Complement factor H genotypes impact risk of age-related macular degeneration by interaction with oxidized phospholipids

Abstract

The rs1061170T/C variant encoding the Y402H change in complement factor H (CFH) has been identified by genome-wide association studies as being significantly associated with age-related macular degeneration (AMD). However, the precise mechanism by which this CFH variant impacts the risk of AMD remains largely unknown. Oxidative stress plays an important role in many aging diseases, including cardiovascular disease and AMD. A large amount of oxidized phospholipids (oxPLs) are generated in the eye because of sunlight exposure and high oxygen content. OxPLs bind to the retinal pigment epithelium and macrophages and strongly activate downstream inflammatory cascades. We hypothesize that CFH may impact the risk of AMD by modulating oxidative stress. Here we demonstrate that CFH binds to oxPLs. The CFH 402Y variant of the protective rs1061170 genotype binds oxPLs with a higher affinity and exhibits a stronger inhibitory effect on the binding of oxPLs to retinal pigment epithelium and macrophages. In addition, plasma from non-AMD subjects with the protective genotype has a lower level of systemic oxidative stress measured by oxPLs per apolipoprotein B (oxPLs/apoB). We also show that oxPL stimulation increases expression of genes involved in macrophage infiltration, inflammation, and neovascularization in the eye. OxPLs colocalize with CFH in drusen in the human AMD eye. Subretinal injection of oxPLs induces choroidal neovascularization in mice. In addition, we show that the CFH risk allele confers higher complement activation and cell lysis activity. Together, these findings suggest that CFH influences AMD risk by modulating oxidative stress, inflammation, and abnormal angiogenesis.

Fig. 1.

(A) Plasma CFH binds preferentially to oxLDL rather than to native LDL. (B) Plasma CFH binding to oxLDL stratified by Y402H genotypes. (C) Differential binding of recombinant CFH402Y and CFH402H to oxidative epitopes of POVPC and oxLDL with BSA and native LDL as controls. Data are expressed in RLU and as mean ± SEM. ***P < 0.001. (D) Plasma oxPLs/apoB level detected by anti-PC antibody T15 stratified by CFHY492H genotypes. (E) Complement-mediated hemolysis stratified by CFH Y402H genotypes. Heat-inactivated normal human sera that contained inactive CFH were used as a negative control for complement activation inhibition. Data are shown as Z values and as mean ± SEM.

Fig. 2.

(A and B) Gene expression as assayed by quantitative PCR on RNA from ARPE19 (A) or J774 (B) macrophages treated with 50 μg/mL of oxLDL for 6 h. Relative mRNA levels were calculated by normalizing results with GAPDH and are expressed relative to untreated samples. n = 4. (C and D) Inhibition of binding of oxLDL to RPE (C) and J774 (D) macrophages by CFH rs1061170 (Y402H) alleles. Cultured RPE or mouse J774 macrophages were incubated with biotin-oxLDL premixed with serial dilutions of plasma of the CFH CC (risk) allele (402H, n = 7) or the TT (protective) allele (402Y, n = 7) genotypes. The biotin-oxLDL bond was detected with NeutrAvidin-AP. The data are expressed as the ratio of B/B_0, where B is binding with CFH and B₀ is binding without CFH as a competitor. Nat-LDL was used as a negative control. Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

(A–C) Induction of CNV by subretinal injection of oxLDL. (A) Following subretinal injection of oxLDL, marked neovascularization was found in an injection site. (B) Subretinal injection of oxLDL also induced increased vascular activity in the subretinal space, which was closely associated with F4/80-positive macrophages (arrows). Vascularity activity was visualized with isolectin staining (green), and macrophages were visualized with F4/80 staining (red). Cell nuclei were counterstained with DAPI (blue). (C) As a negative control, nat-LDL injection did not promote CNV or macrophage activation. (D–I) Localization of VEGF in CNV. Double labeling of neovascularization by isolectin (green, D and G), and VEGF (red, E and H) demonstrates that VEGF was localized within a neovascular complex. G, H, and I show magnified images of the boxed area in D. (Scale bars: 100 μm.)

Fig. 4.

Colocalization of oxPC with CFH in human AMD lesions. Immunohistochemistry of serial sections of an AMD eye stained with antibodies to CFH (B, blue color) or oxPC (D, pink color). Omission of the first antibody served as a negative control (A and C). (Scale bar: 50 μm.)