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.

4D label-free proteomics analysis of oxygen-induced retinopathy with or without anti-VEGF treatment.

Oxygen-induced retinopathy (OIR) animal model is widely used for retinopathy of prematurity (ROP) researches. The purpose of this study was to identify proteins and related pathways of OIR with or without anti-vascular endothelial growth factor (VEGF) treatment, for use as biomarkers in diagnosing and treating ROP. Nine samples were subjected to proteomic analysis. Retina specimens were collected from 3 OIR mice, 3 OIR mice with anti-VEGF treatment and 3 normal mice (control group). Liquid chromatography-tandem mass spectrometry analysis was performed using the 4D label-free technique. Statistically significant differentially expressed proteins, gene ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway representations, InterPro (IPR) and protein interactions were analyzed. In total, 4585 unique proteins were identified as differentially expressed proteins (DEPs). Enrichment analysis of the GO and KEGG indicated functional clusters related to peptide biosynthetic and metabolic process, cellular macromolecule biosynthetic process and nucleic acid binding in OIR group. For anti-VEGF treatment group, DEPs were clustered in DNA replication, PI3K/Akt signaling pathway and Jak/STAT signaling pathway. Proteomic profiling is useful for the exploration of molecular mechanisms of OIR and mechanisms of anti-VEGF treatment. These findings may be useful for identification of novel biomarkers for ROP pathogenesis and treatment.

Icariin alleviates oxygen-induced retinopathy by targeting microglia hexokinase 2.

Retinopathy of prematurity (ROP) is a retinal disease-causing retinal neovascularization that can lead to blindness. Oxygen-induced retinopathy (OIR) is a widely used ROP animal model. Icariin (ICA) has anti-oxidative and anti-inflammation properties; however, whether ICA has a regulatory effect on OIR remains unclear. In this study, ICA alleviated pathological neovascularization, microglial activation and blood-retina barrier (BRB) damage in vivo. Further results indicated that endothelial cell tube formation, migration and proliferation were restored by ICA treatment in vitro. Proteomic microarrays and molecular mimicry revealed that ICA can directly bind to hexokinase 2 (HK2) and decrease HK2 protein expression in vivo and in vitro. In addition, ICA inhibited the AKT/mTOR/HIF1α pathway activation. The effects of ICA on pathological neovascularization, microglial activation and BRB damage disappeared after HK2 overexpression in vivo. Similarly, the endothelial cell function was revised after HK2 overexpression. HK2 overexpression reversed ICA-induced AKT/mTOR/HIF1α pathway inhibition in vivo and in vitro. Therefore, ICA prevented pathological angiogenesis in OIR in an HK2-dependent manner, implicating ICA as a potential therapeutic agent for ROP.

IC100, a humanized therapeutic monoclonal anti-ASC antibody alleviates oxygen-induced retinopathy in mice.

BACKGROUND: Retinopathy of prematurity (ROP), which often presents with bronchopulmonary dysplasia (BPD), is among the most common morbidities affecting extremely premature infants and is a leading cause of severe vision impairment in children worldwide. Activations of the inflammasome cascade and microglia have been implicated in playing a role in the development of both ROP and BPD. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly. Utilizing mouse models of both oxygen-induced retinopathy (OIR) and BPD, this study was designed to test the hypothesis that hyperoxia induces ASC speck formation, which leads to microglial activation and retinopathy, and that inhibition of ASC speck formation by a humanized monoclonal antibody, IC100, directed against ASC, will ameliorate microglial activation and abnormal retinal vascular formation. METHODS: We first tested ASC speck formation in the retina of ASC-citrine reporter mice expressing ASC fusion protein with a C-terminal citrine (fluorescent GFP isoform) using a BPD model that causes both lung and eye injury by exposing newborn mice to room air (RA) or 85% O2 from postnatal day (P) 1 to P14. The retinas were dissected on P14 and retinal flat mounts were used to detect vascular endothelium with AF-594-conjugated isolectin B4 (IB4) and citrine-tagged ASC specks. To assess the effects of IC100 on an OIR model, newborn ASC citrine reporter mice and wildtype mice (C57BL/6 J) were exposed to RA from P1 to P6, then 75% O2 from P7 to P11, and then to RA from P12 to P18. At P12 mice were randomized to the following groups: RA with placebo PBS (RA-PBS), O2 with PBS (O2-PBS), O2 + IC100 intravitreal injection (O2-IC100-IVT), and O2 + IC100 intraperitoneal injection (O2-IC100-IP). Retinal vascularization was evaluated by flat mount staining with IB4. Microglial activation was detected by immunofluorescence staining for allograft inflammatory factor 1 (AIF-1) and CD206. Retinal structure was analyzed on H&E-stained sections, and function was analyzed by pattern electroretinography (PERG). RNA-sequencing (RNA-seq) of the retinas was performed to determine the transcriptional effects of IC100 treatment in OIR. RESULTS: ASC specks were significantly increased in the retinas by hyperoxia exposure and colocalized with the abnormal vasculature in both BPD and OIR models, and this was associated with increased microglial activation. Treatment with IC100-IVT or IC100-IP significantly reduced vaso-obliteration and intravitreal neovascularization. IC100-IVT treatment also reduced retinal microglial activation, restored retinal structure, and improved retinal function. RNA-seq showed that IC100 treatment corrected the induction of genes associated with angiogenesis, leukocyte migration, and VEGF signaling caused by O2. IC100 also corrected the suppression of genes associated with cell junction assembly, neuron projection, and neuron recognition caused by O2. CONCLUSION: These data demonstrate the crucial role of ASC in the pathogenesis of OIR and the efficacy of a humanized therapeutic anti-ASC antibody in treating OIR mice. Thus, this anti-ASC antibody may potentially be considered in diseases associated with oxygen stresses and retinopathy, such as ROP.

Mitochondrial control of hypoxia-induced pathological retinal angiogenesis.

OBJECTIVE: Pathological retinal neovascularization is vision-threatening. In mouse oxygen-induced retinopathy (OIR) we sought to define mitochondrial respiration changes longitudinally during hyperoxia-induced vessel loss and hypoxia-induced neovascularization, and to test interventions addressing those changes to prevent neovascularization. METHODS: OIR was induced in C57BL/6J mice and retinal vasculature was examined at maximum neovessel formation. We assessed total proteome changes and the ratio of mitochondrial to nuclear DNA copy numbers (mtDNA/nDNA) of OIR vs. control retinas, and mitochondrial oxygen consumption rates (OCR) in ex vivo OIR vs. control retinas (BaroFuse). Pyruvate vs. vehicle control was supplemented to OIR mice either prior to or during neovessel formation. RESULTS: In OIR vs. control retinas, global proteomics showed decreased retinal mitochondrial respiration at peak neovascularization. OCR and mtDNA/nDNA were also decreased at peak neovascularization suggesting impaired mitochondrial respiration. In vivo pyruvate administration during but not prior to neovessel formation (in line with mitochondrial activity time course) suppressed NV. CONCLUSIONS: Mitochondrial energetics were suppressed during retinal NV in OIR. Appropriately timed supplementation of pyruvate may be a novel approach in neovascular retinal diseases.

Suppression of retinal neovascularization by intravitreal injection of cryptotanshinone.

Neovascular eye diseases, including proliferative diabetic retinopathy and retinopathy of prematurity, is a major cause of blindness. Laser ablation and intravitreal anti-VEGF injection have shown their limitations in treatment of retinal neovascularization. Identification of a new therapeutic strategies is in urgent need. Our study aims to assess the effects of Cryptotanshinone (CPT), a natural compound derived from Salvia miltiorrhiza Bunge, in retina neovascularization and explore its potential mechanism. Our study demonstrated that CPT did not cause retina tissue toxicity at the tested concentrations. Intravitreal injections of CPT reduced pathological angiogenesis and promoted physical angiogenesis in oxygen-induced retinopathy (OIR) model. CPT improve visual function in OIR mice and reduced cell apoptosis. Moreover, we also revealed that CPT diminishes the expression of inflammatory cytokines in the OIR retina. In vitro, the administration of CPT effectively inhibited endothelial cells proliferation, migration, sprouting, and tube formation induced by the stimulation of human retinal vascular endothelial cells (HRVECs) with VEGF165. Mechanistically, CPT blocking the phosphorylation of VEGFR2 and downstream targeting pathway. After all, the findings demonstrated that CPT exhibits potent anti-angiogenic and anti-inflammatory effects in OIR mice, and it has therapeutic potential for the treatment of neovascular retinal diseases.

Environmental oxygen regulates astrocyte proliferation to guide angiogenesis during retinal development.

Angiogenesis in the developing mammalian retina requires patterning cues from astrocytes. Developmental disorders of retinal vasculature, such as retinopathy of prematurity (ROP), involve arrest or mispatterning of angiogenesis. Whether these vascular pathologies involve astrocyte dysfunction remains untested. Here, we demonstrate that the major risk factor for ROP - transient neonatal exposure to excess oxygen - disrupts formation of the angiogenic astrocyte template. Exposing newborn mice to elevated oxygen (75%) suppressed astrocyte proliferation, whereas return to room air (21% oxygen) at postnatal day 4 triggered extensive proliferation, massively increasing astrocyte numbers and disturbing their spatial patterning prior to the arrival of developing vasculature. Proliferation required astrocytic HIF2α and was also stimulated by direct hypoxia (10% oxygen), suggesting that astrocyte oxygen sensing regulates the number of astrocytes produced during development. Along with astrocyte defects, return to room air also caused vascular defects reminiscent of ROP. Strikingly, these vascular phenotypes were more severe in animals that had larger numbers of excess astrocytes. Together, our findings suggest that fluctuations in environmental oxygen dysregulate molecular pathways controlling astrocyte proliferation, thereby generating excess astrocytes that interfere with retinal angiogenesis.

Role of myeloid cells in ischemic retinopathies: recent advances and unanswered questions.

Myeloid cells including microglia and macrophages play crucial roles in retinal homeostasis by clearing cellular debris and regulating inflammation. These cells are activated in several blinding ischemic retinal diseases including diabetic retinopathy, where they may exert both beneficial and detrimental effects on neurovascular function and angiogenesis. Myeloid cells impact the progression of retinal pathologies and recent studies suggest that targeting myeloid cells is a promising therapeutic strategy to mitigate diabetic retinopathy and other ischemic retinal diseases. This review summarizes the recent advances in our understanding of the role of microglia and macrophages in retinal diseases and focuses on the effects of myeloid cells on neurovascular injury and angiogenesis in ischemic retinopathies. We highlight gaps in knowledge and advocate for a more detailed understanding of the role of myeloid cells in retinal ischemic injury to fully unlock the potential of targeting myeloid cells as a therapeutic strategy for retinal ischemia.

Cone cell dysfunction attenuates retinal neovascularization in oxygen-induced retinopathy mouse model.

Aberrant neovascularization is the most common feature in retinopathy of prematurity (ROP), which leads to the retinal detachment and visual defects in neonates with a low gestational age eventually. Understanding the regulation of inappropriate angiogenic signaling benefits individuals at-risk. Recently, neural activity originating from the specific neural activity has been considered to contribute to retinal angiogenesis. Here, we explored the impact of cone cell dysfunction on oxygen-induced retinopathy (OIR), a mouse model commonly employed to understand retinal diseases associated with abnormal blood vessel growth, using the Gnat2cpfl3 (cone photoreceptor function loss-3) strain of mice (regardless of the sex), which is known for its inherent cone cell dysfunction. We found that the retinal avascular area, hypoxic area, and neovascular area were significantly attenuated in Gnat2cpfl3 OIR mice compared to those in C57BL/6 OIR mice. Moreover, the HIF-1α/VEGF axis was also reduced in Gnat2cpfl3 OIR mice. Collectively, our results indicated that cone cell dysfunction, as observed in Gnat2cpfl3 OIR mice, leads to attenuated retinal neovascularization. This finding suggests that retinal neural activity may precede and potentially influence the onset of pathological neovascularization.

Astrocytic expression of Yes-associated protein (YAP) regulates retinal neovascularization in a mouse model of oxygen-induced retinopathy.

Pathological neovascularization is the hallmark of many vascular oculopathies. There is still a great deal of uncertainty surrounding retinal neovascularization research. A working hypothesis that astrocytic Yes-associated protein (YAP) act as a key factor in retinal neovascularization was proposed. And our study was conducted to verified this hypothesis. In vivo, we successfully generated mice deficient in YAP in astrocytes (YAPf/f GFAP-Cre mice) and set up oxygen-induced retinopathy (OIR) model. Pathological neovascularization was evaluated by immunofluorescence staining and western blotting. In vitro, cultured retinal astrocytes were transfected with YAP siRNA. Enzyme-linked immunosorbent assay (ELISA) and western blot were used to determine the proteins in the supernatants and cells. The results showed that YAP was upregulated and activated in the OIR mice retinas. Conditional ablation of YAP aggravated pathological neovascularization, along with the upregulation of vascular endothelial growth factor A (VEGF-A) and monocyte chemoattractant protein-1 (MCP-1). Studies in vitro confirmed that the knockdown of YAP in astrocytes lead to increases in VEGF-A and MCP-1 levels, thus enhancing pro-angiogenic capability of YAP-deficit astrocytes. In conclusion, astrocytic YAP alleviates retinal pathological angiogenesis by inhibiting the over-activation of astrocytes, which suppresses excessive VEGF-A production and neuroinflammation.

Targeting adenosine A2A receptors for early intervention of retinopathy of prematurity.

Retinopathy of prematurity (ROP) continues to pose a significant threat to the vision of numerous children worldwide, primarily owing to the increased survival rates of premature infants. The pathologies of ROP are mainly linked to impaired vascularization as a result of hyperoxia, leading to subsequent neovascularization. Existing treatments, including anti-vascular endothelial growth factor (VEGF) therapies, have thus far been limited to addressing pathological angiogenesis at advanced ROP stages, inevitably leading to adverse side effects. Intervention to promote physiological angiogenesis during the initial stages could hold the potential to prevent ROP. Adenosine A2A receptors (A2AR) have been identified in various ocular cell types, exhibiting distinct densities and functionally intricate connections with oxygen metabolism. In this review, we discuss experimental evidence that strongly underscores the pivotal role of A2AR in ROP. In particular, A2AR blockade may represent an effective treatment strategy, mitigating retinal vascular loss by reversing hyperoxia-mediated cellular proliferation inhibition and curtailing hypoxia-mediated neovascularization in oxygen-induced retinopathy (OIR). These effects stem from the interplay of endothelium, neuronal and glial cells, and novel molecular pathways (notably promoting TGF-ß signaling) at the hyperoxia phase. We propose that pharmacological targeting of A2AR signaling may confer an early intervention for ROP with distinct therapeutic benefits and mechanisms than the anti-VEGF therapy.

Succinate-induced macrophage polarization and RBP4 secretion promote vascular sprouting in ocular neovascularization.

Pathological neovascularization is a pivotal biological process in wet age-related macular degeneration (AMD), retinopathy of prematurity (ROP) and proliferative diabetic retinopathy (PDR), in which macrophages (Mφs) play a key role. Tip cell specialization is critical in angiogenesis; however, its interconnection with the surrounding immune environment remains unclear. Succinate is an intermediate in the tricarboxylic acid (TCA) cycle and was significantly elevated in patients with wet AMD by metabolomics. Advanced experiments revealed that SUCNR1 expression in Mφ and M2 polarization was detected in abnormal vessels of choroidal neovascularization (CNV) and oxygen-induced retinopathy (OIR) models. Succinate-induced M2 polarization via SUCNR1, which facilitated vascular endothelial cell (EC) migration, invasion, and tubulation, thus promoting angiogenesis in pathological neovascularization. Furthermore, evidence indicated that succinate triggered the release of RBP4 from Mφs into the surroundings to regulate endothelial sprouting and pathological angiogenesis via VEGFR2, a marker of tip cell formation. In conclusion, our results suggest that succinate represents a novel class of vasculature-inducing factors that modulate Mφ polarization and the RBP4/VEGFR2 pathway to induce pathological angiogenic signaling through tip cell specialization.

Role of acyl-coenzyme A: cholesterol transferase 1 (ACAT1) in retinal neovascularization.

BACKGROUND: We have investigated the efficacy of a new strategy to limit pathological retinal neovascularization (RNV) during ischemic retinopathy by targeting the cholesterol metabolizing enzyme acyl-coenzyme A: cholesterol transferase 1 (ACAT1). Dyslipidemia and cholesterol accumulation have been strongly implicated in promoting subretinal NV. However, little is known about the role of cholesterol metabolism in RNV. Here, we tested the effects of inhibiting ACAT1 on pathological RNV in the mouse model of oxygen-induced retinopathy (OIR). METHODS: In vivo studies used knockout mice that lack the receptor for LDL cholesterol (LDLR-/-) and wild-type mice. The wild-type mice were treated with a specific inhibitor of ACAT1, K604 (10 mg/kg, i.p) or vehicle (PBS) during OIR. In vitro studies used human microglia exposed to oxygen-glucose deprivation (OGD) and treated with the ACAT1 inhibitor (1 µM) or PBS. RESULTS: Analysis of OIR retinas showed that increased expression of inflammatory mediators and pathological RNV were associated with significant increases in expression of the LDLR, increased accumulation of neutral lipids, and formation of toxic levels of cholesterol ester (CE). Deletion of the LDLR completely blocked OIR-induced RNV and significantly reduced the AVA. The OIR-induced increase in CE formation was accompanied by significant increases in expression of ACAT1, VEGF and inflammatory factors (TREM1 and MCSF) (p < 0.05). ACAT1 was co-localized with TREM1, MCSF, and macrophage/microglia makers (F4/80 and Iba1) in areas of RNV. Treatment with K604 prevented retinal accumulation of neutral lipids and CE formation, inhibited RNV, and decreased the AVA as compared to controls (p < 0.05). The treatment also blocked upregulation of LDLR, ACAT1, TREM1, MCSF, and inflammatory cytokines but did not alter VEGF expression. K604 treatment of microglia cells also blocked the effects of OGD in increasing expression of ACAT1, TREM1, and MCSF without altering VEGF expression. CONCLUSIONS: OIR-induced RNV is closely associated with increases in lipid accumulation and CE formation along with increased expression of LDLR, ACAT1, TREM1, and MCSF. Inhibiting ACAT1 blocked these effects and limited RNV independently of alterations in VEGF expression. This pathway offers a novel strategy to limit vascular injury during ischemic retinopathy.

RhoA/ROCK Signaling Is Involved in Pathological Retinal Neovascularization.

OBJECTIVE: The aim of the study was to evaluate the effect of the RhoA/ROCK inhibitor Fasudil on retinal neovascularization (NV) in vivo and angiogenesis in vitro. METHODS: C57BL/6 was used to establish an OIR model. First, RhoA/ROCK expression was first examined and compared between OIR and healthy controls. Then, we evaluated the effect of Fasudil on pathological retinal NV. Whole-mount retinal staining was performed. The percentage of NV area, the number of neovascular tufts (NVT), and branch points (BP) were quantified. Finally, human umbilical vein endothelial cells (HUVECs) were used to investigate the effect of Fasudil on angiogenesis. RESULTS: Real-time PCR and Western blotting showed that ROCK expression in retinal tissue was statistically upregulated in OIR. Furthermore, we found that Fasudil attenuated the percentage of NV area, the number of NVT, and BP significantly. In addition, Fasudil could suppress the proliferation and migration of HUVECs induced by VEGF. CONCLUSIONS: RhoA/ROCK might be involved in the pathogenesis of OIR. And its inhibitor Fasudil could suppress retinal NV in vivo and angiogenesis in vitro. Fasudil may be a potential treatment strategy for retinal vascular diseases.

Down-regulation of HuR inhibits pathological angiogenesis in oxygen-induced retinopathy.

HuR (also known as ELAV1), a ubiquitous RNA-binding protein, is implicated in the pathogenesis of diverse diseases via the mechanism of post-transcriptional regulation. Whether it is involved in pathological angiogenesis in oxygen-induced retinopathy is not clear. In this study, we detected HuR expression was increased in the retina of mouse model of oxygen-induced retinopathy (OIR) as well as in vascular endothelial cells exposed to hypoxia. With gain-of-function and loss-of-function studies using adenovirus infection, we found HuR over-expression promoted while HuR knockdown inhibited the migration, proliferation and tube formation of vascular endothelial cells. Moreover, HuR regulated the expression of VEGFA in vascular endothelial cells. We also found the retinal pathological angiogenesis in mouse OIR model was greatly reduced with HuR knockdown using recombinant AAV expressing HuR specific shRNA which was administered by intravitreal injection. The results of this study suggest HuR is involved in pathological angiogenesis via regulating angiogenic behaviors of endothelial cells, providing a potential target for the treatment of retinopathy of prematurity.

AMP kinase activation by Omega-3 polyunsaturated fatty acid protects the retina against ischemic insult: An in vitro and in vivo study.

PURPOSE: To investigate the possible beneficial effects of omega-3 polyunsaturated fatty acids (ω3-PUFAs) in ischemic retinal angiogenesis and whether AMP-activated protein kinase (AMPK) is involved. METHODS: Human retinal microvascular endothelial cells (hRMECs) were exposed to dimethyloxalylglycine (DMOG), a hypoxia-inducible factor hydroxylase inhibitor, in the presence or absence of docosahexaenoic acid (DHA) and small interfering RNA (siRNA) for AMPKα for 24 h. Ischemic factors, endothelial mesenchymal transition marker, endothelial barrier integrity, cell migration, and tube formation were evaluated. Neonatal AMPKα2-/- and control wild-type (WT) mice were submitted to an oxygen-induced retinopathy (OIR) protocol; their nursing mother mice were either fed ω3-PUFAs or not. In the end, ischemic markers and endothelial cell proliferation were evaluated in neonatal mouse retinal tissue through immunohistochemical or immunofluorescent assays among all studied groups. RESULTS: Cells exposed to DMOG displayed increased expressions of hypoxic and endothelial mesenchymal transition (vimentin) markers and barrier disarrangement of Zonula Occludens-1 compared to the control, accompanied by increased cellular migration and tube formation (p < 0.05). AMPK activity was significantly decreased. Supplementation with DHA restored the mentioned alterations compared to DMOG (p<0.05). In siRNAAMPKα-treated cells, the beneficial effects observed with DHA were abolished. DHA upregulated G-protein receptor-120 (GPR120), which promptly increased intracellular levels of calcium (p ≤ 0.001), which consequently increased Calcium/calmodulin-dependent protein kinase kinase ß expression (CaMKKß) thus phosphorylating AMPKThr172. AMPKα2-/- and wild-type (WT) OIR mice exhibited similar retinal ischemic changes, and the oral supplementation with ω3-PUFA efficiently prevented the noticed ischemic alterations only in WT mice, suggesting that AMPKα2 is pivotal in the protective effects of ω3-PUFA. CONCLUSIONS: ω3-PUFAs protect the retina from the effects of ischemic conditions, and this effect occurs via the GPR120-CaMKKß-AMPK axis. A better understanding of this mechanism might improve the control of pathological angiogenesis in retinal ischemic diseases.

Ferrostatin-1 attenuates pathological angiogenesis in oxygen-induced retinopathy via inhibition of ferroptosis.

Retinopathy of prematurity (ROP) is a vision-threatening ocular disease that occurs in premature infants, but the underlying mechanism is still unclear. Since oxidative stress has been well documented in the ROP development, we aimed to investigate whether ferroptosis, a new type of cell death characterized by lipid peroxidation and iron overload, is also involved in ROP. We detected the lipid peroxidation, oxidative stress and the expression of ferroptosis markers in the retina of mouse model of oxygen-induced retinopathy. After ferroptosis inhibitor, ferrostatin-1, was administered by intravitreal injection, ferroptosis marker, lipid peroxidation, retinal vasculature and glial cell activation were examined. We found decreased expression of SLC7A11 and GPX4, increased expression of FTH1 and TFRC, as well as increase of lipid peroxidation in the retina of OIR mice. Ferrostatin-1 administration significantly reduced lipid peroxidation, and also reversed the change of ferroptosis marker. Neovascular area and avascular area were suppressed and the pathological vasculature changes including acellular vessels and ghost pericytes were decreased. Microglial cell and Müller cell activation was not evidently influenced by ferrostatin-1 treatment. Our findings suggest that ferroptosis is involved in the pathological angiogenesis and might be a promising target for ROP therapy.

SIRT2 inhibition attenuates the vasculopathy and vision impairment via Akt signaling in retinopathy of prematurity.

Despite decades of researches, the underlying mechanism of retinopathy of prematurity (ROP) remains unclear. The role of Sirt2, which is involved in both angiogenesis and inflammation, both pivotal in ROP, was investigated in an animal model of ROP known as oxygen-induced retinopathy (OIR). Our study found that Sirt2 was overexpressed and colocalized with microglia in OIR. Furthermore, it demonstrated that the level of Sirt2 was upregulated in hypoxia microglia BV-2 in vitro. Subsequently, our results elucidated that administration of the Sirt2 antagonist AGK2 attenuated the avascular and neovascular area and downregulated the expression of IGF-1. The phosphorylation of Akt and the expression of IGF-1 were upregulated in hypoxia BV-2 and conditional media collected from BV-2 under hypoxia promoted the migration and tube formation of retinal capillary endothelial cells, which were suppressed with AGK2. Notably, our findings are the first to demonstrate the deleterious role of Sirt2 in ROP, as Sirt2 inhibition led to the downregulation of Akt/IGF-1 and ameliorated vasculopathy, ultimately improving visual function. These results suggest that Sirt2 may be a promising therapeutic target for ROP.

TAK1 blockade as a therapy for retinal neovascularization.

Retinal neovascularization, or pathological angiogenesis in the retina, is a leading cause of blindness in developed countries. Transforming growth factor-ß-activated kinase 1 (TAK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) activated by TGF-ß1 and other proinflammatory cytokines. TAK1 is also a key mediator of proinflammatory signals and plays an important role in maintaining vascular integrity upon proinflammatory cytokine stimulation such as TNFα. However, its role in pathological angiogenesis, particularly in retinal neovascularization, remains unclear. Here, we investigate the regulatory role of TAK1 in human endothelial cells responding to inflammatory stimuli and in a rat model of oxygen-induced retinopathy (OIR) featured retinal neovascularization. Using TAK1 knockout human endothelial cells that subjected to inflammatory stimuli, transcriptome analysis revealed that TAK1 is required for activation of NFκB signaling and mediates its downstream gene expression related to endothelial activation and angiogenesis. Moreover, pharmacological inhibition of TAK1 by 5Z-7-oxozeaenol attenuated angiogenic activities of endothelial cells. Transcriptome analysis also revealed enrichment of TAK1-mediated NFκB signaling pathway in the retina of OIR rats and retinal neovascular membrane from patients with proliferative diabetic retinopathy. Intravitreal injection of 5Z-7-oxozeaenol significantly reduced hypoxia-induced inflammation and microglial activation, thus attenuating aberrant retinal angiogenesis in OIR rats. Our data suggest that inhibition of TAK1 may have therapeutic potential for the treatment of retinal neovascular pathologies.

The ß3 adrenoceptor in proliferative retinopathies: "Cinderella" steps out of its family shadow.

In the retina, hypoxic condition leads to overgrowing leaky vessels resulting in altered metabolic supply that may cause impaired visual function. Hypoxia-inducible factor-1 (HIF-1) is a central regulator of the retinal response to hypoxia by activating the transcription of numerous target genes, including vascular endothelium growth factor, which acts as a major player in retinal angiogenesis. In the present review, oxygen urge by the retina and its oxygen sensing systems including HIF-1 are discussed in respect to the role of the beta-adrenergic receptors (ß-ARs) and their pharmacologic manipulation in the vascular response to hypoxia. In the ß-AR family, ß1- and ß2-AR have long been attracting attention because their pharmacology is intensely used for human health, while ß3-AR, the third and last cloned receptor is no longer increasingly emerging as an attractive target for drug discovery. Here, ß3-AR, a main character in several organs including the heart, the adipose tissue and the urinary bladder, but so far a supporting actor in the retina, has been thoroughly examined in respect to its function in retinal response to hypoxia. In particular, its oxygen dependence has been taken as a key indicator of ß3-AR involvement in HIF-1-mediated responses to oxygen. Hence, the possibility of ß3-AR transcription by HIF-1 has been discussed from early circumstantial evidence to the recent demonstration that ß3-AR acts as a novel HIF-1 target gene by playing like a putative intermediary between oxygen levels and retinal vessel proliferation. Thus, targeting ß3-AR may implement the therapeutic armamentarium against neovascular pathologies of the eye.