Secreted Phosphoprotein 1 Expression in Retinal Mononuclear Phagocytes Links Murine to Human Choroidal Neovascularization

Age-related macular degeneration (AMD) represents the most common cause of blindness in the elderly in the Western world. An impairment of the outer blood-retina barrier and a localized inflammatory microenvironment cause sprouting of choroidal neovascular membranes (CNV) in neovascular AMD that are in intimate contact with surrounding myeloid cells, such as retinal microglia, and ultimately lead to visual impairment. The discovery of novel target molecules to interfere with angiogenesis and inflammation is vital for future treatment approaches in AMD patients. To explore the transcriptional profile and the function of retinal microglia at sites of CNV, we performed a comprehensive RNA-seq analysis of retinal microglia in the mouse model of laser-induced choroidal neovascularization (mCNV). Here, we identified the angiogenic factor Osteopontin (Opn), also known as “secreted phosphoprotein 1” (Spp1), as one of the most highly expressed genes in retinal microglia in the course of CNV formation. We confirmed the presence of SPP1 at the lesion site in recruited retinal microglia in Cx3cr1CreER:Rosa26-tdTomato reporter mice by confocal microscopy and in whole retinal tissue lysates by ELISA highlighting a massive local production of SPP1. Inhibition of SPP1 by intravitreal injection of an anti-SPP1 antibody significantly increased the lesion size compared to IgG-treated control eyes. In line with our results in rodents, we found an increased SPP1 mRNA expression in surgically extracted human choroidal neovascular (hCNV) membranes by the quantitative RNA-seq approach of massive analysis of cDNA ends (MACE). Numerous IBA1+SPP1+ myeloid cells were detected in human CNV membranes. Taken together, these results highlight the importance of SPP1 in the formation of CNV and potentially offer new opportunities for therapeutic intervention by modulating the SPP1 pathway.

Download Full-text

Immunosenescence in Choroidal Neovascularization (CNV)—Transcriptional Profiling of Naïve and CNV-Associated Retinal Myeloid Cells during Aging

International Journal of Molecular Sciences ◽

10.3390/ijms222413318 ◽

2021 ◽

Vol 22 (24) ◽

pp. 13318

Author(s):

Anja Schlecht ◽

Adrian Thien ◽

Julian Wolf ◽

Gabriele Prinz ◽

Hansjürgen Agostini ◽

...

Keyword(s):

Choroidal Neovascularization ◽

Transcriptional Profiling ◽

Myeloid Cells ◽

Age Related Macular Degeneration ◽

Physiological Aging ◽

Future Studies ◽

Signature Genes ◽

Age Related ◽

Age Dependent

Immunosenescence is considered a possible factor in the development of age-related macular degeneration and choroidal neovascularization (CNV). However, age-related changes of myeloid cells (MCs), such as microglia and macrophages, in the healthy retina or during CNV formation are ill-defined. In this study, Cx3cr1-positive MCs were isolated by fluorescence-activated cell sorting from six-week (young) and two-year-old (old) Cx3cr1GFP/+ mice, both during physiological aging and laser-induced CNV development. High-throughput RNA-sequencing was performed to define the age-dependent transcriptional differences in MCs during physiological aging and CNV development, complemented by immunohistochemical characterization and the quantification of MCs, as well as CNV size measurements. These analyses revealed that myeloid cells change their transcriptional profile during both aging and CNV development. In the steady state, senescent MCs demonstrated an upregulation of factors contributing to cell proliferation and chemotaxis, such as Cxcl13 and Cxcl14, as well as the downregulation of microglial signature genes. During CNV formation, aged myeloid cells revealed a significant upregulation of angiogenic factors such as Arg1 and Lrg1 concomitant with significantly enlarged CNV and an increased accumulation of MCs in aged mice in comparison to young mice. Future studies need to clarify whether this observation is an epiphenomenon or a causal relationship to determine the role of immunosenescence in CNV formation.

Download Full-text

Absence of TGFβ signaling in retinal microglia induces retinal degeneration and exacerbates choroidal neovascularization

eLife ◽

10.7554/elife.42049 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 24

Author(s):

Wenxin Ma ◽

Sean M Silverman ◽

Lian Zhao ◽

Rafael Villasmil ◽

Maria M Campos ◽

...

Keyword(s):

Choroidal Neovascularization ◽

Microglial Activation ◽

Retinal Disease ◽

Age Related Macular Degeneration ◽

Tgfβ Signaling ◽

Adult Mice ◽

Age Related ◽

Retinal Microglia ◽

Tgfβ Receptor ◽

Activation Status

Constitutive TGFβ signaling is important in maintaining retinal neurons and blood vessels and is a factor contributing to the risk for age-related macular degeneration (AMD), a retinal disease involving neurodegeneration and microglial activation. How TGFβ signaling to microglia influences pathological retinal neuroinflammation is unclear. We discovered that ablation of the TGFβ receptor, TGFBR2, in retinal microglia of adult mice induced abnormal microglial numbers, distribution, morphology, and activation status, and promoted a pathological microglial gene expression profile. TGFBR2-deficient retinal microglia induced secondary gliotic changes in Müller cells, neuronal apoptosis, and decreased light-evoked retinal function reflecting abnormal synaptic transmission. While retinal vasculature was unaffected, TGFBR2-deficient microglia demonstrated exaggerated responses to laser-induced injury that was associated with increased choroidal neovascularization, a hallmark of advanced exudative AMD. These findings demonstrate that deficiencies in TGFβ-mediated microglial regulation can drive neuroinflammatory contributions to AMD-related neurodegeneration and neovascularization, highlighting TGFβ signaling as a potential therapeutic target.

Download Full-text

Associations between the Complement System and Choroidal Neovascularization in Wet Age-Related Macular Degeneration

International Journal of Molecular Sciences ◽

10.3390/ijms21249752 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9752

Author(s):

Emilie Grarup Jensen ◽

Thomas Stax Jakobsen ◽

Steffen Thiel ◽

Anne Louise Askou ◽

Thomas J. Corydon

Keyword(s):

Macular Degeneration ◽

Choroidal Neovascularization ◽

Complement System ◽

Complex Disease ◽

Vision Loss ◽

Alternative Pathway ◽

Age Related Macular Degeneration ◽

Western World ◽

Age Related ◽

The Complement System

Age-related macular degeneration (AMD) is the leading cause of blindness affecting the elderly in the Western world. The most severe form of AMD, wet AMD (wAMD), is characterized by choroidal neovascularization (CNV) and acute vision loss. The current treatment for these patients comprises monthly intravitreal injections of anti-vascular endothelial growth factor (VEGF) antibodies, but this treatment is expensive, uncomfortable for the patient, and only effective in some individuals. AMD is a complex disease that has strong associations with the complement system. All three initiating complement pathways may be relevant in CNV formation, but most evidence indicates a major role for the alternative pathway (AP) and for the terminal complement complex, as well as certain complement peptides generated upon complement activation. Since the complement system is associated with AMD and CNV, a complement inhibitor may be a therapeutic option for patients with wAMD. The aim of this review is to (i) reflect on the possible complement targets in the context of wAMD pathology, (ii) investigate the results of prior clinical trials with complement inhibitors for wAMD patients, and (iii) outline important considerations when developing a future strategy for the treatment of wAMD.

Download Full-text

Promiscuous Chemokine Antagonist (BKT130) Suppresses Laser-Induced Choroidal Neovascularization by Inhibition of Monocyte Recruitment

Journal of Immunology Research ◽

10.1155/2019/8535273 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12

Author(s):

Shira Hagbi-Levi ◽

Michal Abraham ◽

Liran Tiosano ◽

Batya Rinsky ◽

Michelle Grunin ◽

...

Keyword(s):

Choroidal Neovascularization ◽

Ex Vivo ◽

Mononuclear Phagocytes ◽

Age Related Macular Degeneration ◽

Therapeutic Modality ◽

Mrna Levels ◽

Age Related ◽

Chemokine Signaling ◽

Monocyte Recruitment

Background. Age-related macular degeneration (AMD), the most common cause of blindness in the developed world, usually affects individuals older than 60 years of age. The majority of visual loss in this disease is attributable to the development of choroidal neovascularization (CNV). Mononuclear phagocytes, including monocytes and their tissue descendants, macrophages, have long been implicated in the pathogenesis of neovascular AMD (nvAMD). Current therapies for nvAMD are based on targeting vascular endothelial growth factor (VEGF). This study is aimed at assessing if perturbation of chemokine signaling and mononuclear cell recruitment may serve as novel complementary therapeutic targets for nvAMD. Methods. A promiscuous chemokine antagonist (BKT130), aflibercept treatment, or combined BKT130+aflibercept treatment was tested in an in vivo laser-induced model of choroidal neovascularization (LI-CNV) and in an ex vivo choroidal sprouting assay (CSA). Quantification of CD11b+ cell in the CNV area was performed, and mRNA levels of genes implicated in CNV growth were measured in the retina and RPE-choroid. Results. BKT130 reduced the CNV area and recruitment of CD11b+ cells by 30-35%. No effect of BKT130 on macrophages’ proangiogenic phenotype was demonstrated ex vivo, but a lower VEGFA and CCR2 expression was found in the RPE-choroid and a lower expression of TNFα and NOS1 was found in both RPE-choroid and retinal tissues in the LI-CNV model under treatment with BKT130. Conclusions. Targeting monocyte recruitment via perturbation of chemokine signaling can reduce the size of experimental CNV and should be evaluated as a potential novel therapeutic modality for nvAMD.

Download Full-text

Progranulin deficiency in Iba-1+ myeloid cells exacerbates choroidal neovascularization by perturbation of lysosomal function and abnormal inflammation

Journal of Neuroinflammation ◽

10.1186/s12974-021-02203-1 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Kei Takahashi ◽

Shinsuke Nakamura ◽

Wataru Otsu ◽

Masamitsu Shimazawa ◽

Hideaki Hara

Keyword(s):

Choroidal Neovascularization ◽

Myeloid Cells ◽

Peritoneal Macrophages ◽

Age Related Macular Degeneration ◽

Subretinal Space ◽

Lysosomal Function ◽

Exudative Amd ◽

Hypoxic Conditions ◽

Age Related ◽

Associated Proteins

Abstract Background Age-related macular degeneration (AMD) is the principal cause of permanent blindness among elderly individuals worldwide. Chronic inflammation in the subretinal space is associated with a progression of exudative AMD. Progranulin (PGRN) is a growth factor secreted from myeloid cells and plays an important role in controlling the lysosomal function. A deficiency in PGRN leads to inflammation of the neurons in the central nervous system. The purpose of this study was to investigate the role played by PGRN in the size of the choroidal neovascularization (CNV) in laser-induced CNV mice. Methods CNVs were induced in C57BL/6J mice by laser photocoagulation of the retina. The expression of PGRN and the accumulation of Iba-1+ cells around the sites of the CNVs were determined. Grn−/−, Grn+/−, and Grn+/+ mice with laser-induced CNVs were also studied. To evaluate the effect of macrophages on the inflammation, we used a macrophage cell line (RAW264.7) in which the expression of PGRN was knocked down by RNA interference and peritoneal macrophages derived from Grn−/− and Grn+/+ mice. These cells were incubated under hypoxic conditions (1% O2). Results Iba-1+ myeloid cells migrated and accumulated in the photocoagulation-induced CNV areas, and the CNV lesions secreted high levels of PGRN in Grn+/+ mice. The size of the CNVs was larger in Grn−/− mice than in Grn+/− and Grn+/+ mice. In Grn−/− mice, the number of ocular-infiltrating Iba-1+ cells around the CNV was higher, and these cells produced more VEGF-A than the cells in the Grn+/+ mice. PGRN-silencing of RAW264.7 cells led to abnormal activation of the cells. In addition, hypoxic conditions promoted the production of proangiogenic and proinflammatory cytokines from PGRN-deficient macrophages. Interestingly, the expression level of lysosome-associated proteins and the number of activated lysosomes increased in PGRN-deficient macrophages. Conclusions These findings indicate that PGRN deficiency in Iba-1+ cells activates the lysosomal function that then leads to abnormal inflammation. The aberrant activation of Iba-1+ myeloid cells might contribute to the progression of the CNV and the regulation of these cells might be a novel therapeutic target for exudative AMD.

Download Full-text

A Review of Completed and Ongoing Complement Inhibitor Trials for Geographic Atrophy Secondary to Age-Related Macular Degeneration

Journal of Clinical Medicine ◽

10.3390/jcm10122580 ◽

2021 ◽

Vol 10 (12) ◽

pp. 2580

Author(s):

Omar A. Halawa ◽

Jonathan B. Lin ◽

Joan W. Miller ◽

Demetrios G. Vavvas

Keyword(s):

Macular Degeneration ◽

Geographic Atrophy ◽

Age Related Macular Degeneration ◽

Western World ◽

Complement Factor ◽

Complement Protein ◽

Pivotal Phase ◽

Complement Inhibition ◽

Exudative Amd ◽

Age Related

Age-related macular degeneration (AMD) is a leading cause of irreversible blindness among older adults in the Western world. While therapies exist for patients with exudative AMD, there are currently no approved therapies for non-exudative AMD and its advanced form of geographic atrophy (GA). The discovery of genetic variants in complement protein loci with increased susceptibility to AMD has led to the investigation of the role of complement inhibition in AMD with a focus on GA. Here, we review completed and ongoing clinical trials evaluating the safety and efficacy of these studies. Overall, complement inhibition in GA has yielded mixed results. The inhibition of complement factor D has failed pivotal phase 3 trials. Studies of C3 and C5 inhibition meeting their primary endpoint are limited by high rates of discontinuation and withdrawal in the treatment arm and higher risks of conversion to exudative AMD. Studies evaluating other complement members (CFB, CFH, CFI and inhibitors of membrane attack complex—CD59) are ongoing and could offer other viable strategies.

Download Full-text