Rat Retinal Cells Research Articles

Effect of BDNF-TrKB pathway on apoptosis of retinal ganglion cells in glaucomatous animal model.

The aim of this paper is to investigate the effect of BDNF-TrKB pathway on AOH by accessing its regulatory role in retinal ganglion cell apoptosis. Acute ocular hypertension (AOH) model in rats was established by anterior chamber perfusion to increase intraocular tension. Rats were randomly divided into AOH group, control group and k252a group, with ten rats in each group. Rats were sacrificed 72 h after animal procedures and eyeballs were harvested. HE staining was used to observe retinal structural changes at different time points. Immunohistochemical staining was used to observe the BDNF-positive cells in retinal tissues. TUNEL staining was conducted to measure apoptosis of retinal ganglion cells. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and Western blot were performed to detect mRNA and protein levels of BDNF, TrKB, PI3K and ERK1 in retinal tissues, respectively. HE and TUNEL staining showed significant pathological changes and abundant apoptotic cells in rat retina of AOH group and k252a group compared with those of the control group (p<0.05). The number of survived retinal ganglion cells in the AOH group was lower than that of the control group (p<0.05). K252a group had the lowest number of survived retinal ganglion cells. Immunohistochemical results showed that BDNF was rarely expressed in rat retinal tissues of the control group, which was remarkably pronounced in the AOH group and k252a group. The number of BDNF-positive cells in the k252a group was higher than that in the AOH group (p<0.05). RT-PCR and Western blot indicated that mRNA and protein levels of relative genes in BDNF-TrKB and PI3K/ERK1 pathways were upregulated in AOH group (p<0.05), but were significantly downregulated in k252a group (p<0.05). BNDF-TrKB pathway exerts a protective effect on retina against acute ocular hypertension by reducing retinal cell apoptosis.

The Role of the Inflammatory Mediators TNF‐α and IFN‐γ in the Hyperglycemia‐induced PECAM‐1 Ubiquitination and Degradation in Diabetic Retinopathy

ObjectiveThe aim of this study was to investigate the role of TNF‐α and IFN‐γ in PECAM‐1 ubiquitination and subsequent degradation under hyperglycemic conditions in the diabetic retina.IntroductionInflammation is a major contributor to the development of diabetic retinopathy (DR). Two of the key mediators of inflammation, tumor necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ), have been shown to contribute to the pathophysiological changes leading to the progression of DR. However, their role in the hyperglycemia‐induced loss of retinal PECAM‐1 is not known. Platelet endothelial cell adhesion molecule‐1 (PECAM‐1) is a cell surface protein that is heavily expressed on vascular endothelial cells, and has an important role in the maintenance of the endothelium and its functions. We have previously found a significant decrease in PECAM‐1 levels in the diabetic retina and cultured rat retinal microvascular endothelial cells (RRMECs) grown under high glucose conditions. Thus, an investigation into the possible mechanism/s of PECAM‐1 loss may help in understanding the molecular processes leading to DR, and provide a possible therapeutic target.Materials and MethodsFor our in vitro studies, primary RRMECs were grown in normal glucose (NG, 5 mM glucose) or high glucose (HG, 25 mM) media for six days, with TNF‐α and/or IFN‐γ (20 ng/ml and 30 ng/ml, respectively) added 24 hrs prior to harvesting cells for analysis. For our animal studies, age‐matched male Wistar rats were injected with either vehicle (control), or Streptozotocin (STZ) (diabetic, 30 mg/kg/day) for three consecutive days in a model of type I diabetes. Retinas and plasma were collected eight weeks post‐STZ injection for analysis. PECAM‐1, TNF‐α, IFN‐γ, and ubiquitin levels were assessed using western blotting, ELISA, Immunofluorescence, and immunoprecipitation assays.ResultsTotal ubiquitination levels were significantly increased in the diabetic retina and in RRMECs grown under HG conditions (35% and 40% increase, respectively, p&lt;0.05). Moreover, PECAM‐1 ubiquitination levels were significantly increased in RRMECs grown under hyperglycemic conditions (3‐fold, p&lt;0.05). TNF‐α levels were significantly increased in diabetic STZ plasma and RRMECs grown under HG conditions (31%, 74.4% increase, respectively, p&lt;0.05). Additionally, IFN‐γ levels were significantly increased in the diabetic retina (35% increase, p&lt;0.05). PECAM‐1 levels were significantly decreased in RRMECs when treated with TNF‐α and/or IFN‐γ (60–75% decrease, p&lt;0.05), with a significant increase in total ubiquitination levels (20–28% increase, p&lt;0.05).ConclusionThese results indicate a possible role for TNF‐α and IFN‐γ in the hyperglycemia‐induced loss of PECAM‐1 in retinal endothelial cells, possibly by upregulating PECAM‐1 ubiquitination and subsequent degradation.Support or Funding InformationThis work was supported by funding from the National Institutes of Health (NIH) EY025632This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MiR-204-5p promotes diabetic retinopathy development via downregulation of microtubule-associated protein 1 light chain 3.

Diabetic retinopathy (DR) is a chronic and progressive complication of diabetes mellitus. DR impairs sight due to neuronal and vascular dysfunction in the retina. It is critical to investigate the pathogenesis of DR to develop effective treatment. In the present study, a streptozotocin (STZ)-induced diabetic rat model was constructed and the expression of microRNA (miR)-204-5p and vascular endothelial growth factor (VEGF) were determined. Immunohistochemistry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were employed to detect the effects of miR-204-5p on the expression of microtubule-associated protein 1 light chain 3 (LC3B). RT-qPCR analysis demonstrated that miR-204-5p and VEGF were significantly upregulated in the retina tissue of diabetic rats compared with the control group (P<0.01). Immunohistochemistry and western blotting revealed that the protein expression levels of LC3B-II and the ratio of LC3B-II/LC3B-I were significantly suppressed in the diabetes group compared with the control (P<0.01). In retinal tissues, anti-miR-204-5p treatment significantly enhanced the protein expression levels of LC3B-II and the ratio of LC3B-II/LC3B-I and these levels were significantly reduced in response to miR-204-5p mimic treatment compared with the negative miR control (P<0.01). In rat retinal endothelial cells isolated from diabetic rats, anti-miR-204-5p treatment increased the number of autophagic vacuoles, and significantly promoted LC3B-II expression and the LC3B-II/LC3B-I ratio compared with the negative control (P<0.01). The results of the present study revealed that miR-204-5p downregulated the expression of LC3B-II to inhibit autophagy in DR. Therefore, miR-204-5p may be considered as a novel effective therapeutic target during the development of DR.

Loss of Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) in the Diabetic Retina: Role of Matrix Metalloproteinases.

PurposeTo test the hypothesis that high glucose and matrix metalloproteinases (MMPs) contribute to the diabetes-induced loss of platelet endothelial cell adhesion molecule-1 (PECAM-1) in the retinal microvasculature.MethodsPECAM-1 and MMP protein, activity, and interactions with PECAM-1 were assessed using western blotting, zymography, immunofluorescence, or coimmunoprecipitation assays. These assays were conducted using primary rat retinal microvascular endothelial cells (RRMECs) grown either in normal glucose (5 mM) or high glucose (25 mM) conditions and using retinas collected from streptozotocin-induced diabetic or control rats. The broad-spectrum MMP inhibitor GM6001 was administered in vivo and in vitro to ascertain the role of MMPs in the hyperglycemia-induced loss of PECAM-1.ResultsA dramatic decrease in PECAM-1 (western blotting, immunofluorescence) was observed in both the diabetic retina and in hyperglycemic RRMECs. The decrease in PECAM-1 was accompanied by a significant increase in the presence and activity of matrix metalloproteinase-2 (MMP-2) (but not matrix metalloproteinase-9 [MMP-9]) in the diabetic plasma (P < 0.05) and in hyperglycemic RRMECs (P < 0.05). Moreover, RRMEC PECAM-1 significantly decreased when treated with plasma collected from diabetic rats. Several MMP-2 cleavage sites on PECAM-1 were identified using in silico analysis. Moreover, PECAM-1/MMP-2 interactions were confirmed using coimmunoprecipitation. PECAM-1 was significantly decreased in RRMECs treated with MMP-2 (P < 0.05), but became comparable to controls with the MMP inhibitor GM6001 in both the diabetic retina and hyperglycemic RRMECs.ConclusionsThese results indicate a possible role of MMP-2 in hyperglycemia-induced PECAM-1 loss in retinal endothelial cells.

Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

Retinal ganglion cells (RGCs) undergo rapid cell death by apoptosis after injury but can be rescued by suppression of caspase-2 (CASP2) using an siRNA to CASP2 (siCASP2). Pigment epithelium-derived factor (PEDF), has neuroprotective and anti-angiogenic functions and protects RGC from death. The purpose of this study was to investigate if suppression of CASP2 is a possible mechanism of neuroprotection by PEDF in RGC. Adult rat retinal cells were treated in vitro with sub-optimal and optimal concentrations of siCASP2 and PEDF and levels of CASP2 mRNA and RGC survival were then quantified. Optic nerve crush (ONC) injury followed by intravitreal injections of siCASP2 or PEDF and eye drops of PEDF-34 were also used to determine CASP2 mRNA and protein reduction. Results showed that PEDF and PEDF-34 significantly suppressed CASP2 mRNA in culture, by 1.85- and 3.04-fold, respectively, and increased RGC survival by 63.2 ± 3.8% and 81.9 ± 6.6%, respectively compared to cells grown in Neurobasal-A alone. RGC survival was significantly reduced in glial proliferation inhibited and purified RGC cultures suggesting that some of the effects of PEDF were glia-mediated. In addition, intravitreal injection of PEDF and eye drops of PEDF-34 after ONC also suppressed CASP2 mRNA levels by 1.82- and 3.89-fold and cleaved caspase-2 (C-CASP2) protein levels by 4.98- and 8.93-fold compared to ONC + PBS vehicle groups, respectively, without affecting other executioner caspases. Treatment of retinal cultures with PEDF and PEDF-34 promoted the secretion of neurotrophic factors (NTF) into the culture media, of which brain-derived neurotrophic factor (BDNF) caused the greatest reduction in CASP2 mRNA and C-CASP2 protein. The neuroprotective effects of PEDF were blocked by a polyclonal antibody and PEDF suppressed key elements in the apoptotic pathway. In conclusion, this study shows that some of the RGC neuroprotective effects of PEDF is regulated through suppression of CASP2 and downstream apoptotic signalling molecules.

Blockade of microglial adenosine A2A receptor suppresses elevated pressure-induced inflammation, oxidative stress, and cell death in retinal cells.

Glaucoma is a retinal degenerative disease characterized by the loss of retinal ganglion cells and damage of the optic nerve. Recently, we demonstrated that antagonists of adenosine A2A receptor (A2AR) control retinal inflammation and afford protection to rat retinal cells in glaucoma models. However, the precise contribution of microglia to retinal injury was not addressed, as well as the effect of A2AR blockade directly in microglia. Here we show that blocking microglial A2AR prevents microglial cell response to elevated pressure and it is sufficient to protect retinal cells from elevated pressure‐induced death. The A2AR antagonist SCH 58261 or the knockdown of A2AR expression with siRNA in microglial cells prevented the increase in microglia response to elevated hydrostatic pressure. Furthermore, in retinal neural cell cultures, the A2AR antagonist decreased microglia proliferation, as well as the expression and release of pro‐inflammatory mediators. Microglia ablation prevented neural cell death triggered by elevated pressure. The A2AR blockade recapitulated the effects of microglia depletion, suggesting that blocking A2AR in microglia is able to control neurodegeneration in glaucoma‐like conditions. Importantly, in human organotypic retinal cultures, A2AR blockade prevented the increase in reactive oxygen species and the morphological alterations in microglia triggered by elevated pressure. These findings place microglia as the main contributors for retinal cell death during elevated pressure and identify microglial A2AR as a therapeutic target to control retinal neuroinflammation and prevent neural apoptosis elicited by elevated pressure.

The Effect of Drug Pre-treatment on Taurine Transport at the Inner Blood-Retinal Barrier Under Variable Conditions.

Taurine is essential for the development and function of the central nervous system, retina, and cardiovascular system. It is a naturally occurring amino acid, abundantly found in the retina. It has been shown to exhibit antioxidant, neuroprotective, and osmoregulatory functions in the retina. We used conditionally immortalized rat retinal capillary endothelial cells (TR-iBRB), in vitro, to investigate the effects of oxidative stress, high glucose (HG) and hypertonic conditions on taurine transport. TR-iBRB cells pre-treated with tumor necrosis factor alpha (TNF-α) showed a significant increase in [3H]taurine uptake rate, which, however, decreased when treated with taurine (50mM). Addition of paeonol and propranolol to TNF-α pre-treated cells had no significant effect on [3H]taurine uptake, but the addition of 10mM taurine caused a reduction. The uptake rate decreased under HG conditions, in contrast to that under hypertonic conditions. [3H]Taurine uptake increased with pre-incubation time. Additionally, uptake of [3H]taurine and mRNA expression of taurine transporter (TauT) decreased significantly under hypertonic and HG conditions, following pre-incubation with 10mM taurine, 1mM paeonol, and 0.1mM propranolol. [3H]Taurine uptake was significantly inhibited in the presence of taurine transporters such as taurine and β-alanine. Results indicate that oxidative stress and hypertonic conditions increased taurine uptake in iBRB cell lines, whereas HG conditions reduced the uptake rate. Taurine may be useful in stabilizing the microenvironment in cells affected by oxidative stress as well as hypertonic and HG conditions. Moreover, taurine may play a key role in maintaining taurine concentrations in the taurine transporter system of retinal cells.

LncRNA-H19 Induces Retinal Müller Cell Apoptosis via MiR-29b/FOXO4 Axis in Diabetic Retinopathy

Background: The pathological development of Diabetic retinopathy (DR) is an intricate process with multiple steps, and implicates a battery of dysregulated genes and regulators. We aimed to investigate the pathological mechanism of DR involving the regulatory association among H19, miR-29b and FOXO4. Methods: Sprague-Dawley (SD) rats were used to establish the diabetes mellitus (DM) model by injection streptozotocin. The levels of miR-29b, H19 and the mRNA level of FOXO4 were detected by qRT-PCR. The expression of FOXO4 at protein level was assessed by Western blot assay. The apoptosis of rMC-1 cells were analyzed by flow cytometry. To further investigate the relationship between H19 and miR-29b, the RIP and pull down assays were performed. Results: The expression of H19 and FOXO4 were enhanced obviously, and the level of miR-29b was decreased in the retina tissues of DM rats and high glucose (HG)-treated rMC-1 cells. HG, as well as over-expression of H19, stimulated the apoptosis of rat retinal Muller cells. Knockdown of H19 reversed HG stimulation on cell apoptosis and FOXO4 up-regulation. RIP assay and RNA pull-down assay indicated that H19 was a target of miR-29b and inhibition of miR-29b reversed H19 down-regulation effect on cell apoptosis and FOXO4 expression. Down-regulation of FOXO4 reversed the effect of miR-29b knockdown on cell apoptosis. Conclusion: MiR-29b targeting lncRNA-H19 mediates the apoptosis of rat retinal Muller cells via regulating FOXO4 in diabetic retinopathy. Key words: LncRNA-H19, miR-29b, FOXO4, Retinal Muller cell apoptosis, Diabetic retinopathy

Resveratrol exhibits an effect on attenuating retina inflammatory condition and damage of diabetic retinopathy via PON1

Diabetic retinopathy (DR), an obstacle of the visual microvascular system, is a serious complication of diabetic patients. Paraoxonase 1 (PON1) has been extensively evaluated as a genetic candidate for diabetic microvascular complications, and PON1 is associated with DR. In this study, the biological functions of PON1 and its related proteins were determined via gene ontology (GO) enrichment analysis; we demonstrated that treatment with resveratrol alleviated retinal inflammatory activities to evaluate its protective effects on streptozotocin (STZ)-induced diabetic rats and high-glucose (HG) stimulated rat retinal endothelial cells (RRECs). The GO enrichment analysis suggested that PON1 may regulate inflammatory responses and microvascular complications in DR. In an in vivo study, resveratrol significantly recovered the insulin level and PON1 expression and activity, as well as clearly reduced the retinal vascular permeability, retinal AGEs, LDL, Ox-LDL, caspase3 activity, retinal damage, IL-1β, IL-6, TNFα, VEGF, IFNγ and MCP-1 in STZ-diabetic rats. Moreover, resveratrol reduced the caspase3 activity and Ox-LDL expression in HG stimulated RRECs. However, its protective effect was a deficiency in PON1-silenced RRECs. PON1 is a pivotal modulator in the role of resveratrol in reversing the RREC damage induced by HG. Furthermore, we found that resveratrol exhibits an effect on attenuating the retinal inflammatory condition and damage of DR via PON1. Our study suggests that resveratrol-induced PON1 in the retina may be a promising therapeutic strategy to prevent diabetes-related retinopathy.

Eriodictyol inhibits high glucose-induced oxidative stress and inflammation in retinal ganglial cells.

Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes mellitus and is considered as a leading cause of blindness. Oxidative stress and inflammation are significant drivers for the development of DR. Eriodictyol, a flavonoid compound, was proved to possess anti-inflammatory, antioxidative, and antidiabetic activities. However, the role of eriodictyol in DR has not been unveiled. In the current study, we explored the protective effects of eriodictyol on high glucose (HG)-induced rat retinal ganglial cells (RGCs). The results suggested that eriodictyol improved cell viability of HG-induced rat RGC-5 cells in a dose-dependent manner. Eriodictyol reduced the reactive oxygen species production and increased the activities of superoxide dismutase, glutathione peroxidase and catalase in rat RGC-5 cells in response to HG stimulation. The production of proinflammatory cytokines including tumor necrosis factor alpha and interleukin-8 was diminished after eriodictyol treatment. Eriodictyol also suppressed cell apoptosis induced HG in rat RGC-5 cells. Furthermore, eriodictyol enhanced the nuclear translocation of nuclear factor erythroid-2(E2)-related factor 2 (Nrf2) and elevated the expression of antioxidant enzyme heme-oxygenase-1 (HO-1). These findings suggested that eriodictyol protects the RGC-5 cells from HG-induced oxidative stress, inflammation, and cell apoptosis through regulating the activation of Nrf2/HO-1 pathway.

Inhibition of Cx43 gap junction uncoupling prevents high glucose-induced apoptosis and reduces excess cell monolayer permeability in retinal vascular endothelial cells

The aim of this study was to investigate whether inhibition of connexin 43 gap junction-uncoupling is sufficient to prevent retinal vascular cell loss under high glucose condition and reduce cell monolayer permeability. Rat retinal endothelial cells were grown for 3, 5, and 7 days in normal (5 mM) or high glucose (30 mM) medium; in parallel, cells grown in high glucose medium were exposed for 3, 5, and 7 days to 100 nM danegaptide, which stabilizes connexin 43-mediated cell coupling. Additionally, cells grown in normal medium were treated with a connexin 43 blocker as a negative control. To determine gap junction intercellular communication, scrape load dye transfer assay was performed at the three time points. Cells were assessed for apoptosis and cell monolayer permeability by differential dye staining and in vitro permeability assays, respectively. Cells treated with danegaptide preserved gap junction intercellular communication, decreased cell death, and reduced cell monolayer permeability. Scrape load dye transfer assay indicated that cells exposed to danegaptide for 3, 5, and 7 days under high glucose condition maintained gap junction intercellular communication. Importantly, danegaptide significantly prevented high glucose-induced apoptosis at all three time points, and inhibited cell monolayer permeability by day 5. Cells exposed to a connexin 43 blocker, which decreased cell coupling, showed excess apoptosis and cell monolayer permeability. These findings suggest that prevention of high glucose-induced compromised cell-cell coupling may be a useful strategy for inhibiting apoptosis and excess vascular permeability associated with diabetic retinopathy.

PKC delta activation increases neonatal rat retinal cells survival in vitro: Involvement of neurotrophins and M1 muscarinic receptors

Protein kinase C (PKC) is a family of serine/threonine kinases related to several phenomena as cell proliferation, differentiation and survival. Our previous data demonstrated that treatment of axotomized neonatal rat retinal cell cultures for 48 h with phorbol 12-myristate 13-acetate (PMA), a PKC activator, increases retinal ganglion cells (RGCs) survival. Moreover, this treatment decreases M1 receptors (M1R) and modulates BDNF levels. The aim of this work was to assess the possible involvement of neurotrophins BDNF and NGF in the modulation of M1R levels induced by PKC activation, and its involvement on RGCs survival. Our results show that PMA (50 ng/mL) treatment, via PKC delta activation, modulates NGF, BDNF and M1R levels. BDNF and NGF mediate the decrease of M1R levels induced by PMA treatment. M1R activation is essential to PMA neuroprotective effect on RGCs as telenzepine (M1R selective antagonist) abolished it. Based on our results we suggest that PKC delta activation modulates neurotrophins levels by a signaling pathway that involves M1R activation and ultimately leading to an increase in RGCs survival in vitro.

The Role of Matrix Metalloproteinase 2 (MMP2) In Diabetes‐induced Loss of PECAM‐1 in The Retina: Direct and Indirect Mechanisms

ObjectiveMatrix metalloproteinases (MMPs) are calcium‐dependent, zinc‐containing endopeptidases that are responsible for degrading extracellular matrix proteins, and for cleavage of various substrates such as cell surface receptors, proteoglycans, chemokines and cytokines. The aim of this study was to investigate the molecular mechanisms by which MMP2 may lead to PECAM‐1 loss in the diabetic retina, either directly through cleavage, or indirectly by modulating tumor necrosis factor‐α (TNF‐α) levels and activity.BackgroundPlatelet endothelial cell adhesion molecule‐1 (PECAM‐1) is a cell surface protein that is heavily expressed on vascular endothelial cells, and has major roles in barrier function, endothelial cell‐endothelial cell adhesion, and cell signaling. We have previously found a significant decrease (50–90%, P&lt;0.05) in PECAM‐1 levels in the diabetic retina, and in cultured rat retinal microvascular endothelial cells (RRMECs) grown under high glucose conditions (30–40%, P&lt;0.05). The decrease in PECAM‐1 levels was accompanied by a significant increase in MMP2 levels and activity in the diabetic plasma (72% and 78.5% respectively, P&lt;0.05), and in RRMECs grown under high glucose conditions (12‐fold increase, P&lt;0.05).Materials and MethodsFor our in vitro studies, primary RRMECs were grown in normal glucose (NG, 5 mM glucose) or high glucose (HG, 25 mM) media for six days, with treatments added 6–24 hrs prior to harvesting cells for analysis. For our animal studies, age‐matched male Wistar rats were injected with either vehicle (control), or Streptozotocin (STZ) (diabetic, 30 mg/kg/day) for three consecutive days in a model of type I diabetes. GM6001, a broad spectrum MMP inhibitor, was started at week six post‐STZ injection for two weeks (4 mM, 25 μl/eye, in sterile eye drops), and retinas were collected eight weeks post‐STZ injection for analysis. PECAM‐1, MMP2, and TNF‐α levels and interactions were assessed using western blotting, zymography, immunofluorescence, immunoprecipitation, or ELISA assays. In silico analysis for possible protease cleavage sites was done using PROSPER, provided by Monash University, Australia.ResultsSeveral PECAM‐1 cleavage sites by MMP2 were identified using in silico analysis. Moreover, we confirmed PECAM‐1/MMP2 interactions using immunoprecipitation. PECAM‐1 levels were significantly decreased in RRMECs treated with MMP2 (81%, P&lt;0.05), with a significant increase in TNF‐α levels (118.2%, P&lt;0.05). Moreover, RRMECs treated with TNF‐α had a significant decrease in PECAM‐1 levels (34%, P&lt;0.05). Furthermore, TNF‐α levels were significantly increased in diabetic STZ plasma, and in RRMECs grown under HG conditions (31%, 74.4% respectively, P&lt;0.05). To investigate the protective effect of MMP2 inhibition on PECAM‐1 levels, we treated diabetic rats with GM6001 eye drops, and saw an increase of PECAM‐1 levels (77.2%, P&lt;0.05) when compared to untreated rats.ConclusionThese results indicate a possible role of MMP2 in the diabetes‐induced loss of PECAM‐1 in the retina, which can be via direct cleavage of PECAM‐1, or indirectly via upregulation of TNF‐α, with the two mechanisms evaluated in future studies.Support or Funding InformationNIH EY025632, and AHA Predoctoral Fellowship 16PRE30080003This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

GRGM-13 comprising 13 plant and animal products, inhibited oxidative stress induced apoptosis in retinal ganglion cells by inhibiting P2RX7/p38 MAPK signaling pathway

ObjectiveTo determine the effectiveness of GRGM-13 on oxidative stress induced apoptosis of retinal ganglion cells (RGCs) and revealed its possible mechanism.Materials and methods: Caspase-3 activity, MDA level, and glutathione peroxidase level were detected by Caspase-3 assay kit, Lipid Peroxidation MDA Assay Kit, and Total Glutathione Peroxidase Assay Kit, respectively. Protein levels of Bax, Bcl-2, p-p38 and p38 were observed by Western Blot. Reactive oxygen species assay kit was used to determine intracellular ROS level. Apoptotic cells were measured by flow cytometry. ResultsGRGM-13 inhibited apoptosis of RGCs and ROS level in rat retinal tissue and RGC-5 cells, and the decrease degree strengthened with the increase of GRGM-13 concentration. In addition, ROS upregulated p-p38 expression, while GRGM-13 reversed this effect. We also found that p38 inhibitor SB202190 did not change L-glutamate (Glu) or H2O2-induced ROS level, while SB202190 inhibited apoptosis of RGC-5 cells. Finally, we observed that P2 × 7R agonist BzATP reversed the inhibition effect of GRGM-13 on RGC-5 cell apoptosis, ROS level and p-p38 expression, while si-P2 × 7R inhibited oxidative stress-induced phosphorylation of p38. ConclusionGRGM-13 could inhibit oxidative stress-induced RGCs apoptosis via inhibiting P2RX7/p38 MAPK pathway, which revealed the possible mechanism of GRGM-13 on stress-induced RGCs apoptosis and provided new Chinese medicine for the treatment of glaucoma.

Retinal ganglion cell-conditioned medium and surrounding pressure alters gene expression and differentiation of rat retinal progenitor cells.

Loss of retinal ganglion cells is implicated in glaucoma and high intraocular pressure. Factors that affect the differentiation of retinal progenitor cells into retinal ganglion cells remain unclear. The present study aimed to investigate the effects of retinal ganglion cell-conditioned medium on gene expression and differentiation in retinal progenitor cells, and the effects of surrounding pressure on the survival and differentiation of retinal progenitor cells. Retinal progenitor cells and retinal ganglion cells were isolated from rats. Immunofluorescence staining of Nestin and Thy1 was performed to identify rat retinal progenitor cells and retinal ganglion cells, respectively. Retinal progenitor cells and ganglion cells were cultured for 48 h under surrounding pressure of 0, 20, 40, 60 and 80 mmHg. Cellular apoptosis was detected using a caspase-3 assay kit. In addition, the culture supernatant of rat retinal ganglion cells was collected. Retinal progenitor cells were cultured in the presence or absence of retinal ganglion-conditioned medium for 72 h under normal pressure. Gene expression of Nestin, paired box protein 6 (PAX6), Thy1 and brain-specific homeobox/POU domain protein 3 (Brn-3) in retinal progenitor cells was detected by reverse transcription-quantitative polymerase chain reaction. Retinal progenitor cells were cultured in retinal ganglion-conditioned medium for 72 h under surrounding pressure of 0 and 40 mmHg, respectively, and flow cytometry was utilized to evaluate the effects of pressure on the differentiation of retinal progenitor cells into retinal ganglion cells. The results demonstrated that isolated retinal progenitor cells were Nestin-positive and retinal ganglion cells were Thy1-positive, suggesting successful isolation. The activity of caspase-3 increased in retinal progenitor cells and retinal ganglion cells in a pressure-dependent manner. When the surrounding pressure reached 40, 60 and 80 mmHg, the activity of caspase-3 in retinal progenitor cells and ganglion cells increased significantly compared with cells that were not under pressure. Compared with retinal progenitor cells cultured without ganglion-conditioned medium, those cultured with ganglion-conditioned medium had significantly decreased expression levels of Nestin and PAX6, and increased expression levels of Thy1 and Brn3. Compared with 0 mmHg pressure, retinal progenitor cells cultured in ganglion-conditioned medium under 40 mmHg pressure had increased percentages of Thy1-positive cells. In conclusion, the apoptosis of rat retinal progenitor cells and retinal ganglion cells was pressure-dependent. Retinal ganglion cell-conditioned medium increased the differentiation of retinal progenitor cells into retinal ganglion-like cells, and the differentiation increased as surrounding pressure increased. Current study provides insights that may contribute to the efforts of developing a treatment for glaucoma.

Proteolytic cleavage of vascular adhesion protein-1 induced by vascular endothelial growth factor in retinal capillary endothelial cells.

To investigate the mechanism of soluble vascular adhesion protein-1 (sVAP-1) accumulation induced by vascular endothelial growth factor (VEGF) in the vitreous of patients with diabetic retinopathy (DR). Experimental. Protein levels of sVAP-1 and N epsilon-(hexanoyl)lysine (HEL), an oxidative stress marker, in the vitreous samples from patients with proliferative diabetic retinopathy (PDR) with or without intravitreal bevacizumab (IVB) injection were determined by ELISA. The effect of VEGF on both mRNA expression of Vap-1 and secretion of sVAP-1 in rat retinal capillary endothelial cells (TR-iBRB2) was analyzed by real-time PCR and western blotting, respectively. In addition, the impact of VEGF on production and activation ratios of matrix metalloproteinase (MMP)-2 and MMP-9 was examined by gelatin zymography. Hydrogen peroxide production and reactive oxygen species (ROS) levels were assessed in the supernatants of TR-iBRB2 cells treated with VEGF. IVB injection decreased vitreous levels of sVAP-1 and HEL in patients with PDR. VEGF stimulation released sVAP-1 protein from TR-iBRB2 cells as a consequence of membrane-anchored VAP-1 shedding by MMP-2 and MMP-9. In addition, VEGF increased hydrogen peroxide generation and ROS augmentation through spermine oxidation by sVAP-1 as semicarbazide-sensitive amine oxidase (SSAO) in the supernatant of cultured endothelial cells. The current data demonstrate that proangiogenic factor VEGF induces sVAP-1 release from retinal capillary endothelial cells and facilitates hydrogen peroxide generation via enzymatic property of sVAP-1, followed by the increase of oxidative stress, one of the crucial factors in the pathogenesis of DR.

The novel chalcone analog L2H17 protects retinal ganglion cells from oxidative stress-induced apoptosis.

Chalcone is a plant metabolite widely found in fruits, vegetables, spices and tea, and has anti-tumor, anti-inflammation, immunomodulation, antibacterial and anti-oxidation activities, as well as many other pharmacological and biological effects. Our team has shown that its analogs have antioxidant activity, and oxidative stress is a pathological hallmark of retinal ischemia/reperfusion injury that can lead to retinal damage and visual loss. This investigation aims to identify a chalcone that protects retinal ganglion cells in vitro from the effects of oxidative stress and examine its mechanism. Rat retinal ganglion cell-5 cells were pretreated with chalcones and then exposed to tert-butyl hydroperoxide that causes oxidative damage. Controls received dimethyl sulfoxide only or tert-butyl hydroperoxide in dimethyl sulfoxide. Only (E)-3,4-dihydroxy-2'-methylether ketone (L2H17), of the five chalcone analogs, markedly increased the survival rate of oxidatively injured RGC-5 cells. Thus, subsequent experiments only analyzed the results of the L2H17 intervention. Cell viability and apoptosis were measured. Intracellular superoxide dismutase and reactive oxygen species levels were used to assess induced oxidative stress. The mechanism of action by L2H17 was explored by measuring the ER stress/UPR pathway and the expression and localization of Nrf2. All results demonstrated that L2H17 could reduce the apoptosis of oxidatively injured cells, inhibit caspase-3 activity, increase Bcl-2 expression, decrease Bad expression, increase the activity of superoxide dismutase, inhibit the production of reactive oxygen species, increase Nrf2 immunoreactivity, and reduce the activating transcription factor 4, phospho-eukaryotic initiation factor 2 and CHOP expression. L2H17 protects retinal ganglion cells induced by oxidative stress by regulating Nrf2, which indicates that it has the potential to become a drug for retinal ischemia/reperfusion.

Insulin-like growth factor-1 activates PI3K/Akt signalling to protect human retinal pigment epithelial cells from amiodarone-induced oxidative injury.

Amiodarone is one of the most effective anti-arrhythmic drugs available, but its clinical applications are limited by toxic side effects including optic toxicity. The purpose of this study was to investigate the toxic effect of amiodarone on D407 cells (a human retinal pigmented epithelial (RPE) cell line) and the mechanisms of the protective effect of insulin-like growth factor-1 (IGF-1). The involvement of the kinases, Akt and ERK, was analysed by Western blot. Intracellular accumulation of ROS was measured using fluorophotometric quantification. A pharmacological approach with inhibitors was used to investigate the pathways involved in the protective action of IGF-1. Amiodarone concentration-dependently augmented the production of ROS, lipid peroxidation and apoptosis in D407 cells. IGF-1 time- and concentration-dependently reversed these effects of amiodarone and protected D407 cells from amiodarone-mediated toxicity. Amiodarone inhibited the pAkt but not pErk, and IGF-1 reversed this inhibitory effect of amiodarone. However, IGF-1 failed to suppress amiodarone-induced cytotoxicity in the presence of PI3K/Akt inhibitor LY294002 suggesting the direct involvement of the PI3K/Akt pathway. Furthermore, in vivo rat flash electroretinogram (FERG) recordings showed that IGF-1 reverses the amiodarone-induced decrease in a- and b-waves. The immunocytochemistry findings confirmed that vitreous IGF-1 injections promote the survival of RPE cells in rat retina treated with amiodarone. IGF-1 can protect RPE cells from amiodarone-mediated injury via the PI3K/Akt pathway in vivo and in vitro. IGF-1 has potential as a protective drug for the prevention and treatment of amiodarone-induced optic toxicity.

Notoginsenoside R1 attenuates high glucose-induced endothelial damage in rat retinal capillary endothelial cells by modulating the intracellular redox state.

The aim of this study was to examine whether Notoginsenoside R1 (NR1) attenuates high glucose-induced cell damage in rat retinal capillary endothelial cells (RCECs) and to explore the mechanisms involved. The exposure of rat RCECs to high concentration of glucose (30 mM) for 72 h led to significant cytotoxicity, including decreased cell viability, reduced mitochondrial DNA copy number, increased lactate dehydrogenase release and elevated apoptosis. NR1, when present in the culture medium, markedly attenuated the high glucose-induced cytotoxicity in rat RCECs. Moreover, high glucose also induced a significant increase in intracellular reactive oxygen species and subsequently increased the activity of NADPH oxidase and poly-ADP (ribose) polymerase, whereas the activity of catalase decreased. The addition of NR1 to the medium significantly reduced the generation of reactive oxygen species, inhibited NADPH oxidase and poly-ADP (ribose) polymerase activities and increased catalase activity in RCECs, accompanied by a reduced cellular nitrotyrosine level. To explore the underlying mechanisms involved, the cellular redox status was monitored. Both the cellular NAD+ and NADPH levels decreased significantly in high glucose medium, which resulted in a marked decrease in the NAD+/NADH and NADPH/NADP+ ratios. High glucose stimulation also enhanced the accumulation of GSSG, maintaining the GSH/GSSG ratio lower than that in the control group with 5.5 mM glucose. When treated with NR1, the cellular NAD+, NADPH and GSH concentrations increased, and the ratios of NAD+/NADH, NADPH/NADP+ and GSH/GSSG increased, similar to the control group. These results demonstrate that NR1 attenuates high glucose-induced cell damage in RCECs. Therefore, NR1 may exert its protective effects via mechanisms that involve changes in the cellular redox state.

Exposing primary rat retina cell cultures to γ-rays: An in vitro model for evaluating radiation responses

Retinal tissue can receive incidental γ-rays exposure during radiotherapy either of tumors of the eye and optic nerve or of head-and-neck tumors, and during medical diagnostic procedures. Healthy retina is therefore at risk of suffering radiation-related side effects and the knowledge of pathophysiological response of retinal cells to ionizing radiations could be useful to design possible strategies of prevention and management of radiotoxicity.In this study, we have exploited an in vitro model (primary rat retinal cell culture) to study an array of biological effects induced on retinal neurons by γ-rays. Most of the different cell types present in retinal tissue - either of the neuronal or glial lineages - are preserved in primary rat retinal cultures. Similar to the retina in situ, neuronal cells undergo in vitro a maturational development shown by the formation of polarized neuritic trees and operating synapses.Since 2 Gy is the incidental dose received by the healthy retina per fraction when the standard treatment is delivered to the brain, retina cell cultures have been exposed to 1 or 2 Gy of γ-rays at different level of neuronal differentiation in vitro: days in vitro (DIV)2 or DIV8. At DIV9, retinal cultures were analyzed in terms of viability, apoptosis and characterized by immunocytochemistry to identify alterations in neuronal differentiation. After irradiation at DIV2, MTT assay revealed an evident loss of cell viability and βIII-tubulin immunostaining highlighted a marked neuritic damage, indicating that survived neurons showed an impaired differentiation. Differentiated cultures (DIV8) appeared to be more resistant with respect to undifferentiated, DIV2 cultures, both in terms of cell viability and differentiation. Apoptosis evaluated with TUNEL assay showed that irradiation at both DIV2 and DIV8 induced a significant increase in the apoptotic rate. To further investigate the effects of γ-rays on retinal neurons, we evaluated the expression of synaptic proteins, such as SNAP25 and synaptophysin. WB and immunofluorescence analysis showed an altered expression of these proteins in particular when cultures were irradiated at DIV2. To evaluate the effect of γ-rays on photoreceptors, we studied the expression of rhodopsin in WB analysis and immunofluorescence. Our results confirm data from the literature that differentiated photoreceptors appear to be more resistant to irradiation respect to other retinal cell types present in cultures.The results obtained suggest that γ-rays exposure of primary retinal cultures may contribute to shed further light on the mechanisms involved in γ-radiation-induced neurodegeneration.