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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm

Review

Age-related macular degeneration and the role of the complement system Selina McHarg a,b,1 , Simon J. Clark a,b,1 , Anthony J. Day c , Paul N. Bishop a,b,d,∗ a

Centre for Ophthalmology & Vision Sciences, Institute of Human Development, University of Manchester, Manchester, UK Centre for Advanced Discovery & Experimental Therapeutics, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, UK c Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, UK d Manchester Royal Eye Hospital, Central Manchester University Hospitals NHS Foundation Trust, Manchester, UK b

a r t i c l e

i n f o

Article history: Received 19 January 2015 Received in revised form 26 February 2015 Accepted 27 February 2015 Available online xxx Keywords: Age-related macular degeneration Complement system Alternative pathway

a b s t r a c t Age-related macular degeneration (AMD) is a leading cause of visual impairment. It is characterised by damage to a tissue complex composed of the retinal pigment epithelium, Bruch’s membrane and choriocapillaris. In early AMD extracellular debris including drusen accumulates in Bruch’s membrane and then in late AMD geographic atrophy and/or neovascularisation develop. Variants in genes encoding components of the alternative pathway of the complement cascade have a major influence on AMD risk, especially at the RCA locus on chromosome 1, which contains CFH and the CFHR genes. Immunohistochemical studies have demonstrated complement components in unaffected and AMD macular tissue. Whilst other factors, including oxidative stress, play important roles in AMD pathogenesis, evidence for the central role played by complement dysregulation is discussed in this review. © 2015 Published by Elsevier Ltd.

1. Introduction Age-related macular degeneration (AMD) is a slow and progressive disease of the macula, i.e. the central part of the retina, and the leading cause of irreversible visual loss in the Western world (Coleman et al., 2008). Globally, AMD accounts for 8.7% of all blindness and is predicted to affect 196 million people by 2020; it is more prevalent in populations of European descent than those of Asian and African descent (Wong et al., 2014). With the loss of central vision frequently involving both eyes, AMD is a debilitating condition affecting daily tasks such as reading and driving, and ultimately having severe consequences on independence and quality of life (Coleman et al., 2010). Patients with sight loss from AMD have an increased incidence of depression and anxiety, impaired mobility and isolation (Dawson et al., 2014). Based upon clinical appearance AMD is categorised into early, intermediate and late stage disease (Ferris et al., 2013). Early and intermediate AMD are characterised by medium sized or large

∗ Corresponding author at: Centre for Ophthalmology & Vision Sciences, Institute of Human Development, University of Manchester, Manchester, UK. Tel.: +44 1612755755. E-mail address: [email protected] (P.N. Bishop). 1 These authors contributed equally to this work.

soft drusen and pigmentary changes at the macula, with little or no visual loss. In late AMD there is visual loss and it comprises two forms i.e. neovascular AMD (also called ‘wet’ or ‘exudative’ AMD) and geographic atrophy (also called ‘dry’ AMD). Currently there are no treatments for geographic atrophy, but neovascular AMD is treated with vascular endothelial growth factor (VEGF) inhibitors and these, whilst not curative, are often effective in preventing severe visual loss (Scott and Bressler, 2013). Prior to the widespread adoption of anti-VEGF drugs in England and Wales (between April 2007 and March 2008), 56% of registrations for visual impairment (over 13,000) were due to AMD; of these geographic atrophy accounted for 49%, neovascular AMD 35% and 16% were unclassified (Rees et al., 2014). 2. The macula The retina is located in the posterior part of the eye and consists of the neurosensory retina (a multilayered cellular structure containing the photoreceptors, other neurons, glial cells and vasculature) and the retinal pigment epithelium (RPE). Posterior to the RPE is a sheet of extracellular matrix (ECM) called Bruch’s membrane and posterior to this is a vascular layer called the choroid (Fig. 1). The central part of the retina is called the macula; this is a circular region that is approximately 6 mm in diameter (Fig. 2). At the centre of the macula is the fovea, this is about 1.5 mm in

http://dx.doi.org/10.1016/j.molimm.2015.02.032 0161-5890/© 2015 Published by Elsevier Ltd.

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Fig. 1. TCC (C5b-9), FHL-1 and FH proteins localise with distinct patterns in the macula. (A) Schematic diagram showing a heat map of TCC (C5b-9) (blue), FHL-1 (green) and FH (red) localisation within the different layers of human macula. TCC (referred to as MAC) deposition was observed in the choriocapillaris and Bruch’s membrane even at a young age, but increases with age and is increased further in eyes with AMD (Whitmore et al., 2014). In addition, eyes from donors homozygous for 402H variant of FH have more TCC than eyes with the 402Y variant. FHL-1 is able to penetrate Bruch’s membrane, while FH is unable to and is retained in the choroid along with patches being present at the interface between the RPE and Bruch’s membrane; this FH presumably having been secreted by the RPE (Clark et al., 2014). (B) A fluorescent microscopy image of a human macula section with FHL-1 (green) shown to accumulate in Bruch’s membrane and within drusen, whereas FH (red) appears to coat the outside of the drusen. The FH that coats the outside of drusen is presumed to be derived from the RPE as FH derived from the choroid cannot cross Bruch’s membrane. Scale bar represents 5 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

diameter and contains densely-packed cone photoreceptors that subserve central (detailed) vision. The neurosensory retina has a dual vascular supply with the retinal vasculature supplying the inner part of the neurosensory retina and the choroidal vasculature, via the RPE, supporting the outer neurosensory retina (which contains the photoreceptors). The retina, particularly at the macula, is the most metabolically active tissue in the body (Wangsa-Wirawan and Linsenmeier, 2003). The RPE forms a continuous monolayer of hexagonally-shaped, melanin-containing cells which have microvilli on their apical surface that physically associate with the photoreceptor outer segments. The RPE has a number of functions including the transport of oxygen and metabolites from the choroid to the photoreceptors and transport of waste products back to the choroidal circulation, along with phagocytosis of outer segments as they are shed by the photoreceptors (Sparrow et al., 2010). The RPE is attached to Bruch’s membrane, a sheet of ECM that is around 2–4 ␮M thick. Bruch’s membrane has 5 structurally distinct layers including a central elastin layer, flanked by two collagen layers then the RPE basement membrane forms the most anterior layer, whilst choriocapillaris endothelial cell basement membranes form the most posterior layer. The choroid is an extensive vascular network, consisting of an outer macrovascular layer and its supporting stromal tissue, and an inner capillary layer called the choriocapillaris that merges into Bruch’s membrane (Bhutto and Lutty, 2012). The choriocapillaris has fenestrated capillaries that allow the leakage of proteins and other molecules out of the circulation that then bathe the choroid and Bruch’s membrane.

The main barrier to the movement of fluid and metabolites from the choroidal circulation to the neurosensory retina is the tight junctions of the RPE, which form the outer blood–retinal barrier. In addition, both Bruch’s membrane and the fenestrations of the choriocapillaris present barriers to the movement of large macromolecules between the choroidal circulation and the RPE. Indeed, we have shown that the immune regulatory protein factor H (FH) cannot cross Bruch’s membrane, whereas a much smaller splice variant called factor H like protein-1 (FHL-1) can (Clark et al., 2014). The accumulation of debris/drusen in Bruch’s membrane can impede the movement of smaller molecules and fluid across the membrane and this is thought to contribute to the pathogenesis of AMD. 3. Pathological features of AMD Early and intermediate AMD is characterised by soft drusen which can be observed by fundoscopic examination as pale yellow deposits (Fig. 2), morphologically these raised lesions are located between the RPE basal lamina and the anterior collagenous layer of Bruch’s membrane. Drusen can be subdivided into soft and hard drusen based upon their size and morphology. Soft drusen range in size from 63 ␮M upwards and have more indistinct borders than hard drusen. Increases in the number and size of soft drusen are associated with increased AMD risk (Hageman et al., 2011). Hard drusen, which are smaller in diameter, whiter and have more clearly defined borders, are common in the elderly population and do not confer AMD risk. In addition to the clinically visible drusen,

Fig. 2. Clinical presentation of AMD. The macula is the central part of the retina and is about 6 mm in circumference (A). Clinical photographs of posterior retina demonstrate the different features of AMD: (B) soft drusen, the black circle indicates the macula; (C) geographic atrophy and (D) subretinal haemorrhage from neovascular AMD.

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histological examination of post-mortem eyes can demonstrate lipid-rich, basal linear deposits between the inner collagenous layer of Bruch’s membrane and the RPE basement membrane, soft drusen represent focal aggregations of this material (Sarks et al., 2007). Drusen and basal linear deposits are composed, in part, of lipids derived from the incomplete breakdown products of photoreceptor outer segments which are then extruded by the RPE (Curcio and Millican, 1999). The accumulation of hydrophobic material in and around Bruch’s membrane is thought to impede transport across Bruch’s membrane and contribute to late AMD, especially geographic atrophy. Dysfunction of the RPE is a feature of early/intermediate AMD that manifests clinically as changes in pigmentation of the RPE layer. Morphological changes include accumulation of autofluorescent lipofuscin, detachment from Bruch’s membrane and cell death (Sparrow et al., 2010). Death of cells in the RPE monolayer results in patchy depigmentation due to adjacent cells spreading to maintain the monolayer and resulting in dilution of their pigmentation. Recently it has become apparent that there are also early changes in the choroidal layer including thinning and atrophy of the choriocapillaris at the macula (Whitmore et al., 2014). The impaired vascular supply may result in damage to the RPE and a compromised ability to clear extracellular debris from Bruch’s membrane. Conversely, the choriocapillaris relies on trophic support from the RPE, so damage to the RPE can result in a secondary loss of the choriocapillaris (Bhutto and Lutty, 2012). Geographic atrophy is characterised by a loss of choroid, RPE and neurosensory retina; often the area of atrophy is initially observed in a horseshoe shape around the fovea, but as this progresses it involves the central fovea. Geographic atrophy frequently develops in areas of the macula containing confluent drusen, but once the atrophy has developed the drusen regress (Toy et al., 2013). Neovascular AMD occurs when new blood vessels grow from the choroid, pierce through Bruch’s membrane and then proliferate in the retinal layers i.e. between Bruch’s membrane and the RPE and/or between the RPE and the neurosensory retina. These blood vessels are leaky resulting in fluid accumulation in the retina and often bleed (Fig. 2). Eventually they form a fibrovascular ‘disciform’ scar that results in severe visual loss unless the condition has been treated. Overproduction of VEGF by the RPE, driven by ischaemia and possibly complement activation, induces choroidal neovascularisation (Nozaki et al., 2006). 4. Risk factors associated with AMD 4.1. Environmental and clinical risk factors AMD risk factors have been studied intensively in recent years and it has become apparent that risk is associated with genetic, environmental and clinical factors. The most strongly associated environmental and clinical factors are increasing age, cigarette smoking and previous cataract surgery, whilst moderate associations include high body mass index, history of cardiovascular disease, hypertension and high plasma fibrinogen (Chakravarthy et al., 2010). Dietary factors have been associated with increased AMD risk include a high fat intake (Seddon, 2013). There have been a large number of studies investigating the role of micronutrients and the effects of dietary supplementation on modifying AMD risk, but the results of these studies remain controversial (Evans and Lawrenson, 2013). 4.2. Genetic risk factors Recent studies have demonstrated that genetic variants play a major role in determining AMD risk, for recent review see Schramm et al. (2014). The first major breakthrough in AMD genetics came

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in 2005 when, in a series of publications, an association was demonstrated between a polymorphism in the Complement Factor H gene (CFH), that results in a Y402H amino acid substitution in FH, and AMD (Edwards et al., 2005; Hageman et al., 2005; Haines et al., 2005; Klein et al., 2005). This polymorphism is present in 30–35% of individuals of European descent and a meta-analysis of 26 separate studies concluded that the 402H variant was associated with a twofold higher incidence of late AMD per copy within this ethnic population (Sofat et al., 2012). Following the discovery of the CFH association, further large scale genetic studies identified a series of genetic loci associated with modified AMD risk (Fritsche et al., 2013). If these susceptibility loci are stratified according to their importance, at least in populations of European descent, the two most important are on chromosome 1 (1q32) involving CFH and Complement Factor H Related genes (CFHR) 1–5 in the RCA cluster (this region contains the Y402H polymorphism discussed above) and the other is on chromosome 10 (10q31) near the ARMS2/HTRA1 genes. Variants associated with a moderate effect on AMD risk have been identified in the region of the C3, Complement Factor I (CFI) genes and at Complement Factor B (CFB)/C2, where CFB is most probably implicated (Fagerness et al., 2009; Gold et al., 2006; Yates et al., 2007). Several other more minor associations have been identified, but the importance of many of these remains to be confirmed (Fritsche et al., 2013). Recently, rare variants have been identified that are associated with AMD in genes encoding members of the alternative complement pathway including CFH, C3, and CFI (Helgason et al., 2013; Raychaudhuri et al., 2011; Seddon et al., 2013; Zhan et al., 2013; Yu et al., 2014); an AMD associated variant of C9, which is part of the terminal complement pathway, has also been reported. Therefore, many of the genes that are strongly or moderately associated with AMD risk encode components of the alternative complement pathway, the one major exception being ARMS2/HTRA1. The ARMS2/HTRA1 locus is poorly understood and it is unclear which gene is implicated. One recent study found an association between the ARMS2 risk genotype and raised systemic complement activation (Smailhodzic et al., 2012), but further research is required to understand the role of this locus in AMD. Whilst it is clear that genetic alterations at the chromosome 1 locus containing the RCA cluster are important in determining AMD risk, the mechanisms at a biochemical level are less well understood. As a result of extensive linkage disequilibrium in this region a number of haplotypes that span across the CFH and CFHR genes have been defined that modify AMD risk (Raychaudhuri et al., 2011; Seddon et al., 2013). The rs1061170 SNP, that causes the Y402H polymorphism in FH, is present in a common haplotype that is associated with increased AMD risk (Hageman et al., 2005) and there is strong biochemical evidence that the Y402H polymorphism itself is responsible for this risk (see below) (Clark et al., 2010a,b). The SNP rs800292 is contained within haplotypes that are relatively protective, this SNP causes a V62I amino acid substitution in FH that may provide protection due to superior cofactor activity (Tortajada et al., 2009). Haplotypes containing a common deletion of CFHR1 and CFHR3 are protective, but it is not clear whether altered levels of FHR-1 and FHR-3 are responsible for this effect (Hageman et al., 2005; Ansari et al., 2013): It has been shown that the intronic CFH SNP rs6677604 and the CFHR1 and CFHR3 deletion are strongly correlated with each other and with significantly raised plasma FH concentration (Ansari et al., 2013). Further research is required to understand the functional consequences of these various haplotypes. Recently algorithms have been developed for predicting risk of late AMD that are based primarily on genetic analysis, and in some models environmental and clinical parameters are added. In one study the risk of developing neovascular AMD was estimated using genetic markers alone (a 13 SNP panel) and was found to have 82% sensitivity and 63% specificity, with a area under the curve (AUC) of

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0.8 (Hageman et al., 2011). A similar result of AUC 0.82 was obtained in another study using 6 SNPs, but adding in environmental risk factors (Seddon et al., 2009). A prediction model for late AMD that included 26 SNPs, lifestyle data and baseline retinal examination achieved an AUC of 0.87 (Buitendijk et al., 2013). The importance of these observations is that they demonstrate that genetic analysis alone of SNPs in and around genes encoding the alternative complement pathway and at the ARMS2/HTRA1 locus is of high predictive value. It raises the exciting possibility that, in the future, patients can be identified who are at high risk and that strategies can be developed to prevent them from developing late AMD. 5. The alternative pathway of the complement system Given the SNPs in complement genes that modify AMD risk are mostly involved in the alternative pathway of complement activation (Fig. 3), it is understandable that this particular pathway has become a major focus in AMD research. Of the three complement activation pathways, unlike the classical or lectin pathways, the alternative pathway is constitutively active with a low level ‘tick over’ and therefore requires tight control by regulatory molecules on cells, in ECM and within the fluid phase; i.e. to prevent opsonisation of surfaces with C3b. Failure to do so leads to amplification of complement (i.e. via the alternative pathway’s positive feedback loop) with the increased production of pro-inflammatory

mediators, such as the C3a and C5a, that attract leukocytes (e.g. macrophages). The anaphylatoxins C3a and C5a are small proteins that have a number of important effects including mediating chemotaxis, inflammation, and also promote the generation of cytotoxic oxygen radicals, along with inducing smooth muscle contraction, histamine release from mast cells, and enhanced vascular permeability (Klos et al., 2013). In addition, complement activation results in the formation of the terminal complement complex (TCC), comprising complement components C5b-9. TCC can be in a soluble form (SC5b-9) or membrane bound where it forms a scaffold for multiple C9 proteins to assemble onto to produce the membrane attack complex (MAC) that mediates cellular damage. Immunohistochemical and ELISA assays are discussed below that utilised antibodies to recognise the TCC, but these probably do not distinguish between SC5b-9 and MAC. There are a number of cell membrane bound regulators of complement (e.g. complement receptor 1 or membrane cofactor protein), but as a blood borne protein FH can regulate complement activation in a host’s ECM. Synthesised by the liver, as well as locally in the human eye by RPE cells (Chen et al., 2007), FH comprises twenty complement control protein (CCP) domains (Ripoche et al., 1988). An important mechanism whereby FH localises to the tissues that it protects is by binding to glycosaminoglycans (GAGs), and FH has two major GAG binding sites, one in CCPs6-8 and the other in CCPs19-20 (Meri and Pangburn, 1990; Blackmore et al.,

Fig. 3. Flow diagram showing the three pathways of complement activation. Three separate pathways, the lectin, classical or alternative pathways, can each activate complement. All three pathways of activation converge on the complement protein C3 (red box) and its breakdown into C3b, which itself feeds into the amplification loop (highlighted by thick black arrows). This in turn generates a number of anaphylatoxins such as C3a and C5a (pink boxes) and ultimately results in the deposition of the terminal membrane attack complex (MAC). Complement activation can be fine-tuned on a surface through the action of various complement factors (blue boxes), where activation can be inhibited (factor H, FHL-1, factor I) or encouraged (factor B, factor D, properdin). A number of genetic alterations have been associated with AMD risk and most of these affect genes the alternative pathways of complement (highlighted with red circles). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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1996, 1998; Clark et al., 2006; Herbert et al., 2006; Prosser et al., 2007). This initial work mainly investigated binding to heparin (a GAG made by mast cells); however, more recent studies have demonstrated binding of these regions of FH to heparan sulfate (HS), a related GAG found widely in tissues (Clark et al., 2010a,b, 2013). Without the protection of sufficient FH binding, tissues may be vulnerable to complement attack, resulting in the immune and inflammatory responses (Clark et al., 2010a; Langford-Smith et al., 2014). The AMD associated Y402H polymorphism arises in CCP7 (Day et al., 1988) and this alters the binding of FH to a number of ligands including CRP, necrotic cells (Clark et al., 2010a), MDA (Weismann et al., 2011) and HS (Clark et al., 2006; Prosser et al., 2007). Factor H-like protein 1 (FHL-1) is a protein that arises from alternative splicing of the CFH gene (Ripoche et al., 1988). Identical for the first seven CCP domains, FHL-1 terminates in a unique four amino acid C-terminal tail. FHL-1 retains almost all the regulatory functions of FH, although it does not have the GAG and sialic acid binding sites in CCPs19-20 that are believed to be important for FH anchoring to some tissues such as in the kidney (Blaum et al., 2015; Clark et al., 2013). It does, however, contain the GAG binding site in CCP7 that is subject to the Y402H polymorphism. 6. Complement in the ageing macula and AMD Prior to the discovery of the genetic associations between complement genes and AMD, analyses of donor eye tissue had demonstrated the presence of complement proteins in eyes with AMD. Soft drusen were shown to contain C3 and C5. TCC (C5b-9) was observed in the choriocapillaris and Bruch’s membrane, but was rarely associated with RPE cells unless they were “compromised” (Anderson et al., 2002). Further studies have highlighted that TCC (referred to as MAC) is very frequently present in the choriocapillaris (particularly in the intercapillary pillars) and Bruch’s membrane in eyes unaffected by AMD (Fig. 3) and that the amount of TCC increases with age (Mullins et al., 2014; Seth et al., 2008). Surprisingly TCC can be observed in young donor eyes in the choriocapillaris, being frequently present by 21 years of age and it has been detected in eyes as young as 5 years of age (Mullins et al., 2014). Furthermore, choriocapillaris and Bruch’s membrane from individuals with AMD had higher levels than age-matched controls of TCC and those who were homozygous for the 402H variant of FH had higher levels than those homozygous for the 402Y variant (Mullins et al., 2011; Whitmore et al., 2014). Antibodies that can differentially detect FH and FHL-1 demonstrated FH is in the choroid and variably present at the interface between the RPE and Bruch’s membrane, but was not detected within Bruch’s membrane (Fig. 1) (Clark et al., 2014). By contrast, FHL-1 is within Bruch’s membrane and around the choriocapillaris, but labelling did not extend into the outer layers of the choroid. It was also found that FH coats the surface of drusen, whereas FHL-1 was present in the central parts of drusen. Some of this FHL-1 is likely to be derived from the choroidal circulation. However, FHL1 is also synthesised by the RPE and this could then pass through Bruch’s membrane and into the ECM surrounding the choriocapillaris. Whilst the RPE also synthesises full-length FH, this is too large to penetrate Bruch’s membrane and it becomes trapped between the basal surface of the RPE and Bruch’s membrane. FH, and most likely FHL-1, are the only members of the complement system known to inhibit complement activation via the alternative pathway on host ECM and function in conjunction with other inhibitors on cell surfaces. We demonstrated that the GAGs HS and DS are major ligands for FH in Bruch’s membrane and the choriocapillaris and that the binding is mediated largely through the GAG binding domain in CCP7, whilst the CCP19-20 region of FH has a relatively minor role (Clark et al., 2010a,b, 2013,

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2014; Langford-Smith et al., 2014). Furthermore, we found that because the 402H form of FH preferentially binds highly sulfated HS, whereas the 402Y form has a broader HS specificity, there are less binding sites for the 402H variant to Bruch’s membrane. We have also observed an age-related decrease in the amount of HS in Bruch’s membrane in the human eye (Keenan et al., 2014). Therefore, the relatively poor binding of the 402H form of FH (and probably FHL-1) to Bruch’s membrane and ECM of the choriocapillaris combined with an age-related loss of HS binding sites may result in excessive complement activation which eventually leads to AMD. 7. Other biochemical pathways associated with AMD In addition to aberrant complement activation, evidence suggests other biochemical processes such as oxidative stress may cause tissue damage in AMD (Handa, 2012; Langford-Smith et al., 2014). Retinal metabolic demands require a highly oxygen-rich micro-environment that combined with photo-oxidation waste products generated during normal retinal function means the RPE is continuously exposed to oxidative stress. Adding to this prooxidant environment is an age-related impairment of autophagy and degradation of shed photoreceptor outer segments which results in the accumulation of lipofuscin in RPE lysosomes (Mitter et al., 2012). Lipofuscin is a pro-oxidant compound that produces reactive oxygen species (Boulton et al., 1993) and its accumulation is also thought to reduce RPE phagocytic ability and cause apoptosis (Suter et al., 2000). Oxidative damage to RPE cells may promote complement activation and combined with increased cytokine production could result in the recruitment of inflammatory cells. These pathological changes represent a component of a ‘local inflammation model of AMD’ (Hageman, 2001; Anderson et al., 2002, 2010). The increased production of reactive oxygen species contributes to the formation of protein modifications including advancedglycation-endproducts and advanced-lipoxidation-endproducts. Enhanced lipid peroxidation results in the formation of malondialdehyde (MDA), which is pro-inflammatory and accumulates in AMD. FH binds to MDA and can block the uptake of MDA-modified proteins by macrophages and MDA-induced pro-inflammatory effects in mice (Weismann et al., 2011). However, the 402H polymorphism results in a marked decrease in the ability of FH to bind MDA, which may result in an inflammatory response that predisposes to AMD. Another product of enhanced lipid peroxidation, carboxyethylpyrrole (CEP), has been detected in drusen and shown to be capable of initiating a potent immune response in a mouse model that produced lesions resembling geographic atrophy (Hollyfield et al., 2008). Reactive oxygen species may also account for the enhanced AMD risk associated with cigarette smoking as the inhaled toxins are known to cause oxidative damage of proteins (Woodell and Rohrer, 2014). There is also evidence for the enhanced accumulation of iron within ocular tissues of AMD patients which may contribute to pathology by inducing oxidative stress via the Fenton reaction (Wong et al., 2007; Ugarte et al., 2013). However, despite evidence suggesting the involvement of oxidative stress, anti-oxidant supplementation trials for AMD patients have produced inconsistent outcomes (Evans and Lawrenson, 2013). 8. Conclusions Genetic and other studies highlight the central importance of dysregulation of the alternative pathway of the complement cascade in AMD pathogenesis. There is also ample evidence of damaging ‘oxidative stress’ in AMD with high metabolic activity and oxygen levels, along with light being focussed on the macula, predisposing this part of the retina to AMD. Pathological studies

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indicate that the initial site of AMD pathogenesis is the RPE/Bruch’s membrane/choriocapillaris complex with the loss of photoreceptor function being secondary to this. Questions remain as to how the various data can be married together to create an overarching hypothesis on AMD pathogenesis. The RPE and choriocapillaris are interdependent upon one another. The choroidal blood supply provides oxygen and nutrients to the RPE and the RPE provides trophic support for the choriocapillaris. Bhutto and Lutty (2012) suggest that in geographic atrophy the preceding large confluent drusen and pigmentary changes in the RPE indicate primary dysfunction of the RPE with secondary effects on the choroid, whereas in neovascular AMD the loss of choroidal vasculature is the primary insult. Whitmore et al. (2014) emphasise the importance of the MAC deposition in the choriocapillaris and subsequent damage to this capillary layer, suggesting that this drives all forms of late AMD, with effects on the RPE being secondary. At a biochemical level it is possible that complement activation and oxidative stress act independently on the macula tissue to drive AMD pathology, or the pathways may interact at various levels. Our recent finding that the main regulator of complement activation within Bruch’s membrane is FHL-1 and that this protein is likely to also be contributing to the protection of the choriocapillaris may be significant. It is possible that any disruption to the presence of FHL-1 in Bruch’s membrane may lead to a reduced capacity to deal with deposited C3b within the ECM, whether that be through changes in binding capacity (i.e. the Y402H polymorphism) or changes in protein synthesis by the RPE cells driven by genetic or environmental factors. This, in turn, is likely to allow an increased level of complement turnover that would otherwise exist, leading to low level inflammation at this site and perhaps even contributing to the formation of, or aggravating the growth of, drusen. The loss of heparan sulfate in Bruch’s membrane/choriocapillaris associated with age, a binding partner of FHL-1 and one of the main binding ligands of FH to extracellular matrix, would also contribute to this reduced complement regulation and could predispose to AMD. These are exciting times in AMD research as we move closer to understanding the molecular pathology of the disease, and this knowledge in turn promises to deliver new treatments for the condition including preventive interventions. Acknowledgements We would like to thank Dr. Caroline Milner for reviewing the manuscript. SJC is a recipient of a Medical Research Council (MRC) Career Development Fellowship (MR/K024418/1) and we also acknowledge other recent research funding from MRC (G0900538 and K004441), Fight for Sight (1866) and The Macular Society. References Anderson, D.H., Mullins, R.F., Hageman, G.S., Johnson, L.V., 2002. A role for local inflammation in the formation of drusen in the aging eye. Am. J. Ophthalmol. 134, 411–431, http://dx.doi.org/10.1016/S0002-9394(02)01624-0. Anderson, D.H., Radeke, M.J., Gallo, N.B., Chapin, E.A., Johnson, P.T., Curletti, C.R., Hancox, L.S., Hu, J., Ebright, J.N., Malek, G., Hauser, M.A., Rickman, C.B., Bok, D., Hageman, G.S., Johnson, L.V., 2010. The pivotal role of the complement system in aging and age-related macular degeneration: hypothesis re-visited. Prog. Retin. Eye Res. 29, 95–112, http://dx.doi.org/10.1016/j.preteyeres.2009.11.003. Ansari, M., McKeigue, P.M., Skerka, C., Hayward, C., Rudan, I., Vitart, V., Polasek, O., Armbrecht, A.-M., Yates, J.R.W., Vatavuk, Z., Bencic, G., Kolcic, I., Oostra, B.A., Van Duijn, C.M., Campbell, S., Stanton, C.M., Huffman, J., Shu, X., Khan, J.C., Shahid, H., Harding, S.P., Bishop, P.N., Deary, I.J., Moore, A.T., Dhillon, B., Rudan, P., Zipfel, P.F., Sim, R.B., Hastie, N.D., Campbell, H., Wright, A.F., 2013. Genetic influences on plasma CFH and CFHR1 concentrations and their role in susceptibility to age-related macular degeneration. Hum. Mol. Genet. 22, 4857–4869, http://dx.doi.org/10.1093/hmg/ddt336. Bhutto, I., Lutty, G., 2012. Understanding age-related macular degeneration (AMD): relationships between the photoreceptor/retinal pigment

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