IOVS
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

(Investigative Ophthalmology and Visual Science. 2006;47:3119-3128.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-1446
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, C.-M.
Right arrow Articles by Curcio, C. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, C.-M.
Right arrow Articles by Curcio, C. A.

Apolipoprotein Localization in Isolated Drusen and Retinal Apolipoprotein Gene Expression

Chuan-Ming Li, Mark E. Clark, Melissa F. Chimento, and Christine A. Curcio

From the Department of Ophthalmology, University of Alabama School of Medicine, Birmingham, Alabama.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
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.

Age-related maculopathy (ARM), the major cause of new, untreatable vision loss among the elderly of industrialized nations, is an obscure degeneration involving the retinal pigment epithelium (RPE), Bruch’s membrane (BrM), and the choriocapillaris, with secondary sight-threatening effects on the photoreceptors. Although the ingrowth of leaky choriocapillary endothelial cells through BrM occurs in a minority of patients, it accounts for most vision loss. Events setting the stage for choroidal neovascularization are poorly understood. Without new information, preventions rather than treatments of complications are difficult to envision.

A prominent clinical and histopathologic sign of ARM is the presence of drusen, histologically defined as focal deposits of heterogeneous debris external to the RPE basal lamina and internal to the inner collagenous layer of BrM.1 2 Drusen increase with age3 4 and in patients with ARM, drusen of large size, sloping sides ("soft"), and confluence are predictors of advancement to neovascular ARM.5 Prophylactic laser photocoagulation of drusen has been advanced as a method to regress these lesions, although trial results have not yet borne out this expectation.6 From a mechanistic perspective, an established research paradigm in ARM pathobiology is the detection of proteins within drusen accompanied by reverse transcription–polymerase chain reaction (RT-PCR) of the RPE and neurosensory retina, to evaluate the potential for intraocular biosynthesis of these proteins, many of which are secreted in abundance by liver or other organs. This approach, validated by proteomics, has identified in drusen acute-phase reactants (vitronectin), markers of inflammation (C-reactive protein), and members of the complement family of innate immunity proteins (e.g., C37 8 9 10 11 12 ). As variants in the gene encoding complement factor H, a druse-associated molecule, also confer increased risk for ARM in US populations,7 13 14 15 this reductionist approach has represented a fruitful avenue for identifying ARM-perturbed pathways with potential as therapeutic routes.

Recent work has spotlighted a role for lipoproteins and neutral lipids in the formation of these characteristic lesions. Lipoproteins are multimolecular assemblies composed of lipid and protein bound by noncovalent forces. Their general structure is that of a microemulsion formed from an outer layer of phospholipids, unesterified cholesterol (UC), and apolipoproteins (Apos), with a core of neutral lipids, predominately esterified cholesterol (EC) and triglycerides (TG). Drusen contain neutral lipid that binds oil red O, EC that binds filipin after extraction with ethanol and hydrolysis with cholesterol esterase, and the Apos A-I, B, and E.9 16 17 18 19 20 21 22 23 Further, RPE expresses mRNA transcripts for genes encoding these Apos, and importantly, also for microsomal triglyceride transfer protein24 (the abetalipoproteinemia gene product), required for the assembly of an ApoB-containing lipoprotein.25 Histochemical and ultrastructural signatures of lipoprotein particles are found in BrM of elderly humans and macaques,19 26 27 suggesting a major constitutive pathway for neutral lipid deposition. Animal models with genetically or diet-induced hypercholesterolemia do not yet share these BrM signatures,28 29 30 31 adding to the circumstantial evidence that a large, possibly novel lipoprotein is produced within the eye. The functional significance of such a particle remains to be determined, but it is a plausible pathway for RPE-mediated release of fatty acids derived from phagocytosed outer segment phospholipids as TG.

In the present study, we used the druse-associated molecule approach to frame a more detailed picture of a postulated lipoprotein that can inform future studies designed to elicit its secretion in culture. Specifically, we used immunofluorescence to label drusen for previously unexamined Apos A-II, C-I, and C-II, as well as already demonstrated Apos A-I, B, E, and J, directing attention to prevalence and staining pattern. To ensure a druse-enriched sample, drusen were manually isolated and pelleted. We sampled extramacular retina (i.e., peripheral to sites used by clinical macular grading systems), where these lesions are abundant32 33 and exhibit the same proteins, if not the same lipid composition and autofluorescence properties, as macula.21 33 We used RT-PCR to examine Apo gene expression in native human RPE, neurosensory retina, the ARPE-19 cell line, and human hepatoma HepG2 cells. We found evidence of local biosynthesis of ApoC-I and C-II, strengthening the inferred evidence of an intraocular lipoprotein. The within-druse distribution of Apos may have consequences for druse visibility in the living fundus and for understanding the different stages of druse formation.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Human Tissues and Preservation of Eyes
The age, gender, and race of 10 eye bank donors with grossly normal maculas are shown in Table 1 . Eyes obtained from donors within 6 hours of death were preserved by immersion in 4% paraformaldehyde in 0.1 M phosphate buffer (PB), for 6 to 16 hours after removal of the cornea, and stored in 1% paraformaldehyde at 4°C until used (median preservation to experiment interval, 18.2 months; range, 10.8–34.6). Use of human tissues in this study was approved by Institutional Review at the University of Alabama at Birmingham and conformed to the guidelines in the Declaration of Helsinki for research involving human tissue.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Starting Sample Size and Predominant Druse Type

 
Isolation and Sectioning of Drusen
Under stereomicroscopic guidance at 50x to 63x magnification, RPE-encased drusen were removed from BrM with an insect pin attached to a dowel and collected with a borosilicate glass pipette heat-stretched to an inner diameter of ≤50 µm and attached to a PC-S3 salt-bridge electrode (E. W. Wright, Guilford, CT). The electrode was in turn attached to polyethylene intramedic tubing (PE-200) and a 5-mL syringe, which provided mild suction to retrieve drusen and place them within 600-µL Beem capsules (Electron Microscopy Sciences, Hatfield, PA). Quadrants of peripheral retina-choroid-sclera with abundant drusen were pinned to a prepared wax surface in a Petri dish. The retina was removed with forceps, and the RPE-choroid–lined scleral cup was flushed and refilled with PB saline (PBS). With the insect pin tool, drusen were cleaved from the RPE/BrM interface. Usually drusen were easily dislodged when lightly pushed with the pin tip. Cleaved drusen were counted, gathered in small groups on the RPE surface (see Fig. 1A ), and carefully drawn into the end of a 0.58-mm-inner-diameter glass pipette (no. 6010; AM Systems, Everett, WA), keeping them from entering the salt bridge. Then, under reverse suction, pipette contents were allowed to fall slowly to the capsule bottom through PBS (Fig. 1B) . From a swath of peripheral RPE-BrM, drusen selected only on the basis of visibility and detachability from BrM were thus removed. Starting sample sizes are indicated in Table 1 . The median number of drusen collected from individual eyes was 181 (range, 82–880).


Figure 1
View larger version (99K):
[in this window]
[in a new window]
  		FIGURE 1. Isolated drusen. (A) A group of drusen, cleaved from BrM, lay on RPE before harvesting. (B) Harvested drusen suspended in PBS in a Beem capsule. (C) Transparent ovoid druse (black arrow), inverted and viewed from its basal aspect. External surface of the druse dome has a thin reflective shell. White arrowheads: indicate other drusen in situ, before cleavage from BrM, also viewed from their apical aspect. (D) Cloudy drusen (white arrowhead) interspersed with small transparent ovoid drusen (black arrowheads), mostly viewed from the basal aspect after inversion (250 µm). (E) Two transparent ovoid drusen, with thin reflective shells, viewed from the basal aspect. A similar druse, viewed from the apical aspect before inversion (black arrows), is barely visible (125 µm). (F) A single cloudy druse, viewed from its basal aspect, exhibits a core substructure (black arrow; 125 µm). Scale bars: (A, B) 500 µm; C, E, F) 125 µm; (D) 250 µm.

 
Drusen clusters were washed in PBS twice, soaked in 1% sodium borohydrate (5 mL PBS, 5 mL deionized H2O, 0.1 g NaBH) for 1 hour to enhance antigenicity,34 35 covered by slow addition of 400 µL of a 0.75% agar/5% sucrose solution, then set aside to congeal. The Beem capsule was slit open, and the agar pellet was trimmed with a razor blade, placed in a rectangular cryomold, infiltrated with ascending concentrations of sucrose in 0.1 M PB (10%, 20%, 30%), 4:1 30% sucrose-mountaing compound (Histoprep SH7512D; Fisher Scientific, Pittsburgh, PA), 2:1 30% sucrose-mounting compound (Histoprep; Fisher Scientific), and frozen in liquid nitrogen. Ten-micrometer-thick sections were collected on slides (SuperFrost Plus; Fisher Scientific) and stored at 20°C until used. The number of drusen visible in a section ranged from 6 to 57 in individual eyes.

Indirect Immunofluorescence
Cryosections were removed from –20°C storage, heated for 30 minutes at 50°C to 55°C, and rinsed briefly with PBS. After 10 minutes’ incubation with 0.2% Triton-100/PBS, slides were incubated with primary antibody against Apos overnight at 4°C. Goat anti-human ApoA-I (1 mg/mL, 1:200), ApoA-II (1 mg/mL, 1:100), ApoB (1 mg/mL, 1:250), ApoC-I (1 mg/mL, 1:100), and ApoJ (1 mg/mL, 1:200) were purchased from Biodesign (Saco, ME). Goat anti-human ApoC-II (1 mg/mL, 1:50) and ApoC-III (1 mg/mL, 1:100) were purchased from Chemicon (Temecula, CA). Goat anti-human ApoE (92 mg/mL, 1:500) was purchased from Calbiochem (San Diego, CA). According to the manufacturers, cross-reactions with other Apos or human serum proteins, as determined by Western blot analysis or ELISA, were negligible. Identical concentrations of goat IgG were used on negative control sections. After three 10-minute washes with 0.1% Tween-20/PBS (PBST), slides were incubated with biotinylated anti-goat IgG 1:500 for 2 hours at room temperature. After three PBST washes, sections were incubated with rhodamine Red-X-conjugated Streptavidin (1:500; Jackson ImmunoResearch, West Grove, PA) for 30 to 60 minutes. After three PBS washes, coverslips were mounted with 1,4-diaza bicyclo[2.2.2]octane in glycerol (Slow Fade Light Antifade Kit; Invitrogen, Eugene, OR).

Immunofluorescence: Processing, Imaging, and Evaluation
Sections were examined by microscope (Eclipse 80i; Nikon, Melville, NY), a 10x planapo or 40x plan fluor objective, and two filter systems (in nanometers, excitation-dichroic-barrier): 540/25–565-630/60 and 480/30–505-535/40 for rhodamine and autofluorescence, respectively. Images were captured with a digital camera (Retiga 4000R Fast; Q Imaging, Burnaby, BC, Canada). Drusen immunofluorescence was evaluated from saved images at 270x on a computer monitor by two independent graders (C-ML, CAC). Immunofluorescence and differential interference contrast (DIC) images of the same field, combined as separate layers (in Photoshop, Adobe Systems, Mountain View, CA) were toggled back and forth to facilitate identifying unlabeled tissue. Each druse, numbered on an image hard copy, was evaluated as positive or negative, with reference to a same-exposure-length image of a control (IgG) section also visible on the monitor. Intensity values at least threefold higher in experimental sections than control sections (determined with Photoshop; Adobe Systems) were considered specific. Individual graders’ scores were combined, and each druse was considered positive (graders agreeing), negative (graders agreeing), or uncertain (graders disagreeing). In reporting the percentage of immunoreactive drusen, positive labeling and uncertain labeling are combined. Digitized images were composited (Photoshop CS; Adobe Systems).

Statistical Analysis
It was apparent that drusen immunoreactivity could be diffusely distributed across the druse or more highly concentrated within a thin rim around the noncleaved druse edge, with or without immunoreactivity in the druse center (see the Results section). To assess the labeling pattern as a function of age, immunoreactivity was assessed as diffuse, rim, both, or other. For analysis, the proportions of diffuse and both were combined, and the proportions of rim and both were combined. Under the null hypothesis that age and ApoC-I and E immunoreactivity patterns are unassociated, the correlation of proportion of drusen with diffuse and rim staining patterns and age were analyzed (JMP Statistical Discovery Software ver. 5.1; SAS Institute Inc., Cary, NC).

Cell Culture
ARPE-19 and HepG2 cell lines were obtained from the American Type Culture Collection (Manassas, VA) at passage 22 and subjected to RT-PCR after two passages. ARPE-19 cells were plated in T-75 flasks or 6-well plates and grown for 4 weeks in Dulbecco’s minimum essential medium (DMEM)/F12 (1:1) supplemented with 10% fetal calf serum (FCS), as described.36 Medium was changed twice weekly. HepG2 cells were grown in six-well plates in DMEM containing 10% FCS for 5 days with a medium change every other day.

Reverse Transcription–Polymerase Chain Reaction
Total RNA was isolated from human retina, RPE, ARPE-19 cells, and HepG2 cells, as described.21 Primers used for RT-PCR are listed in Table 2 . To distinguish between amplified mRNA and genomic DNA, primers were designed to span intron boundaries. Two pairs of primers were designed for each gene. First-strand cDNA was synthesized with reverse transcriptase (Omniscript; Qiagen, Valencia, CA). A commercial PCR system (PCR Core System; Promega, Madison, WI) was used. The cDNA was denatured for 4 minutes at 94°C before cycling. The reaction was amplified through 30 cycles of 45 seconds at 94°C (denaturing), 45 seconds at 55°C to 66°C (annealing), and 1 minute at 72°C (extension), then incubated for 10 minutes at 72°C.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Sequences of PCR Primers and Expected Fragment Sizes*

 

   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Gross Appearance of Isolated Drusen
The gross appearance of isolated drusen and their distributions within different eyes are shown in Figure 1 and Table 1 , respectively. All extramacular drusen, when cleaved, physically inverted, and viewed from their basal aspects, could be classified into two morphologic types. One, ovoid and largely transparent, had a thin and highly reflective shell around the drusenoid dome. This organization was inferred from the highly reflective rim around the basal aspect en face (Fig. 1C 1E) , a feature not visible from the apical surface (Fig. 1E) . The other was irregular in shape and cloudy (Fig. 1D) , with occasional single corelike structures within (Fig. 1F) . These differences could not be discerned among drusen in situ before their cleavage from BrM and inversion (Fig. 1D) . Although the number of eyes examined is too small for confident generalization, it is noteworthy that five of six eyes from donors aged ≥75 years had predominantly cloudy drusen, whereas three of four eyes from donors aged <75 years had predominantly transparent drusen.

Apo Immunofluorescence: Prevalence, Remodeling, and Pattern
Apolipoprotein immunoreactivity was detected with a rhodamine-conjugated secondary antibody, and the specificity of labeling was confirmed by examining each section with filter sets for rhodamine and tissue autofluorescence, referring to DIC images for overall tissue texture (Fig. 2) . Figure 2C shows specific labeling within a druse for ApoC-I (compared with Fig. 2F 2a control section incubated with goat IgG), which did not match either druse or RPE autofluorescence (compared to Fig. 2B ).


Figure 2
View larger version (85K):
[in this window]
[in a new window]
  		FIGURE 2. Specificity of Apo immunoreactivity. Each column shows the same drusen viewed with DIC optics (top), a fluorescein filter set for autofluorescence (middle), and a rhodamine filter set for immunofluorescence (bottom). (A–C) ApoC-I immunoreactivity. The same RPE (arrow) and druse (*) are indicated. (D–F) Control experiment using goat IgG. Scale bar, 40 µm.

 
By these methods, all Apos examined was detectable in at least some drusen (Fig. 3 and Table 3 ). Figure 3 shows each Apo type drusen considered both positive and negative. Immunofluorescence intensity and labeling pattern varied, with some drusen exhibiting diffuse labeling and others with labeling largely confined to the apical rim. RPE fluorescence (e.g., Fig. 3F ) represented autofluorescence detectable through the rhodamine filter set in some cases in which low immunofluorescence necessitated long exposure times. For some drusen, immunoreactivity could be localized to basal laminar deposit by reference to the matching DIC view (e.g., Fig. 3H 3P ). Table 3 indicates that a median of 230 drusen was evaluated by two graders for each Apo (range, 163–263). The percentages of immunoreactive drusen were (in descending order) 99.5% for ApoE, 99.5% for J, 93.1% for C-I, 80.4% for B, 61.0% for A-I, 59.2% for A-II, 57.7% for C-II, and 16.6% for C-III. That all Apos colocalized to individual drusen is supported by five drusen in three eyes that were probed with all antibodies. Positive and negative labeling, designations requiring agreement between two graders, can be considered most definitive, with uncertain labeling an index of fluorescence intensity. ApoE- and J-positive labeling was found in virtually all (98%–99%) drusen. Uncertain labeling was negligible for these Apos, because their fluorescence was intense. Similarly, uncertain labeling for the highly prevalent ApoC-I, moderately prevalent ApoC-II, and slightly prevalent ApoC-III was <10%. Uncertain labeling for ApoA-I, A-II, and B was >15%, because their immunofluorescence was not intense.


Figure 3
View larger version (51K):
[in this window]
[in a new window]
  		FIGURE 3. Apolipoprotein immunoreactivity in isolated drusen. Each row shows the same drusen viewed with DIC optics (left column) and indirect immunofluorescence, with a rhodamine filter set (right column). Prominent RPE fluorescence in (F) and (L) is autofluorescence detectable through the rhodamine filter in cases in which low specific immunofluorescence necessitated long exposure times. (H, P, arrows) indicate basal deposits. Shown are drusen immunoreactive for Apos (A, B) A-I, (C, D) A-II, (E, F) B, (G, H) C-I, (I, J) C-II, (K, L) C-III, (M, N) E, and (O, P) J. Scale bar, 40 µm.

 

View this table:
[in this window]
[in a new window]
  		TABLE 3. Apolipoprotein Prevalence in Isolated Drusen Checked with Immunofluorescence*

 
To evaluate a possible microscopic correlate to the age-related differences in gross drusen appearance (Table 1) , the patterns of ApoC-I and E immunoreactivity, two Apos with strong signals (Table 3) , were analyzed further in 443 drusen (Table 4) . Apolipoprotein immunoreactivity was either diffusely distributed throughout the druse (56.7%), confined to its rim (16.0%), or both (21.2%). The proportion of diffuse labeling decreased significantly with age for ApoE (P = 0.034) and E/C-I combined (P = 0.027), but not for ApoC-I alone (P = 0.131). A trend toward more drusen with rim labeling with age for ApoE (P = 0.06) did not reach significance at {alpha} = 0.05.


View this table:
[in this window]
[in a new window]
  		TABLE 4. Pattern of ApoC-I and ApoE Immunoreactivity in Drusen

 
In 6.1% of drusen, intensified immunoreactivity for all Apos was confined to a shell approximately one fourth of a druse diameter in width and excluded from internal cavities (Figs. 4B 4D 4F 4H 4J) . DIC imaging revealed evidence of organized substructure reminiscent of active remodeling (Figs. 4A 4C 4E 4G 4I) . Similar views obtained at higher resolution using 1-µm sections revealed involution of druse content associated with cellular invasion.


Figure 4
View larger version (54K):
[in this window]
[in a new window]
  		FIGURE 4. Drusen with evidence of remodeling. (A, C, E, G, I) DIC images; (B, D, F, H, J) epifluorescence images, obtained with a rhodamine filter set. Arrows: remodeling in drusen centers. Shown are drusen with Apos (A, B) A-I, (C, D) B, (E, F) C-I, (G, H) E, (I, J) and J, and (K) 1 µm-thick toluidine-O-blue-stained section showing remodeling druse. (*) Druse; (* *) basal laminar deposits. Scale bar, 40 µm.

 
Expression of Apo mRNA Transcripts
We previously reported evidence of ApoB-100 and A-I expression in neurosensory retina, native human RPE, and the ARPE-19 cell line.21 22 24 Figure 5 shows evidence that other Apolipoprotein genes are expressed in these tissues, as assessed by RT-PCR and using genuine lipoprotein-secretor HepG2 cells as a positive control. RT-PCR products of the expected size were found for ApoA-II in retina and HepG2 cells, and for ApoC-III, in HepG2 cells only, consistent with and expanding on our previous report.21 RT-PCR products for ApoC-I, C-II, E, and J were found in retina, RPE, and ARPE-19 cells.


Figure 5
View larger version (133K):
[in this window]
[in a new window]
  		FIGURE 5. Apolipoprotein gene expression. Total RNA was isolated from human retina, RPE, ARPE-19, and HepG2, and RT-PCR was performed. ApoA-II was expressed in retina and HepG2 cells; ApoC-III was expressed in HepG2 cells only; ApoC-I, ApoC-II, ApoE, and ApoJ were expressed in retina (R), RPE (P) and ARPE-19 (19) and HepG2 (H) cells. Expected sizes of RT-PCR products are 304 bp for ApoA-II, 235 bp for ApoC-I, 306 bp for ApoC-II, 515 bp for ApoC-III, 163 bp for ApoE, and 498 bp for ApoJ. M 100 bp DNA ladder. Arrowhead: 500 bp DNA marker.

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
RPE expression of mRNA for ApoB and microsomal triglyceride transfer protein21 24 37 implies that the RPE has the capacity to assemble and secrete an ApoB-containing lipoprotein particle (ApoB-lp). Further, particles that behave appropriately in density gradients and display ApoA-I and B in two density fractions have been isolated from human BrM.22 Our current model of a large, possibly novel, ApoB-lp in human BrM, is shown in Figure 6 , with hepatic very low density lipoprotein (VLDL) and intestinal chylomicrons (CM) for comparison. An isolated peripheral druse, postfixed to reveal neutral lipid, reveals numerous solid 83-nm diameter particles (Fig. 6A 6B 6C) . A large RPE ApoB-lp, on the basis of previous work, probably contained ApoA-I, B (presumed ApoB-100) and ApoE9 21 22 23 (Fig. 6D) . VLDL, at 55 to 75 nm diameter, contains ApoB-100, E, C-I, C-II, and C-III (Fig. 6D) . CMs, at 70 to 400 nm diameter, contain more TG in a larger core than VLDL and exhibit ApoB-48, A-I, E, C-I, C-II, and C-III (Fig. 6D) . Three caveats constrain this model. First, this scheme explicitly omits presumptive smaller, high-density particles of RPE origin (e.g., containing ApoE42 ). Second, unlike VLDL and CM, the RPE ApoB-lp core lipid composition is incompletely understood. The high-TG content implied by particle diameter has not been consistently revealed by direct assays.22 43 44 Third, with the exception of ApoB, the molar masses of other Apos per plasma particle vary with metabolic state. This consideration and differences in antibody efficacy mean that the range of observed immunofluorescence intensities should not be construed as indicating Apo composition of individual RPE-lp particles. Despite limitations of the model, it is nevertheless appropriate to ask how the present data bear on it.


Figure 6
View larger version (69K):
[in this window]
[in a new window]
  		FIGURE 6. RPE lipoprotein: a model. (A) One-micrometer-thick section of an isolated peripheral druse, postfixed with osmium tannic acid paraphenylenediamine (OTAP), exhibits a darker brown stain in areas of neutral lipid accumulation around its rim (double arrowhead) and in a pocket at the base near the site of cleavage from BrM (arrowhead). Box: representative area illustrated at higher magnification in (B) and (C). (B) Electron micrograph showing RPE, nucleus (N), basal laminar deposit (BlamD) overlying a druse exhibiting OTAP enhancement (black arrowhead), and a high concentration of OTAP-negative, presumed-neutral, lipid-rich particles19 38 at the druse periphery (white arrowhead). (C) Higher magnification view of OTAP-positive (black arrowhead) and OTAP-negative (white arrowhead) particles. Diameter of OTAP-positive particles, 83.3 ± 9.8 nm. Scale bars: (A) 10 µm; (B) 500 nm; (C) 200 nm. (D) Schematic of hepatic VLDL (60 nm in diameter), intestinal dietary CM (100 nm), and a hypothetical RPE large lipoprotein (RPE-LP, 100 nm), showing neutral lipid cores and surface Apos. VLDL and CM Apo identities are taken from References 39 40 41 .

 
ApoC-I and C-II are now candidates for inclusion on RPE ApoB-lp, in addition to ApoB-100, A-I, and E (previously demonstrated and herein confirmed, although at a somewhat lower prevalence of ApoB-100 and A-I). An intraocular source cannot be concluded as yet for ApoA-II, expressed in retina but not in RPE and ARPE-19, but appears unlikely. The fact that a significant proportion of drusen contained ApoA-II and C-III immunoreactivity (36.4% and 9.0%, respectively), in the absence of evidence for corresponding RPE mRNA transcripts, underscores the reality that a plasma source for some Apos cannot be excluded. That ApoC-I and C-II, at concentrations of 6 and 4 mg/dL, respectively,45 in normolipemic plasma, were so clearly detected in drusen, whereas ApoC-III, at 12 mg/dL, was negligible (see also Ref. 21 ), implies either a highly specific ApoC-binding mechanism within drusen or strong evidence for local biosynthesis of these minority ApoCs.

Ocular functions of Apos A-I, B, E, and J have been discussed elsewhere,9 21 24 46 47 and we focus herein on the potential functional implications for RPE and choroidal biology of intraocular biosynthesis of ApoC-I and C-II, which, like all Apos, serve as enzyme modulators and receptor ligands in addition to lipid transporters.45 48 The human APOC1 and APOC2 genes are members of a 48-kb cluster on chromosome 19 that includes ApoE and pseudo-ApoC1'. At ~4.7 kb, APOC1 is expressed primarily in liver and, significantly, also in lung, skin, testis, and spleen, where transcription is driven by a promoter different from the one responsible for high-level hepatic transcription. In vitro studies indicate that ApoC-I activates lecithin cholesterol acyl transferase (LCAT), inhibits, among others, lipoprotein (LPL) and hepatic lipases that hydrolyze TG in particle cores, and hinders binding and uptake of VLDL to the LDL and VLDL receptors by displacing ApoE on the ligand particles. Notably, both LCAT and LPL are expressed in RPE and choroid.22 49 Mice expressing human ApoC-I have elevated plasma cholesterol and TG due to elevated VLDL and LDL fractions, elevated plasma nonesterified fatty acids, scaly skin, and hair loss, and ApoC-I deficient mice have 60% higher plasma triglyceride than wild-type and are sensitive to high-fat diets. Of interest, ApoC-I message is reduced, and ApoC-I protein is increased in the brain in Alzheimer disease.50 Regarding ApoC-II, the 3.4-kb APOC2 gene is expressed in liver and intestine only. Its function, revealed by study of human mutations, is clearly one of an LPL activator, although studies in mice suggest that this effect depends on ApoC-I concentration. Recently, ApoC-II has joined Apos A-I, A-II, and E as those readily forming amyloid in the absence of lipid to stabilize their amphipathic {alpha}-helical domains,51 of potential interest because drusen contain dispersed supramolecular amyloid assemblies.52 53 54 The ocular-specific functions of ApoC-I and C-II remain to be determined, but their presence, especially that of ApoC-II, lends further credence to the overall concept of a large ApoB-lp.

That drusen have a life cycle of nucleation, expansion, coalescence, and alternative endpoints of calcification or involution, is most strongly supported by studies employing longitudinal angiography and transmission electron microscopy,55 56 with corroborative results emanating from long-term fundus observation in populations.57 Information on the molecular and cellular basis of these phenomena is sparse. Nevertheless, detailed immunohistochemical analysis of extramacular drusen8 10 58 59 shows immunoglobulin G and vitronectin immunoreactivity homogenously distributed throughout small drusen and concentrated along irregular surfaces and internal cavities in larger drusen. Our Figure 4 demonstrates similar effects with respect to Apo localization that we, like other investigators, interpret as stigmata of druse degeneration and in some cases, phagocytosis by invading cells. Reportedly, 40% of extramacular drusen contain cores associated with CD1a-CD83-CD86-immunoreactive dendritic cells,58 whereas we found <6% of peripheral drusen with morphologic signs of active remodeling, suggesting cells. Despite these quantitative differences, the concept that newly formed drusen are degraded by invading phagocytes remains distinctly possible.10 Furthermore, the fact that EC, a major lipoprotein core component, is prominent in both normal BrM and in drusen of all sizes,19 60 and the Apos in drusen are plausibly associated with EC (Fig. 6) , we argue that the postulated cellular invasion and remodeling are secondary to a primary process of constitutive lipoprotein secretion.

Within peripheral drusen, Apos could be more highly concentrated within a thin exterior shell, a pattern potentially relevant to both a previously reported EC-rich exterior shell19 21 54 and the optical properties of drusen. Of note, in patients with early-adult-onset grouped drusen, drusen visualized in color fundus photographs are larger than they are when visualized in fluorescein angiograms.61 This finding implies that a hydrophilic domain in a druse center is smaller than the actual druse, which, by necessity, contains a hydrophobic shell that is conceivably neutral lipid rich. Those investigators proposed that drusen cores (discussed earlier)62 could account for a hydrophilic center. We propose that an EC-rich, Apo-immunoreactive rim could account for a hydrophobic shell. A shell immunoreactive for complement protein C58 resembles the thin rims we observed, raising the possibility that Apos and proteins involved in inflammatory response colocalize. Note that the druse shell defined by Apo immunoreactivity (Fig. 3) differs from the outer zone present in remodeled drusen (Fig. 4) in that it is much thinner. Finally, we found that drusen acquire more diffuse Apo immunoreactivity with age, a process resulting in reduced contrast between the rim and the interior. It is unclear whether this process is attributable to either more particles, or more likely, to larger particles. It is well known that large TG-rich lipoprotein particles (VLDL and CM) at the top of density gradients form a characteristically cloudy layer. In either case, it may be possible to detect such changes in the living fundus using the clinical imaging methods described elsewhere.61

Strengths of our study include use of an enriched druse preparation, at least two primers for all genes, and the relative consistency of findings across eyes. Limitations include the relatively small number of eyes and the long interval between fixation and experiments that may account for reduced Apo prevalence relative to previous work.21 Finally, we emphasize that several important questions remain unanswered. First, finding Apos colocalized within individual drusen does not necessarily mean that they are colocalized on the same lipoprotein particle. Second, the Apo composition of macular drusen has not been addressed. Finally, the cellular localization and function of Apos expressed in neurosensory retina (ApoA-II, C-I, and C-II) are unknown. These gene products may be expressed in Müller cells, believed to secrete a lipoprotein particle into the vitreous63 64 ; in retinal ganglion cells, which contain ApoB and MTP immunoreactivity24 ; or both. Answering these questions will be the focus of future investigations.


   Acknowledgements
 
The authors thank the Alabama Eye Bank for retrieving donor eyes; Ramon F. Dacheux II, PhD, for guidance in designing the drusen-harvesting pipette; and Nassrin Dashti, PhD, for helpful discussions.


   Footnotes
 
Supported by National Eye Institute Grants EY06109, the International Retinal Research Foundation, unrestricted departmental funds and a Lew R. Wasserman Merit Award from Research to Prevent Blindness, Inc., the EyeSight Foundation of Alabama, and a Roger Johnson Prize in Macular Degeneration Research.

Submitted for publication November 9, 2005; revised December 9, 2005; accepted April 26, 2006.

Disclosure: C.-M. Li, None; M.E. Clark, None; M.F. Chimento, None; C.A. Curcio, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Christine A. Curcio, Department of Ophthalmology, 700 South 18th Street Room H020, Callahan Eye Foundation Hospital, University of Alabama School of Medicine, Birmingham, AL 35294-0009; curcio{at}uab.edu.


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

  1. Sarks SH. Ageing and degeneration in the macular region: a clinico-pathological study. Br J Ophthalmol. 1976;60:324–341.[Abstract/Free Full Text]
  2. Green WR, Enger C. Age-related macular degeneration histopathologic studies: the 1992 Lorenz E. Zimmerman Lecture. Ophthalmology. 1993;100:1519–1535.[ISI][Medline][Order article via Infotrieve]
  3. van der Schaft TL, Mooy CM, de Bruijn WC, Oron FG, Mulder PGH, de Jong PTVM. Histologic features of the early stages of age-related macular degeneration. Ophthalmology. 1992;99:278–286.[ISI][Medline][Order article via Infotrieve]
  4. Klein R, Klein BEK, Linton KLP. Prevalence of age-related maculopathy. Ophthalmology. 1992;99:933–943.[ISI][Medline][Order article via Infotrieve]
  5. Pauleikhoff D, Barondes MJ, Minassian D, Chisholm IH, Bird AC. Drusen as risk factors in age-related macular disease. Am J Ophthalmol. 1990;109:38–43.[ISI][Medline][Order article via Infotrieve]
  6. Hsu J, Maguire MG, Fine SL. Laser prophylaxis for age-related macular degeneration. Can J Ophthalmol. 2005;40:320–331.[ISI][Medline][Order article via Infotrieve]
  7. Hageman GS, Anderson DH, Johnson LV, et al. A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration. Proc Natl Acad Sci USA. 2005;102:7227–7232.[Abstract/Free Full Text]
  8. Anderson DH, Mullins RF, Hageman GS, Johnson LV. A role for local inflammation in the formation of drusen in the aging eye. Am J Ophthalmol. 2002;134:411–431.[CrossRef][ISI][Medline][Order article via Infotrieve]
  9. Anderson DH, Ozaki S, Nealon M, et al. Local cellular sources of apolipoprotein E in the human retina and retinal pigmented epithelium: implications for the process of drusen formation. Am J Ophthalmol. 2001;131:767–781.[CrossRef][ISI][Medline][Order article via Infotrieve]
  10. Johnson LV, Ozaki S, Staples MK, Erickson PA, Anderson DH. A potential role for immune complex pathogenesis in drusen formation. Exp Eye Res. 2000;70:441–449.[CrossRef][ISI][Medline][Order article via Infotrieve]
  11. Hageman GS, Mullins RG, Russell SR, Johnson LV, Anderson DH. Vitronectin is a constituent of ocular drusen and the vitronectin gene is expressed in human retinal pigmented epithelial cells. FASEB J. 1999;13:477–484.[Abstract/Free Full Text]
  12. Crabb JW, Miyagi M, Gu X, et al. Drusen proteome analysis: an approach to the etiology of age-related macular degeneration. Proc Natl Acad Sci USA. 2002;99:14682–14687.[Abstract/Free Full Text]
  13. Klein RJ, Zeiss C, Chew EY, et al. Complement factor H polymorphism in age-related macular degeneration. Science. 2005;308:385–389.[Abstract/Free Full Text]
  14. Haines JL, Hauser MA, Schmidt S, et al. Complement factor H variant increases the risk of age-related macular degeneration. Science. 2005;308:419–421.[Abstract/Free Full Text]
  15. Edwards AO, Ritter R, 3rd, Abel KJ, Manning A, Panhuysen C, Farrer LA. Complement factor H polymorphism and age-related macular degeneration. Science. 2005;308:421–424.[Abstract/Free Full Text]
  16. Pauleikhoff D, Zuels S, Sheraidah GS, Marshall J, Wessing A, Bird AC. Correlation between biochemical composition and fluorescein binding of deposits in Bruch’s membrane. Ophthalmology. 1992;99:1548–1553.[ISI][Medline][Order article via Infotrieve]
  17. Farkas TG, Sylvester V, Archer D, Altona M. The histochemistry of drusen. Am J Ophthalmol. 1971;71:1206–1215.[ISI][Medline][Order article via Infotrieve]
  18. Wolter JR, Falls HF. Bilateral confluent drusen. Arch Ophthalmol. 1962;68:219–226.[Abstract/Free Full Text]
  19. Curcio CA, Millican CL, Bailey T, Kruth HS. Accumulation of cholesterol with age in human Bruch’s membrane. Invest Ophthalmol Vis Sci. 2001;42:265–274.[Abstract/Free Full Text]
  20. Haimovici R, Gantz DL, Rumelt S, Freddo TF, Small DM. The lipid composition of drusen, Bruch’s membrane, and sclera by hot stage polarizing microscopy. Invest Ophthalmol Vis Sci. 2001;42:1592–1599.[Abstract/Free Full Text]
  21. Malek G, Li C-M, Guidry C, Medeiros NE, Curcio CA. Apolipoprotein B in cholesterol-containing drusen and basal deposits in eyes with age-related maculopathy. Am J Pathol. 2003;162:413–425.[Abstract/Free Full Text]
  22. Li CM, Chung BH, Presley JB, et al. Lipoprotein-like particles and cholesteryl esters in human Bruch’s membrane: initial characterization. Invest Ophthalmol Vis Sci. 2005;46:2576–2586.[Abstract/Free Full Text]
  23. Klaver CC, Kliffen M, van Duijn CM, et al. Genetic association of apolipoprotein E with age-related macular degeneration. Am J Hum Genet. 1998;63:200–206.[CrossRef][ISI][Medline][Order article via Infotrieve]
  24. Li C-M, Presley JB, Zhang X, et al. Retina expresses microsomal triglyceride transfer protein: implications for age-related maculopathy. J Lipid Res. 2005;46:628–640.[Abstract/Free Full Text]
  25. Berriot-Varoqueaux N, Aggerbeck LP, Samson-Bouma M, Wetterau JR. The role of the microsomal triglyceride transfer protein in abetalipoproteinemia. Annu Rev Nutr. 2000;20:663–697.[CrossRef][ISI][Medline][Order article via Infotrieve]
  26. Ruberti JW, Curcio CA, Millican CL, Menco BP, Huang JD, Johnson M. Quick-freeze/deep-etch visualization of age-related lipid accumulation in Bruch’s membrane. Invest Ophthalmol Vis Sci. 2003;44:1753–1759.[Abstract/Free Full Text]
  27. Anderson MD, Dawson WW, Curcio CA. Drusenoid lesions and lipid-filled retinal pigment epithelium cells in a rhesus macula. Vet Ophthalmol. .In press
  28. Miceli MV, Newsome DA, Tate DJ, Jr, Sarphie TG. Pathologic changes in the retinal pigment epithelium and Bruch’s membrane of fat-fed atherogenic mice. Curr Eye Res. 2000;20:8–16.[CrossRef][ISI][Medline][Order article via Infotrieve]
  29. Dithmar S, Sharara NA, Curcio CA, et al. Murine high fat diet and laser photochemical model of basal deposits in Bruch’s membrane. Arch Ophthalmol. 2001;119:1643–1649.[Abstract/Free Full Text]
  30. Espinosa-Heidmann DG, Sall J, Hernandez EP, Cousins SW. Basal laminar deposit formation in APO B100 transgenic mice: complex interactions between dietary fat, blue light, and vitamin E. Invest Ophthalmol Vis Sci. 2004;45:260–266.[Abstract/Free Full Text]
  31. Malek G, Johnson LV, Mace BE, et al. Apolipoprotein E allele-dependent pathogenesis: a model for age-related retinal degeneration. Proc Natl Acad Sci USA. 2005;102:11900–11905.[Abstract/Free Full Text]
  32. Lewis H, Straatsma BR, Foos RY. Chorioretinal juncture: multiple extramacular drusen. Ophthalmology. 1986;93:1098–1112.[ISI][Medline][Order article via Infotrieve]
  33. Lengyel I, Tufail A, Hosaini HA, Luthert P, Bird AC, Jeffery G. Association of drusen deposition with choroidal intercapillary pillars in the aging human eye. Invest Ophthalmol Vis Sci. 2004;45:2886–2892.[Abstract/Free Full Text]
  34. Milam AH, Jacobson SG. Photoreceptor rosettes with blue cone opsin immunoreactivity in retinitis pigmentosa. Ophthalmology. 1990;97:1620–1631.[ISI][Medline][Order article via Infotrieve]
  35. Xiao M, Hendrickson A. Spatial and temporal expression of short, long/medium, or both opsins in human fetal cones. J Comp Neurol. 2000;425:545–559.[CrossRef][ISI][Medline][Order article via Infotrieve]
  36. Dunn KC, Aotaki-Keen AE, Putkey FR, Hjelmeland LM. ARPE-19, a human retinal pigment epithelial cell line with differentiated properties. Exp Eye Res. 1996;62:155–162.[CrossRef][ISI][Medline][Order article via Infotrieve]
  37. Mullins RF, Russell SR, Anderson DH, Hageman GS. 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. 2000;14:835–846.[Abstract/Free Full Text]
  38. Curcio CA, Presley JB, Millican CL, Medeiros NE. Basal deposits and drusen in eyes with age-related maculopathy: evidence for solid lipid particles. Exp Eye Res. 2005;80:761–775.[CrossRef][ISI][Medline][Order article via Infotrieve]
  39. Naito H. Coronary artery disease and disorders of lipid metabolism. Kaplan L Pesce A Kazmierczak S eds. Clinical Chemistry: Theory, Analysis, and Correlation. 1996;642–681. Mosby St. Louis.
  40. Havel RJ, Kane JP. Introduction: structure and metabolism of plasma lipoproteins. Scriver CR Beaudet AL Sly WS Valle D eds. The Metabolic and Molecular Basis of Inherited Disease. 2001;2707–2716. McGraw-Hill New York.
  41. Lusis AJ, Fogelman AM, Fonarow GC. Genetic basis of atherosclerosis: part I: new genes and pathways. Circulation. 2004;110:1868–1873.[Free Full Text]
  42. Ishida BY, Bailey KR, Duncan KG, et al. Regulated expression of apolipoprotein E by human retinal pigment epithelial cells. J Lipid Res. 2004;45:263–271.[Abstract/Free Full Text]
  43. Holz FG, Sheraidah G, Pauleikhoff D, Bird AC. Analysis of lipid deposits extracted from human macular and peripheral Bruch’s membrane. Arch Ophthalmol. 1994;112:402–406.[ISI][Medline][Order article via Infotrieve]
  44. Marshall J, Hussain AA, Starita C, Moore DJ, Patmore AL. Aging and Bruch’s membrane. Marmor MF Wolfensberger TJ eds. The Retinal Pigment Epithelium: Function and Disease. 1998;669–692. Oxford University Press New York.
  45. Jong MC, Hofker MH, Havekes LM. Role of ApoCs in lipoprotein metabolism: functional differences between ApoC1, ApoC2, and ApoC3. Arterioscler Thromb Vasc Biol. 1999;19:472–484.[Free Full Text]
  46. Wong P, Pfeffer BA, Bernstein SL, et al. Clusterin protein diversity in the primate eye. Mol Vis. 2000;6:184–191.[ISI][Medline][Order article via Infotrieve]
  47. Akaguchi H, Miyagi M, Shadrach KG, Rayborn ME, Crabb JW, Hollyfield JG. Clusterin is present in drusen in age-related macular degeneration. Exp Eye Res. 2002;74:547–549.[CrossRef][ISI][Medline][Order article via Infotrieve]
  48. Mahley RW, Innerarity TL, Rall SC, Weisgraber KH. Plasma lipoproteins: apolipoprotein structure and function. J Lipid Res. 1984;25:1277–1294.[Abstract]
  49. Casaroli-Marano RP, Peinado-Onsurbe J, Reina M, Staels B, Auwerx J, Vilaro S. Lipoprotein lipase in highly vascularized structures of the eye. J Lipid Res. 1996;37:1037–1044.[Abstract]
  50. Petit-Turcotte C, Stohl SM, Beffert U, et al. Apolipoprotein C-I expression in the brain in Alzheimer’s disease. Neurobiol Dis. 2001;8:953–963.[CrossRef][Medline][Order article via Infotrieve]
  51. Hatters DM, Howlett GJ. The structural basis for amyloid formation by plasma apolipoproteins: a review. Eur Biophys J. 2002;31:2–8.[CrossRef][ISI][Medline][Order article via Infotrieve]
  52. Johnson LV, Leitner WP, Rivest AJ, Staples MK, Radeke MJ, Anderson DH. The Alzheimer’s Aß-peptide is deposited at sites of complement activation in pathologic deposits associated with aging and age-related macular degeneration. Proc Natl Acad Sci USA. 2002;99:11830–11835.[Abstract/Free Full Text]
  53. Dentchev T, Milam AH, Lee VM, Trojanowski JQ, Dunaief JL. Amyloid-beta is found in drusen from some age-related macular degeneration retinas, but not in drusen from normal retinas. Mol Vis. 2003;9:184–190.[ISI][Medline][Order article via Infotrieve]
  54. Anderson DH, Talaga KC, Rivest AJ, Barron E, Hageman GS, Johnson LV. Characterization of beta amyloid assemblies in drusen: the deposits associated with aging and age-related macular degeneration. Exp Eye Res. 2004;78:243–256.[CrossRef][ISI][Medline][Order article via Infotrieve]
  55. Sarks JP, Sarks SH, Killingsworth MC. Evolution of soft drusen in age-related macular degeneration. Eye. 1994;8:269–283.[ISI][Medline][Order article via Infotrieve]
  56. Sarks SH. Drusen patterns predisposing to geographic atrophy of the retinal pigment epithelium. Aust J Ophthalmol. 1982;99:445–459.
  57. Klein R, Klein BE, Tomany SC, Meuer SM, Huang GH. Ten-year incidence and progression of age-related maculopathy: The Beaver Dam Eye Study. Ophthalmology. 2002;109:1767–1779.[CrossRef][ISI][Medline][Order article via Infotrieve]
  58. Hageman GS, Luthert PJ, Chong NHC, Johnson LV, Anderson DH, Mullins RF. 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. Prog Retin Eye Res. 2001;20:705–732.[CrossRef][ISI][Medline][Order article via Infotrieve]
  59. Johnson LV, Leitner WP, Staples MK, Anderson DH. Complement activation and inflammatory processes in drusen formation and age related macular degeneration. Exp Eye Res. 2001;73:887–896.[CrossRef][ISI][Medline][Order article via Infotrieve]
  60. Curcio CA, Presley JB, Medeiros NE, Malek G, Avery DV, Kruth HS. Esterified and unesterified cholesterol in drusen and basal deposits of eyes with age-related maculopathy. Exp Eye Res. 2005;81:731–741.[ISI][Medline][Order article via Infotrieve]
  61. Russell SR, Gupta RR, Folk JC, Mullins RF, Hageman GS. Comparison of color to fluorescein angiographic images from patients with early-adult onset grouped drusen suggests drusen substructure. Am J Ophthalmol. 2004;137:924–930.[CrossRef][ISI][Medline][Order article via Infotrieve]
  62. Mullins RF, Hageman GS. Human ocular drusen possess novel core domains with a distinct carbohydrate composition. J Histochem Cytochem. 1999;47:1533–1539.[Abstract/Free Full Text]
  63. Amaratunga A, Abraham CR, Edwards RB, Sandell JH, Schreiber BM, Fine RE. Apolipoprotein E is synthesized in the retina by Müller glial cells, secreted in the vitreous, and rapidly transported into the optic nerve by retinal ganglion cells. J Biol Chem. 1996;271:5628–5632.[Abstract/Free Full Text]
  64. Shanmugaratnam J, Berg E, Kimerer L, et al. Retinal Müller glia secrete apolipoproteins E and J which are efficiently assembled into lipoprotein particles. Mol Brain Res. 1997;50:113–120.[Medline][Order article via Infotrieve]



This article has been cited by other articles:


Home page
 Arch OphthalmolHome page
R. Simo, M. Higuera, M. Garcia-Ramirez, F. Canals, J. Garcia-Arumi, and C. Hernandez
Elevation of Apolipoprotein A-I and Apolipoprotein H Levels in the Vitreous Fluid and Overexpression in the Retina of Diabetic Patients
Arch Ophthalmol, August 1, 2008; 126(8): 1076 - 1081.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
M. Rudolf, M. E. Clark, M. F. Chimento, C.-M. Li, N. E. Medeiros, and C. A. Curcio
Prevalence and Morphology of Druse Types in the Macula and Periphery of Eyes with Age-Related Maculopathy
Invest. Ophthalmol. Vis. Sci., March 1, 2008; 49(3): 1200 - 1209.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
U. Schmidt-Erfurth, M. Rudolf, M. Funk, C. Hofmann-Rummelt, N.-S. Franz-Haas, Z. Aherrahrou, and U. Schlotzer-Schrehardt
Ultrastructural Changes in a Murine Model of Graded Bruch Membrane Lipoidal Degeneration and Corresponding VEGF164 Detection
Invest. Ophthalmol. Vis. Sci., January 1, 2008; 49(1): 390 - 398.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
S. Kurumada, A. Onishi, H. Imai, K. Ishii, T. Kobayashi, and S. B. Sato
Stage-Specific Association of Apolipoprotein A-I and E in Developing Mouse Retina
Invest. Ophthalmol. Vis. Sci., April 1, 2007; 48(4): 1815 - 1823.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
S. Sarks, S. Cherepanoff, M. Killingsworth, and J. Sarks
Relationship of Basal Laminar Deposit and Membranous Debris to the Clinical Presentation of Early Age-Related Macular Degeneration
Invest. Ophthalmol. Vis. Sci., March 1, 2007; 48(3): 968 - 977.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map