Don H. Anderson

An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch's membrane interface in aging and age-related macular degeneration.

Age-related macular degeneration (AMD) is a blinding disease that afflicts millions of adults in the Western world. Although it has been proposed that a threshold event occurs during normal aging which leads to AMD, the sequelae of biochemical, cellular, and/or molecular events leading to the development of AMD are poorly understood. Although available data provide strong evidence that a significant proportion of AMD has a genetic basis, no gene(s) has yet been identified that causes a significant proportion of AMD. Moreover, no major molecular pathways involved in the etiology of this disease have been elucidated.Drusen, pathological deposits that form between the retinal pigmented epithelium (RPE) and Bruchs membrane, are significant risk factors for the development of AMD. In our view, the development of testable new hypotheses of drusen origins has been hindered significantly by the absence of a comprehensive profile of their molecular composition. In this review, we describe an integrated ultrastructural, histochemical, molecular biological, and biochemical approach to identify specific molecular pathways associated with drusen biogenesis. The implicit assumption underlying these recent investigations has been that a thorough understanding of the composition of drusen and source(s) of drusen-associated material is likely to provide fresh insight into the pathobiology underlying AMD. Significantly, these studies have revealed that proteins associated with inflammation and immune-mediated processes are prevalent among drusen-associated constituents. Transcripts that encode a number of these molecules have been detected in retinal, RPE, and choroidal cells. These data have also lead to the observations that dendritic cells, potent antigen-presenting cells, are intimately associated with drusen development and that complement activation is a key pathway that is active both within drusen and along the RPE-choroid interface. We propose herein a unifying hypothesis of drusen biogenesis that attempts to incorporate a large body of new and previously published structural, histochemical, and molecular data pertaining to drusen composition and development. This theory is put forth with the acknowledgment that numerous AMD genotypes may exist. Thus, only some aspects of the proposed hypothesis may be involved in any given AMD genotype. Importantly, this hypothesis invokes, for the first time, the potential for a direct role of cell- and immune-mediated processes in drusen biogenesis. We acknowledge that the proposed hypothesis clearly represents a paradigm shift in our conceptualization pertaining to pathways that participate in the development of drusen and age-related macular degeneration. It is our hope that other investigators will test, validate and/or refute various aspects of this hypothesis, and in so doing, increase our overall understanding of the biological pathways associated with early AMD.

Drusen associated with aging and age-related macular degeneration contain proteins common to extracellular deposits associated with atherosclerosis, elastosis, amyloidosis, and dense deposit disease

Age‐related macular degeneration (AMD), a blinding disorder that compromises central vision, is characterized by the accumulation of extracellular deposits, termed drusen, between the retinal pigmented epithelium and the choroid. Recent studies in this laboratory revealed that vitronectin is a major component of drusen. Because vitronectin is also a constituent of abnormal deposits associated with a variety of diseases, drusen from human donor eyes were examined for compositional similarities with other extracellular disease deposits. Thirty‐four antibodies to 29 different proteins or protein complexes were tested for immunoreactivity with hard and soft drusen phenotypes. These analyses provide a partial profile of the molecular composition of drusen. Serum amyloid P component, apolipoprotein E, immunoglobulin light chains, Factor X, and complement proteins (C5 and C5b‐9 complex) were identified in all drusen phenotypes. Transcripts encoding some of these molecules were also found to be synthesized by the retina, retinal pigmented epithelium, and/or choroid. The compositional similarity between drusen and other disease deposits may be significant in view of the recently established correlation between AMD and atherosclerosis. This study suggests that similar pathways may be involved in the etiologies of AMD and other age‐related diseases.—Mullins, R. F., Russell, S. R., Anderson, D. H., Hageman, G. S. Drusen associated with aging and age‐related macular degeneration contain proteins common to extracellular deposits associated with atherosclerosis, elastosis, amyloidosis, and dense deposit disease. FASEB J. 14, 835–846 (2000)

The Pivotal Role of the Complement System in Aging and Age-related Macular Degeneration: Hypothesis Re-visited

During the past ten years, dramatic advances have been made in unraveling the biological bases of age-related macular degeneration (AMD), the most common cause of irreversible blindness in western populations. In that timeframe, two distinct lines of evidence emerged which implicated chronic local inflammation and activation of the complement cascade in AMD pathogenesis. First, a number of complement system proteins, complement activators, and complement regulatory proteins were identified as molecular constituents of drusen, the hallmark extracellular deposits associated with early AMD. Subsequently, genetic studies revealed highly significant statistical associations between AMD and variants of several complement pathway-associated genes including: Complement factor H (CFH), complement factor H-related 1 and 3 (CFHR1 and CFHR3), complement factor B (CFB), complement component 2 (C2), and complement component 3 (C3). In this article, we revisit our original hypothesis that chronic local inflammatory and immune-mediated events at the level of Bruchs membrane play critical roles in drusen biogenesis and, by extension, in the pathobiology of AMD. Secondly, we report the results of a new screening for additional AMD-associated polymorphisms in a battery of 63 complement-related genes. Third, we identify and characterize the local complement system in the RPE-choroid complex - thus adding a new dimension of biological complexity to the role of the complement system in ocular aging and AMD. Finally, we evaluate the most salient, recent evidence that bears directly on the role of complement in AMD pathogenesis and progression. Collectively, these recent findings strongly re-affirm the importance of the complement system in AMD. They lay the groundwork for further studies that may lead to the identification of a transcriptional disease signature of AMD, and hasten the development of new therapeutic approaches that will restore the complement-modulating activity that appears to be compromised in genetically susceptible individuals.

Age‐related macular degeneration—emerging pathogenetic and therapeutic concepts

Today, the average life expectancy in developed nations is over 80 years and climbing. And yet, the quality of life during those additional years is often significantly diminished by the effects of age‐related, degenerative diseases, including age‐related macular degeneration (AMD), the leading cause of blindness in the elderly worldwide. AMD is characterized by a progressive loss of central vision attributable to degenerative and neovascular changes in the macula, a highly specialized region of the ocular retina responsible for fine visual acuity. Estimates gathered from the most recent World Health Organization (WHO) global eye disease survey conservatively indicate that 14 million persons are blind or severely visually impaired because of AMD. The disease has a tremendous impact on the physical and mental health of the geriatric population and their families and is becoming a major public health burden. Currently, there is neither a cure nor a means to prevent AMD. Palliative treatment options for the less prevalent, late‐stage ‘wet’ form of the disease include anti‐neovascular agents, photodynamic therapy and thermal laser. There are no current therapies for the more common ‘dry’ AMD, except for the use of antioxidants that delay progression in 20%–25% of eyes. New discoveries, however, are beginning to provide a much clearer picture of the relevant cellular events, genetic factors, and biochemical processes associated with early AMD. Recently, compelling evidence has emerged that the innate immune system and, more specifically, uncontrolled regulation of the complement alternative pathway plays a central role in the pathobiology of AMD. The complement Factor H gene—which encodes the major inhibitor of the complement alternative pathway—is the first gene identified in multiple independent studies that confers a significant genetic risk for the development of AMD. The emergence of this new paradigm of AMD pathogenesis should hasten the development of novel diagnostic and therapeutic approaches for this disease that will dramatically improve the quality of our prolonged lifespan.

The Alzheimer's Aβ-peptide is deposited at sites of complement activation in pathologic deposits associated with aging and age-related macular degeneration

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss in older individuals worldwide. The disease is characterized by abnormal extracellular deposits, known as drusen, that accumulate along the basal surface of the retinal pigmented epithelium. Although drusen deposition is common in older individuals, large numbers of drusen and/or extensive areas of confluent drusen represent a significant risk factor for AMD. Widespread drusen deposition is associated with retinal pigmented epithelial cell dysfunction and degeneration of the photoreceptor cells of the neural retina. Recent studies have shown that drusen contain a variety of immunomodulatory molecules, suggesting that the process of drusen formation involves local inflammatory events, including activation of the complement cascade. Similar observations in Alzheimers disease (AD) have lead to the hypothesis that chronic localized inflammation is an important element of AD pathogenesis, with significant neurodegenerative consequences. Accordingly, the amyloid beta (Aβ) peptide, a major constituent of neuritic plaques in AD, has been implicated as a primary activator of complement in AD. Here we show that Aβ is associated with a substructural vesicular component within drusen. Aβ colocalizes with activated complement components in these “amyloid vesicles,” thereby identifying them as potential primary sites of complement activation. Thus, Aβ deposition could be an important component of the local inflammatory events that contribute to atrophy of the retinal pigmented epithelium, drusen biogenesis, and the pathogenesis of AMD.

Extended haplotypes in the complement factor H (CFH) and CFH‐related (CFHR) family of genes protect against age‐related macular degeneration: Characterization, ethnic distribution and evolutionary implications

Background. Variants in the complement factor H gene (CFH) are associated with age‐related macular degeneration (AMD). CFH and five CFH‐related genes (CFHR1‐5) lie within the regulators of complement activation (RCA) locus on chromosome 1q32. Aims and Methods. In this study, the structural and evolutionary relationships between these genes and AMD was refined using a combined genetic, molecular and immunohistochemical approach. Results. We identify and characterize a large, common deletion that encompasses both the CFHR1 and CFHR3 genes. CFHR1, an abundant serum protein, is absent in subjects homozygous for the deletion. Genotyping analyses of AMD cases and controls from two cohorts demonstrates that deletion homozygotes comprise 1.1% of cases and 5.7% of the controls (chi‐square = 32.8; P = 1.6 E‐09). CFHR1 and CFHR3 transcripts are abundant in liver, but undetectable in the ocular retinal pigmented epithelium/choroid complex. AMD‐associated CFH/CFHR1/CFHR3 haplotypes are widespread in human populations. Conclusion. The absence of CFHR1 and/or CFHR3 may account for the protective effects conferred by some CFH haplotypes. Moreover, the high frequencies of the 402H allele and the delCFHR1/CFHR3 alleles in African populations suggest an ancient origin for these alleles. The considerable diversity accumulated at this locus may be due to selection, which is consistent with an important role for the CFHR genes in innate immunity.

Y402H Polymorphism of Complement Factor H Affects Binding Affinity to C-Reactive Protein

Complement factor H (FH) is an important regulator of the alternative complement pathway. The Y402H polymorphism within the seventh short consensus repeat of FH was recently shown to be associated with age-related macular degeneration, the most common cause of irreversible blindness in the Western world. We examined the effects of this polymorphism on various FH functions. FH purified from sera of age-related macular degeneration patients homozygous for the FH402H variant showed a significantly reduced binding to C-reactive protein (CRP), an acute phase protein, as compared with FH derived from unaffected controls homozygous for the FH402Y variant. Strongly reduced binding to CRP was also observed with a recombinant fragment of FH (short consensus repeat 5–7) containing the same amino acid change. Because the interaction of CRP and FH promotes complement-mediated clearance of cellular debris in a noninflammatory fashion, we propose that the reduced binding of FH402H to CRP could lead to an impaired targeting of FH to cellular debris and a reduction in debris clearance and enhanced inflammation along the macular retinal pigmented epithelium-choroid interface in individuals with age-related macular degeneration.

Local cellular sources of apolipoprotein E in the human retina and retinal pigmented epithelium: implications for the process of drusen formation☆

PURPOSE The inheritance of specific apolipoprotein E allelles has been linked to atherosclerosis, Alzheimer disease, and, most recently, to the incidence of age-related macular degeneration. Apolipoprotein E is a common component of the extracellular plaques and deposits characteristic of these disorders, including drusen, which are a hallmark of age-related macular degeneration. Accordingly, we assessed the potential biosynthetic contribution of local ocular cell types to the apolipoprotein E found in drusen. METHODS We measured apolipoprotein E mRNA levels in human donor tissues using a quantitative assay of apolipoprotein E transcription, and we localized apolipoprotein E protein to specific cell types and compartments in the neural retina, retinal pigmented epithelium, and choroid using laser scanning confocal immunofluorescence microscopy. RESULTS Apolipoprotein E immunoreactivity is associated with photoreceptor outer segments, the retinal ganglion cell layer, the retinal pigmented epithelium basal cytoplasm and basal lamina, and with both collagenous layers of Bruch membrane. Apolipoprotein E appears to be a ubiquitous component of drusen, irrespective of clinical phenotype. It also accumulates in the cytoplasm of a subpopulation of retinal pigmented epithelial cells, many of which overlie or flank drusen. Mean levels of apolipoprotein E mRNA in the adult human retina are 45% and 150% of the levels measured in liver and adult brain, the two most abundant biosynthetic sources of apolipoprotein E. Apolipoprotein E mRNA levels are highest in the inner retina, and lowest in the outer retina where photoreceptors predominate. Significant levels of apolipoprotein E mRNA are also present in the retinal pigmented epithelium/choroid complex and in cultured human retinal pigmented epithelial cells. CONCLUSIONS Apolipoprotein E protein is strategically located at the same anatomic locus where drusen are situated, and the retinal pigmented epithelium is the most likely local biosynthetic source of apolipoprotein E at that location. Age-related alteration of lipoprotein biosynthesis and/or processing at the level of the retinal pigmented epithelium and/or Bruch membrane may be a significant contributing factor in drusen formation and age-related macular degeneration pathogenesis.

Vitronectin is a constituent of ocular drusen and the vitronectin gene is expressed in human retinal pigmented epithelial cells

Age‐related macular degeneration (AMD) leads to dysfunction and degeneration of retinal photoreceptor cells. This disease is characterized, in part, by the development of extracellular deposits called drusen. The presence of drusen is correlated with the development of AMD, although little is known about drusen composition or biogenesis. Drusen form within Bruchs membrane, a stratified extracellular matrix situated between the retinal pigmented epithelium and choriocapillaris. Because of this association, we sought to determine whether drusen contain known extracellular matrix constituents. Antibodies directed against a battery of extracellular matrix molecules were screened on drusen‐containing sections from human donor eyes, including donors with clinically documented AMD. Antibodies directed against vitronectin, a plasma protein and extracellular matrix component, exhibit intense and consistent reactivity with drusen; antibodies to the conformationally distinct, heparin binding form of human vitronectin are similarly immunoreactive. No differences in vitronectin immunoreactivity between hard and soft drusen, or between macular and extramacular regions, have been observed. RT‐PCR analyses revealed that vitronectin mRNA is expressed in the retinal pigmented epithelium (RPE)‐choroidal complex and cultured RPE cells. These data document that vitronectin is a major constituent of human ocular drusen and that vitronectin mRNA is synthesized locally. Based on these data, we propose that vitronectin may participate in the pathogenesis of AMD.—Hageman, G. S., Mullins, R. F., Russell, S. R., Johnson, L. V., Anderson, D. H. Vitronectin is a constituent of ocular drusen and the vitronectin gene is expressed in human retinal pigmented epithelial cells. FASEB J. 13, 477–484 (1999)

Individuals homozygous for the age-related macular degeneration risk-conferring variant of complement factor H have elevated levels of CRP in the choroid

Polymorphisms in the complement factor H gene (CFH) are associated with a significantly increased risk for, or protection against, the development of age-related macular degeneration (AMD). The most documented risk-conferring single-nucleotide polymorphism results in a tyrosine-to-histidine substitution at position 402 (Y402H) of the CFH protein. In this work, we examined the ocular distributions and relative abundance of CFH, several CFH-binding proteins, and abundant serum proteins in the retinal pigmented epithelium (RPE), Bruchs membrane, and choroid (RPE–choroid) in CFH homozygotes possessing either the “at-risk” 402HH or “normal” 402YY variants. Although CFH immunoreactivity is high in the choroid and in drusen, no differences in CFH-labeling patterns between genotypes are apparent. In contrast, at-risk individuals have significantly higher levels of the CFH-binding protein, C-reactive protein (CRP), in the choroidal stroma. Immunoblots confirm that at-risk individuals have ≈2.5-fold higher levels of CRP in the RPE–choroid; no significant differences in the levels of CFH or other serum proteins are detected. Similarly, we find no differences in CFH transcription levels in the RPE–choroid nor evidence for local ocular CRP transcription. Increased levels of CRP in the choroid may reflect a state of chronic inflammation that is a by-product of attenuated CFH complement-inhibitory activity in those who possess the CFH at-risk allele. Because the CRP-binding site in CFH lies within the domain containing the Y402H polymorphism, it is also possible that the AMD risk-conferring allele alters the binding properties of CFH, thereby leading to choroidal CRP deposition, contributing to AMD pathogenesis.