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

ABSTRACT: 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 Cx3cr1 ^CreER: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.

SUBMITTER: Schlecht A

PROVIDER: S-EPMC7876283 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Schlecht Anja A Zhang Peipei P Wolf Julian J Thien Adrian A Rosmus Dennis-Dominik DD Boneva Stefaniya S Schlunck Günther G Lange Clemens C Wieghofer Peter P

Frontiers in cell and developmental biology 20210128

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 inf ...[more]

PMID: 33585455

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Project description:Inhibition of chemokine C-C motif receptor 3 (CCR3) signaling has been considered as treatment for neovascular age-related macular degeneration (AMD). However, CCR3 is expressed in neural retina from aged human donor eyes. Therefore, broad CCR3 inhibition may be harmful to the retina. We assessed the effects of CCR3 inhibition on retina and choroidal endothelial cells (CECs) that develop into choroidal neovascularization (CNV). In adult murine eyes, CCR3 colocalized with glutamine-synthetase labeled M?ller cells. In a murine laser-induced CNV model, CCR3 immunolocalized not only to lectin-stained cells in CNV lesions but also to the retina. Compared to non-lasered controls, CCR3 mRNA was significantly increased in laser-treated retina. An intravitreal injection of a CCR3 inhibitor (CCR3i) significantly reduced CNV compared to DMSO or PBS controls. Both CCR3i and a neutralizing antibody to CCR3 increased TUNEL+ retinal cells overlying CNV, compared to controls. There was no difference in cleaved caspase-3 in laser-induced CNV lesions or in overlying retina between CCR3i- or control-treated eyes. Following CCR3i, apoptotic inducible factor (AIF) was significantly increased and anti-apoptotic factor BCL2 decreased in the retina; there were no differences in retinal vascular endothelial growth factor (VEGF). In cultured human M?ller cells exposed to eotaxin (CCL11) and VEGF, CCR3i significantly increased TUNEL+ cells and AIF but decreased BCL2 and brain derived neurotrophic factor, without affecting caspase-3 activity or VEGF. CCR3i significantly decreased AIF in RPE/choroids and immunostaining of phosphorylated VEGF receptor 2 (p-VEGFR2) in CNV with a trend toward reduced VEGF. In cultured CECs treated with CCL11 and/or VEGF, CCR3i decreased p-VEGFR2 and increased BCL2 without increasing TUNEL+ cells and AIF. These findings suggest that inhibition of retinal CCR3 causes retinal cell death and that targeted inhibition of CCR3 in CECs may be a safer if CCR3 inhibition is considered as a therapy for neovascular AMD.

Project description:BackgroundAge-related macular degeneration (AMD) is a leading cause of visual impairment in the elderly. The neovascular (wet) form of AMD can be treated with intravitreal injections of different anti-vascular endothelial growth factor (VEGF) agents. Placental growth factor (PGF) is another member of the VEGF family of cytokines with pro-angiogenic and pro-inflammatory effects. Here, we aimed to compare single and combined inhibition of VEGF-A and PGF in the laser-induced mouse model of choroidal neovascularization (CNV) with a focus on the effects on retinal mononuclear phagocytes.MethodsCNV was induced in C57BL/6J mice using a YAG-Laser. Immediately after laser damage antibodies against VEGF-A (aVEGF), anti-PGF (aPGF), aVEGF combined with aPGF, aflibercept, or IgG control were injected intravitreally in both eyes. Three and 7 days after laser damage, the vascular leakage was determined by fluorescence angiography. Lectin staining of retinal and RPE/choroidal flat mounts was used to monitor CNV. In situ mRNA co-expression of Iba1, VEGF and PGF were quantified using in situ hybridization. Retinal and RPE/choroidal protein levels of VEGF and PGF as well as the pro-inflammatory cytokines IL-6, IL1-beta, and TNF were determined by ELISA.ResultsEarly (day 3) and intermediate (day 7) vascular leakage and CNV were significantly inhibited by PGF and VEGF-A co-inhibition, most effectively with the trap molecule aflibercept. While VEGF-A blockage alone had no effects, trapping PGF especially with aflibercept prevented the accumulation of reactive microglia and macrophages in laser lesions. The lesion-related mRNA expression and secretion of VEGF-A and PGF by mononuclear phagocytes were potently suppressed by PGF and partially by VEGF-A inhibition. Protein levels of IL-6 and IL1-beta were strongly reduced in all treatment groups.ConclusionsRetinal inhibition of PGF in combination with VEGF-A prevents vascular leakage and CNV possibly via modulating their own expression in mononuclear phagocytes. PGF-related, optimized strategies to target inflammation-mediated angiogenesis may help to increase efficacy and reduce non-responders in the treatment of wet AMD patients.

Project description:PurposeChoroidal and retinal neovascularization plays an essential role in various ocular diseases. In this study, we examined the role of nestin in this process. Nestin is an intermediate filament protein known to play several roles, including as a marker of neural progenitor and proliferating endothelial cells.MethodsWe used Brown Norway rats, in which choroidal and retinal neovascularization was induced using intraocular laser impacts. The role of nestin was examined using angiography, western blot from the second to the 14th day after laser impacts, and intraocular injection of nestin siRNA. The localization of the protein was specified by co-immunoreactivity with glial fibrillary protein (GFAP), glutamine synthetase (GS), and von Willebrand factor (vWF).ResultsIn the control retina, nestin was found principally in glial structures in the ganglion cell layer, as confirmed by nestin/GFAP immunolabeling. Two days after the laser impacts, the nestin expression extended to numerous radial processes at the site of the impacts. With Bruch's membrane ruptured, these processes penetrated into the choroid. Nestin immunolabeling remained high from the third to the seventh day but appeared reduced on the 14th day. The nature of these processes was not clearly defined, but co-immunolabeling with GFAP suggested that they were principally in activated Müller cells from the third day after the laser impacts. However, the co-immunoreactivity of nestin and GS, a marker of mature functional Müller cells, could be observable only from the seventh day. Nestin was also observed in some vascular cells, as demonstrated by the co-immunoreactivity of the protein with vWF in the choroid and retina. As observed on angiography, the numbers of choroidal and retinal blood vessels were significantly increased (principally on the seventh day) after the laser impacts. An intraocular injection of nestin siRNAs led to a significant decrease in the number of blood vessels.ConclusionsOur results confirmed the presence of nestin in glial (e.g., astrocytes), reactive Müller, and endothelial cells. They demonstrated their critical involvement in a rat model of retinal and choroidal neovascularization experimentally induced using ocular laser impacts.

Dataset Information

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

Publications

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

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