Role of Factor H and Related Proteins in Regulating Complement Activation in the Macula, and Relevance to Age-Related Macular Degeneration.
ABSTRACT: The recent revolution in age-related macular degeneration (AMD) genetics has demonstrated that genetic alterations affecting the alternative pathway of the complement cascade have a major influence on AMD risk. One of the two most important genetic loci is on chromosome 1 and contains genes encoding complement factor H (FH) and the factor H related proteins (FHR proteins). In macular tissue, especially Bruch's membrane, relatively high levels of a truncated splice variant of FH called factor H-like protein 1 (FHL-1) are present. Here we discuss how genetic variations may alter the amounts, or by altering their protein sequences, the functions of these proteins. In particular, the common Y402H polymorphism affects the ability of FHL-1 and FH to localize to Bruch's membrane and the inner choroid because it alters the ability of these complement regulators to bind heparan sulphate (HS) in these structures. In addition, there is an age-related loss of HS from Bruch's membrane. We hypothesize that a combination of poor binding of the 402H variants of FHL-1 and FH to Bruch's membrane, combined with a decrease in binding due to age-related HS loss, eventually results in insufficient FHL-1 and FH binding to Bruch's membrane. This could result in complement activation, inflammation and thereby predispose to AMD.
Project description:The tight regulation of innate immunity on extracellular matrix (ECM) is a vital part of immune homeostasis throughout the human body, and disruption to this regulation in the eye is thought to contribute directly to the progression of age-related macular degeneration (AMD). The plasma complement regulator factor H (FH) is thought to be the main regulator that protects ECM against damaging complement activation. However, in the present study we demonstrate that a truncated form of FH, called FH-like protein 1 (FHL-1), is the main regulatory protein in the layer of ECM under human retina, called Bruch's membrane. Bruch's membrane is a major site of AMD disease pathogenesis and where drusen, the hallmark lesions of AMD, form. We show that FHL-1 can passively diffuse through Bruch's membrane, whereas the full sized, glycosylated, FH cannot. FHL-1 is largely bound to Bruch's membrane through interactions with heparan sulfate, and we show that the common Y402H polymorphism in the CFH gene, associated with an increased risk of AMD, reduces the binding of FHL-1 to this heparan sulfate. We also show that FHL-1 is retained in drusen whereas FH coats the periphery of the lesions, perhaps inhibiting their clearance. Our results identify a novel mechanism of complement regulation in the human eye, which highlights potential new avenues for therapeutic strategies.
Project description:Age-related macular degeneration (AMD) is the predominant cause of blindness in the industrialized world where destruction of the macula, i.e. the central region of the retina, results in loss of vision. AMD is preceded by the formation of deposits in the macula, which accumulate between the Bruch's membrane and the retinal pigment epithelium (RPE). These deposits are associated with complement-mediated inflammation and perturb retinal function. Recent genetic association studies have demonstrated that a common allele (402H) of the complement factor H (CFH) gene is a major risk factor for the development of AMD; CFH suppresses complement activation on host tissues where it is believed to bind via its interaction with polyanionic structures. We have shown previously that this coding change (Y402H; from a tyrosine to histidine residue) alters the binding of the CFH protein to sulfated polysaccharides. Here we demonstrate that the AMD-associated polymorphism profoundly affects CFH binding to sites within human macula. Notably, the AMD-associated 402H variant binds less well to heparan sulfate and dermatan sulfate glycosaminoglycans within Bruch's membrane when compared with the 402Y form; both allotypes exhibit a similar level of binding to the RPE. We propose that the impaired binding of the 402H variant to Bruch's membrane results in an overactivation of the complement pathway leading to local chronic inflammation and thus contributes directly to the development and/or progression of AMD. These studies therefore provide a putative disease mechanism and add weight to the genetic association studies that implicate the 402H allele as an important risk factor in AMD.
Project description:Age-related macular degeneration (AMD) is a leading cause of blindness. Genetic variants at the chromosome 1q31.3 encompassing the complement factor H (CFH, FH) and CFH related genes (CFHR1-5) are major determinants of AMD susceptibility, but their molecular consequences remain unclear. Here we demonstrate that FHR-4 plays a prominent role in AMD pathogenesis. We show that systemic FHR-4 levels are elevated in AMD (P-value?=?7.1?×?10-6), whereas no difference is seen for FH. Furthermore, FHR-4 accumulates in the choriocapillaris, Bruch's membrane and drusen, and can compete with FH/FHL-1 for C3b binding, preventing FI-mediated C3b cleavage. Critically, the protective allele of the strongest AMD-associated CFH locus variant rs10922109 has the highest association with reduced FHR-4 levels (P-value?=?2.2?×?10-56), independently of the AMD-protective CFHR1-3 deletion, and even in those individuals that carry the high-risk allele of rs1061170 (Y402H). Our findings identify FHR-4 as a key molecular player contributing to complement dysregulation in AMD.
Project description:Age-related macular degeneration (AMD) is a major cause of vision loss. It is associated with development of characteristic plaque-like deposits (soft drusen) in Bruch's membrane basal to the retinal pigment epithelium (RPE). A sequence variant (Y402H) in short consensus repeat domain 7 (SCR7) of complement factor H (CFH) is associated with risk for "dry" AMD. We asked whether the eye-targeting of this disease might be related to specific interactions of CFH SCR7 with proteins expressed in the aging human RPE/choroid that could contribute to protein deposition in drusen. Yeast 2-hybrid (Y2H) screens of a retinal pigment epithelium/choroid library derived from aged donors using CFH SCR7 baits detected an interaction with EFEMP1/Fibulin 3 (Fib3), which is the locus for an inherited macular degeneration and also accumulates basal to macular RPE in AMD. The CFH/Fib3 interaction was validated by co-immunoprecipitation of native proteins. Quantitative Y2H and ELISA assays with different recombinant protein constructs both demonstrated higher affinity for Fib3 for the disease-related CFH 402H variant. Immuno-labeling revealed colocalization of CFH and Fib3 in globular deposits within cholesterol-rich domains in soft drusen in two AMD donors homozygous for CFH 402H (H/H). This pattern of labeling was quite distinct from those seen in examples of eyes with Y/Y and H/Y genotypes. The CFH 402H/Fib3 interaction could contribute to the development of pathological aggregates in soft drusen in some patients and as such might provide a target for therapeutic intervention in some forms of AMD.
Project description:An imbalance between activation and inhibition of the complement system has been implicated in the etiologies of numerous common diseases. Allotypic variants of a key complement fluid-phase regulatory protein, complement factor H (CFH), are strongly associated with age-related macular degeneration (AMD), a leading cause of worldwide visual dysfunction, although its specific role in AMD pathogenesis is still not clear. CFH was isolated from individuals carrying combinations of two of the nonsynonymous coding variants most strongly associated with AMD risk, V62/H402 (risk haplotype variants), I62/Y402 (nonrisk haplotype variants), and V62/Y402. These proteins were used in two functional assays (cell surface- and fluid-phase-based) measuring cofactor activity of CFH in the factor I-mediated cleavage of C3b. Although no variant-specific differences in the cofactor activity were detected, when heparan sulfate (HS) was added to these assays, it accelerated the rate of C3b cleavage, and this effect could be modulated by degree of HS sulfation. Bruch's membrane/choroid, a site of tissue damage in AMD, contains high concentrations of glycosaminoglycans, including HS. Addition of human Bruch's membrane/choroid to the fluid-phase assay accelerated the C3b cleavage, and this effect was lost posttreatment of the tissue with heparinase III. Binding of CFH variants to Bruch's membrane/choroid isolated from elderly, non-AMD donor eyes, was similar, as was the functional activity of bound CFH. These findings refine our understanding of interactions of HS and complement and support the hypothesis that these interactions play a role in the transition between normal aging and AMD in Bruch's membrane/choroid.
Project description:Retinal inflammation plays a key role in the progression of age-related macular degeneration (AMD), a condition that leads to loss of central vision. The deposition of the acute phase pentraxin C-reactive protein (CRP) in the macula activates the complement system, thereby contributing to dysregulated inflammation. The complement protein factor H (FH) can bind CRP and down-regulate an inflammatory response. However, it is not known whether a truncated form of FH, called factor H-like protein 1 (FHL-1), which plays a significant regulatory role in the eye, also interacts with CRP. Here, we compare the binding properties of FHL-1 and FH to both CRP and the related protein pentraxin-3 (PTX3). We find that, unlike FH, FHL-1 can bind pro-inflammatory monomeric CRP (mCRP) as well as the circulating pentameric form. Furthermore, the four-amino acid C-terminal tail of FHL-1 (not present in FH) plays a role in mediating its binding to mCRP. PTX3 was found to be present in the macula of donor eyes and the AMD-associated Y402H polymorphism altered the binding of FHL-1 to PTX3. Our findings reveal that the binding characteristics of FHL-1 differ from those of FH, likely underpinning independent immune regulatory functions in the context of the human retina.
Project description:Age-related macular degeneration (AMD) is the leading cause of blindness in the western world and affects nearly 200 million people globally. Local inflammation driven by complement system dysregulation is currently a therapeutic target. Bruch's membrane (BrM) is a sheet of extracellular matrix that separates the retina from the underlying choroid, a highly vascularized layer that supplies oxygen and nutrition to the outer retina. Here, we show that most complement proteins are unable to diffuse through BrM, although FHL-1, factor D and C5a can. AMD-associated lipid deposition in BrM decreases FHL-1 diffusion. We show that this impermeability of BrM creates two separate semi-independent compartments with respect to complement activation and regulation. Complement proteins synthesized locally on either side of BrM, or on the choroidal side if derived from the circulation, predominantly remain on their side of origin. As previous studies suggest that complement activation in AMD is confined to the choroidal side of BrM, we propose a model whereby complement activation in the choriocapillaris layer of the choroid generates C5a, which crosses BrM to interact with its specific receptor on RPE cells to initiate an inflammatory response in the retina. Understanding mechanisms underpinning AMD is essential for developing therapeutics that target the right molecule in the right anatomical compartment.
Project description:A common allele (402H) of the complement factor H (FH) gene is the major risk factor for age-related macular degeneration (AMD), the leading cause of blindness in the elderly population. Development and progression of AMD involves vascular and inflammatory components partly by deregulation of the alternative pathway of the complement system (AP). The loss of central vision results from atrophy and/or from abnormal neovascularization arising from the choroid. The functional link between FH, the main inhibitor of AP, and choroidal neovascularization (CNV) in AMD remains unclear. In a murine model of CNV used as a model for neovascular AMD (nAMD), intraocular human recombinant FH (recFH) reduced CNV as efficiently as currently used anti-VEGF (vascular endothelial growth factor) antibody, decreasing deposition of C3 cleavage fragments, membrane attack complex (MAC), and microglia/macrophage recruitment markers in the CNV lesion site. In sharp contrast, recFH carrying the H402 risk variant had no effect on CNV indicating a causal link to disease etiology. Only the recFH NTal region (recFH1-7), containing the CCPs1-4 C3-convertase inhibition domains and the CCP7 binding domain, exerted all differential biological effects. The CTal region (recFH7-20) containing the CCP7 and CCPs19-20 binding domains was antiangiogenic but did not reduce the microglia/macrophage recruitment. The antiangiogenic effect of both recFH1-20 and recFH-CCP7-20 resulted from thrombospondin-1 (TSP-1) upregulation independently of the C3 cleavage fragments generation. This study provides insight on the mechanistic role of FH in nAMD and invites to reconsider its therapeutic potential.
Project description:To determine the association of high-risk alleles in the complement factor H (CFH; Y402H, rs1061170) and age-related maculopathy susceptibility (ARMS2; A69S, rs10490924) genes with reticular macular disease (RMD), a major clinical subphenotype of age-related macular degeneration (AMD).Using retinal images from the Columbia Macular Genetics Study, we identified 67 subject individuals with RMD. A comparison group of 64 subjects with AMD without RMD was matched by ethnicity, age, sex, and AMD clinical stage.In the RMD group, 53 of 67 subjects (79.1%) were female, the mean age was 83 years, and 47 of 67 (70.1%) had late AMD, with closely matched values in the non-RMD group. The frequencies of the CFH 402H allele were 39.6% in the RMD group (53 of 134 individuals) and 58.6% in the non-RMD group (75 of 128 individuals) (?(2) = 8.8; P = .003; odds ratio, 0.46 [95% confidence interval, 0.28-0.76]). The corresponding frequencies of the risk allele for ARMS2 were 44.0% (40 of 128 individuals) and 31.3% (40 of 128 individuals), respectively (?(2) = 4.0; P = .045; odds ratio, 1.73 [95% confidence interval, 1.04-2.90]). Homozygosity for 402H was particularly associated with the absence of RMD, occurring in 8 of 67 subjects (11.9%) with RMD vs 24 of 64 subjects (37.5%) without RMD (P < .001). Retinal macular disease also was associated with hypertension among male patients.The AMD-associated CFH 402H risk variant is significantly associated with the absence of RMD but enhanced risk for RMD is conferred by the ARMS2 69S AMD risk allele. These results are consistent with the hypothesis that 402H may confer a survival benefit against certain infections, some of which may cause RMD.Reticular macular disease may be genetically distinct from the rest of AMD.
Project description:Complement factor H (CFH) is a major susceptibility gene for age-related macular degeneration (AMD); however, its impact on AMD pathobiology is unresolved. Here, the role of CFH in the development of AMD pathology in vivo was interrogated by analyzing aged Cfh(+/-) and Cfh(-/-) mice fed a high-fat, cholesterol-enriched diet. Strikingly, decreased levels of CFH led to increased sub-retinal pigmented epithelium (sub-RPE) deposit formation, specifically basal laminar deposits, following high-fat diet. Mechanistically, our data show that deposits are due to CFH competition for lipoprotein binding sites in Bruch's membrane. Interestingly and despite sub-RPE deposit formation occurring in both Cfh(+/-) and Cfh(-/-) mice, RPE damage accompanied by loss of vision occurred only in old Cfh(+/-) mice. We demonstrate that such pathology is a function of excess complement activation in Cfh(+/-) mice versus complement deficiency in Cfh(-/-) animals. Due to the CFH-dependent increase in sub-RPE deposit height, we interrogated the potential of CFH as a previously unidentified regulator of Bruch's membrane lipoprotein binding and show, using human Bruch's membrane explants, that CFH removes endogenous human lipoproteins in aged donors. Thus, advanced age, high-fat diet, and decreased CFH induce sub-RPE deposit formation leading to complement activation, which contributes to RPE damage and visual function impairment. This new understanding of the complicated interactions of CFH in AMD-like pathology provides an improved foundation for the development of targeted therapies for AMD.