Figure 1.

Complement regulation and selective diffusion of complement proteins across macular enriched Bruch’s membrane (BrM). (A) When C3b links onto surfaces, it either interacts with factor B (FB) and the complex is cleaved by factor D (FD) to form a C3 convertase (C3bBb) which feeds into the amplification loop, or it interacts with factor H (FH) (or FHL-1) which allows C3b cleavage by factor I (FI), thus preventing a complement response. Complement activation leads to increased release of the anaphylatoxins C3a and C5a, and results in the terminal complement complex which itself can become inserted into cell membranes (referred to as the membrane attack complex) causing cell lysis. (B) Schematic of the human eye highlighting the location of the macula. (C) BrM resides between the retinal pigment epithelium (RPE) cell layer and the choroid. Hallmark lesions of age-related macular degeneration, termed drusen along with diffuse lipid rich deposits called basal linear deposits, form within the BrM and disrupt nutrient flow from the choroid to the RPE cells. (D) An Ussing chamber was used in diffusion experiments, where a: enriched macula BrM; b: sampling access points; and c: magnetic stirrers, are shown. (E) Cropped images of western blots to detect soluble complement proteins in whole human serum and following diffusion through enriched BrM. Weaker band seen in the diffusate chamber for FD could imply an interaction with an unidentified serum partner that impedes its diffusion. Note that the antibodies used in these western blots only recognize one chain of either C3b or FI, thus only a single band is seen. (F) Cropped images of Instant Blue stained polyacrylamide gels following the introduction of purified complement proteins to the sample chamber, showing that the same complement proteins crossed enriched BrM as when serum was used in the experiments, but in this case, similar concentrations of FD were similar in the sample and diffusion chambers.