Role In Pathogenesis Of Age-related Macular Degeneration Research Articles

A mega-analysis of expression quantitative trait loci (eQTL) provides insight into the regulatory architecture of gene expression variation in liver

Genome-wide association studies (GWAS) have identified numerous genetic variants in the human genome associated with diseases and traits. Nevertheless, for most loci the causative variant is still unknown. Expression quantitative trait loci (eQTL) in disease relevant tissues is an excellent approach to correlate genetic association with gene expression. While liver is the primary site of gene transcription for two pathways relevant to age-related macular degeneration (AMD), namely the complement system and cholesterol metabolism, we explored the contribution of AMD associated variants to modulate liver gene expression. We extracted publicly available data and computed the largest eQTL data set for liver tissue to date. Genotypes and expression data from all studies underwent rigorous quality control. Subsequently, Matrix eQTL was used to identify significant local eQTL. In total, liver samples from 588 individuals revealed 202,489 significant eQTL variants affecting 1,959 genes (Q-Value < 0.001). In addition, a further 101 independent eQTL signals were identified in 93 of the 1,959 eQTL genes. Importantly, our results independently reinforce the notion that high density lipoprotein metabolism plays a role in AMD pathogenesis. Taken together, our study generated a first comprehensive map reflecting the genetic regulatory landscape of gene expression in liver.

Cellular Senescence in Age-Related Macular Degeneration: Can Autophagy and DNA Damage Response Play a Role?

Age-related macular degeneration (AMD) is the main reason of blindness in developed countries. Aging is the main AMD risk factor. Oxidative stress, inflammation and some genetic factors play a role in AMD pathogenesis. AMD is associated with the degradation of retinal pigment epithelium (RPE) cells, photoreceptors, and choriocapillaris. Lost RPE cells in the central retina can be replaced by their peripheral counterparts. However, if they are senescent, degenerated regions in the macula cannot be regenerated. Oxidative stress, a main factor of AMD pathogenesis, can induce DNA damage response (DDR), autophagy, and cell senescence. Moreover, cell senescence is involved in the pathogenesis of many age-related diseases. Cell senescence is the state of permanent cellular division arrest and concerns only mitotic cells. RPE cells, although quiescent in the retina, can proliferate in vitro. They can also undergo oxidative stress-induced senescence. Therefore, cellular senescence can be considered as an important molecular pathway of AMD pathology, resulting in an inability of the macula to regenerate after degeneration of RPE cells caused by a factor inducing DDR and autophagy. It is too early to speculate about the role of the mutual interplay between cell senescence, autophagy, and DDR, but this subject is worth further studies.

Molecular mechanisms of cell death in retina during development of age-related macular degeneration

Age-related macular degeneration (AMD) is a chronic progressive disease characterized by damage to the central retina zone. Changes in choriocapillaries, retinal pigment epithelium (RPE), and Bruch’s membrane (typical for aging) underlie AMD pathogenesis; however, the mechanisms launching the transfer of typical age-related changes in the pathological process are unknown. The death of photoreceptors and irreversible loss of vision become the results of pathological changes in RPE and choroid. In spite of intensive studies of AMD pathogenesis, information on molecular genetic preconditions of the events leading to the death of photoreceptors (as well as about the pathways of their death) is extremely limited; this complicates the search for efficient methods of AMD treatment (first of all, the most widely spread atrophic (“dry”) form of the disease). Recent studies demonstrated that not only apoptotic but also autophagic and necrotic signaling cascades are involved in the cellular death of retinal cells. The published data on three main forms of the programmed cell death (apoptosis, necrosis, and autophagy) and their role in AMD pathogenesis are summarized in the present review.

RETINAL VASCULAR PLEXUSES' CHANGES IN DRY AGE-RELATED MACULAR DEGENERATION, EVALUATED BY MEANS OF OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY.

To investigate alteration in superficial and deep retinal vascular densities and choroidal thickness, in patients affected by early and intermediate age-related macular degeneration (AMD). All patients had undergone optical coherence tomography angiography (OCTA). All eyes were grouped into two stages: "early AMD" and "intermediate AMD." Outcome measures were superficial vessel density, deep vessel density, and choroidal thickness. A control group of healthy subjects was selected for the statistical comparisons. A total of 37 eyes of 37 dry AMD patients were enrolled for the study. Fourteen of 37 eyes were classified as having early AMD, the remaining 23 of 37 eyes were classified as being affected by intermediate AMD. Superficial and deep vessel densities were 39.21% ± 10.67% and 43.84% ± 11.57%, respectively, in the control group and 28.30% ± 10.73% and 36.41% ± 12.30%, respectively, in AMD patients (P = 0.001 and P = 0.017, respectively). Choroidal thickness was significantly reduced in AMD patients. In the last years, several studies have reported vascular factors playing an important role in AMD pathogenesis. We demonstrated that both superficial and deep retinal plexuses are altered among patients affected by AMD. Interestingly, this alteration starts immediately at the intermediate AMD stage and also the choroidal thickness reduction.

Dysregulation and activation of immune system in age-related macular degeneration pathogenesis

Dysregulation and activation of immune processes are important in age-related macular degeneration (AMD) pathogenesis. The single nucleotide polymorphism of complement factor H is widely recognized as a risk factor to AMD. Over-activation of nod-like receptor3 and polymorphism of Toll-Like Receptor 3 also associated with AMD. Except for innate immune processes, adaptive immunity also play a critical role in AMD, a growing body of evidence supports that auto-antibodies and T cells are related with AMD. Additionally A2E and lipid oxidation byproducts might also have a role in AMD pathogenesis. Key words: Macular degeneration/etiology; Immunocompetence; Immunomodulation; Review

Recent advance research progress on complement factor I and age-related macular degeneration

Age-related macular degeneration (AMD) is one of the most important diseases that cause vision loss among older patients in developed countries. As the progress of population aging in China, the incidence of AMD is increasing. The activation of the complement system plays an important role in AMD pathogenesis. Complement factor Ⅰ (CFI) is an important complement regulator. In recent years, there have been numerous studies about CFI single-nucleotide polymorphisms (SNPs) and AMD. The mechanism of CFI and the association between CFI polymorphisms and AMD were reviewed in the article. Key words: Age-related macular degeneration; Complement system; Complement factor Ⅰ; Single-nucleotide polymorphisms

Genetic variants of interleukin 17A are functionally associated with increased risk of age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in elderly populations worldwide. Inflammation, among many factors, has been suggested to play an important role in AMD pathogenesis. Interleukin 17 (IL-17) is a proinflammatory cytokine that has been implicated in the pathogenesis of various autoimmune diseases. In the current study, we examined two single nucleotide polymorphisms (SNPs), rs2275913G/A and rs3748067C/T, in the IL-17A gene between AMD patients and healthy controls. Results showed that rs2275913AA genotype and rs3748067TT genotype were associated with increased susceptibility to AMD (hazard ratio [HR], 1.75; 95 % confidence interval [CI], 1.07 to 3.02; P=0.023, and HR, 2.12; 95 % CI, 1.26 to 4.01; P=0.004; data were adjusted for age and sex). Next, we investigated the functional relevance of the two SNPs. In vitro stimulated peripheral blood mononuclear cells (PBMCs) from subjects possessing the rs2275913AA genotype produced significantly more IL-17 than those with the GG genotype. However, PBMCs with rs3748067TT genotype revealed significantly higher IL-17 production than those with rs3748067CC genotype only in AMD patients but not in controls. These data indicate IL-17A polymorphisms are associated with increased risk of AMD probably by affecting gene expression.

Oxidized LDL Induces Apoptosis of Human Retinal Pigment Epithelium Through Activation of ERK-Bax/Bcl-2 Signaling Pathways

Purpose: Retinal pigment epithelium (RPE) cell dysfunction and death play a vital role in the pathogenesis of age-related macular degeneration (AMD). We previously reported that oxidized low-density lipoprotein (OX-LDL) induces retinal degeneration in vivo. In this study, we investigated the role of the ERK-Bax/Bcl-2 signaling pathways in OX-LDL-induced apoptosis in human RPE.Methods: ARPE-19 cells were incubated with 10–100 mg/mL n-LDL or OX-LDL for 24 h. Cell viability was assessed using the Cell Titer 96 Aqueous One Solution cell proliferation assay. RPE apoptosis was measured with a flow cytometer. Reverse transcription polymerase chain reaction was used to detect Bcl-2 and Bax mRNA levels in RPE cells. Bcl-2 and Bax protein expression was measured by western blotting. Activation of extracellular signal-regulated kinase (ERK) protein was evaluated by western blot analysis. One-way analysis of variance was used to compare differences.Results: OX-LDL treatment decreased ARPE-19 cell viability in a dose-dependent manner, whereas n-LDL had no effect. Compared with the control group, OX-LDL significantly increased the apoptosis of RPE, 10 mg/mL, 50 mg/mL, 100 mg/mL apoptosis rate was 6.43 ± 0.19%, 5.12 ± 0.27%, 5.53 ± 0.35%, respectively. OX-LDL also increased Bcl-2 expression and decreased Bax expression significantly. The Bcl-2 to Bax ratio was elevated after OX-LDL treatment. Inhibition of ERK downregulated Bax and was associated with RPE apoptosis.Conclusions: Our data suggest that apoptosis induced by OX-LDL in RPE partly depends on Erk-Bax/Bcl-2 signaling pathway activation. These results may provide further information regarding the effects of OX-LDL in human RPE and their potential role in AMD pathogenesis.

Does toll-like receptor-3 (TLR-3) have any role in Indian AMD phenotype?

Age-related macular degeneration (AMD) is a devastating disease that results in irreversible central vision loss. TLRs signaling pathway has been found to play an important role in AMD pathogenesis as evidenced by several studies. The objective of the study was to determine the single nucleotide polymorphism (SNP) changes in TLR3 in North Indian AMD patients. We recruited 176 patients comprising 115 AMD patients and 61 controls. Real time PCR was used to evaluate the SNP changes at rs3775291 locus. Pearson's χ(2) test was used evaluate association between various groups. No significant association in genotype and allele frequency was found in AMD patients as compared to control. The results suggest that AMD pathology in North Indian AMD patients is not affected by TLR3 signaling but it could be influenced by other genetic or environmental factors unique to North India.

T Cells and Macrophages Responding to Oxidative Damage Cooperate in Pathogenesis of a Mouse Model of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a major disease affecting central vision, but the pathogenic mechanisms are not fully understood. Using a mouse model, we examined the relationship of two factors implicated in AMD development: oxidative stress and the immune system. Carboxyethylpyrrole (CEP) is a lipid peroxidation product associated with AMD in humans and AMD-like pathology in mice. Previously, we demonstrated that CEP immunization leads to retinal infiltration of pro-inflammatory M1 macrophages before overt retinal degeneration. Here, we provide direct and indirect mechanisms for the effect of CEP on macrophages, and show for the first time that antigen-specific T cells play a leading role in AMD pathogenesis. In vitro, CEP directly induced M1 macrophage polarization and production of M1-related factors by retinal pigment epithelial (RPE) cells. In vivo, CEP eye injections in mice induced acute pro-inflammatory gene expression in the retina and human AMD eyes showed distinctively diffuse CEP immunolabeling within RPE cells. Importantly, interferon-gamma (IFN-γ) and interleukin-17 (IL-17)-producing CEP-specific T cells were identified ex vivo after CEP immunization and promoted M1 polarization in co-culture experiments. Finally, T cell immunosuppressive therapy inhibited CEP-mediated pathology. These data indicate that T cells and M1 macrophages activated by oxidative damage cooperate in AMD pathogenesis.

Oxidative Stress, Hypoxia, and Autophagy in the Neovascular Processes of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is the leading cause of severe and irreversible loss of vision in the elderly in developed countries. AMD is a complex chronic neurodegenerative disease associated with many environmental, lifestyle, and genetic factors. Oxidative stress and the production of reactive oxygen species (ROS) seem to play a pivotal role in AMD pathogenesis. It is known that the macula receives the highest blood flow of any tissue in the body when related to size, and anything that can reduce the rich blood supply can cause hypoxia, malfunction, or disease. Oxidative stress can affect both the lipid rich retinal outer segment structure and the light processing in the macula. The response to oxidative stress involves several cellular defense reactions, for example, increases in antioxidant production and proteolysis of damaged proteins. The imbalance between production of damaged cellular components and degradation leads to the accumulation of detrimental products, for example, intracellular lipofuscin and extracellular drusen. Autophagy is a central lysosomal clearance system that may play an important role in AMD development. There are many anatomical changes in retinal pigment epithelium (RPE), Bruch's membrane, and choriocapillaris in response to chronic oxidative stress, hypoxia, and disturbed autophagy and these are estimated to be crucial components in the pathology of neovascular processes in AMD.

Complement system in pathogenesis of AMD: dual player in degeneration and protection of retinal tissue.

Age-related macular degeneration (AMD) is the most common cause of blindness among the elderly, especially in Western countries. Although the prevalence, risk factors, and clinical course of the disease are well described, its pathogenesis is not entirely elucidated. AMD is associated with a variety of biochemical abnormalities, including complement components deposition in the retinal pigment epithelium-Bruch's membrane-choriocapillaris complex. Although the complement system (CS) is increasingly recognized as mediating important roles in retinal biology, its particular role in AMD pathogenesis has not been precisely defined. Unrestricted activation of the CS following injury may directly damage retinal tissue and recruit immune cells to the vicinity of active complement cascades, therefore detrimentally causing bystander damage to surrounding cells and tissues. On the other hand, recent evidence supports the notion that an active complement pathway is a necessity for the normal maintenance of the neurosensory retina. In this scenario, complement activation appears to have beneficial effect as it promotes cell survival and tissue remodeling by facilitating the rapid removal of dying cells and resulting cellular debris, thus demonstrating anti-inflammatory and neuroprotective activities. In this review, we discuss both the beneficial and detrimental roles of CS in degenerative retina, focusing on the diverse aspects of CS functions that may promote or inhibit macular disease.

Role of antioxidant enzymes and small molecular weight antioxidants in the pathogenesis of age-related macular degeneration (AMD)

Cells in aerobic condition are constantly exposed to reactive oxygen species (ROS), which may induce damage to biomolecules, including proteins, nucleic acids and lipids. In normal circumstances, the amount of ROS is counterbalanced by cellular antioxidant defence, with its main components—antioxidant enzymes, DNA repair and small molecular weight antioxidants. An imbalance between the production and neutralization of ROS by antioxidant defence is associated with oxidative stress, which plays an important role in the pathogenesis of many age-related and degenerative diseases, including age-related macular degeneration (AMD), affecting the macula—the central part of the retina. The retina is especially prone to oxidative stress due to high oxygen pressure and exposure to UV and blue light promoting ROS generation. Because oxidative stress has an established role in AMD pathogenesis, proper functioning of antioxidant defence may be crucial for the occurrence and progression of this disease. Antioxidant enzymes play a major role in ROS scavenging and changes of their expression or/and activity are reported to be associated with AMD. Therefore, the enzymes in the retina along with their genes may constitute a perspective target in AMD prevention and therapy.

Mitochondrial and Nuclear DNA Damage and Repair in Age-Related Macular Degeneration

Aging and oxidative stress seem to be the most important factors in the pathogenesis of age-related macular degeneration (AMD), a condition affecting many elderly people in the developed world. However, aging is associated with the accumulation of oxidative damage in many biomolecules, including DNA. Furthermore, mitochondria may be especially important in this process because the reactive oxygen species produced in their electron transport chain can damage cellular components. Therefore, the cellular response to DNA damage, expressed mainly through DNA repair, may play an important role in AMD etiology. In several studies the increase in mitochondrial DNA (mtDNA) damage and mutations, and the decrease in the efficacy of DNA repair have been correlated with the occurrence and the stage of AMD. It has also been shown that mitochondrial DNA accumulates more DNA lesions than nuclear DNA in AMD. However, the DNA damage response in mitochondria is executed by nucleus-encoded proteins, and thus mutagenesis in nuclear DNA (nDNA) may affect the ability to respond to mutagenesis in its mitochondrial counterpart. We reported that lymphocytes from AMD patients displayed a higher amount of total endogenous basal and oxidative DNA damage, exhibited a higher sensitivity to hydrogen peroxide and UV radiation, and repaired the lesions induced by these factors less effectively than did cells from control individuals. We postulate that poor efficacy of DNA repair (i.e., is impaired above average for a particular age) when combined with the enhanced sensitivity of retinal pigment epithelium cells to environmental stress factors, contributes to the pathogenesis of AMD. Collectively, these data suggest that the cellular response to both mitochondrial and nuclear DNA damage may play an important role in AMD pathogenesis.

JNK inhibition reduces apoptosis and neovascularization in a murine model of age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of registered blindness among the elderly and affects over 30 million people worldwide. It is well established that oxidative stress, inflammation, and apoptosis play critical roles in pathogenesis of AMD. In advanced wet AMD, although, most of the severe vision loss is due to bleeding and exudation of choroidal neovascularization (CNV), and it is well known that vascular endothelial growth factor (VEGF) plays a pivotal role in the growth of the abnormal blood vessels. VEGF suppression therapy improves visual acuity in AMD patients. However, there are unresolved issues, including safety and cost. Here we show that mice lacking c-Jun N-terminal kinase 1 (JNK1) exhibit decreased inflammation, reduced CNV, lower levels of choroidal VEGF, and impaired choroidal macrophage recruitment in a murine model of wet AMD (laser-induced CNV). Interestingly, we also detected a substantial reduction in choroidal apoptosis of JNK1-deficient mice. Intravitreal injection of a pan-caspase inhibitor reduced neovascularization in the laser-induced CNV model, suggesting that apoptosis plays a role in laser-induced pathological angiogenesis. Intravitreal injection of a specific JNK inhibitor decreased choroidal VEGF expression and reduced pathological CNV. These results suggest that JNK1 plays a key role in linking oxidative stress, inflammation, macrophage recruitment apoptosis, and VEGF production in wet AMD and pharmacological JNK inhibition offers a unique and alternative avenue for prevention and treatment of AMD.

An association of transferrin gene polymorphism and serum transferrin levels with age-related macular degeneration

Age-related macular degeneration (AMD) is a degenerative disease of the eye, triggered by the damage of the macular cells. In the Western world it is the most frequent cause of blindness in the elderly. Oxidative stress is proved to play a key role in AMD pathogenesis and since iron accumulation has been found in AMD maculas, it may accelerate the oxidative processes in this tissue. In the present work we investigated the association between four polymorphisms of the transferrin gene (rs8177178; rs8177179; rs4481157; rs1130459) and AMD in dependence on the transferrin protein and iron serum levels. We employed PCR-RFLP (polymerase chain reaction – restriction fragment length polymorphism) for genotype determination, ELISA assay for serum transferrin evaluation and colorimetric assay for measurement of iron concentration in the serum. We found that advanced age and AMD family history may be independent risk factors for AMD (1.02, p < 0.05 and 8.88, p < 0.001, respectively). At the rs4481157 site The GG genotype of the rs4481157 polymorphism decreased the risk of dry AMD (OR 0.50; p < 0.05), while the GA increased this risk (OR 1.07; p < 0.05). Moreover, the GA genotype of this polymorphism decreased the risk of progression to the wet form (OR 0.63; p < 0.05). The analysis of the gene-environment interactions showed that the rs4481157 polymorphism modulates the AMD risk among obese (BMI above 30) individuals. In the former smokers group we observed a moderate association between rs4481157 polymorphism and AMD risk while this association in current smokers was stronger. We found also that the serum level of transferrin was higher in the AMD group (p < 0.001) than in the control, but the total serum iron levels did not differ between both groups. We found that the serum transferrin was associated with the rs8177178 (p < 0.001) and rs4481157 (p < 0.01) polymorphisms, and the common variant (GG) of both sites was related to a lower level of transferrin. Presented data may contribute to the involvement of iron homeostasis in AMD risk.

Enhanced apoptosis in retinal pigment epithelium under inflammatory stimuli and oxidative stress

Age-related macular degeneration (AMD) is a neurodegenerative disease that causes irreversible central vision loss in the elderly. Retinal pigment epithelium (RPE) has been found to be a key component in AMD pathogenesis. The Ccl2(-/-)/Cx3cr1(-/-) (DKO) mouse on Crb1(rd8) background is created as an AMD model, developing AMD-like retinal lesions. Our study aimed to examine RPE apoptosis in DKO mouse and human ARPE-19 cell line. DKO RPE expressed higher apoptotic proteins when compared with age-matched wild type (WT) RPE in physiological conditions. Apoptosis of primary cultured mouse RPE was evaluated under stimulation with lipopolysaccharide for inflammatory stimulation and 2,3,7,8-tetrachlorodibenzo-p-dioxin or H(2)O(2) for oxidative stress. Compared with WT RPE, DKO RPE was more susceptible to Fas ligand (FasL)-mediated apoptosis under both inflammatory and oxidative stress, with less cell viability and higher expression of apoptotic transcripts and proteins. Decreased cell viability was also observed in ARPE-19 cells under each stimulus. Furthermore, we also investigated the anti-apoptotic effects of decoy receptor 3 (DcR3), a decoy receptor for FasL, on ARPE-19 cells under inflammatory and oxidative stress. DcR3 pre-incubated ARPE-19 cells showed decreased apoptosis, with increased cell viability and decreased expression of apoptotic transcripts and proteins under the stimuli. On the contrary, knockdown of DcR3 in ARPE-19 cells showed totally opposite results. Our study demonstrates that FasL-mediated RPE apoptosis may play a pivotal role in AMD pathogenesis.

Chronic photo-oxidative stress and subsequent MCP-1 activation as causative factors for age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly in developed countries. Although pathogenic factors, such as oxidative stress, inflammation and genetics are thought to contribute to the development of AMD, little is known about the relationships and priorities between these factors. Here, we show that chronic photo-oxidative stress is an environmental factor involved in AMD pathogenesis. We first demonstrated that exposure to light induced phospholipid oxidation in the mouse retina, which was more prominent in aged animals. The induced oxidized phospholipids led to an increase in the expression of monocyte chemoattractant protein-1, which then resulted in macrophage accumulation, an inflammatory process. Antioxidant treatment prevented light-induced phospholipid oxidation and the subsequent increase of monocyte chemoattractant protein-1 (also known as C-C motif chemokine 2; CCL2), which are the beginnings of the light-induced changes. Subretinal application of oxidized phospholipids induced choroidal neovascularization, a characteristic feature of wet-type AMD, which was inhibited by blocking monocyte chemoattractant protein-1. These findings strongly suggest that a sequential cascade from photic stress to inflammatory processes through phospholipid oxidation has an important role in AMD pathogenesis. Finally, we succeeded in mimicking human AMD in mice with low-level, long-term photic stress, in which characteristic pathological changes, including choroidal neovascularization formation, were observed. Therefore, we propose a consecutive pathogenic pathway involving photic stress, oxidation of phospholipids and chronic inflammation, leading to angiogenesis. These findings add to the current understanding of AMD pathology and suggest protection from oxidative stress or suppression of the subsequent inflammation as new potential therapeutic targets for AMD.

Macrophage polarization in the maculae of age‐related macular degeneration: A pilot study

Macrophages can be polarized to exhibit either pro-inflammatory M1 or pro-angiogenic M2 phenotypes, but have high phenotypic plasticity. This pilot study investigated macrophage polarization in the macular retina and choroid of age-related macular degeneration (AMD) and non-AMD subjects, as well as in AMD choroidal neovascular membranes (CNVM). All specimens were evaluated for routine histopathology. Quantitative real-time polymerase chain reaction for representative M1 (CXCL11) and M2 (CCL22) transcripts were performed on macular choroidal trephines (MCT) of 19 AMD and nine non-AMD eye bank eyes, on the microdissected macular retinal cells from the archived slides of five geographic atrophic AMD, five exudative/neovascular AMD, and eight normal autopsied eyes, and on microdissected inflammatory cells from two surgically removed CNVM that did not respond to anti-vascular endothelial growth factor (VEGF) therapy. High M2-chemokine transcript and a low ratio of M1 to M2 chemokine transcript were found in aging non-AMD MCT. Advanced AMD maculae had a higher M1 to M2 chemokine transcript ratio compared to normal autopsied eyes. Macrophages in the two CNVM of patients unresponsive to anti-VEGF therapy were polarized toward either M1 or M2 phenotypes. The number of M2 macrophages was increased compared to M1 macrophages in normal aging eyes. A pathological shift of macrophage polarization may play a potential role in AMD pathogenesis.

Heparan Sulfate, Including That in Bruch’s Membrane, Inhibits the Complement Alternative Pathway: Implications for Age-Related Macular Degeneration

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.