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(The FASEB Journal. 2000;14:835-846.)
© 2000 FASEB

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

ROBERT F. MULLINS, STEPHEN R. RUSSELL, DON H. ANDERSON* and GREGORY S. HAGEMAN1

The University of Iowa Center for Macular Degeneration, Department of Ophthalmology and Visual Sciences, The University of Iowa, Iowa City, Iowa 52242, USA; and
* Center for the Study of Macular Degeneration, Neuroscience Research Institute, University of California, Santa Barbara, California 93106, USA

1Correspondence: Department of Ophthalmology and Visual Sciences, The University of Iowa, 11190E PFP, 200 Hawkins Dr., Iowa City, Iowa 52240, USA. E-mail address: gregory-hageman{at}uiowa.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUMMARY
 REFERENCES
 
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.


Key Words: retina • retinal pigmented epithelium • amyloid • vitronectin • soft drusen


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUMMARY
 REFERENCES
 
AGE-RELATED MACULAR DEGENERATION (AMD) is a blinding disease that adversely affects vision in the elderly. The disease is characterized by degeneration of the macular retina and choroid by atrophy or detachment and scarring caused by choroidal neovascularization. AMD appears to be heritable as an autosomal dominant trait in a significant proportion of afflicted individuals (1 2 3 4 5 6 7 8) . Early clinical signs of AMD include pigmentary changes at the level of the retinal pigmented epithelium (RPE) and an accumulation of abnormal extracellular deposits, called drusen, adjacent to the basal surface of the RPE. Histologically, drusen are located between the basal lamina of the RPE and the inner collagenous layer of Bruch’s membrane (9 10 11 12) . Drusen size, number, and degree of confluence are significant risk factors for the development of AMD (9 , 11) . It has been proposed that drusen, as well as other age-related changes that can occur in the vicinity of Bruch’s membrane, may lead to dysfunction and/or degeneration of the RPE and retina by inducing ischemia and/or by restricting the exchange of nutrients and waste products between the neural retina and choroid (13) . In addition, drusen themselves may have a detrimental effect on vision, particularly with respect to contrast sensitivity (14 15 16 17 18) . Significantly, a few recent studies have shown that laser photocoagulation treatment of soft drusen can elicit drusen regression and, in some cases, improvement of visual acuity (19 20 21 22 23) .

In view of the strong correlation between drusen deposition and the development of AMD, a more thorough understanding of drusen composition and origin is likely to provide new insight into the pathobiology of this disease. Comprehensive data pertaining to drusen composition and to potential compositional differences between drusen phenotypes are lacking. The few histochemical and immunohistochemical studies (24 25 26 27 28 29 30 31) of drusen that have been performed frequently used small numbers of human donor eyes often with long postmortem times, did not consider potential compositional differences between drusen phenotypes, used relatively few probes, did not typically consider the effects of tissue processing on drusen composition, and in some cases provided conflicting data.

We recently identified vitronectin, a multifunctional plasma and extracellular matrix protein, as a major component of drusen (32) . Vitronectin is a multifunctional glycoprotein that participates in cell adhesion, maintenance of hemostasis, and inhibition of complement-induced cell lysis (33) . Vitronectin is also a component of pathological extracellular deposits associated with atherosclerosis (34 , 35) , amyloidosis (36) , elastosis (37) , and dense deposit disease associated with membranoproliferative glomerulonephritis type II (38) . Like drusen, all of these deposits form within the extracellular matrix and are associated with advancing age. One epidemiological study has provided data suggesting a strong correlation between advanced AMD and atherosclerosis of carotid arteries (39) , while another identified a significant correlation between elastotic degeneration of nonsolar-exposed dermis and choroidal neovascularization in AMD patients (40) . Based on these data, one can speculate that these diseases may share similar genetic/environmental risk factors, developmental pathways, and/or etiologies.

In this study, we demonstrate that there is a high degree of compositional similarity between ocular drusen and the extracellular deposits characteristic of other age-related diseases. We used information pertaining to the established compositions of amyloid deposits, elastotic plaques, atherosclerotic plaques, and dense deposits as a baseline to further refine our understanding of drusen composition and to assess the degree of similarity between drusen and these pathological deposits. The results provide additional insight into drusen composition and the pathobiology of AMD.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 SUMMARY
 REFERENCES
 
Tissue and drusen classification
The 63 human donor eyes used in this study were obtained from The University of Iowa Lions Eye Bank (Iowa City, Iowa) within 4 h of death; donor ages ranged from 45 to 96 years. Drusen were categorized as hard or soft as described previously (41) . Tissues from a minimum of five donors (at least two of whom had clinically documented AMD) were assayed with each antibody used, and each drusen phenotype was examined in at least two donors. Institutional Review Board committee approval for the use of human donor tissues was obtained from the Human Subjects Committee at The University of Iowa.

Immunohistochemistry
Posterior poles, or wedges of posterior poles spanning between the ora serrata and macula, were fixed in 4% (para)formaldehyde in 100 mM sodium cacodylate, pH 7.4. After 2–4 h of fixation, eyes were transferred to 100 mM sodium cacodylate and rinsed (3x10 min), infiltrated, and embedded in acrylamide, as described previously (41) . These tissues were subsequently embedded in OCT, snap frozen in liquid nitrogen, and stored at -80°C. Unfixed posterior poles or wedges thereof were embedded directly in OCT, without acrylamide infiltration or embedment. Both fixed and unfixed tissues were sectioned to a thickness of 6–8 µm on a cryostat. The presence and type(s) of drusen were documented on adjacent sections stained with hematoxylin/eosin, periodic acid Schiff reagent, and Sudan black B (1% in 70% ethanol).

Immunolabeling was performed as described previously (32) . Adjacent sections were incubated with secondary antibody alone to serve as negative controls. Some immunolabeled specimens were viewed by confocal laser scanning microscopy, as described previously (42) .

Antibodies were obtained from Accurate (Westbury, N.Y.), Boehringer Mannheim (Indianapolis, Ind.), Calbiochem (La Jolla, Calif.), Chemicon (Temecula, Calif.), Dako (Carpinteria, Calif.), StressGen Biotechnologies (Victoria, B.C.), and ICN (Costa Mesa, Calif.) (Table 1 ). Since most of the antibodies used in this study react with serum proteins, retinal antigens, and/or choroidal antigens, positive controls for many of the antibodies were based on appropriate labeling of these tissues. Other control tissues included human liver, cornea, and ocular skeletal muscle, as appropriate.


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  		Table 1. Immunoreactivity of drusena

Amyloid histochemistry
Sections containing hard and soft drusen phenotypes were stained for amyloid with crystal violet (Allied Chemical; New York, N.Y.), Congo red (Sigma Chemical; St. Louis, Mo.), and thioflavin T (Sigma) using established protocols (43) . Human liver and cornea were used as a positive control tissue for amyloid staining. Birefringence was assessed by viewing Congo red-stained sections under polarized light. Thioflavin T-stained sections were examined using ultraviolet fluorescence optics (Olympus UG-1 filter set).

Reverse transcription-polymerase chain reaction (RT-PCR) analyses
To determine whether drusen-associated molecules are synthesized by local ocular sources, total RNA was isolated from neural retina, RPE, or combined RPE and choroid using the RNeasy system (Qiagen; Valencia, Calif.). Liver and peripheral blood leukocyte RNA, as well as genomic DNA, served as positive and negative controls, respectively. RT-PCR was performed as described previously (32) . Primers to nucleotide sequences of the 15 molecules examined in this study were used to amplify cDNA molecules from these tissue sources. Primer sequences are depicted in Table 2 . Reaction mix without template and/or omission of reverse transcriptase during the RT reaction were used as negative controls. PCR amplification products were separated by agarose gel electrophoresis and stained with ethidium bromide for visualization.


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  		Table 2. Expression results from retina, RPE/choroid, and livera


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 SUMMARY
 REFERENCES
 
Immunohistochemistry
Sections of human donor eyes possessing various drusen phenotypes (44) were incubated with a battery of antibodies directed against proteins known to be present in abnormal deposits associated with amyloidosis, atherosclerosis, elastosis, and dense deposit disease. Hard and soft drusen, and their respective subclasses, exhibit nearly identical immunohistochemical reactions with the majority of antibodies used (Table 1) . All reactions were specific based on positive reactivity with the appropriate control tissue (see Fig. 1 ). All drusen phenotypes examined were bound by antibodies directed against amyloid P component and apolipoprotein E (Fig. 1C, D ), two plasma proteins also present in abnormal extracellular deposits associated with atherosclerosis and various amyloidoses (45 46 47 48 49) . These antibodies bound homogeneously to both hard and soft drusen. The immunofluorescence associated with drusen was consistently more intense than that of the adjacent choroid. Antibodies directed against complement component C5 (Fig. 1A, B ), the terminal C5b-9 complement complex, and Factor X also reacted homogeneously with all classes of hard and soft drusen. Immunoglobulin lambda chain and transthyretin (prealbumin) frequently bound drusen, although this labeling was less consistent (~75% of donors: 9 of 13 and 9 of 12, respectively). This labeling did not correspond to specific ultrastructural drusen phenotypes. Strong transthyretin immunoreactivity was invariably noted in the RPE, however (Fig. 2A ).



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  		Figure 1. Fluorescence light micrographs depicting examples of drusen (asterisks) immunoreactivity to various antibodies (see Table 1 for complete profile). Immunolabeling is green (red in panel G) and RPE autofluorescence is yellow. Drusen immunoreactivity is consistently observed with anti-C5 antibodies (A); a secondary antibody control is depicted in panel B. Both small, ‘hard’ (C) and large, ‘soft’ (D) drusen also react with antibodies to apolipoprotein E. Labeling was not generally observed with C1q (E) and albumin (J) antibodies (see also ref 32 ). Heterogeneous labeling is noted with a number of antibodies that exhibit variable drusen immunoreactivity, including antifibrinogen (F), which occasionally labels concentric rings within drusen, prothrombin (G), and amyloid A (I), which label spherical profiles within some drusen. Double-labeling with an antivitronectin antibody (H), which labels drusen homogeneously, reveals that vesicular labeling of prothrombin (G) does not correspond to an overall condensation of drusen constituents, but to actual heterogeneous distributions of some drusen-associated molecules. Magnification 120x (A–D, G, H); 180x (E, F, I, J):



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  		Figure 2. Laser scanning confocal micrographs depicting different labeling patterns in aging human eyes. Transthyretin (A) was invariably detected in the RPE, but its labeling in drusen (asterisk) was variable; in this field, intense labeling of the basal RPE is noted, whereas the associated druse (asterisk) is negative. Weak, variable immunoreactivity is noted with some antibodies, including those directed against C-reactive protein, which binds to some smaller drusen (B). Antibodies directed against HLA-DR react intensely with drusen (asterisk), either homogeneously (as noted in a small druse, C) or in globular or core-like domains (not shown). RPE autofluorescence appears red in panels B and C. Approximate magnification x500.

Weak and/or inconsistent drusen labeling of antibodies directed against the protease inhibitors {alpha}1-antitrypsin and {alpha}1-antichymotrypsin was observed. Labeling with complement C3c antibody was more variable than C5 antibody labeling; strong choroidal immunoreactivity was consistently observed with antibodies directed against C3c, whereas drusen reactivity ranged from negative to strongly positive. Drusen that did not react with C3 antibodies, however, invariably exhibited C5 immunoreactivity in adjacent tissue sections, as did all drusen phenotypes examined. Weak or inconsistent immunoreactivity was also noted in some donors with antibodies directed against amyloid A component, fibrin, immunoglobulin kappa chain, cystatin C, apolipoprotein B, complement C1q, C-reactive protein, and ß2-microglobulin (see examples of negative reactions in Figs. 1 and 2 ). Ubiquitin reactivity was noted in small punctate regions of drusen in one donor, but typically was not observed.

No reaction of antibodies to the primary amyloid proteins keratin, apolipoprotein A-I, gelsolin, calcitonin, atrial natriuretic factor, ß-peptide, tau, or amyloid precursor protein was observed (Table 1) . Antibodies directed against human serum albumin and haptoglobin bound strongly to the choroidal stroma, but not to hard or soft drusen (Fig. 1 ; see also ref 32 ).

Immunoreactivity of some drusen-associated proteins was frequently observed in distinct, heterogeneous patterns. For example, drusen binding by prothrombin and amyloid A antibodies was often localized to vesicular profiles within drusen (Fig. 1G, I ). Double labeling of the same drusen with vitronectin (Fig. 1H ) revealed that the localization of prothrombin and amyloid A is not an artifact of condensation of drusen constituents. Drusen were labeled occasionally by anti-fibrinogen antibodies (four of seven donors); this binding was generally confined to peripheral regions and/or concentric bands within drusen (Fig. 1F ). In one donor, this labeling was confined to large drusen, whereas smaller drusen were nonreactive.

In addition, strong immunoreactivity of drusen to two monoclonal antibodies directed against human leukocyte antigen-DR (HLA-DR) molecules was noted (Fig. 2C ). This labeling was often confined to globular or core-like domains within drusen near the inner collagenous zone (data not shown). This pattern of labeling is similar to that described for peanut agglutinin after neuraminidase treatment (50) .

As has been described previously, some drusen stain pale red with Congo red (28) ; this is also observed with amyloid deposits. However, no drusen phenotype examined exhibits the characteristic apple green birefringence of amyloids stained with Congo red when observed under polarized light. Like amyloids, drusen stained with crystal violet (although without obvious metachromasia) and under UV illumination displayed an intense white fluorescence after staining with thioflavin T (data not shown). Thioflavin T staining also revealed amyloid-like material surrounding blood vessels in the inner retina of some eyes.

Transmission electron microscopy
In ongoing ultrastructural studies (44) we have failed to identify any electron microscopic evidence of nonbranching, 10–15 nm diameter fibrils that are characteristic of amyloid deposits in over 320 donors examined (data not shown). In an attempt to expose drusen-associated fibrils that might be masked by lipids and/or membranous debris, drusen-containing tissues were pretreated with DMSO, chloroform/methanol/HCl, or urea/Nonidet P-40 prior to embedding and sectioning. No fibrillar substructures were observed within drusen after such treatments (data not shown).

RT-PCR
Ocular cells were identified as sources for transcripts encoding a number of drusen-associated molecules (see Table 2 and Fig. 3 ). The RPE contained appropriately sized PCR fragments for albumin, alpha-1-antichymotrypsin, apolipoprotein E, complement C3, complement C5, and HLA-DR. Neural retina and/or choroid possessed transcripts coding for Factor X, apolipoproteins B and E, complement proteins C3, C5, and C9, immunoglobulins kappa and lambda, HLA-DR, fibrinogen, and albumin. It is likely that some of these transcripts are synthesized by vascular retinal and/or choroidal leukocytes. Prothrombin and amyloid P transcripts of the appropriate sizes were detected only in the liver, suggesting that these drusen-associated proteins may be derived from extraocular tissue.



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  		Figure 3. RT-PCR analyses of various drusen-associated molecules in retina (R), RPE/choroid (RC), and RPE (PE) derived from donors with (+) and without (-) clinically documented AMD. The primers used in these reactions are depicted in Table 2 . Transcripts for complement proteins C3 and C5, apolipoprotein E, and albumin were detected in neural retina, RPE/choroid, RPE, and liver (L); no amplification of genomicDNA was observed (G). Complement C9, immunoglobulin lambda, Factor X, and apolipoprotein B transcripts were detected in neural retina and RPE/choroid, but not in isolated RPE cells. Immunoglobulin lambda and kappa chain messages were also detected in peripheral blood leukocytes (B). Kappa light chain and fibrinogen cDNAs amplified from RPE/choroid. Amplicons of the appropriate size for the drusen-associated molecules amyloid P component and prothrombin were detected only in liver; the higher molecular weight band (651 bp, as opposed to 536 bp in liver) in other lanes with amyloid P primers is due to amplification of genomic DNA. Lane B depicts a 100 bp DNA ladder in the gel showing fibrinogen.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
SUMMARY
 REFERENCES
 
Drusen develop between the RPE basal lamina and the inner collagenous layer of Bruch’s membrane, a stratified extracellular matrix situated between the choriocapillaris and RPE. Based on their location, we had previously reasoned that drusen might be composed largely of extracellular matrix components. To test this supposition, we had screened drusen-containing tissue sections with antibodies directed against a comprehensive panel of extracellular matrix proteins (32) . Although surprisingly few of these antibodies reacted with drusen, those directed against vitronectin reacted intensely and consistently with all ultrastructural phenotypes of hard and soft drusen (44) .

Vitronectin is a known component of extracellular deposits associated with nonocular, age-related diseases including atherosclerosis, amyloidosis, dense deposit disease, and dermal elastotic degeneration (see Table 3 ; 35 , 37 , 38 , 51 ). In view of the documented clinical correlations between the incidence of AMD and that of atherosclerosis (39) and dermal elastotic degeneration (40) , we screened drusen with a second panel of antibodies directed against molecules known to colocalize with vitronectin in pathological extracellular matrix deposits associated with these extraocular diseases. We also examined drusen for histochemical and ultrastructural evidence of other features diagnostic for amyloid deposits. The results revealed that both hard and soft drusen phenotypes contain additional molecular species associated with abnormal deposits found in other age-related diseases (Table 3) .


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  		Table 3. Compositional comparison of extracellular disease depositsa

Relationship between drusen and amyloid deposits
Histochemically, amyloid deposits are eosinophilic, metachromatic after being stained with crystal violet, fluorescent after being stained with thioflavin T, and birefringent under polarized light after exposure to Congo red (52) . Amyloid deposits characteristically contain a fibrillar protein that exhibits a high degree of antiparallel beta-sheet conformation, heparan sulfate proteoglycans, and amyloid P component (45 , 53) . Plaques associated with Alzheimer’s disease contain amyloid ß, apolipoprotein E, {alpha}-1-chymotrypsin, immunoglobulin G (IgG), complement proteins, amyloid P, glycosaminoglycans, and Sp40,40 (54) .

Drusen share some of these histochemical and compositional properties, but not others. They stain positively with crystal violet, thioflavin T (see also ref 55 ), and (to a lesser degree) with Congo red, but do not exhibit metachromasia or emit green birefringence. In addition, drusen are eosinophilic and show strong evidence of amyloid P component immunoreactivity. Amyloid P is also a component of nonamyloid deposits associated with atherosclerosis (34) , keratin intermediate filament aggregates (56) , and dense deposits characteristic of glomerulonephropathy (47) . Amyloid P component associates with elastic fibers, where it may function as a protease inhibitor (57) . This protein is also a normal component of Bruch’s membrane, where it may protect the elastic lamina against enzymatic degradation (58) . It is possible that the accumulation of amyloid P, TIMP3 (59) and, less frequently, {alpha}1-antichymotrypsin (an inhibitor of serine proteases) in drusen may act to counterbalance attempts by RPE or choroidal cells to clear drusen proteolytically. Whether or not sulfated proteoglycans, ubiquitous components of amyloids, are present in drusen remains to be firmly established (27 , 30) , although we have identified weak reaction of heparan sulfate antibodies with some drusen (32) .

Although one investigation has reported a reaction of drusen with monoclonal antibodies directed against ß-peptide (28) , a principal component of cerebral amyloid in Alzheimer’s disease, we did not detect immunoreactivity for ß-peptide or its parent molecule, amyloid precursor protein, in drusen. We did, however, identify a number of molecules that serve as precursors for amyloid fibrils as components of drusen in some eyes. These include transthyretin, immunoglobulin light chains, and amyloid A. Other known precursors of amyloid fibrils, including ß2-microglobulin, apolipoprotein A1, atrial natriuretic factor, calcitonin, gelsolin, or cystatin C, were not detected as components of drusen. Furthermore, ultrastructural analyses of drusen do not reveal any classic, nonbranching, 10–15 nm diameter amyloid fibrils within drusen even after pretreatment of tissues with lipid-solubilizing compounds.

On balance, our evidence strongly suggests that although drusen exhibit some of the characteristics of amyloid and contain several amyloid-associated proteins, they do not contain fibrils characteristic of true amyloids. Thus, drusen do not appear to be an ocular manifestation of any of the identified forms of amyloidoses, nor do they appear to be an ocular-specific form of amyloidosis. There are certainly alternative explanations for the presence of amyloid-associated molecules in drusen. For example, since transthyretin is the major carrier of vitamin A in the blood, its accumulation in some drusen could result from decreased permeability between the choroid and RPE. It is intriguing to speculate that the accumulation of transthyretin that sometimes occurs in drusen and other sub-RPE deposits might result in a reduction of available vitamin A to the RPE/retina, ultimately leading to the photoreceptor/RPE cell degeneration observed in AMD. Alternatively, since transthyretin has been reported to be synthesized by RPE cells (60) , its presence in some drusen might support hypotheses suggesting that drusen originate from RPE cells (61) .

Relationship between drusen and elastotic lesions
Elastosis is a heterogeneous group of disease conditions in which the elastic fibers in the dermis undergo hyperplasia and/or rearrangement. Whereas sun-exposed skin typically develops elastotic lesions by the second decade of life, these accumulations are less common and severe in sun-protected areas of the skin (62) . The presence of elastotic degeneration of sun-protected dermis has been shown to correlate with the incidence of neovascular AMD (40) . Deposits that comprise elastotic lesions may be either eosinophilic (elastosis perforans serpiginosa)—like drusen—or basophilic (actinic elastosis). Elastotic lesions typically contain vitronectin (37) , amyloid P component (63) , and complement proteins (64) , all of which have been identified as components of drusen in this study. Although drusen contain a vitronectin and amyloid P component, they do not appear to contain elastin or elastin-associated fibrillin (32) , suggesting that they are not derived from hyperplasia and/or rearrangement of the elastic fibers in Bruch’s membrane. As such, it is unlikely that they are related to elastotic deposits.

However, one should not conclude that processes similar to those that occur in dermal elastosis/degeneration are not responsible for changes in the elastic lamina of Bruch’s membrane observed with aging and in patients with AMD (65 , 66) . Marked changes in the structure of elastic fibers are observed in aging and in a variety of disease processes such as atherosclerosis, emphysema, and some heritable diseases affecting the skin. In pseudoxanthoma elasticum, the prototypical heritable elastin disease (67 , 68) , the integrity of Bruch’s membrane is profoundly affected, leading to the formation of angioid streaks (69) . It is conceivable that at least some forms of AMD could stem from such pathological changes within Bruch’s membrane that are induced by specific gene mutations or biological processes also involved in elastosis.

Relationship between drusen and dense deposits
Abnormal basement membrane-associated deposits also appear in inherited and acquired disorders involving the kidney glomerulus. Dense deposit disease (a manifestation of membranoproliferative glomerulonephritis type II) is characterized histologically and ultrastructurally by the appearance of amorphous, electron dense deposits that accumulate in kidney basement membranes (70) . Extrarenal sites of deposition have also been observed in patients with dense deposit disease. These include deposits in the spleen (71) and in Bruch’s membrane, which resemble typical (72) or basal laminar (73) drusen funduscopically. Patients with dense deposit disease frequently develop subretinal neovascular membranes (73 , 74) , suggesting that the defect(s) responsible for dense deposit disease can elicit neovascular changes similar to AMD. Kidney dense deposits are not amyloids, since they fail to stain with Congo red and lack ß-pleated fibrillar components. Like drusen, dense deposits are immunoreactive with antibodies against vitronectin, immunoglobulin, and complement C5 (38) . However, though dense deposits share some compositional features with drusen, the former are electron dense, osmiophilic structures that characteristically contain complement C3 (which is diagnostic of dense deposits; ref 75 ), complement C1q, type VI collagen, and IgM as major components. Collagen VI and IgM have not been shown to be drusen constituents in a number of studies (32 , 76) (although some Ig light chains were detected in this study) and labeling of drusen with anti-C3 antibodies is variable. Collectively, these data suggest that dense deposits are compositionally similar, although not identical, to drusen.

Relationship between drusen and atherosclerotic plaques
Our results, when combined with those from other laboratories, suggest that drusen share a number of molecular constituents with atherosclerotic plaques, including lipids (77) , vitronectin (35) , apolipoprotein E (48 , 78) , calcium (79) , and complement components (34) . In view of the documented clinical correlation between advanced AMD and carotid artery atherosclerosis (39) , it is conceivable that a similar pathogenic mechanism may be involved. Like drusen, atherosclerotic plaques form adjacent to an elastic layer, in the vicinity of vascular endothelial cells and pericytes (80) . Like drusen, atherosclerotic plaques are composed of a number of plasma proteins. However, apolipoprotein B, a major component of atherosclerotic plaques (81) , was not consistently detected in drusen, although antibodies directed against apolipoprotein B reacted with blood vessels in the retina and choroidal stroma. Collectively, these results suggest that although drusen share some major components with atherosclerotic plaques (e.g., vitronectin, apolipoprotein E, complement, and lipid), some compositional differences also exist. We propose that future studies aimed at examining the compositional relationships between specific drusen phenotypes and specific forms of arterial disease may help to identify common pathogenic pathways that contribute to drusen and atherosclerotic plaque formation.

Drusen contain immune- and inflammatory-related proteins
A few studies have indicated a potential role for immune-mediated processes in the development of AMD. Most significantly, autoantibodies have been detected in the sera of some AMD patients (82 , 83) . Data derived from this investigation provide additional support for the concept that immune- and inflammatory-mediated processes may be involved in the development and/or regression of drusen. Drusen contain several acute-phase reactants, coagulation proteins, immunoglobulin, activated complement, and proteins that are involved in the induction or activation of the immune response (see Table 1 ). Vitronectin, for example, is capable of inhibiting complement-mediated cytolysis (33) . It may also be significant that macrophages are observed in the choroid adjacent to drusen (22 , 84) . These cells have been implicated in the development of choroidal neovascularization (85 86 87 88) , but might also contribute to drusen accretion, directly or indirectly, by synthesizing and secreting drusen-associated molecules such as complement C5, apolipoprotein E, Factor X, and prothrombin. In light of the suggestion that humoral immune processes may be associated with the etiology of AMD, it is interesting that drusen of all phenotypes exhibit intense HLA-DR immunoreactivity. Further studies will be required to determine the source(s) of drusen-associated HLA-DR and the putative role of immune-mediated processes in drusen biogenesis and the etiology of AMD.

Drusen origin and biogenesis
These data suggest that the process of protein accumulation in drusen is selective. For example, drusen do not contain albumin or haptoglobin, which, like vitronectin, are abundant plasma proteins (see also ref 32 ) that are present within the choroidal stroma and vascular lumens (89) . This contention is strengthened further by the observation that drusen-associated plasma proteins are not removed after extensive rinsing and that drusen-associated vitronectin is present in both its heparin binding and multimeric forms (32) .

The precise origin(s) of drusen-associated proteins remains to be resolved. Some drusen constituents (e.g., plasma proteins such as amyloid P component and prothrombin) may pass out of choroidal vessels and into the extracellular space adjacent to the RPE, where they might bind to one or more ligands associated with developing drusen. Other drusen constituents might be secreted by local retinal, RPE and/or choroidal cells. Significantly, this study provides compelling data that the messages for vitronectin (32) , complement proteins C3, C5, and C9, Factor X, and apolipoproteins B and E are expressed locally by retinal, RPE, and/or choroidal cells (Fig. 3 , Table 2 ; see also refs 90 91 92 ). Thus, if these messages are translated and the encoded proteins secreted, local cell types may play a significant role in drusen biogenesis.


   SUMMARY
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUMMARY
REFERENCES
 
These data, when combined with those of other investigators, provide us with fresh insight into the cellular processes and molecular events that may govern drusen biogenesis. Specifically, this study suggests that 1) clinically distinct hard and soft drusen may share a common origin; 2) the formation and growth of drusen is most likely an active, selective process and not one attributable solely to passive accumulation of proteins and lipids, as is commonly held; 3) distinct biological pathways, including immune-mediated processes, may be involved in drusen biogenesis; 4) some drusen-associated constituents may be synthesized locally by ocular cells; and 5) similar mechanisms of pathogenesis may be involved in the biogenesis of drusen and the characteristic extracellular deposits associated with amyloidosis, atherosclerosis, elastosis, and dense deposit disease. The hypothesis that drusen may develop as a result of specific biological processes, including immune-mediated events, should stimulate testable new hypotheses regarding the role of drusen in AMD pathogenesis.


   ACKNOWLEDGMENTS
 
The authors are sincerely grateful to all of the donors and their families who gave unselfishly for the advancement of science, and to the staff of the Iowa Lions Eye Bank (Ms. Sara Baker, Ms. Barb Elias, Ms. Jill Hageman, Ms. Heather Luders, Ms. Pat Mason, Ms. LouAnn Mercier, Mr. Paul Ogg, and Dr. John Sutphin) for their untiring dedication to our research program. The technical assistance of Ms. Lisa Hancox, Ms. Bobbie Schneider, and Mr. Cory Speth and the secretarial assistance of Ms. Linda Koser are appreciated. Conversations with, and advice from, Markus Kuehn and Dr. Marilyn Kincaid are also gratefully acknowledged. Supported in part by NIH grants EY06463 (GSH), EY11515 (GSH), EY11521 (DHA), AG00214 (The University of Iowa Center on Aging), the David Woods Kemper Memorial Foundation (GSH), a Lew R. Wassarman Merit Award (GSH), and an unrestricted grant to the University of Iowa Department of Ophthalmology and Visual Sciences from Research to Prevent Blindness, Inc.


   FOOTNOTES
 
Received for publication July 26, 1999. Revised for publication December 2, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUMMARY
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