The IL-23/IL-17 axis promotes the formation of retinal neovascularization by activating the NLRP3 inflammasome in macrophages in an experimental retinopathy mouse model.

Retinal neovascularization (RNV), a pathological process shared among diabetic retinopathy, retinopathy of prematurity and other retinopathies, has been widely studied, but the mechanism remains unclear. In this study, the mechanism by which the interleukin (IL)-23/IL-17 axis regulates RNV in oxygen-induced retinopathy (OIR) model mice and in cell experiments in vitro was characterized. In the retinas of OIR mice, IL-23/IL-17 axis activation was increased and regulated RNV formation, and this effect was accompanied by increased macrophage recruitment and nucleotide-binding domain leucine-rich repeat and pyrin domain containing receptor 3 (NLRP3) inflammasome activation. Moreover, inhibiting the IL-23/IL-17 axis reduced the number of macrophage and the expression and activation of NLRP3 inflammasome. On the other hand, recombinant (r) IL-23p19 and rIL-17A promoted the expression and activation of NLRP3 inflammasome, and the proliferation and migration of macrophages. Furthermore, macrophage elimination decreased the activation of IL-23/IL-17 axis and the expression and activation of NLRP3 inflammasome. In summary, our experiments showed that the IL-23/IL-17 axis promoted the formation of RNV by activating the NLRP3 inflammasome in retinal macrophages of an OIR mouse model.

Etanercept as a TNF-alpha inhibitor depresses experimental retinal neovascularization.

PURPOSE: The formation of retinal neovascularization (RNV) is the primary pathological process underlying retinopathy of prematurity (ROP). Previous studies have shown that inflammatory factors are related to the formation of RNV. Tumor necrosis factor-α (TNF-α), as an important factor in the inflammatory response, is involved in the regulation of RNV formation. However, the mechanism through which TNF-α inhibition reduces RNV formation is not fully clarified. Therefore, the purpose of this study was to explore the effect of etanercept, an inhibitor of TNF-α, on RNV, and its possible mechanism. METHODS: In vivo, an oxygen-induced retinopathy (OIR) mouse model was used to determine the effect of etanercept on the formation of RNV by performing immunostaining. The effect of etanercept on tumor necrosis factor receptor-associated factor 2 (TRAF2), pro-angiogenic-related factors, and pro/anti-inflammatory factors in OIR mice was assessed by real-time PCR and Western blotting. In vitro, the effect of etanercept on TNF-α-induced human retinal microvascular endothelial cell tube formation was evaluated by tube formation assays, and the potential mechanism of etanercept was explored by Western blotting. RESULTS: In vivo, etanercept reduced the area of RNV and decreased the expression of TRAF2 in the OIR mouse model. Etanercept also suppressed the expression of several pro-angiogenic factors and regulated the pro/anti-inflammatory factors. In vitro, etanercept reduced endothelial cell tube formation by inhibiting activation of the NF-κB signaling pathway. CONCLUSION: Etanercept can regulate pro/anti-inflammatory factors and reduce the expression of pro-angiogenic factors by inhibiting NF-κB phosphorylation, thereby reducing RNV formation.

Neutralization of Bombina variegata peptide 8 suppresses retinal neovascularization in two different murine models: The oxygen-induced retinopathy model and the rhodopsin promoter/VEGF transgenic mouse model.

Bombina variegata 8 (Bv8), also known as prokineticin-2 (PK-2), is a potent pro-angiogenic factor. However, its role in retinal neovascularization (RNV) remains unknown. In this study, we explored the role of Bv8 in the pathogenesis of RNV. We found that the expression of Bv8 was significantly increased in two different models of retinal neovascularization: the oxygen-induced retinopathy (OIR) mouse model and the rhodopsin promoter (rho)/VEGF transgenic mouse model. Neutralization of Bv8 by intravitreal injections of its antibody, not only inhibited retinal and subretinal neovascularization but also decreased the mRNA and protein levels of several pro-angiogenic factors. Our in vitro assay showed that recombinant human Bv8 (RhBv8) protein promoted human retinal microvascular endothelial cells (HRECs) tube-formation, cell proliferation and vascular endothelial growth factor receptor 1 (VEGFR1) and receptor 2 (VEGFR2) expression. Our findings suggest that Bv8 could be used as a novel target for the treatment of RNV-related ocular diseases.

Inhibition of integrin α5ß1 ameliorates VEGF-induced retinal neovascularization and leakage by suppressing NLRP3 inflammasome signaling in a mouse model.

PURPOSE: To assess the effect of inhibiting integrin α5ß1 by ATN-161 on vascular endothelial growth factor (VEGF)-induced neovascularization (NV) and leakage causing retinal detachment in adult Tet/opsin/VEGF transgenic mice, and characterize the underlying mechanism of its function. METHOD: Retinas from adult Tet/opsin/VEGF transgenic mice and human retinal endothelial cells (HRECs) exposed to VEGF (treated with ATN-161 or PBS) were used to carry out immunofluorescence, RT-PCR and western blot to examine expression levels of integrin α5ß1 and the NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome. Retinal frozen section analysis was used to assess NV and leakage causing retinal detachment. RESULTS: In comparison to normal-treated mice, doxycycline-treated Tet/opsin/VEGF transgenic mice showed severe retinal detachment and higher integrin α5ß1 expression. Furthermore, the retinal detachment was inhibited significantly by ATN-161. Additionally, ATN-161 treatment was associated with a conspicuous reduction in NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), cleaved caspase-1, and mature interleukin-1ß expression levels in the retinas of Tet/opsin/VEGF transgenic mice treated with doxycycline as well as in HRECs exposed to VEGF. CONCLUSION: ATN-161, an antagonist of integrin α5ß1, is a promising treatment for retinal neovascularization (RNV), and its retinal protection role appears to take effect through inhibition of NLRP3 inflammasome activity.

ATN-161 as an Integrin α5ß1 Antagonist Depresses Ocular Neovascularization by Promoting New Vascular Endothelial Cell Apoptosis.

BACKGROUND ATN-161 (Ac-PHSCN-NH2), an antagonist of integrin α5ß1, has shown an important influence in inhibiting tumor angiogenesis and metastasis of other tumor types. However, the mechanism of action of ATN-161 and whether it can inhibit ocular neovascularization (NV) are unclear. This study investigated the role of ATN-161 in regulating ocular angiogenesis in mouse models and explored the underlying signaling pathway. MATERIAL AND METHODS An oxygen-induced retinopathy (OIR) mouse model and a laser-induced choroidal neovascularization (CNV) mouse model were used to test integrin a5b1 expression and the effect of ATN-161 on ocular NV by immunofluorescence staining, Western blot analysis, and flat-mount analysis. The activation of nuclear factor-κB (NF-κB), matrix metalloproteinase-2/9 (MMP-2/9), and cell apoptosis were detected by immunofluorescence staining, Western blot, real-time RT-PCR, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). The cell proliferation was detected by BrdU labeling. RESULTS In OIR and CNV mice, the protein expression level of integrin α5ß1 increased compared with that in age-matched controls. The mice given ATN-161 had significantly reduced retinal neovascularization (RNV) and CNV. Blocking integrin a5b1 by ATN-161 strongly inhibited nuclear factor-κB (NF-κB) activation and matrix metalloproteinase-2/9 (MMP-2/9) expression and promoted cell apoptosis, but the effect of ATN-161 on proliferation in CNV mice was indirect and required the inhibition of neovascularization. Inhibiting NF-κB activation by ammonium pyrrolidinedithiocarbamate (PDTC) reduced RNV and promoted cell apoptosis in ocular NV. CONCLUSIONS Blocking integrin α5ß1 by ATN-161 reduced ocular NV by inhibiting MMP-2/MMP-9 expression and promoting the cell apoptosis of ocular NV.

Interleukin-17A neutralization alleviated ocular neovascularization by promoting M2 and mitigating M1 macrophage polarization.

Neovascularization (NV), as a cardinal complication of several ocular diseases, has been intensively studied, and research has shown its close association with inflammation and immune cells. In the present study, the role of interleukin-17A (IL-17A) in angiogenesis in the process of ocular NV both in vivo and in vitro was investigated. Also, a paracrine role of IL-17A was demonstrated in the crosstalk between endothelial cells and macrophages in angiogenesis. In the retinas of mice with retinopathy of prematurity, the IL-17A expression increased significantly at postnatal day 15 (P15) and P18 during retinal NV. Mice given IL-17A neutralizing antibody (NAb) developed significantly reduced choroidal NV and retinal NV. Studies on vascular endothelial growth factor (VEGF) over-expressing mice suggested that IL-17A modulated NV through the VEGF pathway. Furthermore, IL-17A deficiency shifted macrophage polarization toward an M2 phenotype during retinal NV with significantly reduced M1 cytokine expression compared with wild-type controls. In vitro assays revealed that IL-17A treated macrophage supernatant gave rise to elevated human umbilical vascular endothelial cell proliferation, tube formation and VEGF receptor 1 and receptor 2 expression. Therefore, IL-17A could potentially serve as a novel target for treating ocular NV diseases. The limitation of this study involved the potential mechanisms, such as which transcription accounted for macrophage polarization and how the subsequent cytokines were modulated when macrophages were polarized. Further studies need to be undertaken to definitively determine the extent to which IL-17A neutralizing anti-angiogenic activity depends on macrophage modulation compared with anti-VEGF treatment.

Neutralization of IL-23 depresses experimental ocular neovascularization.

Interleukin-23 (IL-23) is a heterodimeric cytokine that consists of p19, a novel subunit, and p40, which is shared by IL-12. IL-23 has been demonstrated to play an important role in autoimmunity and tumor growth. However, the role of IL-23 in ocular neovascularization (NV) diseases remains unclear. In this study, we explored the role of IL-23 in the processing of retinal and choroidal neovascularization (RNV and CNV). We found a significantly higher expression of IL-23 in the retinas with oxygen-induced retinopathy (OIR), and after neutralizing IL-23, the mRNA and protein levels of the angiogenic factors vascular endothelial growth factor receptor (VEGFR)1/FLT-1, VEGFR2/FLK-1, placental growth factor (PIGF), endothelial-specific receptor tyrosine kinase (Tie2), inducible nitric-oxide synthase (iNOS), matrix metalloproteinase (MMP) 2 and MMP9 were significantly down regulated, while the opposite trend was found for the anti-angiogenic molecules chemokine (C-X-C motif) ligand (CXCL) 9 and CXCL10. IL-23 blockade caused less NV in both the RNV and CNV mouse models. In addition, our in vitro assay showed that IL-23 alone is able to increase the ability of endothelial cells to form tubes. Our findings suggest that targeting IL-23 could be a potential therapy for RNV and CNV diseases.

Inhibiting NF-κB Signaling Activation Reduces Retinal Neovascularization by Promoting a Polarization Shift in Macrophages.

Purpose: Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling is involved in regulating tumor angiogenesis and metastasis; however, the exact mechanism of action in retinal neovascularization (RNV) remains unclear. The purpose of this study was to determine the role and underlying mechanism of NF-κB in regulating RNV in retinal neovascularization mice. Methods: Expression levels of NF-κB signaling were detected by immunofluorescence staining and western blotting in retinas of oxygen-induced retinopathy (OIR) mice. OIR mice were treated with either pyrrolidinedithiocarbamate (PDTC), a NF-κB signaling inhibitor, or PBS, and retinal flat-mounts were performed to quantify the area of RNV and the recruitment of retinal macrophages by immunofluorescence staining. Macrophage polarization detected by flow cytometric analysis and the expression of macrophage polarization-associated genes were evaluated by immunofluorescence staining, quantitative RT-PCR, and western blotting. Results: Expression levels of phosphorylated IκBα (p-IκBα) and p-p65 increased in OIR mice. Inhibiting NF-κB signaling activation by PDTC significantly reduced RNV. After treatment with PDTC, a reduction in the quantity of macrophages was observed: M1 polarized macrophages decreased, and M2 polarized macrophages increased; the expression of M1 macrophage-associated cytokines decreased and M2 macrophage-associated cytokines increased in the retinas of OIR mice. Conclusions: Blocking activation of NF-κB signaling reduces RNV by promoting polarization of M1 macrophages to M2 macrophages in OIR mice.

Inhibiting the NLRP3 inflammasome with MCC950 ameliorates retinal neovascularization and leakage by reversing the IL-1ß/IL-18 activation pattern in an oxygen-induced ischemic retinopathy mouse model.

Activation of the nucleotide-binding domain leucine-rich repeat and pyrin domain containing receptor 3 (NLRP3) inflammasome plays an important role in ocular neovascularization. In our study, we found that the expression and activation levels of NLRP3 inflammasome components, including NLRP3, an apoptosis-associated speck-like protein (ASC) containing caspase activation and recruitment domain (CARD) and caspase-1 (CAS1), were significantly upregulated. In addition, we found interleukin (IL)-1ß activity increased while IL-18 activity decreased in the retinas of oxygen-induced ischemic retinopathy (OIR) mice. MCC950, an inhibitor of NLRP3, reversed the IL-1ß/IL-18 activation pattern, inhibited the formation of retinal neovascularization (RNV), decreased the number of acellular capillaries and reduced leakage of retinal vessels. Moreover, MCC950 could regulate the expression of endothelial cell- and pericyte function-associated molecules, such as vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)1, VEGFR2, matrix metalloproteinase (MMP)2, MMP9, tissue inhibitor of metalloproteinases (TIMP)1, TIMP2, platelet-derived growth factor receptor-ß (PDGFR-ß), platelet-derived growth factor-B (PDGF-B), and angiopoietin2 (Ang2). In vitro, recombinant human (r)IL-18 and rIL-1ß regulated the expression of endothelial cell- and pericyte function-associated molecules and the proliferation and migration of endothelial cells and pericytes. We therefore determined that inhibiting the NLRP3 inflammasome with MCC950 can regulate the function of endothelial cells and pericytes by reversing the IL-1ß/IL-18 activation pattern to ameliorate RNV and leakage; thereby opening new avenues to treat RNV-associated ocular diseases.

Endocan Blockade Suppresses Experimental Ocular Neovascularization in Mice.

Purpose: Ocular neovascularization (NV) is a pathologic process characterized by the proliferation and infiltration of various types of cells such as RPE, glial, and endothelial cells, which interact with proangiogenic factors and inflammatory cytokines. Endocan is known to be enriched in retinal endothelial tip cells under hypoxia, but the effect of endocan on ocular NV progression is largely unknown. In this study, we investigated the role of endocan in the ocular NV pathologic process and the possible mechanisms involved. Methods: In the eyes of mice with oxygen-induced retinopathy (OIR); choroidal NV (CNV); and rhodopsin promoter (rho)/VEGF transgenic mice, endocan expression was assessed by quantitative real-time PCR (RT-PCR) and Western blot. In vivo, a specific functional antibody was used to neutralize endocan and ocular NV levels were evaluated by RT-PCR, Western blot and immunostaining of flat-mounts. In vitro, the effect of endocan on human retinal microvascular endothelial cell (HREC) tube formation was observed using a routine method. Results: Endocan was significantly elevated in these three experimental mice models. Endocan blockade with the neutralizer intravitreal injection not only suppressed the area of retinal, choroidal and subretinal NV, but also resulted in a decrease in several angiogenesis-associated molecules. Recombinant endocan protein (rhEndocan) was found to induce tube formation on HRECs directly. Conclusions: The current data suggest that endocan is a potential therapeutic or an additional target for retinal and subretinal NV diseases.

PEDF mediates pathological neovascularization by regulating macrophage recruitment and polarization in the mouse model of oxygen-induced retinopathy.

Macrophages have been demonstrated to play a proangiogenic role in retinal pathological vascular growth. Pigment epithelium-derived factor (PEDF) works as a powerful endogenous angiogenesis inhibitor, but its role in macrophage recruitment and polarization is largely unknown. To explore the underlying mechanisms, we first evaluated macrophage polarization in the retinas of the oxygen-induced retinopathy (OIR) mouse model. Compared to that in normal controls, M1- and M2-like macrophages were all abundantly increased in the retinas of OIR mice. In addition, both M1 and M2 subtypes significantly promoted neovascularization in vitro and in vivo. In addition, we found that PEDF inhibited retinal neovascularization by dampening macrophage recruitment and polarization. Furthermore, PEDF inhibited macrophage polarization through adipose triglyceride lipase (ATGL) by regulating the activation of MAPKs and the Notch1 pathway, as we found that the phosphorylation of MAPKs, including p38MAPK, JNK and ERK, as well as the accumulation of Notch1 were essential for hypoxia-induced macrophage polarization, while PEDF significantly dampened M1 subtype-related iNOS and M2 subtype-related Arg-1 expression by inhibiting hypoxia-induced activation of Notch1 and MAPKs through ATGL. These findings reveal a protective role of PEDF against retinal neovascularization by regulating macrophage recruitment and polarization.

Identification of different macrophage subpopulations with distinct activities in a mouse model of oxygen-induced retinopathy.

The aim of the present study was to characterize the phenotypic shift, quantity and role changes in different subgroups of retinal macrophages in a mouse model of oxygen-induced retinopathy (OIR). The mRNA expression levels of macrophage M1 and M2 subgroup marker genes and polarization-associated genes were analyzed by RT-qPCR. The number of M1 and M2 macrophages in our mouse model of OIR was analyzed by flow cytometry at different time points during the progression of OIR. Immunofluorescence whole mount staining of the retinas of mice with OIR was performed at different time points to examine the influx of macrophages, as well as the morphological characteristics and roles of M1 and M2 macrophages. An increased number of macrophages was recruited during the progression of angiogenesis in the retinas of mice with OIR due to the pro-inflammatory microenvironment containing high levels of cell adhesion and leukocyte transendothelial migration molecules. RT-qPCR and flow cytometric analysis at different time points revealed a decline in the number of M1 cells from a significantly high level at post-natal day (P)13 to a relatively normal level at P21, as well as an increase in the number of M2 cells from P13 to P21 in the mice with OIR, implicating a shift of macrophage polarization towards the M2 subtype. Immunofluorescence staining suggested that the M1 cells interacted with endothelial tip cells at the vascular front, while M2 cells embraced the emerging vessels and bridged the neighboring vessel sprouts. Thus, our data indicate that macrophages play an active role in OIR by contributing to the different steps of neovascularization. Our findings indicate that tissue macrophages may be considered as a potential target for the anti-angiogenic therapy of ocular neovascularization disease.