Extracellular vesicles released by human retinal pigment epithelium mediate increased polarised secretion of drusen proteins in response to AMD stressors.

Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key contributors to the etiology of AMD and the ability to modulate drusen biogenesis could lead to therapeutic strategies to slow or halt AMD progression. The mechanisms underlying drusen biogenesis, however, remain mostly unknown. Here we demonstrate that under homeostatic conditions extracellular vesicles (EVs) secreted by retinal pigment epithelium (RPE) cells are enriched in proteins associated with mechanisms involved in AMD pathophysiology, including oxidative stress, immune response, inflammation, complement system and drusen composition. Furthermore, we provide first evidence that drusen-associated proteins are released as cargo of extracellular vesicles secreted by RPE cells in a polarised apical:basal mode. Notably, drusen-associated proteins exhibited distinctive directional secretion modes in homeostatic conditions and, differential modulation of this directional secretion in response to AMD stressors. These observations underpin the existence of a finely-tuned mechanism regulating directional apical:basal sorting and secretion of drusen-associated proteins via EVs, and its modulation in response to mechanisms involved in AMD pathophysiology. Collectively, our results strongly support an active role of RPE-derived EVs as a key source of drusen proteins and important contributors to drusen development and growth.

Complement Activation Products and Cytokines in Pachychoroid Neovasculopathy and Neovascular Age-Related Macular Degeneration.

Purpose: To investigate the characteristics of complement activation products and angiogenic cytokines in the aqueous humor in eyes with pachychoroid neovasculopathy (PNV) and neovascular age-related macular degeneration (nAMD). Methods: This was a prospective, comparative, observational study. All patients with choroidal neovascularization were classified as PNV without polyps, PNV with polyps (polypoidal choroidal vasculopathy [PCV]), or drusen-associated nAMD according to the presence or absence of pachychoroid features and soft drusen. This study included a total of 105 eyes. Aqueous humor samples were collected from 25 eyes with PNV without polyps, 23 eyes with PCV, and 24 eyes with drusen-associated nAMD before intravitreal anti-vascular endothelial growth factor (VEGF) injection and cataract surgery in 33 control eyes. Clinical samples were measured for complement component 3a (C3a), C4a, C5a, VEGF, and macrophage chemoattractant protein 1 (MCP-1) using a bead-based immunoassay. Results: C3a and MCP-1 levels were significantly higher in PCV (P = 0.032 and P = 0.039, respectively) and drusen-associated nAMD (P = 0.01 for both comparisons) than in controls, and no difference was seen in C3a and MCP-1 levels between PNV and controls (P = 0.747 and P = 0.294, respectively). VEGF levels were significantly higher in PNV (P = 0.016), PCV (P = 0.009), and drusen-associated nAMD (P = 0.043) than in controls. In PNV, the VEGF levels elevated without elevated C3a and MCP-1. Conclusions: PNV, PCV, and drusen-associated nAMD had significantly distinct profiles of complement activation products and cytokines in the aqueous humor.

Novel Programmed Cell Death as Therapeutic Targets in Age-Related Macular Degeneration?

Age-related macular degeneration (AMD) is a leading cause of severe visual loss among the elderly. AMD patients are tormented by progressive central blurring/loss of vision and have limited therapeutic options to date. Drusen accumulation causing retinal pigment epithelial (RPE) cell damage is the hallmark of AMD pathogenesis, in which oxidative stress and inflammation are the well-known molecular mechanisms. However, the underlying mechanisms of how RPE responds when exposed to drusen are still poorly understood. Programmed cell death (PCD) plays an important role in cellular responses to stress and the regulation of homeostasis and diseases. Apart from the classical apoptosis, recent studies also discovered novel PCD pathways such as pyroptosis, necroptosis, and ferroptosis, which may contribute to RPE cell death in AMD. This evidence may yield new treatment targets for AMD. In this review, we summarized and analyzed recent advances on the association between novel PCD and AMD, proposing PCD's role as a therapeutic new target for future AMD treatment.

Soft Drusen in Age-Related Macular Degeneration: Biology and Targeting Via the Oil Spill Strategies.

AMD is a major cause of legal blindness in older adults approachable through multidisciplinary research involving human tissues and patients. AMD is a vascular-metabolic-inflammatory disease, in which two sets of extracellular deposits, soft drusen/basal linear deposit (BLinD) and subretinal drusenoid deposit (SDD), confer risk for end-stages of atrophy and neovascularization. Understanding how deposits form can lead to insights for new preventions and therapy. The topographic correspondence of BLinD and SDD with cones and rods, respectively, suggest newly realized exchange pathways among outer retinal cells and across Bruch's membrane and the subretinal space, in service of highly evolved, eye-specific physiology. This review focuses on soft drusen/BLinD, summarizing evidence that a major ultrastructural component is large apolipoprotein B,E-containing, cholesterol-rich lipoproteins secreted by the retinal pigment epithelium (RPE) that offload unneeded lipids of dietary and outer segment origin to create an atherosclerosis-like progression in the subRPE-basal lamina space. Clinical observations and an RPE cell culture system combine to suggest that soft drusen/BLinD form when secretions of functional RPE back up in the subRPE-basal lamina space by impaired egress across aged Bruch's membrane-choriocapillary endothelium. The soft drusen lifecycle includes growth, anterior migration of RPE atop drusen, then collapse, and atrophy. Proof-of-concept studies in humans and animal models suggest that targeting the "Oil Spill in Bruch's membrane" offers promise of treating a process in early AMD that underlies progression to both end-stages. A companion article addresses the antecedents of soft drusen within the biology of the macula.

Polarized Exosome Release from the Retinal Pigmented Epithelium.

The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier and provides nutrients and recycling of visual pigment to the photoreceptors, among many other functions. The RPE is also a key site of pathophysiological changes in age-related macular degeneration, making it an important focus of study in both visual health and disease. Exosomes are nanometer-sized vesicles that are released by cells in a controlled fashion and mediate a range of extra- and intercellular activities. Some key exosome actions include cell-cell communication, immune modulation, extracellular matrix turnover, stem cell division/differentiation, neovascularization, and cellular waste removal. While much is known about their role in cancer and cardiovascular disease, exosome function in the many specialized tissues of the eye is just beginning to undergo rigorous study. Here we review current knowledge of the functions and roles of exosomes and other small extracellular vesicles released from the RPE. In particular, we discuss the potential role and importance of polarized exosome release from the RPE.

Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model.

Purpose: Extracellular deposits containing hydroxyapatite, lipids, proteins, and trace metals that form between the basal lamina of the RPE and the inner collagenous layer of Bruch's membrane are hallmarks of early AMD. We examined whether cultured RPE cells could produce extracellular deposits containing all of these molecular components. Methods: Retinal pigment epithelium cells isolated from freshly enucleated porcine eyes were cultured on Transwell membranes for up to 6 months. Deposit composition and structure were characterized using light, fluorescence, and electron microscopy; synchrotron x-ray diffraction and x-ray fluorescence; secondary ion mass spectroscopy; and immunohistochemistry. Results: Apparently functional primary RPE cells, when cultured on 10-µm-thick inserts with 0.4-µm-diameter pores, can produce sub-RPE deposits that contain hydroxyapatite, lipids, proteins, and trace elements, without outer segment supplementation, by 12 weeks. Conclusions: The data suggest that sub-RPE deposit formation is initiated, and probably regulated, by the RPE, as well as the loss of permeability of the Bruch's membrane and choriocapillaris complex associated with age and early AMD. This cell culture model of early AMD lesions provides a novel system for testing new therapeutic interventions against sub-RPE deposit formation, an event occurring well in advance of the onset of vision loss.

Aquaporin-1 Expression in Retinal Pigment Epithelial Cells Overlying Retinal Drusen.

PURPOSE: In the outer retina, age-related macular degeneration (AMD) results in reduced hydraulic conductivity in Bruch's membrane, possibly leading to altered water transport in retinal pigment epithelial (RPE) cells. We hypothesize that RPE cells may express aquaporin-1 (AQP1) to compensate for these changes. Therefore, we wanted to investigate the expression of AQP1 in RPE cells of human eyes with age-related maculopathy (ARM) and AMD, and eyes with tumour-associated drusen. METHODS: Nine human eyes with ARM, 6 eyes with AMD and 9 eyes with choroidal malignant melanoma were examined for immunoreactivity to AQP1. AQP1 labelling in the RPE cells was evaluated for each drusen and grouped according to size and AQP1 labelling. AQP1 labelling in the RPE outside drusen was also evaluated. RESULTS: AQP1 labelling was observed in the apical membrane of the RPE cells situated above drusen in all three groups. There was a significant association between AQP1 labelling and drusen size (p < 0.001), and AQP1 labelling was more frequently observed in large drusen. CONCLUSION: AQP1 was expressed in RPE cells covering drusen but not in RPE cells outside drusen. We suggest that AQP1 expression is upregulated in the cell membranes of RPE cells above drusen in order to alleviate the increased need for fluid transport across the growing drusen.

[Molecular genetic analysis and clinical phenotype of a pedigree with familial dominant drusen].

OBJECTIVE: To analyze clinical features and mutations of EFEMP1 gene in a Chinese pedigree with familial dominant drusen. METHODS: Clinical features of the pedigree were studied with fundus photography, fundus fluorescein angiography and optical coherence tomography. Molecular genetic analysis was performed on the patients and unaffected individuals from the family. All coding exons of the EFEMP1 gene were amplified by polymerase chain reaction (PCR) and sequenced. The results were compared with wild-type sequences from NCBI. The proband who had suffered from choroidal neovascularization and preretinal hemorrhage received an intravitreal injection of an anti-vascular endothelial growth factor (VEGF) preparation. RESULTS: A heterozygous mutation C>T (R345W) was identified in exon 10 of the EFEMP1 gene in two affected individuals from the family. The same mutation was not detected in unaffected family members and 100 healthy individuals. Postoperative follow-up of the patient receiving intravitreal injection of anti-VEGF drug showed that visual acuity was improved and fundus appeared to be stable. CONCLUSION: The R345W mutation in EFEMP1 is responsible for the dominant drusen in this family. Intravitreal injection of anti-VEGF drug is a promising treatment for the improvement in vision.

REFRACTILE DRUSEN: Clinical Imaging and Candidate Histology.

PURPOSE: To evaluate eyes with refractile drusen using clinical imaging and to identify candidate histologic correlates of refractile drusen. METHODS: Refractile drusen were defined as drusenoid material containing small refractile spherules. Retrospective analysis of color, autofluorescence, and spectral domain optical coherence tomography images of eyes with refractile drusen was performed to characterize the morphology and topography of these lesions. Macular sections from donor eyes were processed with a von Kossa stain for calcium phosphate and viewed by light microscopy. Punches of retinal pigment epithelium-choroid from donors with geographic atrophy were prepared for transmission electron microscopy. RESULTS: Fundus findings of 14 eyes of 10 patients with age-related macular degeneration (age, 82.9 ± 5.6 years) were evaluated. A generalized loss of autofluorescence signal over refractile drusen appeared to spread over a larger area than each druse, for drusen located centrally. By color fundus photography, refractile drusen showed corresponding depigmentation around drusen that were located in the center of the macula. Optical coherence tomography imaging of refractile drusen showed hyperreflective dots. In the histologic specimens, drusen contained many small spherules rich in calcium phosphate. Ultrastructural examination of the spherules showed complex assemblies consisting of concentric shells containing thin layers of calcium. CONCLUSION: Refractile drusen appear to be a stage of drusen regression marked by loss of retinal pigment epithelium, thus contributing to the development of geographic atrophy. Calcium-containing spherules appear to account for the glistening appearance.

The drusenlike phenotype in aging Ccl2-knockout mice is caused by an accelerated accumulation of swollen autofluorescent subretinal macrophages.

PURPOSE: Drusen, which are defined clinically as yellowish white spots in the outer retina, are cardinal features of age-related macular degeneration (AMD). Ccl2-knockout (Ccl2(-/-)) mice have been reported to develop drusen and phenotypic features similar to AMD, including an increased susceptibility to choroidal neovascularization (CNV). This study was conducted to investigate the nature of the drusenlike lesions in vivo and further evaluate the Ccl2(-/-) mouse as a model of AMD. METHODS: The eyes of 2- to 25-month-old Ccl2(-/-) and C57Bl/6 mice were examined in vivo by autofluorescence scanning laser ophthalmoscopy (AF-SLO) and electroretinography, and the extent of laser-induced CNV was measured by fluorescein fundus angiography. The retinal morphology was also assessed by immunohistochemistry and quantitative histologic and ultrastructural morphometry. RESULTS: The drusenlike lesions of Ccl2(-/-) mice comprised accelerated accumulation of swollen CD68(+), F4/80(+) macrophages in the subretinal space that were apparent as autofluorescent foci on AF-SLO. These macrophages contained pigment granules and phagosomes with outer segment and lipofuscin inclusions that may account for their autofluorescence. Only age-related retinal pigment epithelium (RPE) damage, photoreceptor loss, and sub-RPE deposits were observed but, despite the accelerated accumulation of macrophages, we identified no spontaneous development of CNV in the senescent mice and found a reduced susceptibility to laser-induced CNV in the Ccl2(-/-) mice. CONCLUSIONS: These findings suggest that the lack of Ccl2 leads to a monocyte/macrophage-trafficking defect during aging and to an impaired recruitment of these cells to sites of laser injury. Other, previously described features of Ccl2(-/-) mice that are similar to AMD may be the result of aging alone.

Drusenoid maculopathy in rhesus monkeys: autofluorescence, lipofuscin and drusen pathogenesis.

PURPOSE: To examine patterns of retinal pigment epithelial autofluorescence and lipofuscin accumulation in relation to drusen and to explore the pathogenesis of drusen in rhesus monkeys. METHODS: The macular areas of six rhesus monkeys, euthanized at 19 to 28 years of age, were studied by bright field and fluorescence light microscopy and transmission electron microscopy. RESULTS: There was strong autofluorescence in the retinal epithelium that tended to diminish over drusen. Electron microscopy revealed that all retinal epithelial cells had large concentrations of lipofuscin bodies. The epithelial cells overlying drusen, however, tended to have less lipofuscin than epithelial cells not associated with drusen. Electron microscopy revealed that the epithelial cells overlying drusen were losing segments of cytoplasm containing lipofuscin bodies. Macrophage-like cells were consistently present in Bruch's membrane microns away from this lipofuscin-containing cytoplasmic material. CONCLUSIONS: Retinal epithelial cells overlying drusen have less lipofuscin than neighboring epithelial cells. The loss of lipofuscin seems due to a loss of cytoplasm containing lipofuscin that contributes to drusen formation. Macrophages in Bruch's membrane may be responsible for removing this lipofuscin debris. The results support in vivo studies showing reduced autofluorescence over drusen and support the "budding" of epithelial cytoplasm as a source of drusen material.

Altered function of factor I caused by amyloid beta: implication for pathogenesis of age-related macular degeneration from Drusen.

The results of recent studies have implicated local inflammation and complement activation as the processes involved in the pathogenesis of age-related macular degeneration (AMD). We have demonstrated that amyloid beta (Abeta), which is deposited in drusen, causes an imbalance in the angiogenesis-related factors in retinal pigment epithelial cells. We have also shown that neprilysin gene-disrupted mice accumulate Abeta, and develop several features of AMD. The purpose of this study was to investigate the mechanisms involved in the development of AMD that are triggered by Abeta. Our results showed that Abeta binds to complement factor I which inhibits the ability of factor I to cleave C3b to inactivated iC3b. Factor H and factor I are soluble complement-activation inhibitors, and preincubation of factor I with Abeta in the presence of factor H abolished the ability of Abeta to cleave C3b, and also abolished the ability of factor I to cleave FGR-AMC. In contrast, Abeta did not affect the function of factor H even after binding. The production of iC3b was significantly decreased when C3b and factor H were incubated with the eyes from neprilysin gene-disrupted mice as compared with when C3b and factor H were incubated with eyes from age-matched wild-type mice. These results suggest that Abeta activates the complement system within drusen by blocking the function of factor I leading to a low-grade, chronic inflammation in subretinal tissues. These findings link four factors that have been suggested to be associated with AMD: inflammation, complement activation, Abeta deposition, and drusen.

Glycoproteins of drusen and drusen-like lesions.

Drusen are a marker of age-related macular degeneration (AMD). Lesions similar to drusen, both in histology and their clinical appearance, are also seen in choroidal tumours, chronic inflammatory and degenerative conditions of the eye, and in mesangiocapillary glomerulonephritis type II (MCGN-II). This study aims to compare the saccharide composition of these drusen-like lesions in the various ocular pathological groups and in MCGN-II. Formalin fixed and paraffin wax embedded tissue from 21 eyes was studied. The histological diagnoses included AMD, retinal detachment, phthisis bulbi following failed retinal detachment surgery, malignant melanoma, long-standing uveitis, glaucoma and MCGN II. Glycosylation was examined using a panel of twenty biotinylated lectins and an avidin-peroxidase DAB-cobalt revealing system, with and without neuraminidase pre-treatment. High mannose, bi/tri-nonbisected and bisected complex N-glycan, N-acetyl glucosaminyl, galactosyl and sialyl residues were found to be expressed by drusen, while treatment with neuraminidase exposed subterminal N-acetyl galactosamine and galactosyl residues. Similar binding patterns were found in the various pathological groups studied. As there was no significant difference in the lectin-binding pattern in drusen in different pathologies, a common pathogenesis or at least a final common pathway for the elaboration of carbohydrate components of drusen is suggested.

Clinicopathological findings of retinal angiomatous proliferation.

BACKGROUND: Neovascular membranes obtained from surgical excision of neovascularization for retinal angiomatous proliferation (RAP) were examined histopathologically in an attempt to elucidate the pathogenic mechanism of RAP. METHODS: Nine eyes of eight patients (mean age, 79 +/- 6 years) who underwent neovascularization excision were studied. Three eyes had stage II with RPE detachment, six had stage III. Immunohistochemical studies were performed to identify von Willebrand factor, vascular endothelial factor (VEGF), CD68 and hypoxia inducible factors (HIF-1 alpha and HIF-2 alpha). RESULTS: Multiple soft drusen were present in the macular area in all patients. In one stage II eye, we observed intraretinal neovascularization as a VEGF-positive mass, CD68-positive macrophage migration and HIF expression. In another stage II eye, neovascularization had extended above the RPE, while VEGF-positive fibroblasts were observed below the RPE. Therefore, in stage II, neither angiographic nor histopathological examinations identified choroidal neovascularization. In one phase III eye, angiography demonstrated choroidal neovascularization and chorioretinal anastomosis. Histopathologically, chorio-retinal communication was observed in the region where the RPE was destroyed, and VEGF-positive neovascularization was also seen below the RPE. CONCLUSIONS: The findings of multiple drusen in elderly patients together with macrophage migration and HIF expression surrounding VEGF-positive retinal neovascularization suggest ischemic and inflammatory factors to be associated with the development and progression of RAP.

Secreted proteome profiling in human RPE cell cultures derived from donors with age related macular degeneration and age matched healthy donors.

Age-related macular degeneration (AMD) is characterized by progressive loss of central vision, which is attributed to abnormal accumulation of macular deposits called "drusen" at the interface between the basal surface of the retinal pigment epithelium (RPE) and Bruch's membrane. In the most severe cases, drusen deposits are accompanied by the growth of new blood vessels that breach the RPE layer and invade photoreceptors. In this study, we hypothesized that RPE secreted proteins are responsible for drusen formation and choroidal neovascularization. We used stable isotope labeling by amino acids in cell culture (SILAC) in combination with LC-MS/MS analysis and ZoomQuant quantification to assess differential protein secretion by RPE cell cultures prepared from human autopsy eyes of AMD donors (diagnosed by histological examinations of the macula and genotyped for the Y402H-complement factor H variant) and age-matched healthy control donors. In general, RPE cells were found to secrete a variety of extracellular matrix proteins, complement factors, and protease inhibitors that have been reported to be major constituents of drusen (hallmark deposits in AMD). Interestingly, RPE cells from AMD donors secreted 2 to 3-fold more galectin 3 binding protein, fibronectin, clusterin, matrix metalloproteinase-2 and pigment epithelium derived factor than RPE cells from age-matched healthy donors. Conversely, secreted protein acidic and rich in cysteine (SPARC) was found to be down regulated by 2-fold in AMD RPE cells versus healthy RPE cells. Ingenuity pathway analysis grouped these differentially secreted proteins into two groups; those involved in tissue development and angiogenesis and those involved in complement regulation and protein aggregation such as clusterin. Overall, these data strongly suggest that RPE cells are involved in the biogenesis of drusen and the pathology of AMD.

Apolipoprotein localization in isolated drusen and retinal apolipoprotein gene expression.

PURPOSE: To evaluate apolipoprotein (Apo) gene expression in native human retinal pigment epithelium (RPE) and neurosensory retina and to detect apolipoproteins within age-related, extramacular drusen. METHOD: Drusen were isolated manually from 10 eyes of 10 donors (age range, 58-93 years) with grossly normal maculas that were preserved in 4% paraformaldehyde within 6 hours of death. In cryosections of druse-enriched pellets (6-57 drusen per eye), the Apos A-I, A-II, B, C-I, C-II, C-III, E, and J were detected by indirect immunofluorescence. Two graders assessed the prevalence and pattern of immunoreactivity. mRNA transcripts were detected by reverse-transcription polymerase chain reaction (RT-PCR), with human hepatoma HepG2 cells as the positive control. RESULTS: Extramacular drusen were classified in two groups on gross appearance: transparent with a reflective shell and cloudy. The proportion of the latter increased significantly with age. All Apos examined were detectable, in descending order of prevalence: ApoE (99.5%), J (99.5%), C-I (93.1%), B (80.4%), A-I (61.0%), A-II (59.2%), C-II (57.7%), and C-III (16.6%). Immunoreactivity was either diffusely distributed throughout the drusen (56.7%), confined to the druse rim (16.0%), or both (21.2%). Six percent displayed evidence of organized substructure reminiscent of active remodeling. The proportion of diffusely labeled drusen decreased significantly with age for ApoE (P=0.034) and ApoE/C-I combined (P=0.027). RT-PCR products for Apos C-I, C-II, E, and J were found in retina and RPE; for ApoA-II in the retina only. The ApoC-III message was undetectable. CONCLUSIONS: To an emerging model of an RPE-secreted large lipoprotein particle implied by previous work, this study adds ApoC-I and ApoC-II, major modulators of lipoprotein lipase activity, and confirms previously demonstrated Apos A-I, B-100, and E. It is possible that a neutral lipid-rich druse shell containing Apos will be visible in the living fundus.

Drusen complement components C3a and C5a promote choroidal neovascularization.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in industrialized nations, affecting 30-50 million people worldwide. The earliest clinical hallmark of AMD is the presence of drusen, extracellular deposits that accumulate beneath the retinal pigmented epithelium. Although drusen nearly always precede and increase the risk of choroidal neovascularization (CNV), the late vision-threatening stage of AMD, it is unknown whether drusen contribute to the development of CNV. Both in patients with AMD and in a recently described mouse model of AMD, early subretinal pigmented epithelium deposition of complement components C3 and C5 occurs, suggesting a contributing role for these inflammatory proteins in the development of AMD. Here we provide evidence that bioactive fragments of these complement components (C3a and C5a) are present in drusen of patients with AMD, and that C3a and C5a induce VEGF expression in vitro and in vivo. Further, we demonstrate that C3a and C5a are generated early in the course of laser-induced CNV, an accelerated model of neovascular AMD driven by VEGF and recruitment of leukocytes into the choroid. We also show that genetic ablation of receptors for C3a or C5a reduces VEGF expression, leukocyte recruitment, and CNV formation after laser injury, and that antibody-mediated neutralization of C3a or C5a or pharmacological blockade of their receptors also reduces CNV. Collectively, these findings establish a mechanistic basis for the clinical observation that drusen predispose to CNV, revealing a role for immunological phenomena in angiogenesis and providing therapeutic targets for 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.