Fig. 5.

CFH levels regulate lipoprotein binding and remove endogenous human lipoproteins in BrM. (A and B) Six-millimeter porcine RPE/BrM tissue explants were incubated with doubling concentrations of CFH (62 nM to 4 μM) in the presence of a 200 μM excess of albumin. (A) The amount of CFH bound to the tissue explant was determined by Western blot of the tissue lysate to determine EC₅₀ binding. (B) Similar porcine tissue explants were incubated 2 μL of lipoprotein and increasing concentrations of CFH (0, 60, 120 nM), and lipoprotein binding to the explants was assessed by Western blot analysis for ApoB-100 and ApoE. Data are expressed as mean ± SE. The asterisk (*) indicates P < 0.05 for ApoE and ApoB-100 by ANOVA compared with 0 nM CFH. Data presented are a representative experiment from three independent experiments (n = 12–15). (C) Because aged human BrM contains endogenous sub-RPE lipoproteins, we tested the ability of CFH to remove these accumulated lipoproteins ex vivo. Anti-CFH (green) immunohistochemistry of aged human BrM donor tissue, before (Top) and after (Bottom) overnight incubation with 1 μM exogenous CFH, shows accumulation of CFH in BrM. (Scale bar: 5 μm.) (D) FPLC fractionation of human BrM lysates shows endogenous lipoproteins present in aged BrM tissue are removed with the addition of CFH (1 μM CFH, green trace). The asterisk (*) indicates P < 0.05 for total cholesterol in each fraction comparing 0 μM CFH to 1 μM CFH. Three independent experiments each with an n = 3 were performed to confirm these results of human BrM FPLC experiments. Actin served as a loading control in A and B.