Laser-induced Choroidal Neovascularization Research Articles

Beta-alanine promotes angiogenesis in laser-induced choroidal neovascularization mice models.

To investigate the effect of β-alanine (BA) on laser-induced choroidal neovascularization (CNV) mice models. Laser-induced CNV mice models were established, and BA was administrated for one week and two weeks in advance, separately. Furthermore, retinal pigment epithelium (RPE)-choroid flat mounts were separated, and immunohistochemical staining was performed. The laser-induced CNV lesion areas were measured and compared. In addition, liver and kidney morphologies were observed to identify potential hepatorenal toxicity. Enlarged CNV lesion areas were observed in the BA treated group. No significant differences were observed in the liver and kidney sections between groups. BA treatment increase CNV lesion areas, suggesting the detrimental effects of BA as a nutritional supplement in age-related macular degeneration (AMD) population.

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.

CBX7 promotes choroidal neovascularization by activating the HIF-1α/VEGF pathway in choroidal vascular endothelial cells

Vascular endothelial growth factor (VEGF) signaling is crucial for choroidal neovascularization (CNV), a major pathological feature of neovascular age-related macular degeneration (nAMD). Gene transcription of VEGF is mainly regulated by hypoxia-inducible factor 1-alpha (HIF-1α). The chromobox (CBX) family polycomb protein (Pc) subgroup includes CBX2, CBX4, CBX6, CBX7, and CBX8. CBX4 enhances hypoxia-induced VEGF expression and angiogenesis in hepatocellular carcinoma (HCC) cells by increasing HIF-1α′s transcriptional activity. The objective of the study was to examine the functions of members of the CBX family Pc subgroup in choroidal vascular endothelial cells (CVECs) during CNV. CBX4 and CBX7 expression was up-regulated in hypoxic human choroidal vascular endothelial cells (HCVECs). In HCVECs, CBX7 facilitated HIF-1α transcription and expression, while CBX4 did not. In HCVECs, CBX7 stimulated HIF-1α′s nuclear translocation and transcriptional activity, which in turn stimulated VEGF transcription and expression. The CBX7/HIF-1α/VEGF pathway promoted the migration, proliferation, and tube formation of HCVECs. The CBX7/HIF-1α/VEGF pathway was up-regulated in CVECs and in the mouse model with laser-induced CNV. Mouse CNV was lessened by the blockade of CBX7 through the down-regulation of HIF-1α/VEGF. In conclusion, CBX7 enhanced pro-angiogenic behaviors of hypoxic CVECs by up-regulating the HIF-1α/VEGF pathway, which contributing to the formation of mouse laser-induced CNV.

Targeted 8-arm PEG Nanosystems for Localization of Choroidal Neovascularization Macular Degeneration Model.

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

PDK4-mediated metabolic reprogramming is a potential therapeutic target for neovascular age-related macular degeneration

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

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.

Effects of Topic Delivery of an Inhibitor of Serine Racemase on Laser-Induced Choroidal Vasculopathy.

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

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.

In vivo multi-contrast depth-resolved choroidal imaging of a mouse using polarization-diversity optical coherence tomography.

The results of depth-resolved multi-contrast in vivo mouse choroidal imaging using a polarization-diversity optical coherence tomography (PD-OCT) system are presented. A selectively chosen depth of focus that was fine-tuned with a sensorless adaptive optics technique and a simple segmentation based on the degree of polarization uniformity signal visualizes the detailed features of a mouse choroid from the OCT angiography images. A comprehensive image analysis of the choroid revealed the distinctive pathological characteristics of the laser-induced choroidal neovascularization mouse.

Stable inhibition of choroidal neovascularization by adeno-associated virus 2/8-vectored bispecific molecules.

Neovascular age-related macular degeneration (nAMD) causes severe visual impairment. Pigment epithelium-derived factor (PEDF), soluble CD59 (sCD59), and soluble fms-like tyrosine kinase-1 (sFLT-1) are potential therapeutic agents for nAMD, which target angiogenesis and the complement system. Using the AAV2/8 vector, two bi-target gene therapy agents, AAV2/8-PEDF-P2A-sCD59 and AAV2/8-sFLT-1-P2A-sCD59, were generated, and their therapeutic efficacy was investigated in laser-induced choroidal neovascularization (CNV) and Vldlr-/- mouse models. After a single injection, AAV2/8-mediated gene expression was maintained at high levels in the retina for two months. Both AAV2/8-PEDF-P2A-sCD59 and AAV2/8-sFLT-1-P2A-sCD59 significantly reduced CNV development for an extended period without side effects and provided efficacy similar to two injections of current anti-vascular endothelial growth factor monotherapy. Mechanistically, these agents suppressed the extracellular signal-regulated kinase and nuclear factor-κB pathways, resulting in anti-angiogenic activity. This study demonstrated the safety and long-lasting effects of AAV2/8-PEDF-P2A-sCD59 and AAV2/8-sFLT-1-P2A-sCD59 in CNV treatment, providing a promising therapeutic strategy for nAMD.

PlGF and VEGF-A/PlGF Heterodimer are Crucial for Recruitment and Activation of Immune Cells During Choroid Neovascularization.

Recruitment and activation of inflammatory cells, such as retinal microglia/macrophages, in the subretinal space contribute significantly to the pathogenesis of age-related macular degeneration (AMD). This study aims to explore the functional role of vascular endothelial growth factor (VEGF-A), placental growth factor (PlGF) and VEGF-A/PlGF heterodimer in immune homeostasis and activation during pathological laser-induced choroidal neovascularization (CNV). To investigate these roles, we utilized the PlGF-DE knockin (KI) mouse model, which is the full functional knockout (KO) of PlGF. In this model, mice express a variant of PlGF, named PlGF-DE, that is unable to bind and activate VEGFR-1 but can still form heterodimer with VEGF-A. Our findings demonstrate that, although there is no difference in healthy conditions, PlGF-DE-KI mice exhibit decreased microglia reactivity and reduced recruitment of both microglia and monocyte-macrophages, compared to wild-type mice during laser-induced CNV. This impairment is associated with a reduction in VEGF receptor 1 (VEGFR-1) phosphorylation in the retinae of PlGF-DE-KI mice compared to C57Bl6/J mice. Corroborating these data, intravitreal delivery of PlGF or VEGF-A/PlGF heterodimer in PlGF-DE-KI mice rescued the immune cell response at the early phase of CNV compared to VEGF-A delivery. In summary, our study suggests that targeting PlGF and the VEGF-A/PlGF heterodimer, thereby preventing VEGFR-1 activation, could represent a potential therapeutic approach for the management of inflammatory processes in diseases such as AMD.

Botulinum neurotoxin serotype A inhibited ocular angiogenesis through modulating glial activation via SOCS3.

Pathological angiogenesis causes significant vision loss in neovascular age-related macular degeneration and other retinopathies with neovascularization (NV). Neuronal/glial-vascular interactions influence the release of angiogenic and neurotrophic factors. We hypothesized that botulinum neurotoxin serotype A (BoNT/A) modulates pathological endothelial cell proliferation through glial cell activation and growth factor release. A laser-induced choroidal NV (CNV) was employed to investigate the anti-angiogenic effects of BoNT/A. Fundus fluorescence angiography, immunohistochemistry, and real-time PCR were used to assess BoNT/A efficacy in inhibiting CNV and the molecular mechanisms underlying this inhibition. Neuronal and glial suppressor of cytokine signaling 3 (SOCS3) deficient mice were used to investigate the molecular mechanisms of BoNT/A in inhibiting CNV via SOCS3. In laser-induced CNV mice with intravitreal BoNT/A treatment, CNV lesions decreased > 30%; vascular leakage and retinal glial activation were suppressed; and Socs3 mRNA expression was induced while vascular endothelial growth factor A (Vegfa) mRNA expression was suppressed. The protective effects of BoNT/A on CNV development were diminished in mice lacking neuronal/glial SOCS3. BoNT/A suppressed laser-induced CNV and glial cell activation, in part through SOCS3 induction in neuronal/glial cells. BoNT/A treatment led to a decrease of pro-angiogenic factors, including VEGFA, highlighting the potential of BoNT/A as a therapeutic intervention for pathological angiogenesis in retinopathies.

SPI1-mediated macrophage polarization aggravates age-related macular degeneration.

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

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.

Contrast-enhanced near-infrared photoacoustic microscopy and optical coherence tomography imaging of rat fundus

Abstract In this paper, we design a multimodal visible/near-infrared photoacoustic microscopy and optical coherence tomography (VIS/NIR-PAM-OCT) system for imaging both retina and retinal pigment epithelium (RPE)/choroid complex layer. F127 and DSPE-PEG-cRGD encapsulated IR-1048 nanoparticles (FINPs) exhibiting absorption peak up to 1,064 nm were utilized as contrast agents to enhance NIR-PAM for in vivo imaging of fundus tissues. The fundus structure and vessels are clearly visualized by the multimodal imaging, and their parameters were quantitatively analyzed. NIR-PAM and OCT imaging of fundus were time-serially monitored over 60 min following the intravenous injection of FINPs into rats. The results indicated a 134 % increase in image signals in PAM at 1 min, along with an 8.23 % intensity enhancement in OCT. Moreover, laser-induced choroidal neovascularization (CNV) was specifically detected and accurately quantified using VIS/NIR-PAM-OCT. Lastly, FINPs demonstrated excellent biocompatibility in hematology analysis and pathology testing.

Dual anti-angiogenic and anti-inflammatory action of tRNA-Cys-5-0007 in ocular vascular disease

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

An adeno-associated virus variant enabling efficient ocular-directed gene delivery across species

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

Role of NLRP3 Inflammasomes in Monocyte and Microglial Recruitments in Choroidal Neovascularization.

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

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.

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.