Choroidal Endothelial cells synthesize CFH locally to protect against complement mediated injury.

Abstract

Purpose : Age related macular degeneration (AMD), the most prevalent form of central vision loss worldwide, is a complex disease. Genetic risk factors have been identified in multiple complement pathway genes, most notably a polymorphism in the CFH gene that encodes a Y402H substitution. CFH protein is a negative regulator of the alternative complement cascade and Y402H is thought to impair its function at blocking complement activation. Consequently, dysregulation of complement may lead to choroidal endothelial cell (CEC) death via membrane attack complex (MAC) formation contributing to AMD pathogenesis. Given the abundance of MAC in the choroid we hypothesized that CECs produce CFH to counteract complement activation. We further tested whether CFH genotype affects MAC formation on iPSC derived CECs.

Methods : Human donor eyes (n=3) were obtained and the macular RPE was mechanically separated from the choroid, prior to RNA isolation from both fractions. Purity of RPE/choroid separation was confirmed by qPCR of choroidal markers, vWF and CA4, and RPE markers, BEST1 and RLBP1. We then examined levels of CFH and CFHL1 mRNA in RPE and choroid. We generated iPSC-derived CECs from individuals with different CFH risk genotypes, which were then treated with: 25% human serum; 25% heat-inactivated serum; serum-free medium; or 25% C2-depleted human serum and formation of MAC was quantified by immunofluorescence using a microplate reader.

Results : Separation of RPE from choroid cells was successfully confirmed by positive enrichment of transcripts. The choroid expressed higher levels of both CFH (8 fold change, p < 0.0001) and CFHL1 (9 fold change, p < 0.0001) compared to the RPE. Given the high levels of CFH mRNA in the choroid, we assessed whether individuals with high-risk CFH genotypes were more susceptible to complement activation on iPSC derived CECs compared to cells with low risk genotypes. The abundance of MAC was significantly elevated on cells harboring the CFH Y402H risk allele compared to cells with low risk genotypes (p < 0.05).

Conclusions : The choroid is the major source of CFH production in the human posterior pole. Individuals with high risk CFH genotypes are more susceptible to MAC formation, consistent with findings in genotyped donor eyes. A greater understanding of the mechanism leading to MAC mediated CEC loss may aid in the identification of therapeutic targets to prevent AMD progression.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.