September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Permeability of Bruch’s membrane to complement proteins: Implications for translational medicine in AMD
Author Affiliations & Notes
  • Simon John Clark
    Faculty of Medicine and Human Sciences, University of Manchester, Manchester, Greater Manchester, United Kingdom
    Central Manchester University Hospitals NHS Foundation Trust, Centre for Advanced Discovery and Experimental Therapeutics, Manchester, United Kingdom
  • Selina McHarg
    Faculty of Medicine and Human Sciences, University of Manchester, Manchester, Greater Manchester, United Kingdom
  • Nicole Brace
    Faculty of Medicine and Human Sciences, University of Manchester, Manchester, Greater Manchester, United Kingdom
  • Paul N Bishop
    Faculty of Medicine and Human Sciences, University of Manchester, Manchester, Greater Manchester, United Kingdom
    Central Manchester University Hospitals NHS Foundation Trust, Centre for Advanced Discovery and Experimental Therapeutics, Manchester, United Kingdom
  • Footnotes
    Commercial Relationships   Simon Clark, None; Selina McHarg, None; Nicole Brace, None; Paul Bishop, None
  • Footnotes
    Support  MRC Career Development Fellowship MR/K024418/1
Investigative Ophthalmology & Visual Science September 2016, Vol.57, No Pagination Specified. doi:
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      Simon John Clark, Selina McHarg, Nicole Brace, Paul N Bishop; Permeability of Bruch’s membrane to complement proteins: Implications for translational medicine in AMD. Invest. Ophthalmol. Vis. Sci. 201657(12):.

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      © ARVO (1962-2015); The Authors (2016-present)

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  • Supplements
Abstract

Purpose : The dysregulation of the complement system is implicated in the pathogenesis of age-related macular degeneration (AMD), where variants in genes encoding complement proteins are known to greatly modify risk of disease. Complement proteins are mainly synthesized in the liver, but can also be made locally by retinal pigment epithelial (RPE) cells. The relative contribution made between systemic and locally produced complement on immune homeostasis at the RPE cell/Bruch’s membrane interface, and the underlying choriocapillaris, remains unknown. Here, we investigate the ability of complement proteins to diffuse across Bruch’s membrane and reach their respective site of function within the eye.

Methods : Using modified Ussing chambers and Bruch’s membrane enriched from human donor eyes, we assess the passive diffusion of complement across Bruch's membrane. Western blots identify the presence of complement components in the diffusate chamber. Functional tests (i.e. C3b breakdown assays) are used to assess if protein below our level of detection is still capable of diffusing across Bruch’s membrane and mediating C3b breakdown. Also, the local expression of complement proteins by RPE cells is investigated using primary adult RPE cells harvested from postmortem eyes.

Results : We demonstrate for the first time that specific complement proteins cannot diffuse through Bruch’s membrane in either direction. In some cases this may be due to the glycosylation state of the complement protein. Factor I is not able to diffuse across Bruch’s membrane and mediate C3b breakdown. Furthermore, we demonstrate local expression of complement proteins necessary for maintaining immune homeostasis on Bruch’s membrane by primary adult RPE cells.

Conclusions : While certain systemic complement proteins will contribute to immune homeostasis of the choriocapillaris and surrounding stroma, they cannot help protect from the natural tick-over of the alternative pathway of complement on the Bruch’s membrane interface with the RPE cell monolayer, and vice versa. This means that targeting complement turnover at this site (i.e. the site of drusen formation) must be directed via RPE cell synthesis directly. Similarly, readdressing complement turnover in the choriocapillaris must be mediated via the choroid. These findings must be taken into account when developing any complement mediated therapeutic strategies for treating AMD.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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