Abstract
Purpose :
Macrophages are key pathogenic cells in neovascular age-related macular degeneration. However, macrophages are heterogeneous cells whose function depends upon their microenvironment and/or origin. Choroidal macrophages are derived from blood monocytes, and classical monocyte-derived macrophages (MDMs) are necessary for laser-induced choroidal neovascularization (CNV). We hypothesized that a subtype of classical MDMs stimulates angiogenesis during laser-induced CNV.
Methods :
Female wildtype (WT) and Ccr2-/- mice underwent no treatment or laser injury. On Day 3, eyes were harvested, digested into single cell suspensions, and CD45+ cells were isolated by fluorescence-activated cell sorting for single cell RNA-sequencing. Cell Ranger was used for genome alignment and empty droplet exclusion. The data were loaded into Seurat v3 for quality control metrics and cell clustering. Hypergeometric distribution analysis determined which genotype and treatment contributed to each cluster. Differential expression (DE) analysis was performed between each macrophage cluster and all other clusters. GOrilla gene ontology analysis was used to determine the function of each macrophage cluster.
Results :
We sequenced 34,215 cells with an average 36,890 reads per cell. We identified 12 total macrophage clusters, including 6 microglia subtypes and 6 macrophage subsets. DE and GO analysis found that only one cluster was enriched for positive regulation of angiogenesis (6.3-fold, q<0.05). DE genes contributing to positive regulation of angiogenesis included Vegfa (vascular endothelial growth factor), Anxa1/2/4/5 (annexins), Il1b (interleukin-1 beta), Fn1 (fibronectin), Mmp12/14 (metalloproteinases), and Spp1 (osteopontin). This Spp1+ cluster was specifically derived from the WT + laser group. In addition, Spp1+ MDMs were enriched for glycolysis and lipid catabolism GO terms, two processes implicated in macrophage-driven angiogenesis. Finally, Spp1+ MDMs specifically express the CD11c receptor.
Conclusions :
Spp1+ MDMs express a transcriptome including VEGF and cytokine signaling, matrix metalloproteinases that degrade basement membranes, annexins to increase endothelial sprouting, and fibronectin for scaffolding that stimulates angiogenesis by multiple pathways. Spp1+ MDMs express specific cell surface markers like CD11c, which can be used for therapeutic ablation.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.