Choroidal Neovascularization Formation Research Articles

Essential contribution of macrophage Tie2 signal mediated autophagy in laser-induced choroidal neovascularization

Autophagy plays critical roles in various ocular diseases, including age-related macular degeneration (AMD). Tie2-expressing macrophages (TEMs) play crucial roles in angiogenesis. To investigate the role of TEMs and autophagy in the development of AMD, we employed macrophage-specific Tie2 knockout mice and used a laser-induced choroidal neovascularization (CNV). The results showed that TEMs can promote CNV formation by up-regulating the level of autophagy. These results were further verified by in vitro cell experiments that peritoneal macrophages from Tie2 knockout mice can inhibit the expression of autophagy-related factors and inhibit the expression of angiogenic factor of VEGF by activating AMPK signaling pathway. Our results suggest that TEMs and macrophage Tie2 signal mediated-autophagy play critical role in experimental CNV, and they may be novel preventive targets for AMD treatment.

Clinical and angiographic characterization of choroidal neovascularization in diabetic retinopathy.

To report the clinical and angiographic characteristics of choroidal neovascularization in patients with diabetic retinopathy. Patients of type 2 diabetes mellitus with presence of choroidal neovascularization in at least one eye were retrospectively analyzed. The study eyes were divided into three groups based on presence (active or scarred) or absence of choroidal neovascularization (fellow eyes). Imaging characteristics of active choroidal neovascularization were recorded using optical coherence tomography, fluorescein, and indocyanine angiography. Central macular thickness, subfoveal choroidal thickness, and large choroidal vessel layer thickness were compared at baseline and final visit. Our study reports the prevalence rate of choroidal neovascularization in eyes with diabetic retinopathy (0.27%; 36 out of 13,382 eyes). A total of 64 eyes of 32 patients (age, mean ± standard deviation: 68.5 ± 9.3 years) with baseline visual acuity of 0.69 ± 0.69 logarithm of minimum angle of resolution (Snellen equivalent 20/100) were included. Nonproliferative diabetic retinopathy (57 eyes) comprised the majority followed by proliferative diabetic retinopathy (7 eyes). Eyes with choroidal neovascularization (36, 56.25%) included both active (25) and scarred (11) choroidal neovascularization, with bilateral choroidal neovascularization in 4 patients. Type 1 choroidal neovascularization was the most common subtype of choroidal neovascularization on optical coherence tomography. Common etiologies for active choroidal neovascularization included age-related macular degeneration (3; 12%), myopia (1; 4%), and inflammatory choroidal neovascularization secondary to chorioretinitis (1; 4%). In the remaining 20 eyes, choroidal neovascularization formation was primarily due to diabetic choroidopathy. The prevalence of choroidal neovascularization in eyes with diabetic retinopathy is very low, with a lower prevalence of age-related macular degeneration. Diabetic choroidopathy plays a significant role in formation of choroidal neovascularization in eyes with diabetic retinopathy.

CRB1rd8 mutation influences the age-related macular degeneration phenotype of NRF2 knockout mice and favors choroidal neovascularization

PurposeWe examined the influence of retinal degeneration 8 (rd8) mutation of crumbs homolog 1 (CRB1) gene on age-related macular degeneration (AMD) phenotype in nuclear factor E2-related factor 2 knock out (NRF2−/−) mouse model. MethodsCRB1rd8 mutation genotype was determined by polymerase chain reaction from tail clips in 73 NRF2−/− mice originating from C57BL/6J background on mixed C57BL/6J and C57BL/6N ancestry. The clinical grade of AMD-like fundus alterations was determined by funduscopy, optical coherence tomography (OCT) and fluorescein angiography (FLA) at the age of 9 or 12 months. ResultsTwelve NRF2−/− mice were wildtype CRB1+/+, 61 NRF2−/− were homozygous CRB1rd8/rd8. NRF2−/−CRB1rd8/rd8 mice had a significantly higher probability to show an advanced grade (grade 4 and 5) of AMD-like fundus alterations known to appear in NRF2−/− mice. Choroidal neovascularization (CNV) was only detected in NRF2−/−CRB1rd8/rd8 homozygous mice. ConclusionsHomozygous CRB1rd8/rd8 mutation is common in commercial vendor mice strains of C57BL/6J origin if partly on C57BL/6N ancestry. The mutation has an influence on the extent of AMD-like retinal alterations in NRF2−/− mice and favors CNV formation.

TSP1 ameliorates age-related macular degeneration by regulating the STAT3-iNOS signaling pathway

Age-related macular degeneration is a progressive ocular disease that is the leading cause of vision loss among elderly. AMD usually is divided into two types: wet and dry AMD, which is linked with inflammation. Choroidal Neovascularization (CNV) formation or wet AMD is also associated with oxidative stress. Previously, TSP1 has been shown to have a significant alleviating effect on CNV in TSP1 knockout (TSP1−/−) mice. However, the mechanism by which TSP1 ameliorates CNV remains unclear. Here we report that TSP1 reduces nitric oxide production to prevent cells from forming tubes formation and reduced the levels of vascular endothelial growth factor (VEGF) and lipid peroxides (LPO) during oxidative stress. We measured RF/6A cell viability by CCK-8 assay and apoptosis by flow cytometry. RF/6A cell were transfected with TSP1 and STAT3 overexpression, and then the mRNA and protein levels of TSP1 and also the signal pathways were detected by qRT-PCR and Western blot analysis. Migration assays were performed using a transwell system. Co-Immunoprecipitation was used to analyze the binding relationship between CD47 and SHP-2. The results show that overexpression of TSP1 alleviated the damage of oxidative stress to RF/6A cells including increased cell activity and migration, decreased apoptosis and reduced migration compared to the control group. SHP-2 was activated by TSP1 through its receptor CD47 and STAT3 phosphorylation was reduced by activation of SHP-2, thereby blocking STAT3-iNOS pathway and reducing NO concentration in RF/6A cells ultimately protecting them from oxidative stress. Finally, the CNV mice model confirmed that TSP1 overexpression could protect the mice against CNV in vivo, modified the antioxidants levels and decreased the expression of TNF-α and IL-6 under laser irradiation. These results indicate a potential mechanism of TSP1 to slow down formation of CNV in wet AMD, which may bring hope for new treatment strategies.

Species differences in ocular pharmacokinetics and pharmacological activities of regorafenib and pazopanib eye-drops among rats, rabbits and monkeys.

Age‐related macular degeneration (AMD) is the leading cause of severe vision impairment in patients over the age of 60 years. Choroidal neovascularization (CNV) is the hallmark of neovascular AMD and vascular endothelial growth factor (VEGF) plays a causal role in the formation of CNV. Although regorafenib and pazopanib, small molecule VEGF receptor (VEGFR) inhibitors, were developed as eye‐drops, their efficacies were insufficient in clinical. In this study, we evaluated ocular pharmacokinetics and pharmacological activities of regorafenib and pazopanib after ocular instillation in multiple animal species. In rats, both regorafenib and pazopanib showed high enough concentrations in the posterior eye tissues to inhibit VEGFR. In laser‐induced rat CNV model, regorafenib showed clear reduction in CNV area. On the other hand, the concentrations of regorafenib and pazopanib in the posterior eye tissues were much lower after ocular instillation in rabbits and monkeys compared to those in rats. Pazopanib did not show any improvement in monkey model. Regorafenib was nano‐crystalized to improve its drug delivery to the posterior eye tissues. The nano‐crystalized formulation of regorafenib showed higher concentrations in the posterior segments in rabbits compared to its microcrystal suspension. From these studies, large interspecies differences were found in ocular delivery to the posterior segments after ocular instillation. Such large interspecies difference could be the reason for the insufficient efficacies of regorafenib and pazopanib in clinical studies. Nano‐crystallization was suggested to be one of the effective ways to overcome this issue.

Chitinase-3-Like-1 Promotes M2 Macrophage Differentiation and Induces Choroidal Neovascularization in Neovascular Age-Related Macular Degeneration.

Choroidal neovascularization (CNV) is the principal pathological factor contributing to blindness in neovascular age-related macular degeneration (nAMD). Infiltration of M2 macrophage is thought to contribute to CNV progress, although the way that regulates its differentiation remains unclear. Here, we investigate the role of CHI3L1 in M2 differentiation and angiogenesis in CNV. Serums from nAMD patients were tested for CHI3L1 expression. Mice were subjected to laser injury to induce CNV, and lesion expansion were tracked using fundus fluorescence angiography (FFA) and immunofluorescence analysis. Several strategies were taken to verify the contribution of M2 macrophage and CHI3L1: macrophage depletion by clodrosome, local CHI3L1 inhibition using intravitreally injection neutralize antibody (mAY), and depletion of CHI3L1 receptor (IL13-Ra2) by small-interfering RNA (siRNA). Tuber analysis was used to further determine angiogenetic effect of CHI3L1. Anti-VEGFA was used as positive control for mAY. Serum levels of CHI3L1 were highly elevated in nAMD patients. CHI3L1 was expressed by infiltrating M2 macrophages and was elevated as CNV progress in a mice model. System macrophage depletion and local suppression of CHI3L1 alleviated CNV formation while enhancing anti-VEGFA therapeutic effect. Stimulation of macrophage with recombinant CHI3L1 activated MAPK signaling cascade and induced transition to M2, while siRNA knockdown of IL13-Ra2 abolished it. In an in vitro coculture system, supernatants from CHI3L1-stimulated M2 macrophages and promoted tube vascularization. These results unveil novel angiogenic regulation of CHI3L1 and M2 polarized macrophages in CNV development. These mechanistic insights may point to CHI3L1 as a new therapeutic target for treatment for nAMD.

MiR-505 promotes M2 polarization in choroidal neovascularization model mice by targeting transmembrane protein 229B.

We aimed to analyse the relative abundance of miR-505 in age-related macular degeneration (AMD) and elucidate its underlying mechanisms. Relative expression of miR-505 was analysed by real-time polymerase chain reaction (PCR). Macrophage polarization was characterized by measurement of molecular markers including Ym-1, Arg-1, TNF-α and iNOS via both real-time PCR and Western blot. Vascular endothelial growth factor (VEGF) content was determined by enzyme-linked immunosorbent assay. Choroidal neovascularization (CNV) formation was evaluated by choroidal flat mount technique. The regulatory action of miR-505-5p on 3'UTR of Transmembrane Protein 229B (TMEM229B) was interrogated by luciferase reporter assay. miR-505 was aberrantly upregulated in both AMD and laser-induced choroidal neovascularization mouse model. Administration with miR-505 specific inhibitor suppressed M2 polarization in CNV mice as indicated by decreasing both Ym-1 and Arg-1. Meanwhile, VEGF expression and CNV formation were greatly suppressed by miR-505 inhibition as well. The similar phenotype was consolidated in Prostaglandin E2 (PGE2)-stimulated bone marrow-derived macrophages. At the molecular level, miR-505-5p directly targeted and negatively regulated TMEM229B expression, while forced ectopic expression of TMEM229B significantly rescued miR-505-imposed M2 polarization. Our data have uncovered the critical contribution of miR-505 in AMD, which is predominantly mediated by downregulation of TMEM229B.

Platelet‑derived growth factor‑BB promotes proliferation and migration of retinal microvascular pericytes by up‑regulating the expression of C‑X‑C chemokine receptor types 4

Stromal cell-derived growth factor (SDF)-1α acts as a ligand to C-X-C chemokine receptors 4 (CXCR4) and 7 (CXCR7), which are involved in the formation of choroidal neovascularization. Previous studies have demonstrated crosstalk between the platelet-derived growth factor (PDGF)-BB/PDGF receptor (PDGFR)-β and SDF-1α/CXCR4 axes during tumor neovascularization by increasing the recruitment of pericytes. However, the effects of interactions between these two signaling pathways in retinal microvascular pericytes remain poorly understood. Western blotting and reverse transcription-quantitative PCR were used to measure CXCR4 and CXCR7 expression in PDGF-BB-treated pericytes, whilst Cell Counting Kit-8 and Transwell migration assays were used to investigate cell viability and migration following PDGF-BB pretreatment on SDF-1α-treated pericytes. Exogenous PDGF-BB enhanced CXCR4 and CXCR7 expression through PDGFR-β in a dose- and time-dependent manners. In addition, PDGF-BB increased cell viability and migration in SDF-1α-treated pericytes, which were inhibited by AMD3100 and niclosamide, inhibitors for CXCR4 and STAT3 respectively. Crosstalk between PDGF-BB/PDGFR-β and SDF-1α/CXCR4/CXCR7 were involved in the JAK2/STAT3 signaling pathway. PDGF-BB treatment enhanced CXCR4, CXCR7 and PDGFR-βexpression, which may be associated with the phosphorylation of STAT3. siRNA-PDGFR-β transfection reduced CXCR4 and CXCR7 expression in pericytes. Therefore, PDGF-BB directly targets PDGFR-β and serves an important role in regulating CXCR4 and CXCR7 expression, ultimately affecting viability and migration in SDF-1α-treated pericytes. Therefore, targeting CXCR4/CXCR7 may serve as a potential therapeutic strategy for fundus diseases.

Brivanib, a multitargeted small-molecule tyrosine kinase inhibitor, suppresses laser-induced CNV in a mouse model of neovascular AMD.

In age-related macular degeneration (AMD), choroidal neovascularization (CNV), a major pathologic feature of neovascular AMD (nAMD), affects 10% of patients, potentially causing serious complications, including vision loss. Vascular endothelial growth factor receptor 2 (VEGFR2) and fibroblast growth factor receptor 1 (FGFR1) contribute to the pathogenesis of CNV. Brivanib is an oral selective dual receptor tyrosine kinase (RTK) inhibitor of FGFRs and VEGFRs, especially VEGFR2 and FGFR1. In this study, brivanib inhibited zebrafish embryonic angiogenesis without impairing neurodevelopment. In a mouse CNV model, brivanib intravitreal injection blocked phosphorylation of FGFR1 and VEGFR2 and reduced CNV leakage, area, and formation without causing intraocular toxicity. Moreover, brivanib oral gavage reduced CNV leakage and area. Accordingly, brivanib remained at high concentrations (above 14,000 ng/ml) in retinal/choroidal/scleral tissues following intravitreal injection. Similarly, brivanib remained at high concentrations (over 10,000 ng/ml) in retinal/choroidal/scleral tissues following oral gavage. Finally, in vitro cell experiments demonstrated that brivanib inhibited the proliferation, migration and tube formation of microvascular endothelial cells. In conclusion, our study suggested that brivanib treatment could be a novel therapeutic strategy for nAMD.

En-Face Optical Coherence Tomography Angiography for Longitudinal Monitoring of Retinal Injury.

A customized Optical Coherence Tomography Angiography (OCTA) algorithm and Orthogonal OCT (en-face and B-Scans) were used for longitudinal assessment of retina murine vascular and tissue remodeling comparing photoreceptor ablation and laser-induced Choroidal Neovascularization (CNV). In the mouse model, we utilized a combined OCTA/OCT technique to image and quantify morphological and vascular features of laser lesions over time. This approach enabled us to monitor and correlate the dynamics of retina vascular and tissue remodeling as evidenced by swelling, edema, and scarring. From the OCT B-Scans, three stages of inflammatory progression were identified: the early response occurring within hours to day 3, the transition phase from 3–7 days, and the late stage of 7–21 days entering either the resolving phase or chronic phase, respectively. For the case of CNV, en-face OCTA revealed a transient non-perfusion of inner retina capillaries, specifically Deep Vascular Plexus (DVP), which corresponded to growth in lesions of a height of 200 μm or greater. Non-perfusion first occurred at 24 hours, persisted during edema and CNV formation days 7–14. In contrast, the acute inflammation induced photoreceptor damage, but no detectable alterations to the microvasculature were observed. We demonstrated that the en-face OCTA system is capable of visualizing capillary networks (~5 μm) and the corresponding tissue remodeling and growth dynamics allowing for separating acute injury from CNV. For the first time, by using OCTA we observed the presence of the 5–10 μm capillary non-perfusion present in DVP as part of CNV formation and the associated wound healing in the retina.

Suppression of Choroidal Neovascularization and Fibrosis by a Novel RNAi Therapeutic Agent against (Pro)renin Receptor

The receptor-associated prorenin system refers to the pathogenic mechanism whereby prorenin binding to (pro)renin receptor [(P)RR] dually activates the tissue renin-angiotensin system (RAS) and RAS-independent signaling, and its activation contributes to the molecular pathogenesis of various ocular diseases. We recently developed a new single-stranded RNAi agent targeting both human and mouse (P)RR ((P)RR-proline-modified short hairpin RNA [(P)RR-PshRNA]), and confirmed its therapeutic effect on murine models of ocular inflammation. Here, we investigated the efficacy of (P)RR-PshRNA against laser-induced choroidal neovascularization (CNV) and subretinal fibrosis, both of which are involved in the pathogenesis of age-related macular degeneration (AMD). Administration of (P)RR-PshRNA in mice significantly reduced CNV formation, together with the expression of inflammatory molecules, macrophage infiltration, and extracellular signal-regulated kinase (ERK) 1/2 activation. In addition, (P)RR-PshRNA attenuated subretinal fibrosis, together with epithelial-mesenchymal transition (EMT)-related markers including phosphorylated SMAD2. The suppressive effect of (P)RR-PshRNA is comparable with aflibercept, an anti-vascular endothelial growth factor drug widely used for AMD therapy. AMD patient specimens demonstrated (P)RR co-localization with phosphorylated ERK1/2 in neovascular endothelial cells and retinal pigment epithelial cells. These results indicate that (P)RR contributes to the ocular pathogenesis of both inflammation-related angiogenesis and EMT-driven fibrosis, and that (P)RR-PshRNA is a promising therapeutic agent for AMD.

Optical coherence tomography angiography (OCT-A) in an animal model of laser-induced choroidal neovascularization

Aim of the study was to compare optical coherence tomography angiography (OCT-A) and conventional fluorescein angiography (FA) for quantitative analysis of the retinal and choroidal vasculature in the animal model of laser-induced choroidal neovascularization (CNV). Therefore, Dark Agouti rats underwent argon laser photocoagulation to induce CNV at D0. In vivo imaging using combined confocal scanner laser ophthalmoscopy (cSLO)-based FA and OCT-A (Heidelberg Engineering GmbH, Heidelberg, Germany) was performed before and immediately after laser treatment as well as at day 2, 7, 14 and 21. OCT-A en-face images were compared to cSLO images obtained by conventional FA topographic uptake recorded using a series of different pre-defined focus settings. For a quantitative comparison of CNV imaging by OCT-A and FA, CNV area, vessel density, number of vessel junctions, total vessel length and number of vessel end points were analyzed. Subsequent ex vivo analyses of the CNV included immunofluorescence staining of vessels in retinal and RPE/choroidal/scleral flatmount preparations. We found, that OCT-A allowed for high-resolution non-invasive imaging of the superficial, intermediate and deep retinal capillary plexus as well as the choroidal blood vessels in rats. Compared with OCT-A, visualization of CNV progression by invasive FA was less accurate, in particular the deep vascular plexus was visualized in more detail by OCT-A. The area of neovascularization was mainly detected in the deep retinal vascular plexus, outer nuclear layer (ONL), ellipsoid zone (EZ) and the choroid. Within the laser lesions, signs of CNV formation occurred at day 7 with progression in size and number of small vessels until day 21. Due to leakage and staining effects, CNV areas appeared significantly larger in FA compared to OCT-A images (p ≤ 0.0001 for all tested layers). Vessel density, number of vessel junctions, total vessel length and number of vessel end points were significantly higher in intermediate vascular plexus (IVP) and deep vascular plexus (DVP) in OCT-A compared to FA images. Overall, CNV area in flatmounts was similar to OCT-A results and much smaller compared to the area of dye leakage by FA. This study demonstrates that in vivo OCT-A imaging in small animals is feasible and allows for precise analysis of the formation of new blood vessel formation in the animal model of laser-induced CNV. Given its superior axial resolution, sensitivity and non-invasiveness compared to conventional FA imaging, OCT-A opens the door for a more detailed evaluation of CNV development in such a model and, thus, enables the analysis of the response to novel therapeutic interventions in longitudinal in vivo studies.

Fenofibrate-Loaded Biodegradable Nanoparticles for the Treatment of Experimental Diabetic Retinopathy and Neovascular Age-Related Macular Degeneration.

Fenofibrate is a peroxisome proliferator-activated receptor α (PPARα) agonist and has been shown to have therapeutic effects on diabetic retinopathy (DR). However, the effects of fenofibrate through systemic administration are not as potent as desired due to inefficient drug delivery to the retina. The present study aimed to explore the sustained therapeutic effects of fenofibrate-loaded biodegradable nanoparticles (NP) on both DR and neovascular age-related macular degeneration (AMD). Fenofibrate was successfully encapsulated into poly(lactic- co-glycolic acid) (PLGA) NP (Feno-NP), and Feno-NP were optimized by varying polymer composition to achieve high drug loading and prolonged drug release. The Feno-NP made of PLGA 34 kDa demonstrated a drug content of 6% w/w and a sustained drug release up to 60 days in vitro. Feno-NP (PLGA 34 kDa) was selected for following in vivo studies, and one single intravitreal (IVT) injection of Feno-NP into rat eyes with a 30G fine needle maintained sustained fenofibric acid drug level in the eye for more than 60 days. The efficacy of Feno-NP in DR and neovascular AMD was investigated using streptozotocin (STZ)-induced diabetic rats, laser-induced choroidal neovascularization (CNV) rats, and very low-density lipoprotein receptor knockout ( Vldlr -/-) mice. Therapeutic effects of Feno-NP were evaluated by measuring electroretinogram (ERG), retinal vascular leakage, leukostasis, CNV size, and retinal levels of vascular endothelial growth factor (VEGF) and intracellular adhesion molecule-1 (ICAM-1). In diabetic rats, Feno-NP ameliorated retinal dysfunctions, reduced retinal vascular leakage, inhibited retinal leukostasis, and downregulated the overexpression of VEGF and ICAM-1 at 8 weeks after one IVT injection. In addition, Feno-NP reduced retinal vascular leakage and CNV formation in both CNV rats and Vldlr -/- mice. Moreover, no toxicity of Feno-NP or Blank-NP to retinal structure and function was detected. Feno-NP exhibited good physiochemical characteristics and controlled drug release profile, conferring prolonged beneficial effects on DR and neovascular AMD.

Association of CD11b+ Monocytes and Anti–Vascular Endothelial Growth Factor Injections in Treatment of Neovascular Age-Related Macular Degeneration and Polypoidal Choroidal Vasculopathy

CD11b+ immune cells have been implicated in the formation of choroidal neovascularization in experimental studies on animals and disease-association studies on humans. However, the clinical importance of such observations remains unknown. To investigate whether the proportion of CD11b+ circulating monocytes is associated with the number of anti-vascular endothelial growth factor (anti-VEGF) injections in neovascular age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV). These observational cohort studies collected data from January 1, 2010, through December 31, 2013, and from January 1, 2015, through December 31, 2018. Fresh venous blood samples were acquired for flow cytometric immune studies in patients with neovascular AMD or PCV receiving treatment with aflibercept or ranibizumab as needed for 36 months. Patients (n = 81) without immune diseases were consecutively recruited from a single center in Denmark. Proportion of CD11b+ circulating monocytes. The estimation of the number of intravitreal anti-VEGF injections given at 12, 24, and 36 months by the proportion of CD11b+ circulating monocytes and the correlation between these values. The angiogenic role of CD11b+ circulating monocytes was further evaluated by investigating the expression of the known proangiogenic receptor CCR2. Eighty-one patients were included in the analysis (54% women; mean [SD] age, 76 [7] years). The proportion of CD11b+ monocytes at baseline positively estimated the future number of anti-VEGF injections at 12 (ρ = 0.77; 95% CI, 0.35-0.93; P = .004), 24 (ρ = 0.82; 95% CI, 0.44-0.95; P = .002), and 36 (ρ = 0.78; 95% CI, 0.34-0.94; P = .005) months. This association was also found retrospectively in a larger sample of patients with neovascular AMD at 12 (ρ = 0.46; 95% CI, 0.16-0.68; P = .004), 24 (ρ = 0.49; 95% CI, 0.20-0.70; P = .002), and 36 (ρ = 0.65; 95% CI, 0.41-0.80; P < .001) months and patients with PCV at 12 (ρ = 0.27; 95% CI, -0.28 to 0.68; P = .30), 24 (ρ = 0.60; 95% CI, 0.12-0.85; P = .02), and 36 (ρ = 0.70; 95% CI, 0.27-0.90; P = .005) months, suggesting that this association is not specific to AMD but rather reflects VEGF activity in neovascularization. CD11b+ monocytes highly coexpressed CCR2, an important monocytic marker of proangiogenic activity. Results of this study demonstrated that the proportion of circulating CD11b+ monocytes estimated and correlated with the number of anti-VEGF injections in patients with neovascular AMD and PCV. Additional longitudinal studies are needed to determine whether these findings have clinical relevance to influence treatment algorithms or provide novel targets for medical therapy.

TIMP-3 suppression induces choroidal neovascularization by moderating the polarization of macrophages in age-related macular degeneration

PurposeTo investigate the role of tissue inhibitor of metalloproteinases–3 (TIMP-3) as a key moderator of macrophage polarization in choroidal neovascularization (CNV) lesions of model mice and in bone marrow-derived macrophage (BMDM). MethodWe used siR-TIMP-3 to transfect BMDM and gave an intravitreal injection of siR-TIMP-3 to laser-induced CNV mice model, real time-PCR and western blot were applied for detecting the expressions of TIMP-3 and macrophages’ biomarker. Besides, CNV lesions in different treatment groups of animal model were examined by the optical coherence tomography angiography (OCTA). ResultsOur experimental data showed that lack of TIMP-3 stimulated M2 polarization proved by real time-PCR and western blot in BMDMs and CNV mice model. Moreover, intravitreal injection of siR-TIMP-3 accelerated CNV formation using OCTA, which indicated that TIMP-3 suppression is related to pro-angiogenesis of M2 macrophage. ConclusionWe showed that the absence of TIMP-3 leads to a more pro-angiogenic microenvironment, playing a key role in CNV formation by positively modulating M2 polarization. The role of TIMP-3 in the regulating inflammation and novel therapeutic target of nAMD needs to be further studied.

Preclinical SPECT Imaging of Choroidal Neovascularization in Mice Using Integrin-Binding [99mTc]IDA-D-[c(RGDfK)]2.

Integrin ɑvβ3, an adhesion molecule overexpressed in neovascular endothelial cells, is involved in ocular angiogenesis. Integrin ɑvβ3-binding arginine-glycine-aspartic acid (RGD) peptide has been used to target and visualize new vessels. We explored the use of integrin ɑvβ3-targeted RGD peptide ([99mTc]IDA-D-[c(RGDfK)]2) for in vivo molecular imaging of choroidal neovascularization (CNV). To induce CNV in animals, the right eyes of C57BL/6 mice were treated with retinal argon laser photocoagulation. CNV formation was confirmed on immunohistopathological examination of retinal and choroidal tissues. To explore the association of integrin with angiogenesis, integrin mRNA expression in the retinal and choroidal tissues was measured using real-time reverse transcriptase-polymerase chain reaction. For in vivo imaging, mice were intravenously injected with [99mTc]IDA-D-[c(RGDfK)]2 and single-photon emission computed tomography (SPECT) images of [99mTc]IDA-D-[c(RGDfK)]2 were obtained before laser induction (baseline) and at 1, 3, 7, and 14days post-induction. CNV-induced regional alterations were measured using radiotracer uptake count. Immunohistopathological examination revealed that CNV lesions showed intense fluorescein isothiocyanate (FITC)-D-[c(RGDfK)]2 immunofluorescence, in contrast to the normal retina and choroid. Retinal integrin mRNA expression peaked at day 1 following CNV induction. On SPECT images using [99mTc]IDA-D-[c(RGDfK)]2, the radio-uptake count in eyes with CNV was significantly higher than in normal controls on days 1-7 (all p < 0.05), with a peak at day 3 representing the highest angiogenic activity. Our preclinical data demonstrated that [99mTc]IDA-D-[c(RGDfK)]2 can detect CNV and its associated angiogenesis in an animal model of CNV. SPECT imaging using an integrin ɑvβ3-targeted RGD peptide radiotracer may be a useful tool for in vivo functional molecular imaging of CNV.

Leukotriene B4 promotes neovascularization and macrophage recruitment in murine wet-type AMD models.

Age-related macular degeneration (AMD), a progressive chronic disease of the central retina, is associated with aging and is a leading cause of blindness worldwide. Here, we demonstrate that leukotriene B4 (LTB4) receptor 1 (BLT1) promotes laser-induced choroidal neovascularization (CNV) in a mouse model for wet-type AMD. CNV was significantly less in BLT1-deficient (BLT1-KO) mice compared with BLT1-WT controls. Expression of several proangiogenic and profibrotic factors was lower in BLT1-KO eyes than in BLT1-WT eyes. LTB4 production in the eyes was substantially increased in the early phase after laser injury. BLT1 was highly expressed in M2 macrophages in vitro and in vivo, and ocular BLT1+ M2 macrophages were increased in the aged eyes after laser injury. Furthermore, M2 macrophages were rapidly attracted by LTB4 and subsequently produced VEGF-A- through BLT1-mediated signaling. Consequently, intravitreal injection of M2 macrophages augmented CNV formation, which was attenuated by BLT1 deficiency. Thus, laser-induced injury to the retina triggered LTB4 production and attracted M2 macrophages via BLT1, leading to development of CNV. A selective BLT1 antagonist (CP105696) and 3 LTB4 inhibitors (zileuton, MK-886, and bestatin) reduced CNV in a dose-dependent manner. CP105696 also inhibited the accumulation of BLT1+ M2 macrophages in the laser-injured eyes of aged mice. Together, these results indicate that the LTB4-BLT1 axis is a potentially novel therapeutic target for CNV of wet-type AMD.

Inhibition of TRAF6 alleviates choroidal neovascularization in vivo

Choroidal neovascularization (CNV) is a type of wet age-related macular degeneration (AMD) which is a major cause of blindness in elder patients. Tumor necrosis factor receptor-associated factor 6 (TRAF6) promotes tumor angiogenesis via upregulating the expression of hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF). Additionally, TRAF6 facilitates the inflammatory response in macrophages and microglia. Here, using mouse laser-induced CNV model and TRAF6 siRNA, the study shows that TRAF6 is a critical player in CNV. The expression of TRAF6, HIF-1α, and VEGF increased in the model. TFAF6 siRNA intravitreal (IVT) injection inhibited CNV formation, as well as expression of HIF-1α and VEGF, activation of macrophages and microglia. Together, our data suggest that TFAF6 inhibition reduces CNV formation via down-regulating expression of HIF-1α and VEGF and activation of macrophages and microglia, demonstrating the unique advantages of TRAF6 inhibition in the alleviation of AMD.

Development of an inducible anti-VEGF rAAV gene therapy strategy for the treatment of wet AMD

Vascular endothelial growth factor (VEGF) is a key mediator in the development and progression of choroidal neovascularization (CNV) in patients with wet age-related macular degeneration (AMD). As a consequence, current treatment strategies typically focus on the administration of anti-VEGF agents, such as Aflibercept (Eylea), that inhibit VEGF function. While this approach is largely successful at counteracting CNV progression, the treatment can require repetitive (i.e. monthly) intravitreal injections of the anti-VEGF agent throughout the patient’s lifetime, imposing a substantial financial and medical burden on the patient. Moreover, repetitive injection of anti-VEGF agents over a period of years may encourage progression of retinal and choroidal atrophy in patients with AMD, leading to a decrease in visual acuity. Herein, we have developed a single-injection recombinant adeno-associated virus (rAAV)-based gene therapy treatment for wet AMD that prevents CNV formation through inducible over-expression of Eylea. First, we demonstrate that by incorporating riboswitch elements into the rAAV expression cassette allows protein expression levels to be modulated in vivo through oral supplementation on an activating ligand (e.g. tetracycline). We subsequently utilized this technology to modulate the intraocular concentration of Eylea following rAAV delivery, leading to nearly complete (p = 0.0008) inhibition of clinically significant CNV lesions in an established mouse model of wet AMD. The results shown in this study pave the way for the development of a personalized gene therapy strategy for the treatment of wet AMD that is substantially less invasive and more clinically adaptable than the current treatment paradigm of repetitive bolus injections of anti-VEGF agents.

Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo

choroidal neovascularization (CNV), a characteristic of wet age-related macular degeneration (AMD), causes severe vision loss among elderly patients. TANK-binding kinase 1 (TBK1) is a ubiquitously expressed serine–threonine kinase and is found to induce endothelial cells proliferation, represent a novel mediator of tumor angiogenesis and exert pro-inflammatory effect. However, the role of TBK1 in choroidal neovascularization has not been investigated so far. In this study, we found that the expression of TBK1 and VEGF was up-regulated in RF/6 A cells chemical hypoxia model and laser-induced mouse CNV model. Silencing of TBK1 suppressed the proliferation and tube formation activity of RF/6 A cells. Intravitreal injection of anti-TBK1 monoclonal antibody ameliorates CNV formation. Taken together, these findings exhibit a proangiogenic role for TBK1 via upregulating the expression of VEGF, and may suggest that TBK1 inhibition offers a unique and alternative method for prevention and treatment of AMD.