RNA sequencing reveals altered gene expression signatures in complement challenged primate choroidal endothelial cells

Abstract

Purpose: We hypothesized that activation of the complement membrane attack complex (MAC) within the choroid may alter homeostatic gene expression in endothelial cells, creating an environment conducive to AMD. To test this hypothesis, we challenged primate choroidal endothelial cells with MAC and measured differential gene expression by RNA-Seq.

Methods: Rhesus macaque RF/6A choroidal endothelial cells were grown in six-well culture plates and treated with either 50% non-heated (n=3) or heat inactivated (n=3) human serum for 4 hours. Additional wells were used for MAC immunocytochemistry. Following RNA extraction, samples were prepared by The University of Iowa DNA Core Facility for sequencing on an Illumina HiSeq 2000. Sequence reads were mapped to the Ensembl macaque genome build MMUL_1 using Tophat2 (ver. 2.0.6), transcript abundance was estimated using Cufflinks (ver. 2.0.2), and differential expression was assessed using CuffDiff (ver. 2.0.2) and cummeRbund (ver. 2.2.0). Functional annotation clustering was performed with DAVID (ver. 6.7), using the MINT, UP_TISSUE, and UCSC_TFBS datasets in addition to the default human annotations.

Results: Formation of the MAC was confirmed on cells exposed to normal, but not heat inactivated, serum. RNA-Seq analysis showed 850 differentially expressed genes (q-value <= 0.001 and two fold expression up or down) of 11,732 reliably expressed genes (FPKM >= 1 in all replicates of either treated or control cells). Of differentially expressed genes, we submitted the top 200 genes (ordered on q-value; ties ordered on decreasing magnitude of fold change) to DAVID. Genes with increased expression in MAC-injured endothelial cells included molecules (a) implicated in angiogenesis and blood vessel development, (b) involved in TGF-beta or BMP pathways, and (c) secreted as signaling proteins. Genes with decreased expression in MAC-injured cells included those (a) bound by thirteen transcription factors, (b) containing SH3-domains, and (c) containing fibronectin-3 domains.

Conclusions: Complement activation of choroidal endothelial cells alters a wide range of molecular pathways, including a robust increase in genes associated with angiogenesis.

Keywords: 412 age-related macular degeneration • 452 choroid • 533 gene/expression