Choroidal Macrophages Research Articles

The choroidal nervous system: a link between mineralocorticoid receptor and pachychoroid.

Central serous chorioretinopathy (CSCR) belongs to the pachychoroid spectrum, a pathological phenotype of the choroidal vasculature, in which blood flow is under the choroidal nervous system (ChNS) regulation. The pathogenesis of CSCR is multifactorial, with the most recognised risk factor being intake of glucocorticoids, which activate both the gluco- and the mineralocorticoid (MR) receptors. As MR over-activation is pathogenic in the retina and choroid, it could mediate the pathogenic effects of glucocorticoids in CSCR. But the role of MR signalling in pachychoroid is unknown and whether it affects the ChNS has not been explored. Using anatomo-neurochemical characterisation of the ChNS in rodents and humans, we discovered that beside innervation of arteries, choroidal veins and choriocapillaris are also innervated, suggesting that the entire choroidal vasculature is under neural control. The numerous synapses together with calcitonin gene-related peptide (CGRP) vesicles juxtaposed to choroidal macrophages indicate a neuro-immune crosstalk. Using ultrastructural approaches, we show that transgenic mice overexpressing human MR, display a pachychoroid-like phenotype, with signs of choroidal neuropathy including myelin abnormalities, accumulation and enlargement of mitochondria and nerves vacuolization. Transcriptomic analysis of the RPE/choroid complex in the transgenic mice reveals regulation of corticoids target genes, known to intervene in nerve pathophysiology, such as Lcn2, rdas1/dexras1, S100a8 and S100a9, rabphilin 3a (Rph3a), secretogranin (Scg2) and Kinesin Family Member 5A (Kif5a). Genes belonging to pathways related to vasculature development, hypoxia, epithelial cell apoptosis, epithelial mesenchymal transition, and inflammation, support the pachychoroid phenotype and highlight downstream molecular targets. Hypotheses on the imaging phenotype of pachychoroid in humans are put forward in the light of these new data. Our results provide evidence that MR overactivation causes a choroidal neuropathy that could explain the pachychoroid phenotype found in transgenic mice overexpressing human MR. In patients with pachychoroid and CSCR in which systemic dysautonomia has been demonstrated, MR-induced choroidal neuropathy could be the missing link between corticoids and pachychoroid.

Assessment of Choroidal Vasculature and Innate Immune Cells in the Eyes of Albino and Pigmented Mice.

The visualization of choroidal vasculature and innate immune cells in the eyes of pigmented mice has been challenging due to the presence of a retinal pigment epithelium (RPE) layer separating the choroid and retina. Here, we established methods for visualizing the choroidal macrophages, mast cells, and vasculature in eyes of albino and pigmented mice using cell type-specific staining. We were able to visualize the choroidal arterial and venous systems. An arterial circle around the optic nerve was found in mice similar to the Zinn–Haller arterial circle that exists in humans and primates. Three different structural patterns of choriocapillaris were observed throughout the whole choroid: honeycomb-like, maze-like, and finger-like patterns. Choroidal mast cells were relatively few but dense around the optic nerve. Mast cell distribution in the middle and periphery was different among strains. Macrophages were found in all layers of the choroid. Thus, utilizing the simple and reliable methods described herein will allow the evaluation of transgenic and preclinical mouse models of ocular diseases that affect the choroid, including age-related macular degeneration (AMD), diabetic choroidopathy, and retinopathy of prematurity. These studies will advance our understanding of the pathophysiology, and molecular and cellular mechanisms that can be targeted therapeutically, in these diseases.

Animal models for studies of HIV-1 brain reservoirs.

The HIV-1 often evades a robust antiretroviral-mediated immune response, leading to persistent infection within anatomically privileged sites including the CNS. Continuous low-level infection occurs in the presence of effective antiretroviral therapy (ART) in CD4+ T cells and mononuclear phagocytes (MP; monocytes, macrophages, microglia, and dendritic cells). Within the CNS, productive viral infection is found exclusively in microglia and meningeal, perivascular, and choroidal macrophages. MPs serve as the principal viral CNS reservoir. Animal models have been developed to recapitulate natural human HIV-1 infection. These include nonhuman primates, humanized mice, EcoHIV, and transgenic rodent models. These models have been used to study disease pathobiology, antiretroviral and immune modulatory agents, viral reservoirs, and eradication strategies. However, each of these models are limited to specific component(s) of human disease. Indeed, HIV-1 species specificity must drive therapeutic and cure studies. These have been studied in several model systems reflective of latent infections, specifically in MP (myeloid, monocyte, macrophages, microglia, and histiocyte cell) populations. Therefore, additional small animal models that allow productive viral replication to enable viral carriage into the brain and the virus-susceptible MPs are needed. To this end, this review serves to outline animal models currently available to study myeloid brain reservoirs and highlight areas that are lacking and require future research to more effectively study disease-specific events that could be useful for viral eradication studies both in and outside the CNS.

Role of Erythropoietin Receptor Signaling in Macrophages or Choroidal Endothelial Cells in Choroidal Neovascularization.

Erythropoietin (EPO) has been proposed to reduce the progression of atrophic age-related macular degeneration (AMD) due to its potential role in neuroprotection. However, overactive EPO receptor (EPOR) signaling increased laser-induced choroidal neovascularization (CNV) and choroidal macrophage number in non-lasered mice, which raised the question of whether EPOR signaling increased CNV through the recruitment of macrophages to the choroid that released pro-angiogenic factors or through direct angiogenic effects on endothelial cells. In this study, we addressed the hypothesis that EPOR signaling increased CNV by direct effects on macrophages or endothelial cells. We used tamoxifen-inducible macrophage-specific or endothelial cell-specific EPOR knockout mice in the laser-induced CNV model, and cultured choroidal endothelial cells isolated from adult human donors. We found that macrophage-specific knockout of EPOR influenced laser-induced CNV in females only, whereas endothelial-specific knockout of EPOR reduced laser-induced CNV in male mice only. In cultured human choroidal endothelial cells, knockdown of EPOR reduced EPO-induced signal transducer and activator of transcription 3 (STAT3) activation. Taken together, our findings suggest that EPOR signaling in macrophages or choroidal endothelial cells regulates the development of CNV in a sex-dependent manner. Further studies regarding the role of EPO-induced signaling are required to assess EPO safety and to select or develop appropriate therapeutic approaches.

Choroidal endothelial and macrophage gene expression in atrophic and neovascular macular degeneration.

The human choroid is a heterogeneous, highly vascular connective tissue that dysfunctions in age-related macular degeneration (AMD). In this study, we performed single-cell RNA sequencing on 21 human choroids, 11 of which were derived from donors with early atrophic or neovascular AMD. Using this large donor cohort, we identified new gene expression signatures and immunohistochemically characterized discrete populations of resident macrophages, monocytes/inflammatory macrophages and dendritic cells. These three immune populations demonstrated unique expression patterns for AMD genetic risk factors, with dendritic cells possessing the highest expression of the neovascular AMD-associated MMP9 gene. Additionally, we performed trajectory analysis to model transcriptomic changes across the choroidal vasculature, and we identified expression signatures for endothelial cells from choroidal arterioles and venules. Finally, we performed differential expression analysis between control, early atrophic AMD, and neovascular AMD samples, and we observed that early atrophic AMD samples had high expression of SPARCL1, a gene that has been shown to increase in response to endothelial damage. Likewise, neovascular endothelial cells harbored gene expression changes consistent with endothelial cell damage and demonstrated increased expression of the sialomucins CD34 and ENCM, which were also observed at the protein level within neovascular membranes. Overall, this study characterizes the molecular features of new populations of choroidal endothelial cells and mononuclear phagocytes in a large cohort of AMD and control human donors.

Guardians of the eye: new tales about retinal microglia and other ocular macrophages.

Guardians of the eye: new tales about retinal microglia and other ocular macrophages.

Prevalence of Ocular Diseases in Human Donor Eyes in New Zealand: A Study Based on Clinical and Histological Imaging.

Fundus pathology in donor eyes was correlated with cross-sectional Optical Coherence Tomography (OCT) images and histological assessment was performed to determine the prevalence of retinal diseases without the constraints imposed during in vivo clinical imaging. A fundus camera and OCT imaging system was adapted to enable posterior segment imaging of the entire post-mortem human eye. Retinas from 59 donors (57 retina pairs and two single globes) were imaged in a seven-field imaging format and cross-sectional analysis was done using OCT. To confirm that the signs observed represented true disease incidence analysis of disease markers including gliosis (Glial Fibrillary Acidic Protein), hemichannel expression (Connexin43), Müller cell activation (vimentin) and choroidal endothelial cells (CD-31) and macrophages (CD-68 marker) was performed. Pathological signs were correlated with clinical diagnoses in eyes from 25 donors (donor ages 45-87 years) but lesions were also found in 23 eyes (donor ages 39- 83 years) with no previously reported clinical diagnosis. Retinas from six donors aged 21-89 years of age were unremarkable. Of all donors, five donors had signs of age related macular degeneration (AMD) and 14 had signs of diabetic retinopathy (DR). Their lesions correlated with OCT and histopathology showed signs of activated microglia, Müller cell hyper-reactivity, increased Cx43 expression and choroidal inflammation. These data indicate that with over 8% of donors showing signs of AMD and 24% of donors showing signs of DR the incidence of AMD may be 1.7 times higher and DR up to 1.6 times higher than clinically reported. The detection of pathological signs characteristic of AMD and DR in donors suggests a higher prevalence of posterior segment abnormalities amongst New Zealanders donors than previously reported. A more detailed evaluation protocol of the posterior segment in patients will aid detection of lesions that are none the less pathological signs.

Systemic dendrimer nanotherapies for targeted suppression of choroidal inflammation and neovascularization in age-related macular degeneration

Inflammation and neovascularization are key pathological events in human age-related macular degeneration (AMD). Activated microglia/macrophages (mi/ma) and retinal pigmented epithelium (RPE) play an active role in every stage of disease progression. Systemic therapies that can target these cells and address both inflammation and neovascularization will broaden the impact of existing therapies and potentially open new avenues for early AMD where there are no viable therapies. Utilizing a clinically relevant rat model of AMD that mirrors many aspects that of human AMD pathological events, we show that systemic hydroxyl-terminated polyamidoamine dendrimer-triamcinolone acetonide conjugate (D-TA) is selectively taken up by the injured mi/ma and RPE (without the need for targeting ligands). D-TA suppresses choroidal neovascularization significantly (by >80%, >50-fold better than free drug), attenuates inflammation in the choroid and retina, by limiting macrophage infiltration in the pathological area, significantly suppressing pro-inflammatory cytokines and pro-angiogenic factors, with minimal side effects to healthy ocular tissue and other organs. In ex vivo studies on human postmortem diabetic eyes, the dendrimer is also taken up into choroidal macrophages. These results suggest that the systemic hydroxyl dendrimer-drugs can offer new avenues for therapies in treating early/dry AMD and late/neovascular AMD alone, or in combination with current anti-VEGF therapies. This hydroxyl dendrimer platform but conjugated to a different drug is undergoing clinical trials for severe COVID-19, potentially paving the way for faster clinical translation of similar compounds for ocular and retinal disorders.

CSF1/CSF1R-mediated Crosstalk Between Choroidal Vascular Endothelial Cells and Macrophages Promotes Choroidal Neovascularization.

PurposeThis study examined the role of the CSF1/CSF1Raxis in the crosstalk between choroidal vascular endothelial cells (CVECs) and macrophages during the formation of choroidal neovascularization (CNV).MethodsQuantitative reverse transcriptase (QRT)-PCR, Western blot and ELISA measured the production and release of CSF1 from human choroidal vascular endothelial cells (HCVECs) under hypoxic conditions. Western blot detected CSF1 released from HCVECs under hypoxic conditions that activated the PI3K/AKT/FOXO1 axis in human macrophages via binding to CSF1R. Transwell migration assay, qRT-PCR, and Western blot detected the effect of CSF1 released from HCVECs on macrophage migration and M2 polarization via the CSF1R/PI3K/AKT/FOXO1 pathway. Incorporation of 5-ethynyl-20-deoxyuridine, transwell migration, and tube formation assays detected the effects of CSF1/CSF1R on the behaviors of HCVECs. Fundus fluorescein angiography (FFA), indocyanine green angiography (ICGA), and immunofluorescence detected the effect of blockade of CSF1/CSF1R on mouse laser-induced CNV. Color fundus photograph, ICGA, and FFA detected CNV lesions in neovascular AMD (nAMD) patients. ELISA detected CSF1 and CSF1R in the aqueous humor of age-related cataract and nAMD patients.ResultsCSF1 released from HCVECs under hypoxic conditions activated the PI3K/AKT/FOXO1 axis in human macrophages via binding to CSF1R, promoting macrophage migration and M2 polarization via up-regulation of the CSF1R/PI3K/AKT/FOXO1 pathway. Human macrophages promoted the proliferation, migration, and tube formation of HCVECs in a CSF1/CSFR1-dependent manner under hypoxic conditions. CSF1/CSF1R blockade ameliorated the formation of mouse laser-induced CNV. CSF1 and CSF1R were increased in the aqueous humor of nAMD patients.ConclusionsOur results affirmed the crucial role of CSF1/CSF1R in boosting the formation of CNV and offered potential molecular targets for the treatment of nAMD.

Ocular macrophage origin and heterogeneity during steady state and experimental choroidal neovascularization

BackgroundNeovascular age-related macular degeneration (nAMD) commonly causes vision loss from aberrant angiogenesis, termed choroidal neovascularization (CNV). Macrophages are heterogeneous cells that are necessary for experimental CNV, present in human CNV samples, and can display diverse functions, which are dependent upon both their origin and tissue microenvironment. Despite these associations, choroidal macrophage heterogeneity remains unexplored.MethodsWe performed multi-parameter flow cytometry on wildtype (WT) and Ccr2−/− mice after laser injury to identify macrophage subtypes, and determine which subsets originate from classical monocytes. To fate map tissue resident macrophages at steady state and after laser injury, we used the Cx3cr1CreER/+ ; Rosa26zsGFP/+ mouse model. We reanalyzed previously published single-cell RNA-seq of human choroid samples from healthy and nAMD patients to investigate human macrophage heterogeneity, disease association, and function.ResultsWe identified 4 macrophage subsets in mice: microglia, MHCII+CD11c−, MHCII+CD11c+, and MHCII−. Microglia are tissue resident macrophages at steady state and unaffected by laser injury. At steady state, MHCII− macrophages are long lived, tissue resident macrophages, while MHCII+CD11c− and MHCII+CD11c+ macrophages are partially replenished from blood monocytes. After laser injury, MHCII+CD11c− macrophages are entirely derived from classical monocytes, MHCII− macrophages originate from classical monocytes (90%) and an expansion of tissue resident macrophages (10%), and MHCII+CD11c+ macrophages are derived from classical monocytes (70%), non-classical monocytes (10%), and an expansion of tissue resident macrophages (20%). Single-cell RNA-seq analysis of human choroid found 5 macrophage subsets: two MHCII+CD11C− and three MHCII+CD11C+ populations. One MHCII+CD11C+ subset was 78% derived from a patient with nAMD. Differential expression analysis identified up-regulation of pro-angiogenic gene expression in one MHCII+CD11C− and two MHCII+CD11C+ subsets, including the disease-associated cluster. The upregulated MHCII+CD11C− pro-angiogenic genes were unique compared to the increased MHCII+CD11C+ angiogenesis genes.ConclusionsMacrophage origin impacts heterogeneity at steady state and after laser injury in mice. Both mice and human patients demonstrate similar macrophage subtypes. Two discrete pro-angiogenic macrophage populations exist in the human choroid. Targeting specific, pro-angiogenic macrophage subsets is a potential novel therapeutic for nAMD.

Fruquintinib inhibits VEGF/VEGFR2 axis of choroidal endothelial cells and M1-type macrophages to protect against mouse laser-induced choroidal neovascularization

Wet age-related macular degeneration, which is characterized by choroidal neovascularization (CNV) and induces obvious vision loss. Vascular endothelial growth factor (VEGF) family member VEGF-A (also named as VEGF) and its receptor VEGFR2 contribute to the pathogenesis of CNV. Choroidal endothelial cells (CECs) secret C–C motif chemokine ligand 2 (CCL2), which attracts macrophages to CNV lesion and promotes macrophage M1 polarization. Accordingly, infiltrating macrophages secret inflammatory cytokines to promote CNV. In vivo, intravitreal injection of fruquintinib (HMPL-013), an antitumor neovascularization drug, alleviated mouse CNV formation without obvious ocular toxicity. Meanwhile, HMPL-013 inhibited VEGF/VEGFR2 binding in CECs and macrophages, as well as macrophage M1 polarization. In vitro, noncontact coculture of human choroidal vascular endothelial cells (HCVECs) and macrophages under hypoxia conditions was established. HMPL-013 downregulated VEGF/VEGFR2/phosphoinositide-3-kinase/protein kinase B (AKT)/nuclear factor kappa B pathway and CCL2 secretion in HCVECs, as well as VEGF/VEGFR2-induced macrophage M1 polarization under hypoxia condition. In addition, HMPL-013 inhibited HCEVC derived CCL2-induced macrophage migration and M1 polarization, along with macrophage M1 polarization-induced HCVECs proliferation, migration, and tube formation. Altogether, HMPL-013 alleviated CNV formation might via breaking detrimental cross talk between CECs and macrophages.

CSF1R blockade induces macrophage ablation and results in mouse choroidal vascular atrophy and RPE disorganization.

The choroid, which provides vascular supply to the outer retina, demonstrates progressive degeneration in aging and age-related macular degeneration (AMD). However mechanisms that maintain or compromise choroidal homeostasis are obscure. We discovered that the ablation of choroidal macrophages via CSF1R blockade was associated with choroidal vascular atrophy and retinal pigment epithelial (RPE) changes including structural disruption, downregulation of visual cycle genes, and altered angiogenic factor expression. Suspending CSF1R blockade following ablation enabled spontaneous macrophage regeneration, which fully restored original macrophage distributions and morphologies. Macrophage regeneration was accompanied by arrested vascular degeneration and ameliorated pathological RPE alterations. These findings suggest that choroidal macrophages play a previously unappreciated trophic role in maintaining choroidal vasculature and RPE cells, implicating insufficiency in choroidal macrophage function as a factor in aging- and AMD-associated pathology. Modulating macrophage function may constitute a strategy for the therapeutic preservation of the choroid and RPE in age-related retinal disorders.

In Vivo Choroidal Neovascularization and Macrophage Studies Provide Further Evidence for a Broad Role of Prostacyclin in Angiogenesis.

The purpose of these studies was (1) to investigate the ability of human M1 phenotype macrophages to secrete vascular endothelial growth factor (VEGF) and the influence of prostacyclin receptor (IP) stimulation (2) to evaluate the contribution of the proangiogenic prostanoid prostacyclin to experimental choroidal neovascularization Methods: Human macrophages derived from primary blood mononuclear cells were functionally biased toward the M1 phenotype by using tumor necrosis factor α (TNFα). Experimental choroidal neovascularization was produced by laser photocoagulation. Antagonist drugs RO-3244794 (IP antagonist) and GW 627368 (EP4 antagonist) were administered according to an optimal dosing regimen that was predetermined by bioavailability studies. IP receptor stimulation had diametrically opposed effects on VEGF release compared with reported data on cytokine/chemokine secretion from human macrophages. For example, the IP agonist cicaprost stimulated VEGF secretion although it inhibits monocyte chemoattractant protein-1 (MCP-1) secretion: both would favor a proangiogenic effect. The IP receptor antagonist RO-3244794 produced an ∼20% statistically significant reduction in the neovascularized lesion area in the choroidal neovascularization model, which was a similar level to that produced by the EP4 antagonist GW 627368. Combining the 2 drugs produced a statistically significant reduction in neovascularization but only of slightly greater magnitude than that obtained with each antagonist administered alone. IP receptor stimulation potently and highly efficaciously promoted VEGF release from human M1 macrophages, indicating a possible contribution of the M1 macrophage subtype to VEGF-induced choroidal neovascularization. Studies in living animals suggest that prostacyclin and its target IP receptor contribute to choroidal neovascularization, although to a more modest extent than might have been expected.

Erythropoietin Signaling Increases Choroidal Macrophages and Cytokine Expression, and Exacerbates Choroidal Neovascularization

Erythropoietin (EPO) is recognized for neuroprotective and angiogenic effects and has been associated with aging and neovascular age-related macular degeneration (AMD). We hypothesized that systemic EPO facilitates the development of choroidal neovascularization (CNV). Wild type mice expressed murine EPOR (mWtEPOR) in RPE/choroids at baseline and had significantly increased serum EPO after laser treatment. To test the role of EPO signaling, we used human EPOR knock-in mice with the mWtEPOR gene replaced by either the human EPOR gene (hWtEPOR) or a mutated human EPOR gene (hMtEPOR) in a laser-induced choroidal neovascularization (LCNV) model. Loss-of-function hWtEPOR mice have reduced downstream activation, whereas gain-of-function hMtEPOR mice have increased EPOR signaling. Compared to littermate controls (mWtEPOR), hMtEPOR with increased EPOR signaling developed larger CNV lesions. At baseline, hMtEPOR mice had increased numbers of macrophages, greater expression of macrophage markers F4/80 and CD206, and following laser injury, had greater expression of cytokines CCL2, CXCL10, CCL22, IL-6, and IL-10 than mWtEPOR controls. These data support a hypothesis that injury from age- and AMD-related changes in the RPE/choroid leads to choroidal neovascularization through EPOR-mediated cytokine production.

Comprehensive analysis of mouse retinal mononuclear phagocytes.

The innate immune system is activated in a number of degenerative and inflammatory retinal disorders such as age-related macular degeneration (AMD). Retinal microglia, choroidal macrophages, and recruited monocytes, collectively termed 'retinal mononuclear phagocytes', are critical determinants of ocular disease outcome. Many publications have described the presence of these cells in mouse models for retinal disease; however, only limited aspects of their behavior have been uncovered, and these have only been uncovered using a single detection method. The workflow presented here describes a comprehensive analysis strategy that allows characterization of retinal mononuclear phagocytes in vivo and in situ. We present standardized working steps for scanning laser ophthalmoscopy of microglia from MacGreen reporter mice (mice expressing the macrophage colony-stimulating factor receptor GFP transgene throughout the mononuclear phagocyte system), quantitative analysis of Iba1-stained retinal sections and flat mounts, CD11b-based retinal flow cytometry, and qRT-PCR analysis of key microglia markers. The protocol can be completed within 3 d, and we present data from retinas treated with laser-induced choroidal neovascularization (CNV), bright white-light exposure, and Fam161a-associated inherited retinal degeneration. The assays can be applied to any of the existing mouse models for retinal disorders and may be valuable for documenting immune responses in studies for immunomodulatory therapies.

Distribution and Quantification of Choroidal Macrophages in Human Eyes With Age-Related Macular Degeneration.

PurposeIncreasing evidence suggests a role for macrophages in the pathogenesis of age-related macular degeneration (AMD). This study examined choroidal macrophages and their activation in postmortem eyes from subjects with and without AMD.MethodsChoroids were incubated with anti-ionized calcium-binding adapter molecule 1 (anti-IBA1) to label macrophages, anti-human leukocyte antigen-antigen D-related (anti-HLA-DR) as a macrophage activation marker, and Ulex europaeus agglutinin lectin to label blood vessels. Whole mounts were imaged using confocal microscopy. IBA1- and HLA-DR–positive (activated) cells were counted in submacula, paramacula, and nonmacula, and cell volume and sphericity were determined using computer-assisted image analysis.ResultsIn aged control eyes, the mean number of submacular IBA1+ and HLA-DR+ macrophages was 433/mm2 and 152/mm2, respectively. In early AMD eyes, there was a significant increase in IBA1+ and HLA-DR+ cells in submacula compared to those in controls (P = 0.0015 and P = 0.008, respectively). In eyes with neovascular AMD, there were significantly more HLA-DR+ cells associated with submacular choroidal neovascularization (P = 0.001). Mean cell volume was significantly lower (P ≤ 0.02), and sphericity was significantly higher (P ≤ 0.005) in all AMD groups compared to controls.ConclusionsThe average number of IBA1+ macrophages in submacular and paramacular choroid was significantly higher in early/intermediate AMD compared to that in aged controls. HLA-DR+ submacular macrophages were significantly increased in all stages of AMD, and they were significantly more round and smaller in size in the submacular AMD choroid, suggesting their activation. These findings support the concept that AMD is an inflammatory disease.

IKK2 inhibition using TPCA-1-loaded PLGA microparticles attenuates laser-induced choroidal neovascularization and macrophage recruitment.

The inhibition of NF-κB by genetic deletion or pharmacological inhibition of IKK2 significantly reduces laser-induced choroid neovascularization (CNV). To achieve a sustained and controlled intraocular release of a selective and potent IKK2 inhibitor, 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1) (MW: 279.29), we developed a biodegradable poly-lactide-co-glycolide (PLGA) polymer-delivery system to further investigate the anti-neovascularization effects of IKK2 inhibition and in vivo biosafety using laser-induced CNV mouse model. The solvent-evaporation method produced spherical TPCA-1-loaded PLGA microparticles characterized with a mean diameter of 2.4 ¼m and loading efficiency of 80%. Retrobulbar administration of the TPCA-1-loaded PLGA microparticles maintained a sustained drug level in the retina during the study period. No detectable TPCA-1 level was observed in the untreated contralateral eye. The anti-CNV effect of retrobulbarly administrated TPCA-1-loaded PLGA microparticles was assessed by retinal fluorescein leakage and isolectin staining methods, showing significantly reduced CNV development on day 7 after laser injury. Macrophage infiltration into the laser lesion was attenuated as assayed by choroid/RPE flat-mount staining with anti-F4/80 antibody. Consistently, laser induced expressions of Vegfa and Ccl2 were inhibited by the TPCA-1-loaded PLGA treatment. This TPCA-1 delivery system did not cause any noticeable cellular or functional toxicity to the treated eyes as evaluated by histology and optokinetic reflex (OKR) tests; and no systemic toxicity was observed. We conclude that retrobulbar injection of the small-molecule IKK2 inhibitor TPCA-1, delivered by biodegradable PLGA microparticles, can achieve a sustained and controllable drug release into choroid/retina and attenuate laser-induced CNV development without causing apparent systemic toxicity. Our results suggest a potential clinical application of TPCA-1 delivered by microparticles in treatment of CNV in the patients with age-related macular degeneration and other retinal neovascularization diseases.

JNK inhibition reduces apoptosis and neovascularization in a murine model of age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of registered blindness among the elderly and affects over 30 million people worldwide. It is well established that oxidative stress, inflammation, and apoptosis play critical roles in pathogenesis of AMD. In advanced wet AMD, although, most of the severe vision loss is due to bleeding and exudation of choroidal neovascularization (CNV), and it is well known that vascular endothelial growth factor (VEGF) plays a pivotal role in the growth of the abnormal blood vessels. VEGF suppression therapy improves visual acuity in AMD patients. However, there are unresolved issues, including safety and cost. Here we show that mice lacking c-Jun N-terminal kinase 1 (JNK1) exhibit decreased inflammation, reduced CNV, lower levels of choroidal VEGF, and impaired choroidal macrophage recruitment in a murine model of wet AMD (laser-induced CNV). Interestingly, we also detected a substantial reduction in choroidal apoptosis of JNK1-deficient mice. Intravitreal injection of a pan-caspase inhibitor reduced neovascularization in the laser-induced CNV model, suggesting that apoptosis plays a role in laser-induced pathological angiogenesis. Intravitreal injection of a specific JNK inhibitor decreased choroidal VEGF expression and reduced pathological CNV. These results suggest that JNK1 plays a key role in linking oxidative stress, inflammation, macrophage recruitment apoptosis, and VEGF production in wet AMD and pharmacological JNK inhibition offers a unique and alternative avenue for prevention and treatment of AMD.

Macrophage markers and C3d in the central & peripheral choroid of young, aged and amd eyes

AbstractPurpose To investigate the distribution and expression of macrophage phenotypes and the complement activation protein, C3d, in human choroid/retina in young, aged and AMD eyes.Methods Paraffin sections of central and peripheral choroid/retina from young (<40 years, n=6), aged (>70 years, n=4) and AMD (>70 years, n=8) human post‐mortem eyes were co‐immunolabelled with antibodies to leukocyte and macrophage markers (CD68, CD163, Iba1 & CD45) and complement activation component, C3d. Localisation and distribution of antibodies was assessed using confocal microscopy. Counts of macrophage sub‐populations were compared in central and peripheral choroid for each group.Results Heterogeneous populations of macrophages were found in the choroid/retina of all groups. Iba1+ and CD45+ retinal microglia and macrophages, and choroidal macrophages were observed in all eyes. In AMD eyes, Iba1+ and CD45+ cells were seen in the subretinal space amongst photoreceptors, as well as in inner retina. Compared to young eyes, aged and AMD specimens showed increased numbers of CD163+ macrophages (M2, proangiogenic) in choroid, and in central vs peripheral retina. Fewer CD68+ cells were seen in all specimens, regardless of age or location. C3d was expressed in young, aged and AMD eyes, localised to Bruch’s membrane and choriocapillaris, with more extensive immunostaining in aged and AMD eyes. C3d was also seen in drusen in aged and AMD eyes, and more peripherally, compared to young eyes.Conclusion Age‐related changes in macrophage phenotypes and in complement pathway activation at the choroidal/retinal interface may play a role in development and progression of AMD. Supported by Sydney Foundation for Medical Research.

Bruch's membrane and choroidal macrophages in early and advanced age-related macular degeneration

AimTo determine the sub-macular Bruch's membrane (BrM) macrophage count and the choroidal and BrM macrophage immunophenotype in normal eyes and in eyes with early and advanced age-related macular degeneration (AMD).MethodsBrM...