Adipocytes influence choroidal neovascularization via PRDM16

Vascular endothelial growth factor (VEGF) signaling via VEGF receptor 2 (VEGFR2) plays a crucial role in pathologic ocular neovascularization. In this study, we investigated the antiangiogenic effect of apatinib, a pharmacologic inhibitor of VEGFR2 tyrosine kinase, against oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) in mice. Western blotting and in vitro angiogenesis assays were performed using human retinal microvascular endothelial cells (HRMECs). OIR was induced in neonatal mice by exposure to 75% oxygen from postnatal day (P) 7 to P12 and to room air from P12 to P17. Experimental CNV was induced in mice using laser photocoagulation. Apatinib was intravitreally and orally administered to mice. Neovascularization and phosphorylation of VEGFR2 were evaluated by immunofluorescence staining. Apatinib inhibited VEGF-mediated activation of VEGFR2 signaling and substantially reduced VEGF-induced proliferation, migration, and cord formation in HRMECs. A single intravitreal injection of apatinib significantly attenuated retinal or choroidal neovascularization in mice with OIR or laser injury-induced CNV, respectively. Retinal or choroidal tissues of the eyes treated with apatinib exhibited substantially lower phosphorylation of VEGFR2 than those of controls injected with vehicle. Intravitreal injection of apatinib did not cause noticeable ocular toxicity. Moreover, oral administration of apatinib significantly reduced laser-induced CNV in mice. Our study demonstrates that apatinib inhibits pathologic ocular neovascularization in mice with OIR or laser-induced CNV. Apatinib may, therefore, be a promising drug for the prevention and treatment of ischemia-induced proliferative retinopathy and neovascular age-related macular degeneration.

Role of interleukin 33 in neovascular age-related macular degeneration

The present study investigated the interactions among the complement membrane attack complex (MAC), CCL2, and VEGF that occur in vivo during the development of choroidal neovascularization (CNV). We first investigated the sequential expression of MAC, CCL2, and VEGF during laser-induced CNV in C57BL/6 mice. Increased MAC deposition was detected at 1 h, CCL2 increased at 3 h, and VEGF was up-regulated at day 3 post-laser treatment. These results suggested that during laser-induced CNV, MAC, CCL2 and VEGF are formed and/or expressed in the following order: MAC → CCL2 → VEGF. To determine the cross-talk between MAC, CCL2, and VEGF during laser-induced CNV, neutralizing antibodies were injected both systemically and locally to block the bioactivity of each molecule. Blocking MAC formation inhibited CCL2 and VEGF expression and also limited CNV formation, whereas neutralization of CCL2 bioactivity did not affect MAC deposition; however, it reduced VEGF expression and CNV formation. When bioactivity of VEGF was blocked, CNV formation was significantly inhibited, but MAC deposition was not affected. Together, our results demonstrate that MAC is an upstream mediator and effect of MAC on the development of laser-induced CNV can be attributed to its direct effect on VEGF as well as its effect on VEGF that is mediated by CCL2. Understanding the interplay between immune mediators is critical to gain insight into the pathogenesis of CNV.

Interleukin-6 Receptor-Mediated Activation of Signal Transducer and Activator of Transcription-3 (STAT3) Promotes Choroidal Neovascularization

PurposeAnti-VEGF resistance represents a major unmet clinical need in the management of choroidal neovascularization (CNV). We have previously reported that a combination of AIBP, apoA-I, and an anti-VEGF antibody overcomes anti-VEGF resistance in laser-induced CNV in old mice in prevention experiments. The purpose of this work is to conduct a more clinically relevant study to assess the efficacy of the combination of AIBP, apoA-I, and aflibercept in the treatment of anti-VEGF resistance of experimental CNV at different time points after laser photocoagulation.MethodsTo understand the pathobiology of anti-VEGF resistance, we performed comprehensive examinations of the vascular morphology of laser-induced CNV in young mice that are highly responsive to anti-VEGF treatment, and in old mice that are resistant to anti-VEGF therapy by indocyanine green angiography (ICGA), fluorescein angiography (FA), optical coherence tomography (OCT), and Alexa 568 isolectin labeled choroid flatmounts. We examined the efficacy of the combination therapy of AIBP, apoA-I, and aflibercept intravitreally delivered at 2, 4, and 7 days after laser photocoagulation in the treatment of CNV in old mice.ResultsLaser-induced CNV in young and old mice exhibited cardinal features of capillary and arteriolar CNV, respectively. The combination therapy and the aflibercept monotherapy were equally effective in treating capillary CNV in young mice. In old mice, the combination therapy was effective in treating anti-VEGF resistance by potently inhibiting arteriolar CNV, whereas aflibercept monotherapy was ineffective.ConclusionsCombination therapy of AIBP, apoA-I, and aflibercept overcomes anti-VEGF resistance in experimental CNV in old mice by inhibiting arteriolar CNV.

Purpose: Mononuclear phagocytes (MNPs) are present in neovascular age-related macular degeneration (nv AMD) which is also called choroidal neovascularization (CNV). The number and phenotype of the MNPs depend upon the local environment in the CNV and effect of nv AMD therapy. We investigated ocular cell infiltration and conditions that modulate angiogenesis in a laser-induced mouse CNV model.Methods: We developed assays to quantify MNPs in our established mouse CNV model. One MNP assay quantified the number of subretinal cells peripheral to the CNV lesions. A second assay semiquantitatively assesses the number of MNPs localized to the CNV lesion. We used these assays to measure the effect of toll-like receptor-2 (TLR-2) activation, anti-vascular endothelial growth factor (VEGF) therapy, and chemokine (C-C motif) ligand 2 (Ccl2) genetic deletion on MNP infiltration after laser injury.Results: Laser injury induced blood vessel growth and infiltration of MNPs. Systemic administration of a TLR-2 activating peptide increased laser-induced CNV area, MNP cell numbers, and MNP density over the CNV lesions. Systemic administration of a VEGF antibody reduced CNV area, while Ccl2 genetic deletion increased CNV area. Despite the change in amount of angiogenesis, MNP infiltration was, surprisingly, unchanged in these 2 conditions.Conclusions: MNP quantification provides biological insights for candidate AMD therapies. The number of infiltrating MNP cells does not correlate with the amount of laser-induced CNV area.

Novel CCR3 Antagonists Are Effective Mono- and Combination Inhibitors of Choroidal Neovascular Growth and Vascular Permeability

To evaluate the anti-angiogenic effects of integrin alphavbeta3 specific antagonist BS-1417 on laser-induced choroidal neovascularization (CNV) in mice. Male C57BL/6 mice were treated with daily intraperitoneal injections of BS-1417 or saline starting at the onset (day 0) of experiments. CNV was induced by laser photocoagulation the next day. Fluorescein angiograms (FA) and choroidal flatmount FITC-dextran perfusion were performed on experimental day 8. Histological and immunohistochemical examinations were performed with consecutive cryosections. Sub-confluent human vascular endothelial cells (HUVEC) were grown in vitro under various concentrations (0-10 microg/ml) of BS-1417 and the numbers of cell were measured at 48 hours of incubation. After fixation, immunocytochemistry was performed. BS-1417 significantly decreased the area of dye leakage determined by FA (40% of control, p = 0.0008) and reduced the CNV size found on choroidal flatmount (18% of control, p = 0.007). In histological findings, BS-1417 apparently suppressed the size of laser-induced CNV. Immunoreactivities for VEGF and integrin alphav were remarkably attenuated with BS-1417 compared to control. BS-1417 inhibited the growth and VEGF expression of HUVEC in vitro. The integrin alphavbeta3 may play a key role in the induction of laser-induced CNV. The antagonists for integrin alphavbeta3 may have therapeutic effects in CNV associated diseases such as age-related macular degenerations.

Pathological angiogenesis is a major cause of irreversible blindness in individuals with neovascular age-related macular degeneration (nAMD). Macrophages and microglia (MΦ) contribute to aberrant ocular angiogenesis. However, the role of glucose metabolism of MΦ in nAMD is still undefined. Here, we have investigated the involvement of glycolysis, driven by the kinase/phosphatase PFKFB3, in the development of choroidal neovascularization (CNV). CNV was induced in mice with laser photocoagulation. Choroid/retinal pigment epithelium (RPE) complexes and MΦ were isolated for analysis by qRT-PCR, western blot, flow cytometry, immunostaining, metabolic measurements and angiogenesis assays. MΦ accumulated within the CNV of murine nAMD models and expressed high levels of glycolysis-related enzymes and M1/M2 polarization markers. This phenotype of hyper-glycolytic and activated MΦ was replicated in bone marrow-derived macrophages stimulated by necrotic RPE in vitro. Myeloid cell-specific knockout of PFKFB3, a key glycolytic activator, attenuated pathological neovascularization in laser-induced CNV, which was associated with decreased expression of MΦ polarization markers and pro-angiogenic factors, along with decreased sprouting of vessels in choroid/RPE complexes. Mechanistically, necrotic RPE increased PFKFB3-driven glycolysis in macrophages, leading to activation of HIF-1α/HIF-2α and NF-κB, and subsequent induction of M1/M2 markers and pro-angiogenic cytokines, finally promoting macrophage reprogramming towards an angiogenic phenotype to facilitate development of CNV. The PFKFB3 inhibitor AZ67 also inhibited activation of HIF-1α/HIF-2α and NF-κB signalling and almost completely prevented laser-induced CNV in mice. Modulation of PFKFB3-mediated macrophage glycolysis and activation is a promising strategy for the treatment of nAMD.

PurposeThe purpose of this study was to evaluate the pathological involvement of erythropoietin (EPO) in experimental choroidal neovascularization (CNV) and its association with neovascular age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV) in the Chinese population.MethodsTreatment effect of recombinant EPO protein were assessed by human umbilical vein endothelial cell (HUVEC) proliferation, migration, and tube formation, and ex vivo choroid-sprouting ability. The effect of intravitreal injection of Epo siRNA against neovascularization was evaluated in the laser-induced CNV mouse model. In addition, the association of EPO variants with neovascular AMD and PCV was determined.ResultsExogenous supplementation of EPO significantly enhanced the migration and tube formation of HUVECs and promoted ex vivo choroid sprouting in mouse retinal pigment epithelium (RPE)-choroid-sclera complex culture. In the experimental CNV mouse model, Epo expression was found to be significantly upregulated by 3.5-folds in RPE-choroid-sclera complex at day 10 after laser induction as compared to the baseline. Immunofluorescence analysis showed that Epo was mainly expressed around the vascular endothelial cells in the RPE-choroid-sclera complex. Intravitreal injection of siRNA targeting Epo reduced 40% Epo expression and 40% CNV lesion areas as compared to the scramble control. However, EPO variants were not associated with neovascular AMD nor PCV in the Chinese population.ConclusionsThis study revealed the promotion of human endothelial cell tube formation in vitro and choroid sprouting ex vivo by EPO, and the reduction of laser-induced CNV in vivo by Epo RNA interference.Translational RelevanceTargeting EPO could be a potential additional treatment for CNV-related diseases.

Quantitative Optical Coherence Tomography Angiography of Choroidal Neovascularization in Age-Related Macular Degeneration

To investigate therapeutic effects of traditional Chinese medicine formulations, Hexuemingmu (HXMM) on laser-induced choroidal neovascularization (CNV) and follow-up effect in mice. C57BL/6 mice of 8-week-old were used and CNV was induced with 577 nm laser photocoagulation. Animals were randomly divided into groups and different doses of HXMM were administered daily. One, four, and eight weeks after the intervention, the electroretinogram (ERG), fundus fluorescence angiography, choroidal flat mount and immunofluorescence staining were preformed to evaluate the function and CNV formation. The expression levels of angiogenic proteins were determined by Western blotting and immunofluorescence staining. An analysis of variance and Kruskal-Wallis test were used to test the differences among the groups. The results showed that HXMM effectively increased amplitude of ERG of mice (P<0.05), alleviated fundus CNV leakage (P<0.05), and reduced the area of neovascularization and the expression of angiogenic proteins (P<0.05) after laser-induced CNV. HXMM can protect the retinal function of mice after laser-induced CNV, and inhibit the CNV development.

Laser-induced choroidal neovascularization (CNV) is a widely used model to mimic many features of CNV resulting from wet AMD. Macrophages have been implicated in the pathogenesis of AMD. Class A scavenger receptors, scavenger receptor-A (SR-A) and macrophage receptor with collagenous domain (MARCO), are expressed on macrophages and are associated with macrophage function. The goal of this study is to examine the role of macrophage scavenger receptors in immune cell recruitment and the formation of CNV. Laser photocoagulation was performed in wild-type and knockout mice with deletion of SR-A (SR-A(-/-)), MARCO (MARCO(-/-)), or both SR-A and MARCO double knockout (DKO). Immune cell recruitment at different time points and CNV lesions at 14 days after laser treatment were evaluated through immunostaining and confocal microscopy. Microarray analysis was performed in eyes 1 day after laser injury. Wild-type eyes showed higher chemokine/receptor expression compared with knockout eyes after laser injury. Scavenger receptor deficiency markedly impaired the recruitment of neutrophils and macrophages to CNV lesions at 1- and 3-days post laser injury, respectively. Significantly reduced CNV volumes were found in the eyes from scavenger receptor knockout mice compared with wild-type mice. The deficiency of scavenger receptors impairs the formation of CNV and immune cell recruitment. Our findings suggest a potential role for scavenger receptors in contributing to CNV formation and inflammation in AMD.

Nintedanib inhibits neovascularization and subretinal fibrosis in a laser-induced choroidal neovascularization mouse model.

ObjectiveChoroidal neovascularization (CNV) is a major cause of vision loss in retinal diseases such as age-related macular degeneration (AMD). Previously, we demonstrated that semaphorin3A (Sema3A), which is a chemorepellent guidance molecule, inhibited the formation of retina neovascularization. In the present study, we investigated the antiangiogenic effects of Sema3A on transforming growth factor beta (TGF-β) in vitro and in vivo.MethodsEnzyme-linked immunosorbent assays (ELISAs) were used to measure the TGF-β levels in the vitreous humor of patients with AMD and controls. Human umbilical vein endothelial cells (HUVECs) were used for the in vitro study, and a laser-induced CNV mouse model was prepared for the in vivo study. The HUVECs were incubated with TGF-β and Sema3A. The proliferation, migration, apoptosis, and tube formation of the cells were then measured using BrdU, Transwell, flow cytometry, and Matrigel assays, respectively, and the SMAD2/3 signaling pathways were analyzed using western blot analysis. The C57BL/6J mouse retina was exposed to a laser to induce choroidal neovascularization (CNV), and Sema3A was injected intravitreously. After 14 days, fundus fluorescein angiography was performed to evaluate the leakage area of the CNV. The vascular endothelial growth factor (VEGF) and TGF-β concentrations in the retina-choroid complex were measured with ELISA. Components of the p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase-1/2 (ERK1/2), c-Jun NH2-terminal kinase (JNK), and SMAD2/3 signaling pathways in the Sema3A-treated groups were analyzed using western blotting.ResultsIn this study, we first verified that the vitreous TGF-β level was higher in patients with neovascular AMD than in the controls. We also showed that Sema3A inhibited TGF-β-induced HUVEC proliferation, migration, and tube formation and inhibited the downstream SMAD2/3 signaling pathway. Sema3A also induced TGF-β-stimulated HUVEC apoptosis and inhibited the response of TGF-β in vitro. In vivo, the TGF-β level was increased in the CNV mouse model. Sema3A not only inhibited laser-induced CNV formation but also inhibited the uptake of VEGF and TGF-β. In the western blot analysis, Sema3A was shown to inhibit the phosphorylation of p38 MAPK, ERK1/2, and JNK and to inhibit the SMAD2/3 signaling pathway after Sema3A treatment in CNV mice.ConclusionsSema3A can be applied as a useful, adjunctive therapeutic strategy for preventing CNV formation.