Abstract
Purpose :
To investigate the expression and localisation of complement cascade components in the RPE/Bruch’s membrane/choroid complex.
Methods :
Human donor eye tissue was obtained from Manchester Eye Tissue Repository. RNA was isolated separately from the RPE and choroid and analysed by RNA-Seq following ribosomal depletion. RNA-Seq was performed on cultured primary RPE cells, ARPE19 and hTERT cells. Immunofluorescent localisation studies were performed on frozen sections of lightly fixed, macular RPE/Bruch’s membrane/choroid using antibodies against FH, FHL-1, FHR-4, C3 and C5b-9.
Results :
The gene expression studies demonstrated that only some components of the complement cascade are expressed to a significant extent by RPE (analysing both directly isolated RNA and RNA from cultured RPE cells) and the choroid. C3 and CFH were expressed at high levels by both the RPE and choroid. By contrast, the expression of the CFHR genes, lectin pathway and terminal pathway (with the exception of C7) genes was absent or negligible. Immunolocalisation studies demonstrated FH, FHL-1, FHR-4, C3 and C5b-9 in the extracellular matrix of the choriocapillaris including the intercapillary septa, but these were not localised within Bruch’s membrane, apart from FHL-1. However, in eyes with early AMD, these complement components were observed within drusen and the underlying Bruch’s membrane.
Conclusions :
Complement expression is limited to specific components of the cascade within the RPE and choroid. Complement components including FHR-4 and terminal pathway proteins reach the extracellular matrix around the choriocapillaris from the circulation where complement activation occurs. Bruch’s membrane normally provides a barrier to their further diffusion into the retina, but in early AMD there is focal breakdown of the barrier properties of Bruch’s membrane permitting these complement proteins to contribute towards drusen formation.
This is a 2020 ARVO Annual Meeting abstract.