Retinal Cell Apoptosis Research Articles

Senescence marker protein 30 (SMP30) protects against high glucose-induced apoptosis, oxidative stress and inflammatory response in retinal ganglion cells by enhancing Nrf2 activation via regulation of Akt/GSK-3β pathway

Senescence marker protein 30 (SMP30) is an aging-related protein that participates in the regulation of tissue damage under various pathological conditions. However, the role of SMP30 in mediating high glucose (HG)-induced injury of retinal ganglion cells (RGCs) has not been fully determined. We found that SMP30 expression declined during HG stimulation in RGCs. Cellular functional studies showed that the up-regulation of SMP30 dramatically prohibited HG-evoked apoptosis, oxidative stress and inflammatory response in RGCs. Mechanism research reported that SMP30 overexpression led to the enhancement of nuclear factor erythroid 2-related factor (Nrf2) activation in HG-stimulated RGCs. Moreover, SMP30 overexpression enhanced the phosphorylation of Akt and glucogen synthase kinase-3β (GSK-3β), and the suppression of Akt markedly abolished SMP30-mediated Nrf2 activation in HG-stimulated RGCs. Additionally, the suppression of Nrf2 substantially reversed SMP30-overexpression-induced anti-HG injury effects in RGCs. Overall, these findings suggest that SMP30 protects against HG injury of RGCs by potentiating Nrf2 through regulation of the Akt/GSK-3β pathway. Our work underscores that SMP30/Akt/GSK-3β/Nrf2 may exert a vital role in mediating the injury of RGCs during diabetic retinopathy.

Resveratrol inhibits neural apoptosis and regulates RAX/P-PKR expression in retina of diabetic rats

ABSTRACT Purpose This study was to investigate the effect of resveratrol (RSV) administration on diabetes-induced neural apoptosis and on RNA-dependent-protein-kinase (PKR)-associated protein X (RAX), PKR and phosphorylated PKR (P-PKR) expression and distribution in retina of diabetic rats. Methods Retina was obtained from normal and diabetic Sprague–Dawley rats with or without RSV (5 and 10 mg/kg/d) treatment at 30-, 32-, 34- and 36-weeks. Apoptosis of retinal neural cells and distribution of RAX/P-PKR was assessed by TUNEL and immunofluorescence methods. Expression of RAX, PKR and P-PKR was evaluated by qRT-PCR and western-blotting methods. Results Our study showed that the TUNEL-positive cells were mainly localized in ganglion cells layer (GCL), inner nuclear layer (INL) and outer nuclear layer (ONL) of the diabetic rat's retina at 30-, 32-, 34- and 36-weeks. RSV administration effectively suppressed the neural apoptosis in GCL, INL and ONL. Almost no TUNEL-positive cells were observed in retina of normal control and RSV-treated normal control rats. Our study also showed that the expression level of RAX, P-PKR in diabetic rats retina at 30-, 32-, 34-, and 36-weeks was elevated. With supplementation of 5 and 10 mg/kg/d RSV, the expression level of RAX and P-PKR was decreased (P < 0.05). The expression level of RAX and P-PKR in the retina of normal control rats was not altered by RSV. The expression level of PKR was not altered by streptozotocin injection and RSV treatment. Conclusions Our results suggested that RSV attenuates retinal neural apoptosis in diabetic rats retina may be via regulation RAX/P-PKR expression.

Endogenous SO2 Controls Cell Apoptosis: The State-of-the-Art.

SO2, previously known as the product of industrial waste, has recently been proven to be a novel gasotransmitter in the cardiovascular system. It is endogenously produced from the metabolism pathway of sulfur-containing amino acids in mammalians. Endogenous SO2 acts as an important controller in the regulation of many biological processes including cardiovascular physiological and pathophysiological events. Recently, the studies on the regulatory effect of endogenous SO2 on cell apoptosis and its pathophysiological significance have attracted great attention. Endogenous SO2 can regulate the apoptosis of vascular smooth muscle cells, endothelial cells, cardiomyocytes, neuron, alveolar macrophages, polymorphonuclear neutrophils and retinal photoreceptor cells, which might be involved in the pathogenesis of hypertension, pulmonary hypertension, myocardial injury, brain injury, acute lung injury, and retinal disease. Therefore, in the present study, we described the current findings on how endogenous SO2 is generated and metabolized, and we summarized its regulatory effects on cell apoptosis, underlying mechanisms, and pathophysiological relevance.

Treatment during a developmental window prevents NF1-associated optic pathway gliomas by targeting Erk-dependent migrating glial progenitors.

The mechanism of vulnerability to pediatric low-grade gliomas (pLGGs)-the most common brain tumor in children-during development remains largely unknown. Using mouse models of neurofibromatosis type 1 (NF1)-associated pLGGs in the optic pathway (NF1-OPG), we demonstrate that NF1-OPG arose from the vulnerability to the dependency of Mek-Erk/MAPK signaling during gliogenesis of one of the two developmentally transient precursor populations in the optic nerve, brain-derived migrating glial progenitors (GPs), but not local progenitors. Hyperactive Erk/MAPK signaling by Nf1 loss overproduced GPs by disrupting the balance between stem-cell maintenance and gliogenesis of hypothalamic ventricular zone radial glia (RG). Persistence of RG-like GPs initiated NF1-OPG, causing Bax-dependent apoptosis in retinal ganglion cells. Removal of three Mek1/Mek2 alleles or transient post-natal treatment with a low-dose MEK inhibitor normalized differentiation of Nf1-/- RG-like GPs, preventing NF1-OPG formation and neuronal degeneration. We provide the proof-of-concept evidence for preventing pLGGs before tumor-associated neurological damage enters an irreversible phase.

LncRNA LINC00673 is Downregulated in Diabetic Retinopathy and Regulates the Apoptosis of Retinal Pigment Epithelial Cells via Negatively Regulating p53.

BackgroundLong noncoding RNA (LncRNA) LINC00673 has been proven to play critical roles in cancer biology, while its role in other diseases is unknown. It has been reported that LINC00673 could interact with p53, a critical player in diabetes and diabetic complications, suggesting that LINC00673 may also participate in diabetic retinopathy (DR). This study aimed to investigate the role of LINC00673 in DR.MethodsThe present study included 3 groups of participants, including DR group, diabetes (DB) group, and healthy control (Control) group. Flow cytometry was utilized to determine cell apoptosis. Proteins and messenger RNAs (mRNAs) were estimated by Western blot and quantitative reverse transcription PCR (qRT-PCR), respectively.ResultsLINC00673 was downregulated in plasma samples of DR patients (n=60) in comparison with the healthy controls (n=60) and negatively correlated with p53 only across DR patients but not across the healthy controls. In retinal pigment epithelial cells (RPECs), high glucose treatment downregulated LINC00673. Moreover, LINC00673 overexpression downregulated p53 and decreased RPEC apoptosis, while LINC00673 silencing upregulated p53 and increased RPEC apoptosis. In addition, p53 overexpression reduced the effects of LINC00673 overexpression.ConclusionLINC00673 is downregulated in DR patients and regulates RPEC apoptosis via negatively regulating p53.

Endoplasmic reticulum stress is involved in retinal injury induced by repeated transient spikes of intraocular pressure.

Clinically, a large proportion of glaucoma patients undergo repeated intraocular pressure (IOP) spike (Spike IOP) attacks during their sleep, which may facilitate retinopathy. In this study, we established a mouse model of repeated transient Spike IOP to investigate the direct damage to the retina following Spike IOP attacks, and elucidated the underlying molecular mechanism. We analyzed the changes in the number of retinal ganglion cells (RGCs) via immunofluorescence. Thereafter, we detected retinal cell apoptosis via terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) staining, and performed RNA sequencing (RNA-seq) to reveal the underlying molecular mechanism. Finally, we validated the expression of key molecules in the endoplasmic reticulum (ER) stress pathway using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis. Results revealed a time-dependent RGC loss in Spike IOP, evidenced by a reduction in the number of Brn3a-positive RGCs in experimental eyes following a 7-d continuous treatment with Spike IOP. In addition, TUNEL staining indicated that apoptosis of retinal cells started in the outer nuclear layer (ONL), and then spread to the ganglion cell layer (GCL) with time. RNA-seq analysis revealed that ER stress might be involved in Spike IOP-induced retinal injury. This result was corroborated by western blot, which revealed upregulation of ER stress-related proteins including binding immunoglobulin protein/glucose-regulated protein 78 (BiP/GRP78), phosphorylated inositol-requiring enzyme 1 (p-IRE1), unspliced X-box-binding protein 1 (XBP1-u), spliced X-box-binding protein 1 (XBP1-s), phosphorylated c-Jun N-terminal kinase (p-JNK), C/EBP-homologous protein (CHOP), and B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax). These findings indicate that repeated IOP transients are detrimental to the retina, while ER stress plays an important role in retinal cell apoptosis in this situation. Notably, repeated Spike IOP among glaucoma patients is a crucial factor for progressive retinopathy.

Retraction Note: Mst1 facilitates hyperglycemia-induced retinal pigmented epithelial cell apoptosis by evoking mitochondrial stress and activating the Smad2 signaling pathway.

Retraction Note: Mst1 facilitates hyperglycemia-induced retinal pigmented epithelial cell apoptosis by evoking mitochondrial stress and activating the Smad2 signaling pathway.

Soluble tumor necrosis factor-alpha-induced hyperexcitability contributes to retinal ganglion cell apoptosis by enhancing Nav1.6 in experimental glaucoma

BackgroundNeuroinflammation plays an important role in the pathogenesis of glaucoma. Tumor necrosis factor-alpha (TNF-α) is a major pro-inflammatory cytokine released from activated retinal glial cells in glaucoma. Here, we investigated how TNF-α induces retinal ganglion cell (RGC) hyperexcitability and injury.MethodsWhole-cell patch-clamp techniques were performed to explore changes in spontaneous firing and evoked action potentials, and Na+ currents in RGCs. Both intravitreal injection of TNF-α and chronic ocular hypertension (COH) models were used. Western blotting, immunofluorescence, quantitative real-time polymerase chain reaction (q-PCR), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) techniques were employed to investigate the molecular mechanisms of TNF-α effects on RGCs.ResultsIntravitreal injection of soluble TNF-α significantly increased the spontaneous firing frequencies of RGCs in retinal slices. When the synaptic transmissions were blocked, more than 90% of RGCs still showed spontaneous firing; both the percentage of cells and firing frequency were higher than the controls. Furthermore, the frequency of evoked action potentials was also higher than the controls. Co-injection of the TNF-α receptor 1 (TNFR1) inhibitor R7050 eliminated the TNF-α-induced effects, suggesting that TNF-α may directly act on RGCs to induce cell hyperexcitability through activating TNFR1. In RGCs acutely isolated from TNF-α-injected retinas, Na+ current densities were upregulated. Perfusing TNF-α in RGCs of normal rats mimicked this effect, and the activation curve of Na+ currents shifted toward hyperpolarization direction, which was mediated through p38 MAPK and STAT3 signaling pathways. Further analysis revealed that TNF-α selectively upregulated Nav1.6 subtype of Na+ currents in RGCs. Similar to observations in retinas of rats with COH, intravitreal injection of TNF-α upregulated the expression of Nav1.6 proteins in both total cell and membrane components, which was reversed by the NF-κB inhibitor BAY 11-7082. Inhibition of TNFR1 blocked TNF-α-induced RGC apoptosis.ConclusionsTNF-α/TNFR1 signaling induces RGC hyperexcitability by selectively upregulating Nav1.6 Na+ channels, thus contributing to RGC apoptosis in glaucoma.

Retinoprotective Effects of PACAP Eye Drops in Microbead-Induced Glaucoma Model in Rats.

Glaucoma is associated with increased intraocular pressure (IOP), causing the apoptosis of retinal ganglion cells (RGCs) and the loss of their axons leading to blindness. Pituitary adenylate cyclase activating polypeptide (PACAP) is neuroprotective in several neural injuries, including retinopathies. The aim of this study was to investigate the effects of PACAP1-38 eye drops in a model of glaucoma. IOP was elevated bilaterally by injections of microbeads to block the aqueous humor outflow. The control groups received the same volume of saline. Animals were treated with PACAP1-38 (1 µg/drop, 3 × 1 drop/day) or vehicle for 4 weeks starting one day after the injections. Retinal morphology by histology and optical coherence tomography, function by electroretinography, and IOP changes were analyzed. Animals were sacrificed 8 weeks after the injections. Microbeads injections induced a significant increase in the IOP, while PACAP1-38 treatment lowered it to normal levels (~10 mmHg). Significant retinal degeneration and functional impairment were observed in the microbead-injected group without PACAP1-38 treatment. In the microbeads + PACAP1-38 group, the retinal morphology and functionality were close to the normal values. In summary, our results show that PACAP1-38, given in form of eye drops, is neuroprotective in glaucoma, providing the basis for potential future therapeutic administration.

MicroRNA-145-5p targeting of TRIM2 mediates the apoptosis of retinal ganglion cells via the PI3K/AKT signaling pathway in glaucoma.

There is accumulating evidence to suggest that microRNAs (miRNAs) are associated with the progressive optic neuropathy including glaucoma. Apoptosis of retinal ganglion cells (RGCs) is a hallmark of glaucoma. The present study focused on the effects of miR-145-5p on RGC apoptosis in glaucoma. We established a glaucoma rat model by intraocular injection of N-methyl-d-aspartic acid (NMDA). RGCs were isolated from newborn rats and treated with NMDA. Hematoxylin and eosin staining was performed to detect morphological changes in the retinas of rats. The expression of miR-145-5p and tripartite motif-containing 2 (TRIM2) in RGCs was measured by RT-qPCR. The viability of RGCs was measured by MTT assay. Flow cytometry analysis and TUNEL assays were conducted to assess the apoptosis of RGCs. The interaction between miR-145-5p and TRIM2 was investigated using a luciferase reporter assay. Rats injected with NMDA showed a thinner ganglion cell layer (GCL) and inner plexiform layer (IPL) as well as increased expression of miR-145-5p. Silencing of miR-145-5p significantly increased the GCL and IPL in the glaucoma rat model. Moreover, miR-145-5p expression was upregulated in RGCs ex vivo in response to NMDA. Silencing of miR-145-5p promoted cell viability and suppressed apoptosis in NMDA-treated RGCs. Mechanistically, miR-145-5p targeted the TRIM2 3' untranslated region to suppress its expression. TRIM2 was upregulated in NMDA-treated RGCs and protected RGCs against NMDA-induced apoptosis. Furthermore, miR-145-5p suppressed the PI3K/AKT pathway by downregulating TRIM2 in NMDA-treated RGCs. Suppression of miR-145-5p inhibited the apoptosis of RGCs via TRIM2-mediated activation of the PI3K/AKT signaling pathway in NMDA-induced glaucoma.

Astragalus Polysaccharide Regulates miR-182/Bcl-2 Axis to Relieve Metabolic Memory through Suppressing Mitochondrial Damage-Mediated Apoptosis in Retinal Pigment Epithelial Cells

Introduction: Metabolic memory is one of the causes of diabetic retinopathy, and astragalus polysaccharide (APS) has great advantages in the treatment of diabetes. However, the effect of APS on metabolic memory remains to be investigated. Methods: Retinal pigment epithelial cell line ARPE-19 and primary retinal pigment epithelial cells were used to verify the effect of APS on mitochondria damage and apoptosis induced by high glucose-induced metabolic memory. The relationship between miR-182 and Bcl-2 was confirmed by a luciferase activity assay. Western blotting and quantitative reverse-transcriptase polymerase chain reaction were conducted to investigate the changes in mitochondrial damage- and apoptosis-associated markers. The cell mitochondrial membrane potential was assessed by JC-1 fluorescence. Terminal deoxynucleotidyl transferase dUTP nick end labelling staining and flow cytometry assays were performed to determine the occurrence of apoptosis. Results: Treatment with high glucose followed by normal glucose significantly upregulated the expression of miR-182 and downregulated the expression of its target Bcl-2, and APS treatment reversed the above effects. Additionally, APS treatment restored mitochondrial function and inhibited apoptosis in cells in a state of metabolic memory. The effects of APS against mitochondrial damage and apoptosis were partially inhibited after miR-182 overexpression. Conclusion: APS alleviated mitochondrial damage and apoptosis induced by metabolic memory by regulating the miR-182/Bcl-2 axis, which might serve as a new strategy for the treatment of diabetic retinopathy.

Downregulation of General Control Nonderepressible 2/Eukaryotic Initiation Factor 2α Attenuates Inflammation, Oxidative Stress and Cell Apoptosis in Retinal Cells Induced by High Glucose

Background: Diabetic retinopathy (DR), the frequent complication of diabetes mellitus, has been the main factor of clinical blindness. It is of great clinical significance to seek a novel therapeutic target of DR. The present study aims to investigate the important role of GCN2/eIF2α in DR and the underlying mechanism. Materials and Methods: The expression levels of GCN2 and p-eIF2α were measured by western blot assay and q-PCR. The inflammation levels were assessed by ELISA assay and oxidative stress was measured by colorimetric method. Then, the key proteins related to the function of endothelial cell were measured by western blot assay. Cell apoptotic rate was detected by flow cytometry and proteins related to cell apoptosis were detected by western blot assay. Results: High glucose activated GCN2/eIF2α signaling pathway in HRCECs. Downregulation of GCN2 attenuated HG-induced cell apoptosis, inflammatory and oxidative stress in HRCECs. Meanwhile, downregulation of GCN2 ameliorated HG-induced endothelial cell dysfunction. Inhibition of GCN2 inhibited p-eIF2α, ATF4, CHOP and activated UCP2. Conclusion: The results in this study proved that knockdown of GCN2 could significantly mitigate HG-induced injury, suggesting GCN2/eIF2α as a potential target for DR therapy.

Intravitreally Injected Methylene Blue Protects Retina against Acute Ocular Hypertension in Rats

ABSTRACT Purpose To assess the neuroprotective effects of methylene blue (MB) in a rat model of acute ocular hypertension (AOH) and explore its possible mechanisms. Methods Our AOH rat model was obtained with anterior chamber perfusion for 60 min. After that, 100 μM MB was injected into the vitreous cavity immediately after injury. Electroretinogram, fundus photography, optical coherence tomography (OCT) and retina morphology examination were utilized to quantify retinal damage before surgery, as well as 7, 14 and 28 days after. The average number of surviving retinal ganglion cells (RGCs) was counted after fluorescent retrograde labelling with 4% DiI. And TUNEL assay was used to investigate retinal cell apoptosis at 24 hours after AOH. Nrf2 and BACE1 in the retina were determined by RT-qPCR analysis. Results AOH did produce a severe degeneration effect on the whole retinal layer. Intravitreally injected MB maintained certain retinal thickness after AOH, reduced the destruction of electroretinograms, and enhanced RGCs survival. The average number of TUNEL-labelled cells statistically reduced in the MB-treated retina tissue compared with retina treated with normal saline. The relative mRNA level of Nrf2 was also much higher in the MB-treated retinas after AOH, and the expression of BACE1 had a decline in the AOH + MB group. Conclusions MB can protect the retina from AOH injury and the possible mechanism might involve the inhibition of BACE1 expression and the activation of Nrf2 antioxidant pathway.

AUY922 induces retinal toxicity through attenuating TRPM1

BackgroundOcular adverse events are common dose-limiting toxicities in cancer patients treated with HSP90 inhibitors, such as AUY922; however, the pathology and molecular mechanisms that mediate AUY922-induced retinal toxicity remain undescribed.MethodsThe impact of AUY922 on mouse retinas and cell lines was comprehensively investigated using isobaric tags for relative and absolute quantitation (iTRAQ)‑based proteomic profiling and pathway enrichment analysis, immunohistochemistry and immunofluorescence staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, MTT assay, colony formation assay, and western blot analysis. The effect of AUY922 on the Transient Receptor Potential cation channel subfamily M member 1 (TRPM1)-HSP90 chaperone complex was characterized by coimmunoprecipitation. TRPM1-regulated gene expression was analyzed by RNAseq analysis and gene set enrichment analysis (GSEA). The role of TRPM1 was assessed using both loss-of-function and gain-of-function approaches.ResultsHere, we show that the treatment with AUY922 induced retinal damage and cell apoptosis, dysregulated the photoreceptor and retinal pigment epithelium (RPE) layers, and reduced TRPM1 expression. Proteomic profiling and functional annotation of differentially expressed proteins reveals that those related to stress responses, protein folding processes, regulation of apoptosis, cell cycle and growth, reactive oxygen species (ROS) response, cell junction assembly and adhesion regulation, and proton transmembrane transport were significantly enriched in AUY922-treated cells. We found that AUY922 triggered caspase-3-dependent cell apoptosis, increased ROS production and inhibited cell growth. We determined that TRPM1 is a bona fide HSP90 client and characterized that AUY922 may reduce TRPM1 expression by disrupting the CDC37-HSP90 chaperone complex. Additionally, GSEA revealed that TRPM1-regulated genes were associated with retinal morphogenesis in camera-type eyes and the JAK-STAT cascade. Finally, gain-of-function and loss-of-function analyses validated the finding that TRPM1 mediated the cell apoptosis, ROS production and growth inhibition induced by AUY922.ConclusionsOur study demonstrates the pathology of AUY922-induced retinal toxicity in vivo. TRPM1 is an HSP90 client, regulates photoreceptor morphology and function, and mediates AUY922-induced cytotoxicity.

Diterpene Ginkgolides Meglumine Injection inhibits apoptosis induced by optic nerve crush injury via modulating MAPKs signaling pathways in retinal ganglion cells

Ethnopharmacological relevanceDiterpene Ginkgolides Meglumine Injection (DGMI) is made of extracts from Ginkgo biloba L, including Ginkgolides A, B, and K and some other contents, and has been widely used as the treatment of cerebral ischemic stroke in clinic. It can be learned from the “Compendium of Materia Medica” that Ginkgo possesses the effect of “dispersing toxin”. The ancient Chinese phrase “dispersing toxin” is now explained as elimination of inflammation and oxidative state in human body. And it led to the original ideas for today's anti-oxidation studies of Ginkgo in apoptosis induced by optic nerve crush injury. Aim of the studyTo investigate the underlying molecular mechanism of the DGMI in retinal ganglion cells (RGCs) apoptosis. Materials and methodsTUNEL staining was used to observe the anti-apoptotic effects of DGMI on the adult rat optic nerve injury (ONC) model, and flow cytometry and hoechst 33,342 staining were used to observe the anti-apoptotic effects of DGMI on the oxygen glucose deprivation (OGD) induced RGC-5 cells injury model. The regulation of apoptosis and MAPKs pathways were investigated with Immunohistochemistry and Western blotting. ResultsThis study demonstrated that DGMI is able to decrease the conduction time of F-VEP and ameliorate histological features induced by optic nerve crush injury in rats. Immunohistochemistry and TUNEL staining results indicated that DGMI can also inhibit cell apoptosis via modulating MAPKs signaling pathways. In addition, treatment with DGMI markedly improved the morphological structures and decreased the apoptotic index in RGC-5 cells. Mechanistically, DGMI could significantly inhibit cell apoptosis by inhibiting p38, JNK and Erk1/2 activation. ConclusionThe study shows that DGMI and ginkgolides inhibit RGCs apoptosis by impeding the activation of MAPKs signaling pathways in vivo and in vitro. Therefore, the present study provided scientific evidence for the underlying mechanism of DGMI and ginkgolides on optic nerve crush injury.

Circ_0000615 promotes high glucose-induced human retinal pigment epithelium cell apoptosis, inflammation and oxidative stress via miR-646/YAP1 axis in diabetic retinopathy.

Diabetic retinopathy (DR), a common complication of diabetes mellitus, is the major cause of visual impairment and blindness. Circ_0000615 was found to be elevated in retina samples of diabetic patients. Hence, the detailed effects and molecular mechanisms of circ_0000615 in DN progression were explored. The levels of circ_0000615, microRNA (miR)-646 and YAP1 (yes-associated protein 1) were detected using quantitative real-time polymerase chain reaction and Western blot assays. Cell viability, apoptosis, inflammation and reactive oxygen species (ROS) generation were determined using cell counting kit-8 assay, flow cytometry, caspase3 activity analysis, Western blot, enzyme-linked immunosorbent assay (ELISA) and Dichlorofluorescein diacetate (DCFH-DA) assay, respectively. The binding interaction between miR-646 and circ_0000615 or YAP1 was determined using dual-luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays. Circ_0000615 was elevated in high glucose (HG)-induced human retinal pigment epithelium (HRPE) cells. Knockdown of circ_0000615 attenuated HG-triggered HRPE cell apoptosis, inflammation, and ROS generation. Mechanistically, miR-646 was confirmed to be a target of circ_0000615, inhibition of miR-646 reversed the protective effects of circ_0000615 knockdown on HG-evoked HRPE cell dysfunction. MiR-646 was verified to target YAP1, overexpression of YAP1 abolished the impairment induced by miR-646 on HG-induced HRPE cell damage. Besides that, we confirmed that circ_0000615 could regulate YAP1 expression via miR-646. Circ_0000615 contributed to HG-induced HRPE cell dysfunction via miR-646/YAP1 axis, suggesting a novel insight into the pathogenesis of DR and a potential candidate for DR treatment.

Protective role and molecular mechanism of action of Nesfatin-1 against high glucose-induced inflammation, oxidative stress and apoptosis in retinal epithelial cells.

Diabetic retinopathy (DR) is a major complication of diabetes mellitus that may cause severe visual impairment. It has been reported that the levels of nesfatin-1 in the serum and vitreous humor were negatively correlated with DR; however, its role in DR has not been fully elucidated. Therefore, the present study was performed to investigate the effect of nesfatin-1 on high glucose-treated human retinal epithelial cells (ARPE-19) and explore the underlying mechanism. The effects of nesfatin-1 on cell viability, inflammation, oxidative stress and apoptosis were examined under high glucose conditions. The Cell Counting Kit-8 assay was used to determine cell viability. The levels of inflammatory cytokines were evaluated using ELISA kits. The reactive oxygen species and malondialdehyde content was estimated using commercial assay kits. Flow cytometry was performed to detect apoptotic cells and western blot analysis was employed to evaluate the expression of apoptosis-associated proteins. Moreover, the levels of NF-κB, NACHT, LRR and PYD domains-containing protein 3 (NLRP3) and high-mobility group protein B1 (HMGB1) were determined via western blot analysis. The results revealed that nesfatin-1 enhanced cell viability and suppressed inflammation, oxidative stress and apoptosis in the presence of high glucose concentration. Moreover, the activation of the NF-κB/NLRP3 inflammasome signaling and the expression of HMGB1 were inhibited by nesfatin-1. Furthermore, HMGB1 overexpression partially abrogated the inactivation of the NF-κB/NLRP3 inflammasome pathway caused by nesfatin-1. Taken together, these findings demonstrated that nesfatin-1 inhibited the activation of the NF-κB/NLRP3 inflammasome signaling via modulating HMGB1 and exerted a protective effect on ARPE-19 cells against high glucose-induced inflammation, oxidative stress and apoptosis.

Reduced Levels of Drp1 Protect against Development of Retinal Vascular Lesions in Diabetic Retinopathy.

High glucose (HG)-induced Drp1 overexpression contributes to mitochondrial dysfunction and promotes apoptosis in retinal endothelial cells. However, it is unknown whether inhibiting Drp1 overexpression protects against the development of retinal vascular cell loss in diabetes. To investigate whether reduced Drp1 level is protective against diabetes-induced retinal vascular lesions, four groups of mice: wild type (WT) control mice, streptozotocin (STZ)-induced diabetic mice, Drp1+/− mice, and STZ-induced diabetic Drp1+/− mice were examined after 16 weeks of diabetes. Western Blot analysis indicated a significant increase in Drp1 expression in the diabetic retinas compared to those of WT mice; retinas of diabetic Drp1+/− mice showed reduced Drp1 level compared to those of diabetic mice. A significant increase in the number of acellular capillaries (AC) and pericyte loss (PL) was observed in the retinas of diabetic mice compared to those of the WT control mice. Importantly, a significant decrease in the number of AC and PL was observed in retinas of diabetic Drp1+/− mice compared to those of diabetic mice concomitant with increased expression of pro-apoptotic genes, Bax, cleaved PARP, and increased cleaved caspase-3 activity. Preventing diabetes-induced Drp1 overexpression may have protective effects against the development of vascular lesions, characteristic of diabetic retinopathy.

Multifactorial Pathogenic Processes of Retinal Ganglion Cell Degeneration in Glaucoma towards Multi-Target Strategies for Broader Treatment Effects.

Glaucoma is a chronic neurodegenerative disease characterized by apoptosis of retinal ganglion cell (RGC) somas, degeneration of axons, and loss of synapses at dendrites and axon terminals. Glaucomatous neurodegeneration encompasses multiple triggers, multiple cell types, and multiple molecular pathways through the etiological paths with biomechanical, vascular, metabolic, oxidative, and inflammatory components. As much as intrinsic responses of RGCs themselves, divergent responses and intricate interactions of the surrounding glia also play decisive roles for the cell fate. Seen from a broad perspective, multitarget treatment strategies have a compelling pathophysiological basis to more efficiently manipulate multiple pathogenic processes at multiple injury sites in such a multifactorial neurodegenerative disease. Despite distinct molecular programs for somatic and axonal degeneration, mitochondrial dysfunction and glia-driven neuroinflammation present interdependent processes with widespread impacts in the glaucomatous retina and optic nerve. Since dysfunctional mitochondria stimulate inflammatory responses and proinflammatory mediators impair mitochondria, mitochondrial restoration may be immunomodulatory, while anti-inflammatory treatments protect mitochondria. Manipulation of these converging routes may thus allow a unified treatment strategy to protect RGC axons, somas, and synapses. This review presents an overview of recent research advancements with emphasis on potential treatment targets to achieve the best treatment efficacy to preserve visual function in glaucoma.

Semaphorin3A increases M1-like microglia and retinal ganglion cell apoptosis after optic nerve injury

BackgroundThe mechanisms leading to retinal ganglion cell (RGC) death after optic nerve injury have not been fully elucidated. Current evidence indicates that microglial activation and M1- and M2-like dynamics may be an important factor in RGC apoptosis after optic nerve crush (ONC). Semaphorin3A (Sema3A) is a classic axonal guidance protein,which has been found to have a role in neuroinflammation processes. In this study, we investigated the contribution of microglial-derived Sema3A to progressive RGC apoptosis through regulating paradigm of M1- and M2-like microglia after ONC.Method A mouse ONC model and a primary microglial-RGC co-culture system were used in the present study. The expression of M1- and M2-like microglial activation markers were assessed by real-time polymerase chain reaction (RT-qPCR). Histological and Western blot (WB) analyses were used to investigate the polarization patterns of microglia transitions and the levels of Sema3A. RGC apoptosis was investigated by TUNEL staining and caspase-3 detection.ResultsLevels of Sema3A in the mouse retina increased after ONC. Treatment of mice with the stimulating factor 1 receptor antagonist PLX3397 resulted in a decrease of retinal microglia. The levels of CD16/32 (M1) were up-regulated at days 3 and 7 post-ONC. However, CD206 (M2) declined on day 7 after ONC. Exposure to anti-Sema3A antibodies (anti-Sema3A) resulted in a decrease in the number of M1-like microglia, an increase in the number of M2-like microglia, and the amelioration of RGC apoptosis.ConclusionsAn increase in microglia-derived Sema3A in the retina after ONC partially leads to a continuous increase of M1-like microglia and plays an important role in RGC apoptosis. Inhibition of Sema3A activity may be a novel approach to the prevention of RGC apoptosis after optic nerve injury.