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 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.

The role of interferon regulatory factor 8 for retinal tissue homeostasis and development of choroidal neovascularisation

“…This suggests that Irf8 -deficient MG are functionally sufficient to accompany physiological retinal development and that minor developmental disturbances, which we cannot completely rule out, can be compensated over time. The fact that Irf8 -deficient microglia exhibit transcriptional changes that reduce their responsiveness while maintaining a normal retinal phenotype, provides an ideal setting to study the role of microglia in the development of choroidal neovascularisation, which is known to be associated with significant microglia activation and migration [ 35 , 52 , 59 ].…”

“…During adulthood, rMG interact closely with synapses to maintain synaptic structure and electroretinal function and continuously scan the local environment for danger signals associated with injury or pathogens [ 56 ]. In response to tissue damage or infection, rMG rapidly attain an activated phenotype, migrate towards the site of injury and contribute to phagocytosis, inflammation and pathological events [ 4 , 52 , 59 ]. As such, activated microglia cells have been found in the subretinal space of patients with age-related macular degeneration (AMD) and in particular at sites of choroidal neovascularisation (CNV) in neovascular AMD [ 11 , 20 ] which is a common cause of irreversible blindness in the elderly [ 10 , 60 ].…”

Subretinal fibrosis in neovascular age-related macular degeneration: current concepts, therapeutic avenues, and future perspectives

“…Histological studies showed that choroidal neovascular membranes are composed of ECM components such as collagen, fibronectin, and laminin (Grossniklaus and Green 1998;Grossniklaus et al 1994;Hinton et al 1998a, b;Lopez et al 1996). Recent RNA sequencing analysis of human CNV membranes furthermore revealed increased COL6A1, COL3A1, SPP1, and SPARC expression in human CNV membranes compared to control tissue (Schlecht et al 2020). Furthermore, periostin, which is a ligand for several integrins and thus supports epithelial cell adhesion and migration, is an important ECM component in human MNV (Yoshida et al 2019) and has been discussed as a possible anti-fibrotic therapeutic target (Nakama et al 2015).…”

“…3). Most recently, these investigations were complemented by RNA sequencing and in silico analyses of human CNV specimens, providing additional insight into the cellular composition (Schlecht et al 2020). Further evidence on the cellular mechanisms underlying CNV formation is based on experimental CNV models.…”

“…However, they can also adopt an anti-inflammatory phenotype that promotes tissue remodelling and repair through removal of cellular and myelin debris, degradation of scar tissue, and production of neurotrophic factors in later stages (D'Ambrosi and Apolloni 2020). For example, resident microglia contribute to the formation of the gliotic barrier by expressing the matricellullar protein Osteopontin (OPN, gene name SPP1) at sites of human and murine CNV (Schlecht et al 2020(Schlecht et al , 2021. OPN is expressed by myeloid cells after ischemic or traumatic brain injury and contributes to the formation of the gliotic barrier by binding to α (v) β 3 -integrin on astrocytes (Ellison et al 1998).…”