Oxygen-induced Retinopathy Mice Research Articles

Upregulation of HMOX1 associated with M2 macrophage infiltration and ferroptosis in proliferative diabetic retinopathy

The roles of immune cell infiltration and ferroptosis in the progression of proliferative diabetic retinopathy (PDR) remain unclear. To identify upregulated molecules associated with immune infiltration and ferroptosis in PDR, GSE60436 and GSE102485 datasets were downloaded from the Gene Expression Omnibus (GEO). Genes associated with immune cell infiltration were examined through Weighted Gene Co-expression Network Analysis (WGCNA) and CIBERSORT algorithm. Common differentially expressed genes (DEGs) were intersected with ferroptosis-associated and immune cell infiltration-related genes. Localization of cellular expression was confirmed by single-cell analysis of GSE165784 dataset. Findings were validated by qRT-PCR, ELISA, Western blotting, and immunofluorescence staining. As a result, the infiltration of M2 macrophages was significantly elevated in fibrovascular membrane samples from PDR patients than the retinas of control subjects. Analysis of DEGs, M2 macrophage-related genes and ferroptosis-related genes identified three hub intersecting genes, TP53, HMOX1 and PPARA. qRT-PCR showed that HMOX1 was significantly higher in the oxygen-induced retinopathy (OIR) mouse model retinas than in controls. Single-cell analysis confirmed that HMOX1 was located in M2 macrophages. ELISA and western blotting revealed elevated levels of HMOX1 in the vitreous humor of PDR patients and OIR retinas, and immunofluorescence staining showed that HMOX1 co-localized with M2 macrophages in the retinas of OIR mice. This study offers novel insights into the mechanisms associated with immune cell infiltration and ferroptosis in PDR. HMOX1 expression correlated with M2 macrophage infiltration and ferroptosis, which may play a crucial role in PDR pathogenesis.

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

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. 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/6J) 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. 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. 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.

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.

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.

GBP2 inhibits pathological angiogenesis in the retina via the AKT/mTOR/VEGFA axis

Pathological retinal angiogenesis is not only the hallmark of retinopathies, but also a major cause of blindness. Guanylate binding protein 2 (GBP2) has been reported to be associated with retinal diseases such as diabetic retinopathy and hypoxic retinopathy. However, GBP2-mediated pathological retinal angiogenesis remains largely unknown. The present study aimed to investigate the role of GBP2 in pathological retinal angiogenesis and its underlying molecular mechanism. In this study, we established oxygen-induced retinopathy (OIR) mice model for in vivo study and hypoxia-induced angiogenesis in ARPE-19 cells for in vitro study. We demonstrated that GBP2 expression was markedly downregulated in the retina of mice with OIR and ARPE-19 cells treated with hypoxia, which was associated with pathological retinal angiogenesis. The regulatory mechanism of GBP2 in ARPE-19 cells was studied by GBP2 silencing and overexpression. The regulatory mechanism of GBP2 in the retina was investigated by overexpressing GBP2 in the retina of OIR mice. Mechanistically, GBP2 downregulated the expression and secretion of vascular endothelial growth factor (VEGFA) in ARPE-19 cells and retina of OIR mice. Interestingly, overexpression of GBP2 significantly inhibited neovascularization in OIR mice, conditioned medium of GBP2 overexpressing ARPE-19 cells inhibited angiogenesis in human umbilical vein endothelial cells (HUVECs). Furthermore, we confirmed that GBP2 downregulated VEGFA expression and angiogenesis by inhibiting the AKT/mTOR signaling pathway. Taken together, we concluded that GBP2 inhibited pathological retinal angiogenesis via the AKT/mTOR/VEGFA axis, thereby suggesting that GBP2 may be a therapeutic target for pathological retinal angiogenesis.

Decreasing mitochondrial fission ameliorates HIF-1α-dependent pathological retinal angiogenesis.

Angiogenesis plays a critical role in many pathological processes, including irreversible blindness in eye diseases such as retinopathy of prematurity. Endothelial mitochondria are dynamic organelles that undergo constant fusion and fission and are critical signalling hubs that modulate angiogenesis by coordinating reactive oxygen species (ROS) production and calcium signalling and metabolism. In this study, we investigated the role of mitochondrial dynamics in pathological retinal angiogenesis. We showed that treatment with vascular endothelial growth factor (VEGF; 20 ng/ml) induced mitochondrial fission in HUVECs by promoting the phosphorylation of dynamin-related protein 1 (DRP1). DRP1 knockdown or pretreatment with the DRP1 inhibitor Mdivi-1 (5 μM) blocked VEGF-induced cell migration, proliferation, and tube formation in HUVECs. We demonstrated that VEGF treatment increased mitochondrial ROS production in HUVECs, which was necessary for HIF-1α-dependent glycolysis, as well as proliferation, migration, and tube formation, and the inhibition of mitochondrial fission prevented VEGF-induced mitochondrial ROS production. In an oxygen-induced retinopathy (OIR) mouse model, we found that active DRP1 was highly expressed in endothelial cells in neovascular tufts. The administration of Mdivi-1 (10 mg·kg-1·d-1, i.p.) for three days from postnatal day (P) 13 until P15 significantly alleviated pathological angiogenesis in the retina. Our results suggest that targeting mitochondrial fission may be a therapeutic strategy for proliferative retinopathies and other diseases that are dependent on pathological angiogenesis.

Photoreceptors inhibit pathological retinal angiogenesis through transcriptional regulation of Adam17 via c-Fos.

Pathological retinal angiogenesis profoundly impacts visual function in vascular eye diseases, such as retinopathy of prematurity (ROP) in preterm infants and age-related macular degeneration in the elderly. While the involvement of photoreceptors in these diseases is recognized, the underlying mechanisms remain unclear. This study delved into the pivotal role of photoreceptors in regulating abnormal retinal blood vessel growth using an oxygen-induced retinopathy (OIR) mouse model through the c-Fos/A disintegrin and metalloprotease 17 (Adam17) axis. Our findings revealed a significant induction of c-Fos expression in rod photoreceptors, and c-Fos depletion in these cells inhibited pathological neovascularization and reduced blood vessel leakage in the OIR mouse model. Mechanistically, c-Fos directly regulated the transcription of Adam17 a shedding protease responsible for the production of bioactive molecules involved in inflammation, angiogenesis, and cell adhesion and migration. Furthermore, we demonstrated the therapeutic potential by using an adeno-associated virus carrying a rod photoreceptor-specific short hairpin RNA against c-fos which effectively mitigated abnormal retinal blood vessel overgrowth, restored retinal thickness, and improved electroretinographic (ERG) responses. In conclusion, this study highlights the significance of photoreceptor c-Fos in ROP pathology, offering a novel perspective for the treatment of this disease.

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.

Amelioration of oxygen-induced retinopathy in neonatal mice with fetal growth restriction.

Introduction: With the aim of optimizing the balance of maintaining a safe oxygen saturation and reducing the risk of retinopathy of prematurity in human neonates with fetal growth restriction (FGR), the present study investigated the distinct effects of oxygen supplementation on the retinal neovasculature using a murine premature neonatal oxygen-induced retinopathy (OIR) model with or without fetal growth restriction. Methods: For comparison with normal birth-weight neonates, maternal low-protein diet-induced FGR neonates were subjected to fluctuating oxygen levels to generate oxygen-induced retinopathy. The retinal neovasculature was histologically evaluated, and comprehensive transcriptome analysis was conducted. Results: Compared to OIR neonates with normal birth weight, significant amelioration of the neovasculature, as indicated by decreases in the number of branch junctions, vascular distribution, maximal vascular radius and microaneurysm-like tufts, was observed in OIR mice with FGR. The results of retinal RNA-sequencing revealed downregulation of angiogenic factors that trigger pathological retinal neovascularization, such as the mitogen-activated protein kinase pathway and corresponding upstream signaling pathways in OIR mice with FGR. Conclusion: Our findings demonstrated that FGR neonates have a higher capacity for retinal oxygen stress, and the risk of OIR development is attenuated compared to that in mature neonates with normal birth weight.

M2 microglia-derived exosomes promote vascular remodeling in diabetic retinopathy

Diabetic retinopathy (DR) is a vision-threatening diabetic complication that is characterized by microvasculature impairment and immune dysfunction. The present study demonstrated that M2 microglia intensively participated in retinal microangiopathy in human diabetic proliferative membranes, mice retinas, retinas of mice with oxygen-induced retinopathy (OIR) mice, and retinas of streptozotocin-induced DR mice. Further in vivo and in vitro experiments showed that exosomes derived from M2 polarized microglia (M2-exo) could reduce pericyte apoptosis and promote endothelial cell proliferation, thereby promoting vascular remodeling and reducing vascular leakage from the diabetic retina. These effects were further enhanced by M2-exo that facilitated M2 polarization of retinal microglia. Collectively, the study demonstrated the capability of M2-exo to induce retinal microvascular remodeling, which may provide a new therapeutic strategy for the treatment of DR.Graphical

The Anti-Diabetic Drug Metformin Suppresses Pathological Retinal Angiogenesis via Blocking the mTORC1 Signaling Pathway in Mice (Metformin Suppresses Pathological Angiogenesis)

Purpose Metformin, a biguanide antihyperglycemic drug, can exert various beneficial effects in addition to its glucose-lowering effect. The effects of metformin are mainly mediated by AMP-activated protein kinase (AMPK)-dependent pathway. AMPK activation interferes with the action of the mammalian target of rapamycin complex 1 (mTORC1), and blockade of mTORC1 pathway suppresses pathological retinal angiogenesis. Therefore, in this study, we examined the effects of metformin on pathological angiogenesis and mTORC1 activity in the retinas of mice with oxygen-induced retinopathy (OIR). Methods OIR was induced by exposing the mice to 80% oxygen from postnatal day (P) 7 to P10. The OIR mice were treated with metformin, rapamycin (an inhibitor of mTORC1), or the vehicle from P10 to P12 or P14. The formation of neovascular tufts, revascularization in the central avascular areas, expression of vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) 2, and phosphorylated ribosomal protein S6 (pS6), a downstream indicator of mTORC1 activity, were evaluated at P10, P13, or P15. Results Neovascular tufts and vascular growth in the central avascular areas were observed in the retinas of P15 OIR mice. The formation of neovascular tufts, but not the revascularization in the central avascular areas, was attenuated by metformin administration from P10 to P14. Metformin had no significant inhibitory effect on the expression of VEGF and VEGFR2, but it reduced the pS6 immunoreactivity in vascular cells at the sites of angiogenesis. Rapamycin completely blocked the phosphorylation of ribosomal protein S6 and markedly reduced the formation of neovascular tufts. Conclusions These results suggest that metformin partially suppresses the formation of neovascular tufts on the retinal surface by blocking the mTORC1 signaling pathway. Metformin may exert beneficial effects against the progression of ocular diseases in which abnormal angiogenesis is associated with the pathogenesis.

Acrizanib as a Novel Therapeutic Agent for Fundus Neovascularization via Inhibitory Phosphorylation of VEGFR2.

The present study aimed to evaluate the effect of acrizanib, a small molecule inhibitor targeting vascular endothelial growth factor receptor 2 (VEGFR2), on physiological angiogenesis and pathological neovascularization in the eye and to explore the underlying molecular mechanisms. We investigated the potential role of acrizanib in physiological angiogenesis using C57BL/6J newborn mice, and pathological angiogenesis using the mouse oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) models. Moreover, vascular endothelial growth factor (VEGF)-treated human umbilical vein endothelial cells (HUVECs) were used as an in vitro model for studying the molecular mechanism underlying acrizanib's antiangiogenic effects. The intravitreal injection of acrizanib did not show a considerable impact on physiological angiogenesis and retinal thickness, indicating a potentially favorable safety profile. In the mouse models of OIR and CNV, acrizanib showed promising results in reducing pathological neovascularization, inflammation, and vascular leakage, indicating its potential efficacy against pathological angiogenesis. Consistent with in vivo results, acrizanib blunted angiogenic events in VEGF-treated HUVECs such as proliferation, migration, and tube formation. Furthermore, acrizanib inhibited the multisite phosphorylation of VEGFR2 to varying degrees and the activation of its downstream signal pathways in VEGF-treated HUVECs. This study suggested the potential efficacy and safety of acrizanib in suppressing fundus neovascularization. Acrizanib functioned through inhibiting multiple phosphorylation sites of VEGFR2 in endothelial cells to different degrees. These results indicated that acrizanib might hold promise as a potential candidate for the treatment of ocular vascular diseases.

G protein subunit alpha i2's pivotal role in angiogenesis.

Here we explored the potential role of Gαi2 (G protein subunit alpha i2) in endothelial cell function and angiogenesis. Methods: Genetic methodologies such as shRNA, CRISPR/Cas9, dominant negative mutation, and overexpression were utilized to modify Gαi2 expression or regulate its function. Their effects on endothelial cell functions were assessed in vitro. In vivo, the endothelial-specific Gαi2 shRNA adeno-associated virus (AAV) was utilized to silence Gαi2 expression. The impact of this suppression on retinal angiogenesis in control mice and streptozotocin (STZ)-induced diabetic retinopathy (DR) mice was analyzed. Results: Analysis of single-cell RNA sequencing data revealed Gαi2 (GNAI2) was predominantly expressed in retinal endothelial cells and expression was increased in retinal endothelial cells following oxygen-induced retinopathy (OIR) in mice. Moreover, transcriptome analysis linking Gαi2 to angiogenesis-related processes/pathways, supported by increased Gαi2 expression in experimental OIR mouse retinas, highlighted its possible role in angiogenesis. In various endothelial cell types, shRNA-induced silencing and CRISPR/Cas9-mediated knockout (KO) of Gαi2 resulted in substantial reductions in cell proliferation, migration, invasion, and capillary tube formation. Conversely, Gαi2 over-expression in endothelial cells induced pro-angiogenic activities, enhancing cell proliferation, migration, invasion, and capillary tube formation. Furthermore, our investigation revealed a crucial role of Gαi2 in NFAT (nuclear factor of activated T cells) activation, as evidenced by the down-regulation of NFAT-luciferase reporter activity and pro-angiogenesis NFAT-targeted genes (Egr3, CXCR7, and RND1) in Gαi2-silenced or -KO HUVECs, which were up-regulated in Gαi2-overexpressing endothelial cells. Expression of a dominant negative Gαi2 mutation (S48C) also down-regulated NFAT-targeted genes, slowing proliferation, migration, invasion, and capillary tube formation in HUVECs. Importantly, in vivo experiments revealed that endothelial Gαi2 knockdown inhibited retinal angiogenesis in mice, with a concomitant down-regulation of NFAT-targeted genes in mouse retinal tissue. In contrast, Gαi2 over-expression in endothelial cells enhanced retinal angiogenesis in mice. Single-cell RNA sequencing data confirmed increased levels of Gαi2 specifically in retinal endothelial cells of mice with streptozotocin (STZ)-induced diabetic retinopathy (DR). Importantly, endothelial Gαi2 silencing ameliorated retinal pathological angiogenesis in DR mice. Conclusion: Our study highlights a critical role for Gαi2 in NFAT activation, endothelial cell activation and angiogenesis, offering valuable insights into potential therapeutic strategies for modulating these processes.

Transcriptional Comparison of Human and Murine Retinal Neovascularization.

Retinal neovascularization (RNV) is the leading cause of vision loss in diseases like proliferative diabetic retinopathy (PDR). A significant failure rate of current treatments indicates the need for novel treatment targets. Animal models are crucial in this process, but current diabetic retinopathy models do not develop RNV. Although the nondiabetic oxygen-induced retinopathy (OIR) mouse model is used to study RNV development, it is largely unknown how closely it resembles human PDR. We therefore performed RNA sequencing on murine (C57BL/6J) OIR retinas (n = 14) and human PDR RNV membranes (n = 7) extracted during vitrectomy, each with reference to control tissue (n=13/10). Differentially expressed genes (DEG) and associated biological processes were analyzed and compared between human and murine RNV to assess molecular overlap and identify phylogenetically conserved factors. In total, 213 murine- and 1223 human-specific factors were upregulated with a small overlap of 94 DEG (7% of human DEG), although similar biological processes such as angiogenesis, regulation of immune response, and extracellular matrix organization were activated in both species. Phylogenetically conserved mediators included ANGPT2, S100A8, MCAM, EDNRA, and CCR7. Even though few individual genes were upregulated simultaneously in both species, similar biological processes appeared to be activated. These findings demonstrate the potential and limitations of the OIR model to study human PDR and identify phylogenetically conserved potential treatment targets for PDR.

Aurora kinase B disruption suppresses pathological retinal angiogenesis by affecting cell cycle progression

PurposeThe detrimental effects of pathological angiogenesis on the visual function are indisputable. Within a prominent role in chromosome segregation and tumor progression, aurora kinase B (AURKB) assumes a prominent role. However, its role in pathological retinal angiogenesis remains unclear. This study explores this latent mechanism. MethodsTo inhibit AURKB expression, we designed specific small interfering RNAs targeting AURKB and transfected them into vascular endothelial cells. Barasertib was selected as the AURKB inhibitor. The anti-angiogenic effects of both AURKB siRNA and barasertib were assessed in vitro by cell proliferation, transwell migration, and tube formation. To evaluate the angiogentic effects of AURKB in vivo, neonatal mice were exposed to 75% oxygen followed by normoxic repositioning to establish an oxygen-induced retinopathy (OIR) model. Subsequently, phosphate-buffered saline and barasertib were administered into OIR mice via intravitreal injection. The effects of AURKB on cell cycle proteins were determined by western blot analysis. ResultsWe found that AURKB was overexpressed during pathological angiogenesis. AURKB siRNA and barasertib significantly inhibited endothelial cell proliferation, migration, and tube formation in vitro. Furthermore, AURKB inhibition attenuated retinal angiogenesis in the OIR model. A possible mechanism is the disruption of cell cycle by AURKB inhibition. ConclusionIn conclusion, AURKB significantly influenced pathological retinal angiogenesis, thereby presenting a promising therapeutic target in ocular neovascular diseases.

IL1R2 promotes retinal angiogenesis to participate in retinopathy of prematurity by activating the HIF1α/PFKFB3 pathway

Retinopathy of prematurity (ROP) is the leading cause of blindness in children, but there is no safe and effective treatment available. Interleukin-1 receptor type 2 (IL1R2) acts as a decoy receptor for IL-1 may affect ROP progression. This study aimed to investigate the role of IL1R2 in ROP. A microglial cell model was established under hypoxia conditions and co-cultured with choroidal endothelial cells, while an oxygen-induced retinopathy (OIR) model was also established. Microglial activation and IL1R2 levels in retinal tissues were analyzed using immunofluorescence assay. Endothelial cell migration was evaluated by Transwell assay and scratch test, angiogenesis was assessed using ELISA and tube formation assay, and proliferation was evaluated by EdU assay. The HIF1α/PFKFB3 pathway was analyzed by western blot. We observed that IL1R2 expression was predicted to be upregulated in ROP and was increased in hypoxia-treated BV2 cells. Additionally, IL1R2 levels were upregulated in the retinal tissues of OIR mice and correlated with microglial activation. In vitro experiments, we found that hypoxia promoted endothelial cell migration, angiogenesis, proliferation, and activated the HIF1α/PFKFB3 pathway, which were rescued by IL1R2 knockdown. Moreover, NHWD-870 (a HIF1α/PFKFB3 pathway inhibitor) suppressed endothelial cell migration, angiogenesis, and proliferation induced by IL1R2 overexpression. In conclusion, IL1R2 facilitates the migration, angiogenesis, and proliferation of choroidal endothelial cells by activating the HIF1α/PFKFB3 pathway to regulate ROP progression.

Method to Regulate Monocyte Function by Silencing HIF-1α mRNA in a Model of Retinal Neovascularization.

Circulating monocytes migrate into the retina in response to inflammation and neovascularization. Furthermore, under inflammatory conditions such as diabetes, healthy monocytes become activated in the circulation. However, the contribution of activated monocytes to neovascularization is largely unknown. HIF-1α has been shown to contribute to the pathogenesis of neovascularization. We describe here the synthesis of a hybrid nanomaterial for targeted delivery and gene silencing in activated monocytes that are associated with pathological neovascularization. To test the gene silencing ability of AS-shRNA-lipids in vitro, we used the probe to inhibit HIF-1α mRNA induced in mouse monocytes by exposing them to hypoxia. In addition, we tested AS-shRNA-lipids for inhibition of neovascularization in vivo using the mouse model of oxygen-induced retinopathy (OIR). Significant reduction of neovascularization was achieved in mouse OIR by targeting activated monocytes using intraperitoneal injections of AS-shRNA-lipids. Expression of HIF-1α and CD14 mRNA were both inhibited in circulating cells, suggesting normalization of the activated monocytes in P17 OIR animals treated with AS-shRNA-lipids. We hypothesized that inhibition of HIF-1α mRNA in activated monocytes may have a direct impact on VEGF expression in the retinal tissues in vivo. We observed that VEGF mRNA expression was inhibited in P17 retinal tissues after systemic treatment with HIF-1α-targeted AS-shRNA-lipids. These findings may provide a framework for a strategy to inhibit retinal neovascularization by targeting circulating activated monocytes.

AAV2-antiVEGFscFv gene therapy for retinal neovascularization

Retinal neovascularization (NV) may lead to irreversible vision impairment, the main treatment for which is the inhibition of vascular endothelial growth factor (VEGF). Existing drugs show limited clinical benefits because of their high prices and short half-lives, which increase the financial burden and medical risks to patients. Gene therapy on the basis of adeno-associated viruses is a promising approach to overcome these limitations because ofthe nonintegrative nature, low immunogenicity, and potential long-term gene expression of adeno-associated viruses. In this study, we constructed a novel recombinant adeno-associated virus with the single-chain fragment variable (scFv) fragment ofthe anti-VEGF antibody, AAV2-antiVEGFscFv, consisting ofthe VH and VL structural domains of IgG. AAV2-antiVEGFscFv effectively inhibited NV, retinal leakage, and retinal detachment in oxygen-induced retinopathy (OIR) mice, Tet/opsin/VEGF double-transgenic mice, and VEGF-induced rabbit NV models. AAV2-antiVEGFscFv also significantly suppressed VEGF-induced inflammation. Furthermore, we showed that AAV2-antiVEGFscFv could be sustainably expressed for a prolonged period and exhibited low immunotoxicity invivo. This study indicates that AAV2-antiVEGFscFv could be a potential approach for NV treatment and provides strong support for preclinical research.

HIF-1α Reduction by Lowering Intraocular Pressure Alleviated Retinal Neovascularization.

Hypoxia-induced retinal neovascularization is a leading cause of blindness worldwide. Oxygen-induced retinopathy (OIR) mouse, a well-established angiogenesis model, has been extensively used to evaluate the effect of anti-angiogenic agents through intravitreal injection. Here, we serendipitously found that the needles used for intravitreal injection caused an unexpected "anti-angiogenic" effect in the OIR mice. To evaluate the effects of various intravitreal puncture sizes on retinal neovascularization and explore the potential underlying mechanism, intravitreal punctures using 0.5 mm (25 G), 0.3 mm (30 G), or 0.21 mm (33 G) needles were performed in OIR mice. Compared with 0.3 mm and 0.21 mm puncture, the 0.5 mm puncture remarkably suppressed the formation of pathological angiogenesis, inhibited vascular leakage, and remodeled the retinal vasculature. Mechanistically, the 0.5 mm puncture induced a substantial reduction in intraocular pressure (IOP), leading to an improvement in oxygen partial pressure (pO2) and significant reduction in Hif1a expression, resulting in resolution of angiogenic and inflammatory responses. Furthermore, IOP-lowering drugs, Travatan or Azarga, also promoted the alleviation of hypoxia and exhibited a potent anti-angiogenesis efficacy. Our study revealed an acute and significant reduction in IOP caused by a large puncture, which could remarkably suppress HIF-1α-mediated retinal neovascularization, indicating that lowering IOP may be a promising therapeutic avenue for treating retinal neovascular diseases.

METTL3-Mediated RNA m6A Modification Regulates the Angiogenic Behaviors of Retinal Endothelial Cells by Methylating MMP2 and TIE2.

N6-methyladenosine (m6A) is a commonly occurring modification of mRNAs, catalyzed by a complex containing methyltransferase like 3 (METTL3). Our research aims to explore how METTL3-dependent m6A modification affects the functions of retinal endothelial cells (RECs). An oxygen-induced retinopathy (OIR) mouse model was established, and RECs were isolated using magnetic beads method. Human retinal microvascular endothelial cells (HRMECs) were treated with normoxia (21% O2) or hypoxia (1% O2). Dot blot assay determined m6A modification levels. Quantitative RT-PCR and Western blot detected the mRNA and protein expression levels of the target candidates, respectively. Genes were knocked down by small interfering RNA transfection. Matrigel-based angiogenesis and transwell assays evaluated the abilities of endothelial tube formation and migration, respectively. Methylated RNA immunoprecipitation-qPCR determined the levels of m6A modification in the target genes. The m6A modification levels were significantly upregulated in the retinas and RECs of OIR mice. Exposure to hypoxia significantly elevated both METTL3 expression and m6A modification levels in HRMECs. METTL3 knockdown curtailed endothelial tube formation and migration in vitro under both normoxic and hypoxic conditions. Concurrently, this knockdown in HRMECs resulted in reduced m6A modification levels of MMP2 and TIE2 transcripts, subsequently leading to a decrease in their respective protein expressions. Notably, knockdown of MMP2 and TIE2 also markedly inhibited the angiogenic activities of HRMECs. METTL3-mediated m6A modification promotes the angiogenic behaviors of RECs by targeting MMP2 and TIE2, suggesting its significance in retinal angiogenesis and METTL3 as a potential therapeutic target.