gms | German Medical Science

Purpose: Although genome-wide association studies, animal models, and cell culture systems have yielded important insights into the pathogenesis of neovascular age-related macular degeneration (nAMD), the underlying molecular pathways remain ill defined. The current study aims to explore the transcriptome of human CNV membranes via comparative RNA-sequencing-based transcriptional analysis.

Methods: Massive Analysis of cDNA Ends (MACE) RNA sequencing was performed on four formalin-fixed and paraffin-embedded (FFPE) CNV membranes extracted from patients with nAMD during vitreoretinal surgery between 1992 and 1999. Four age-matched FFPE RPE-choroidal specimens obtained from the macular region of enucleated eyes suffering from ciliary body melanoma served as controls. Transcriptome profiles were generated for CNV membranes and control tissue and statistical and bioinformatic Methods were employed to identify disease-associated gene signatures. Calprotectin (S100A8/A9) protein expression was investigated by immunohistochemistry on paraffin sections and ELISA on vitreous samples of four patients with nAMD and six patients without AMD.

Results: We identified 777 DEG (log2FC > 2, FDR <0.05, mean counts in human CNV samples > 100) that were significantly increased in CNV membranes compared to control tissue. Gene ontology (GO) enrichment analysis demonstrated that most of these DEG contributed to biological processes, such as Symbiont Process (p=3.0E-9), Blood Vessel Development (p= 1.7E-7), Extracellular Structure Organization (p= 4.3E-18), Leucocyte Degranulation (p= 2.9E-12) and Response to Wounding (p= 4.9E-8). The S100 calcium-binding protein A8 (S100A8) and S100A9 emerged among the top differentially-expressed genes in human CNV membranes, confirmed by immunohistochemistry on CNV tissue samples and protein analysis of the S100A8/A9 heterodimer (Calprotectin) in vitreous and plasma samples of nAMD patients (35.86 ± 15.06) and controls (2.856 ± 0.586, p = 0.02).

Conclusions: This study provides a high-resolution RNA-sequencing-based transcriptional signature of choroidal neovascular membranes in AMD patients and reveals S100A8/A9 as a novel biomarker and promising target for AMD-directed therapeutics and diagnostics.