A University of Manchester led team of scientists has discovered that the most common form of adult blindness is probably caused by a failure of at least one of five proteins to regulate the immune system.
The breakthrough could one day herald the development of transformative treatments for developing age-related macular degeneration (AMD), which affects 600,000 people in the UK alone.
A test to determine which patients are at risk of developing the disease could also emerge, as the team estimate that 40-50% of patients have elevated levels of at least one of the five proteins, although more work still needs to be done.
The research, funded by Medical Research Council and a collaboration between scientists in Manchester, London and Tübingen in Germany, is published in the American Journal of Human Genetics.
The discovery of new drug targets could help scientists develop therapies which lower levels of what are known as FHR proteins—and therefore the risk of developing or exacerbating AMD.
Scientists have long known that inflammation at the back of the eye plays a role in AMD development.
A series of genes thought to regulate the activity of the complement pathway—a key player in our immune defense against pathogens—were identified in previous research as candidates that affect a person’s risk of developing the disease.
However, until now the role of these genes—Complement Factor H (CFH) and Complement Factor H-Related 1 to 5 (CFHR-1 to CFHR-5) – has been unclear.
But by studying the levels of the products of these genes—FH and FHR-1 to FHR-5 proteins– in 604 blood plasma samples using a method called mass spectrometry, the team was able to show for the first time that all five FHR proteins are at higher levels in people with AMD than in those without.
Part of the innate immune system, the complement pathway is our first line of defense against infections and clears damaged cells by marking them for destruction and recruiting and activating other immune cells.
In AMD, the complement pathway is over activated in the back of the eye, promoting an inappropriate and damaging inflammatory response.
Dr. Richard Unwin from The University of Manchester’s Stoller Biomarker Discovery Center said: “This is a hugely important study for people with AMD. Measuring the levels of these FHR proteins has been a big challenge over the last few years and is technically quite challenging as they are present at low levels in the blood and are very similar to each other.
“By using state-of-the-art mass spectrometry methods we can now confidently measure these proteins and show for the first time what is an important, if not the most important, factor in how AMD develops.
“This opens up whole new areas for improving patient care, through the development of new treatments targeted at these proteins or in simply monitoring levels to discover who has higher levels of complement activation and as such will benefit from complement-modifying treatments.
“It’s important to stress that studies over time need to be carried out before we can say with authority that these proteins are able to predict risk—and that will take time.
“We’re confident about our results: a second study by Dr. Laura Lorés-Motta and Prof Anneke den Hollander at Radboud University in the Netherlands has in parallel arrived to the same conclusion about FHR proteins using a different measuring technique.”
In its early stages, AMD starts to damage the back of the eye forming deposits after which then patients go on to develop two form of the disease: wet or dry AMD. But the team hope that intervening at an early stage could stop its progression.
Dr. Valentina Cipriani, a Lecturer in Statistical Genomics at Queen Mary University of London who led the data analysis said “For more than 15 years the focus for AMD has been on Complement Factor H and its protein FH. Our analysis clearly points beyond FH.”
“By using an approach called a genome-wide association study that looks at genetic variants across the genomes of people with and without the disease, we were able to identify genetic variants that determine both higher FHR protein levels and increased risk of AMD.”
Prof Simon Clark, Helmut Ecker Endowed Professor of AMD at Eberhard Karls University of Tübingen who co-supervised the work said “This really marks a step-change in our understanding around the driving mechanisms behind specific types of AMD.
“It follows on from our original discovery last year around FHR-4, but while all higher levels of at least one of the five FHR proteins are now known to be associated with AMD risk not all patients will have their disease driven by this route.
“Therefore, being able to measure these proteins in patients’ blood will be vital in identifying patients who will react to FHR-targeting therapies sometime in the future.”
Age-related macular degeneration (AMD) is a chronic and progressive degenerative disease of the retina, which culminates in blindness and affects mainly the elderly population. AMD pathogenesis and pathophysiology are incredibly complex due to the structural and cellular complexity of the retina, and the variety of risk factors and molecular mechanisms that contribute to disease onset and progression. AMD is driven by a combination of genetic predisposition, natural ageing changes and lifestyle factors, such as smoking or nutritional intake. The mechanism by which these risk factors interact and converge towards AMD are not fully understood and therefore drug discovery is challenging, where no therapeutic attempt has been fully effective thus far. Genetic and molecular studies have identified the complement system as an important player in AMD. Indeed, many of the genetic risk variants cluster in genes of the alternative pathway of the complement system and complement activation products are elevated in AMD patients. Nevertheless, attempts in treating AMD via complement regulators have not yet been successful, suggesting a level of complexity that could not be predicted only from a genetic point of view. In this review, we will explore the role of complement system in AMD development and in the main molecular and cellular features of AMD, including complement activation itself, inflammation, ECM stability, energy metabolism and oxidative stress.
Age-related macular degeneration (AMD) as a multifactorial disease
AMD is a complex, multi-factorial disease of the elderly. Although genetic studies have been quite successful in identifying genes and processes underlying AMD risk, the understanding of how these genetic variants drive AMD progression is still largely incomplete . As of today, there are strong reasons to believe that the complement system plays a central role in AMD pathogenesis and over activation of the alternative complement pathway is one of the main drivers for disease. Indeed, genetic and epidemiologic studies have been able to pinpoint more than 35 genetic variants conferring risk for developing AMD, many of them mapping to the complement system . Consequently, a number of therapeutic pipelines and clinical trials are currently focusing on regulating the complement system as a therapeutic strategy to treat AMD. However, AMD remains an incurable disease and despite great progress in uncovering the genetic links of AMD, treatment has remained symptomatic. This is, at least in part, due to the fact that AMD is not entirely a genetic disease and indeed, epidemiology studies pinpoint other environmental and lifestyle factors that define the individual risk for disease. In the recent years, a plethora of additional, but just as important, risk factors have been identified, including physiological changes that occur with age and lifestyle, such as smoking and nutrition, which can alter retinal health [3, 4]. At the genetic level, beside inflammation associated with gene variants mapping into the complement pathway, turnover of extracellular matrix (ECM) components and lipid handling are all likely to be important in AMD pathogenesis. At the molecular level, the combination of risk factors result in events defining the disease, including age-related deposition of lipids and protein underneath the retinal pigment epithelial (RPE) cells, metabolic and oxidative stress in RPE cells, changes within the retinal ECM and Bruch’s membrane, complement activation and chronic inflammation [5, 6]. Genetic and environmental risks are likely to converge into critical disease pathways, which may differ in discrete subgroups of patients.
The involvement of different cellular and extracellular components and controllable/uncontrollable risks, makes understanding the generation, pathogenesis and manifestation of a complex disease, such as AMD one of the big challenges in medical research. Subsequently turning this knowledge into prediction, prevention and treatment is yet another imposing task. Capturing the next level of complexity is required in order to understand the interconnection of distinct genetic risks, the interaction with lifestyle factors, the non-response to treatment and the effect of age on the development and progression of the various forms of AMD. This review specifically focusses on the complement pathway as a main driver of disease.
The complement system
The complement system is a protein cascade composed of more than fifty proteins, which are found in both the fluid phase and bound to cell membranes. The main role of complement is to recognise and mediate the removal of pathogens, debris and dead cells . Proteins of the complement system can be rapidly converted into active forms via a proteolytic cascade triggered by any of the three activating pathways: the classical, lectin and alternative pathways (see Fig. 1) [8, 9]. The classical complement pathway is triggered by the antibody-mediated binding of complement component 1q (C1q) to pathogen surfaces. Activation through the lectin pathway relies on the recognition of pathogen-associated molecular patterns (PAMPs) (d-mannose, N-acetyl-d-glucosamine or acetyl groups), on the surface of pathogens or to apoptotic or necrotic cells, by the pattern-recognition molecules mannose-binding lectin (MBL), ficolins and collectins . The alternative pathway of the complement system presents a unique characteristic, since unlike the other initiating pathways, it is constitutively active at low levels due to spontaneous hydrolysis of Complement component 3 (C3) to C3(H20): a process referred to as complement ‘tick-over’.
All three pathways converge in the formation of a protein complex, the C3 convertase, which cleaves C3 into the anaphylatoxin C3a and the central protein in the complement amplification loop, C3b (see Fig. 1). The C3 convertase of the lectin and classical activation pathways is composed of cleaved complement components -4 and -2 (C4bC2b). In the alternative pathway, factor B (FB) can bind C3(H20) and is cleaved by factor D (FD) to form a distinct C3 convertase termed C3(H2O)Bb; which, can continuously produce C3b and provide an amplification loop for the complement system activation, independent of the initial trigger. Indeed, C3b is needed to form the downstream C5 convertase, a complex that is responsible for the cleavage of complement component 5 (C5) into the second anaphylatoxin C5a and C5b. Formation of C5b is a prerequisite for the assembly of the membrane attack complex (MAC) composed of C5b, complement components -6 (C6), -7 (C7), -8 (C8) and numerous -9 (polyC9). The function of this complex is to form a pore on a pathogen/hostile material to mediate its cell lysis [8, 9].
The alternative pathway of the complement system needs to be tightly regulated to avoid excessive activation. The serine protease, complement factor I (FI), can cleave C3b deposited on any surface into inactive C3b (iC3b). iC3b cannot contribute to the amplification loop of complement and therefore the action of FI directly both slows down and prevents complement activation. However, FI cannot perform this function alone: it requires the presence of a co-factor protein. There are a number of cell membrane complement regulators, such as: membrane cofactor protein (MCP, CD46), a cofactor for FI; decay-accelerating factor (DAF, CD55), with C3-convertase decay activity; and complement receptor 1 (CR1, CD35), which possesses both cofactor and decay activities . However, the blood borne co-factors complement factor H (FH), and its alternative splice variant factor H-like protein 1 (FHL-1), are the only two complement regulators that will allow FI-mediated C3b cleavage on acellular surfaces, such as ECM. Moreover, FH, by physically displacing FB from C3b, also accelerates the decay of the C3bBb convertase . In addition, other cell-bound regulators inhibit the terminal pathway of complement, by interfering with the formation of MAC on cellular membranes and the subsequent cell lysis, such as clusterin (CLU), vitronectin (VTN) and CD59 [13, 14].
Both FH and FHL-1 are expressed by the single CFH gene found in the region of complement activation (RCA) cluster on chromosome 1 [15, 16]. Downstream of the CFH gene are five factor H-related genes (CFHR1-5) and in contrast to the FH and FHL-1 regulatory proteins, the five FH-related proteins (FHR1-5) are believed to act as positive activators of the complement system [15, 17]. The FHR proteins compete for binding to C3b, but do not themselves share the FI co-factor domain possessed by FH and FHL-1, and therefore actively prevent FI-mediated C3b breakdown, leading to increased complement turnover . As the activated complement system is turning over, the release of the anaphylatoxins C3a and C5a exert specific additional functions. These small peptides act as chemoattractants, which recruit circulating immune cells to the site of complement activation, and by binding to specific cell receptors (i.e., C3aR and C5aR respectively) promote degranulation and release of pro-inflammatory cytokines .
Clearly, tight regulation of complement activation is required in order to maintain tissue homeostasis and prevent unnecessary inflammation and tissue damage. Over-activation of the complement system is associated with driving the pathogenesis of a number of systemic and organ specific, diseases. Most recently, a strong genetic and biochemical link have been made between poor regulation of complement activation in the back of the eye and the blinding disease AMD.
More information: Valentina Cipriani et al, Beyond factor H: The impact of genetic-risk variants for age-related macular degeneration on circulating factor-H-like 1 and factor-H-related protein concentrations, The American Journal of Human Genetics (2021). DOI: 10.1016/j.ajhg.2021.05.015