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Neovascular age-related macular degeneration (nvAMD) is the leading cause of severe vision loss in the elderly in the developed world. While the introduction of therapies targeting vascular endothelial growth factor (VEGF) has provided the first opportunity to significantly improve vision in patients with nvAMD, many patients respond inadequately to current anti-VEGF therapies. It was recently demonstrated that expression of a second angiogenic mediator, angiopoietin-like 4 (ANGPTL4), synergizes with VEGF to promote choroidal neovascularization (CNV) in mice and correlates with reduced response to anti-VEGF therapy in patients with nvAMD. Here, we report that expression of ANGPTL4 in patients with nvAMD increases following treatment with anti-VEGF therapy and that this increase is dependent on accumulation of hypoxia-inducible factor (HIF)-1α in response to inhibition of VEGF/KDR signaling in the retinal pigment epithelium (RPE). We therefore explored HIF-1 inhibition with 32-134D, a recently developed pharmacologic HIF-inhibitor, for the treatment of nvAMD. 32-134D prevented the expression of both VEGF and ANGPTL4 and was at least as effective as aflibercept in treating CNV in mice. Moreover, by preventing the increase in HIF-1α accumulation in the RPE in response to anti-VEGF therapy, combining 32-134D with aflibercept was more effective than either drug alone for the treatment of CNV. Collectively, these results help explain why many patients with nvAMD respond inadequately to anti-VEGF therapy and suggest that the HIF inhibitor 32-134D will be an effective drug-alone or in combination with current anti-VEGF therapies-for the treatment of patients with this blinding disease., Competing Interests: Competing interests statement:G.L.S. and A.S. are co-founders of and hold equity in HIF Therapeutics, Inc. S.S., Y.H., G.L.S., and A.S. are inventors on provisional patent application PCT/US2022/039883. This arrangement has been reviewed and approved by the Johns Hopkins University in accordancewith its conflict of interest policies.

Purpose: Evaluate the ocular pharmacodynamics (PD) of intravitreal faricimab, a bispecific inhibitor of angiopoietin-2 (Ang-2) and vascular endothelial growth factor-A (VEGF-A), in patients with neovascular age-related macular degeneration (nAMD) or diabetic macular edema (DME)., Methods: Aqueous humor (AH) samples (1025 free Ang-2 concentrations and 1345 free VEGF-A concentrations) were collected from approximately 300 faricimab-treated patients with nAMD or DME in phase 2/3 trials. A population pharmacokinetic pharmacodynamic (popPKPD) model was developed to describe the dynamic effect of faricimab on free AH Ang-2 and VEGF-A., Results: Mean baseline Ang-2 concentrations were 8.1 and 13.4 pg/mL in patients with nAMD and DME, respectively. The corresponding mean baseline VEGF-A concentrations were 58 and 135 pg/mL, respectively. Overall, approximately 79% of Ang-2 (84% within 8 weeks postdose and 55% beyond 12 weeks postdose) and 7% of VEGF-A postdose observations were below the lower limit of quantification. Model-derived Ang-2 and VEGF-A concentration-time profiles for patients on every 4-week/every 8-week dosing were predicted to maintain greater than 50% suppression of Ang-2 concentrations for the entire dosing period. Patients on every 12-week/16-week dosing were predicted to have greater than 50% Ang-2 suppression for 12 or more weeks, whereas 50% VEGF-A suppression was maintained for 9 to 10 weeks. At 8 weeks postdose, the median Ang-2 concentrations remained suppressed by approximately 80%. At 16 weeks postdose, the median VEGF-A concentrations returned to baseline, but median Ang-2 levels remained below baseline., Conclusions: A popPKPD analysis demonstrated faricimab's rapid and sustained suppression of AH Ang-2 and VEGF-A., Translational Relevance: A popPKPD analysis suggested that sustained suppression of ocular Ang-2 contributes to faricimab's extended durability, observed in clinical trials.

Age-related macular degeneration (AMD) is a prominent cause of vision loss, characterized by two different types, dry (atrophic) and wet (neovascular). Dry AMD is distinguished by the progressive deterioration of retinal cells, which ultimately causes a decline in vision. In contrast, wet AMD is defined by the abnormal development of blood vessels underneath the retina, leading to a sudden and severe vision impairment. The course of AMD is primarily driven by chronic inflammation and pathological angiogenesis, in which the NF-κB signaling pathway plays a crucial role. The activation of NF-κB results in the generation of pro-inflammatory cytokines, chemokines, and angiogenic factors like VEGF, which contribute to inflammation and the formation of new blood vessels in AMD. This review analyzes the intricate relationship between NF-κB signaling, inflammation, and angiogenesis in AMD and assesses the possibility of using NF-κB as a target for therapy. The evaluation involves a comprehensive examination of preclinical and clinical evidence that substantiates the effectiveness of NF-κB inhibitors in treating AMD by diminishing inflammation and pathological angiogenesis., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Purpose: This study explores the immune cells' role in anti-VEGF resistance in nAMD patients, and the potential of Zi-Yin-Jiang-Huo-Tang (ZYJHT), a Traditional Chinese Medicine formula, as complementary therapy., Methods: Aqueous humor proteomics data from 10 nAMD patients with anti-VEGF resistance and 10 nAMD patients without anti-VEGF resistance were analyzed, investigating immune cells's role in anti-VEGF resistance and its underlying mechanism. Network pharmacology methods are employed to analyze the active ingredients in ZYJHT that contribute to therapeutic effects and their mechanisms. Real-time PCR (polymerase chain reaction) was used to detect changes in the expression of SOD1 (superoxide dismutase 1) after treatment with compounds targeting SOD1 in ARPE-19 cells., Results: nAMD patients with anti-VEGF resistance showed enhancement of biological processes linked to the positive regulation of immune function, along with decreased cellular resistance to oxidative stress. Infiltration of B cells memory, plasma cells, CD8 + and γδ-T cells were higher in nAMD patients with anti-VEGF resistance. SOD1 was identified as a hub gene in the occurrence of anti-VEGF resistance and a core therapeutic target of ZYJHT, negatively correlated with B and T cell infiltration. Compounds diosgenin, naringenin, and liquiritin in ZYJHT can bind to SOD1 and upregulating SOD1 expression in ARPE-19 cells.

Age-related macular degeneration (AMD) is a common retinal pathology characterized by degeneration of macula's retinal pigment epithelium (RPE) and photoreceptors, visual impairment, or loss. Compared to wet AMD, dry AMD is more common, but lacks cures; therefore, identification of new potential therapeutic targets and treatments is urgent. Increased oxidative stress and declining antioxidant, detoxifying systems contribute to the pathophysiologic mechanisms underlying AMD. The present work shows that the Embryonic Lethal Abnormal Vision-Like 1/Human antigen R (ELAVL1/HuR) and the Vascular Endothelial Growth Factor (VEGF) protein levels are higher in the RPE of both dry and wet AMD patients compared to healthy subjects. Moreover, increased HuR protein levels are detected in the retina, and especially in the RPE layer, of a dry AMD model, the nuclear factor erythroid 2-related factor 2 (Nrf2) / peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) double knock-out mouse. The crosstalk among Nrf2, HuR and VEGF has been also studied in ARPE-19 cells in basal and stressful conditions related to the AMD context (i.e., oxidative stress, autophagy impairment, Nrf2 deficit), offering new evidence of the mutual influence between Nrf2 and HuR, of the dependence of VEGF expression and secretion by these two factors, and of the increased susceptibility of cells to stressful conditions in Nrf2- or HuR-impaired contexts. Overall, this study shows evidence of the interplay among Nrf2, HuR and VEGF, essential factors for RPE homeostasis, and represents an additional piece in the understanding of the complex pathophysiologic mechanisms underlying AMD., Competing Interests: Declaration of Competing Interest I declare that potential competing interests do not exist and there is no financial/personal interest, (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: The objective of this study was to ascertain metabolic biomarkers and investigate the metabolic alterations associated with aqueous humor (AH) in wet age-related macular degeneration (AMD)., Methods: AH samples were collected from a total of 20 participants, including 10 individuals diagnosed with wet AMD and 10 individuals undergoing cataract surgery, serving as the control group. Metabolomics analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify and quantify metabolites., Results: A total of 155 metabolites were identified in the AH samples. Among them, 10 metabolites emerged as potential biomarkers capable of differentiating patients with wet AMD from the control group. In the AH of wet AMD patients, there was increased expression of Cardiolipin (CL) (72:5), Diglyceride (DG) (18:3_18:2), DG (36:5e) and Triglyceride (TG) (24:7), while the expression of Ceramides (Cer) (d32:0), Cer (d34:0), Cer (d36:0), Monogalactosyldiacylglycerol (MGDG) (16:1_18:3), Sphingosine (SPH) (d18:0) and TG (16:0_10:4_16:0) was down regulated., Conclusion: Through metabolomics analysis of AH, this study successfully uncovered valuable metabolic biomarkers linked to wet AMD. These findings contribute to a more comprehensive understanding of the pathogenesis of wet AMD and offer potential avenues for the development of innovative treatment strategies for this condition., Competing Interests: The authors declare that they have no competing interests., (© 2024 Wei et al.)

Purpose: To evaluate the effect of intravitreal bevacizumab on aqueous levels of a panel of 12 inflammatory cytokines in patients with neovascular age-related macular degeneration (nAMD) and correlate response to treatment, as measured by change in the central subfovea thickness (CST), with cytokine levels., Methods: Thirty-three treatment-naïve patients with nAMD received a loading dose of intravitreal bevacizumab consisting of three injections at six weekly intervals. The aqueous samples prior to the first (baseline), second (week 6), and third (week 12) injections were analyzed for cytokine levels. Participants were subgrouped based on changes in CST on spectral-domain optical coherence tomography (SD-OCT) at 12 weeks. Group 1 included patients with a decrease in CST (responders; n = 27). Group 2 included patients who had no decrease in CST (poor responders; n = 6)., Results: Aqueous IL-8 was the only cytokine to demonstrate a significant difference in levels between responders and poor responders, with higher interleukin-8 (IL-8) at week 12 in the poor responder group. Aqueous IL-6 and IL-8 levels showed a positive correlation with CST on SD-OCT (Spearman r = 0.45 and 0.55, respectively). There was a temporal increase overall in cytokine concentration accompanying bevacizumab treatment., Conclusions: Aqueous IL-6 and IL-8 may be important markers of treatment response or poor response in nAMD. Future therapeutic strategies may include targeted treatment against both vascular endothelial cell growth factor (VEGF) and IL-6 and/or IL-8 in patients who do not respond to anti-VEGF treatment alone.

Purpose: To examine the changes in aqueous humor cytokine levels and clinical outcomes of switching from aflibercept to faricimab in eyes with neovascular age-related macular degeneration (nAMD)., Methods: Fifty-four eyes of 54 patients with AMD undergoing treatment with aflibercept under a treat-and-extend (TAE) regimen were switched to faricimab and studied prospectively. Best-corrected visual acuity (BCVA; in logarithm of the minimum angle of resolution), central retinal thickness (CRT), central choroidal thickness (CCT), and exudative status were analyzed using optical coherence tomography. Aqueous humor was collected before and after the switch, and angiopoietin-2 (Ang-2), placental growth factor (PlGF), and vascular endothelial growth factor (VEGF) A levels were measured., Results: After switching from aflibercept to faricimab, exudative changes improved in 28 eyes (52%), remained stable in eight eyes (15%), and worsened in 18 eyes (33%). BCVA changed from 0.27 ± 0.31 to 0.26 ± 0.29 (P = 0.46), CRT decreased from 306.2 ± 147.5 µm to 278.6 ± 100.4 µm (P = 0.11), and CCT changed from 189.5 ± 92.8 µm to 186.8 ± 93.9 µm (P = 0.21). VEGF-A levels were below the detection sensitivity in many cases throughout the pre- and post-switching periods. Ang-2 significantly decreased from 23.8 ± 23.5 pg/mL to 16.4 ± 21.9 pg/mL (P < 0.001), and PlGF significantly increased from 0.86 ± 0.85 pg/mL to 1.72 ± 1.39 pg/mL (P < 0.001)., Conclusions: Switching from aflibercept to faricimab in patients with nAMD may not only suppress VEGF-A but also Ang-2 and reduce exudative changes.

Purpose: This study aimed to investigate the potential of microRNAs (miRNAs) in tears, blood, and aqueous humor as biomarkers for predicting treatment response in wet age-related macular degeneration (AMD) patients undergoing anti-vascular endothelial growth factor (anti-VEGF) therapy., Methods: In a single-center prospective cohort study, treatment-naïve wet AMD patients and age-matched controls were enrolled. Clinical data and miRNA levels (miR-199a-3p, miR-365-3p, miR-200b-3p, miR-195-5p, miR-335-5p, and miR-185-5p) in tears, blood, and aqueous humor were collected. Treatment response was categorized into responders and non-responders based on visual acuity and central subfield thickness. MiRNA levels were quantified using reverse-transcription PCR. Statistical analyses were performed, including ROC analysis, to evaluate predictive accuracy., Results: Dysregulated miRNA profiles were observed in wet AMD tears and blood compared to controls. Specifically, miR-199a-3p, miR-195-5p, and miR-185-5p were upregulated, while miR-200b-3p was downregulated in tears. All six miRNAs were elevated in wet AMD blood samples. Notably, responders showed higher tear expression of miR-195-5p and miR-185-5p. Combining these miRNAs yielded the highest predictive power (AUC = 0.878, p = 0.006) for anti-VEGF responders., Conclusions: Dysregulated miRNA profiles in tears and blood suggest their potential as biomarkers for wet AMD. MiR-195-5p and miR-185-5p in tears demonstrate predictive value for anti-VEGF treatment responders. This study underscores the non-invasive prediction potential of miRNA tear analysis in wet AMD treatment responses., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: To investigate aqueous humor cytokine levels in neovascular age-related macular degeneration (nAMD) patients with subretinal fibrosis and to explore the relationship between cytokine levels and disease severity., Methods: The aqueous humor samples were collected from 16 eyes with subretinal fibrosis due to nAMD (SRFi group), 33 eyes with nAMD without subretinal fibrosis (nAMD group) and 28 eyes with cataract patients (control group). Clinical samples were analyzed for 5 cytokines,including vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), basic fibroblast growth factor (bFGF), transforming growth factor-α (TGF-α), platelet-derived growth factor-BB (PDGF-BB)., Results: Aqueous humor cytokines VEGF and bFGF were significantly higher in nAMD patients than controls (all P < 0.05), and VEGF, bFGF and TGF-α levels were significantly higher in SRFi patients than controls (all P < 0.05). No significant differences in 4 cytokine levels were observed between nAMD and SRFi patients in aqueous humor. We also identified a positive correlation between the aqueous humor levels of IL-6 and VEGF in the SRFi group, while bFGF and TGF-α in the nAMD group. Moreover, VEGF levels were strongly related to BCVA, and bFGF levels were positively related to the maximum thickness of subretinal hyperreflective material (SHRM) in fibrosis due to nAMD., Conclusion: VEGF and bFGF levels in aqueous humor were elevated in macular neovascularization with and without subretinal fibrosis. TGF-α levels exclusively differed in neovascular AMD with fibrosis. Cytokines are distributed differently and play a synergistic role in different stages (angiogenesis and fibrogenesis) of nAMD. The bFGF levels could predict the negative prognosis in fibrosis due to nAMD., (© 2024. The Author(s).)

Purpose: The purpose of this study was to investigate the molecular mechanisms underlying anti-vascular endothelial growth factor (anti-VEGF) efficacy and response variability in neovascular age-related macular degeneration (nAMD) using longitudinal proteomic and metabolomic analysis alongside three-dimensional lesion measurements., Methods: In this prospective study, 54 treatment-naive patients with nAMD underwent "3+ pro re nata" (3+PRN) anti-VEGF regimens followed for at least 12 weeks. Aqueous humors were collected pre- and post-treatment for proteomic and metabolomic analysis. Three-dimensional optical coherence tomography (OCT) and OCT angiography assessed different types of nAMD lesion volumes and areas., Results: There were 1350 proteins and 1268 metabolites that were identified in aqueous humors, with 301 proteins and 353 metabolites significantly altered during anti-VEGF treatment, enriched in pathways of angiogenesis, energy metabolism, signal transduction, and neurofunctional regulation. Sixty-seven changes of (Δ) molecules significantly correlated with at least one type of ΔnAMD lesion. Notably, proteins FGA, TALDO1, and ASPH significantly decreased during treatment, with their reductions correlating with greater lesion regression in at least two lesion types. Conversely, despite that YIPF3 also showed significant downregulation, its decrease was associated with poorer regression in total nAMD lesion and subretinal hyper-reflective material., Conclusions: This study identifies FGA, TALDO1, and ASPH as potential key molecules in the efficacy of anti-VEGF therapy, whereas YIPF3 may be a key factor in poor response. The integration of longitudinal three-dimensional lesion analysis with multi-omics provides valuable insights into the mechanisms and response variability of anti-VEGF treatment in nAMD.

Purpose: Retinal neovascularization is a significant feature of advanced age-related macular degeneration (AMD) and a major cause of blindness in patients with AMD. However, the underlying mechanism of this pathological neovascularization remains unknown. Iron metabolism has been implicated in various biological processes. This study was conducted to investigate the effects of iron metabolism on retinal neovascularization in neovascular AMD (nAMD)., Methods: C57BL/6J and very low-density lipoprotein receptor (VLDLR) knockout (Vldlr-/-) mice, a murine model of nAMD, were used in this study. Bulk-RNA sequencing was used to identify differentially expressed genes. Western blot analysis was performed to test the expression of proteins. Iron chelator deferiprone (DFP) was administrated to the mice by oral gavage. Fundus fluorescein angiography was used to evaluate retinal vascular leakage. Immunofluorescence staining was used to detect macrophages and iron-related proteins., Results: RNA sequencing (RNA-seq) results showed altered transferrin expression in the retina and RPE of Vldlr-/- mice. Disrupted iron homeostasis was observed in the retina and RPE of Vldlr-/- mice. DFP mitigated iron overload and significantly reduced retinal neovascularization and vascular leakage. In addition, DFP suppressed the inflammation in Vldlr-/- retinas. The reduced signals of macrophages were observed at sites of neovascularization in the retina and RPE of Vldlr-/- mice after DFP treatment. Further, the IL-6/JAK2/STAT3 signaling pathway was activated in the retina and RPE of Vldlr-/- mice and reversed by DFP treatment., Conclusions: Disrupted iron metabolism may contribute to retinal neovascularization in nAMD. Restoring iron homeostasis by DFP could be a potential therapeutic approach for nAMD.

Purpose: To investigate differences in volume and distribution of the main exudative biomarkers across all types and subtypes of macular neovascularization (MNV) using artificial intelligence (AI)., Design: Cross-sectional study., Methods: An AI-based analysis was conducted on 34,528 OCT B-scans consisting of 281 (250 unifocal, 31 multifocal) MNV3, 55 MNV2, and 121 (30 polypoidal, 91 non-polypoidal) MNV1 treatment-naive eyes. Means (SDs), medians and heat maps of cystic intraretinal fluid (IRF), subretinal fluid (SRF), pigment epithelial detachments (PED), and hyperreflective foci (HRF) volumes, as well as retinal thickness (RT) were compared among MNV types and subtypes., Results: MNV3 had the highest mean IRF with 291 (290) nL, RT with 357 (49) µm, and HRF with 80 (70) nL, P ≤ .05. MNV1 showed the greatest mean SRF with 492 (586) nL, whereas MNV3 exhibited the lowest with 218 (382) nL, P ≤ .05. Heat maps showed IRF confined to the center, whereas SRF was scattered in all types. SRF, HRF, and PED were more distributed in the temporal macular half in MNV3. Means of IRF, HRF, and PED were higher in the multifocal than in the unifocal MNV3 with 416 (309) nL,114 (95) nL, and 810 (850) nL, P ≤ .05. Compared to the non-polypoidal subtype, the polypoidal subtype had greater means of SRF with 695 (718) nL, HRF 69 (63) nL, RT 357 (45) µm, and PED 1115 (1170) nL, P ≤ .05., Conclusions: This novel quantitative AI analysis shows that SRF is a biomarker of choroidal origin in MNV1, whereas IRF, HRF, and RT are retinal biomarkers in MNV3. Polypoidal MNV1 and multifocal MNV3 present with higher exudation compared to other subtypes., (Copyright © 2024 The Authors. Published by Elsevier Inc. 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 assess the impact of ocular confounding factors on aqueous humor (AH) proteomic and metabolomic analyses for retinal disease characterization., Methods: This study recruited 138 subjects (eyes): 102 with neovascular age-related macular degeneration (nAMD), 18 with diabetic macular edema (DME), and 18 with cataract (control group). AH samples underwent analysis using Olink Target 96 proteomics and Metabolon's metabolomics platform Data analysis included correlation, differential abundance, and gene-set analysis., Results: In total, 756 proteins and 408 metabolites were quantified in AH. Total AH protein concentration was notably higher in nAMD (3.2-fold) and DME (4.1-fold) compared to controls. Pseudophakic eyes showed higher total AH protein concentrations than phakic eyes (e.g., 1.6-fold in nAMD) and a specific protein signature indicative of matrix remodeling. Unexpectedly, pupil-dilating drugs containing phenylephrine/tropicamide increased several AH proteins, notably interleukin-6 (5.4-fold in nAMD). Correcting for these factors revealed functionally relevant protein correlation clusters and disease-relevant, differentially abundant proteins across the groups. Metabolomics analysis, for which the relevance of confounder adjustment was less apparent, suggested insufficiently controlled diabetes and chronic hyperglycemia in the DME group., Conclusions: AH protein concentration, pseudophakia, and pupil dilation with phenylephrine/tropicamide are important confounding factors for AH protein analyses. When these factors are considered, AH analyses can more clearly reveal disease-relevant factors., Translational Relevance: Considering AH protein concentration, lens status, and phenylephrine/tropicamide administration as confounders is crucial for accurate interpretation of AH protein data.

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.)

Background: This analysis evaluated aqueous humour (AH) interleukin (IL)-6 concentrations and the association between AH IL-6 and visual outcomes in patients with neovascular age-related macular degeneration (nAMD) or diabetic macular oedema (DMO) receiving anti-vascular endothelial growth factor (VEGF) monotherapy., Methods: Post hoc analysis of the multicentre, double-masked, randomised HARBOR (NCT00891735) and READ-3 (NCT01077401) trials. HARBOR enrolled treatment-naïve nAMD patients. READ-3 enrolled treatment-naïve/previously treated DMO patients. HARBOR patients received ranibizumab 0.5 or 2.0 mg monthly or as needed; AH samples were collected at month 2, after two previous intravitreal injections. READ-3 patients received ranibizumab 0.5 or 2.0 mg as needed; AH samples were collected at baseline and months 3, 6, 9, and 12., Main Outcome Measure: association between AH IL-6 concentrations and month 24 best-corrected visual acuity (BCVA)., Results: In both trials (HARBOR, N = 36; READ-3, N = 137), patients with higher AH IL-6 concentrations had worse visual outcomes. HARBOR patients with low AH IL-6 concentrations at month 2 had a mean (95% CI) BCVA change at month 24 of +2.9 (-2.6, 8.3) letters, whereas patients with high AH concentrations had a mean (95% CI) BCVA change of -9.0 (-22.7, 4.7) letters. READ-3 patients with low AH concentrations at baseline had a mean (95% CI) BCVA change at month 12 of +9.3 (7.4, 11.3) letters, whereas patients with high AH concentrations had a mean (95% CI) BCVA change of +5.6 (2.2, 9.1) letters., Conclusions: Higher IL-6 AH concentrations may predict suboptimal visual responses to anti-VEGF monotherapy in patients with nAMD/DMO., (© 2024. The Author(s).)

The aim of this study is to investigate the relationship between age-related macular degeneration (AMD) and lymphangiogenesis biomarkers, namely LYVE-1, Podoplanin, VEGF-C, VEGFR-2 and VEGFR-3. This prospective and interventional study includes 30 patients with AMD which may be dry or wet type and 30 controls for whom vitrectomy and phacoemulsification was indicated due to additional pathologies (epiretinal membrane, macular hole, retinal detachment, and cataract). 0.1-0,2 ml of aqueous humor and 0.5-1 ml of vitreous sample was taken during the operations. Before the operations 1 tube serum was also taken. All the lymphangiogenesis biomarkers in the study are examined by ELISA method. LYVE-1 (p = 0.001) and Podoplanin (p = 0.004) levels in the vitreous for the patient group are found to be significantly lower than the control group. Serum (p = 0.019), vitreous (p = 0.001), aqueous (p < 0.001) levels of VEGF-C for the patient group are significantly higher than the control group. VEGF-C/VEGFR-2 (p < 0.001), VEGF-C/VEGFR-3 (p < 0.001) ratios in the vitreous for the patient group are found to be significantly higher than the control group. Especially in wet AMD patients, LYVE-1 level is significantly lower in the vitreous (p = 0.002) and aqueous (p = 0.002) than the control group. In addition, Podoplanin level is observed as significantly lower in the vitreous (p = 0.014) and serum (p = 0.002) in comparison to control group. In the wet AMD group, VEGF-C level in the vitreous (p < 0.001), aqueous (p < 0.001) and serum (p = 0.001) is higher than the control group. The result of this study indicates a valid relationship between the weakening of lymphangiogenesis and the pathophysiology of AMD, especially for the wet type. It is observed that the levels of receptors that bind VEGF-C (VEGFR-2 and VEGFR-3) do not increase at the same rate as VEGF-C to compensate for the increase in VEGF-C. The absence of an increase in VEGFR-3, which is especially necessary for lymphangiogenesis, also suggests that lymphangiogenesis is weakened or decreased in AMD. In the future interventional studies with larger series, examination of lymphangiogenic biomarkers in inflammatory retinal diseases and glaucoma may reveal unexplored details., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Wet age-related macular degeneration (wet AMD) is a primary contributor to visual impairment and severe vision loss globally, but 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 (ECs) is mainly dictated by angiogenic growth factors. Even though treatments targeting vascular endothelial growth factor (VEGF), like ranibizumab, are widely administered, more than half of patients still exhibit inadequate or null responses, suggesting the involvement of other pathogenic mechanisms. With advances in research in recent years, it has become well recognized that EC metabolic regulation plays an active rather than merely passive responsive role in angiogenesis. Disturbances of these metabolic pathways may lead to excessive neovascularization in angiogenic diseases such as wet AMD, therefore targeted modulation of EC metabolism represents a promising therapeutic strategy for wet AMD. In this review, we comprehensively discuss the potential applications of EC metabolic regulation in wet AMD treatment from multiple perspectives, including the involvement of ECs in wet AMD pathogenesis, the major endothelial metabolic pathways, and novel therapeutic approaches targeting metabolism for wet AMD.

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.)

Purpose: To investigate cytokine levels in the tear fluid of patients receiving serial intravitreal injections (IVI) with anti-vascular endothelial growth factor (anti-VEGF) for neovascular age-related macular degeneration (nAMD)., Methods: Concentrations of six cytokines (IFN-γ, IL-1β, IL-6, IL-8, TNF and VEGF) in tears of patients receiving anti-VEGF in one eye were assayed using multiplex cytometric bead array. The fellow untreated eye served as control. Tear sampling was performed on a single occasion at a minimum of four weeks after IVI. Patients underwent a pre-IVI antisepsis protocol with povidone-iodine., Results: Tear fluid from thirty patients with a mean age of 78.8 years (range 58-90) was assayed. Subjects received a median of 43.5 (range 22-106) IVI in one eye. The median level of IFN-γ was 0.33 (interquartile range (IQR) 0.22-0.52) pg/mg of total protein in injected eyes versus 0.41 (IQR 0.21-1.05) pg/mg in fellow eyes (p = 0.017). For TNF, a median level of 0.12 (IQR 0.08-0.18) pg/mg of total protein was found in injected eyes versus 0.14 (IQR 0.07-0.33) pg/mg of total protein in fellow eyes (p = 0.019). There were no differences between injected and fellow eyes regarding the levels of IL-1β, IL-6, IL-8 and VEGF., Conclusion: Tear fluid in eyes receiving serial IVI with anti-VEGF and preoperative povidone-iodine antisepsis constitutes lower levels of the pro-inflammatory cytokines IFN-γ and TNF compared to fellow eyes. This provides biochemical support of previous findings of reduced signs of inflammation and healthier tear film parameters in patients treated with serial IVI., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Age-related macular degeneration (AMD), along with other age-associated ophthalmologies, is a significant cause of loss of visual functions among the elderly, but the relationship of exudative AMD with lacrimal fluid chemokines has not been sufficiently studied. The aim of the work was to study the relationship of exudative AMD with lacrimal fluid chemokines in elderly patients. The content of chemokines in the lacrimal fluid was studied in 92 patients (184 eyes) 60-74 years old with exudative AMD and 84 patients (168 eyes) 60-74 years old without ophthalmopathology using a certified MAGRIX (USA) immunoassay device with Luminex technology on the xMAX platform. AMD diagnosis was performed based on the results of a comprehensive ophthalmological examination and in accordance with the classification criteria (Age-Related Eye Disease Study). Regression analysis and the program Statistica 10.0 were used in the processing of the obtained data. An increase in the production of most of the considered chemokines in patients with exudative AMD in lacrimal fluid was found in relation to representatives of the same age without ophthalmopathology, namely: MCP-1/CCL2 to 712,4±3,4 pg/ml reference 598,6±3,1 pg/ml (p<0,01), IP-10/CXCL10 to 37,9±2,6 pg/ml reference 11,3±2,3 pg/ml (p<0,001), SDF-1α/CXCL12 to 262,5±4,3 pg/ml reference 208,2±4,7 pg/ml (p<0,001), Eotaxin/CCL11 to 6,2±0,4 pg/ml reference 4,3±0,6 pg/ml (p<0,01), RANTES/CCL5 to 1,7±0,3 versus 0,6±0,2 pg/ml (p<0,001). On the contrary, the content of MIP-1a/CCL3 chemokines decreased to 2±0,3 versus 4,1±0,5 pg/ml (p<0,001), GROa/CXCL1 to 7,2±0,9 versus 15,8±2,1 pg/ml (p<0,001). The coefficients of the performed multidimensional regression analysis turned out to be the highest for IP-10/CXCL10 (β=+3,538), RANTES/CCL5 - (β=+2,859) and GROa/CXCL1 - (β=-2,419), which indicates the greatest association of these lacrimal fluid chemokines with the development of exudative AMD. These lacrimal fluid chemokines can be used as additional diagnostic markers of exudative AMD.

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.

Wet age-related macular degeneration (AMD), characterized by leaky neovessels emanating from the choroid, is a main cause of blindness. As current treatments for wet AMD require regular intravitreal injections of anti-vascular endothelial growth factor (VEGF) biologics, there is a need for the development of less invasive treatments. Here, we designed an allosteric inhibitor of end binding-3 (EB3) protein, termed EBIN, which reduces the effects of environmental stresses on endothelial cells by limiting pathological calcium signaling. Delivery of EBIN via eye drops in mouse and non-human primate (NHP) models of wet AMD prevents both neovascular leakage and choroidal neovascularization. EBIN reverses the epigenetic changes induced by environmental stresses, allowing an activation of a regenerative program within metabolic-active endothelial cells comprising choroidal neovascularization (CNV) lesions. These results suggest the therapeutic potential of EBIN in preventing the degenerative processes underlying wet AMD., Competing Interests: Declaration of interests Y.A.K., U.S., M.I.R., and A.B.M. have two international patents on EBIN; Y.A.K., M.I.R., and A.B.M. had served the role of consultants for Laurel Therapeutics, Inc.; and M.S. is the CEO of Laurel Therapeutics, Inc. M.S.L. is the CEO of Virscio, Inc., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Age-related macular degeneration (AMD) is a leading cause of vision loss among elderly people in developed countries. Neovascular AMD (nAMD) accounts for more than 90% of AMD-related vision loss. At present, intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) is widely used as the first-line therapy to decrease the choroidal and retinal neovascularizations, and thus to improve or maintain the visual acuity of the patients with nAMD. However, about 1/3 patients still progress to irreversible visual impairment due to subretinal fibrosis even with adequate anti-VEGF treatment. Extensive literatures support the critical role of epithelial-mesenchymal transformation (EMT) of retinal pigment epithelium (RPE) in the pathogenesis of subretinal fibrosis in nAMD, but the underlying mechanisms still remain largely unknown. This review summarized the molecular pathogenesis of subretinal fibrosis in nAMD, especially focusing on the transforming growth factor-β (TGF-β)-induced EMT pathways. It was also discussed how these pathways crosstalk and respond to signals from the microenvironment to mediate EMT and contribute to the progression of nAMD-related subretinal fibrosis. Targeting EMT signaling pathways might provide a promising and effective therapeutic strategy to treat subretinal fibrosis secondary to nAMD., Competing Interests: Declaration of Competing Interest The authors have no conflicting financial interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Disordered immune responses and cholesterol metabolism have been implicated in age-related macular degeneration (AMD), the leading cause of blindness in elderly individuals. SULT2B1, the key enzyme of sterol sulfonation, plays important roles in inflammation and cholesterol metabolism. However, the role and underlying mechanism of SULT2B1 in AMD have not been investigated thus far. Here, we report that SULT2B1 is specifically expressed in macrophages in choroidal neovascularization lesions. Sutl2b1 deficiency significantly reduced leakage areas and inhibited pathological angiogenesis by inhibiting M2 macrophage activation in vivo and in vitro. Mechanistically, loss of Sult2b1 activated LXRs and subsequently increased ABCA1 and ABCG1 (ABCA1/G1)-mediated cholesterol efflux from M2 macrophages. LXR inhibition (GSK2033 treatment) in Sult2b1 -/- macrophages reversed M2 polarization and decreased intracellular cholesterol capacity to promote pathological angiogenesis. In contrast to SULT2B1, STS, an enzyme of sterol desulfonation, protected against choroidal neovascularization development by activating LXR-ABCA1/G1 signalling to block M2 polarization. Collectively, these data reveal a cholesterol metabolism axis related to macrophage polarization in neovascular AMD., (© 2023 Wang et al.)

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.

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.)

Background: The basis of Age-related macular degeneration (AMD) genetic risk has been well documented; however, few studies have looked at genetic biomarkers of disease progression or treatment response within advanced AMD patients. Here we report the first genome-wide analysis of genetic determinants of low-luminance vision deficit (LLD), which is seen as predictive of visual acuity loss and anti-VEGF treatment response in neovascular AMD patients., Methods: AMD patients were separated into small- and large-LLD groups for comparison and whole genome sequencing was performed. Genetic determinants of LLD were assessed by common and rare variant genetic analysis. Follow-up functional analysis of rare coding variants identified by the burden test was then performed in vitro., Results: We identified four coding variants in the CIDEC gene. These rare variants were only present in patients with a small LLD, which has been previously shown to indicate better prognosis and better anti-VEGF treatment response. Our in vitro functional characterization of these CIDEC alleles revealed that all decrease the binding affinity between CIDEC and the lipid droplet fusion effectors PLIN1, RAB8A and AS160. The rare CIDEC alleles all cause a hypomorphic defect in lipid droplet fusion and enlargement, resulting in a decreased fat storage capability in adipocytes., Conclusions: As we did not detect CIDEC expression in the ocular tissue affected by AMD, our results suggest that the CIDEC variants do not play a direct role in the eye and influence low-luminance vision deficit via an indirect and systemic effect related to fat storage capacity., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: at the time of the study, all authors were full time employees of Genentech/Roche with stock and stock options in Roche. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Kim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Choroidal neovascularization (CNV) is a pathological angiogenesis of the choroidal plexus of the retina and is a key feature in the wet form of age-related macular degeneration. Mononuclear phagocytic cells (MPCs) are known to accumulate in the subretinal space, generating a chronic inflammatory state that promotes the growth of the choroidal neovasculature. However, how the MPCs are recruited and activated to promote CNV pathology is not fully understood. Using genetic and pharmacological tools in a mouse model of laser-induced CNV, we demonstrate a role for the p75 neurotrophin receptor (p75 NTR ) in the recruitment of MPCs, in glial activation, and in vascular alterations. After laser injury, expression of p75 NTR is increased in activated Muller glial cells near the CNV area in the retina and the retinal pigmented epithelium (RPE)-choroid. In p75 NTR knockout mice (p75 NTR KO) with CNV, there is significantly reduced recruitment of MPCs, reduced glial activation, reduced CNV area, and the retinal function is preserved, as compared to wild type mice with CNV. Notably, a single intravitreal injection of a pharmacological p75 NTR antagonist in wild type mice with CNV phenocopied the results of the p75 NTR KO mice. Our results demonstrate that p75 NTR is etiological in the development of CNV.

Intravitreal injections of anti-vascular endothelial growth factor (VEGF) agents are used to treat wet age-related macular degeneration (wAMD); however, they are associated with a considerable treatment burden and poor real-world outcomes. The molecular size and charge of anti-VEGF agents influence drug pharmacokinetics in the vitreous and peak drug efficacy. This article reviews the established and novel strategies to prolong drug action, in the vitreal cavity, and thus reduce dosing frequency. Increased ocular residency can be attained by increasing drug size as with large molecules, such as KSI-301; adding polyethylene glycol to pegcetacoplan (APL-2) or avacincaptad pegol to increase molecular size; or binding to other targets that increase molecular size, such as vitreal albumin in the case of BI-X. Faricimab is a bispecific antibody in which the fragment crystallizable portion is engineered to prolong ocular residency and reduce systemic exposure. Conversely, small VEGF-binding molecules, such as brolucizumab, can be administered at higher clinical doses, with the potential for prolonged clinical activity versus larger molecules. Other important considerations include sustained drug delivery routes, such as the ranibizumab port delivery system or subconjunctival or suprachoroidal injection. More effective and longer-lasting treatments are needed for wAMD to prolong drug action and reduce dosing frequency. Several strategies are under investigation and the prevention of vision loss in patients with AMD or other retinal diseases may be attainable in the near future., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

Age-related macular degeneration (AMD) is an eye disease, which causes impaired vision that can lead to blindness. The incidence of AMD increases with age. Retinal pigment epithelial (RPE) cells maintain retinal homeostasis and support the functionality of photoreceptors. In the pathogenesis of AMD, the degeneration of the RPE cells precedes photoreceptor cell death. RPE cells are susceptible to oxidative stress, and chronic inflammation involving nucleotide-binding domain, leucine-rich repeat and pyrin domain 3 (NLRP3) inflammasome activation and impaired autophagy are challenges faced by aged RPE cells in AMD. There are two types of AMD, dry (85-90%) and wet (10-15%) disease forms. Choroidal neovascularization is typical for wet AMD, and anti-vascular endothelial growth factor (anti-VEGF) injections are used to prevent the progression of the disease but there is no curative treatment. There is no cure for the dry disease form, but antioxidants have been proposed as a potential treatment option. Ageing is the most important risk factor of AMD, and tobacco smoke is the most important environmental risk factor that can be controlled. Hydroquinone is a cytotoxic, immunotoxic, carcinogenic and pro-oxidative component of tobacco smoke. The aim of this PhD thesis was to study hydroquinone-induced oxidative stress and NLRP3 inflammasome activation in human RPE cells (ARPE-19 cells). An age-related eye disease study (AREDS) formulation (incl. omega-3 fatty acids, vitamin C and E, copper, zinc, lutein and zeaxanthin), which is clinically investigated p.o. dosing combination of dietary supplements for AMD patients, has been evaluated as a possible treatment and restraining option for AMD. Resvega (4.1.1, Table 2) is a similar kind of product to AREDS with added resveratrol, and many of the components incorporated within Resvega can be considered as belonging to the normal antioxidative defence system of the retina. Another aim was to evaluate the effects of Resvega on hydroquinone-induced oxidative stress or NLRP3 inflammasome activation induced by impaired protein clearance. The results of this study reveal that hydroquinone elevated the activity of NADPH oxidase which subsequently mediated the production of reactive oxygen species (ROS) and predisposed RPE cells to degeneration by reducing levels of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). Hydroquinone induced an NLRP3-independent IL-18 release and NLRP3 accumulation inside the IL-1α-primed cells. Resvega treatment reduced the extent of hydroquinone-induced ROS production and NLRP3 inflammasome activation evoked by impaired protein clearance. Thus, Resvega alleviated hydroquinone- and impaired protein clearance-induced stress in human RPE cells, but more studies are needed, for example, to reveal the most optimal route of administration for targeting the cells in the retina, since both oxidative stress and NLRP3 inflammasome activation are important contributors to the development of AMD and represent significant treatment targets., (© 2022 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Age-related macular degeneration (AMD) is the leading cause of visual impairment in the aging population with a limited understanding of its pathogenesis and the number of patients are all the time increasing. AMD is classified into two main forms: dry and neovascular AMD (nAMD). Dry AMD is the most prevalent form (80-90%) of AMD cases. Neovascular AMD (10-20% of AMD cases) is treated with monthly or more sparsely given intravitreal anti-vascular endothelial growth factor inhibitors, but unfortunately, not all patients respond to the current treatments. A clinical hallmark of nAMD is choroidal neovascularization. The progression of AMD is initially characterized by atrophic alterations in the retinal pigment epithelium, as well as the formation of lysosomal lipofuscin and extracellular drusen deposits. Cellular damage caused by chronic oxidative stress, protein aggregation and inflammatory processes may lead to advanced geographic atrophy and/or choroidal neovascularization and fibrosis. Currently, it is not fully known why different AMD phenotypes develop. In this review, we connect angiogenesis and inflammatory regulators in the development of nAMD and discuss therapy challenges and hopes.

Muller glia (MG) play a central role in reactive gliosis, a stress response associated with rare and common retinal degenerative diseases, including age-related macular degeneration (AMD). The posttranslational modification citrullination​ targeting glial fibrillary acidic protein (GFAP) in MG was initially discovered in a panocular chemical injury model. Here, we report in the paradigms of retinal laser injury, a genetic model of spontaneous retinal degeneration (JR5558 mice) and human wet-AMD tissues that MG citrullination is broadly conserved. After laser injury, GFAP polymers that accumulate in reactive MG are citrullinated in MG endfeet and glial cell processes. The enzyme responsible for citrullination, peptidyl arginine deiminase-4 (PAD4), localizes to endfeet and associates with GFAP polymers. Glial cell-specific PAD4 deficiency attenuates retinal hypercitrullination in injured retinas, indicating PAD4 requirement for MG citrullination. In retinas of 1-mo-old JR5558 mice, hypercitrullinated GFAP and PAD4 accumulate in MG endfeet/cell processes in a lesion-specific manner. Finally, we show that human donor maculae from patients with wet-AMD also feature the canonical endfeet localization of hypercitrullinated GFAP. Thus, we propose that endfeet are a "citrullination bunker" that initiates and sustains citrullination in retinal degeneration., Competing Interests: Competing interest statement: J.A. is a cofounder of iVeena Holdings, iVeena Delivery Systems, and Inflammasome Therapeutics and has been a consultant for Allergan, Boehringer-Ingelheim, Immunovant, Olix Pharmaceuticals, Retinal Solutions, and Saksin LifeSciences unrelated to this work. J.A. and B.D.G. are cofounders of DiceRx., (Copyright © 2022 the Author(s). Published by PNAS.)

Age-related macular degeneration (AMD), a degenerative disease affecting the retinal pigment epithelium (RPE) and photoreceptors in the macula, is the leading cause of central blindness in the elderly. AMD progresses to advanced stages of the disease, atrophic AMD (aAMD), or in 15% of cases "wet" or neovascular AMD (nAMD), associated with substantial vision loss. Whilst there has been advancement in therapies treating nAMD, to date, there are no licenced effective treatments for the 85% affected by aAMD, with disease managed by changes to diet, vitamin supplements, and regular monitoring. AMD has a complex pathogenesis, involving highly integrated and common age-related disease pathways, including dysregulated complement/inflammation, impaired autophagy, and oxidative stress. The intricacy of AMD pathogenesis makes therapeutic development challenging and identifying a target that combats the converging disease pathways is essential to provide a globally effective treatment. Interleukin-33 is a cytokine, classically known for the proinflammatory role it plays in allergic disease. Recent evidence across degenerative and inflammatory disease conditions reveals a diverse immune-modulatory role for IL-33, with promising therapeutic potential. Here, we will review IL-33 function in disease and discuss the future potential for this homeostatic cytokine in treating AMD., (© 2021. The Author(s).)

Aim: To objectively assess disease activity and treatment response in patients with retinal vein occlusion (RVO), neovascular age-related macular degeneration (nAMD) and centre-involved diabetic macular oedema (DME), using artificial intelligence-based fluid quantification., Methods: Posthoc analysis of 2311 patients (11 151 spectral-domain optical coherence tomography volumes) from five clinical, multicentre trials, who received a flexible antivascular endothelial growth factor (anti-VEGF) therapy over a 12-month period. Fluid volumes were measured with a deep learning algorithm at baseline/months 1, 2, 3 and 12, for three concentric circles with diameters of 1, 3 and 6 mm (fovea, paracentral ring and pericentral ring), as well as four sectors surrounding the fovea (superior, nasal, inferior and temporal)., Results: In each disease, at every timepoint, most intraretinal fluid (IRF) per square millimetre was present at the fovea, followed by the paracentral ring and pericentral ring (p<0.0001). While this was also the case for subretinal fluid (SRF) in RVO/DME (p<0.0001), patients with nAMD showed more SRF in the paracentral ring than at the fovea up to month 3 (p<0.0001). Between sectors, patients with RVO/DME showed the highest IRF volumes temporally (p<0.001/p<0.0001). In each disease, more SRF was consistently found inferiorly than superiorly (p<0.02). At month 1/12, we measured the following median reductions of initial fluid volumes. For IRF: RVO, 95.9%/97.7%; nAMD, 91.3%/92.8%; DME, 37.3%/69.9%. For SRF: RVO, 94.7%/97.5%; nAMD, 98.4%/99.8%; DME, 86.3%/97.5%., Conclusion: Fully automated localisation and quantification of IRF/SRF over time shed light on the fluid dynamics in each disease. There is a specific anatomical response of IRF/SRF to anti-VEGF therapy in all diseases studied., Competing Interests: Competing interests: MM, MF, PS, AS, BHN and HB: none; UMS-E: Bayer/Novartis/Böhringer-Ingelheim/Alcon (C) and BSG: Novartis (C) and IDx (F)., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose/aim of the Study: Vascular endothelial growth factor (VEGF)-antagonists are given over long time periods in the clinic, but the long-term effects on retinal pigment epithelium (RPE) cells are not fully investigated. This study aims to investigate these effects with two clinical relevant VEGF antagonists, bevacizumab and aflibercept, on the function of primary RPE cells., Materials and Methods: All tests were conducted with primary porcine RPE. Cells were stimulated with bevacizumab or aflibercept (both 250 µg/ml) for 1 day, 7 days or 4 weeks. Cell viability was tested in MTT Assay. Secretion of TGF-ß was tested in ELISA, phagocytosis in a microscopic assay, migration in a scratch assay, and expression of RPE65 in Western blot. Barrier function was tested for bevacizumab in transwell-cultured cells by measuring transepithelial electrical resistance for up to 3 days., Results: Viability was reduced by both antagonists at all time points tested. TGF-ß secretion was not altered by any treatment. Phagocytosis was not significantly reduced by any treatment. Wound healing ability was not significantly altered by any treatment. The expression of RPE65 was reduced by bevacizumab but not aflibercept after 4 weeks. Transepithelial electrical resistance was not altered., Conclusions: Long-term treatment with anti VEGF may affect viability of RPE cells, and treatment with bevacizumab may have effects on RPE function in long-term treatment.

Purpose: This trial was conducted to investigate the clinical equivalence of the proposed biosimilar FYB201 and reference ranibizumab in patients with treatment-naive, subfoveal choroidal neovascularization caused by neovascular age-related macular degeneration (nAMD)., Design: This was a prospective, multicenter, evaluation-masked, parallel-group, 48-week, phase III randomized study., Participants: A total of 477 patients were randomly assigned to receive FYB201 (n = 238) or reference ranibizumab (n = 239)., Methods: Patients received FYB201 or reference ranibizumab 0.5 mg by intravitreal (IVT) injection in the study eye every 4 weeks., Main Outcome Measures: The primary end point was change from baseline in best-corrected visual acuity (BCVA) by Early Treatment Diabetic Retinopathy Study (ETDRS) letters at 8 weeks before the third monthly IVT injection. Biosimilarity of FYB201 to its originator was assessed via a 2-sided equivalence test, with an equivalence margin in BCVA of 3 ETDRS letters., Results: The BCVA improved in both groups, with a mean improvement of +5.1 (FYB201) and +5.6 (reference ranibizumab) ETDRS letters at week 8. The analysis of covariance (ANCOVA) least squares mean difference for the change from baseline between FYB201 and reference ranibizumab was -0.4 ETDRS letters with a 90% confidence interval (CI) of -1.6 to 0.9. Primary end point was met as the 90% CI was within the predefined equivalence margin. Adverse events were comparable between treatment groups., Conclusions: FYB201 is biosimilar to reference ranibizumab in terms of clinical efficacy and ocular and systemic safety in the treatment of patients with nAMD., (Copyright © 2021 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Aberrant angiogenesis lies at the heart of a wide range of ocular pathologies such as proliferative diabetic retinopathy, wet age-related macular degeneration and retinopathy of prematurity. This study explores the anti-angiogenic activity of a novel small molecule investigative compound capable of inhibiting profilin1-actin interaction recently identified by our group. We demonstrate that our compound is capable of inhibiting migration, proliferation and angiogenic activity of microvascular endothelial cells in vitro as well as choroidal neovascularization (CNV) ex vivo. In mouse model of laser-injury induced CNV, intravitreal administration of this compound diminishes sub-retinal neovascularization. Finally, our preliminary structure-activity relationship study (SAR) demonstrates that this small molecule compound is amenable to improvement in biological activity through structural modifications., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Choroidal neovascularization (CNV) is a key characteristic of wet age-related macular degeneration (AMD) that can lead to severe vision loss in the elderly. Anti-VEGF therapy is currently the premier strategy for wet AMD, but it has limited efficacy. Previous studies have shown that chitinase-3-like-1 (YKL-40) can promote microangiogenesis and inflammation, but its effect on CNV formation has not yet been studied. Here, we investigated the potential role of YKL-40 in wet AMD and the underlying mechanism(s). We report that the serum expression of YKL-40 in wet AMD patients was significantly higher than that in control patients and was positively correlated with VEGF expression, indicating that YKL-40 may participate in the development of wet AMD. In addition, YKL-40 and VEGF expression levels were observed to be increased and the ERK1/2 pathway activated in the neuroretinal (NR) and RPE/choroid tissues of mice with laser-induced CNV. The YKL-40 and phosphorylated protein levels of the ERK1/2 pathway were decreased after intravitreal injection with an anti-YKL-40 antibody, suggesting that anti-YKL-40 could inhibit the activation of the ERK1/2 pathway. These results indicate that YKL-40 may serve as a novel target for the diagnosis and treatment of wet AMD., (© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Age-related macular degeneration (AMD) is the leading cause of vision loss in geriatric population. Intravitreal (IVT) injections are popular clinical option. Biologics and small molecules offer efficacy but relatively shorter half-life after intravitreal injections. To address these challenges, numerous technologies and therapies are under development. Most of these strategies aim to reduce the frequency of injections, thereby increasing patient compliance and reducing patient-associated burden. Unlike IVT frequent injections, molecular therapies such as cell therapy and gene therapy offer restoration ability hence gained a lot of traction. The recent approval of ocular gene therapy for inherited disease offers new hope in this direction. However, until such breakthrough therapies are available to the majority of patients, antibody therapeutics will be on the shelf, continuing to provide therapeutic benefits. The present review aims to highlight the status of pre-clinical and clinical studies of neovascular AMD treatment modalities including Anti-VEGF therapy, upcoming bispecific antibodies, small molecules, port delivery systems, photodynamic therapy, radiation therapy, gene therapy, cell therapy, and combination therapies.

Purpose: Age-related macular degeneration (AMD) is associated with altered gene and protein expression in the retina. We characterize the aqueous humor (AH) proteome in AMD to gain insight into the pathogenesis of the disease and identify potential biomarkers., Methods: AH was collected from age and gender matched neovascular AMD (nvAMD; n = 10) patients and controls (n = 10). AH was pooled to create two samples (nvAMD and control), followed by intensity-based label-free quantification (MS1). Functional and bioinformatic analysis were then performed. A validation set (20 controls, 15 atrophic AMD and 15 nvAMD) was tested via multiplex ELISA for nine differentially expressed proteins according to the MS1 findings., Results: MS1 identified 674 proteins in the AH. 239 proteins were upregulated in nvAMD (nvAMD/control > 2, peptide tags (PT) > 2), and 86 proteins were downregulated (nvAMD/control < 0.5, PT > 2). Functional analysis of proteins upregulated in AMD demonstrated enrichment for platelet degranulation (enrichment score (ES):28.1), negative regulation of endopeptidase activity (ES:18.8), cellular protein metabolic process (ES:11.8), epidermal growth factor-like domain (ES:10.3), sushi/SCR/CCP (ES:10.1), and complement/coagulation cascades (ES:9.2). AMD protein clusters were upregulated for 3/6 (χ2 < 0.05 compared to randomization). Validation via ELISA confirmed MS1 in 2/9 proteins (Clusterin and Serpin A4, P < 0.05), while 3/9 showed differential expression between aAMD and nvAMD (Clusterin, Serpin A4, and TF P < 0.05). Receiver operating characteristic curve calculation identified the area under the curve of 0.82 for clusterin as a biomarker for distinction of AMD., Conclusions: AH proteomics in AMD patients identified several proteins and functional clusters with altered expression. Further research should confirm if these proteins may serve as biomarkers or therapeutic target for the disease.

Age-related macular degeneration (AMD) is the most common cause of blindness among the elderly and can be classified either as dry or as neovascular (or wet). Neovascular AMD is characterized by a strong immune response and the inadequate release of cytokines triggering angiogenesis and induction of photoreceptor death. The pathomechanisms of AMD are only partly understood. Here, we identify the endolysosomal two-pore cation channel TPC2 as a key factor of neovascularization and immune activation in the laser-induced choroidal neovascularization (CNV) mouse model of AMD. Block of TPC2 reduced retinal VEGFA and IL-1β levels and diminished neovascularization and immune activation. Mechanistically, TPC2 mediates cationic currents in endolysosomal organelles of immune cells and lack of TPC2 leads to reduced IL-1β levels in areas of choroidal neovascularization due to endolysosomal trapping. Taken together, our study identifies TPC2 as a promising novel therapeutic target for the treatment of AMD., (© 2021 Li et al.)

We previously demonstrated that the bile acid taurocholic acid (TCA) inhibits features of age-related macular degeneration (AMD) in vitro. The purpose of this study was to determine if the glycine-conjugated bile acids glycocholic acid (GCA), glycodeoxycholic acid (GDCA), and glycoursodeoxycholic acid (GUDCA) can protect retinal pigment epithelial (RPE) cells against oxidative damage and inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis in choroidal endothelial cells (CECs). Paraquat was used to induce oxidative stress and disrupt tight junctions in HRPEpiC primary human RPE cells. Tight junctions were assessed via transepithelial electrical resistance and ZO-1 immunofluorescence. GCA and GUDCA protected RPE tight junctions against oxidative damage at concentrations of 100-500 µM, and GDCA protected tight junctions at 10-500 µM. Angiogenesis was induced with VEGF in RF/6A macaque CECs and evaluated with cell proliferation, cell migration, and tube formation assays. GCA inhibited VEGF-induced CEC migration at 50-500 µM and tube formation at 10-500 µM. GUDCA inhibited VEGF-induced CEC migration at 100-500 µM and tube formation at 50-500 µM. GDCA had no effect on VEGF-induced angiogenesis. None of the three bile acids significantly inhibited VEGF-induced CEC proliferation. These results suggest glycine-conjugated bile acids may be protective against both atrophic and neovascular AMD.

Purpose: To evaluate retinal fluid volume data extracted from optical coherence tomography (OCT) scans by artificial intelligence algorithms in the treatment of neovascular age-related macular degeneration (NV-AMD)., Design: Perspective., Methods: A review was performed of retinal image repository datasets from diverse clinical settings., Settings: Clinical trial (HARBOR) and trial follow-on (Age-Related Eye Disease Study 2 10-year Follow-On); real-world (Belfast and Tel-Aviv tertiary centers)., Patients: 24,362 scans of 1,095 eyes (HARBOR); 4,673 of 880 (Belfast); 1,470 of 132 (Tel-Aviv); 511 of 511 (Age-Related Eye Disease Study 2 10-year Follow-On). ObservationProcedures: Vienna Fluid Monitor or Notal OCT Analyzer applied to macular cube scans. OutcomeMeasures: Intraretinal fluid (IRF), subretinal fluid (SRF), and pigment epithelial detachment (PED) volumes., Results: The fluid volumes measured in neovascular AMD were expressed efficiently in nanoliters. Large ranges that differed by population were observed at the treatment-naïve stage: 0-3,435 nL (IRF), 0-5,018 nL (SRF), and 0-10,022 nL (PED). Mean volumes decreased rapidly and consistently with anti-vascular endothelial growth factor therapy. During maintenance therapy, mean IRF volumes were highest in Tel-Aviv (100 nL), lower in Belfast and HARBOR-Pro Re Nata, and lowest in HARBOR-monthly (21 nL). Mean SRF volumes were low in all: 30 nL (HARBOR-monthly) and 48-49 nL (others)., Conclusions: Quantitative measures of IRF, SRF, and PED are important biomarkers in NV-AMD. Accurate volumes can be extracted efficiently from OCT scans by artificial intelligence algorithms to guide the treatment of exudative macular diseases. Automated fluid monitoring identifies fluid characteristics in different NV-AMD populations at baseline and during follow-up. For consistency between studies, we propose the nanoliter as a convenient unit. We explore the advantages of using these quantitative metrics in clinical practice and research., (Published by Elsevier Inc.)

Inflammatory cytokines are involved in the pathogenesis of neovascular age-related macular degeneration (nAMD) and have been shown to be useful as diagnostic and predictive biomarkers. Given the heterogeneity of data within the literature, we aimed to quantitatively summarize data related to inflammatory cytokines in nAMD. A systematic search without year limitation was performed up to 13 April 2020. Studies were included if they provided data on aqueous or vitreous cytokine concentrations in patients with nAMD. Data were extracted from 95 studies that encompassed 3105 study eyes with nAMD and 1209 control eyes. Effect sizes were generated as standardized mean differences (SMDs) of cytokine concentrations between patients with nAMD and controls. Among the 4314 eyes in 95 studies, aqueous concentrations (standard mean difference, 95% confidence interval and p-value) of MCP-1 (0.43, 0.09 to 0.77 and p = 0.01), MIG (0.63, 0.31 to 0.94 and p = 0.0001), TGF-β (0.45, 0.07 to 0.82 and p = 0.02) and VEGF (0.64, 0.31 to 0.98 and p = 0.0001) were significantly higher in patients with nAMD compared to healthy controls. No differences, failed sensitivity analyses or insufficient data were found between patients with nAMD and healthy controls for the concentrations of the remaining cytokines and with all vitreous samples. Previous studies had shown conflicting associations with nAMD for all 27 cytokines assessed. Our analysis indicates multiple candidate cytokines other than VEGF that are implicated in nAMD and adds clarity to the previous literature. This will help focus translational research in nAMD investigating biomarkers and therapeutic targets., (© 2020 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Background : Age-related macular degeneration (AMD) is a type of macular degeneration disease, and amyloid beta (aβ) is the main component of vitreous warts in AMD patients. Neovascular AMD (nAMD) is the most serious type of AMD, but its pathogenesis remains unclear. The aim of this study was to detect the expression of aβ42 in the aqueous humor of nAMD patients and to evaluate whether aβ42 expression of aqueous humor is correlated with cognitive function in these patients. Methods : A total of 70 patients were enrolled in this study, including 50 nAMD patients (nAMD group) and 20 patients with cataract (control group). The cognitive function of the patients was assessed using the Mini-Mental State Examination and Montreal Cognitive Assessment Scale, and based on their scores, 50 patients with nAMD were divided into two subgroups: the p -nAMD group (18 nAMD patients with normal cognition) and the ci-nAMD group (32 nAMD patients with cognitive impairment). An immunofluorescence microsphere probe technique was used to detect the aβ42 expression of aqueous humor in all patients. Pearson correlation analysis was used. Results : The aβ42 expression of aqueous humor was significantly higher in the nAMD group (124.56 ± 41.93 pg/mL) as compared with the control group (82.94 ± 33.75 pg/mL; P < .01). There was no significant difference in aβ42 expression of aqueous humor between the p -nAMD group (136.42 ± 51.68 pg/mL) and ci-nAMD group (117.90 ± 34.46 pg/mL; P = .14). Conclusion : In nAMD patients, aβ42 was highly expressed in the aqueous humor but was not correlated with cognitive function.

Runt-related transcription factor 1 (RUNX1) acts as a mediator of aberrant retinal angiogenesis and has been implicated in the progression of proliferative diabetic retinopathy (PDR). Patients with PDR, retinopathy of prematurity (ROP), and wet age-related macular degeneration (wet AMD) have been found to have elevated levels of Tumor Necrosis Factor-alpha (TNF-α) in the eye. In fibrovascular membranes (FVMs) taken from patients with PDR RUNX1 expression was increased in the vasculature, while in human retinal microvascular endothelial cells (HRMECs), TNF-α stimulation causes increased RUNX1 expression, which can be modulated by RUNX1 inhibitors. Using TNF-α pathway inhibitors, we determined that in HRMECs, TNF-α-induced RUNX1 expression occurs via JNK activation, while NF-κB and p38/MAPK inhibition did not affect RUNX1 expression. JNK inhibitors were also effective at stopping high D-glucose-stimulated RUNX1 expression. We further linked JNK to RUNX1 through Activator Protein 1 (AP-1) and investigated the JNK-AP-1-RUNX1 regulatory feedback loop, which can be modulated by VEGF. Additionally, stimulation with TNF-α and D-glucose had an additive effect on RUNX1 expression, which was downregulated by VEGF modulation. These data suggest that the downregulation of RUNX1 in conjunction with anti-VEGF agents may be important in future treatments for the management of diseases of pathologic ocular angiogenesis., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)