Macrophages induce choroidal senescence in an in vitro model of 3D RPE/choroid to cause neovascular AMD-like phenotypes

Abstract

Purpose : Purpose: Activated macrophages in the outer blood-retinal barrier (oBRB) environment have been noticed as one of the key immunological factors of AMD. In order to provide a model that allows discovering the effect of immune cells, we integrated activated macrophages in our established in vitro 3D-bioprinted oBRB model. Here we aim to investigate the mechanisms behind neovascular AMD (nAMD) initiation and progression in this 3D-oBRB model with activated M1 and M2 macrophages.

Methods : Methods: GFP expressing endothelial cells (ECs), choroidal fibroblasts, and pericytes were mixed in a fibrin-based hydrogel (choroidal-bioink) and 3D-bioprinted on the bottom side of degradable poly-(lactic-co-glycolic) acid (PLGA) scaffold. Polarized M1 and M2 macrophages differentiated from human primary peripheral blood monocytes were added to the basal (non-RPE) side of 3D-bioprinted “choroid”. One week after capillary formation in the 3D-choroid with or without addition of macrophages, iPSC-derived RPEs were seeded on the top side of PLGA scaffold. Confocal microscopy, trans-epithelial electrical resistance (TER) measurements, and vascular maturations were used to analyze 3D tissue.

Results : Results: When in vitro activated M1 and M2 macrophages were mixed in either 1:1, 2:1 or 1:2 ratios with the choroidal-bioink, increased capillary growth in the choroid space was observed at one-week post printing, compared to the no macrophage or M1 or M2 macrophage only added tissues. Five weeks post printing, mixed 2:1 M1:M2 macrophage treatment caused type-I choroidal neovascularization (CNV) with the 2:1 M1:M2 ratio causing the most severe increase. Interestingly, the RPE monolayer demonstrated the most decreased TER value when 1:2 M1:M2 macrophages were added to the tissue. Macrophages take on “bloated” foam-cell like morphologies, similar to what is seen in patients with advanced AMD. Differential expressions of genes related to the senescence and angiogenic pathways were observed in the 2:1 M1:M2 group compared to no macrophage or M2 only macrophage groups.

Conclusions : Conclusions: Macrophages have beneficial or detrimental effects on capillary development or pathology based on the ratio of M1 and M2 macrophages – likely via senescent changes in the choroid. This 3D tissue model allows for the study of ocular immune populations, and their role in oBRB homeostasis and subsequent RPE physiology.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.