Abstract
Purpose:
Complement activation and oxidative stress has been increasingly implicated in the pathogenesis of AMD. We previously reported a new model whereby oxidative stress sensitizes RPE cells to complement attack by the alternative complement pathway. Herein, we pursued proteomic analyses to screen potential candidate molecules/pathways to better understand the mechanisms of RPE injury in this model.
Methods:
Cultured hRPE cells were stimulated with or without hydroquinone (HQ) for 1.5 hours, primed with an anti-ARPE-19 antibody for 30 minutes followed by incubation for 6 hours with either 6% C1q-depleted human serum (C1q-Dep) to initiate complement attack, or 6% heat inactive C1q-Dep. Quantified proteins were electrophoresed. Stained bands were excised and subjected to in-gel tryptic digestion. The peptide mixes were subjected to a LC-MS/MS. Three independent protein quantifications were conducted by three spectra counting methods: the normalized spectral abundance factor, the normalized weighted spectra, and the exponentially modified protein abundance index. Percentage of CV (SD/average) per protein of 4 treatments greater than 100% (≥2 average of CV%) from three methods were considered as detected altered proteins.
Results:
Over 1000 proteins were compared. As expected, and as controls for the model, complement components such as C3, C5 and C9 were deposited in cells incubated with complement or HQ+complement, but not in control- or HQ-treated cells. Heme oxygenase-1 (HO-1) and hexokinase-1 were elevated in cells stimulated with HQ and HQ+complement when compared to controls and complement alone. HO-2 levels were not altered by any of the treatments. Detected proteins altered by complement alone were predominantly involved in calcium homeostasis, regulation of cell signaling processes and energy metabolism. Proteins altered by HQ alone were involved in cellular protein metabolic process, apoptosis and cellular iron ion homeostasis. Proteins altered by the combination of complement and HQ were involved signal transduction, gene expression and response to stress.
Conclusions:
Complement and oxidative stress altered several proteins that are involved in key cellular processes. These results may help to improve our understanding of complement- and oxidative stress-mediated RPE injury, and provide insight into potential therapeutic targets.
Keywords: 412 age-related macular degeneration •
701 retinal pigment epithelium •
663 proteomics