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Choroidal neovascularization (CNV) is a leading cause of visual impairment in wet age-related macular degeneration (wAMD). Recent investigations have validated the potential of reducing glutamine synthetase (GS) to inhibit neovascularization formation, offering prospects for treating various neovascularization-related diseases. In this study, we devised a CRISPR/Cas9 delivery system employing the nucleic acid aptamer AS1411 as a targeting moiety and exosome-liposome hybrid nanoparticles as carriers (CAELN). Exploiting the binding affinity between AS1411 and nucleolin on endothelial cell surfaces, the delivery system was engineered to specifically target the glutamine synthetase gene (GLUL), thereby attenuating GS levels and continuously suppressing CNV. CAELN exhibited spherical and uniform dispersion. In vitro cellular investigations demonstrated gene editing efficiencies of CAELN ranging from 42.05 to 55.02% and its capacity to inhibit neovascularization in HUVEC cells. Moreover, in vivo pharmacodynamic studies conducted in CNV rabbits revealed efficacy of CAELN in restoring the thickness of intra- and extranuclear tissues. The findings suggest that GS is a novel target for the inhibition of pathological CNV, while the development of AS1411-modified exosome-liposome hybrid nanoparticles represents a novel delivery method for the treatment of neovascular-related diseases., Competing Interests: Declarations Ethics approval and consent to participate All the experimental procedures that had been conducted adhered to ethical guidelines and were approved by the Laboratory Animal Ethics and Welfare Committee of Jilin University (Approval No. SY202306034). Consent for publication All authors read and approved the final manuscript. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Abnormal growth of blood vessels (choroidal neovascularization) can lead to age-related macular degeneration (AMD) and eventually cause vision loss due to detachment of the retinal pigmented epithelium. This indicates that choroidal neovascularization is important for the treatment of AMD. The circadian clock in the mammalian retina is responsible for controlling various functions of the retina, enabling it to adjust to changes in light and darkness. Recent studies have revealed a potential connection between the circadian clock and eye diseases, although a cause-and-effect relationship has not been definitively established. C57BL/6J male mice (aged 6 weeks) were randomly divided into two groups (Control group: 9:00-21:00 light period (300 lx); Jet lag group: 8-hour phase advance once every 4 days). A laser-induced CNV model was created after 2 weeks of feeding in a controlled or jet-lagged environment. Then, full transcriptome sequencing was performed. The pathways regulated by differentially expressed mRNAs were identified by GO analysis and GSEA. Further protein networks were constructed with the STRING database and Cytoscape software. WGCNA was used to further explore the co-expression modules of these differential genes and the correlation between these differential genes and phenotypes. ceRNA networks were constructed with miRanda and TargetScan. The pathways associated with the overlapping differentially expressed mRNAs in the ceRNA network were identified, and the hub genes were validated by qPCR. A total of 661 important DEGs, 31 differentially expressed miRNAs, 106 differentially expressed lncRNAs and 87 differentially expressed circRNAs were identified. GO and GSEA showed that the upregulated DEGs were mainly involved in reproductive structure development and reproductive system development. The STRING database and Cytoscape were used to determine the protein interaction relationships of these DEGs. WGCNA divided the expression of these genes into several modules and screened the hub genes of each module separately. Furthermore, a ceRNA network was constructed. GO analysis and GSEA showed that these target DEmRNAs mainly function in wound healing, cell spreading, epiboly involved in wound healing, epiboly, and morphogenesis of an epithelial sheet. Finally, ten key genes were identified, and their expression patterns were confirmed by real-time qPCR. In this study, we investigated the regulatory mechanism of ceRNAs in choroidal neovascularization according to different light-dark cycles in the eyeball., (© 2024. The Author(s).)

Inflammasome activation is implicated in diseases of aberrant angiogenesis such as age-related macular degeneration (AMD), though its precise role in choroidal neovascularization (CNV), a characteristic pathology of advanced AMD, is ill-defined. Reports on inhibition of inflammasome constituents on CNV are variable and the precise role of inflammasome in mediating pathological angiogenesis is unclear. Historically, subretinal injection of inflammasome agonists alone has been used to investigate retinal pigmented epithelium (RPE) degeneration, while the laser photocoagulation model has been used to study pathological angiogenesis in a model of CNV. Here, we report that the simultaneous introduction of any of several disease-relevant inflammasome agonists (Alu or B2 RNA, Alu cDNA, or oligomerized amyloid β (1-40)) exacerbates laser-induced CNV. These activities were diminished or abrogated by genetic or pharmacological targeting of inflammasome signaling constituents including P2rx7, Nlrp3, caspase-1, caspase-11, and Myd88, as well as in myeloid-specific caspase-1 knockout mice. Alu RNA treatment induced inflammasome activation in macrophages within the CNV lesion, and increased accumulation of macrophages in an inflammasome-dependent manner. Finally, IL-1β neutralization prevented inflammasome agonist-induced chemotaxis, macrophage trafficking, and angiogenesis. Collectively, these observations support a model wherein inflammasome stimulation promotes and exacerbates CNV and may be a therapeutic target for diseases of angiogenesis such as neovascular AMD., Competing Interests: Declarations Competing interests J.A: co-founder of DiceRx, iVeena Holdings, iVeena Delivery Systems and Inflammasome Therapeutics, and, unrelated to this work, has been a consultant for Abbvie/Allergan, Boehringer-Ingelheim, Janssen, Olix Pharmaceuticals, Retinal Solutions, and Saksin LifeSciences. B.D.G: co-founder of DiceRx. J.A., S.W., B.D.G.: inventors on matter-related patent applications filed by the University of Virginia or Kentucky., (© 2024. The Author(s).)

Background: Pathological angiogenesis causes significant vision loss in neovascular age-related macular degeneration and other retinopathies with neovascularization (NV). Neuronal/glial-vascular interactions influence the release of angiogenic and neurotrophic factors. We hypothesized that botulinum neurotoxin serotype A (BoNT/A) modulates pathological endothelial cell proliferation through glial cell activation and growth factor release., Methods: A laser-induced choroidal NV (CNV) was employed to investigate the anti-angiogenic effects of BoNT/A. Fundus fluorescence angiography, immunohistochemistry, and real-time PCR were used to assess BoNT/A efficacy in inhibiting CNV and the molecular mechanisms underlying this inhibition. Neuronal and glial suppressor of cytokine signaling 3 (SOCS3) deficient mice were used to investigate the molecular mechanisms of BoNT/A in inhibiting CNV via SOCS3., Findings: In laser-induced CNV mice with intravitreal BoNT/A treatment, CNV lesions decreased > 30%; vascular leakage and retinal glial activation were suppressed; and Socs3 mRNA expression was induced while vascular endothelial growth factor A (Vegfa) mRNA expression was suppressed. The protective effects of BoNT/A on CNV development were diminished in mice lacking neuronal/glial SOCS3., Conclusion: BoNT/A suppressed laser-induced CNV and glial cell activation, in part through SOCS3 induction in neuronal/glial cells. BoNT/A treatment led to a decrease of pro-angiogenic factors, including VEGFA, highlighting the potential of BoNT/A as a therapeutic intervention for pathological angiogenesis in retinopathies., Competing Interests: Declarations Competing interests The authors declare no competing interests. Disclosures Y.S., L.E.H.S, and T.W. are inventors on patent applications filed by Boston Children’s Hospital. The remaining authors declare no competing interests., (© 2024. The Author(s).)

Neovascular age-related macular degeneration (nAMD) is characterized by abnormal blood vessel growth from the choroid, leading to complications and eventual blindness. Despite anti-VEGF therapy, subretinal fibrosis remains a major concern, as VEGF/VEGF receptor-2 (VEGFR2) signaling can contribute to both angiogenesis and fibrosis. For the identification of the aqueous humor proteome, we performed liquid chromatography with tandem mass spectrometry analysis. To investigate the potential therapeutic effects of targeting the VEGF signaling pathway using apatinib, a highly selective VEGFR2 tyrosine kinase inhibitor, this study employed in vitro (THP-1 conditioned media-treated ARPE-19 cells) and in vivo (laser-induced choroidal neovascularization mouse) models of nAMD. This study revealed elevated VEGFR2 protein levels in the aqueous humor of nAMD patients, suggesting a potential target to mitigate neovascularization and fibrosis in nAMD. Apatinib effectively reduced VEGFA and αSMA levels in both in vitro and in vivo models. Moreover, apatinib showed improvement in laser-induced subretinal hyper-reflective lesions. The action mechanism was linked to the inhibition of VEGFR2 activation, leading to the suppression of both angiogenesis and fibrosis through the downregulation of STAT3 phosphorylation. Therefore, the VEGFR2 signaling pathway appears to play a central role in the development of nAMD by regulating both angiogenesis and fibrosis., (© 2024. The Author(s).)

Neovascular age-related macular degeneration (nAMD), a leading cause of blindness in older adults, presents a challenging pathophysiology involving choroidal neovascularization (CNV) and retinal degeneration. Current treatments relying on intravitreal (IVT) administration of anti-angiogenic agents are costly and of moderate effectiveness. Metformin, the common anti-diabetic drug, has been associated with decreased odds of developing AMD. Studies have shown that metformin can mitigate cellular aging, neoangiogenesis, and inflammation across multiple diseases. This preclinical study assessed metformin's impact on vessel growth using choroidal explants before exploring IVT metformin's effects on laser-induced CNV and light-induced retinal degeneration in C57BL/6J and BALB/cJ mice, respectively. Metformin reduced new vessel growth in choroidal explants in a dose-dependent relationship. Following laser induction, IVT metformin suppressed CNV and decreased peripheral infiltration of IBA1 + macrophages/microglia. Furthermore, IVT metformin protected against retinal thinning in response to light-induced degeneration. IVT metformin downregulated genes in the choroid and retinal pigment epithelium which are associated with angiogenesis and inflammation, two key processes that drive nAMD progression. These findings underscore metformin's capacity as an anti-angiogenic and neuroprotective agent, demonstrating this drug's potential as an accessible option to help manage nAMD.

'Wet' age-related macular degeneration (AMD) is characterized by pathologic choroidal neovascularization (CNV) that destroys central vision. Abundant evidence points to inflammation and immune cell dysfunction in the progression of CNV in AMD. Mast cells are resident immune cells that control the inflammatory response. Mast cells accumulate and degranulate in the choroid of patients with AMD, suggesting they play a role in CNV. Activated mast cells secrete various biologically active mediators, including inflammatory cytokines and proteolytic enzymes such as tryptase. We investigated the role of mast cells in AMD using a model of CNV. Conditioned media from activated mast cells exerts proangiogenic effects on choroidal endothelial cells and choroidal explants. Laser-induced CNV in vivo was markedly attenuated in mice genetically depleted of mast cells (Kit W-sh/W-sh ) and in wild-type mice treated with mast cell stabilizer, ketotifen fumarate. Tryptase was found to elicit pronounced choroidal endothelial cell sprouting, migration and tubulogenesis; while tryptase inhibition diminished CNV. Transcriptomic analysis of laser-treated RPE/choroid complex revealed collagen catabolism and extracellular matrix (ECM) reorganization as significant events correlated in clusters of mast cell activation. Consistent with these analyses, compared to wildtype mice choroids of laser-treated mast cell-deficient mice displayed less ECM remodelling evaluated using collagen hybridizing peptide tissue binding. Findings herein provide strong support for mast cells as key players in the progression of pathologic choroidal angiogenesis and as potential therapeutic targets to prevent pathological neovascularization in 'wet' AMD., (© 2024. The Author(s).)

Vascular endothelial growth factor (VEGF) signaling is crucial for choroidal neovascularization (CNV), a major pathological feature of neovascular age-related macular degeneration (nAMD). Gene transcription of VEGF is mainly regulated by hypoxia-inducible factor 1-alpha (HIF-1α). The chromobox (CBX) family polycomb protein (Pc) subgroup includes CBX2, CBX4, CBX6, CBX7, and CBX8. CBX4 enhances hypoxia-induced VEGF expression and angiogenesis in hepatocellular carcinoma (HCC) cells by increasing HIF-1α's transcriptional activity. The objective of the study was to examine the functions of members of the CBX family Pc subgroup in choroidal vascular endothelial cells (CVECs) during CNV. CBX4 and CBX7 expression was up-regulated in hypoxic human choroidal vascular endothelial cells (HCVECs). In HCVECs, CBX7 facilitated HIF-1α transcription and expression, while CBX4 did not. In HCVECs, CBX7 stimulated HIF-1α's nuclear translocation and transcriptional activity, which in turn stimulated VEGF transcription and expression. The CBX7/HIF-1α/VEGF pathway promoted the migration, proliferation, and tube formation of HCVECs. The CBX7/HIF-1α/VEGF pathway was up-regulated in CVECs and in the mouse model with laser-induced CNV. Mouse CNV was lessened by the blockade of CBX7 through the down-regulation of HIF-1α/VEGF. In conclusion, CBX7 enhanced pro-angiogenic behaviors of hypoxic CVECs by up-regulating the HIF-1α/VEGF pathway, which contributing to the formation of mouse laser-induced CNV., Competing Interests: Declaration of competing interest All authors assert that there are no potential conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Rationale: Very-low-density lipoprotein receptor (VLDLR) involves in ocular neovascularization, a major cause of severe vision loss. However, the underlying molecular mechanisms were not completely clarified. Here, we aimed to investigate roles of circular RNAs (circRNAs) in VLDLR-associated ocular neovascularization., Methods: Vldlr knockout (Vldlr-/-, ko), Robo4 knockout (Robo4-/-, ko) and wild-type (WT) mice were used. Mouse model of oxygen induced retinopathy (OIR) and high-throughput sequence were performed to profile the differential expression of circRNA and transcripts. RNase R treatment, Sanger PCR sequencing and quantitative polymerase chain reaction (qPCR) were used to validate candidate circRNAs and their expression patterns. Choroidal sprouting assay ex vivo and laser induction choroid neovascularization were used to determine the expression and functions of QKI/CircSlc17a5 on choroidal neovascularization., Results: In macrophage and ocular tissues derived from Vldlr (Vldlr-/-,Vldlr ko) or Robo4 (Robo4-/-,Robo4 ko) deficiency as well as wild-type (WT) mice, Quaking (Qki) expression was significantly down-regulated in Vldlr deficiency compared to WT and Robo4 deficiency groups. Ectopic VLDLR expression or Reelin stimulation increased expression of QKI in bEnd.3 cells. Circular RNA sequencing uncovered that VLDLR regulated the biogenesis of certain circular RNAs, including the circSlc17a5. The number of Circular RNAs increased in mice treated with OIR. QKI mediated the biogenesis of circSlc17a5, which was an important regulator of choroidal angiogenesis., Conclusion: CircSlc17a5 regulated by VLDLR/QKI plays important roles in the choroidal angiogenesis., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

In the aging population, choroidal vessels grow through the Bruch's membrane, resulting in a loss of central vision due to choroidal neovascularization (CNV). During active neovascularization, CNV is associated with inappropriate levels of apoptosis in multiple cell types, including choroidal endothelial cells (ChECs). Bim is a pro-apoptotic member of the Bcl-2 family. It is essential for cell apoptosis due to exposure to drugs such as dexamethasone or decreased pro-survival factors, including vascular endothelial growth factor (VEGF). To better elucidate the cell autonomous contribution of Bim expression in the integrity and neovascularization of the choroidal vasculature, we isolated ChECs from wild-type and Bim-deficient (Bim -/- ) mice. ChECs lacking Bim expression demonstrated increased expression of VEGF, osteopontin, and the inflammatory cytokines Rantes/Ccl5 and IL6. Bim -/- ChECs were more proliferative and demonstrated an increased capacity to undergo capillary morphogenesis. Anti-VEGF had a diminished capacity to disrupt capillary morphogenesis in Bim -/- ChECs. In vivo, utilizing the mouse laser photocoagulation model, anti-VEGF treatment mitigated CNV in wild-type but not Bim -/- mice. We also tested other modalities that are thought to not require the intrinsic death pathway for their function and showed that propranolol, anti-CTGF, and the TSP1-mimetic peptide ABT898 mitigated CNV in mice lacking Bim expression to varying degrees. Thus, in ChECs, Bim expression could impact the effectiveness of treatment modalities that require the intrinsic death pathway to mitigate CNV.

To investigate quantitative associations between AI-assessed disease activity and optical coherence tomography angiography (OCTA)-derived parameters in patients with neovascular age-related macular degeneration (nAMD) undergoing anti-VEGF therapy. OCTA and SD-OCT images obtained from multicenter, randomized study data were evaluated. A deep learning algorithm (RetInSight) was used to detect and quantify macular fluid on SD-OCT. Mixed effects models were applied to evaluate correlations between fluid volumes, macular neovascularization (MNV)-type and OCTA-derived MNV parameters; lesion size (LS) and vessel area (NVA). 230 patients were included. A significant positive correlation was observed between SRF and NVA (estimate = 199.8 nl/mm 2 , p = 0.023), while a non-significant but negative correlation was found between SRF and LS (estimate = - 71.3 nl/mm 2 , p = 0.126). The presence of Type I and Type II MNV was associated with significantly less intraretinal fluid (IRF) compared to Type III MNV (estimate type I:- 52.1 nl, p = 0.019; estimate type II:- 51.7 nl, p = 0.021). A significant correlation was observed between pigment epithelial detachment (PED) and the interaction between NVA and LS (estimate:28.97 nl/mm 2 ; p = 0.012). Residual IRF at week 12 significantly correlated to baseline NVA (estimate:38.1 nl/mm 2 ; p = 0.015) and LS (estimate:- 22.6 nl/mm 2 ; p = 0.012). Fluid in different compartments demonstrated disparate associations with MNV OCTA features. While IRF at baseline was most pronounced in type III MNV, residual IRF was driven by neovascular MNV characteristics. Greater NVA in proportion to LS was associated with higher amounts of SRF and PED. The correlation between these parameters may represent MNV maturation and can be used as a biomarker for resolution of disease activity. AI-based OCT analysis allows for a deeper understanding of neovascular disease in AMD and the potential to adjust therapeutic strategies to optimize outcomes through precision medicine., (© 2024. The Author(s).)

8-arm PEG (polyethylene-glycol) is a highly promising nanoplatform due to its small size (<10 nm), ease-of-conjugation (many functionalized variants are readily available with "click-like" properties), biocompatibility, and optical inactivity. This study evaluates 8-arm PEG uptake into cells ( in vitro ) and localization and clearance in vasculature ( in vivo ) for targeting of choroidal neovascularization in mice, an animal model of macular degeneration. 8-arm PEG nanoparticles were labeled with fluorescein isothiocyanate (FITC) and functionalized in the absence or presence of pentameric Ar-Gly-Asp (RGD; 4 RGD motifs and a PGC linker), one of the most common peptide motifs used for active targeting. In vitro studies show that RGD-conjugated 8-arm PEG nanoparticles exhibit enhanced cellular uptake relative to non-RGD-conjugated control NPs at 34% ± 9%. Laser-induced choroidal neovascularization (CNV) was performed in a mouse model to measure 8-arm PEG localization and clearance to model macular degeneration lesions in vivo . It was determined that both RGD-conjugated and non-RGD-conjugated (nRGD) 8-arm PEG particles localized to CNV lesions, with a half-life around 24 h. In vivo experiments showed that RGD-conjugated nanoparticles exhibited enhanced localization by 15-20% relative to without RGD controls. Exhibiting a high rate of localization and fast clearance relative to larger nanoparticles, targeted 8-arm PEG nanoparticles with a conjugated RGD-peptide could be a promising modality for macular degeneration diagnosis and therapy.

Neovascular age-related macular degeneration (nvAMD) is the leading cause of blindness in the elderly population. Although it is known that nvAMD is associated with focal inflammation, understanding of the precise immune components governing this process remains limited. Here, we identified natural killer (NK) cells as a prominent lymphocyte population infiltrating the perivascular space of choroidal neovascularization (CNV) lesions in patients with nvAMD and in mouse models. Olink proteomic analysis and single-cell RNA sequencing combined with knockout studies demonstrated the involvement of C-C chemokine receptor 5 (CCR5) in NK cell recruitment and extravasation at the CNV sites of mice. Depletion of NK cells or inhibition of activating receptor NK group 2, member D (NKG2D) inhibited the formation of neutrophil extracellular traps, increased vascular leakage, and exacerbated pathological angiogenesis, indicating that NK cells restrain pathogenesis in this mouse model. Age is the strongest risk factor for AMD, and we show that NK cells from aged human donors exhibited a less cytotoxic phenotype. NK cells from old mice exhibited compromised protective effects in the CNV mouse model. In addition, interleukin-2 complex-mediated expansion of NK cells improved CNV formation in mice. Collectively, our study highlights NK cells as a potential therapeutic target for patients with nvAMD.

Age-related macular degeneration (AMD) causes severe blindness in the elderly due to choroidal neovascularization (CNV), which results from the dysfunction of the retinal pigment epithelium (RPE). While normal RPE depends exclusively on mitochondrial oxidative phosphorylation for energy production, the inflammatory conditions associated with metabolic reprogramming of the RPE play a pivotal role in CNV. Although mitochondrial pyruvate dehydrogenase kinase (PDK) is a central node of energy metabolism, its role in the development of CNV in neovascular AMD has not been investigated. In the present study, we used a laser-induced CNV mouse model to evaluate the effects of Pdk4 gene ablation and treatment with pan-PDK or specific PDK4 inhibitors on fluorescein angiography and CNV lesion area. Among PDK isoforms, only PDK4 was upregulated in the RPE of laser-induced CNV mice, and Pdk4 gene ablation attenuated CNV. Next, we evaluated mitochondrial changes mediated by PDK1-4 inhibition using siRNA or PDK inhibitors in inflammatory cytokine mixture (ICM)-treated primary human RPE (hRPE) cells. PDK4 silencing only in ICM-treated hRPE cells restored mitochondrial respiration and reduced inflammatory cytokine secretion. Likewise, GM10395, a specific PDK4 inhibitor, restored oxidative phosphorylation and decreased ICM-induced upregulation of inflammatory cytokine secretion. In a laser-induced CNV mouse model, GM10395 significantly alleviated CNV. Taken together, we demonstrate that specific PDK4 inhibition could be a therapeutic strategy for neovascular AMD by preventing mitochondrial metabolic reprogramming in the RPE under inflammatory conditions., (© 2024. The Author(s).)

Subretinal fibrosis is associated with worse visual outcomes in patients with neovascular age-related macular degeneration. As there is a lack of optimal biomarkers and no method that directly detects collagen in the back of the eye, novel tools that monitor fibrosis-related changes in neovascular age-related macular degeneration are needed. Here, using two mouse models (the laser-induced choroidal neovascularization model, and the JR5558 mouse presenting with spontaneous subretinal neovascularization with fibrosis), we imaged active fibrotic lesions using fluorescently labeled collagen hybridizing peptides (CHPs), short peptides that bind to single α-chain collagen structures during collagen remodeling. JR5558 retinal pigment epithelium/choroid flat mounts showed CHP co-staining with fibrosis and epithelial mesenchymal transition-related markers; additionally, CHP histopathology staining correlated with in vivo CHP imaging. After laser-induced choroidal neovascularization, in vivo CHP binding correlated with laser intensity, histopathology CHP and fibronectin staining. Laser-induced choroidal neovascularization showed decreased CHP intensity over time in healing/regressing versus active scars in vivo, whereas increased CHP binding correlated with elevated fibrosis in JR5558 mouse eyes with age. In bispecific angiopoietin 2/vascular endothelial growth factor antibody-treated JR5558 mice, CHPs detected significantly decreased collagen remodeling versus immunoglobulin G control. These results demonstrate the first use of CHPs to directly image remodeling collagen in the eye and as a potential clinical optical biomarker of active subretinal fibrosis associated with ocular neovascularization., (© 2024. The Author(s).)

Purpose: Intravitreal injection of anti-VEGF antibodies remains the primary therapy for exudative age-related macular degeneration (exAMD), although its efficacy is limited. Previous research has demonstrated that both a loss-of-function mutation of srr and the intravenous injection of a serine racemase inhibitor, L-aspartic acid β-hydroxamate (L-ABH), significantly inhibit laser-induced choroidal neovascularization (CNV) in mice. Given that L-ABH is a small molecule, this study investigated the effects of L-ABH administered via eye drops on CNV, aiming to develop a noninvasive treatment strategy for exAMD., Methods: CNV models in mice and rhesus macaques were established through laser photocoagulation. Seven monkeys were randomly assigned to receive either saline solution or L-ABH eye drops. Intraperitoneal or intravenous injection of fluorescein characterized CNV in both mice and monkeys. Fluorescein fundus angiography was used to assess leakage, whereas optical coherence tomography measured retinal thickness in the monkeys., Results: L-ABH eye drops significantly reduced fluorescein leakage in laser-injured mice (P < 0.001 compared to saline). In laser-injured rhesus macaques, the average percent changes in leakage areas treated with L-ABH were 2.5% ± 25.8% (P = 0.004) and 1.5% ± 75.7% (P = 0.023 compared to saline solution) on day 14 and day 28, respectively. However, L-ABH eye drops did not significantly affect the number of grade IV laser spots or retinal thickness, whereas bevacizumab did., Conclusions: This study demonstrates the potential efficacy of an SRR inhibitor in two animal models of laser-induced CNV., Translational Relevance: This represents the first investigation into the effects of topical delivery of an SRR inhibitor on CNV.

Age-related macular degeneration (AMD) is a common retinal neurodegenerative disease among the elderly. Neovascular AMD (nAMD), a leading cause of AMD-related blindness, involves choroidal neovascularization (CNV), which can be suppressed by anti-angiogenic treatments. However, current CNV treatments do not work in all nAMD patients. Here we investigate a novel target for AMD. Granzyme B (GzmB) is a serine protease that promotes aging, chronic inflammation and vascular permeability through the degradation of the extracellular matrix (ECM) and tight junctions. Extracellular GzmB is increased in retina pigment epithelium (RPE) and mast cells in the choroid of the healthy aging outer retina. It is further increased in donor eyes exhibiting features of nAMD and CNV. Here, we show in RPE-choroidal explant cultures that exogenous GzmB degrades the RPE-choroid ECM, promotes retinal/choroidal inflammation and angiogenesis while diminishing anti-angiogenic factor, thrombospondin-1 (TSP-1). The pharmacological inhibition of either GzmB or mast-cell degranulation significantly reduces choroidal angiogenesis. In line with our in vitro data, GzmB-deficiency reduces the extent of laser-induced CNV lesions and the age-related deterioration of electroretinogram (ERG) responses in mice. These findings suggest that targeting GzmB, a serine protease with no known endogenous inhibitors, may be a potential novel therapeutic approach to suppress CNV in nAMD., (© 2024. The Author(s).)

We aimed to determine the role of cathepsin S (CTSS) in modulating oxidative stress-induced immune and inflammatory reactions and angiogenesis in age-related macular degeneration. Human retinal pigment epithelium cells line ARPE-19 (immature) were maintained and treated with H 2 O 2 . The expression of CTSS, inflammatory cytokines, and complement factors induced by oxidative stress was compared between cells incubated without (control) and with CTSS knockdown (using small interfering ribonucleic acid; siRNA). To evaluate the role of CTSS in angiogenesis, we assayed tube formation using human umbilical vein endothelial cells and conditioned medium from ARPE-19 cells. We also used a mouse model of laser-induced choroidal neovascularization. CTSS levels were higher in ARPE-19 cells treated with H 2 O 2 than in control cells. Oxidative stress-induced CTSS resulted in significantly elevated transcription of nuclear factor kappa B-dependent inflammatory cytokines, complement factors C3a and C5a, membrane attack complex (C5b-9), and C3a and C5a receptors. siRNA-mediated knockdown of CTSS reduced the number of inflammatory signals. Furthermore, oxidative stress-induced CTSS regulated the expression of peroxisome proliferator-activated receptor γ and vascular endothelial growth factor A/Akt serine/threonine kinase family signaling, which led to angiogenesis. Tube formation assays and mouse models of choroidal neovascularization revealed that CTSS knockdown ameliorated angiogenesis in vitro and in vivo. The present findings suggest that CTSS modulates the complement pathway, inflammatory reactions, and neovascularization, and that CTSS knockdown induces potent immunomodulatory effects. Hence, it could be a promising target for the prevention and treatment of early- and late-stage age-related macular degeneration., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Age-related macular degeneration (AMD), a prevalent and progressive degenerative disease of the macula, is the leading cause of blindness in elderly individuals in developed countries. The advanced stages include neovascular AMD (nAMD), characterized by choroidal neovascularization (CNV), leading to subretinal fibrosis and permanent vision loss. Despite the efficacy of anti-vascular endothelial growth factor (VEGF) therapy in stabilizing or improving vision in nAMD, the development of subretinal fibrosis following CNV remains a significant concern. In this review, we explore multifaceted aspects of subretinal fibrosis in nAMD, focusing on its clinical manifestations, risk factors, and underlying pathophysiology. We also outline the potential sources of myofibroblast precursors and inflammatory mechanisms underlying their recruitment and transdifferentiation. Special attention is given to the potential role of mast cells in CNV and subretinal fibrosis, with a focus on putative mast cell mediators, tryptase and granzyme B. We summarize our findings on the role of GzmB in CNV and speculate how GzmB may be involved in the pathological transition from CNV to subretinal fibrosis in nAMD. Finally, we discuss the advantages and drawbacks of animal models of subretinal fibrosis and pinpoint potential therapeutic targets for subretinal fibrosis., Competing Interests: Author DG is a Co-Founder and serves as the Chief Scientific Officer for viDA Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Gill, Yoo, Chakravarthy, Granville and Matsubara.)

Neovascular age-related macular degeneration (nAMD) remains a major cause of visual impairment and puts considerable burden on patients and health care systems. l-DOPA-treated Parkinson's disease (PD) patients have been shown to be partially protected from nAMD, but the mechanism remains unknown. Using murine models that combine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced (MPTP-induced) PD and laser-induced nAMD with standard PD treatment of l-DOPA/DOPA-decarboxylase inhibitor or specific dopamine receptor inhibitors, we here demonstrate that l-DOPA treatment-induced increase of dopamine-mediated dopamine receptor D2 (DRD2) signaling inhibits choroidal neovascularization independently of MPTP-associated nigrostriatal pathway lesion. Analyzing a retrospective cohort of more than 200,000 patients with nAMD receiving anti-VEGF treatment from the French nationwide insurance database, we show that DRD2 agonist-treated PD patients have a significantly delayed age of onset of nAMD and reduced need for anti-VEGF therapies, similar to the effects of the l-DOPA treatment. While providing a mechanistic explanation for an intriguing epidemiological observation, our findings suggest that systemic DRD2 agonists might constitute an adjuvant therapy to delay and reduce the need for anti-VEGF therapy in patients with nAMD.

This study aimed to develop a quantitative analysis program of blood flow velocity by vessel diameter in neovascular age-related macular degeneration (nAMD) subjects using high-speed swept-source optical coherence tomography angiography. This retrospective, observational, cross-sectional study included 10 eyes of healthy volunteers and 4 eyes of patients with representative nAMD. Novel scan patterns and variable interscan time analysis were utilized to measure the flow parameter, a surrogate marker of blood flow velocity, by vessel diameter within different depths. Detected vessels at superficial and deep as well as outer retinal regions were categorized into three vessel diameters (major vessels (> 40 μm), medium vessels (20-40 μm), and capillaries (< 20 μm)). The flow parameter increased with enlarged vessel diameter in all participants at superficial and deep layer. All nAMD subjects, except for type 3 macular neovascularization (MNV), contained a structure dominated by medium vessels at outer retinal region. The mean flow parameter at outer retinal region was type 1 MNV (1.46 ms -1 ), type 1 + 2 MNV (0.98 ms -1 ), and polypoidal choroidal vasculopathy, including branching vascular networks (1.46 ms -1 ). This program provides the possibility to extract the blood flow information at different depths by vessel diameter types, which is considered to be useful tool for evaluating nAMD pathology and activity., (© 2024. The Author(s).)

The choroid, a vascularized tissue situated between the retina and the sclera, plays a crucial role in maintaining ocular homeostasis. Despite its significance, research on choroidal abnormalities and the establishment of effective in vitro models have been limited. In this study, we developed an in vitro choroid model through the co-culture of human induced pluripotent stem cells (hiPSC)-derived endothelial cells (ECs) and mouse choroidal fibroblasts (msCFs) with hiPSC-derived retinal pigment epithelial (RPE) cells via a permeable membrane. This model, inclusive of ECs, CFs, and RPE cells, exhibited similarities with in vivo choroidal vessels, as confirmed through immunohistochemistry of extracellular matrix markers and vascular-related markers, as well as choroid angiogenesis sprouting assay analysis. The effectiveness of our in vitro model was demonstrated in assessing vascular changes induced by drugs targeting vasoregulation. Our model offers a valuable tool for gaining insights into the pathological mechanisms underlying choroid development and the progression of choroidal vascular diseases., (© 2024. The Author(s).)

Metabolic dysfunction is recognized as a contributing factor in the pathogenesis of wet age-related macular degeneration (wAMD). However, the specific metabolism-related proteins implicated in wAMD remain elusive. In this study, we assessed the expression profiles of 92 metabolism-related proteins in aqueous humor (AH) samples obtained from 44 wAMD patients and 44 cataract control patients. Our findings revealed significant alterations in the expression of 60 metabolism-related proteins between the two groups. Notably, ANGPTL7 and METRNL displayed promising diagnostic potential for wAMD, as evidenced by area under the curve values of 0.88 and 0.85, respectively. Subsequent validation studies confirmed the upregulation of ANGPTL7 and METRNL in the AH of wAMD patients and in choroidal neovascularization (CNV) models. Functional assays revealed that increased ANGPTL7 and METRNL played a pro-angiogenic role in endothelial biology by promoting endothelial cell proliferation, migration, tube formation, and spouting in vitro . Moreover, in vivo studies revealed the pro-angiogenic effects of ANGPTL7 and METRNL in CNV formation. In conclusion, our findings highlight the association between elevated ANGPTL7 and METRNL levels and wAMD, suggesting their potential as novel predictive and diagnostic biomarkers for this condition. These results underscore the significance of ANGPTL7 and METRNL in the context of wAMD pathogenesis and offer new avenues for future research and therapeutic interventions.

Neovascular age-related macular degeneration, also known as exudative or wet age-related macular degeneration, is the leading cause of blindness in the developed world. Photobiomodulation has the potential to target the up-stream hypoxic and pro-inflammatory drivers of choroidal neovascularization. This study investigated whether photobiomodulation attenuates characteristic pathological features of choroidal neovascularization in a rodent model. Experimental choroidal neovascularization was induced in Brown Norway rats with laser photocoagulation. A custom-designed, slit-lamp-mounted, 670 nm laser was used to administer retinal photobiomodulation every 3 days, beginning 6 days prior to choroidal neovascularization induction and continuing until the animals were killed 14 days later. The effect of photobiomodulation on the size of choroidal neovascular membranes was determined using isolectin-B4 immunohistochemistry and spectral domain-optical coherence tomography. Vascular leakage was determined with fluorescein angiography. The effect of treatment on levels of vascular endothelial growth factor expression was quantified with enzyme-linked immunosorbent assay. Treatment with photobiomodulation was associated with choroidal neovascular membranes that were smaller, had less fluorescein leakage, and a diminished presence of inflammatory cells as compared to sham eyes. These effects were not associated with a statistically significant difference in the level of vascular endothelial growth factor when compared to sham eyes. The data shown herein indicate that photobiomodulation attenuates pathological features of choroidal neovascularization in a rodent model by mechanisms that may be independent of vascular endothelial growth factor., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: To investigate the levels of angiogenesis and inflammatory cytokines in individuals with myopic choroidal neovascularization (mCNV) and the changes in these factors following intravitreal anti-VEGF injection., Methods: Aqueous humor samples were gathered from eyes with mCNV, those with single macular bleeding (SMB) without mCNV in highly myopic eyes, and those with age-related cataracts. Using a multiplex bead immunoassay, we analyzed 28 angiogenesis and inflammatory factors in the aqueous humor. Furthermore, clinical data were documented for correlation analysis., Results: In this study, the levels of vascular endothelial growth factor A (VEGF-A), interleukin 8 (IL-8), and fibroblast growth factors 1 (FGF-1) were significantly elevated in mCNV compared to SMB eyes (p < 0.05). Their odds ratios for mCNV occurrence were 1.05, 3.45, and 2.64, respectively. Hepatocyte growth factor (HGF) and VEGF-C were notably higher in mCNV than in cataract patients (p < 0.05), and VEGF-C correlated to the degree of myopic atrophic maculopathy (p = 0.024). Axial length exhibited a negative correlation with VEGF-A and positive correlations with VEGF-C, HGF, and MCP-1 (p < 0.01). Following anti-VEGF treatment, a reduction in VEGF-A, endothelin-1, and FGF-2 was noted in mCNV patients (p < 0.05), but MCP-1 levels increased., Conclusion: Our findings highlight the predominant role of angiogenesis and inflammation factors in mCNV pathogenesis. VEGF-C's correlation with axial length and atrophy suggests its involvement in the process of myopic atrophic maculopathy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The microglia-mediated neuroinflammation have been shown to play a crucial role in the ocular pathological angiogenesis process, but specific immunotherapies for neovascular ocular diseases are still lacking. This study proposed that targeting GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) might be a novel immunotherapy for these angiogenesis diseases. We found a significant upregulation of CGAS and STING genes in the RNA-seq data derived from retinal tissues of the patients with proliferative diabetic retinopathy. In experimental models of ocular angiogenesis including laser-induced choroidal neovascularization (CNV) and oxygen-induced retinopathy (OIR), the cGAS-STING pathway was activated as angiogenesis progressed. Either genetic deletion or pharmacological inhibition of STING resulted in a remarkable suppression of neovascularization in both models. Furthermore, cGAS-STING signaling was specifically activated in myeloid cells, triggering the subsequent RIP1-RIP3-MLKL pathway activation and leading to necroptosis-mediated inflammation. Notably, targeted inhibition of the cGAS-STING pathway with C-176 or SN-011 could significantly suppress pathological angiogenesis in CNV and OIR. Additionally, the combination of C-176 or SN-011 with anti-VEGF therapy led to least angiogenesis, markedly enhancing the anti-angiogenic effectiveness. Together, our findings provide compelling evidence for the importance of the cGAS-STING-necroptosis axis in pathological angiogenesis, highlighting its potential as a promising immunotherapeutic target for treating neovascular ocular diseases., (© 2024. The Author(s).)

In this retrospective case series on neovascular age-related macular degeneration (nAMD), we aimed to improve Choroidal Neovascularization (CNV) visualization in Optical Coherence Tomography Angiography (OCTA) scans by addressing segmentation errors. Out of 198 eyes, 73 OCTA scans required manual segmentation correction. We compared uncorrected scans to those with minimal (2 corrections), moderate (10 corrections), and detailed (50 corrections) efforts targeting falsely segmented Bruch's Membrane (BM). Results showed that 55% of corrected OCTAs exhibited improved quality after manual correction. Notably, minimal correction (2 scans) already led to significant improvements, with additional corrections (10 or 50) not further enhancing expert grading. Reduced background noise and improved CNV identification were observed, with the most substantial improvement after two corrections compared to baseline uncorrected images. In conclusion, our approach of correcting segmentation errors effectively enhances image quality in OCTA scans of nAMD. This study demonstrates the efficacy of the method, with 55% of resegmented OCTA images exhibiting enhanced quality, leading to a notable increase in the proportion of high-quality images from 63 to 83%., (© 2024. The Author(s).)

Background: Intravitreal injections of angiogenesis inhibitors have proved efficacious in the majority of patients with ocular angiogenesis. However, one-fourth of all treated patients fail to derive benefits from intravitreal injections. tRNA-derived small RNA (tsRNA) emerges as a crucial class of non-coding RNA molecules, orchestrating key roles in the progression of human diseases by modulating multiple targets. Through our prior sequencing analyses and bioinformatics predictions, tRNA-Cys-5-0007 has shown as a potential regulator of ocular angiogenesis. This study endeavors to elucidate the precise role of tRNA-Cys-5-0007 in the context of ocular angiogenesis., Methods: Quantitative reverse transcription PCR (qRT-PCR) assays were employed to detect tRNA-Cys-5-0007expression. EdU assays, sprouting assays, transwell assays, and Matrigel assays were conducted to elucidate the involvement of tRNA-Cys-5-0007 in endothelial angiogenic effects. STZ-induced diabetic model, OIR model, and laser-induced CNV model were utilized to replicate the pivotal features of ocular vascular diseases and evaluate the influence of tRNA-Cys-5-0007 on ocular angiogenesis and inflammatory responses. Bioinformatics analysis, luciferase activity assays, RNA pull-down assays, and in vitro studies were employed to elucidate the anti-angiogenic mechanism of tRNA-Cys-5-0007. Exosomal formulation was employed to enhance the synergistic anti-angiogenic and anti-inflammatory efficacy of tRNA-Cys-5-0007., Results: tRNA-Cys-5-0007 expression was down-regulated under angiogenic conditions. Conversely, tRNA-Cys-5-0007 overexpression exhibited anti-angiogenic effects in retinal endothelial cells, as evidenced by reduced proliferation, sprouting, migration, and tube formation abilities. In diabetic, laser-induced CNV, and OIR models, tRNA-Cys-5-0007 overexpression led to decreased ocular vessel leakage, inhibited angiogenesis, and reduced ocular inflammation. Mechanistically, these effects were attributed to the targeting of vascular endothelial growth factor A (VEGFA) and TGF-β1 by tRNA-Cys-5-0007. The utilization of an exosomal formulation further potentiated the synergistic anti-angiogenic and anti-inflammatory efficacy of tRNA-Cys-5-0007., Conclusions: Concurrent targeting of tRNA-Cys-5-0007 for anti-angiogenic and anti-inflammatory therapy holds promise for enhancing the effectiveness of current anti-angiogenic therapy., (© 2024. The Author(s).)

This retrospective observational study aimed to investigate the difference in 4-year outcomes of ranibizumab or aflibercept therapy for macular neovascularization (MNV) with high myopia between pathologic myopia (PM) and non-PM. This study was conducted at Kyoto University Hospital and included consecutive treatment-naïve eyes with active myopic MNV, in which a single intravitreal ranibizumab or aflibercept injection was administered, followed by a pro re nata (PRN) regimen for 4 years. Based on the META-PM study classification, eyes were assigned to the non-PM and PM groups. This study analyzed 118 eyes of 118 patients (non-PM group, 19 eyes; PM group, 99 eyes). Baseline, 1-year, and 2-year best-corrected visual acuity (BCVA) were significantly better in the non-PM group (P = 0.02, 0.01, and 0.02, respectively); however, the 3-year and 4-year BCVA were not. The 4-year BCVA course was similar in both groups. However, the total number of injections over 4 years was significantly higher in the non-PM than in the PM group (4.6 ± 2.6 vs. 2.9 ± 2.6, P = 0.001). Four-year BCVA significantly correlated only with baseline BCVA in both non-PM (P = 0.047, β = 0.46) and PM groups (P < 0.001, β = 0.59). In conclusion, over the 4-year observation period, the BCVA course after anti-VEGF therapy for myopic MNV was similar in the eyes with non-PM and those with PM; however, more additional injections in a PRN regimen were required in the eyes with non-PM compared to those with PM. Thus, more frequent and careful follow-up is required for the eyes with non-PM compared with those with PM to maintain long-term BCVA., (© 2024. The Author(s).)

Purpose: The aim of this study was to elucidate the role of Sema4D in the pathogenesis of senescence-associated choroidal neovascularization (CNV) and to explore its underlying mechanisms., Methods: In this study, we utilized a model of laser-induced CNV in both young (3 months old) and old (18 months old) mice, including those with or without Sema4D knockout. The expression and localization of Sema4D in CNV were assessed using PCR, Western blot, and immunostaining. Subsequently, the morphological and imaging examinations were used to evaluate the size of CNV and vascular leakage. Finally, the expression of M2 markers, senescence-related markers, and molecules involved in the RhoA/ROCK pathway was detected., Results: We found that Sema4D was predominantly expressed in macrophages within CNV lesions, and both the mRNA and protein levels of Sema4D progressively increased following laser photocoagulation, a trend more pronounced in old mice. Moreover, Sema4D knockout markedly inhibited M2 polarization in senescent macrophages and reduced the size and leakage of CNV, particularly in aged mice. Mechanistically, aging was found to upregulate RhoA/ROCK signaling, and knockout of Sema4D effectively suppressed the activation of this pathway, with more significant effects observed in aged mice., Conclusions: Our findings revealed that the deletion of Sema4D markedly inhibited M2 macrophage polarization through the suppression of the RhoA/ROCK pathway, ultimately leading to the attenuation of senescence-associated CNV. These data indicate that targeting Sema4D could offer a promising approach for gene editing therapy in patients with neovascular age-related macular degeneration.

Age-related macular degeneration (AMD) disease has become a worldwide senile disease, and frequent intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) is the mainstream treatment in the clinic, which is associated with sight-threatening complications. Herein, nintedanib, an inhibitor of angiogenesis, and lutein, a potent antioxidant, can co-assemble into nanoparticles through multiple noncovalent interactions. Interestingly, the co-assembled lutein/nintedanib nanoparticles (L/N NPs) exhibit significantly improved stability and achieve long-term sustained release of two drugs for at least two months in mice. Interestingly, in rabbit eyeball with a more complete barrier system, the L/N NPs still successfully distribute in the retina and choroid for a month. In the laser-induced mouse choroidal neovascularization model, the L/N NPs after a minimally invasive subconjunctival administration can successfully inhibit angiogenesis and achieve comparable and even better therapeutic results to that of standard intravitreal injection of anti-VEGF. Therefore, the subconjunctival injection of L/N NPs with long-term sustained drug release behavior represents a promising and innovative strategy for AMD treatment. Such minimally invasive administration together with the ability to effectively inhibit angiogenesis reduce inflammation and counteract oxidative stress and holds great potential for improving patient outcomes and quality of life in those suffering from this debilitating eye condition., (© 2024 Wiley‐VCH GmbH.)

The prolonged intravitreal administration of anti-vascular endothelial growth factor (VEGF) drugs is prone to inducing aberrant retinal vascular development and causing damage to retinal neurons. Hence, we have taken an alternative approach by designing and synthesizing a series of cyclic peptides targeting CC motif chemokine receptor 3 (CCR3). Based on the binding mode of the N-terminal region in CCR3 protein to CCL11, we used computer-aided identification of key amino acid sequence, conformational restriction through different cyclization methods, designed and synthesized a series of target cyclic peptides, and screened the preferred compound IB-2 through affinity. IB-2 exhibits excellent anti-angiogenic activity in HRECs. The apoptosis level of 661W cells demonstrated a significant decrease with the escalating concentration of IB-2. This suggests that IB-2 may have a protective effect on photoreceptor cells. In vivo experiments have shown that IB-2 significantly reduces retinal vascular leakage and choroidal neovascularization (CNV) area in a laser-induced mouse model of CNV. These findings indicate the potential of IB-2 as a safe and effective therapeutic agent for AMD, warranting further development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Choroidal neovascularization (CNV), characterized as a prominent feature of wet age-related macular degeneration (AMD), is a primary contributor to visual impairment and severe vision loss globally, while the prevailing treatments are often unsatisfactory. The development of conventional treatment strategies has largely been based on the understanding that the angiogenic switch of endothelial cells is dictated by angiogenic growth factors alone. Even though treatments targeting vascular endothelial growth factor (VEGF), like Ranibizumab, are widely administered, more than half of the patients still exhibit inadequate or null responses, emphasizing the imperative need for solutions to this problem. Here, aiming to explore therapeutic strategies from a novel perspective of endothelial cell metabolism, a biocompatible nanomedicine delivery system is constructed by loading RGD peptide-modified liposomes with 2-deoxy-D-glucose (RGD@LP-2-DG). RGD@LP-2-DG displayed good targeting performance towards endothelial cells and excellent in vitro and in vivo inhibitory effects on neovascularization were demonstrated. Moreover, our mechanistic studies revealed that 2-DG interfered with N-glycosylation, leading to the inhibition of vascular endothelial growth factor receptor 2 (VEGFR2) and its downstream signaling. Notably, the remarkable inhibitory effect on neovascularization and biocompatibility of RGD@LP-2-DG render it a highly promising and clinically translatable therapeutic candidate for the treatment of wet AMD and other angiogenic diseases, particularly in patients who are unresponsive to currently available treatments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Choroidal neovascularization (CNV) is the principal driver of blindness in neovascular age-related macular degeneration (nvAMD). Increased activity of telomerase, has been associated with endothelial cell proliferation, survival, migration, and invasion in the context of tumor angiogenesis. Expanding on this knowledge, we investigated the role of telomerase in the development of CNV in mouse model. We observed increased gene expression and activity of telomerase in mouse CNV. Genetic deficiency of the telomerase components, telomerase reverse transcriptase (Tert) and telomerase RNA component (Terc) suppressed laser-induced CNV in mice. Similarly, a small molecule inhibitor of TERT (BIBR 1532), and antisense oligonucleotides (ASOs) targeting Tert and Terc reduced CNV growth. Bone marrow chimera studies suggested that telomerase activity in non-bone marrow-derived cells is crucial for the development of CNV. Comparison of BIBR 1532 with VEGF neutralizing therapeutic strategy in mouse revealed a comparable level of angiosuppressive activity. However, when BIBR and anti-VEGF antibodies were administered as a combination at sub-therapeutic doses, a statistically significant suppression of CNV was observed. These findings underscore the potential benefits of combining sub-therapeutic doses of BIBR and anti-VEGF antibodies for developing newer therapeutic strategies for NV-AMD. Telomerase inhibition with BIBR 1532 suppressed induction of multiple cytokines and growth factors critical for neovascularization. In conclusion, our study identifies telomerase as a promising therapeutic target for treating neovascular disease of the eye and thus provides a proof of principle for further exploration of telomerase inhibition as a novel treatment strategy for nvAMD., Competing Interests: Declaration of competing interest N.K. is named as an inventor on a patent application on non-canonical signaling activity of cGAMP filed by the University of Virginia. N.K. and J.A., are named as inventors on patent applications on macular degeneration filed by the University of Virginia or the University of Kentucky. J.A., is a co-founder of DiceRx, iVeena Holdings, iVeena Delivery Systems and Inflammasome Therapeutics, and, unrelated to this work, he has been a consultant for Abbvie/Allergan, Boehringer-Ingelheim, Janssen, Olix Pharmaceuticals, Retinal Solutions, and Saksin LifeSciences. The other authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Choroidal neovascularization (CNV) can be seen in many fundus diseases, and lead to fundus exudation, bleeding, or vision loss. miRNAs are vital regulator in CNV. miR-199a-5p has been proved to be involved in regulating vascular formation of endothelial cells, but its role in CNV remains unclear. This study aims to study the role of miR-199a-5p in CNV. Laser irradiation was used to induce CNV model. The lesion area of CNV was calculated by high-resolution angiography with fluorescein isothiocyanate-dextran. Wnt family member 7b (Wnt7b), β-catenin, and Wnt pathway proteins was measured by western blot. Immunofluorescence was performed to test Wnt7b, β-catenin, CD31, and p-p65. miR-199a-5p and Wnt7b mRNA were tested by reverse transcription real-time polymerase chain reaction. Cell count kit-8, wound healing, Transwell, tube formation, and flow cytometry were used to detect the function of miR-199a-5p and Wnt7b on human retinal microvascular endothelial cells (HRMEC). TargetScan database and dual-luciferase reporter assay verified the interaction between miR-199a-5p and Wnt7b. The results revealed that Wnt7b increased in CNV rats. Knocking down Wnt7b repressed cell proliferation, migration, invasion, and angiogenesis, and accelerated cell apoptosis of HRMEC. Dual-luciferase reporter assay verified that miR-199a-5p targeted Wnt7b. Overexpression of miR-199a-5p inhibited the angiogenesis of HRMEC and promoted cell apoptosis by inhibiting Wbt7b. In vivo experiment found that Wnt7b rescued the promotion of miR-199a-5p inhibition on CNV lesion of rats. In addition, Wnt7b positively regulated Wnt/β-catenin signaling pathway and promoted the angiogenesis of HRMEC. In conclusion, overexpression of miR-199a-5p inhibited the angiogenesis of HRMEC by regulating Wnt7b/Wnt/β-catenin signaling pathway, which may serve as a promising therapy target of CNV., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Sustainable retinal codelivery poses significant challenges technically, although it is imperative for synergistic treatment of wet age-related macular degeneration (wAMD). Here, a microemulsion-doped hydrogel (Bor/PT-M@TRG) is engineered as an intravitreal depot composing of temperature-responsive hydrogel (TRG) and borneol-decorated paeoniflorin (PF) & tetramethylpyrazine (TMP)-coloaded microemulsions (Bor/PT-M). Bor/PT-M@TRG, functioning as the "ammunition depot", resides in the vitreous and continuously releases Bor/PT-M as the therapeutic "bullet", enabling deep penetration into the retina for 21 days. A single intravitreal injection of Bor/PT-M@TRG yields substantial reductions in choroidal neovascularization (CNV, a hallmark feature of wAMD) progression and mitigates oxidative stress-induced damage in vivo. Combinational PF&TMP regulates the "reactive oxygen species/nuclear factor erythroid-2-related factor 2/heme oxygenase-1" pathway and blocks the "hypoxia inducible factor-1α/vascular endothelial growth factor" signaling in retina, synergistically cutting off the loop of CNV formation. Utilizing fluorescence resonance energy transfer and liquid chromatography-mass spectrometry techniques, they present compelling multifaceted evidence of sustainable retinal codelivery spanning formulations, ARPE-19 cells, in vivo eye balls, and ex vivo section/retina-choroid complex cell levels. Such codelivery approach is elucidated as the key driving force behind the exceptional therapeutic outcomes of Bor/PT-M@TRG. These findings highlight the significance of sustainable retinal drug codelivery and rational combination for effective treatment of wAMD., (© 2024 Wiley‐VCH GmbH.)

The role of mast cells in physiologic and pathological processes extends far beyond the allergy processes: they are involved in wound healing, chronic inflammation, and tumor growth. This short article emphasizes the role played by mast cells in age-related macular degeneration (AMD). Mast cells can induce angiogenesis and are present around Bruch's membrane during the early and late stages of choroidal neovascularization in AMD. Proteolytic enzymes released by mast cells lead to thinning of the choroid in AMD as well as degradation of vascular basement membranes and Bruch's membrane, which in turn could result in retinal pigment epithelial death and choriocapillaris degeneration in geographical atrophy and exudative AMD., (© 2024. The Author(s).)

Although the pathogenesis of choroidal neovascularization (CNV) is largely unknown in age-related macular degeneration (AMD), inflammasomes may contribute to CNV development and progression. To understand the role NLRP3 inflammasomes in CNV, we used Ccr2RFPCx3cr1GFP dual-reporter mice and immunostaining techniques to confirm localization of NLRP3 inflammasomes in the laser-induced CNV (LCNV) lesions. Confocal microscopy was used to image and quantify LCNV volumes. MCC950 was used as NLRP3 inhibitor. ELISA and quantitative RT-PCR were used to confirm the activation of NLRP3 by monitoring the expression of IL-1β protein and mRNA in choroidal tissues from LCNV mice. In addition, NLRP3 (-/-) LCNV mice were used to investigate whether NLRP3 inflammasomes contribute to the development of LCNV lesions. We observed that red fluorescent protein (RFP)-positive monocyte-derived macrophages and GFP-positive microglia-derived macrophages, in addition to other cell types, were localized in LCNV lesions at day 7 post-laser injury. In addition, NLRP3 inflammasomes are associated with LCNV lesions. Inhibition of NLRP3 inflammasomes, using MCC950, caused an increased Ccr2RFP-positive macrophages, Cx3cr1GFP-positive microglia, and other cells, resulting in an increase in total lesion size. NLRP3 (-/-) LCNV mice showed significantly increased lesion size compared with age-matched controls. Inhibition of NLRP3 resulted in decreased IL-1β mRNA and protein expression in the choroidal tissues, suggesting that increased lesion size may not be directly related to IL-1β., (Copyright © 2024 The Authors.)

Choroidal neovascularization (CNV) is a hallmark of neovascular age-related macular degeneration (nAMD) and a major contributor to vision loss in nAMD cases. However, the identification of specific cell types associated with nAMD remains challenging. Herein, we performed single-cell sequencing to comprehensively explore the cellular diversity and understand the foundational components of the retinal pigment epithelium (RPE)/choroid complex. We unveiled 10 distinct cell types within the RPE/choroid complex. Notably, we observed significant heterogeneity within endothelial cells (ECs), fibroblasts, and macrophages, underscoring the intricate nature of the cellular composition in the RPE/choroid complex. Within the EC category, four distinct clusters were identified and EC cluster 0 was tightly associated with choroidal neovascularization. We identified five clusters of fibroblasts actively involved in the pathogenesis of nAMD, influencing fibrotic responses, angiogenic effects, and photoreceptor function. Additionally, three clusters of macrophages were identified, suggesting their potential roles in regulating the progression of nAMD through immunomodulation and inflammation regulation. Through CellChat analysis, we constructed a complex cell-cell communication network, revealing the role of EC clusters in interacting with fibroblasts and macrophages in the context of nAMD. These interactions were found to govern angiogenic effects, fibrotic responses, and inflammatory processes. In summary, this study reveals noteworthy cellular heterogeneity in the RPE/choroid complex and provides valuable insights into the pathogenesis of CNV. These findings will open up potential avenues for deep understanding and targeted therapeutic interventions in nAMD., Competing Interests: Declaration of competing interest The authors declare that there are no competing interests associated with the manuscript., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Objective: Choroidal neovascularization (CNV) represents the predominant form of advanced wet Age-related Macular Degeneration (wAMD). Macrophages play a pivotal role in the pathological progression of CNV. Meteorin-like (Metrnl), a novel cytokine known for its anti-inflammatory properties in macrophages, is the focus of our investigation into its mechanism of action and its potential to impede CNV progression., Methods: Cell viability was evaluated through CCK-8 and EdU assays following Metrnl treatment. Expression levels of inflammatory cytokines and proteins were assessed using quantitative reverse-transcription polymerase chain reaction(qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and western blot techniques. Protein-protein interactions were identified through protein mass spectrometry and co-immunoprecipitation (Co-IP). Additionally, in vivo and in vitro neovascularization models were employed to evaluate angiogenesis., Results: Our results revealed downregulated Metrnl levels in the choroid-sclera complex of CNV mice, the aqueous humor of wAMD patients, and activated macrophages. Metrnl overexpression demonstrated a reduction in pro-inflammatory cytokine production, influenced endothelial cell function, and suppressed angiogenesis in choroid explants and CNV models. Through protein mass spectrometry and Co-IP, we confirmed Metrnl binds to UCHL-1 to modulate the NF-κB signaling pathway. This interaction inhibited the transcription and expression of pro-inflammatory cytokines, ultimately suppressing angiogenesis., Conclusion: In summary, our findings indicate that Metrnl down-regulates macrophage pro-inflammatory cytokine secretion via the UCHL-1/NF-κB signaling pathway. This mechanism alleviates the inflammatory microenvironment and effectively inhibits choroidal neovascularization., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhang, Li, Wu, Shen, Zeng, Liu, Zhang, Yang, Liu, Tang, Ou and Fang.)

Ocular abnormal angiogenesis and edema are featured in several ocular diseases. S1P signaling via S1P1 likely is part of the negative feedback mechanism necessary to maintain vascular health. In this study, we conducted pharmacological experiments to determine whether ASP4058, a sphingosine 1-phosphate receptor 1/5 (S1P1/5) agonist, is useful in abnormal vascular pathology in the eye. First, human retinal microvascular endothelial cells (HRMECs) were examined using vascular endothelial growth factor (VEGF)-induced cell proliferation and hyperpermeability. ASP4058 showed high affinity and inhibited VEGF-induced proliferation and hyperpermeability of HRMECs. Furthermore, S1P1 expression and localization changes were examined in the murine laser-induced choroidal neovascularization (CNV) model, a mouse model of exudative age-related macular degeneration, and the efficacy of ASP4058 was verified. In the CNV model mice, S1P1 tended to decrease in expression immediately after laser irradiation and colocalized with endothelial cells and Müller glial cells. Oral administration of ASP4058 also suppressed vascular hyperpermeability and CNV, and the effect was comparable to that of the intravitreal administration of aflibercept, an anti-VEGF drug. Next, efficacy was also examined in a retinal vein occlusion (RVO) model in which retinal vascular permeability was increased. ASP4058 dose-dependently suppressed the intraretinal edema. In addition, it suppressed the expansion of the perfusion area observed in the RVO model. ASP4058 also suppressed the production of VEGF in the eye. Collectively, ASP4058 can be a potential therapeutic agent that normalizes abnormal vascular pathology, such as age-related macular degeneration and RVO, through its direct action on endothelial cells., (© 2024. The Author(s).)

Introduction: Age-related macular degeneration (AMD) is a prevalent, chronic and progressive retinal degenerative disease characterized by an inflammatory response mediated by activated microglia accumulating in the retina. In this study, we demonstrate the therapeutically effects and the underlying mechanisms of microglial repopulation in the laser-induced choroidal neovascularization (CNV) model of exudative AMD., Methods: The CSF1R inhibitor PLX3397 was used to establish a treatment paradigm for microglial repopulation in the retina. Neovascular leakage and neovascular area were examined by fundus fluorescein angiography (FFA) and immunostaining of whole-mount RPE-choroid-sclera complexes in CNV mice receiving PLX3397. Altered cellular senescence was measured by beta-galactosidase (SA-β-gal) activity and p16INK4a expression. The effect and mechanisms of repopulated microglia on leukocyte infiltration and the inflammatory response in CNV lesions were analyzed., Results: We showed that ten days of the CSF1R inhibitor PLX3397 treatment followed by 11 days of drug withdrawal was sufficient to stimulate rapid repopulation of the retina with new microglia. Microglial repopulation attenuated pathological choroid neovascularization and dampened cellular senescence in CNV lesions. Repopulating microglia exhibited lower levels of activation markers, enhanced phagocytic function and produced fewer cytokines involved in the immune response, thereby ameliorating leukocyte infiltration and attenuating the inflammatory response in CNV lesions., Discussion: The microglial repopulation described herein are therefore a promising strategy for restricting inflammation and choroidal neovascularization, which are important players in the pathophysiology of AMD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Song, Liao, Liu, Chen, Wang, Zhou, Zhang and Li.)

Objective: Investigate retinal characteristics of pathologic myopia (PM) among patients self-identifying as Black., Design: Retrospective cohort single-institution retrospective medical record review., Methods: Adult patients between January 2005 and December 2014 with International Classification of Diseases (ICD) codes consistent with PM and given 5-year follow-up were evaluated. The Study Group consisted of patients self-identifying as Black, and the Comparison Group consisted of those not self-identifying as Black. Ocular features at study baseline and 5-year follow-up visit were evaluated., Results: Among 428 patients with PM, 60 (14%) self-identified as Black and 18 (30%) had baseline and 5-year follow-up visits. Of the remaining 368 patients, 63 were in the Comparison Group. For the study (n = 18) and Comparison Group (n = 29), median (25th percentile, 75th percentile) baseline visual acuity was 20/40 (20/25, 20/50) and 20/32 (20/25, 20/50) in the better-seeing eye and 20/70 (20/50, 20/1400) and 20/100 (20/50, 20/200), respectively, in the worse-seeing eye. In the eyes that did not have choroidal neovascularization (CNV) in the study and Comparison Group, median study baseline optical coherence tomography central subfield thickness was 196 μm (169, 306 μm) and 225 μm (191, 280 μm), respectively, in the better-seeing eye and 208 μm (181, 260 μm) and 194 μm (171, 248 μm), respectively, in the worse-seeing eye. Baseline prevalence of CNV was 1 Study Group eye (3%) and 20 Comparison Group eyes (34%). By the 5-year visit, zero (0%) and 4 (15%) additional eyes had CNV in the study and Comparison Group, respectively., Conclusion: These findings suggest that the prevalence and incidence of CNV may be lower in patients with PM self-identifying as Black when compared with individuals of other races., (Copyright © 2023 Canadian Ophthalmological Society. Published by Elsevier Inc. All rights reserved.)

Introduction: New insights on polypoidal choroidal vasculopathy (PCV) have shed light regarding its pathophysiology and associations. However, PCV characterization is still incomplete in Caucasians, which is due to presumed lower prevalence in this population. Features typically associated with AMD such as drusen, retinal pigmentary changes or atrophy are seen in PCV, as precursors and in the fellow eye. Pachychoroid spectrum, predisposing to PCV, also presents with chronic changes in the retinal pigment epithelium (RPE), such as drusen-like deposits (DLD), and in the choroid. The purpose of this study is to perform a multimodal imaging characterization of unaffected fellow eyes in a sample of Caucasian patients with unilateral PCV., Methods: Multicenter retrospective cohort study with a sample of 55 unaffected fellow eyes from patients diagnosed with unilateral PCV confirmed by indocyanine green angiography. The sample was characterized in the baseline by color fundus photography, spectral domain optical coherence tomography (SD-OCT), fluorescein angiography and indocyanine green angiography. Morphological characteristics of both the retina and the choroid were evaluated. The SD-OCT of the last follow-up visit was also evaluated in order to exclude evolution to PCV or choroidal neovascularization. All images captured underwent evaluation by two independent graders. Informed consent was obtained from all participants., Results: Fifty-five patients (median age, 74 ± 15 years) were included. After 15.5 ± 6.4 months of follow-up, only one developed disease (1.9%). Soft and/or hard drusen were present in 60% and pachydrusen in 23.6%. Pachychoroid signs were present in 47.2%, the double-layer sign in 36.4%, disruption of the RPE changes in 16.4% and RPE atrophy in 10.9%. ICGA revealed choroidal vascular dilation in 63.6% and punctiform hyperfluorescence in 52.7%. Branching vascular networks were identified in only 1.9% of cases., Conclusion: The identification of pachychoroid signs in the OCT and ICGA were present in over half of the cases and the presence of the double-layer sign in more than a third provide crucial insights for enhanced characterization of this pathology and deeper understanding of its pathogenesis. These findings contribute significantly to the current knowledge, offering valuable markers to discern various phases of the pathology's progression., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Aims: To investigate the incidence of macular neovascularisation (MNV) subtypes of neovascular age-related macular degeneration (nAMD) and summarise these subtypes' clinical features in the Chinese population using multimodal imaging., Methods: We retrospectively analysed 506 consecutive treatment-naïve nAMD patients (582 eyes). Incidence of MNV subtypes and clinical features were recorded based on their multimodal images. The classification of MNV subtypes in nAMD patients were referred to Consensus on Neovascular Age-related Macular Degeneration Nonmenclature (CONAN) study group classifications., Results: 460 eyes of 389 nAMD patients were included in our study. 68.5% (315/460) of nAMD eyes were from male. According to CONAN, we identified type 1 macular neovascularisation (MNV) in 61.1% of eyes (281/460), type 2 MNV in 16.3% of eyes (75/460), type 3 MNV in 2.0% of eyes (9/460), mixed type 1 and type 2 MNV in 20.6% of eyes (95/460). 58% of eyes (267/460) were diagnosed as polypoidal choroidal vasculopathy lesions (PCV). 45.2% of eyes (208/460) with PCV lesions were type 1 MNV and 12.8% of eyes (59/460) with PCV lesions were co-occurred with type 2 MNV., Conclusion: Based on the consensus anatomical classification system developed by the CONAN Study Group, we updated the incidence of MNV subtypes and found that PCV was the most common subtype and type 3 MNV was the least common subtype among Chinese nAMD patients. In addition, the co-occurrence of PCV and type 2 MNV was typically observed, and its frequency was reported in our study., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: To determine prevalence of anisomyopia (axial length (AL) difference ≥2.5 mm) among high myopes ((HMs), defined by spherical equivalent of ≤6.0 diopters or AL ≥ 26.5 mm). To characterise the shorter anisomyopic eye (SAE) and evaluate if pathologic myopia (PM) in the longer anisomyopic eye (LAE) was associated with increased risk of PM in the SAE., Methods: 1168 HMs were recruited from Singapore National Eye Centre clinic for this cross-sectional study. Biometry, fundus photography and swept-source optical coherence tomography were performed. Patients with high axial anisomyopia were identified. Structural characteristics and presence of PM were described. Stepwise multivariate regression explored associations between PM in the LAE and pathology in the SAE, controlling for confounding variables., Results: Prevalence of anisomyopia was 15.8% (184 of 1168 patients). Anisomyopic patients (age 65.8±13.5 years) had mean AL of 30.6±2.0 mm and 26.2±2.3 mm in the LAE and SAE, respectively. 52.7% of SAEs had AL < 26.5 mm. Prevalence of myopic macular degeneration, macula-involving posterior staphyloma (PS), myopic traction maculopathy (MTM) and myopic choroidal neovascularisation (mCNV) in the SAE was 52.2%, 36.5%, 13.0% and 8.2%, respectively. Macular hole in the LAE was associated with increased risk of MTM in the SAE (OR=4.88, p=0.01). mCNV in the LAE was associated with mCNV in the SAE (OR=3.57, p=0.02). PS in the LAE was associated with PS in the SAE (OR=4.03, p<0.001)., Conclusions: Even when controlled for AL, PM complications in the LAE predict similar PM complications in the SAE. Patients with high axial anisometropia with PM in the LAE should be monitored carefully for complications in the SAE., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: To report three cases of aneurysmal type 2 neovascularization (AT2), a novel entity within the pachychoroid disease (PD) spectrum., Methods: We conducted an observational retrospective study of three patients with subretinal polyps treated with intravitreal aflibercept. We reviewed clinical and imaging data of the three patients. Best corrected visual acuity (BCVA), central macular thickness (CMT), choroidal subfoveal thickness, choroidal thickness under the polyps and the presence of a dry macula were assessed at baseline and throughout the follow-up., Results: All of the patients showed granular hypoautofluorescence on fundus autofluorescence. Indocyanine green angiography revealed prominent hyperfluorescent branching vascular networks ending in multiple aneurysmal dilatations. Optical coherence tomography (OCT) demonstrated that the aneurysmal lesions were localized in the subretinal space. Additionally, OCT showed retinal pigment epithelial microtears, the double-layer sign and pachyvessels. En face OCT-A perfectly defined prominent telangiectatic branching vascular networks in all the patients, but only revealed polyps in two out of the three patients. Cross-sectional OCT-A demonstrated polyps as patchy circular hypoflow signals in each case. After the intravitreal treatment, BCVA remained unimproved in all of the patients, despite decreased CMT and achievement of a dry macula, as a result of the development of subretinal fibrosis., Conclusion: In summary, we describe a new entity within the spectrum of PD, which we have termed AT2. This novel disease is characterized by the presence of aneurysmal dilatations in the subretinal space, along with the typical features of PD, such as choroidal vascular hyperpermeability, thickening of the choroid and pachyvessels., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Objective: Three-dimensional (3D) reconstruction using swept-source OCT angiography (SS-OCTA) can provide insights into the nature and structure of polypoidal choroidal vasculopathy (PCV) and its component parts, the polypoidal lesion (PL) and the branching neovascular network (BNN). This study aims to describe novel observations of PCV using 3D reconstruction of SS-OCTA, and to compare these observations with similar images of type I macular neovascularization (MNV) typical neovascular age-related macular degeneration (nAMD)., Design: Clinical case series., Subjects: Patients with PCV in either eye from clinical studies conducted in a tertiary retina center., Methods: Images with prespecified SS-OCTA imaging protocol were obtained and reconstructed in 3D. Forty neovascularization lesions (30 PCV and 10 typical nAMD) based on SS-OCTA were analyzed., Main Outcome Measures: The following 3 specific features were evaluated: (1) the pattern of flow signal within the PLs as either homogenous or showing internal vascular architecture; (2) the configuration of the BNN as hypermature, mature, or immature; and (3) the spatial arrangement of the PLs in relation to the BNN. Comparisons were made between PCV and typical nAMD., Results: All PLs exhibited internal vascular architecture in the form of coil-like loops and none exhibited homogenous flow. Small focal nodules were present within this internal vascular architecture in 70% of PLs. Branching neovascular networks exhibited a hypermature/mature configuration (100 vs. 50%, P < 0.01) and were associated with thicker choroid compared with typical nAMD type 1 MNV (238.7 ± 104.3 vs. 155.6 ± 49.2, P = 0.02). The BNN and PL were located at distinct anteroposterior planes in 81% of the eyes., Conclusions: We identified proliferating vasculature in both the PL and the BNN. Comparison of the configuration suggests that the BNN represents a more chronic and inactive lesion than the PL., Financial Disclosure(s): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article., (Copyright © 2023 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

A variety of techniques exist to investigate retinal and choroidal vascular changes in experimental mouse models of human ocular diseases. While all have specific advantages, a method for evaluating the choroidal vasculature in pigmented mouse eyes has been more challenging especially for whole mount visualization and morphometric analysis. Here we report a simple, reliable technique involving bleaching pigment prior to immunostaining the vasculature in whole mounts of pigmented mouse choroids. Eyes from healthy adult pigmented C57BL/6J mice were used to establish the methodology. The retina and anterior segment were separated from the choroid. The choroid with retinal pigment epithelial cells (RPE) and sclera was soaked in 1% ethylenediaminetetraacetic acid (EDTA) to remove the RPE. Tissues were fixed in 2% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Choroids were subjected to melanin bleaching with 10% hydrogen peroxide (H 2 O 2 ) at 55 °C for 90 min, washed in PBS and then immunostained with anti-podocalyxin antibody to label vascular endothelium followed by Cy3-AffiniPure donkey anti-goat IgG at 4 °C overnight. Images of immunostained bleached choroids were captured using a Zeiss 710 confocal microscope. In addition to control eyes, this method was used to analyze the choroids from subretinal sodium iodate (NaIO 3 ) RPE atrophy and laser-induced choroidal neovascularization (CNV) mouse models. The H 2 O 2 pretreatment effectively bleached the melanin, resulting in a transparent choroid. Immunolabeling with podocalyxin antibody following bleaching provided excellent visualization of choroidal vasculature in the flat perspective. In control choroids, the choriocapillaris (CC) displayed different anatomical patterns in peripapillary (PP), mid peripheral (MP) and far peripheral (FP) choroid. Morphometric analysis of the vascular area (VA) revealed that the CC was most dense in the PP region (87.4 ± 4.3% VA) and least dense in FP (79.9 ± 6.7% VA). CC diameters also varied depending on location from 11.4 ± 1.97 mm in PP to 15.1 ± 3.15 mm in FP. In the NaIO 3 -injected eyes, CC density was significantly reduced in the RPE atrophic regions (50.7 ± 5.8% VA in PP and 45.8 ± 6.17% VA in MP) compared to the far peripheral non-atrophic regions (82.8 ± 3.8% VA). CC diameters were significantly reduced in atrophic regions (6.35 ± 1.02 mm in PP and 6.5 ± 1.2 mm in MP) compared to non-atrophic regions (14.16 ± 2.12 mm). In the laser-induced CNV model, CNV area was 0.26 ± 0.09 mm 2 and luminal diameters of CNV vessels were 4.7 ± 0.9 mm. Immunostaining on bleached choroids with anti-podocalyxin antibody provides a simple and reliable tool for visualizing normal and pathologic choroidal vasculature in pigmented mouse eyes for quantitative morphometric analysis. This method will be beneficial for examining and evaluating the effects of various treatment modalities on the choroidal vasculature in mouse models of ocular diseases such as age-related macular degeneration, and degenerative genetic diseases., Competing Interests: Declaration of competing interest The authors declare no relevant conflicts of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Previous studies on the biomarkers of pathologic myopia choroidal neovascularization (pmCNV) development merely detected limited types of proteins and provide a meagre illustration of the underlying pathways. Hence, a landscape of protein changes in the aqueous humor (AH) of pmCNV patients is lacking. Here, to explore the potential mechanisms and biomarkers of pmCNV, we analyzed the clinical data and protein profile among atrophic (A) lesions, tractional lesions (T) and neovascular (N) lesions in myopic patients based on the ATN grading system for myopic maculopathy (MM)., Results: After investigating demographic data of our patients, a correlation was found between A and N lesions (R = 0.5753, P < 0.0001). Accordingly, groups were divided into patients without MM, patients with myopic atrophic maculopathy (MAM), and patients with pmCNV (N2a lesion). In proteomics analysis, the increased protein level of GFAP and complement-associated molecules in AH samples of the 3 groups also indicated that MAM and pmCNV shared similar characteristics. The GO enrichment and KEGG pathway analysis were performed, which mapped that differential expressed proteins mainly engaged in JAK-STAT pathway between the pmCNV group and two controls. Furthermore, we identified several potential biomarkers for pmCNV, including FCN3, GFAP, EGFR, SFRP3, PPP2R1A, SLIT2, and CD248., Conclusions: Atrophic lesions under pathologic myopic conditions demonstrated similarities to neovascularization development. Potential biomarkers including GFAP were associated with the pathogenesis of pmCNV. In summary, our study provides new insights for further research on pmCNV development., (© 2023. The Author(s).)