In our analyses, we found an independent relationship between activation fragments Bb and C5a and advanced AMD. Our study provides new information regarding the association between the complement system and AMD in several ways. We evaluated each complement component or fragment, both with and without seven AMD genotypes, controlling for other covariates including age, sex, smoking, and BMI. We found a significant association between median levels of fragments Bb and C5a and advanced AMD as well as the GA group separately. There was a significant inverse association between the median level of CFH component and the total AMD group and the GA group separately. Median C3a level was significantly higher among GA cases, but this association was not significant after adjustment for other genetic and nongenetic factors. Some associations were stronger in GA cases than NV cases, compared with controls. Reasons for this are unclear, and additional studies may clarify the biologic mechanisms responsible for these differences in advanced AMD.
Bb is a catalytic domain of the alternative pathway C3 convertase, C3bBb. Upon decay or disassociation of this two-part protein complex by complement regulators such as the membrane protein decay accelerating factor (DAF, CD55) or the plasma protein factor H, Bb is liberated into the surrounding milieu. Thus, it is a marker of alternative pathway activation and increased levels provide strong evidence for engagement of the alternative pathway in AMD patients. Of note, elevated levels of Bb were also recently reported in preeclampsia, another condition in which excessive alternative pathway activation has been implicated in etiopathogenesis.25
Preeclampsia is characterized by small vessel angiopathy and rare mutations in the complement regulatory proteins of the alternative pathway have also been identified in this condition and in the hemolytic uremic syndromes, another thrombomicroangiopathy.25,26
We also found associations between higher BMI and activation fragments C3a, iC3b, and component CFH among the control subjects, as well as associations between BMI and components C3, CFB, and CFH among the entire study population, when controlling for AMD. Higher BMI is a known risk factor for the development of AMD,14,17,24
it has been shown to be related to higher CRP,15,16
and the independent association between BMI and complement components and fragments found in this study is also noteworthy. We found a significant inverse association between C5a fragment and LOC387715/ARMS2
genotypes, and significant positive trend between C3
genotypes and higher C5a fragment. We calculated ROC curves and C
statistics for C3a, Bb, and C5a which included demographic and environmental covariates and seven SNPs. Significant increases in C
statistics were observed when these fragments were added to the prediction model, especially when they were considered together, with discrimination between advanced AMD and a control increasing to as high as 94%. These findings add to the evidence that the complement system, in particular the alternative pathway, is activated and involved in the development of advanced AMD, such as through chronic low-level activation of complement in the retina.
In AMD, there is increased complement deposition in Bruch’s membrane and in drusen.27
Photo-oxidation of bisretinoid lipofuscin in cultured RPE cells has been shown to lead to complement activation and release of fragments iC3b and C3a in vitro.28
This finding supports the thinking that increased complement activation in AMD may occur as a result of photo-oxidation within RPE. Having insufficient functional CFH to dampen the complement-induced injury in the outer retina may then lead to pathologic features in advanced AMD. This line of thinking also is supported by the inverse relationship we found between CFH and AMD for analyses that were not controlled by genotype. The major predisposing effect is thus one of decreased regulation of activation of the complement cascade in the retina, such as through dysfunctional CFH gene protein or from insufficient production of protein. Whether there is systemic activation of the complement system or the elevated levels reflect systemically circulating fragments from local activation in the eye (as is observed in rheumatoid arthritis with complement activation in a joint) is not clear.29
These are not mutually exclusive possibilities, in that both systemic activation and local (i.e., retinal) complement activation could play a role. The latter is established based on findings of complement deposition in drusen.27
Biologically, the same outcome would occur if CFH were present in lower than normal amounts or were not fully functional. Complement activation, although probably not the initiating cause of the injury in AMD, can nevertheless substantially contribute to subsequent tissue damage. Animal models of laser induced damage to Bruch’s membrane provide evidence of the importance of C5a and other components of alternative pathway in the development of choroidal neovascularization.30,31
Elevated plasma activation fragments Bb, C3a, and C5a are consistent with continuous, low-level alternative pathway activation in patients with advanced AMD. In sum, we hypothesize that subtle alterations in the efficiency of activation and/or in regulatory capacity negatively influence a pathologic process that plays out over the years.
Activation of the complement system may lead to the formation the membrane attack complex (MAC or C5b-9). This terminal pathway begins with the cleavage of C5 to C5a and C5b. SC5b-9 is the fluid phase terminal complex that usually circulates bound to the inhibitor vitronectin. The quantity of SC5b-9 in the circulation is in part a reflection of the MAC that is generated locally. Thus, a fraction is deposited at the site and part is in the fluid phase. Our results and those of others indicate increased activation of C5 based on the elevated C5a levels in the blood and deposition of C5b-9 in the retina in AMD patients. C5a can bind to its receptor, C5aR, and has been shown to be important in models of AMD.32
C5b can be generated by activation of the alternative pathway or by direct cleavage by proteases including trypsin and thrombin. A recent report provided considerable evidence to indicate C5 activation by thrombin in a lung model of immune complex activation.33
In our study, the SC5b-9 was not increased in AMD, although it was in a similar study by Scholl et al.34
That study had few GA patients, and we speculate that the efficiency of C5b-9 deposition and retention in the two forms or the difference in the proportion of the types of advanced disease compared with our sample may account for the differences in results regarding MAC and other differences between the two reports. Another possibility relates to the quantity of local versus systemic activation which could alter the ratio of blood to tissue MAC. We still have much to learn about how the complement system is triggered and regulated, especially in niche locations such as the retina.
BMI is a known risk factor for the development of AMD.14,17,24
The association described in this report between high BMI and elevated C3, CFB, and CFH as well as increased concentrations of complement activation fragments derived from several components of the alternative pathway point to a role of the complement system in obesity. Adipose tissue is the source of acylation-stimulating protein (ASP), which is also C3a desArg. C3a desArg is a C3-derived protein formed by removal of an arginine from the carboxyl terminus of C3a and is the major form of circulating C3a. Before the relationship to C3 was discovered, the lipogenic function of ASP/C3a desArg was discovered.35
Sivaprasad et al.36
studied C3a desArg as an indicator of C3 systemic complement activation among 84 cases of advanced AMD (42 GA, 42 NV), as well as in association with the CFH:rs1061170
genotype. They found no significant association between genotype and C3a desArg, but did find an increase in C3a desArg concentration among cases, which suggests systemic activation. Largely consistent with these data, we found associations with C3a and AMD, and additionally found a new association between a second C3 split product, iC3b and BMI. Adipsin, an adipose-specific factor linked to adipocyte differentiation, was shown to be factor D of the alternative pathway of the complement system.37
We did not find a relationship with plasma factor D, whereas a previous study showed factor D was increased among patients with AMD compared with controls with a different proportion of geographic atrophy to neovascular AMD cases.34
Other noteworthy adipose-complement alterations include altered lipid clearance in C3−/−
mice and partial lipodystrophy in children with an autoantibody that stabilizes the alternative pathway C3 convertase.38,39
Also, there are a few reports of elevated C3 concentrations in individuals with a high BMI.40,41
Our data support increased turnover and activation of the alternative pathway in individuals with high BMI, evidenced by increased activation fragments, Bb, C3a, and iC3b. Enhanced alternative pathway activation in obesity and in AMD may jointly accelerate tissue damage.
Another noteworthy finding was the OR for the relationship between AMD and LOC387715/ARMS2 increased with the addition of C5a into the model. Since the function of ARMS2 is unknown, further research is needed. However, one possibility is that a tissue metalloproteinase directly activates C5 leading to formation of C5a and C5b, with the former binding to its receptor and the latter beginning the MAC.