gms | German Medical Science

Purpose: Myeloid cells (MC), such as resident microglia and infiltrating blood-derived monocytes, are key players in the formation of choroidal neovascularization (CNV). However, their precise function during CNV development remains unclear. In this study, we performed RNA-sequencing analysis (RNASeq) of human CNV (hCNV) membranes and isolated murine MC in the laser-induced CNV model to further unveil MC function in CNV development.

Methods: RNA sequencing was performed on four formalin-fixed-paraffin-embedded (FFPE) hCNV membranes that were extracted during vitreoretinal surgery between 1992 and 1999. Four age-matched FFPE RPE-choroidal specimens that were obtained from the macula of enucleated eyes suffering from ciliary body melanoma served as controls. Furthermore adult C57Bl6/J wildtype mice (n=40) were exposed to the laser-induced CNV model and MC were sorted by flowcytometry and used for RNASeq. Untreated littermates (n=40) served as controls. Transcriptome profiles were generated and bioinformatic methods were employed to identify disease-associated gene signatures. SPP1 (osteopontin) protein analysis was performed by ELISA and immunohistochemistry (IHC).

Results: We identified 1797 genes that were significantly upregulated (p<0.05, log-fold-change (logFC)>2) in human CNV compared to control RPE-choroidal samples. Among the known angiogenic factors, SPP1 belonged to the most strongly upregulated gene transcripts in hCNV compared to control tissue (logFC=4.8, p=2.2E-20). IHC demonstrated that SPP1 was expressed in MC at sites of hCNV. SPP1 was also highly upregulated in murine MC following laser-induced CNV formation (logFC=7.06, p=2.9E-18). Immunohistochemistry and ELISA analysis demonstrated that SPP1 expression was increased in MC around laser-induced CNV and in the choroid of lasered mice compared to controls (p = 0.0003).

Conclusion: Our study demonstrates that SPP1, which is a known secreted angiogenic factor, is highly upregulated in hCNV membranes and in isolated murine retinal MG in laser-induced CNV. Further studies need to investigate the specific role of SPP1 in CNV development to determine its potential as a therapeutic target.