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Aims: Choroidal neovascularisation (CNV) in patients with X-linked retinoschisis (XLRS) has been poorly documented. This study aims to investigate the prevalence and clinical characteristics of CNV in patients with XLRS, as well as analyse the preliminary genotype-phenotype correlation., Methods: A retrospective case series of patients with genetically confirmed XLRS was included. Demographic, clinical and genetic features were analysed, with a comparison between CNV and non-CNV eyes., Results: Among 185 eyes of 129 patients with XLRS, the prevalence of CNV was 8.1% (15/185). The mean diagnostic age of all patients with CNV is 5.1±2.56 years. CNV eyes exhibited a mean best-corrected visual acuity (BCVA) (logarithm of the minimal angle of resolution) of 1.37±0.74. All CNVs were classified as subretinal and active. Peripapillary CNVs accounted for 80.0% (12/15), while subfoveal CNVs accounted for 20.0% (3/15). In CNV eyes, the prevalence of macular atrophy (5/15, 33.3%, p=0.013) and bullous peripheral schisis (14/15, 93.3%, p=0.000) was higher compared with non-CNV eyes. Additionally, CNV eyes exhibited poorer integrity of the outer retina and BCVA (p=0.007) compared with non-CNV eyes. All 15 eyes with CNV underwent anti-vascular endothelial growth factor (anti-VEGF) therapy. Genotype analysis revealed that 7 of 10 patients (70.0%, 10 eyes) were predicted to have missense variants, while 3 of 10 patients (30.0%, 5 eyes) exhibited severe variants., Conclusions: The prevalence of CNV in XLRS eyes was found to be 8.1%. All CNVs secondary to XLRS were active and classified as type 2. CNV eyes demonstrated poorer visual function and compromised retinal structures. Anti-VEGF therapy demonstrated effectiveness in treating XLRS-CNVs. No significant genotype-phenotype correlation was established., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

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.

Purpose: This study aims to investigate the relationship among STRA6, circadian rhythm, and choroidal neovascularization (CNV) formation, as well as the regulatory mechanism of STRA6 in CNV under circadian rhythm disturbances., Methods: C57BL/6J male mice (aged 6 weeks) were randomly divided into control and jet lag groups (using a time shift method every 4 days to disrupt the molecular clock's capacity to synchronize with a stable rhythm). A laser-induced CNV model was established in both the control and the jet lag group after 2 weeks of jet lag. The size of CNV lesions and vascular leakage were detected by morphological and imaging examination on the seventh day post laser. STRA6 was screened by full transcriptome sequencing. Bioinformatics analysis was conducted to assess the variation and association of STRA6 in the GSE29801 dataset. The effects of STRA6 were evaluated both in vivo and in vitro. The pathway mechanism was further elucidated and confirmed through immunofluorescence of paraffin sections and Western blotting., Results: The disturbance of circadian rhythm promotes the formation of CNV. Patients with age-related macular degeneration (AMD) exhibited higher levels of STRA6 expression compared to the control group, and STRA6 was enriched in pathways related to angiogenesis. In addition, CLOCK and BMAL1, which are initiators that drive the circadian cycle, had regulatory effects on STRA6. Knocking down STRA6 reversed the promotion of CNV formation caused by circadian rhythm disturbance in vivo, and it also affected the proliferation, migration, and VEGF secretion of RPE cells without circadian rhythm in vitro, as well as impacting endothelial cells. Through activation of the JAK2/STAT3/VEGFA signaling pathway in unsynchronized RPE cells, STRA6 promotes CNV formation., Conclusions: This study suggests that STRA6 reduces CNV production by inhibiting JAK2/STAT3 phosphorylation after circadian rhythm disturbance. The results suggest that STRA6 may be a new direction for the treatment of AMD.

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.)

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.

Purpose: To analyze the genetic findings, clinical spectrum, and natural history of Best vitelliform macular dystrophy (BVMD) in a cohort of 222 children and adults., Design: Single-center retrospective, consecutive, observational study., Participants: Patients with a clinical diagnosis of BVMD from pedigrees with a likely disease-causing monoallelic sequence variant in the BEST1 gene., Methods: Data were extracted from electronic and physical case notes. Electrophysiologic assessment and molecular genetic testing were analyzed., Main Outcome Measures: Molecular genetic test findings and clinical findings including best-corrected visual acuity (BCVA), choroidal neovascularization (CNV) rates, and electrophysiologic parameters., Results: Two hundred twenty-two patients from 141 families were identified harboring 69 BEST1 variants. Mean age at presentation was 26.8 years (range, 1.3-84.8 years) and most patients (61.5%) demonstrated deterioration of central vision. Major funduscopic findings included 128 eyes (30.6%) with yellow vitelliform lesions, 78 eyes (18.7%) with atrophic changes, 49 eyes (11.7%) with fibrotic changes, 48 eyes (11.5%) with mild pigmentary changes, and 43 eyes (10.3%) showing a vitelliruptive appearance. Mean BCVA was 0.37 logarithm of the minimum angle of resolution (logMAR; Snellen equivalent, 20/47) for the right eye and 0.33 logMAR (Snellen equivalent, 20/43) for the left eye at presentation, with a mean annual loss rate of 0.013 logMAR and 0.009 logMAR, respectively, over a mean follow-up of 9.7 years. Thirty-seven patients (17.3%) received a diagnosis of CNV over a mean follow-up of 8.0 years. Eyes with CNV that received treatment with an anti-vascular endothelial growth factor (VEGF) agent showed better mean BCVA compared with eyes that were not treated with an anti-VEGF agent (0.28 logMAR [Snellen equivalent, 20/38] vs. 0.62 logMAR [Snellen equivalent, 20/83]). Most eyes exhibited a hyperopic refractive error (78.7%), and 13 patients (6.1%) received a diagnosis of amblyopia. Among the 3 most common variants, p.(Ala243Val) was associated with a later age of onset, better age-adjusted BCVA, and less advanced Gass stages compared with p.(Arg218Cys) and p.(Arg218His)., Conclusions: BVMD shows a wide spectrum of phenotypic variability. The disease is very slowly progressive, and the observed phenotype-genotype correlations allow for more accurate prognostication and counselling., Financial Disclosure(s): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article., (Copyright © 2024 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Objective: This study revealed a core regulator and common upstream mechanisms for the multifaceted pathological processes of age-related macular degeneration (AMD) and provided proof-of-concept for this new therapeutic target., Methods: Comprehensive gene expression analysis was performed using RNA sequencing of eye cup from old mice as well as laser-induced choroidal neovascularization (CNV) mouse model. Through integrative analysis and protein-protein interaction (PPI) analysis, common pathways and key transcription factor was identified simultaneously engaged in age-related retinal degeneration and CNV, the two typical pathological process of AMD. Subsequently, the expression changes of Spi1 , the key regulator, as well as the alternation of the downstream mechanisms were validated in both models through qRT-PCR, Elisa, flow cytometry and immunofluorescence. Further, we assessed the impact of Spi1 knockdown in vitro and in vivo using gene intervention vectors carried by adeno-associated virus or lentivirus to test its potential as a therapeutic target., Results: Compared to corresponding controls, we found 1,939 and 1,319 genes differentially expressed in eye cups of old and CNV mice respectively. The integrative analysis identified a total of 275 overlapping DEGs, of which 150 genes were co-upregulated. PPI analysis verified a central transcription factor, SPI1. The significant upregulation of Spi1 expression was then validated in both models, accompanied by macrophage polarization towards the M1 phenotype. Finally, SPI1 suppression significantly inhibited M1 polarization of BMDMs and attenuated neovascularization in CNV mice., Conclusion: This study demonstrates that SPI1 exerts a pivotal role in AMD by regulation of macrophage polarization and innate immune response, offering promise as an innovative target for treating 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 Qi, Zhang, Kong, Bi, Kong, Zhang and Zhao.)

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.

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.)

Recombinant adeno-associated viruses (rAAVs) have emerged as promising gene therapy vectors due to their proven efficacy and safety in clinical applications. In non-human primates (NHPs), rAAVs are administered via suprachoroidal injection at a higher dose. However, high doses of rAAVs tend to increase additional safety risks. Here, we present a novel AAV capsid (AAVv128), which exhibits significantly enhanced transduction efficiency for photoreceptors and retinal pigment epithelial (RPE) cells, along with a broader distribution across the layers of retinal tissues in different animal models (mice, rabbits, and NHPs) following intraocular injection. Notably, the suprachoroidal delivery of AAVv128-anti-VEGF vector completely suppresses the Grade IV lesions in a laser-induced choroidal neovascularization (CNV) NHP model for neovascular age-related macular degeneration (nAMD). Furthermore, cryo-EM analysis at 2.1 Å resolution reveals that the critical residues of AAVv128 exhibit a more robust advantage in AAV binding, the nuclear uptake and endosome escaping. Collectively, our findings highlight the potential of AAVv128 as a next generation ocular gene therapy vector, particularly using the suprachoroidal delivery route., (© 2024. The Author(s).)

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.)

Purpose: The purpose of this study was to report functional and anatomical outcomes of anti-VEGF treatment in eyes with autosomal recessive bestrophinopathy (ARB) presenting in the first decade of life., Methods: The study included case series of four eyes from two siblings with compound heterozygous mutations in the BEST1 gene who were treated with eight monthly intravitreal bevacizumab injections. Response to treatment was analyzed using color fundus photography, fundus autofluorescence, optical coherence tomography, OCT angiography, and microperimetry., Results: Patient 1 (male, age 9 years) had visual acuity of 20/20 in the right eye and 20/50 in the left eye. Patient 2 (female, age 10 years) had visual acuity of 20/25 in the right eye and 20/20 in the left eye. All eyes had multifocal subretinal deposition of lipofuscin and subretinal fluid, and three eyes had choroidal neovascularization (CNV). Lipofuscin material reabsorbed in 2 of 4 eyes, the CNV regressed in 3 of 3 eyes, a bacillary detachment resolved in 1 of 1 eye, but the subretinal fluid did not change. Functional improvement in visual acuity was noted, but MP showed scattered areas of reduced retinal sensitivity. No ocular or systemic side effects were detected., Conclusion: Anti-VEGF treatment of choroidal neovascularization in eyes with ARB resulted in anatomical changes that were only clinically significant in the eye with decreased visual acuity. The hyporeflective subretinal material remained unchanged suggesting a nonexudative cause. These findings provide new insights into the management of ARB, especially in pediatric subjects with CNV., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Opthalmic Communications Society, Inc.)

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.)

Choroidal Neovascularization (CNV) is capable of inciting recurrent hemorrhage in the macular region, severely impairing patients' visual acuity. During the onset of CNV, infiltrating M2 macrophages play a crucial role in promoting angiogenesis. To control this disease, our study utilizes the RNA interference (RNAi)-based gene therapy to reprogram M2 macrophages to the M1 phenotype in CNV lesions. We synthesize the mannose-modified siRNA-loaded liposome specifically targeting M2 macrophages to inhibit the inhibitory kappa B kinase β (IKKβ) gene involved in the polarization of macrophages, consequently modulating macrophage polarization state. In vitro and in vivo, the mannose-modified IKKβ siRNA-loaded liposome (siIKKβ-ML) has been proven to effectively target M2 macrophages to repolarize them to M1 phenotype, and inhibit the progression of CNV. Collectively, our findings elucidate that siIKKβ-ML holds the potential to control CNV by reprogramming the macrophage phenotype, indicating a promising therapeutic avenue for CNV management., Competing Interests: Declaration of competing interest None., (Copyright © 2023. Published by Elsevier Inc.)

Choroidal neovascularization (CNV) is a devastating pathology of numerous ocular diseases, such as wet age-related macular degeneration (wAMD), which causes irreversible vision loss. Although anti-vascular endothelial growth factor (VEGF) therapy has been widely used, poor response or no response still exists in some cases, suggesting that there are other components involved in the angiogenic process. Therefore, the underlying mechanism needs to be clarified and new target of anti-angiogenic therapy is urgently needed. It has been demonstrated that damaged retinal pigment epithelium (RPE) cells can activate inflammasome, driving a degenerative tissue environment and an enhanced pro-angiogenic response, which implies that RPE dysfunction may be a hallmark of the pathogenesis. Previously, we have shown that DNA damage can induce RPE dysfunction, triggering senescence-associated secretory phenotype (SASP) and local inflammation. In this study, we identify that chrysin can reduce DNA damage, especially telomere erosion in vitro, thus compromise the dysfunction of RPE and the decreased expression of SASP factor. Importantly, we find that DNA damage of RPE cells is remarkable in laser-induced CNV lesion, resulting in inflammatory response, which can be ameliorated by chrysin, mainly through IL-17 signaling pathway and its downstream signal transducer and activator of transcription 3 (STAT3) activities. In summary, our results indicate the interplay between DNA damage, perturbed RPE homeostasis, inflammatory response and angiogenesis in laser-induced CNV, and more importantly, chrysin may be an effective therapeutic supplement for CNV., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

Purpose: Choroidal neovascularization (CNV) can constitute the final pathology of many ocular diseases and result in severe vision loss. Studies have demonstrated that DNA methylation is critical in retinal development, aging, and disorders. The current work investigated the effects and underlying mechanism of 5-Aza-2'-deoxycytidine (5-aza-dC), a suppressor of DNA methylation, in the pathological progression of CNV., Methods: The DNA methylation profiles of retinal pigment epithelial (RPE)/choroidal complexes in normal and laser-induced CNV mice were assessed by Arraystar Mouse RefSeq Promoter Arrays. The CNV area and blood flow density and intensity were observed by optical coherence tomography angiography, and fluorescence leakage was examined by fundus fluorescein angiography in CNV mice with systemic administration of 5-aza-dC. The effects of 5-aza-dC on the biological functions of bEnd.3 cells were estimated by related assays. Notum gene promoter methylation was measured using bisulfite sequencing PCR. Methyltransferases and Wnt signaling-related genes were detected in animal and cell culture experiments by real-time PCR and immunoblot., Results: Methyltransferases were upregulated, but Notum (a secretion inhibitor of Wnt signaling) was downregulated in the RPE/choroidal complexes of mice with experimental CNV. Intraperitoneal injection of 5-aza-dC inactivated the Wnt pathway and ameliorated the lesion area and the intensity and density of blood flow, as well as the degree of leakage in CNV. In vitro, vascular endothelial growth factor A (VEGFA) stimulation promoted methyltransferases expression and suppressed Notum expression, consequently activating Wnt signaling, whereas exogenous 5-aza-dC reversed VEGFA-induced hyperpermeability, proliferation, migration, and tube formation in bEnd.3 cells via demethylation of Notum promoter., Conclusions: We observed that 5-aza-dC attenuates the growth of CNV by inhibiting the Wnt signaling pathway via promoter demethylation of the Wnt antagonist Notum. These findings provide a theoretical basis for methylation-based treatment with the Notum gene as a potential target for CNV treatment.

Age-related macular degeneration (AMD) is a leading cause of blindness featuring pathogenic neovascularization of the choroidal vasculature (CNV). Although systemic immunity plays a role in AMD, the ocular signals that recruit and activate immune cells remain poorly defined. Using single-cell RNA sequencing, we prospectively profile peripheral blood mononuclear cells from 65 individuals including AMD and controls, which we integrate with existing choroid data. We generate a network of choroid-peripheral immune interactions dysregulated in AMD, including known AMD-relevant gene vascular endothelial growth factor (VEGF) receptor 2. Additionally, we find CYR61 is upregulated in choroidal veins and may signal to circulating monocytes. In mice, we validate that CYR61 is abundant in endothelial cells within CNV lesions neighboring monocyte-derived macrophages. Mechanistically, CYR61 activates macrophage anti-angiogenic gene expression, and ocular Cyr61 knockdown increases murine CNV size, indicating CYR61 inhibits CNV. This study highlights the potential of multi-tissue human datasets to identify disease-relevant and potentially therapeutically modifiable targets., Competing Interests: Declaration of interests J.B.L., P.A.R., and R.S.A. are listed as inventors on intellectual property filed by Washington University in St. Louis., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: The purpose of this study was to identify key genes and their regulatory networks that are conserved in mouse models of age-related macular degeneration (AMD) and human AMD., Methods: Retinal RNA-Seq was performed in laser-induced choroidal neovascularization (CNV) mice at day 3 and day 7 after photocoagulation. Mass spectrometry-based proteomic analysis was performed with retinas collected at day 3. Retinal RNA-Seq data was further compared among mouse models of laser-induced CNV and NaIO3-induced retinal degeneration (RD) and a large AMD cohort., Results: Retinal RNA-Seq revealed upregulated genes and pathways related to innate immunity and inflammation in mice with CNV, with more profound changes at the early stage (day 3). Proteomic analysis further validated these differentially expressed genes and their networks in retinal inflammation during CNV. Notably, the most evident overlap in the retina of mice with laser-induced CNV and NaIO3-induced RD was the upregulation of inflammation-related genes, pointing to a common vital role of retinal inflammation in the early stage for both mouse AMD models. Further comparative transcriptomic analysis of the mouse AMD models and human AMD identified 48 conserved genes mainly involved in inflammation response. Among them, B2M, C3, and SERPING1 were upregulated in all stages of human AMD and the mouse AMD models compared to controls., Conclusions: Our study demonstrates conserved molecular changes related to retinal inflammation in mouse AMD models and human AMD and provides new insight into the translational application of these mouse models in studying AMD mechanisms and treatments.

This study aims to investigate the effect of a systemic lipopolysaccharide (LPS) stimulus in the course of laser-induced choroidal neovascularization (CNV) in C57BL/6 J mice. A group of CNV-subjected mice received 1 mg/kg LPS via the tail vein immediately after CNV induction. Mouse eyes were monitored in vivo with fluorescein angiography for 2 weeks. In situ hybridization and flow cytometry were performed in the retina at different time points. LPS led to increased fluorescein leakage 3 days after CNV, correlated with a large influx of monocyte-derived macrophages and increase of pro-inflammatory microglia/macrophages in the retina. Additionally, LPS enhanced Vegfα mRNA expression by Glul -expressing cells but not Aif1 positive microglia/macrophages in the laser lesion. These findings suggest that systemic LPS exposure has transient detrimental effects in the course of CNV through activation of microglia/macrophages to a pro-inflammatory phenotype and supports the important role of these cells in the CNV course.

Purpose: To evaluate the genetic associations of different subtypes of central serous chorioretinopathy (CSCR), neovascular age-related macular degeneration (nAMD), and polypoidal choroidal vasculopathy (PCV)., Design: A case-control genetic association study., Methods: This study enrolled 217 CSCR, 341 nAMD, 288 PCV patients, and 1380 controls. The CSCR patients were classified into those with focal or diffuse leakage, with or without pigment epithelial detachment (PED), and with or without macular neovascularization (MNV). Associations between 11 variants from 8 genes, ADAMTS9, ANGPT2, ARMS2, CFH, NR3C2, PGF, TNFRSF10A and VIPR2, and diseases/subtypes were analyzed by logistic regression analysis adjusted for age and sex, and inter-phenotype comparison by heterogeneity test., Results: The CFH rs800292-A conferred a protective effect for CSCR with MNV (OR=0.44, P = 0.002) and a risk effect for CSCR without MNV (OR=1.31, P = 0.023). CSCR patients carrying rs800292-G had a 3.23-fold of increased risk towards developing secondary MNV (P = 1.45 ×10 -4 ). CFH rs3753394, rs800292 and rs1329428 showed similar effects among CSCR with MNV, nAMD and PCV, but opposite effects on CSCR without MNV. TNFRSF10A rs13278062-T was associated with overall CSCR but not with CSCR subtypes, nAMD or PCV. Moreover, CFH and ARMS2 SNPs showed heterogeneous effects in CSCR without MNV against CSCR with MNV, nAMD and PCV., Conclusions: Genetic associations of CSCR with MNV resembled nAMD and PCV compared to CSCR without MNV, indicating differential genetic effects on neovascularization and choroidopathy. Further investigation of the functional roles of CFH, ARMS2, and TNFRSF10A in CSCR, nAMD and PCV should help elucidate the mechanisms of these maculopathies., Competing Interests: Declaration of Competing Interest No conflicting relationship exists for any author., (Copyright © 2023. Published by Elsevier Inc.)

Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are vascular diseases with high prevalence, ranking among the leading causes of blindness and vision loss worldwide. Despite being effective, current treatments for AMD and DR are burdensome for patients and clinicians, resulting in suboptimal compliance and real risk of vision loss. Thus, there is an unmet need for long-lasting alternatives with improved safety and efficacy. Adeno-associated virus (AAV) is the leading vector for ocular gene delivery, given its ability to enable long-term expression while eliciting relatively mild immune responses. Progress has been made in AAV-based gene therapies for not only inherited retinal diseases but also acquired conditions with preclinical and clinical studies of AMD and DR showing promising results. These studies have explored several pathways involved in the disease pathogenesis, as well as different strategies to optimise gene delivery. These include engineered capsids with enhanced tropism to particular cell types, and expression cassettes incorporating elements for a targeted and controlled expression. Multiple-acting constructs have also been investigated, in addition to gene silencing and editing. Here, we provide an overview of strategies employing AAV-mediated gene delivery to treat AMD and DR. We discuss preclinical efficacy studies and present the latest data from clinical trials for both diseases., (© 2023. The Author(s).)

It has previously been reported that antioxidant vitamins can help reduce the risk of vision loss associated with progression to advanced age-related macular degeneration (AMD), a leading cause of visual impairment among the elderly. Nonetheless, how oxidative stress contributes to the development of choroidal neovascularization (CNV) in some AMD patients and geographic atrophy (GA) in others is poorly understood. Here, we provide evidence demonstrating that oxidative stress cooperates with hypoxia to synergistically stimulate the accumulation of hypoxia-inducible factor (HIF)-1α in the retinal pigment epithelium (RPE), resulting in increased expression of the HIF-1-dependent angiogenic mediators that promote CNV. HIF-1 inhibition blocked the expression of these angiogenic mediators and prevented CNV development in an animal model of ocular oxidative stress, demonstrating the pathological role of HIF-1 in response to oxidative stress stimulation in neovascular AMD. While human-induced pluripotent stem cell (hiPSC)-derived RPE monolayers exposed to chemical oxidants resulted in disorganization and disruption of their normal architecture, RPE cells proved remarkably resistant to oxidative stress. Conversely, equivalent doses of chemical oxidants resulted in apoptosis of hiPSC-derived retinal photoreceptors. Pharmacologic inhibition of HIF-1 in the mouse retina enhanced-while HIF-1 augmentation reduced-photoreceptor apoptosis in two mouse models for oxidative stress, consistent with a protective role for HIF-1 in photoreceptors in patients with advanced dry AMD. Collectively, these results suggest that in patients with AMD, increased expression of HIF-1α in RPE exposed to oxidative stress promotes the development of CNV, but inadequate HIF-1α expression in photoreceptors contributes to the development of GA., Competing Interests: Competing interests statement: A.S. is the cofounder of, holds equity in, is a member of the Board of Directors, and serves as the Chief Executive Officer of HIF Therapeutics, Inc. This arrangement has been reviewed and approved by the Johns Hopkins University in accordance with its conflict of interest policies.

Purpose: Myopic choroidal neovascularization (mCNV) is a prevalent cause of vision loss. However, the development of effective therapeutic targets for mCNV has been hindered by the paucity of suitable animal models. Therefore, the aim of this study is to identify potential genes and pathways associated with mCNV and to unearth prospective therapeutic targets that can be utilized to devise efficacious treatments. Methods: Text data mining was used to identify genes linked to choroid, neovascularization, and myopia. g: Profiler was utilized to analyze the biological processes of gene ontology and the Reactome pathways. Protein interaction network analysis was performed using strings and visualized in Cytoscape. MCODE and cytoHubba were used for further screening. Results: Discovery-driven text data mining identified 55 potential genes related to choroid, neovascularization, and myopia. Gene enrichment analysis revealed 11 biological processes and seven Reactome pathways. A protein-protein interaction network with 47 nodes was constructed and analyzed using centrality ranking. Key clusters were identified through algorithm tools. Finally, 14 genes (IL6, FGF2, MMP9, IL10, TNF, MMP2, HGF, MMP3, IGF1, CCL2, CTNNB1, BDNF, NGF, and EDN1), in addition to VEGFA, were evaluated as targets with potential as future therapeutics. Conclusions: This study provides new potential therapeutic targets for mCNV, including IL6, FGF2, MMP9, IL10, TNF, MMP2, HGF, MMP3, IGF1, CCL2, CTNNB1, BDNF, NGF, and EDN1, which correspond to seven potential enriched pathways. These findings provide a basis for further research and offer new possibilities for developing therapeutic interventions for this condition.

We examined the effects of gene ablation and chemical inhibition of transient receptor potential ankyrin 1 (TRPA1) on the growth of experimental argon laser-induced choroidal neovascularization (CNV) in mice. CNV was induced in the eyes of 6- to 8-week-old TRPA1-null (knockout [KO]) and wild-type (WT) mice by argon laser irradiation. Gene expression analysis was performed in laser-injured tissues at days 1 and 3. CNV growth was evaluated at day 14. Reciprocal bone marrow transplantation was performed between each genotype to identify the components responsible for either recipient tissue or bone marrow-derived inflammatory cells. Our results show that laser irradiation successfully induced CNV growth at the site of laser injury. The size of induced CNV was significantly smaller in KO mice than in WT mice at day 14, as determined by angiography with fluorescein isothiocyanate-dextran. Invasion of neutrophils, but not macrophages, was suppressed in association with suppression of the expression of transforming growth factor β1 and interleukin 6 in laser-irradiated KO tissue. Bone marrow transplantation indicated that the genotype of the recipient mouse, but not of inflammatory cells, is attributable to the KO phenotype. Systemic administration of a TRPA1 antagonist also reduced the CNV in a WT mouse. In conclusion, TRPA1 signaling in local cells is involved in growth of laser-induced CNV. The phenotype was not attributable to vascular endothelial cells and inflammatory cells. Blocking TRPA1 signal may therefore be a potential treatment strategy for CNV-related ocular diseases., (Copyright © 2023. Published by Elsevier Inc.)

The upregulation of vascular endothelial growth factor (VEGF) is strongly associated with the development of choroidal neovascularization (CNV) in patients with neovascular age-related macular degeneration (nAMD). Currently, the standard treatment for nAMD involves frequent intravitreal injections of anti-VEGF agents, which inhibit the growth of new blood vessels and prevent leakage. However, this treatment regimen places a significant burden on patients, their families, and healthcare providers due to the need for repeated visits to the clinic for injections. Gene therapy, which enables the sustained expression of anti-VEGF proteins after a single injection, can dramatically reduce the treatment burden. KH631 is a recombinant adeno-associated virus 8 vector that encodes a human VEGF receptor fusion protein, and it is being developed as a long-term treatment for nAMD. In preclinical studies using non-human primates, subretinal administration of KH631 at a low dose of 3 × 10 8 vg/eye resulted in remarkable retention of the transgene product in the retina and prevented the formation and progression of grade IV CNV lesions. Furthermore, sustained transgene expression was observed for more than 96 weeks. These findings suggest that a single subretinal injection of KH631 has the potential to offer a one-time, low-dose treatment for nAMD patients., Competing Interests: Declaration of interests X.K., H.J., Q.L., S.L., Y.Q., J.L., Q.X., and Q.Z. are employees of Chengdu Origen Biotechnology Co., Ltd. X.K., H.J., and Q.Z. are inventors on a patent covering this work, which has been licensed to Chengdu Origen Biotechnology., (Copyright © 2023 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Choroidal neovascularization (CNV) is characterized by the infiltration of immune cells, particularly neutrophils. Neutrophil extracellular trap (NET) facilitates the angiogenesis of pulmonary endothelial cells via activating Toll-like receptor 4 (TLR4). TLR4 promotes the expression of transcription factor hypoxia inducible factor-1α (HIF-1α), which promotes inflammation and angiogenesis via the up-regulation of metalloproteinase-9 (MMP-9) and interleukin-1β (IL-1β). In the present study, we aimed to identify the formation of NET and its role in CNV. Our results showed that NET levels were increased in a mouse laser-induced CNV model via oxidative stress, whereas the inhibition of NET alleviated CNV. In vitro, NET activated the TLR4/HIF-1α pathway in human choroidal endothelial cells (HCECs). Additionally, NET increased the transcription and expression of MMP-9 and IL-1β in HCECs via activating the TLR4/HIF-1α pathway. Meanwhile, NET promoted the inflammatory response accompanied by the proliferation, migration and tube formation of HCECs in a MMP-9- and IL-1β-dependent manner. In conclusion, NET was up-regulated in CNV and promoted the formation of CNV via activating the TLR4/HIF-1α pathway in choroidal endothelial cells. Our data uncovered the novel role of NET in promoting the formation of CNV. The underlying mechanism of NET could be targeted to delay the process of CNV., (© 2023 Federation of European Biochemical Societies.)

Nicotine-induced endoplasmic reticulum (ER) stress in retinal pigment epithelium (RPE) cells is thought to be one pathological mechanism underlying age-related macular degeneration (AMD). ERp29 attenuates tobacco extract-induced ER stress and mitigates tight junction damage in RPE cells. Herein, we aimed to further investigate the role of ERp29 in nicotine-induced ER stress and choroidal neovascularization (CNV). We found that the expression of ERp29 and GRP78 in ARPE-19 cells was increased in response to nicotine exposure. Overexpression of ERp29 decreased the levels of GRP78 and the C/EBP homologous protein (CHOP). Knockdown of ERp29 increased the levels of GRP78 and CHOP while reducing the viability of ARPE-19 cells under nicotine exposure conditions. In the ARPE-19 cell/macrophage coculture system, overexpression of ERp29 decreased the levels of M2 markers and increased the levels of M1 markers. The viability, migration and tube formation of human umbilical vein endothelial cells (HUVECs) were inhibited by conditioned medium from the ERp29-overexpressing group. Moreover, overexpression of ERp29 inhibits the activity and growth of CNV in mice exposed to nicotine in vivo. Taken together, our results revealed that ERp29 attenuated nicotine-induced ER stress, regulated macrophage polarization and inhibited CNV.

Background: Neovascular age-related macular degeneration causes vision loss from destructive angiogenesis, termed choroidal neovascularization (CNV). Cx3cr1 -/- mice display alterations in non-classical monocytes and microglia with increased CNV size, suggesting that non-classical monocytes may inhibit CNV formation. NR4A1 is a transcription factor that is necessary for maturation of non-classical monocytes from classical monocytes. While Nr4a1 -/- mice are deficient in non-classical monocytes, results are confounded by macrophage hyper-activation. Nr4a1 se2/se2 mice lack a transcriptional activator, resulting in non-classical monocyte loss without macrophage hyper-activation., Main Body: We subjected Nr4a1 -/- and Nr4a1 se2/se2 mice to the laser-induced CNV model and performed multi-parameter flow cytometry. We found that both models lack non-classical monocytes, but only Nr4a1 -/- mice displayed increased CNV area. Additionally, CD11c + macrophages were increased in Nr4a1 -/- mice. Single-cell transcriptomic analysis uncovered that CD11c + macrophages were enriched from Nr4a1 -/- mice and expressed a pro-angiogenic transcriptomic profile that was disparate from prior reports of macrophage hyper-activation., Conclusions: These results suggest that non-classical monocytes are dispensable during CNV, and NR4A1 deficiency results in increased recruitment of pro-angiogenic macrophages., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Purpose: Macular neovascularization is a relatively common and potentially visually devastating complication of age-related macular degeneration. In macular neovascularization, pathologic angiogenesis can originate from either the choroid or the retina, but we have limited understanding of how different cell types become dysregulated in this dynamic process., Methods: To study how gene expression is altered in focal areas of pathology, we performed spatial RNA sequencing on a human donor eye with macular neovascularization as well as a healthy control donor. We performed differential expression to identify genes enriched within the area of macular neovascularization and used deconvolution algorithms to predict the originating cell type of these dysregulated genes., Results: Within the area of neovascularization, endothelial cells demonstrated increased expression of genes related to Rho family GTPase signaling and integrin signaling. Likewise, VEGF and TGFB1 were identified as potential upstream regulators that could drive the observed gene expression changes produced by endothelial and retinal pigment epithelium cells in the macular neovascularization donor. These spatial gene expression profiles were compared to previous single-cell gene expression experiments in human age-related macular degeneration as well as a model of laser-induced neovascularization in mice. As a secondary aim, we investigated regional gene expression patterns within the macular neural retina and between the macular and peripheral choroid., Conclusions: Overall, this study spatially analyzes gene expression across the retina, retinal pigment epithelium, and choroid in health and describes a set of candidate molecules that become dysregulated in macular neovascularization.

Abnormal ocular neovascularization, a major pathology of eye diseases, leads to severe visual loss. The role of lens epithelial cell (LEC)-derived exosomes (Lec-exo) is largely unknown. Thus, we aimed to investigate whether Lec-exo can inhibit abnormal ocular neovascularization and explore the possible mechanisms. In our study, we proved the first evidence that exosomes derived from LECs attenuated angiogenesis in both oxygen-induced retinopathy and laser-induced choroidal neovascularization mice models. Further in vitro experiments proved that Lec-exo inhibited proliferation, migration, and tube formation capability of human umbilical vein endothelial cells in high glucose condition. Further high-throughput miRNAs sequencing analysis detected that miR-146a-5p was enriched in Lec-exo. Mechanistically, exosomal miR-146a-5p was delivered to endothelial cells and bound to the NRAS coding sequence, which subsequently inactivated AKT/ERK signaling pathway. We successfully elucidated the function of Lec-exo in inhibiting abnormal ocular neovascularization, which may offer a promising strategy for treatment of abnormal ocular neovascularization., (© 2023 Federation of American Societies for Experimental Biology.)

During diabetic retinopathy (DR) progression, the retina undergoes various metabolic changes, including hypoxia-signalling cascade induction in the cells of retinal pigmented epithelium (RPE). The overexpression of hypoxic inducible factors causes transcription of many target genes including vascular endothelial growth factor (VEGF). The RPE cells form the outer blood retinal barrier (oBRB), a specialized structure that regulates ions and metabolites flux into the retina to maintain a suitable quality of its extracellular microenvironment. VEGF worsens retinal condition since its secretion from the basolateral compartment of RPE cells compromises the barrier's integrity and induces choroidal neovascularization. In this work, we hypothesized that PACAP prevents the damage to oBRB and controls choroidal neovascularization through the induction of ADNP. Firstly, we demonstrated that ADNP is expressed in Streptozotocin (STZ)-induced diabetic animals. To validate our hypothesis, we cultured endothelial cells (H5V) forming vessels-like structures, in a conditioned medium (CM) derived from ARPE-19 cells exposed to hyperglycaemic/hypoxic insult, containing a known VEGF concentration. The involvement of PACAP-ADNP axis on oBRB integrity was evaluated through the measurement of trans-epithelial-electrical resistance and permeability assay performed on ARPE cell monolayer cultured in CM and by analysing the expression of two tight junction forming proteins, ZO1 and occludin. By culturing H5V in CM, we demonstrated that PACAP-ADNP axis counteracted vessels-like structures formation promoted by VEGF. In conclusion, the results suggested a primary role of PACAP/ADNP axis in preventing oBRB damage and in controlling aberrant choroidal neovascularization induced by VEGF secreted from RPE cells exposed to hyperglycaemia/hypoxic insult in DR., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Purpose: Wet AMD (wAMD) is associated with cellular senescence. However, senescent cell-targeted therapies for wAMD have rarely been comprehensively studied. This study aimed to explore the therapeutic effects of senolytic agents on choroidal neovascularization (CNV)., Methods: RNA sequencing datasets were obtained from the Gene Expression Omnibus database and used to explore the association between senescence and wAMD. We explored the effects of senescent adult RPE cell line-19 cells on the proliferation, migration, invasion, and tube formation of human umbilical vein endothelial cells. A laser-induced CNV animal model was used to study wAMD. We studied a senescent cell elimination therapy for CNV progression using two types of senolytics and a transgenic method., Results: Cells in the retinal pigment epithelium-choroid of the CNV model were enriched in senescence, inflammation, and angiogenesis gene sets. AP20187 was used to specifically eliminate senescent cells and proven to alleviate CNV progression in INK-ATTAC transgenic mice. Senescent adult RPE cell line-1 cells produced elevated levels of senescence-associated secretory phenotypes, including VEGFs; they also demonstrated increased proliferation, migration, invasion, and tube formation in human umbilical vein endothelial cells. The number of senescent cells increased in the laser-induced CNV rat model, and intravitreal injections of dasatinib with quercetin reduced the expression of p16 in CNV and alleviated neovascularization., Conclusions: Senescent RPE cells can accelerate pathological neovascularization; thus, senescent cell-targeting therapy has great clinical potential for wAMD.

Purpose: Risk for developing age-related macular degeneration (AMD) is linked to an overactive complement system. In the mouse model of laser-induced choroidal neovascularization (CNV), elevated levels of complement effector molecules, including complement C3, have been identified, and the alternative pathway (AP) is required for pathology. The main soluble AP regular is complement factor H (fH). We have previously shown that AP inhibition via subretinal AAV-mediated delivery of CR2-fH using a constitutive promoter is efficacious in reducing CNV. Here we ask whether the C3 promoter (pC3) effectively drives CR2-fH bioavailability for gene therapy., Methods: Truncated pC3 was used to generate plasmids pC3-mCherry/CR2-fH followed by production of corresponding AAV5 vectors. pC3 activation was determined in transiently transfected ARPE-19 cells stimulated with H2O2 or normal human serum (+/- antioxidant or humanized CR2-fH, respectively). CNV was analyzed in C57BL/6J mice treated subretinally with AAV5-pC3-mCherry/CR2-fH using imaging (optical coherence tomography [OCT] and fundus imaging), functional (electroretinography [ERG]), and molecular (protein expression) readouts., Results: Modulation of pC3 in vitro is complement and oxidative stress dependent, as shown by mCherry fluorescence. AAV5-pC3-CR2-fH were identified as safe and effective using OCT and ERG. CR2-fH expression significantly reduced CNV compared to mCherry and was correlated with reduced levels of C3dg/C3d in the retinal pigment epithelium/choroid fraction., Conclusions: We conclude that complement-dependent regulation of AP inhibition ameliorates AMD pathology as effectively as using a constitutive promoter., Translational Relevance: The goal of anticomplement therapy is to restore homeostatic levels of complement activation, which might be more easily achievable using a self-regulating system.

Age-related macular degeneration (AMD), a leading cause of irreversible blindness in adults, may result in poor central vision, making it difficult to see, read, and drive. AMD is generally classified in either dry or wet types. Milder cases of dry AMD may progress to geographic atrophy (GA), leading to significant visual disability; wet, or neovascular AMD, which involves choroidal neovascularization (CNV), can lead to complete loss of central vision. Adiponectin (APN) discovery in the mid-1990's and, subsequently, its two cognate receptors (AdipoRs) in the early 2000s have led to a remarkable progress in better understanding metabolic disorders, as well as metabolism-associated ocular pathology. APN/AdipoRs signaling plays a central role in a variety of molecular and cellular physiological events, including glucose and lipid metabolism, whole-body energy regulation, immune and inflammation responses, insulin sensitivity and retinal cell biological functions. This review is an amalgamation of recent information related to APN/AdipoRs in the pathophysiology of retinal diseases and furthers its association with AMD and diabetic retinopathy. Additionally, we present our original research, where we designed control peptide and CNV inhibitory peptide from the globular region of APN to see the effect of these peptides on the mouse model of laser-induced CNV. The inhibitory peptide (APN1) inhibited CNV by more than 75% while the control peptide did not inhibit CNV.

Background: Endothelial cell activation is tightly controlled by the balance between VEGF (vascular endothelial cell growth factor) and Notch signaling pathway. VEGF destabilizes blood vessels and promotes neovascularization, which are common features of sight-threatening ocular vascular disorders. Here, we show that BCL6B (B-cell CLL/lymphoma 6 member B protein), also known as BAZF, ZBTB28, and ZNF62, plays a pivotal role in the development of retinal edema and neovascularization., Methods: The pathophysiological physiological role of BCL6B was investigated in cellular and animal models mimicking 2 pathological conditions: retinal vein occlusion and choroidal neovascularization. An in vitro experimental system was used in which human retinal microvascular endothelial cells were supplemented with VEGF. Choroidal neovascularization cynomolgus monkey model was generated to investigate the involvement of BCL6B in the pathogenesis. Mice lacking BCL6B or treated with BCL6B-targeting small-interfering ribose nucleic acid were examined for histological and molecular phenotypes., Results: In retinal endothelial cells, the BCL6B expression level was increased by VEGF. BCL6B-deficient endothelial cells showed Notch signal activation and attenuated cord formation via blockage of the VEGF-VEGFR2 signaling pathway. Optical coherence tomography images showed that choroidal neovascularization lesions were decreased by BCL6B-targeting small-interfering ribose nucleic acid. Although BCL6B mRNA expression was significantly increased in the retina, BCL6B-targeting small-interfering ribose nucleic acid suppressed ocular edema in the neuroretina. The increase in proangiogenic cytokines and breakdown of the inner blood-retinal barrier were abrogated in BCL6B knockout (KO) mice via Notch transcriptional activation by CBF1 (C promotor-binding factor 1) and its activator, the NICD (notch intracellular domain). Immunostaining showed that Müller cell activation, a source of VEGF, was diminished in BCL6B-KO retinas., Conclusions: These data indicate that BCL6B may be a novel therapeutic target for ocular vascular diseases characterized by ocular neovascularization and edema., Competing Interests: Disclosures H. Hara received a research grant from the Carna Biosciences Inc. K. Yunoki and Y. Sato were employees of Carna Biosciences Inc. M. Sawa and K. Yoshino were Chief Scientific Officer and Chief Executive Officer for the Carna Biosciences Inc, respectively, and they were stockholders in Carna Biosciences Inc at the time this study was conducted.

Age-related macular degeneration (AMD) is one of the leading causes of irreversible blindness in the elderly population. Neovascular AMD is the late stage, characterized by choroidal neovascularization (CNV). Non-coding RNAs have been implicated in CNV; however, the role of circular RNAs (circRNAs) has not yet been elucidated. Herein, we comprehensively investigated circRNA profiles in laser-induced CNV mouse models and patient specimens. A novel circRNA, circRNA Uxs1, was identified, and its function in CNV regulation was investigated in the present study. CircRNA Uxs1 was consistently upregulated in CNV patient specimens and CNV mouse models. Knockdown of circRNA Uxs1 interrupted the tube formation, migration, and proliferation of endothelial cells in vitro. Silencing circRNA Uxs1 in vivo alleviated neovascularization formation, as shown by the decreased size of laser spots. Mechanistically, circRNA Uxs1 functioned by binding to miR-335-5p, which further upregulated the expression of placental growth factor (PGF) gene and activated the mammalian target of rapamycin/p70 S6 Kinase (mTOR/p70 S6k) pathway. By subretinal injections of adeno-associated virus (AAV), we demonstrated the anti-angiogenic function of circRNA Uxs1 knockdown in vivo. In conclusion, circRNA Uxs1 promoted CNV by sponging miR-335-5p, which stimulated PGF expression and subsequently activated the mTOR/p70 S6k pathway. Therefore, circRNA Uxs1 may serve as a promising therapeutic target for CNV., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Purpose: Age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV) are sister diseases and have several similar clinical features and still have few genetic differences. The association of HERPUD1 (homocysteine inducible ER protein with ubiquitin like domain 1) gene variant rs2217332 with PCV is known; however, such association with AMD has not been reported in the Indian population. We analyzed the association of rs2217332 with PCV and AMD to identify the preferential association of this variant with these diseases., Methods: This is a population-based case-control study consisting of 422 patients (129 AMD cases; 101 PCV cases, 192 healthy controls) recruited from the vitreoretinal clinic Sankara Nethralaya. The sample size for the study was calculated using appropriate power calculation methods. Genotype was determined using PCR-based Sanger sequencing. The SPSS V23.0 statistical package tool was used to calculate chi-square and ROC to determine the association of rs2217332 with control, AMD, and PCV., Results: Here, we report for the first time the association of this genetic variant (rs2217332) with AMD and PCV in the Indian population. The case-control study shows a significant association of this SNP with PCV (P value = 0.002); however, this variant is not significantly associated with AMD (P value = 0.602). Comparison between AMD (as control) and PCV (as case) also showed significant association of the SNP with PCV (P value = 0.02). Minor allele A conferred to increase the risk of PCV., Conclusions: The study concludes that the genetic variant rs2217332 in HERPUD1 gene is highly significantly associated with PCV and not with AMD in Indian populations., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Adrenomedullin 2 (AM2; also known as intermedin) is a member of the adrenomedullin (AM) peptide family. Similarly to AM, AM2 partakes in a variety of physiological activities. AM2 has been reported to exert protective effects on various organ disorders; however, its significance in the eye is unknown. We investigated the role of AM2 in ocular diseases. The receptor system of AM2 was expressed more abundantly in the choroid than in the retina. In an oxygen-induced retinopathy model, physiological and pathologic retinal angiogenesis did not differ between AM2-knockout (AM2-/-) and wild-type mice. In contrast, in laser-induced choroidal neovascularization, a model of neovascular age-related macular degeneration, AM2-/- mice had enlarged and leakier choroidal neovascularization lesions, with exacerbated subretinal fibrosis and macrophage infiltration. Contrary to this, exogenous administration of AM2 ameliorated the laser-induced choroidal neovascularization-associated pathology and suppressed gene expression associated with inflammation, fibrosis, and oxidative stress, including that of VEGF-A, VEGFR-2, CD68, CTGF, and p22-phox. The stimulation of human adult retinal pigment epithelial (ARPE) cell line 19 cells with TGF-β2 and TNF-α induced epithelial-to-mesenchymal transition (EMT), whereas AM2 expression was also elevated. The induction of EMT was suppressed when the ARPE-19 cells were pretreated with AM2. A transcriptome analysis identified 15 genes, including mesenchyme homeobox 2 (Meox2), whose expression was significantly altered in the AM2-treated group compared with that in the control group. The expression of Meox2, a transcription factor that inhibits inflammation and fibrosis, was enhanced by AM2 treatment and attenuated by endogenous AM2 knockout in the early phase after laser irradiation. The AM2 treatment of endothelial cells inhibited endothelial to mesenchymal transition and NF-κB activation; however, this effect tended to be canceled following Meox2 gene knockdown. These results indicate that AM2 suppresses the neovascular age-related macular degeneration-related pathologies partially via the upregulation of Meox2. Thus, AM2 may be a promising therapeutic target for ocular vascular diseases., (Copyright © 2022 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

The CRISPR/Cas9 system easily edits target genes in various organisms and is used to treat human diseases. In most therapeutic CRISPR studies, ubiquitously expressed promoters, such as CMV, CAG, and EF1α, are used; however, gene editing is sometimes necessary only in specific cell types relevant to the disease. Therefore, we aimed to develop a retinal pigment epithelium (RPE)-specific CRISPR/Cas9 system. We developed a CRISPR/Cas9 system that operates only in retinal pigment epithelium (RPE) by expressing Cas9 under the RPE-specific vitelliform macular dystrophy 2 promoter (pVMD2). This RPE-specific CRISPR/pVMD2-Cas9 system was tested in human retinal organoid and mouse model. We confirmed that this system works specifically in the RPE of human retinal organoids and mouse retina. In addition, the RPE-specific Vegfa ablation using the novel CRISPR-pVMD2-Cas9 system caused regression of choroidal neovascularization (CNV) without unwanted knock-out in the neural retina in laser-induced CNV mice, which is a widely used animal model of neovascular age-related macular degeneration. RPE-specific Vegfa knock-out (KO) and ubiquitous Vegfa KO were comparable in the efficient regression of CNV. The promoter substituted, cell type-specific CRISPR/Cas9 systems can be used in specific 'target cell' therapy, which edits genes while reducing unwanted off- 'target cell' effects., (© 2023. The Author(s).)

Pathological neovascularization in age-related macular degeneration (nvAMD) drives the principal cause of blindness in the elderly. While there is a robust genetic association between genes of innate immunity and AMD, genome-to-phenome relationships are low, suggesting a critical contribution of environmental triggers of disease. Possible insight comes from the observation that a past history of infection with pathogens such as Chlamydia pneumoniae, or other systemic inflammation, can predispose to nvAMD in later life. Using a mouse model of nvAMD with prior C. pneumoniae infection, endotoxin exposure, and genetic ablation of distinct immune cell populations, we demonstrated that peripheral infections elicited epigenetic reprogramming that led to a persistent memory state in retinal CX3CR1+ mononuclear phagocytes (MNPs). The immune imprinting persisted long after the initial inflammation had subsided and ultimately exacerbated choroidal neovascularization in a model of nvAMD. Single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) identified activating transcription factor 3 (ATF3) as a central mediator of retina-resident MNP reprogramming following peripheral inflammation. ATF3 polarized MNPs toward a reparative phenotype biased toward production of proangiogenic factors in response to subsequent injury. Therefore, a past history of bacterial endotoxin-induced inflammation can lead to immunological reprograming within CNS-resident MNPs and aggravate pathological angiogenesis in the aging retina.

Ocular neovascular diseases including neovascular age-related macular degeneration (nvAMD) are widespread causes of blindness. Patients' non-responsiveness to currently used biologics that target vascular endothelial growth factor (VEGF) poses an unmet need for novel therapies. Here, we identify protein arginine methyltransferase 5 (PRMT5) as a novel therapeutic target for nvAMD. PRMT5 is a well-known epigenetic enzyme. We previously showed that PRMT5 methylates and activates a proangiogenic and proinflammatory transcription factor, the nuclear factor kappa B (NF-κB), which has a master role in tumor progression, notably in pancreatic ductal adenocarcinoma and colorectal cancer. We identified a potent and specific small molecule inhibitor of PRMT5, PR5-LL-CM01, that dampens the methylation and activation of NF-κB. Here for the first time, we assessed the antiangiogenic activity of PR5-LL-CM01 in ocular cells. Immunostaining of human nvAMD sections revealed that PRMT5 is highly expressed in the retinal pigment epithelium (RPE)/choroid where neovascularization occurs, while mouse eyes with laser induced choroidal neovascularization (L-CNV) showed PRMT5 is overexpressed in the retinal ganglion cell layer and in the RPE/choroid. Importantly, inhibition of PRMT5 by PR5-LL-CM01 or shRNA knockdown of PRMT5 in human retinal endothelial cells (HRECs) and induced pluripotent stem cell (iPSC)-derived choroidal endothelial cells (iCEC2) reduced NF-κB activity and the expression of its target genes, such as tumor necrosis factor α (TNF-α) and VEGF-A. In addition to inhibiting angiogenic properties of proliferation and tube formation, PR5-LL-CM01 blocked cell cycle progression at G 1 /S-phase in a dose-dependent manner in these cells. Thus, we provide the first evidence that inhibition of PRMT5 impedes angiogenesis in ocular endothelial cells, suggesting PRMT5 as a potential therapeutic target to ameliorate ocular neovascularization., (© 2023. The Author(s).)

Choroidal neovascularization (CNV) causes acute vision loss in neovascular age-related macular degeneration (AMD). Genetic variations of the nuclear receptor RAR-related orphan receptor alpha (RORα) have been linked with neovascular AMD, yet its specific role in pathological CNV development is not entirely clear. In this study, we showed that Rora was highly expressed in the mouse choroid compared with the retina, and genetic loss of RORα in Staggerer mice ( Rorasg/sg ) led to increased expression levels of Vegfr2 and Tnfa in the choroid and retinal pigment epithelium (RPE) complex. In a mouse model of laser-induced CNV, RORα expression was highly increased in the choroidal/RPE complex post-laser, and loss of RORα in Rorasg/sg eyes significantly worsened CNV with increased lesion size and vascular leakage, associated with increased levels of VEGFR2 and TNFα proteins. Pharmacological inhibition of RORα also worsened CNV. In addition, both genetic deficiency and inhibition of RORα substantially increased vascular growth in isolated mouse choroidal explants ex vivo . RORα inhibition also promoted angiogenic function of human choroidal endothelial cell culture. Together, our results suggest that RORα negatively regulates pathological CNV development in part by modulating angiogenic response of the choroidal endothelium and inflammatory environment in the choroid/RPE complex.

Current anti-VEGF-A therapies inhibit choroidal neovascularization (CNV) in a subset of patients with neovascular age-related macular degeneration (NV-AMD). However, long-term treatment with such anti-VEGF-A therapies may impair physiological functions of the choriocapillaris and retina for which VEGF-A is needed. Moreover, disease progression can occur despite continuous anti-VEGF-A treatment. Thus, novel therapies for NV-AMD are urgently needed that target specifically disease-associated mechanisms without impairing growth factors and cellular pathways that are required for homeostatic functions of the retina and choroid. Inhibiting the inflammatory pathways that promote CNV would be such a promising novel approach that would likely not interfere with the normal functions of healthy retinal and choroidal cells. In this context, the inflammasome, a proinflammatory protein complex that promotes pathologic angiogenesis largely through generation of IL-1β and which has been reported to be activated in AMD, has become an area of much interest in the AMD field. However, most studies have focused mainly on the NLRP3 inflammasome in retinal pigment epithelial cells (RPE), and conflicting findings have resulted in an unclear picture of the role of the inflammasome for AMD pathogenesis. Recent data suggest that inflammasome activation in activated macrophages and retinal microglia but not in RPE cells promotes CNV. Furthermore, inflammasome activation can occur in CNV macrophages and microglia despite lack of NLRP3. Thus, activation of both NLRP3 inflammasomes as well as non-NLRP3 inflammasomes in macrophages/microglia at sites of CNV formation likely promote NV-AMD., (© 2021 Federation of European Biochemical Societies.)

TIMP3 mutations are associated with early-onset macular choroidal neovascularisation for which no treatment currently exists. CRISPR base editing, with its ability to irreversibly correct point mutations by chemical modification of nucleobases at DNA level, may be a therapeutic option. We report a bioinformatic analysis of potential therapeutic options in a patient presenting with Sorsby fundus dystrophy. Genetic testing in a 35-year-old gentleman with bilateral macular choroidal neovascularisation revealed the patient to be heterozygous for a TIMP3 variant c.610A>T, p.(Ser204Cys). Using a glycosylase base editor (GBE), another DNA-edit could be introduced that would revert the variant back to wild-type on amino acid level. Alternatively, the mutated residue could be changed to another amino acid that would be better tolerated, and for that, an available 'NG'-PAM site was found to be available for the SpCas9-based adenine base editor (ABE) that would introduce p.(Ser204Arg). In silico analyses predicted this variant to be non-pathogenic; however, a bystander edit, p.Ile205Thr, would be introduced. This case report highlights the importance of considering genetic testing in young patients with choroidal neovascularisation, particularly within the context of a strong family history of presumed wet age-related macular degeneration, and describes potential therapeutic options.

Choroid neovascularization (CNV) is important adverse pathological changes that contributes to the aggravation of hypoxic-ischemic eye diseases, and our preliminary work evidences that the thrombospondin-1 (TSP-1) synthetic polypeptide VR-10 may be the candidate therapeutic agent for the treatment of CNV, but its detailed effects and molecular mechanisms are not fully delineated. In this study, the CNV models in BN rats were established by using the laser photocoagulation method, which were further subjected to VR-10 peptide treatment. The RNA-seq and bioinformatics analysis suggested that VR-10 peptide significantly altered the expression patterns of genes in the rat ocular tissues, and the changed genes were especially enriched in the CD36-associated signal pathways. Next, by performing the Real-Time qPCR and Western Blot analysis, we expectedly found that VR-10 upregulated the anti-angiogenesis biomarker (PEDF) and downregulated pro-angiogenesis biomarkers (VEGF, HIF-1 and IL-17) in rat tissues. In addition, we evidenced that VR-10 downregulated CDK2, CDK4, CDK6, Cyclin D1 and Cyclin D2 to induce cell cycle arrest, upregulated cleaved Caspase-3, Bax and downregulated Bcl-2 to promote cell apoptosis, and increased LC3B-II/I ratio and facilitate p62 degradation to promote cell autophagy in RF/6A cells, which were all reversed by knocking down CD36. Moreover, VR-10 upregulated PEDF, and decreased the expression levels of VEGF, HIF-1 and IL-17 to block angiogenesis of RF/6A cells in a CD36-dependent manner. Taken together, VR-10 peptide interacts with its receptor CD36 to regulate the biological functions of RF/6A cells, and these data suggest that VR-10 peptide may be the putative therapeutic drug for the treatment of CNV in clinic., Competing Interests: Conflict of interest The authors declare no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Neovascularization in ocular vessels causes a major disease burden. The most common causes of choroidal neovascularization (CNV) are age-related macular degeneration and diabetic retinopathy, which are the leading causes of irreversible vision loss in the adult population. Vascular endothelial growth factor (VEGF) is critical for the formation of new vessels and is the main regulator in ocular angiogenesis and vascular permeability through its receptors. Laser therapy and antiangiogenic factors have been used for CNV treatment. Bevacizumab, ranibizumab, and aflibercept are commonly used anti-VEGF agents; however, high costs and the need for frequent intraocular injections are major drawbacks of anti-VEGF drugs. Gene therapy, given the potency of one-time treatment and no need for frequent injections offers the real possibility of such a lasting treatment, with fewer adverse effects and higher patient quality of life. Herein, we reviewed the role of gene therapy in the CNV treatment. In addition, we discuss the advantages and challenges of current treatments compared with gene therapy.

We investigated the effects of lacking TNFα on the development and regression of Argon-laser-induced choroidal neovascularization (CNV) in mice. We lasered ocular fundus for induction of CNV in both wild-type (WT) and TNFα-null (KO) mice. Fluorescence angiography was performed to examine the size of CNV lesions. Gene expression pattern of wound healing-related components was examined. The effects of exogenous TNFα on apoptosis of human retinal microvascular endothelial cells (HRMECs) and on the tube-like structure of the cells were investigated in vitro. The results showed that Argon-laser irradiation-induced CNV was significantly larger in KO mice than WT mice on Day 21, but not at other timepoints. Lacking TNFα increased neutrophil population in the lesion. The distribution of cleaved caspase3-labelled apoptotic cells was more frequently observed in the laser-irradiated tissue in a WT mouse as compared with a KO mouse. Exogenous TNFα induced apoptosis of HRMECs and accelerated regression of tube-like structure of HRMECs in cell culture. Taken together, TNFα gene knockout delays the regression of laser-induced CNV in mice. The mechanism underlying the phenotype might include the augmentation of neutrophil population in the treated tissue and attenuation of vascular endothelial cell apoptosis., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)