In mouse CNV, a role for the alternative complement pathway has been demonstrated by inhibition of the AP by CFB siRNA,
32 CFB gene knockout, and treatment with the inhibitor CR2-fH.
21 In these studies, CFB deficiency or AP inhibition was associated with a significant reduction in the size of CNV following laser injury, preserved retinal function, and decreased VEGF mRNA expression. After siRNA injection, CFB mRNA and protein was reduced to <95% within the eye, liver, and spleen, eliminating the possibility to examine tissue-specific contributions. In patients, the question whether global, liver-derived, or local, RPE-derived, production of complement is relevant for progression of AMD has been addressed in liver transplant patients. Lotery and colleagues
15 published a retrospective study on >200 liver transplant patients, documenting that AMD, 5 years after the transplant, was associated with recipient
CFH genotype. Rather than performing liver transplants, we generated mice expressing CFB uniquely in the RPE (i.e., CFB driven by RPE-specific promoter and crossed onto CFB-KO mice), the results of which demonstrated that local CFB expression and secretion in the eye appears to be sufficient to drive pathology. However, additional contributions by CFB recruited from the bloodstream cannot be excluded under normal conditions, since CFB-KO mice partially reconstituted with CFB-sufficient wild-type mouse serum developed larger CNV lesions than littermates injected with PBS only. After 200-μL serum application, assumed to result in maximum reconstitution of CFB to ∼35% to 43% of wild-type levels (based on a blood volume of 1.2–1.5 mL for an adult mouse and a blood-to-serum ratio of 3:1), mice exhibited ∼20% to 14% of wild-type levels of AP activity and generated 10% to 30% of maximal C3a levels when measured after 4 and 48 hours, respectively. These data suggest that mouse CFB is a relatively stable protein in blood, presumably supplying CFB for consumption in tissues, including the eye. Similar stability has been reported for human CFB, with a half-life of ∼72 hours.
45 However, it is not clear how much of the systemically injected CFB is recruited to the CNV lesions. Likewise, the experiments utilizing the CFB-tg also did not provide a clear answer as to how much CFB is required to drive pathology in the eye, since we could not cleanly separate RPE and choroid in the mouse eye and hence determined the presence of CFB in the combined RPE-choroid samples from nonperfused animals. In addition, the amount generated, or importantly, the amount locally secreted might be significantly higher under CNV conditions; since we showed here that RPE cells from CFB-tg in culture secrete higher levels of CFB under oxidative stress conditions when compared to baseline.