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

Objective: 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. Methods: 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. Results: 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. Conclusion: 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.

Transcriptomic analysis of choroidal neovascularization reveals dysregulation of immune and fibrosis pathways that are attenuated by a novel anti-fibrotic treatment.

Neovascular AMD (nAMD) leads to vision loss and is a leading cause of visual impairment in the industrialised world. Current treatments that target blood vessel growth have not been able to treat subretinal fibrosis and nAMD patients continue to lose vision. The molecular mechanisms involved in the development of fibrotic lesions in nAMD are not well understood. The aim of this study was to further understand subretinal fibrosis in the laser photocoagulation model of choroidal neovascularization (CNV) by studying the whole transcriptome of the RPE/choroid following CNV and the application of an anti-fibrotic following CNV. Seven days after laser induced CNV, RPE and choroid tissue was separated and underwent RNAseq. Differential expression analysis and pathway analysis revealed an over representation of immune signalling and fibrotic associated pathways in CNV compared to control RPE/choroid tissue. Comparisons between the mouse CNV model to human CNV revealed an overlap in upregulated expression for immune genes (Ccl2, Ccl8 and Cxcl9) and extracellular matrix remodeling genes (Comp, Lrcc15, Fndc1 and Thbs2). Comparisons between the CNV model and other fibrosis models showed an overlap of over 60% of genes upregulated in either lung or kidney mouse models of fibrosis. Treatment of CNV using a novel cinnamoyl anthranilate anti-fibrotic (OCX063) in the laser induced CNV model was selected as this class of drugs have previously been shown to target fibrosis. CNV lesion leakage and fibrosis was found to be reduced using OCX063 and gene expression of genes within the TGF-beta signalling pathway. Our findings show the presence of fibrosis gene expression pathways present in the laser induced CNV mouse model and that anti-fibrotic treatments offer the potential to reduce subretinal fibrosis in AMD.

The natural product vioprolide A exerts anti-inflammatory actions through inhibition of its cellular target NOP14 and downregulation of importin-dependent NF-ĸB p65 nuclear translocation.

Chronic inflammation is characterized by persisting leukocyte infiltration of the affected tissue, which is enabled by activated endothelial cells (ECs). Chronic inflammatory diseases remain a major pharmacotherapeutic challenge, and thus the search for novel drugs and drug targets is an ongoing demand. We have identified the natural product vioprolide A (vioA) to exert anti-inflammatory actions in vivo and in ECs in vitro through inhibition of its cellular target nucleolar protein 14 (NOP14). VioA attenuated the infiltration of microglia and macrophages during laser-induced murine choroidal neovascularization and the leukocyte trafficking through the vascular endothelium in the murine cremaster muscle. Mechanistic studies revealed that vioA downregulates EC adhesion molecules and the tumor necrosis factor receptor (TNFR) 1 by decreasing the de novo protein synthesis in ECs. Most importantly, we found that inhibition of importin-dependent NF-ĸB p65 nuclear translocation is a crucial part of the action of vioA leading to reduced NF-ĸB promotor activity and inflammatory gene expression. Knockdown experiments revealed a causal link between the cellular target NOP14 and the anti-inflammatory action of vioA, classifying the natural product as unique drug lead for anti-inflammatory therapeutics.

Dendritic cells play no significant role in the laser-induced choroidal neovascularization model.

Age-related macular degeneration (AMD) is genetically associated with complement. Dendritic cells (DCs) play key roles during innate and adaptive immunity, and express complement components and their receptors. We investigated ocular DC heterogeneity and the role of DCs in the laser-induced choroidal neovascularization (CNV) model. In order to determine the function of DCs, we used two models of DC deficiency: the Flt3-/- and Flt3l-/- mouse. We identified three types of ocular DCs: plasmacytoid DC, classical DC-1, and classical DC-2. At steady-state, classical DCs were found in the iris and choroid but were not detectable in the retina. Plasmacytoid DCs existed at very low levels in iris, choroid, and retina. After laser injury, the number of each DC subset was up-regulated in the choroid and retina. In Flt3-/- mice, we found reduced numbers of classical DCs at steady-state, but each DC subset equally increased after laser injury between wildtype and Flt3-/- mice. In Flt3l-/- mice, each DC subsets was severely reduced after laser injury. Neither Flt3-/- or Flt3l-/- mice demonstrated reduced CNV area compared to wildtype mice. DCs do not play any significant role during the laser-induced CNV model of neovascular AMD.

Natural immunoglobulin M-based delivery of a complement alternative pathway inhibitor in mouse models of retinal degeneration.

PURPOSE: Age-related macular degeneration is a slowly progressing disease. Studies have tied disease risk to an overactive complement system. We have previously demonstrated that pathology in two mouse models, the choroidal neovascularization (CNV) model and the smoke-induced ocular pathology (SIOP) model, can be reduced by specifically inhibiting the alternative complement pathway (AP). Here we report on the development of a novel injury-site targeted inhibitor of the alternative pathway, and its characterization in models of retinal degeneration. METHODS: Expression of the danger associated molecular pattern, a modified annexin IV, in injured ARPE-19 cells was confirmed by immunohistochemistry and complementation assays using B4 IgM mAb. Subsequently, a construct was prepared consisting of B4 single chain antibody (scFv) linked to a fragment of the alternative pathway inhibitor, fH (B4-scFv-fH). ARPE-19 cells stably expressing B4-scFv-fH were microencapsulated and administered intravitreally or subcutaneously into C57BL/6 J mice, followed by CNV induction or smoke exposure. Progression of CNV was analyzed using optical coherence tomography, and SIOP using structure-function analyses. B4-scFv-fH targeting and AP specificity was assessed by Western blot and binding experiments. RESULTS: B4-scFv-fH was secreted from encapsulated RPE and inhibited complement in RPE monolayers. B4-scFv-fH capsules reduced CNV and SIOP, and western blotting for breakdown products of C3α, IgM and IgG confirmed a reduction in complement activation and antibody binding in RPE/choroid. CONCLUSIONS: Data supports a role for natural antibodies and neoepitope expression in ocular disease, and describes a novel strategy to target AP-specific complement inhibition to diseased tissue in the eye. PRECIS: AMD risk is tied to an overactive complement system, and ocular injury is reduced by alternative pathway (AP) inhibition in experimental models. We developed a novel inhibitor of the AP that targets an injury-specific danger associated molecular pattern, and characterized it in disease models.

Distinct effects of complement and of NLRP3- and non-NLRP3 inflammasomes for choroidal neovascularization.

NLRP3 inflammasome activation and complement-mediated inflammation have been implicated in promoting choroidal neovascularization (CNV) in age-related macular degeneration (AMD), but central questions regarding their contributions to AMD pathogenesis remain unanswered. Key open questions are (1) whether NLRP3 inflammasome activation mainly in retinal pigment epithelium (RPE) or rather in non-RPE cells promotes CNV, (2) whether inflammasome activation in CNV occurs via NLRP3 or also through NLRP3-independent mechanisms, and (3) whether complement activation induces inflammasome activation in CNV. Here we show in a neovascular AMD mouse model that NLRP3 inflammasome activation in non-RPE cells but not in RPE cells promotes CNV. We demonstrate that both NLRP3-dependent and NLRP3-independent inflammasome activation mechanisms induce CNV. Finally, we find that complement and inflammasomes promote CNV through independent mechanisms. Our findings uncover an unexpected role of non-NLRP3 inflammasomes for CNV and suggest that combination therapies targeting inflammasomes and complement may offer synergistic benefits to inhibit CNV.

Potential Role of Myeloid-Derived Suppressor Cells (MDSCs) in Age-Related Macular Degeneration (AMD).

Myeloid cells, such as granulocytes/neutrophils and macrophages, have responsibilities that include pathogen destruction, waste material degradation, or antigen presentation upon inflammation. During persistent stress, myeloid cells can remain partially differentiated and adopt immunosuppressive functions. Myeloid-derived suppressor cells (MDSCs) are primarily beneficial upon restoring homeostasis after inflammation. Because of their ability to suppress adaptive immunity, MDSCs can also ameliorate autoimmune diseases and semi-allogenic responses, e.g., in pregnancy or transplantation. However, immunosuppression is not always desirable. In certain conditions, such as cancer or chronically inflamed tissue, MDSCs prevent restorative immune responses and thereby aggravate disease progression. Age-related macular degeneration (AMD) is the most common disease in Western countries that severely threatens the central vision of aged people. The pathogenesis of this multifactorial disease is not fully elucidated, but inflammation is known to participate in both dry and wet AMD. In this paper, we provide an overview about the potential role of MDSCs in the pathogenesis of AMD.

mTORC1 signaling pathway regulates macrophages in choroidal neovascularization.

Macrophages are involved in choroidal neovascularization (CNV). The mechanistic target of rapamycin complex 1 (mTORC1) is a central cell regulator, but mTORC1 function in macrophages in CNV is not fully understood. We explored the effect of mTORC1 pathway regulation on macrophages in CNV. A laser-induced murine CNV model was performed. Expression of phospho-S6 and F4/80 in CNV lesions was analyzed by immunofluorescence. Macrophages in CNV lesions were found at 1 day after laser treatment, reached a peak at 5 days, and decreased at 7 and 14 days. mTORC1 activity of cells in CNV lesions was increased from 3 to 7 days, and deceased at 14 days. Most infiltrating macrophages in CNV lesions had strong mTORC1 activity at 3 and 5 days that subsequently decreased. In vitro, THP-1 macrophages were polarized to M1 or M2 with rapamycin or siRNA treatment. The human retinal pigment epithelium (RPE) cell line ARPE-19 was co-cultured with macrophages. Cytokine expression of macrophages and ARPE-19 cells was detected by quantitative PCR. Inhibiting mTORC1 activity of macrophages reduced M1 and strengthened M2, which was reversed by mTORC1 hyperactivation. Both M1 and M2 macrophages induced RPE cells to express less PEDF and more MMP9, IL-1ß and MCP-1. Inhibiting or enhancing mTORC1 activity of macrophages changed cytokine expression of RPE cells. Together, we demonstrated that macrophage functions in CNV were regulated partly by the mTORC1 pathway, and mTORC1 activity of macrophages influenced the expression of cytokines that are associated with CNV development in RPE cells. This study provides more understanding about the regulatory mechanism of macrophages in CNV.

Comprehensive expression patterns of inflammatory cytokines in aqueous humor of patients with neovascular age-related macular degeneration.

Neovascular age-related macular degeneration (nAMD) is a complex and multi-factorial disease, and low-grade inflammation is associated with pathogenesis of nAMD. Aqueous humor could reflect intraocular immune environments in various eye diseases. The research so far used aqueous humor samples and revealed that inflammation is involved in pathophysiology of nAMD, although immunological roles of cytokines were evaluated inadequately with aspect to individual effects. Here we used 27 kinds of cytokines covering general immunologic reactions, examined specific expression patterns of cytokines, and assessed relationships between inflammation and pathophysiology of nAMD by multivariate analyses. In nAMD eyes, principal component analysis showed that IL-7, MCP-1, MIP-1ß and VEGF had high principal component loadings of over 0.6 in the first principal component constituting 32.6% of all variability of the data. In exploratory factor analysis, IL-6, MCP-1 and MIP-1ß had high factor loadings (FL) of over 0.5 in Factor 1 constituting 32.6% of all variability, while VEGF had FL of over 1.0 in Factor 3 constituting 10.7% of all variability. In hierarchical cluster analysis, MCP-1 and VEGF were located in the cluster of first proximate mutual distance to central retinal thickness. These data could suggest that low-grade inflammation is a principal contributor in nAMD.

Polypoidal Choroidal Vasculopathy Associate With Diminished Regulatory T Cells That Are Polarized Into a T Helper 2-Like Phenotype.

Purpose: To investigate possible roles of T helper (Th) cells, regulatory T cells (Tregs), and the recently mapped Th-like Tregs in patients with polypoidal choroidal vasculopathy (PCV). Methods: In this prospective case-control study, we obtained fresh venous blood from patients with PCV (n = 24), age-matched healthy controls (n = 32), and patients with neovascular AMD (n = 45). All participants underwent a comprehensive ocular examination including fluorescein and indocyanine green angiography for where retinal disease was suspected. Using flow cytometry, we identified Th subsets, Tregs, and Th-like Tregs. Plasma samples were stored at -80°C to investigate plasma cytokines of interest. Results: Compared to healthy controls, patients with PCV had lower percentages of Tregs (8.7% ± 2.8% vs. 7.3% ± 1.7%, P = 0.027), which were significantly more Th2-like polarized (42.6% ± 13.3% vs. 50.5% ± 13.0%, P = 0.029). These changes differed from that observed in neovascular AMD, which compared to healthy controls had fewer Th1/Th17 cells (3.6% ± 2.7% vs. 2.4% ± 2.5%, P = 0.049), comparable Treg levels, and no distinct polarization of Th-like Tregs. Because of these findings, we measured plasma IL-4 and IL-33 levels. Plasma IL-33 in patients with PCV (median 0.30 pg/mL) was twice as high compared to healthy controls (median 0.16 pg/mL; P = 0.037). Conclusions: PCV associate with diminished Tregs that are polarized more into a Th2-like phenotype. This is correlated to IL-33 levels, which we also find increased in patients with PCV. Our findings suggest a possible role for Th2-like Tregs and IL-33 in PCV.

An anti-PLVAP antibody suppresses laser-induced choroidal neovascularization in monkeys.

Plasmalemma vesicle-associated protein (PLVAP, also called PV-1) is the only protein that forms endothelial diaphragms. PLVAP expression is very low in the normal blood-retinal barrier; however, pathological factors such as high glucose and vascular endothelial growth factor (VEGF) induce its expression, leading to the exacerbation of cellular permeability. Because the new blood vessels are fragile and leaky, PLVAP could possibly be considered a therapeutic target against retinovascular diseases. VEGF inhibitors are commonly used for the treatment of such diseases; however, there are several concerns associated with their use, especially in the case of chronic suppression of VEGF. In this study, we investigated the expressional level of PLVAP mRNA in VEGF-treated endothelial cells and the retinas of 2 animal models: streptozotocin-induced diabetic Brown Norway rats and Sprague-Dawley rats with oxygen-induced retinopathy. Among transcellular transport-related genes, the induction of PLVAP mRNA is the most apparent; the increase of PLVAP mRNA levels in the retina is evident during pathological progression. Furthermore, anti-PLVAP antibodies were generated, and their efficacy against laser-induced choroidal neovascularization was tested in cynomolgus monkeys. Although the leakage was exacerbated in the saline-injected group during the progression of neovascularization, the intravitreal injection of anti-PLVAP antibodies significantly ameliorated the exudation. These data imply that the PLVAP inhibition is a promising therapeutic approach against retinal diseases such as diabetic macular edema, retinopathy of prematurity, and wet age-related macular degeneration.

Inhibition of the alternative complement pathway accelerates repair processes in the murine model of choroidal neovascularization.

Age-related macular degeneration (AMD) is the leading cause of blindness in the US. Polymorphisms in complement components are associated with increased AMD risk, and it has been hypothesized that an overactive complement system is partially responsible for AMD pathology. Choroidal neovascularization (CNV) has two phases, injury/angiogenesis and repair/fibrosis. Complement activation has been shown to be involved in the angiogenesis phase of murine CNV, but has not been investigated during repair. Anaphylatoxin (C3a and C5a) signaling in particular has been shown to be involved in both tissue injury and repair in other models. CNV was triggered by laser-induced photocoagulation in C57BL/6 J mice, and lesion sizes measured by optical coherence tomography. Alternative pathway (AP) activation or C3a-receptor (C3aR) and C5a-receptor (C5aR) engagement was inhibited during the repair phase only of CNV with the AP-inhibitor CR2-fH, a C3aR antagonist (N2-[(2,2-diphenylethoxy)acetyl]-l-arginine, TFA), or a C5a blocking antibody (CLS026), respectively. Repair after CNV was also investigated in C3aR/C5aR double knockout mice. CR2-fH treatment normalized anaphylatoxin levels in the eye and accelerated regression of CNV lesions. In contrast, blockade of anaphylatoxin-receptor signaling pharmacologically or genetically did not significantly alter the course of lesion repair. These results suggest that continued complement activation prevents fibrotic scar resolution, and emphasizes the importance of reducing anaphylatoxins to homeostatic levels. This duality of complement, playing a role in injury and repair, will need to be considered when selecting a complement inhibitory strategy for AMD.

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

PURPOSE: To 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). METHOD: We 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). RESULTS: Our 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. CONCLUSION: We 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.

Computational Approach to Investigating Key GO Terms and KEGG Pathways Associated with CNV.

Choroidal neovascularization (CNV) is a severe eye disease that leads to blindness, especially in the elderly population. Various endogenous and exogenous regulatory factors promote its pathogenesis. However, the detailed molecular biological mechanisms of CNV have not been fully revealed. In this study, by using advanced computational tools, a number of key gene ontology (GO) terms and KEGG pathways were selected for CNV. A total of 29 validated genes associated with CNV and 17,639 nonvalidated genes were encoded based on the features derived from the GO terms and KEGG pathways by using the enrichment theory. The widely accepted feature selection method-maximum relevance and minimum redundancy (mRMR)-was applied to analyze and rank the features. An extensive literature review for the top 45 ranking features was conducted to confirm their close associations with CNV. Identifying the molecular biological mechanisms of CNV as described by the GO terms and KEGG pathways may contribute to improving the understanding of the pathogenesis of CNV.

Anaphylatoxin Signaling in Retinal Pigment and Choroidal Endothelial Cells: Characteristics and Relevance to Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in the USA. Polymorphisms in various complement components are associated with an increased risk for AMD, and it has been hypothesized that an overactive complement system is partially responsible for the pathology of AMD. AMD is classified as early, intermediate, or late AMD, depending on the degree of the associated pathologies. Late AMD can be characterized as either lesions associated with neovascular AMD or geographic atrophy. Both sets of lesions are associated with pathology at the RPE/choroid interface, which include a thickening of Bruch's membrane, presence of drusen, and pigmentary alterations, and deterioration of the blood-retina barrier has been reported. These changes can lead to the slow degeneration and atrophy of the photoreceptors in the macula in dry AMD, or progress to choroidal neovascularization (CNV) and leakage of these new vessels in wet AMD. It has been shown previously that complement anaphylatoxins C3a and C5a, signaling via their respective G-protein-coupled receptors, can alter RPE cell function and promote choroidal neovascularization. However, it is important to note these components also play a role in tissue repair. Here we discuss anaphylatoxin signaling in AMD-related target cells and the potential implications for the design of anti-complement therapeutics.

Effect of Anti-C5a Therapy in a Murine Model of Early/Intermediate Dry Age-Related Macular Degeneration.

Purpose: A large body of evidence supports a central role for complement activation in the pathobiology of age-related macular degeneration (AMD), including plasma complement component 5a (C5a). Interestingly, C5a is a chemotactic agent for monocytes, a cell type also shown to contribute to AMD. However, the role monocytes play in the pathogenesis of "dry" AMD and the pharmacologic potential of targeting C5a to regulate these cells are unclear. We addressed these questions via C5a blockade in a unique model of early/intermediate dry AMD and large panel flow cytometry to immunophenotype monocytic involvement. Methods: Heterozygous complement factor H (Cfh+/-) mice aged to 90 weeks were fed a high-fat, cholesterol-enriched diet (Cfh+/-∼HFC) for 8 weeks and were given weekly intraperitoneal injections of 30 mg/kg anti-C5a (4C9, Pfizer). Flow cytometry, retinal pigmented epithelium (RPE) flat mounts, and electroretinograms were used to characterize anti-C5a treatment. Results: Aged Cfh+/- mice developed RPE damage, sub-RPE basal laminar deposits, and attenuation of visual function and immune cell recruitment to the choroid that was accompanied by expression of inflammatory and extracellular matrix remodeling genes following 8 weeks of HFC diet. Concomitant systemic administration of an anti-C5a antibody successfully inhibited local recruitment of mononuclear phagocytes to the choroid-RPE interface but did not ameliorate these AMD-like pathologies in this mouse model. Conclusions: These results show that immunotherapy targeting C5a is not sufficient to block the development of the AMD-like pathologies observed in Cfh+/-∼HFC mice and suggest that other complement components or molecules/mechanisms may be driving "early" and "intermediate" AMD pathologies.

T-cell differentiation and CD56+ levels in polypoidal choroidal vasculopathy and neovascular age-related macular degeneration.

Polypoidal choroidal vasculopathy (PCV) and neovascular age-related macular degeneration (AMD) are prevalent age-related diseases characterized by exudative changes in the macula. Although they share anatomical and clinical similarities, they are also distinctly characterized by their own features, e.g. vascular abnormalities in PCV and drusen-mediated progression in neovascular AMD. PCV remains etiologically uncharacterized, and ongoing discussion is whether PCV and neovascular AMD share the same etiology or constitute two substantially different diseases. In this study, we investigated T-cell differentiation and aging profile in human patients with PCV, patients with neovascular AMD, and age-matched healthy control individuals. Fresh venous blood was prepared for flow cytometry to investigate CD4+ and CD8+ T-cell differentiation (naïve, central memory, effector memory, effector memory CD45ra+), loss of differentiation markers CD27 and CD28, and expression of aging marker CD56. Patients with PCV were similar to the healthy controls in all aspects. In patients with neovascular AMD we found significantly accelerated T-cell differentiation (more CD28-CD27- cells) and aging (more CD56+ cells) in the CD8+ T-cell compartment. These findings suggest that PCV and neovascular AMD are etiologically different in terms of T cell immunity, and that neovascular AMD is associated with T-cell immunosenescence.

Inhibition of Placenta Growth Factor Reduces Subretinal Mononuclear Phagocyte Accumulation in Choroidal Neovascularization.

Purpose: The cellular immune response driven by mononuclear phagocytes (MPs) is crucial for choroidal neovascularization (CNV) progression. Case reports show that a switch from pure anti-vascular endothelial growth factor-A (VEGF-A) intravitreal treatment to aflibercept, a drug with combined anti-VEGF-A and anti-placenta growth factor (PlGF) activity, can be beneficial for patients who do not respond to anti-VEGF-A alone. Since MPs harbor VEGFR1, we hypothesize that the interplay of P1GF/vascular endothelial growth factor receptor 1 (VEGFR1) in immune cells plays a pivotal role for CNV. Methods: CNV was induced with laser, and immune cells and neovascularization were analyzed in vivo and ex vivo. Immunohistochemistry was employed for protein detection. Differential expression of angiogenic factors and macrophage polarization markers were assessed by quantitative PCR (qPCR). One day after laser, intravitreal injection of aflibercept or anti-PlGF was performed. Results: In the early inflammatory phase after laser, Plgf but not Vegfa was significantly upregulated. VEGF-A upregulation is limited to the scar, whereas PlGF shows a wider distribution. M1 (proinflammatory) macrophage markers were upregulated in the early phase of CNV. However, M2 (proangiogenic) markers showed more inconsistent dynamics. We demonstrated that both aflibercept and anti-PlGF treatments decrease the overall amount of activated subretinal MPs, and especially of those expressing PlGF. These data correlated with a reduction in leakage associated to CNV. Aflibercept showed a stronger reduction in both parameters. Conclusions: The results hint at an interplay between PlGF/VEGFR1 and MPs that is important in the early phase of CNV. A combined inhibition of VEGF-A and PlGF is superior to a specific anti-PlGF treatment in terms of subretinal MP recruitment.

Periostin in vitreoretinal diseases.

Proliferative vitreoretinal diseases such as diabetic retinopathy, proliferative vitreoretinopathy (PVR), and age-related macular degeneration are a leading cause of decreased vision and blindness in developed countries. In these diseases, retinal fibro(vascular) membrane (FVM) formation above and beneath the retina plays an important role. Gene expression profiling of human FVMs revealed significant upregulation of periostin. Subsequent analyses demonstrated increased periostin expression in the vitreous of patients with both proliferative diabetic retinopathy and PVR. Immunohistochemical analysis showed co-localization of periostin with α-SMA and M2 macrophage markers in FVMs. In vitro, periostin blockade inhibited migration and adhesion induced by PVR vitreous and transforming growth factor-ß2 (TGF-ß2). In vivo, a novel single-stranded RNAi agent targeting periostin showed the inhibitory effect on experimental retinal and choroidal FVM formation without affecting the viability of retinal cells. These results indicated that periostin is a pivotal molecule for FVM formation and a promising therapeutic target for these proliferative vitreoretinal diseases.

A novel bispecific molecule delivered by recombinant AAV2 suppresses ocular inflammation and choroidal neovascularization.

Elevated vascular endothelial growth factor (VEGF) and complement activation are implicated in the pathogenesis of different ocular diseases. The objective of this study was to investigate the hypothesis that dual inhibition of both VEGF and complement activation would confer better protection against ocular inflammation and neovascularization. In this study, we engineered a secreted chimeric VEGF inhibitor domain (VID), a complement inhibitor domain (CID) and a dual inhibitor (ACVP1). Vectors expressing these three inhibitors were constructed and packaged into AAV2 (sextY-F) particles. The expression and secretion of the proteins were validated by Western blot. The effects of these inhibitors expressed from AAV2 vectors were examined in endotoxin-induced uveitis (EIU), experimental autoimmune uveoretinitis (EAU) and choroidal neovascularization (CNV) mouse models. The AAV2 vectors expressing the CID- and ACVP1-attenuated inflammation in EIU and EAU model, whereas the vector expressing VID showed improved retinal structure damaged by EAU, but not affect the infiltration of inflammatory cells in EAU or EIU eyes. Both VID and CID vectors improved laser-induced retinal and choroid/RPE injuries and CNV, whereas ACVP1 vector provided significantly better protection. Our results suggest that gene therapy targeting VEGF and complement components could provide an innovative and long-term strategy for ocular inflammatory and neovascular diseases.