivotal mediator of many physiological and pathological processes. However, the role of exosomes in proliferative vitreoretinopathy (PVR) has not been reported. In this study, we aimed to investigate the role of exosomes in PVR. Transforming growth factor beta 2 (TGFß‐2) was used to induce epithelial‐mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells, as an in vitro model of PVR. Exosomes from normal and EMTed RPE cells were extracted and identified. We incubated extracted exosomes with recipient RPE cells, and co‐cultured EMTed RPE cells and recipient RPE cells in the presence of the exosome inhibitor GW4869. Both experiments suggested that there are further EMT‐promoting effects of exosomes from EMTed RPE cells. MicroRNA sequencing was also performed to identify the miRNA profiles in exosomes from both groups. We identified 34 differentially expressed exosomal miRNAs (P <. 05). Importantly, miR‐543 was found in exosomes from EMTed RPE cells, and miR‐543‐enriched exosomes significantly induced the EMT of recipient RPE cells. Our study demonstrates that exosomal miRNA is differentially expressed in RPE cells during EMT and that these exosomal miRNAs may play pivotal roles in EMT induction. Our results highlight the importance of exosomes as cellular communicators within the microenvironment of PVR.     

KRT8 phosphorylation regulates the epithelial-mesenchymal transition in retinal pigment epithelial cells through autophagy modulation

Proliferative vitreoretinopathy (PVR) is a severe ocular disease which results in complex retinal detachment and irreversible vision loss. The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is considered to be critical in the pathogenesis of PVR. In this study, we focused on the potential impact of keratin 8 (KRT8) phosphorylation and autophagy on TGF-β2–induced EMT of RPE cells and explored the relationship between them. Using immunofluorescence and Western blot analysis, the co-localization of KRT8 and autophagy marker, as well as the abundance of phosphorylated KRT8 (p-KRT8) expression, was observed within subretinal and epiretinal membranes from PVR patients. Moreover, during TGF-β2–induced EMT process, we found that p-KRT8 was enhanced in RPE cells, which accompanied by an increase in autophagic flux. Inhibition of autophagy with pharmacological inhibitors or specific siRNAs was associated with a reduction in cell migration and the synthesis of several EMT markers. In the meantime, we demonstrated that p-KRT8 was correlated with the autophagy progression during the EMT of RPE cells. Knockdown the expression or mutagenesis of the critical phosphorylated site of KRT8 would induce autophagy impairment, through affecting the fusion of autophagosomes and lysosomes. Therefore, this study may provide a new insight into the pathogenesis of PVR and suggests the potential therapeutic value of p-KRT8 in the prevention and treatment of PVR.     

Yes-associated protein is essential for proliferative vitreoretinopathy development via the epithelial-mesenchymal transition in retinal pigment epithelial fibrosis

This study was aim to investigate whether the progression of proliferative vitreoretinopathy (PVR) depended on the activation of Yes-associated protein (YAP) and the subsequent epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cell. The effect of YAP activation on retinal fibrosis in a PVR mouse model and in human ARPE-19 cells in vitro was studied. After treated with transforming growth factor-β2(TGF-β2), the expressions of fibrogenic molecules, YAP activation and the TGF-β2-Smad signalling pathway in ARPE-19 cells were detected by Western blot and immunocytochemical analyses. The effect of YAP on change in fibrosis and EMT was tested by knockdown experiment using verteporfin (YAP inhibitor). YAP was upregulated in the PVR mouse model and during TGF-β2–induced RPE cell EMT. In an in vivo study, verteporfin attenuated PVR progression in a mouse model. Additionally, YAP knockdown retained phenotype of RPE cells and ameliorated TGF-β2–induced migration, gel contraction and EMT in vitro. YAP knockdown inhibited the TGF-β2–induced upregulation of connective tissue growth factor (CTGF), smooth muscle actin (SMA-α) and fibronectin. YAP was essential for the TGF-β2–induced nuclear translocation and phosphorylation of Smad2/3. Our work provides direct evidence that YAP is an essential regulator of EMT and profibrotic responses in PVR and indicates that YAP inhibition could be a potential target in PVR therapeutic intervention.     

Inhibition of miR-21-5p suppresses high glucose-induced proliferation and angiogenesis of human retinal microvascular endothelial cells by the regulation of AKT and ERK pathways via maspin

The aim of the present study is to investigate the role of miR-21-5p in angiogenesis of human retinal microvascular endothelial cells (HRMECs). HRMECs were incubated with 5 mM glucose, 30 mM glucose or 30 mM mannitol for 24 h, 48 h or 72 h. Then, HRMECs exposed to 30 mM glucose were transfected with miR-21-5p inhibitor. We found that high glucose increased the expression of miR-21-5p, VEGF, VEGFR2 and cell proliferation activity. Inhibition of miR-21-5p reduced high glucose-induced proliferation, migration, tube formation of HRMECs, and reversed the decreased expression of maspin as well as the abnormal activation of PI3K/AKT and ERK pathways. Down-regulation of maspin by siRNA significantly increased the activities of PI3K/AKT and ERK pathways. In conclusion, inhibition of miR-21-5p could suppress high glucose-induced proliferation and angiogenesis of HRMECs, and these effects may partly dependent on the regulation of PI3K/AKT and ERK pathways via its target protein maspin.     

视网膜下液基质金属蛋白酶双向电泳分离酶谱鉴定分析

以增生性玻璃体视网膜病变患者视网膜下液为研究对象,结合单向明胶酶谱法和双向电泳分离技术创建了基质金属蛋白酶双向电泳分离酶谱鉴定技术,该技术具有很高的分辨率和灵敏度,重复性好,能够提供蛋白质的相关修饰信息。通过该技术初步发现在增生性玻璃体视网膜病变患者的视网膜下液中存在有4种基质金属蛋白酶,其中2种是由3或4种分子量相同但等电点不同的同分异构体蛋白质组成。     

Advanced glycation end products-induced apoptosis and overexpression of vascular endothelial growth factor in bovine retinal pericytes.

The influence of advanced glycation end products (AGEs) on apoptotic cell death and vascular endothelial growth factor (VEGF) gene expression in cultured bovine retinal pericytes was investigated. When pericytes were incubated with three immunochemically distinct AGEs, which were prepared in vitro by incubating bovine serum albumin with glucose, glyceraldehyde, or glycolaldehyde, apoptotic cell death and DNA ladder formation were significantly induced. The cytopathic effects of glyceraldehyde- or glycolaldehyde-derived AGEs were significantly enhanced in AGE receptor-transfected pericytes. Furthermore, all of these AGEs were found to upregulate the secretory forms of VEGF mRNA levels in retinal pericytes. These results suggest that AGEs disturbed retinal microvascular homeostasis by inducing pericyte apoptosis and VEGF overproduction and thus were involved in the pathogenesis of early phase diabetic retinopathy.     

Connective tissue growth factor is necessary for retinal capillary basal lamina thickening in diabetic mice.

Experimental prevention of basal lamina (BL) thickening of retinal capillaries ameliorates early vascular changes caused by diabetes. Connective tissue growth factor (CTGF) is upregulated early in diabetes in the human retina and is a potent inducer of expression of BL components. We hypothesize that CTGF is causally involved in diabetes-induced BL thickening of retinal capillaries. To test this hypothesis, we compared the effects of streptozotocin (STZ)-induced diabetes on retinal capillary BL thickness between wild-type mice (CTGF+/+) and mice lacking one functional CTGF allele (CTGF+/-). Differences in BL thickness were calculated by quantitative analysis of electron microscopic images of transversally sectioned capillaries in and around the inner nuclear layer of the retina. We show that BL thickening was significant in diabetic CTGF+/+ mice compared with control CTGF+/+ mice, whereas diabetes did not significantly induce BL thickening in CTGF+/- mice. We conclude that CTGF expression is necessary for diabetes-induced BL thickening and suggest that reduction of CTGF levels may be protective against the development of diabetic retinopathy.     

Elevated lipid peroxides induced angiogenesis in proliferative diabetic retinopathy

Oxidative stress is associated with causation of diabetic vascular complications. A case–control study was undertaken to evaluate the association of platelet thiobarbituric acid reacting substances (TBARS) with the severity of diabetic retinopathy for the first time. Platelet TBARS levels were estimated using standard protocol. Platelet TBARS levels in the cases with non-proliferative diabetic retinopathy, proliferative diabetic retinopathy, and healthy controls were 0.56 ± 0.09, 0.69 ± 0.11 and 0.41 ± 0.1 nmol/h/10⁸ platelets, respectively. A significant increase in platelet TBARS levels was observed in the cases as compared to controls (p < 0.001). Elevated TBARS levels were observed to significantly increase further during the proliferative stage of the disease (p < 0.01). The increase in platelet TBARS levels, and thereby at retinal level, is associated with angiogenesis in diabetic retinopathy. Supplemental anti-oxidant therapy in diabetic retinopathy may prevent ocular angiogenesis resulting as a consequence of oxidative stress.     

RhoB antibody alters retinal vascularization in models of murine retinopathy

Neovascularization in cancer or retinopathy is driven by pathological changes that foster abnormal sprouting of endothelial cells. Mouse genetic studies indicate that the stress-induced small GTPase RhoB is dispensable for normal physiology but required for pathogenic angiogenesis. In diabetic retinopathy, retinopathy of prematurity (ROP) or age-related wet macular degeneration (AMD), progressive pathologic anatomic changes and ischemia foster neovascularization are characterized by abnormal sprouting of endothelial cells. This process is driven by the angiogenic growth factor VEGF, which induces and supports the formation of new blood vessels. While injectable biologics targeting VEGF have been used to treat these pathological conditions, many patients respond poorly, prompting interest in other types of mechanism-based therapy. Here we report the preclinical efficacy of a monoclonal antibody that specifically targets RhoB, a signaling molecule that is genetically dispensable for normal physiology but required for pathogenic retinal angiogenesis. In murine models of proliferative retinal angiogenesis or oxygen-induced retinopathy, administering a monoclonal RhoB antibody (7F7) was sufficient to block neoangiogenesis or avascular pathology, respectively. Our findings offer preclinical proof of concept for antibody targeting of RhoB to limit diabetic retinopathy, ROP or wet AMD and perhaps other diseases of neovasculogenesis such as hemangioma or hemangiosarcoma nonresponsive to existing therapies.     

The influence of hyperglycemia on the safety of ultrasound in retinal pigment epithelial cells

Ultrasound (US) assisted drug delivery is receiving interest in treating posterior eye diseases, such as diabetic retinopathy due to its ability to maximize drug penetration into difficult to reach tissues. Despite its promise, the technique has only been investigated using healthy cell and tissue models, with no evidence to date about its safety in active disease. As a result, the aim of this study was to evaluate the safety of US administration in vitro in retinal pigment epithelial cells under normal and high glucose conditions. US protocols within the presently accepted safety threshold were applied and their influence on cell membrane and tight junction integrity as well as intracellular inflammation was evaluated using lactate dehydrogenase (LDH), zona occludens-1 (ZO-1), fluorescein isothiocyanate (FITC)-dextran dye leak and nuclear factor-kappaB (NF-κB) assays, respectively. Under high glucose conditions, US application increased LDH release and resulted in loss of ZO-1 labeling at 2 h; however, normal levels were restored within 24 h. US within its safety parameters did not induce any FITC-dextran dye leak or NF-κB nuclear translocation in normal or high glucose conditions. In conclusion, our results suggest that while high glucose conditions increase cell susceptibility to US-mediated stress, basal conditions can be restored within 24 h without long-lasting cell damage.     

Association between miRNAs expression and signaling pathways of oxidative stress in diabetic retinopathy

Diabetic retinopathy (DR) is a major cause of vision reduction in diabetic patients. Hyperglycemia is a known instigator for the development of DR, even though the role of oxidative stress pathways in the pathogenesis of DR is established. The studies indicate that microRNAs (miRNAs) are significant to the etiology of DR; changes in miRNAs expression levels may be associated with onset and progression of DR. In addition, miRNAs have emerged as a useful disease marker due to their availability and stability in detecting the severity of DR. The relationship between miRNAs expression levels and oxidative stress pathways has been investigated in several studies. The aim of this study is the examination of function and expression levels of target miRNAs in oxidative stress pathway and pathogenesis of diabetic retinopathy.     

Increased serum urea and creatinine levels correlate with decreased retinal nerve fibre layer thickness in diabetic retinopathy

Abstract

Correlation of increased levels of serum urea and creatinine with retinal nerve fibre layer (RNFL) thinning on spectral domain optical coherence tomography (SD-OCT) was studied in diabetic retinopathy (DR). Sixty consecutive cases and 20 healthy controls were included. Cases were divided into three groups: without DR, non-proliferative DR with macular oedema and proliferative DR with oedema. Serum urea and creatinine were measured using a standard protocol. Average (RNFL) was measured using SD-OCT. Increased severity of DR was associated with decrease in levels of serum urea and serum creatinine levels. RNFL thinning correlated positively with increase in serum urea and creatinine levels.     

MiR-455-5p ameliorates HG-induced apoptosis,oxidative stress and inflammatory via targeting SOCS3 in retinal pigment epithelial cells

Diabetic retinopathy (DR) remains the leading cause of blindness in adults with diabetes mellitus. Numerous microRNAs (miRNAs) have been identified to modulate the pathogenesis of DR. The main purpose of this study was to evaluate the potential roles of miR-455-5p in high glucose (HG)-treated retinal pigment epithelial (RPE) cells and underlying mechanisms. Our present investigation discovered that the expression of miR-455-5p was apparently downregulated in ARPE-19 cells stimulated with HG. In addition, forced expression of miR-455-5p markedly enhanced cell viability and restrained HG-induced apoptosis accompanied by decreased BCL2-associated X protein (Bax)/B-cell leukemia/lymphoma 2 (Bcl-2) ratio and expression of apoptotic marker cleaved caspase-3 during HG challenged. Subsequently, augmentation of miR-455-5p remarkably alleviated HG-triggered oxidative stress injury as reflected by decreased the production of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) content as well as NADPH oxidase 4 expression, concomitant with enhanced the activities of superoxide dismutase, catalase, and GPX stimulated with HG. Furthermore, enforced expression of miR-455-5p effectively ameliorated HG-stimulated inflammatory response as exemplified by repressing the secretion of inflammatory cytokines interleukin 1β (IL-1β), IL-6, and tumour necrosis factor-α in ARPE-19 cells challenged by HG. Most importantly, we successfully identified suppressor of cytokine signaling 3 (SOCS3) as a direct target gene of miR-455-5p, and miR-455-5p negatively regulated the expression of SOCS3. Mechanistically, restoration of SOCS3 abrogated the beneficial effects of miR-455-5p on apoptosis, accumulation of ROS, and inflammatory factors production in response to HG. Taken together, these findings demonstrated that miR-455-5p relieved HG-induced damage through repressing apoptosis, oxidant stress, and inflammatory response by targeting SOCS3. The study gives evidence that miR-455-5p may serve as a new potential therapeutic agent for DR treatment.     

Small molecular weight G-protein, H-Ras, and retinal endothelial cell apoptosis in diabetes

We have demonstrated that the expressions of small molecular weight G-protein, H-Ras, and its effector protein, Raf-1, are increased in the retina in diabetes, and the specific inhibitors of Ras function inhibit glucose-induced apoptosis of retinal capillary cells. This study is to examine the contributory roles for H-Ras in glucose-induced apoptosis of retinal endothelial cells by genetic manipulation of functionally active H-Ras levels. Bovine retinal endothelial cells were transfected with the plasmids of either wild type (WT), constitutively active (V12) or dominant-negative (N17) H-Ras. Glucose-induced increase in apoptosis, nitric oxide (NO) levels and activation of NF-κB and caspase-3 were determined in these genetically manipulated cells. Exposure of bovine retinal endothelial cells to 20 mM glucose significantly increased H-Ras activation as determined by Raf-1 binding assay. Overexpression of V12 in the endothelial cells further increased their glucose-induced apoptosis by 40%, NO levels by about 50%, and activated NF-κB and caspase-3 by about 30–40% compared to the untransfected cells incubated in 20 mM glucose. In contrast, overexpression of the inactive mutant, N17, inhibited glucose-mediated increases in apoptotic cell death, NO levels and NF-κB and caspase-3 activation; the values were significantly different (p < 0.02) compared to those obtained from the untransfected cells incubated under similar conditions. Our findings demonstrate that H-Ras activation is important in the activation of the specific signaling events leading to the accelerated retinal capillary cell apoptosis in hyperglycemic conditions, suggesting the possible use of H-Ras inhibitors to inhibit the pathogenesis of diabetic retinopathy.     

[3H]Serotonin binding sites in goldfish retinal membranes

Serotonin (5HT) binding sites were studied in goldfish retinal membranes by radioligand experiments. The binding site of [³H]5HT was sensitive to pre-treatment of the membranes at 40° or 60° C. 5HT and 5-methoxy-N,N-dimethyltryptamine were the best inhibitors of [³H]5HT binding to retinal membranes. The 5HT₂ agonist, 1-(-naphtyl)piperazine, was also a potent inhibitor, however, (+)-1-2,5-dimethoxy-4-iodopheny1-2-aminopropane was less efficient. The catecholaminergic agents haloperidol and clonidine did not display an important inhibition. Propranolol, also reported as 5HT_1B antagonist, was a relatively potent blocker. Monoamine uptake blockers did not show potent inhibition. The GTP analog, GppNHp, inhibited the binding. The iterative analysis of saturation curves revealed two classes of binding sites, a high affinity component (B_max 2.45 pmol/mg of protein, k_d 6.86 nM), and a low affinity component (B_max 53.46 pmol/mg of protein, K_d 232.07 nM). Analysis of the association and dissociation kinetics suggested a binding site (K_d 2 nM). The semilogarithmic plot of the dissociation kinetics gave curves concave to the upper side. The selectivity of the binding and the inhibition by GppNHp suggest the existance of 5HT₁ receptors in goldfish retina. The low affinity interaction probably represents the transporter of 5HT or a suptype of receptor expressed in glial cells.Abbreviations used B _max maximum binding capacity - CPP, 1 (3 chlorophenyl)piperazine - CLN clonidine - DMI desimipramine - DMT 5-methoxy-N,N-dimethyltryptamine - DOI (+)-1-(2,5-dimethoxy-4-iodophenyl-2-aminopropane - DPAT (+)-8-hydroxy-2-(D1-N-propylamino)tetralin - GppNHp 5-guanylylimidodiphosphate - HAL haloperidol - 5HT serotonin - IC₅₀ concentration of drug producing 50% inhibition of binding - IMI imioramine - K_d equilibrium dissociation constant - MIAN mianserin - NOM nomifensin - NP 1-(1-napthyl)piperazine - PRP propranolol In memory of Dr. Boris Druian who died on Dec. 24, 1991.