Complement dysregulation in AMD: RPE-Bruch's membrane-choroid

Abstract

The question as to why the macula of the retina is prone to an aging disease (age-related macular degeneration) remains unanswered. This unmet challenge has implications since AMD accounts for approximately 54% of blindness in the USA (Swaroop, Chew, Bowes Rickman and Abecasis, 2009). While AMD has onset in the elder years, it likely develops over time. Genetic discovery to date has accounted for approximately 50% of the inheritable component of AMD. The polymorphism that has been most widely studied is the Y402H allele in the complement factor H gene. The implication of this genetic association is that in a subset of AMD cases, unregulated complement activation is permissive for AMD. Given that this gene variant results in an amino acid substitution, it is assumed that this change will have functional consequences although the precise mechanisms are still unknown. Genetic predisposition is not the only factor however, since in this complex disease there is substantial evidence that lifestyle factors such as diet and smoking contribute to risk. Here we provide an overview of current knowledge with respect to factors involved in AMD pathogenesis. Interwoven with these issues is a discussion of the significant role played by aging processes, some of which are unique to the retina and retinal pigment epithelium. One recurring theme is the potential for disease promotion by diverse types of oxidation products.

Fig. 1

A2E photooxidation and photodegradation. UPLC-ESI/MS, (ultra performance liquid chromatography-electrospray ionization/mass spectrometry) detection of A2E and oxidized A2E in ARPE-19 cells that accumulated A2E and were irradiated for 2, 6, 10 and 20 min. The more polar photooxidized forms of A2E elute ahead of the parent A2E and isoA2E compounds. Shown are three chromatographic peaks that on the basis of molecular weight (m/z 608; insets above) can be identified as mono-oxidized A2E (molecular weight of A2E, 592; 592 + 16; m/z 608). Reductions in peak areas of A2E/isoA2E under conditions of 430 nm irradiation are indicative of A2E photooxidation/photodegradation. Note that as the duration of irradiation is extended, A2E/isoA2E peak areas decrease as a result of photooxidation; yet the areas of the m/z 608 peaks do not increase because of photodegradation.

Fig. 2

Vitamin E decreases photooxidation and photodegradation of A2E and suppresses complement activation. ARPE-19 cells that had accumulated A2E were pre-treated or untreated with vitamin E (100 μM) for 24 h before 430 nm irradiation. (A) Pre-treatment with vitamin E reduces A2E photooxidation when cells are irradiated at 430 nm. A2E was quantified by HPLC with reference to known quantities of authentic A2E. The photooxidation of A2E is reflected in the reduced content of A2E in a sample after 430 nm illumination. The loss of A2E was diminished in the presence of vitamin E. (B) iC3b generation in serum overlying A2E-containing RPE was measured by enzyme immunoassay. iC3b production is reduced when the cells had been pre-treated with vitamin E. iC3b content in serum incubated in an otherwise empty well was subtracted as background. Mean ± SEM of five experiments; duplicate wells per experiment.

Fig. 3

Complement activation in the presence of complement factor H (CFH) peptide, CFH protein and CFH Y402H polymorphisms. ARPE-19 cells that had accumulated A2E were irradiated to cause photooxidation and photodegradation of A2E. Complement activation was monitored by measuring production of the C3 cleavage product iC3b in normal human serum overlying the RPE cells; iC3b was measured by enzyme immunoassay as described (Zhou et al., 2006). Serum incubated in empty wells was subtracted as background. Mean ± SEM; three experiments, duplicate wells per experiment; +, presence of condition. (A) A recombinant human peptide encompassing CCP 6, 7 and 8 of CFH (gift of Dr. Michael K. Pangburn, University of Texas Health Science Center) was added to normal human serum before irradiation. The Y402H polymorphism resides in CCP 7 and the peptide carried the Y402 form. (B) Whole human CFH (Quidel, San Diego CA) was added to normal human serum before irradiation. (C) Complement activation in the presence of serum obtained from donors with risk CFH 402H polymorphism (genotype 1277CC) and non-risk CFH Y402 variant (genotype 1277TT).