Laser-induced Choroidal Neovascularization Model Research Articles

The Effects of STRA6 Regulation of the Circadian Rhythm on Choroidal Neovascularization.

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. C57BL/6J male mice (aged 6 weeks) were randomly divided into control and jet lag groups (using a time shift method every 4days 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. 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. 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.

Pre-Clinical Studies of a Novel Bispecific Fusion Protein Targeting C3b and VEGF in Neovascular and Nonexudative AMD Models.

KNP-301 is a bi-specific fragment crystallizable region (Fc) fusion protein, which inhibits both C3b and vascular endothelial growth factor (VEGF) simultaneously for patients with late-stage age-related macular degeneration (AMD). The present study evaluated in vitro potency, in vivo efficacy, intravitreal pharmacokinetics (IVT PK), and injectability of KNP-301. C3b and VEGF binding of KNP-301 were assessed by surface plasmon resonance (SPR) and enzyme-linked immunosorbent assay (ELISA), and cellular bioassays. A laser-induced choroidal neovascularization (CNV) model and a sodium iodate-induced nonexudative AMD model were used to test the in vivo efficacy of mouse surrogate of KNP-301. Utilizing fluorescein angiography (FA) and spectral-domain optical coherence tomography (SD-OCT) scans, the reduction in disease lesions were analyzed in a CNV mouse model. In the nonexudative AMD mouse model, outer nuclear layer (ONL) was assessed by immunofluorescence staining. Lastly, intravitreal pharmacokinetic study was conducted with New Zealand white rabbits via IVT administration of KNP-301 and injectability of KNP-301 was examined by a viscosity test at high concentrations. KNP-301 bound C3b selectively, which resulted in a blockade of the alternative pathway, not the classical pathway. KNP-301 also acted as a VEGF trap, impeding VEGF-mediate signaling. Our dual-blockade strategy was effective in both neovascular and nonexudative AMD models. Moreover, KNP-301 had an advantage of potentially less frequent dosing due to the long half-life in the intravitreal chamber. Our viscosity assessment confirmed that KNP-301 meets the criteria of the IVT injection. Unlike current therapies, KNP-301 is expected to cover patients with late-stage AMD of both neovascular and nonexudative AMD, and its long-term PK profile at the intravitreal chamber would allow convenience in the dosing interval of patients.

In vivo monitoring of active subretinal fibrosis in mice using collagen hybridizing peptides

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

Sema4D Knockout Attenuates Choroidal Neovascularization by Inhibiting M2 Macrophage Polarization Via Regulation of the RhoA/ROCK Pathway.

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. 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. 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. 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.

Sphingosine-1-phosphate receptor 1/5 selective agonist alleviates ocular vascular pathologies

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

Novel CCR3-targeted cyclic peptides as potential therapeutic agents for age-related macular degeneration via inhibiting angiogenesis and reducing retinal photoreceptor damage

The prolonged intravitreal administration of anti-vascular endothelial growth factor (VEGF) drugs is prone to inducing aberrant retinal vascular development and causing damage to retinal neurons. Hence, we have taken an alternative approach by designing and synthesizing a series of cyclic peptides targeting CC motif chemokine receptor 3 (CCR3). Based on the binding mode of the N-terminal region in CCR3 protein to CCL11, we used computer-aided identification of key amino acid sequence, conformational restriction through different cyclization methods, designed and synthesized a series of target cyclic peptides, and screened the preferred compound IB-2 through affinity. IB-2 exhibits excellent anti-angiogenic activity in HRECs. The apoptosis level of 661W cells demonstrated a significant decrease with the escalating concentration of IB-2. This suggests that IB-2 may have a protective effect on photoreceptor cells. In vivo experiments have shown that IB-2 significantly reduces retinal vascular leakage and choroidal neovascularization (CNV) area in a laser-induced mouse model of CNV. These findings indicate the potential of IB-2 as a safe and effective therapeutic agent for AMD, warranting further development.

Microglial repopulation restricts ocular inflammation and choroidal neovascularization in mice.

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

Tocilizumab attenuates choroidal neovascularization by regulating macrophage polarization through the IL-6R/STAT3/VEGF pathway

Globally, age-related macular degeneration (AMD) is the leading cause of irreversible visual impairment. Up to 80% of severe vision loss is caused by AMD, which is characterized by the development of choroidal neovascularization (CNV). Uncertainty exists regarding the precise pathophysiological mechanisms of CNV. It has been suggested that the interleukin (IL) IL-6/IL-6R signaling pathway is crucial in the progression of CNV. Tocilizumab (TCZ), a monoclonal antibody, binds to soluble and membrane-bound IL-6R and competitively inhibits IL-6 downstream signaling. Previous research has demonstrated that TCZ promotes several roles related to inflammation and neovascularization. However, the effects of TCZ on CNV and the underlying mechanism are still unknown. This study found that TCZ administration decreased the area and leakage of CNV lesions in the mice model of laser-induced CNV. Additionally, results demonstrated that TCZ promotes the expression of iNOS, CCL-3, CCL-5, TNF-α and inhibits the expression of Arg-1, IL-10, YM-1 and CD206. Furthermore, TCZ treatment inhibited the signal transducer and activator of transcription (STAT) STAT3/vascular endothelial growth factor (VEGF) pathway, which was activated after CNV formation. Colivelin, a STAT3 agonist, reversed the inhibitory effects of TCZ on CNV formation and macrophage polarization. In a mouse model of laser-induced CNV, our findings demonstrated that TCZ attenuated CNV formation and inhibited the leakage of CNV lesions by regulating macrophage polarization via inhibiting the STAT3/VEGF axis. TCZ is the potential therapeutic strategy for CNV.

Emerging Co-Assembled and Sustained Released Natural Medicinal Nanoparticles for Multitarget Therapy of Choroidal Neovascularization.

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

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

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

Targeting endothelial glycolytic reprogramming by tsRNA-1599 for ocular anti-angiogenesis therapy.

Rationale: Current treatments for ocular angiogenesis primarily focus on blocking the activity of vascular endothelial growth factor (VEGF), but unfavorable side effects and unsatisfactory efficacy remain issues. The identification of novel targets for anti-angiogenic treatment is still needed. Methods: We investigated the role of tsRNA-1599 in ocular angiogenesis using endothelial cells, a streptozotocin (STZ)-induced diabetic model, a laser-induced choroidal neovascularization model, and an oxygen-induced retinopathy model. CCK-8 assays, EdU assays, transwell assays, and matrigel assays were performed to assess the role of tsRNA-1599 in endothelial cells. Retinal digestion assays, Isolectin B4 (IB4) staining, and choroidal sprouting assays were conducted to evaluate the role of tsRNA-1599 in ocular angiogenesis. Transcriptomic analysis, metabolic analysis, RNA pull-down assays, and mass spectrometry were utilized to elucidate the mechanism underlying angiogenic effects mediated by tsRNA-1599. Results: tsRNA-1599 expression was up-regulated in experimental ocular angiogenesis models and endothelial cells in response to angiogenic stress. Silencing of tsRNA-1599 suppressed angiogenic effects in endothelial cells in vitro and inhibited pathological ocular angiogenesis in vivo. Mechanistically, tsRNA-1599 exhibited little effect on VEGF signaling but could cause reduced glycolysis and NAD+/NADH production in endothelial cells by regulating the expression of HK2 gene through interacting with YBX1, thus affecting endothelial effects. Conclusions: Targeting glycolytic reprogramming of endothelial cells by a tRNA-derived small RNA represents an exploitable therapeutic approach for ocular neovascular diseases.

Oral Metformin Inhibits Choroidal Neovascularization by Modulating the Gut-Retina Axis.

Emerging data indicate that metformin may prevent the development of age-related macular degeneration (AMD). Whereas the underlying mechanisms of metformin's anti-aging properties remain undetermined, one proposed avenue is the gut microbiome. Using the laser-induced choroidal neovascularization (CNV) model, we investigate the effects of oral metformin on CNV, retinal pigment epithelium (RPE)/choroid transcriptome, and gut microbiota. Specific pathogen free (SPF) male mice were treated via daily oral gavage of metformin 300mg/kg or vehicle. Male mice were selected to minimize sex-specific differences to laser induction and response to metformin. Laser-induced CNV size and macrophage/microglial infiltration were assessed by isolectin and Iba1 immunostaining. High-throughput RNA-seq of the RPE/choroid was performed using Illumina. Fecal pellets were analyzed for gut microbiota composition/pathways with 16S rRNA sequencing/shotgun metagenomics, as well as microbial-derived metabolites, including small-chain fatty acids and bile acids. Investigation was repeated in metformin-treated germ-free (GF) mice and antibiotic-treated/GF mice receiving fecal microbiota transplantation (FMT) from metformin-treated SPF mice. Metformin treatment reduced CNV size (P < 0.01) and decreased Iba1+ macrophage/microglial infiltration (P < 0.005). One hundred forty-five differentially expressed genes were identified in the metformin-treated group (P < 0.05) with a downregulation in pro-angiogenic genes Tie1, Pgf, and Gata2. Furthermore, metformin altered the gut microbiome in favor of Bifidobacterium and Akkermansia, with a significant increase in fecal levels of butyrate, succinate, and cholic acid. Metformin did not suppress CNV in GF mice but colonization of microbiome-depleted mice with metformin-derived FMT suppressed CNV. These data suggest that oral metformin suppresses CNV, the hallmark lesion of advanced neovascular AMD, via gut microbiome modulation.

Visualization of choroidal vasculature in pigmented mouse eyes from experimental models of AMD

A variety of techniques exist to investigate retinal and choroidal vascular changes in experimental mouse models of human ocular diseases. While all have specific advantages, a method for evaluating the choroidal vasculature in pigmented mouse eyes has been more challenging especially for whole mount visualization and morphometric analysis. Here we report a simple, reliable technique involving bleaching pigment prior to immunostaining the vasculature in whole mounts of pigmented mouse choroids. Eyes from healthy adult pigmented C57BL/6J mice were used to establish the methodology. The retina and anterior segment were separated from the choroid. The choroid with retinal pigment epithelial cells (RPE) and sclera was soaked in 1% ethylenediaminetetraacetic acid (EDTA) to remove the RPE. Tissues were fixed in 2% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Choroids were subjected to melanin bleaching with 10% hydrogen peroxide (H2O2) at 55 °C for 90 min, washed in PBS and then immunostained with anti-podocalyxin antibody to label vascular endothelium followed by Cy3-AffiniPure donkey anti-goat IgG at 4 °C overnight. Images of immunostained bleached choroids were captured using a Zeiss 710 confocal microscope. In addition to control eyes, this method was used to analyze the choroids from subretinal sodium iodate (NaIO3) RPE atrophy and laser-induced choroidal neovascularization (CNV) mouse models. The H2O2 pretreatment effectively bleached the melanin, resulting in a transparent choroid. Immunolabeling with podocalyxin antibody following bleaching provided excellent visualization of choroidal vasculature in the flat perspective. In control choroids, the choriocapillaris (CC) displayed different anatomical patterns in peripapillary (PP), mid peripheral (MP) and far peripheral (FP) choroid. Morphometric analysis of the vascular area (VA) revealed that the CC was most dense in the PP region (87.4 ± 4.3% VA) and least dense in FP (79.9 ± 6.7% VA). CC diameters also varied depending on location from 11.4 ± 1.97 mm in PP to 15.1 ± 3.15 mm in FP. In the NaIO3-injected eyes, CC density was significantly reduced in the RPE atrophic regions (50.7 ± 5.8% VA in PP and 45.8 ± 6.17% VA in MP) compared to the far peripheral non-atrophic regions (82.8 ± 3.8% VA). CC diameters were significantly reduced in atrophic regions (6.35 ± 1.02 mm in PP and 6.5 ± 1.2 mm in MP) compared to non-atrophic regions (14.16 ± 2.12 mm). In the laser-induced CNV model, CNV area was 0.26 ± 0.09 mm2 and luminal diameters of CNV vessels were 4.7 ± 0.9 mm. Immunostaining on bleached choroids with anti-podocalyxin antibody provides a simple and reliable tool for visualizing normal and pathologic choroidal vasculature in pigmented mouse eyes for quantitative morphometric analysis. This method will be beneficial for examining and evaluating the effects of various treatment modalities on the choroidal vasculature in mouse models of ocular diseases such as age-related macular degeneration, and degenerative genetic diseases.

Intravenous 3K3A-Activated Protein C Inhibits Murine Ocular Inflammation and Suppresses Ocular Choroidal Neovascularization

Introduction: Choroidal neovascularization (CNV) is characterized by the growth of newly formed abnormal blood vessels from the choroid extending into the neurosensory retina, leading to vision loss. CNV is a common cause of visual impairment in neovascular age-related macular degeneration (nAMD) patients. Current treatments failed to stop the continuity of the neurodegenerative process. Despite the urgency, no neuroprotective agent has been found yet to treat AMD and other neurodegenerative ocular diseases. 3K3A-activated protein C (APC) is a cell-signaling analog of the endogenous blood serine protease APC. Preclinical studies showed that circulating plasma 3K3A-APC readily crosses the blood-brain barrier (BBB) and reaches neurons due to EPCR-dependent transport. In rodent models of stroke, brain injury, and neurodegenerative disorders, 3K3A-APC exerts neuroprotective, anti-inflammatory, and vasculoprotective activities. Due to its pleiotropic effects, 3K3A-APC has advanced to clinical trial stages for treating acute brain damage and chronic neurodegenerative diseases, which share similarities with retinal neurodegenerative disorders. Notably, the retina is part of the central nervous system (CNS), and the blood-retina barrier shares functional and structural similarities with the BBB. We previously demonstrated that intraocular administration of 3K3A-APC functions as a pleiotropic cytoprotective molecule within the retina, reducing ocular inflammation, suppressing CNV growth, and exhibiting promising therapeutic potential as a neuroprotective agent for treating ocular disorders. Intraocular administration is commonly utilized for delivering therapeutics to treat retinal conditions. However, safety concerns, the burden on the healthcare system, and patient comfort may indicate that systemic administration of 3K3A-APC is a beneficial alternative. Aim: To determine whether intravenously injecting 3K3A-APC into mice tails will cross the blood-retina barrier, induce anti-inflammatory activities in the retina, and suppress CNV. Methods: The efficacy of systemic administration of 3K3A-APC was evaluated using two C57BL/6J murine models: the LPS-induced ocular inflammation and the laser-induced CNV model. CNV was triggered by laser and ocular inflammation was triggered by intravitreal injection of lipopolysaccharide (LPS). Murine recombinant 3K3A-APC was administered systemically via tail injection. Fluorescein isothiocyanate (FITC)-dextran perfusion followed by retinal flatmount staining with anti-CD11b was used to assess inflammatory co-localization of cells to retinal vessels. FITC-dextran perfusion followed by retinal pigment epithelium (RPE)-choroid flatmount was used to assess CNV vascular penetration using 3D confocal imaging. Results: LPS injection triggered robust inflammatory cell infiltration in both the anterior and posterior chambers of the eyes. Systemic administration of 3K3A-APC (0.5 mg/kg, 1 and 4 hours post-LPS) significantly inhibited ocular inflammation. Immunostaining performed 24 hours post LPS revealed that 3K3A-APC reduced the number of CD11b+ monocyte and their extravasation into the retinal parenchyma (Figure). In the laser-induced CNV model, systemic administration of 0.2 mg/kg 3K3A-APC was applied one hour and four days post-laser. Evaluation of retinal specimens ten days post-laser showed that 3K3A-APC treatment significantly suppressed CNV growth, attenuated the abnormal growth of blood vessels from the choroid into the sensory retina, and decreased myeloid cell recruitment to the RPE-choroid area. Conclusion: Our study reveals that systemically administered 3K3A-APC effectively inhibits ocular inflammation and reduces CNV formation. Drawing insights from the neuroprotective activities of 3K3A-APC in the brain and CNS and recognizing the retina, particularly the macula, as an extension of the brain, we propose 3K3A-APC as a potential neuroprotective treatment for AMD and other neurodegenerative retinal pathologies. Given the established safety of systemic 3K3A-APC administration in phase 2 clinical trials for ischemic stroke, our findings support further exploring 3K3A-APC as a novel therapeutic approach in ophthalmology. Further research is warranted to elucidate fully the mechanisms underlying 3K3A-APC's beneficial effects on the retina.

Protective role of IL-17-producing γδ T cells in a laser-induced choroidal neovascularization mouse model

BackgroundVision loss in patients with wet/exudative age-related macular degeneration (AMD) is associated with choroidal neovascularization (CNV), and AMD is the leading cause of irreversible vision impairment in older adults. Interleukin-17A (IL-17A) is a component of the microenvironment associated with some autoimmune diseases. Previous studies have indicated that wet AMD patients have elevated serum IL-17A levels. However, the effect of IL-17A on AMD progression needs to be better understood. We aimed to investigate the role of IL-17A in a laser-induced CNV mouse model.MethodsWe established a laser-induced CNV mouse model in wild-type (WT) and IL-17A-deficient mice and then evaluated the disease severity of these mice by using fluorescence angiography. We performed enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell sorting (FACS) to analyze the levels of IL-17A and to investigate the immune cell populations in the eyes of WT and IL-17A-deficient mice. We used ARPE-19 cells to clarify the effect of IL-17A under oxidative stress.ResultsIn the laser-induced CNV model, the CNV lesions were larger in IL-17A-deficient mice than in WT mice. The numbers of γδ T cells, CD3+CD4+RORγt+ T cells, Treg cells, and neutrophils were decreased and the number of macrophages was increased in the eyes of IL-17A-deficient mice compared with WT mice. In WT mice, IL-17A-producing γδ T-cell numbers increased in a time-dependent manner from day 7 to 28 after laser injury. IL-6 levels increased and IL-10, IL-24, IL-17F, and GM-CSF levels decreased in the eyes of IL-17A-deficient mice after laser injury. In vitro, IL-17A inhibited apoptosis and induced the expression of the antioxidant protein HO-1 in ARPE-19 cells under oxidative stress conditions. IL-17A facilitated the repair of oxidative stress-induced barrier dysfunction in ARPE-19 cells.ConclusionsOur findings provide new insight into the protective effect of IL-17A in a laser-induced CNV model and reveal a novel regulatory role of IL-17A-producing γδ T cells in the ocular microenvironment in wet AMD.

PolySialic acid-nanoparticles inhibit macrophage mediated inflammation through Siglec agonism: a potential treatment for age related macular degeneration.

Age-related macular degeneration (AMD) is a chronic, progressive retinal disease characterized by an inflammatory response mediated by activated macrophages and microglia infiltrating the inner layer of the retina. In this study, we demonstrate that inhibition of macrophages through Siglec binding in the AMD eye can generate therapeutically useful effects. We show that Siglecs-7, -9 and -11 are upregulated in AMD associated M0 and M1 macrophages, and that these can be selectively targeted using polysialic acid (PolySia)-nanoparticles (NPs) to control dampen AMD-associated inflammation. In vitro studies showed that PolySia-NPs bind to macrophages through human Siglecs-7, -9, -11 as well as murine ortholog Siglec-E. Following treatment with PolySia-NPs, we observed that the PolySia-NPs bound and agonized the macrophage Siglecs resulting in a significant decrease in the secretion of IL-6, IL-1β, TNF-α and VEGF, and an increased secretion of IL-10. In vivo intravitreal (IVT) injection of PolySia-NPs was found to be well-tolerated and safe making it effective in preventing thinning of the retinal outer nuclear layer (ONL), inhibiting macrophage infiltration, and restoring electrophysiological retinal function in a model of bright light-induced retinal degeneration. In a clinically validated, laser-induced choroidal neovascularization (CNV) model of exudative AMD, PolySia-NPs reduced the size of neovascular lesions with associated reduction in macrophages. The PolySia-NPs described herein are therefore a promising therapeutic strategy for repolarizing pro-inflammatory macrophages to a more anti-inflammatory, non-angiogenic phenotype, which play a key role in the pathophysiology of non-exudative AMD.

NR4A1 deletion promotes pro-angiogenic polarization of macrophages derived from classical monocytes in a mouse model of neovascular age-related macular degeneration

BackgroundNeovascular 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. Nr4a1se2/se2 mice lack a transcriptional activator, resulting in non-classical monocyte loss without macrophage hyper-activation.Main bodyWe subjected Nr4a1−/− and Nr4a1se2/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.ConclusionsThese results suggest that non-classical monocytes are dispensable during CNV, and NR4A1 deficiency results in increased recruitment of pro-angiogenic macrophages.

Chimpanzee adenovirus-mediated multiple gene therapy for age-related macular degeneration

Neovascular age-related macular degeneration AMD (nAMD) is characterized by choroidal neovascularization (CNV) and could lead to irreversible blindness. However, anti-vascular endothelial growth factor (VEGF) therapy has limited efficacy. Therefore, we generated a chimpanzee adenoviral vector (AdC68-PFC) containing three genes, pigment endothelial-derived factor (PEDF), soluble fms-like tyrosine kinase-1 (sFlt-1), and soluble forms of CD59 (sCD59), to treat nAMD. The results showed that AdC68-PFC mediated a strong onset of PEDF, sFlt-1, and sCD59 expression both invivo and invitro. AdC68-PFC showed preventive and therapeutic effects following intravitreal (IVT) injection in the laser-induced CNV model and very low-density lipoprotein receptor-deficient (Vldlr-/-) mouse model. Invitro assessment indicated that AdC68-PFC had a strong inhibitory effect on endothelial cells. Importantly, the safety test showed no evidence of invivo toxicity of adenovirus in murine eyes. Our findings suggest that AdC68-PFC may be a long-acting and safe gene therapy vector for future nAMD treatments.

Neutrophil extracellular traps boost laser-induced mouse choroidal neovascularization through the activation of the choroidal endothelial cell TLR4/HIF-1α pathway.

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.

Neovascular Progression and Retinal Dysfunction in the Laser-Induced Choroidal Neovascularization Mouse Model.

The mouse model of laser-induced choroidal neovascularization (LI-CNV) has been widely used to study neovascular age-related macular degeneration; however, it still lacks a comprehensive characterization. Here, CNV was induced in the eyes of 12-week-old C57BL/6J male mice by argon laser irradiation. We studied the CNV lesion progression of an LI-CNV mouse cohort by using multimodal imaging (color fundus, optical coherence tomography (OCT), and fluorescence angiography, focal electroretinography features for 14 days, and related cytokines, angiogenic factors, and reactive gliosis for 5 days. CNV lesions involving the rupture of the Bruch's membrane were confirmed using funduscopy and OCT after laser photocoagulation. During the initial stage, from the CNV induction until day 7, CNV lesions presented leakage observed by using fluorescence angiography and a typical hyperreflective area with cell infiltration, subretinal leakage, and degeneration of photoreceptors observed through OCT. This correlated with decreased retinal responses to light. Moreover, inflammatory and angiogenic markers were reduced to basal levels in the first 5 days of CNV progression. In contrast, reactive gliosis and the VEGF expression in retinal sections were sustained, with infiltration of endothelial cells in the subretinal space. In the second stage, between days 7 and 14 post-induction, we observed stabilization of the CNV lesions, a hyperfluorescent area corresponding to the formation of fibrosis, and a partial rescue of retinal function. These findings suggest that the LI-CNV lesion development goes through an acute phase during the first seven days following induction, and then the CNV lesion stabilizes. According to these results, this model is suitable for screening anti-inflammatory and anti-angiogenic drugs in the early stages of LI-CNV. At the same time, it is more convenient for screening anti-fibrotic compounds in the later stages.