Abstract
Purpose :
AMD is a leading cause of vision loss worldwide. Homeostatic disruption of lipid handling, particularly in terms of oxidized phosholipids (oxPLs), disrupts tightly controlled inflammatory suppression and can lead to AMD pathology. Our lab previously demonstrated a putative link between risk-associated variation in complement factor H (CFH) and oxidative stress in that risk genotypes lead to decreased association with oxPLs, which results in decreased protection of RPE from accumulation of lipids and inflammation. As expression of CFHR1 is also associated with increased AMD risk, we hypothesized the conferred risk was a result of similar impaired oxPL interaction.
Methods :
Binding of CFH or CFHR1 protein to native or oxLDL was assessed by ELISA. Lipid/CFH(R1) complex effect on stimulation of pro-inflammatory cytokines was assessed in ARPE-19 cells post 18 hours incubation via qPCR for inflammatory markers. For lipid accumulation studies, ARPE-19 cells were incubated with 25 µg/ml concentration of native or oxLDL (+/- CFH or CFHR1) for 48 hours. After incubation, the cells were fixed and stained with heated Oil red O/propylene glycol and lipid accumulation quantified as lipid droplets/cell per total 100 cells counted.
Results :
CFHR1/oxPL association was not statistically different than CFH (CFH: 98136 RLU/ms ± 11250 v.s. CFHR1: 87643 RLU/ms ± 12054, MEAN ± SD). CFH incubation with oxLDL was able to reduce the inflammatory cytokine IL-6 (OxLDL: 2.35 ± 0.23 v.s. OxLDL/CFH: 1.43 ± 0.16, relative fold change to native LDL normalized with GAPDH). CFHR1 did not elicit the same protective benefits (OxLDL: 2.35 ± 0.23 v.s. OxLDL/CFHR1: 2.23 ± 0.29, relative fold change to native LDL normalized with GAPDH). The lipids droplets number accumulated in ARPE-19 cell were also not impacted by CFHR1 (Nat-LDL: 1.6 ± 0.5 v.s. OxLDL: 13.7 ± 1.6 v.s. OxLDL/CFH: 6.4 ± 0.7 v.s. OxLDL/CFHR1: 12.3 ± 1.4. lipids droplets/cell, Mean ± SD).
Conclusions :
Contrary to our original hypothesis, CFHR1-associated risk for AMD is not due to impaired binding of oxPL molecules. However, CFHR1 interaction with oxPLs does not attenuate inflammation or lipid accumulation in RPE cells. CFHR1 is highly homologous to CFH in its included domains, but is overall smaller and lacks some of the domains contained in CFH. As a result, future studies will dissect the domains within CFH necessary for protective benefits.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.