Inhibition of NLRP3 inflammasome by MCC950 under hypoxia alleviates photoreceptor apoptosis via inducing autophagy in Müller glia.

NLRP3 inflammasome activation has emerged as a critical initiator of inflammatory response in ischemic retinopathy. Here, we identified the effect of a potent, selective NLRP3 inhibitor, MCC950, on autophagy and apoptosis under hypoxia. Neonatal mice were exposed to hyperoxia for 5 days to establish oxygen-induced retinopathy (OIR) model. Intravitreal injection of MCC950 was given, and then autophagy and apoptosis markers were assessed. Retinal autophagy, apoptosis, and related pathways were evaluated by western blot, immunofluorescent labeling, transmission electron microscopy, and TUNEL assay. Autophagic activity in Müller glia after NLRP3 inflammasome inhibition, together with its influence on photoreceptor death, was studied using western blot, immunofluorescence staining, mRFP-GFP-LC3 adenovirus transfection, cell viability, proliferation, and apoptosis assays. Results showed that activation of NLRP3 inflammasome in Müller glia was detected in OIR model. MCC950 could improve impaired retinal autophagic flux and attenuate retinal apoptosis while it regulated the retinal AMPK/mTOR/ULK-1 pathway. Suppressed autophagy and depressed proliferation capacity resulting from hypoxia was promoted after MCC950 treatment in Müller glia. Inhibition of AMPK and ULK-1 pathway significantly interfered with the MCC950-induced autophagy activity, indicating MCC950 positively modulated autophagy through AMPK/mTOR/ULK-1 pathway in Müller cells. Furthermore, blockage of autophagy in Müller glia significantly induced apoptosis in the cocultured 661W photoreceptor cells, whereas MCC950 markedly preserved the density of photoreceptor cells. These findings substantiated the therapeutic potential of MCC950 against impaired autophagy and subsequent apoptosis under hypoxia. Such protective effect might involve the modulation of AMPK/mTOR/ULK-1 pathway. Targeting NLRP3 inflammasome in Müller glia could be beneficial for photoreceptor survival under hypoxic conditions.

Inhibition of vascular endothelial growth factor alleviates neovascular retinopathy with regulated neurotrophic/proinflammatory cytokines through the modulation of DBI-TSPO signaling.

Diazepam binding inhibitor (DBI)-translocator protein (18kDa) (TSPO) signaling in the retina was reported to possess coordinated macroglia-microglia interactions. We investigated DBI-TSPO signaling and its correlation with vascular endothelial growth factor (VEGF), neurotrophic or inflammatory cytokines in neovascular retinopathy, and under hypoxic conditions. The vitreous expression of DBI, VEGF, nerve growth factor (NGF), and interleukin-1beta (IL-1ß) were examined in proliferative diabetic retinopathy (PDR) patients with or without anti-VEGF therapy and nondiabetic controls. Retinal DBI-TSPO signaling and the effect of the anti-VEGF agent were evaluated in a mouse model of oxygen-induced retinopathy (OIR). Interactions between Müller cell-derived VEGF and DBI, as well as cocultured microglial cells under hypoxic conditions, were studied, using Western blot, real-time RT-PCR, enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunofluorescent labeling. Results showed that vitreous levels of DBI, VEGF, NGF, and IL-1ß were significantly higher in PDR patients compared with controls, which further changed after anti-VEGF therapy. A statistical association was found between vitreous DBI and VEGF, NGF, IL-1ß, and age. The application of the anti-VEGF agent in the OIR model induced retinal expression of DBI and NGF, and attenuated inflammation and microglial cell activation. Inhibition of Müller cell-derived VEGF could increase its DBI expression under hypoxic conditions, while the DBI-TSPO signaling pathway is essential for anti-VEGF agents exerting anti-inflammatory and neuroprotective effects, as well as limiting inflammatory magnitude, promoting its neurotrophin production and anti-inflammatory (M2) polarization in microglial cells. These findings suggest the beneficial effect of anti-VEGF therapy on inflammation and neurotrophy of retinal glial cells through modulation of the DBI-TSPO signaling pathway.

Inhibition of Ferroptosis Ameliorates Photoreceptor Degeneration in Experimental Diabetic Mice.

Diabetic retinopathy (DR) is a leading cause of vision impairment in the working-age population worldwide. Various modes of photoreceptor cell death contribute to the development of DR, including apoptosis and autophagy. However, whether ferroptosis is involved in the pathogenesis of photoreceptor degeneration in DR is still unclear. High-glucose (HG)-stimulated 661W cells and diabetic mice models were used for in vitro and in vivo experiments, respectively. The levels of intracellular iron, glutathione (GSH), reactive oxygen species (ROS), lipid peroxidation (MDA), and ferroptosis-related proteins (GPX4, SLC7A11, ACSL4, FTH1, and NCOA4) were quantified to indicate ferroptosis. The effect of ferroptosis inhibition was also assessed. Our data showed the levels of iron, ROS, and MDA were enhanced and GSH concentration was reduced in HG-induced 661W cells and diabetic retinas. The expression of GPX4 and SLC7A11 was downregulated, while the expression of ACSL4, FTH1, and NCOA4 was upregulated in the 661W cells cultured under HG conditions and in the photoreceptor cells in diabetic mice. Furthermore, the administration of the ferroptosis inhibitor ferrostatin-1 (Fer-1) obviously alleviated ferroptosis-related changes in HG-cultured 661W cells and in retinal photoreceptor cells in diabetic mice. Taken together, our findings suggest that ferroptosis is involved in photoreceptor degeneration in the development of the early stages of DR.

Regulation of inflammation by VEGF/BDNF signaling in mouse retinal Müller glial cells exposed to high glucose.

The inflammatory changes seem to play an important role in the development of diabetic retinopathy (DR). Anti-VEGF therapy has been testified to inhibit inflammation in animal models of diabetes, but the detailed mechanisms during this process are not yet clear. Müller glial cells (MGCs) in the mammalian retina are deeply involved in DR, while the BDNF overexpression reduces inflammation in diabetic mice. In this research, we aimed to explore the relationship between VEGF and BDNF in mouse retinal MGCs during inflammation of diabetes. We examined the expression of glutamine-synthetase (GS), glial fibrillary acidic protein (GFAP), vascular-endothelial growth factor (VEGF), interleukin-1beta (IL-1ß), and tumor necrosis factor-alpha (TNF-α) at different time points after mouse retinal MGCs exposed to high glucose (25 mM). We also explored changes in the expression of brain-derived neurotrophic factor (BDNF), nuclear factor kappa B (NF-κB), IL-1ß, and TNF-α in MGCs after treatments with anti-VEGF, VEGF siRNA, BDNF siRNA, BDNF recombination protein, and NF-κB inhibitor. In mouse retinal MGCs exposed to high glucose, BDNF was increased after treatments with anti-VEGF or VEGF siRNA. BDNF was decreased in MGCs from VEGF overexpressed mice. Moreover, the expressions of NF-κB, IL-1ß, and TNF-α changed with BDNF: NF-κB, IL-1ß, and TNF-α were increased after treatments with BDNF siRNA; NF-κB, IL-1ß, and TNF-α were decreased after treatments with BDNF recombination protein. VEGF may regulate cytokines (IL-1ß and TNF-α) by BDNF/NF-κB signaling pathway. The regulation of the VEGF/BDNF/NF-κB signaling pathway may be a significant therapeutic strategy for DR.

Knockdown of thioredoxin interacting protein in Müller cells attenuates photoreceptor apoptosis in streptozotocin-induced diabetic mouse model.

We explored the effect of inhibition of thioredoxin interacting protein (Txnip) on neuroprotection in Müller cells under high glucose. Wild-type (WT) and Txnip knockout (Txnip-/-) mice were used to establish a streptozotocin (STZ)-induced diabetes model and a Müller cells high glucose model. We detected BDNF expression and PI3K/AKT/CREB pathway activation levels in the retina and Müller cells of each group in vivo and in vitro experiments. The Txnip-/- STZ group showed higher expression of BDNF and phosphorylation of PI3K/AKT/CREB in retina, and less retinal photoreceptor apoptosis was observed in Txnip-/- diabetic group than in WT. After using an inhibitor of PI3K signaling pathway, BDNF expression was reduced; In vitro co-cultured with Müller cells in different groups, 661 W cells showed different situations, Txnip-/- Müller cells maximum downregulated Cleaved-caspase 3 expression in 661 W, accompanied by an increase in Bcl-2/Bax ratio. These findings indicate that inhibiting endogenous Txnip in mouse Müller cells can promote their expression and secretion of BDNF, thereby reducing HG induced photoreceptor apoptosis and having important neuroprotective effects on DR. The regulation of BDNF expression by Txnip may be achieved by activating the PI3K/AKT/CREB pathway. This study suggests that regulating Txnip may be a potential target for DR treatment.

Interleukin-17A attenuates photoreceptor cell apoptosis in streptozotocin-induced diabetic mouse model.

Diabetic retinopathy (DR) represents an important microvascular complication of diabetes, which is the top etiology of vision impairment worldwide. Although interleukin (IL)-17A is increasingly implicated in DR development, the underlying cellular mechanisms remain poorly defined. This work aims to evaluate IL-17A levels in the retina of streptozotocin (STZ)-induced diabetic mice and elucidate their potential roles. We found IL-17A was upregulated in diabetic retina after intraperitoneal injection of STZ and high-glucose (HG)-cultured primary Müller cells. IL-17A knockout (IL-17A-/-) downregulated glial fibrillary acidic protein (GFAP) and inhibited the conversion of proneurotrophin-3 (proNT-3) to mature NT-3 in retinal specimens from diabetic mice as well as in Müller cells cultured under HG conditions. Induced apoptosis and upregulated Bax and cleaved caspase-3 were observed in retinal specimens from IL-17A-/- diabetic mice and photoreceptor (661 W) cells after co-culture with IL-17A-/- Müller cells. Moreover, RNA interference-induced gene silencing of tyrosine kinase C receptor (TrkC) in 661 W cells reversed the anti-apoptotic effect of IL-17A under HG conditions. Taken together, our findings suggest that IL-17A/NT-3/TrkC axis regulation suppresses apoptosis in photoreceptor cells, providing a new treatment strategy for DR.

Blocking the interaction between interleukin-17A and endoplasmic reticulum stress in macrophage attenuates retinal neovascularization in oxygen-induced retinopathy.

BACKGROUND: Neovascularization is a leading cause of visual loss typically associated with diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Interleukin-17A (IL-17A) and endoplasmic reticulum (ER) stress both have been demonstrated to play a proangiogenic role in ischemic retinopathies. However, the relationship between IL-17A and ER stress in retinal neovascularization (RNV) under hypoxic conditions and its underlying mechanisms remain unclear. METHODS: In this study, oxygen-induced retinopathy (OIR) mice model was established and intravitreal injections were conducted. Changes of IL-17A and ER stress markers in retinas and cultured primary bone marrow derived macrophage (BMDM) under normoxic or hypoxic conditions were detected. Western blotting, Real-Time RT-PCR, Immunofluorescence assays were conducted to explore the roles and relationship of IL-17A and ER stress in RNV, as well as its underlying mechanisms. RESULTS: Compared to that in normal controls, IL-17A and ER stress markers were all remarkably increased under hypoxic conditions both in vivo and in vitro. Neutralization or knock out of IL-17A decreased ER stress. ER stress inhibitor 4-phenylbutyrate (4-PBA), attenuated the production of IL-17A, suggesting a positive feedback loop between IL-17A and ER stress. Inhibition of IL-17A or ER stress decreased areas of nonperfusion and neovascularization in OIR retinas. As TXNIP/NLRP3 pathway activation has been demonstrated to be involved in increased retinal vascular permeability of ischemic retinopathy, we observed that TXNIP/NLRP3 pathway mediated in the interaction between IL-17A and ER stress under hypoxic conditions. CONCLUSION: The interplay between IL-17A and ER stress contributes to RNV in macrophages via modulation of TXNIP/NLRP3 signaling pathway under hypoxic conditions. The feedback loops may become an innovative and multiple pharmacological therapeutic target for ischemic retinopathy.

Blockade of Adenosine A2A Receptor Protects Photoreceptors after Retinal Detachment by Inhibiting Inflammation and Oxidative Stress.

PURPOSE: Adenosine A2A receptor (A2AR) signaling is neuroprotective in some retinal damage models, but its role in neuronal survival during retinal detachment (RD) is unclear. We tested the hypothesis that A2AR antagonist ZM241385 would prevent photoreceptor apoptosis by inhibiting retinal inflammation and oxidative stress after RD. METHODS: The A2AR antagonist ZM241385 was delivered daily to C57BL/6J mice for three days at a dose (3 mg/kg, i.p.) starting 2 hours prior to creating RD. A2AR expression, microglia proliferation and reactivity, glial fibrillary acidic protein (GFAP) accumulation, IL-1ß expression, and reactive oxygen species (ROS) production were evaluated with immunofluorescence. Photoreceptor TUNEL was analyzed. RESULTS: A2AR expression obviously increased and accumulated in microglia and Müller cells in the retinas after RD. The A2AR antagonist ZM241385 effectively inhibited retinal microglia proliferation and reactivity, decreased GFAP upregulation and proinflammatory cytokine IL-1ß expression of Müller cells, and suppressed ROS overproduction, resulting in attenuation of photoreceptor apoptosis after RD. CONCLUSIONS: The A2AR antagonist ZM241385 is an effective suppressor of microglia proliferation and reactivity, gliosis, neuroinflammation, oxidative stress, and photoreceptor apoptosis in a mouse model of RD. This suggests that A2AR blockade may be an important therapeutic strategy to protect photoreceptors in RD and other CNS diseases that share a common etiology.

Elevated Activating Transcription Factor 4 and Glucose-Regulated 78 Kda Protein Levels Correlate with Inflammatory Cytokines in the Aqueous Humor and Vitreous of Proliferative Diabetic Retinopathy.

PURPOSE: To determine concentrations of endoplasmic reticulum (ER) stress-related factors activating transcription factor 4 (ATF4) and glucose-regulated 78 kDa protein (GRP78) in vitreous and aqueous humor (AqH) of patients with proliferative diabetic retinopathy (PDR) and the correlation of ATF4, GRP78 and inflammatory cytokines interleukin-6(IL-6) and monocyte chemoattractant protein-1 (MCP-1). MATERIALS AND METHODS: AqH and vitreous samples were collected from eyes of patients with PDR and idiopathic macular hole (IMH) which needed vitrectomy. Protein Levels of ATF4, GRP78, and IL-6, MCP-1 in samples were evaluated using enzyme-linked immunosorbent assay (ELISA). RESULTS: ELISA analysis revealed significantly increased levels in both AqH and vitreous of ATF4 and GRP78 in eyes affected with PDR compared to the controls (all p < 0.001). The mean concentrations of IL-6, MCP-1 were also higher in both AqH and vitreous samples from patients with PDR compared to those of IMH (all p < 0.001). (Independent Student t-test, normality test followed with Skewness-Kurtosis Test). In addition, correlations of ATF4 and GRP78 with inflammatory factors IL-6 and MCP-1 in subjects of patients were analyzed. No significant correlation between the AqH concentrations of ATF4/IL-6 and ATF4/MCP-1 was detected in eyes of PDR patients (r = 0.346, p = 0.072 and r = 0.275, p = 0.157). Significant correlations were observed between AqH concentrations of GRP78/IL-6 (r = 0.724, p < 0.001), GRP78/MCP-1 (r = 0.654, p < 0.001) in PDR patients. Significant correlations were observed between vitreous concentrations of ATF4/IL-6 (r = 0.918, p < 0.001), ATF4/MCP-1 (r = 0.921, p < 0.001), GRP78/IL-6 (r = 0.978, p < 0.001), GRP78/MCP-1 (r = 0.979, p < 0.001) in PDR patients. No significant correlations was observed between AqH concentrations of ATF4/IL-6 (r = 0.187, p = 474), ATF4/MCP-1 (r = 0.240, p = 0.353), GRP78/IL-6 (r = 0.321, p = 0.209) and GRP78/MCP-1 (r = 0.169, p = 0.516) in eyes of IMH patients. And also no significant correlation was observed between vitreous concentrations of ATF4/IL-6 (r = 0.130, p = 0.563), ATF4/MCP-1(r = 0.029, p = 0.897), GRP78/IL-6 (r = 0.078, p = 0.717), GRP78/MCP-1 (r = 0.005, p = 0.982) in IMH patients. (Pearson correlation coefficient (two-tailed)). CONCLUSIONS: Our results demonstrated that ATF4 and GRP78 may play an important role in the pathogenesis of PDR and work in concert with inflammatory cytokines IL-6 and MCP-1 in pathological process. ATF4 and GRP78 may be good diagnostic biomarkers and new therapeutic targets for PDR. ABBREVIATIONS: ER stress, endoplasmic reticulum stress; ATF4, activating transcription factor 4; GRP78, glucose-regulated 78 kDa protein; AqH, aqueous humor; PDR, proliferative diabetic retinopathy; IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein-1; IMH, idiopathic macular hole.

PEDF mediates pathological neovascularization by regulating macrophage recruitment and polarization in the mouse model of oxygen-induced retinopathy.

Macrophages have been demonstrated to play a proangiogenic role in retinal pathological vascular growth. Pigment epithelium-derived factor (PEDF) works as a powerful endogenous angiogenesis inhibitor, but its role in macrophage recruitment and polarization is largely unknown. To explore the underlying mechanisms, we first evaluated macrophage polarization in the retinas of the oxygen-induced retinopathy (OIR) mouse model. Compared to that in normal controls, M1- and M2-like macrophages were all abundantly increased in the retinas of OIR mice. In addition, both M1 and M2 subtypes significantly promoted neovascularization in vitro and in vivo. In addition, we found that PEDF inhibited retinal neovascularization by dampening macrophage recruitment and polarization. Furthermore, PEDF inhibited macrophage polarization through adipose triglyceride lipase (ATGL) by regulating the activation of MAPKs and the Notch1 pathway, as we found that the phosphorylation of MAPKs, including p38MAPK, JNK and ERK, as well as the accumulation of Notch1 were essential for hypoxia-induced macrophage polarization, while PEDF significantly dampened M1 subtype-related iNOS and M2 subtype-related Arg-1 expression by inhibiting hypoxia-induced activation of Notch1 and MAPKs through ATGL. These findings reveal a protective role of PEDF against retinal neovascularization by regulating macrophage recruitment and polarization.

Intravitreal Ranibizumab Injection as an Adjuvant in the Treatment of Neovascular Glaucoma Accompanied by Vitreous Hemorrhage after Diabetic Vitrectomy.

Purpose. To determine the efficacy of intravitreal ranibizumab injection as adjuvant therapy in the treatment of neovascular glaucoma (NVG) accompanied by postvitrectomy diabetic vitreous hemorrhage (PDVH). Methods. Eighteen NVG patients (18 eyes) accompanied by PDVH were enrolled in this prospective, monocenter, 12-month, interventional case series. The consecutive 18 patients with an IOP ≥ 25 mmHg despite being treated with the maximum medical therapy were treated with intravitreal ranibizumab injections. Vitreous surgery or/with Ahmed valve implantation were indicated if no clinical improvement in vitreous haemorrhage and uncontrolled IOP was shown. Results. Ten patients got clear vitreous and controlled IOP only with 2.7 ± 1.8 injections of ranibizumab without additional surgery. Vitrectomy or/with Ahmed valve implantation was administered in the other 8 eyes due to uncontrolled VH and IOP. At follow-up month 12, all the 18 eyes gained clear vitreous. At month 12 BCVA improved significantly compared to baseline. The baseline and follow-up at month 12 IOP/medication usage were 36.7 ± 8.1 mmHg on 3.4 ± 0.7 medications and 16.2 ± 4.9 mmHg on 0.67 ± 0.77 medications, respectively. Conclusions. The findings suggest that intravitreal ranibizumab injection as adjuvant therapy for treatment of NVG accompanied by PDVH may be safe and potentially effective. This clinical trial is registered with NCT02647515.

Pigment epithelium-derived factor regulates glutamine synthetase and l-glutamate/l-aspartate transporter in retinas with oxygen-induced retinopathy.

PURPOSE: A predominant function of Müller cells is to regulate glutamate levels, but these cells are compromised in oxygen-induced retinopathy. The aim of this study was to investigate the role of pigment epithelium-derived factor (PEDF) in regulating glutamate levels in retina under hypoxia. MATERIALS AND METHODS: One-week-old C57BL/6J mice were exposed to 75% oxygen for 5 days and then kept in room air for another 5 days to establish the oxygen-induced retinopathy (OIR) mouse model. Mice received intravitreous injections of 2 µg PEDF or vehicle on postnatal (P)12 and P14, respectively. Antibody against interleukin-1Beta (IL-1ß) (IL-1ab) was used to neutralize the activity of IL-1ß, mice received intravitreous injections of 500 ng IL-1ab or vehicle on P12 and P14, respectively, too. At P17, the mice were euthanized and their eyes were enucleated. The expression levels of IL-1ß, glutamine synthetase (GS) and l-glutamate/l-aspartate transporter (GLAST) in retinas with different treatments were detected. In addition, wild-type C57BL/6J mice received intravitreous injections of IL-1ß or PEDF. After 24 h, the expression of GS and GLAST in the retinas was also detected. Furthermore, high-performance liquid chromatography (HPLC) was performed to determine the glutamate concentrations in retinas with different treatments. RESULTS: The expression of IL-1ß and levels of glutamate were increased in retinas with OIR, while the expression of GS and GLAST was decreased. Administration of PEDF ameliorated the characteristic changes in retinas of OIR mice. And neutralization of IL-1ß by administration of IL-1ab increased GS and GLAST expression in retinas with OIR. Moreover, the effects of IL-1ß on GS and GLAST expression and unbalanced glutamate levels were inhibited after receiving intravitreous injections of PEDF in retinas of normal mice. CONCLUSIONS: These results suggested that PEDF might up-regulate GS and GLAST expression and decrease glutamate levels by suppressing the role of IL-1ß as an anti-inflammatory factor under hypoxia, and these functions may underlie the neuroprotective effects of PEDF.