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1 From the Departments of Ophthalmology 2 Physiological Optics, University of Alabama at Birmingham 3 Section on Experimental Atherosclerosis, National Heart Lung and Blood Institute, Bethesda, Maryland.
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Abstract |
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METHODS. Human eyes with grossly normal maculas were preserved <4 hours after donor death. Cryosections of retina and choroid from the macula and temporal equator were stained with filipin to reveal esterified (EC) or unesterified (UC) cholesterol (n = 20, 17–92 years). Filipin fluorescence in Bruch’s membrane was quantified with digital microscopy. Maculas were prepared for lipid-preserving electron microscopy (n = 18, 16–87 years) and for ultrastructural analysis after lipid extraction (n = 2, 85 and 89 years). Punches of macular Bruch’s membrane, 8 mm in diameter, were assayed for cholesterol content by enzymatic fluorometry (n = 10, >70 years).
RESULTS. EC and UC in Bruch’s membrane increased with age in the macula. EC was sevenfold higher in macula than in periphery. Sixty percent of total cholesterol was esterified, and Bruch’s membrane EC was 16- to 40-fold enriched relative to plasma. Solid, 100-nm-diameter particles occupied >30% of the inner collagenous layer in eyes >60 years. Cholesterol accumulated in choroidal arteries and in small age-related drusen.
CONCLUSIONS. Human Bruch’s membrane ages like arterial intima and other connective tissues for which plasma lipoproteins are the known source of extracellular cholesterol. Age-related maculopathy and atherosclerotic cardiovascular disease may share common pathogenic mechanisms.
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Introduction |
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Late age-related maculopathy (ARM), or age-related macular degeneration,15 is the leading cause of untreatable new vision loss in elderly individuals,16 17 18 but its causes are poorly understood. The most prominent clinical and histopathologic lesions of early and late ARM involve Bruch’s membrane, a thin connective tissue between the basal surface of the retinal pigment epithelium (RPE) and the choriocapillaris that is traversed by molecules essential for photoreceptor and RPE function. The only known risk factor for early ARM (i.e., drusen and RPE changes) is advanced age.16 17 18 It is therefore important to understand how age-related changes in Bruch’s membrane predispose some individuals for subsequent disease.19 20 In Bruch’s membrane of the macula there is a progressive accumulation of lipids stainable by oil red O21 , and Bruch’s membrane/choroid extracts contain phospholipids, triglycerides, UC, and EC, in descending order of abundance.22 It is thought that lipid accumulation renders Bruch’s membrane increasingly hydrophobic with age, impeding diffusion between the RPE and choroidal vessels.21 23 24
On the basis of comparison with other tissues, it is logical to hypothesize that the oil red O-positive material that increases with age in human Bruch’s membrane is EC-rich particles. This hypothesis is supported by the presence of numerous small, round electron-lucent droplets in adult Bruch’s membrane21 25 26 27 28 and preliminary polarizing microscopy studies demonstrating birefringence patterns consistent with EC.29 However, only a small proportion of the cholesterol recovered in Bruch’s membrane/choroid extracts is esterified.22 Resolving the apparent discrepancy between morphologic and biochemical data is important for understanding whether lipid deposition in Bruch’s membrane is an ocular manifestation of a systemic process or a phenomenon unique to the eye. We therefore examined Bruch’s membrane cholesterol in normal donor eyes, using the fluorescent probe filipin to reveal EC and UC as a function of age and retinal location in tissue sections,9 30 an enzymatic fluorometric assay to determine the relative proportions of EC and UC of isolated Bruch’s membrane,31 and lipid-preserving electron microscopy to identify EC-rich droplets.11 Our data indicate that Bruch’s membrane is highly enriched in EC.
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Methods |
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Cryosectioning
Seven millimeter-wide samples containing retina, RPE, and
choroid from the macula and periphery were cryosectioned for
histochemistry. Macular samples included the fovea and the temporal
half of the optic nerve head. Peripheral samples included the temporal
equator and ora serrata. Samples were infiltrated in 4:1 and 2:1 30%
sucrose and Histo Prep medium (Fisher, Norcross, GA) for 30
minutes each, frozen in -70°C isopentane, sectioned at 10 µm,
collected on glass slides, and dried at 40 to 60°C.
Filipin Histochemistry
We localized EC and UC using the fluorescent polyene antibiotic
filipin, which binds specifically to sterols and interacts with the
3ß-hydroxy group of cholesterol.30
For EC
detection,9
native UC was extracted from cryosections by
two 5-minute rinses in 70% ethanol, native EC was hydrolyzed with
cholesterol esterase (EC 3.1.1.13; Boehringer Mannheim,
Indianapolis, IN) at a concentration of 1.65 units/ml in 0.1 M
potassium PB (pH 7.4) for 3 hours at 37°C, and UC newly released by
the hydrolysis of EC was stained with filipin (5 mg filipin, dissolved
in 1 ml dimethylformamide and diluted in 100 ml PB saline; Sigma, St.
Louis, MO). Control sections were incubated in the enzyme
vehicle. For UC detection,33
sections were hydrated and
incubated for 30 minutes in the above filipin solution without prior
extraction and hydrolysis. Control sections were incubated in the
filipin vehicle. All sections were counterstained with Mayer’s
hematoxylin.
Photography
Filipin-processed sections were photographed on ASA100 black and
white film (TMax100; Eastman Kodak, Rochester, NY) using an
Optiphot fluorescence microscope (Nikon, Melville, NY) equipped
with a 420-nm excitation filter, 520-nm barrier filter, and a
60x plan apochromat objective. Negative film was scanned to
create composite images using Photoshop (Adobe, San Jose, CA).
Quantification of Filipin Fluorescence
The mean fluorescence intensity of Bruch’s membrane in
filipin-stained and control sections was determined by digital
microscopy. In each section (one per condition per eye), three
240-µm-long segments of Bruch’s membrane were digitized. Sections
were viewed on a Leitz Orthoplan microscope with a 50x Fluotar
oil objective (Wetzlar, Germany) and a Chromatechnology 83000
(Brattleboro, VT) fluorescence filter set (excitation, 346 nm;
barrier, 460 nm). Images were captured at a resolution of 0.18
µm/pixel using a Photometrics CH250 CCD video camera (Roper
Scientific, Tucson, AZ) and IP Lab Spectrum 3.2 software (Scanalytics,
Fairfax, VA). All images were exposed for 2 seconds to prevent
saturation of the camera. To minimize variability due to fluorescence
quenching, digitized sites were separated by 1.5 to 2 mm and were
identified and focused using bright-field illumination. To minimize
variability due to oblique sectioning planes and haze from RPE
autofluorescence, only sites where the RPE was attached to Bruch’s
membrane and the inner edge of Bruch’s membrane and RPE pigment
granules could be focused simultaneously were used. In digitized
images, an observer placed 4.56-µm square sampling windows
over Bruch’s membrane. The sampling window spanned the thick macular
Bruch’s membrane in older eyes and included choriocapillary
endothelium and lumina in addition to thin Bruch’s membrane in other
specimens. Five sampling windows were placed across the length of each
three digitized images per section, with at least two within and three
between intercapillary pillars (total, 15 windows). Sampling windows
avoided drusen and basal deposits. Fluorescence intensities in each
window were summed and expressed in arbitrary units
x10-6. The SEM was <3% for unlabeled control
sections and 0.8% to 8.6% for filipin-labeled sections. Three
sections on the same slide differed by <3%.
Cholesterol Assay
Total cholesterol and UC content was determined with an
enzymatic fluorometric assay.31
Paraformaldehyde-preserved
eyes were used because of availability and because formalin fixation
minimally changes cholesterol content.34
Punches of
macular and peripheral retina, RPE, and choroid were obtained with an
8-mm-diameter trephin. The macular punch was centered on the fovea, and
the peripheral punch was centered on the temporal equator. The retina
was detached, the RPE was removed with a camel hair brush, and major
choroidal vessels were removed by a combination of brush and fine
forceps. Grossly visible large drusen in peripheral retina were
avoided. Small pieces were removed before and after dissection to
assess the completeness of choroid removal in 1-µm histologic
sections. Bruch’s membrane, retina, and choroidal vessels were rinsed
with water, placed in separate pre-weighed 0.25-ml plastic tubes,
lyophilized, weighed, and shipped overnight on dry ice. Lipids were
extracted with chloroform/methanol (2:1, by volume), distributed to
sample tubes, dried by heating, and redissolved with 95% ethanol. An
assay solution was added for 30 minutes at 37°C. The assay for total
cholesterol used cholesterol esterase to hydrolyze native EC,
cholesterol oxidase to generate
H2O2, and peroxidase to
catalyze the reaction of
H2O2 with
p-hydroxyphenylacetic acid. The resulting fluorescent
product excited at 325 nm and emitted at 425 nm. For determination of
UC, cholesterol esterase was omitted. EC was the difference of total
cholesterol and UC. Cholesterol content was expressed as nmol/g dry
weight.
Electron Microscopy
Glutaraldehyde-preserved maculas were divided horizontally
through the fovea. One tissue block was processed
conventionally.28
The other tissue block was processed by
the osmium-tannic acid-paraphenylenediamine (OTAP) method to preserve
small extracellular lipid particles.11
Tissues were
postfixed with 1% osmium tetroxide in 0.1 M sodium cacodylate (NaCaco)
buffer (2.5 hours), 1% tannic acid in 0.05 M NaCaco buffer (30
minutes), and fresh 1% para-phenylenediamine in 70%
ethanol (30 minutes). Silver sections were cut from adjoining block
faces with a diamond knife, collected on mesh grids, and stained with
lead citrate. Electron micrographs spanning the full thickness of
Bruch’s membrane were taken at 7500 to 8000x and enlarged three
times. Five micrographs per preparation were examined, with two within
and three between intercapillary pillars. Other OTAP-processed blocks
from 10 eyes were sectioned in a horizontal plane.
Extraction Studies
Cryosections were extracted with ethanol and chloroform/methanol
(2:1, with 1% hydrochloric acid) before filipin histochemistry and
digital microscopy. Tissue blocks of the macula of two eyes were
subject to the same solvents before osmication and processing for
conventional electron microscopy. The number of electron micrographs
containing lipid-rich components was determined by an observer unaware
of the solvent.
Stereologic Analysis of OTAP Preparations
The proportion of the inner collagenous layer occupied by
lipid-rich particles (area fraction) was measured in 16 eyes of
different ages using point-counting stereology.35
A
transparency containing a 4-mm2 grid was taped to
each of three prints for each eye, and the inner collagenous layer was
delimited. Intersections of the grid overlying lipid particles and
other inner collagenous layer constituents were scored by an observer.
The area fraction was the ratio of intersections overlying particles to
the total number of intersections. The median number of points scored
per eye was 1112 (range, 621-1682), and the SEM was <10%. Results
from younger and older eyes were compared using a t-test.
Diameters of lipid-rich particles were measured in six eyes using a
digitizing tablet.
Statistical Analysis
The relationship among filipin fluorescence intensities and age
in the macula and periphery was analyzed with multiple linear
regression, adjusting for age to examine the independence of
associations (e.g., macular EC and peripheral EC) with the effect of
age removed. We report the slopes of regression lines as crude
parameter estimates and the slopes after adjustment. A P
value of 0.05 was considered significant.
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Ultrastructure
The intensity and diffuse distribution of filipin fluorescence in
Bruch’s membrane (see Fig. 1
) suggests that cholesterol-containing
particles are densely packed and smaller than the resolution limit of
light microscopy. To identify these particles, we used conventional and
lipid-preserving electron microscopy. Conventional electron microscopy
revealed numerous small round particles with electron-lucent interiors
("droplets") in Bruch’s membrane of older eyes (Fig. 4A
). Droplets were scattered throughout the collagenous layers. They were
also grouped within coated membrane-bounded bodies
(CMBBs),27
91% of which occurred in the outer collagenous
layer, and were associated with coiled membranes, the presumed remnants
of CMBBs (Fig. 4A)
. Droplets have been previously described as
vesicles25
42
(i.e., membrane-bounded structures with
aqueous contents). However, in preparations in which lipid-rich
particles are preserved by the OTAP method (Fig. 4B)
, droplets were
solid, electron-dense particles that formed a single size distribution
(minimum/median/maximum diameters; 54/98/156 nm for inner collagenous
layer; 56/112/225 nm for outer collagenous layer). These results
indicate that electron-lucent droplets are not vesicles but rather
solid particles whose lipid-rich contents were extracted by
conventional tissue processing.
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67 years (36% ± 7.0%, n = 10) was
significantly higher than in eyes 
51 year (7% ± 2.7%,
n = 6, t = -11.6, P <
0.0001).
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Discussion |
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Using ultrastructural techniques that preserve lipid particle morphology and extraction experiments, we demonstrated that the electron-lucent droplets recognized in human Bruch’s membrane for more than 30 years21 25 26 27 42 are the EC-rich particles suggested by filipin histochemistry. Droplets resemble the solid, EC-rich particles seen in other tissues with regard to size and alignment along matrix fibers.5 47 A membranous shell of UC and phospholipid12 could account for the vesicular appearance of Bruch’s membrane droplets in tissue examined by conventional electron microscopy. Droplets also occur in coated membrane-bounded bodies,27 46 implicating these enigmatic structures in lipid trafficking. In the maculas of older adults, EC-rich particles occupy one third of the inner collagenous layer and nearly 100% of a narrow sublayer external to the RPE basal lamina. These results provide a basis for the finding that hydraulic conductivity across Bruch’s membrane and choroid isolated from older donors improves markedly when the inner collagenous layer is removed.24
Our data contrast sharply with those of Holz et al.,22 who indicated that the proportion of EC in macular Bruch’s membrane was only 14% and that EC content was unrelated to age. The previous study,22 which used thin-layer chromatography to assay lipids in unfixed tissues and did not remove the choroid from Bruch’s membrane, recovered one tenth as much total cholesterol per unit area as we did. Our higher yield may be due to using tissues that were preserved in paraformaldehyde quickly after death, presumably cross-linking EC-rich particles to a surrounding proteinaceous matrix. Our higher proportion of EC may be due to our removing the choroid. Because the choroid contains many cells and is 50- to 100-fold thicker than Bruch’s membrane, its presence would inflate Bruch’s membrane UC content relative to EC. In humans, two thirds of total plasma cholesterol is transported by LDL,38 and 64% of the cholesterol in LDL is esterified.48 The low proportion of Bruch’s membrane EC found by Holz et al.,22 relative to total lipids, was the basis of that study’s conclusion that plasma was an unlikely source for Bruch’s membrane lipids. Although we did not examine other lipid classes (e.g., triglycerides or phospholipids), our demonstration that EC accounts for a high proportion of total cholesterol suggests that this conclusion should be re-evaluated, at least for cholesterol.7
Although it is thought that the RPE produces many Bruch’s membrane constituents,49 50 there is currently little reason to suspect that the RPE is a source of EC-rich droplets. It is possible that the large-diameter (1–2 µm), oil red O-positive droplets occasionally seen in RPE cells (lipoidal degeneration51 52 53 ) could be released into Bruch’s membrane upon cell death. However, we did not detect EC within RPE cells using filipin, and the uniformly small size of Bruch’s membrane droplets is inconsistent with their originating from the breakdown of larger droplets.12 Another potential source of Bruch’s membrane EC is the photoreceptor outer segment membranes that are regularly ingested by RPE. However, outer segment membranes are poor in UC compared with typical plasma membranes,54 and it is not yet known if the RPE can esterify cholesterol. Although the RPE cannot be conclusively excluded as a source without further data, it is more parsimonious to postulate that cholesterol deposition in Bruch’s membrane reflects a well-described systemic process than to invoke a new mechanism involving the RPE.
We found a highly variable but marked (sevenfold) difference in the degree of cholesterol accumulation of macular and peripheral Bruch’s membrane. This difference is far greater than can be explained by differences in the number of photoreceptors in the macula and at the temporal equator (macula/periphery ratio = 2.7 for rods, 1.9 for cones; calculated from Ref. 55 ). Regional differences in age-related EC accumulation also occur in the cornea, which has higher EC near the limbus (arcus lipoides) than in the center.5 In the cornea this effect is attributed to greater vascular perfusion near the limbus and a tightly packed matrix that retards LDL diffusion toward the center.5 A similar combination of a lipoprotein-retaining milieu and a high blood flow that saturates retentive components could underlie the predilection of macular Bruch’s membrane for extracellular cholesterol. Notably, the choroid has the highest blood flow in the body, and blood flow in the macula is eightfold higher than that in the periphery.56 High blood flow alone is insufficient to account for regional differences in Bruch’s membrane cholesterol, however, because the EC content of the choroidal arteries was low and we could not detect a difference in age-related EC accumulation between macular and peripheral arteries using histochemistry. Understanding the relative roles of blood flow and matrix in EC accumulation will benefit from additional information about regional differences in Bruch’s membrane composition.26 57
Although Bruch’s membrane is the wall of a capillary bed, it is intriguingly similar to the inner wall of an artery. The arterial intima is located between two diffusion barriers: an endothelial cell layer and a dense elastic layer.58 Throughout life intima thickens adaptively to the mechanical stresses of blood flow and wall tension.59 Collagen, elastin, and proteoglycans at sites of intimal thickening specifically interact and bind with plasma LDL.60 61 Similarly, Bruch’s membrane is located between the choriocapillaris endothelium and the RPE component of the blood–retina barrier, it thickens threefold throughout adulthood,27 40 and it contains extracellular matrix molecules that could potentially interact with lipoproteins.49 50 62 63 64 Consistent with this model is our previous demonstration of immunoreactivity for apolipoprotein B (apo B), the principal protein of LDL, in peripheral Bruch’s membrane associated with sub-RPE deposits.45 Little immunoreactivity was detected in normal macular Bruch’s membrane, despite high neutral lipid content,45 suggesting that apo B, if present, is normally degraded and cleared efficiently. EC accumulates in Bruch’s membrane of rabbits with extremely high levels of plasma lipoproteins65 but not in mice with moderately elevated levels.66 67 68 The role played by plasma lipoproteins in normal retinal homeostasis is poorly understood. The RPE has LDL receptor activity,69 and plasma LDL transports docosahexanoic acid destined for photoreceptors.70 However, the choriocapillaris endothelium reportedly excludes molecules as large as LDL,71 72 which is inconsistent with our findings. Studies to clarify these issues are warranted.
If Bruch’s membrane can be conceptualized as an arterial intima, it is appropriate to examine diseases of the intima for insight into ARM. Atherosclerotic cardiovascular disease (CVD), a major cause of morbidity and mortality, is a complex disease that proceeds from the normal infusion of plasma LDL and accumulation of EC in thickened intima through advanced lesions containing increased levels of cholesterol and lipoproteins, culminating in neovascularization, inflammation, and thrombosis.73 Although the cholesterol and lipoprotein content of ARM-specific lesions28 remain to be determined, our study confirmed that small age-related drusen contain EC and UC, extending our previous observation that these drusen contain neutral lipids and apo B45 (but see Ref. 74 ). A plasma origin for neutral lipids in small drusen is therefore likely, consistent with studies demonstrating other plasma proteins in drusen.74 75 EC-rich droplets occur in other age-related and disease-related sub-RPE deposits.76 77 78 In dominant late-onset retinal degeneration, an inherited disorder, a cholesterol-enriched basal laminar deposit also displays intense apo B immunoreactivity.77 Thus, diverse retinal degenerations with sub-RPE debris may involve plasma lipoproteins interacting with a locally retentive matrix material.
Significantly, our study demonstrates that with regard to the deposition of extracellular cholesterol, Bruch’s membrane ages like the intima of large, atherosclerosis-prone arteries. In fact, the proportion of Bruch’s membrane occupied by EC-rich droplets in older adults is much higher than in normal arterial intima at the same age (1%–3%).12 Therefore, it is plausible that the progression from aging to ARM shares pathogenic mechanisms with atherosclerotic CVD. Seeking this link through epidemiologic studies79 is difficult, because the earliest lesions in CVD occur decades earlier than those in ARM.16 44 80 Our data now strengthen the rationale for seeking links between these two diseases at the tissue, cellular, and molecular level. We caution that atherosclerotic CVD and ARM are complex multifactorial diseases, and the geometry, spatial scale, and cellular constituents of arterial intima and Bruch’s membrane differ in important ways. Nevertheless, this analogy can provide a useful conceptual framework for generating testable new hypotheses about the pathogenesis of ARM.
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Acknowledgements |
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Footnotes |
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Submitted for publication July 21, 2000; revised September 22, 2000; accepted September 29, 2000.
Commercial relationships policy: N.
Corresponding author: Christine A. Curcio, Department of Ophthalmology, University of Alabama at Birmingham, Eye Foundation Hospital, Rm H20, 700 South 18th Street, Birmingham, AL 35294-0009. curcio{at}uab.edu
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M. D. Chamberlain, R. H. Guymer, M. Dirani, J. L. Hopper, and P. N. Baird Heritability of Macular Thickness Determined by Optical Coherence Tomography Invest. Ophthalmol. Vis. Sci., January 1, 2006; 47(1): 336 - 340. [Abstract] [Full Text] [PDF]
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G. McGwin Jr, K. Modjarrad, T. A. Hall, A. Xie, and C. Owsley 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors and the Presence of Age-Related Macular Degeneration in the Cardiovascular Health Study Arch Ophthalmol, January 1, 2006; 124(1): 33 - 37. [Abstract] [Full Text] [PDF]
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M Rudolf, B Winkler, Z Aherrahou, L C Doehring, P Kaczmarek, and U Schmidt-Erfurth Increased expression of vascular endothelial growth factor associated with accumulation of lipids in Bruch's membrane of LDL receptor knockout mice Br. J. Ophthalmol., December 1, 2005; 89(12): 1627 - 1630. [Abstract] [Full Text] [PDF]
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J. Tian, K. Ishibashi, K. Ishibashi, K. Reiser, R. Grebe, S. Biswal, P. Gehlbach, and J. T. Handa Advanced glycation endproduct-induced aging of the retinal pigment epithelium and choroid: A comprehensive transcriptional response PNAS, August 16, 2005; 102(33): 11846 - 11851. [Abstract] [Full Text] [PDF]
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C.-M. Li, B. H. Chung, J. B. Presley, G. Malek, X. Zhang, N. Dashti, L. Li, J. Chen, K. Bradley, H. S. Kruth, et al. Lipoprotein-like Particles and Cholesteryl Esters in Human Bruch's Membrane: Initial Characterization Invest. Ophthalmol. Vis. Sci., July 1, 2005; 46(7): 2576 - 2586. [Abstract] [Full Text] [PDF]
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S. Zareparsi, M. Buraczynska, K. E.H. Branham, S. Shah, D. Eng, M. Li, H. Pawar, B. M. Yashar, S. E. Moroi, P. R. Lichter, et al. Toll-like receptor 4 variant D299G is associated with susceptibility to age-related macular degeneration Hum. Mol. Genet., June 1, 2005; 14(11): 1449 - 1455. [Abstract] [Full Text] [PDF]
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M. V. Miceli and S. M. Jazwinski Nuclear Gene Expression Changes Due to Mitochondrial Dysfunction in ARPE-19 Cells: Implications for Age-Related Macular Degeneration Invest. Ophthalmol. Vis. Sci., May 1, 2005; 46(5): 1765 - 1773. [Abstract] [Full Text] [PDF]
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P. Fernandez-Robredo, D. Moya, J. A. Rodriguez, and A. Garcia-Layana Vitamins C and E Reduce Retinal Oxidative Stress and Nitric Oxide Metabolites and Prevent Ultrastructural Alterations in Porcine Hypercholesterolemia Invest. Ophthalmol. Vis. Sci., April 1, 2005; 46(4): 1140 - 1146. [Abstract] [Full Text] [PDF]
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 C.-M. Li, J. B. Presley, X. Zhang, N. Dashti, B. H. Chung, N. E. Medeiros, C. Guidry, and C. A. Curcio Retina expresses microsomal triglyceride transfer protein: implications for age-related maculopathy J. Lipid Res., April 1, 2005; 46(4): 628 - 640. [Abstract] [Full Text] [PDF]
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 N.H. V. Chong, J. Keonin, P. J. Luthert, C. I. Frennesson, D. M. Weingeist, R. L. Wolf, R. F. Mullins, and G. S. Hageman Decreased Thickness and Integrity of the Macular Elastic Layer of Bruch's Membrane Correspond to the Distribution of Lesions Associated with Age-Related Macular Degeneration Am. J. Pathol., January 1, 2005; 166(1): 241 - 251. [Abstract] [Full Text] [PDF]
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H. Itaya, V. Gullapalli, I. K. Sugino, M. Tamai, and M. A. Zarbin Iris Pigment Epithelium Attachment to Aged Submacular Human Bruch's Membrane Invest. Ophthalmol. Vis. Sci., December 1, 2004; 45(12): 4520 - 4528. [Abstract] [Full Text] [PDF]
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I. Lengyel, A. Tufail, H. A. Hosaini, P. Luthert, A. C. Bird, and G. Jeffery Association of Drusen Deposition with Choroidal Intercapillary Pillars in the Aging Human Eye Invest. Ophthalmol. Vis. Sci., September 1, 2004; 45(9): 2886 - 2892. [Abstract] [Full Text] [PDF]
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N. Gordiyenko, M. Campos, J. W. Lee, R. N. Fariss, J. Sztein, and I. R. Rodriguez RPE Cells Internalize Low-Density Lipoprotein (LDL) and Oxidized LDL (oxLDL) in Large Quantities In Vitro and In Vivo Invest. Ophthalmol. Vis. Sci., August 1, 2004; 45(8): 2822 - 2829. [Abstract] [Full Text] [PDF]
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I. R. Rodriguez, S. Alam, and J. W. Lee Cytotoxicity of Oxidized Low-Density Lipoprotein in Cultured RPE Cells Is Dependent on the Formation of 7-Ketocholesterol Invest. Ophthalmol. Vis. Sci., August 1, 2004; 45(8): 2830 - 2837. [Abstract] [Full Text] [PDF]
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D. K. Bowles, C. L. Heaps, J. R. Turk, K. K. Maddali, and E. M. Price Hypercholesterolemia inhibits L-type calcium current in coronary macro-, not microcirculation J Appl Physiol, June 1, 2004; 96(6): 2240 - 2248. [Abstract] [Full Text] [PDF]
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D. G. Espinosa-Heidmann, J. Sall, E. P. Hernandez, and S. W. Cousins Basal Laminar Deposit Formation in APO B100 Transgenic Mice: Complex Interactions between Dietary Fat, Blue Light, and Vitamin E Invest. Ophthalmol. Vis. Sci., January 1, 2004; 45(1): 260 - 266. [Abstract] [Full Text] [PDF]
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P. T. Johnson, G. P. Lewis, K. C. Talaga, M. N. Brown, P. J. Kappel, S. K. Fisher, D. H. Anderson, and L. V. Johnson Drusen-Associated Degeneration in the Retina Invest. Ophthalmol. Vis. Sci., October 1, 2003; 44(10): 4481 - 4488. [Abstract] [Full Text] [PDF]
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S. A. Burns, A. E. Elsner, M. B. Mellem-Kairala, and R. B. Simmons Improved Contrast of Subretinal Structures using Polarization Analysis Invest. Ophthalmol. Vis. Sci., September 1, 2003; 44(9): 4061 - 4068. [Abstract] [Full Text] [PDF]
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G McGwin Jr, C Owsley, C A Curcio, and R J Crain The association between statin use and age related maculopathy Br. J. Ophthalmol., September 1, 2003; 87(9): 1121 - 1125. [Abstract] [Full Text] [PDF]
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R. Klein, B. E. K. Klein, S. C. Tomany, L. G. Danforth, and K. J. Cruickshanks Relation of Statin Use to the 5-Year Incidence and Progression of Age-Related Maculopathy Arch Ophthalmol, August 1, 2003; 121(8): 1151 - 1155. [Abstract] [Full Text] [PDF]
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J. W. Ruberti, C. A. Curcio, C. L. Millican, B. P. M. Menco, J.-D. Huang, and M. Johnson Quick-Freeze/Deep-Etch Visualization of Age-Related Lipid Accumulation in Bruch's Membrane Invest. Ophthalmol. Vis. Sci., April 1, 2003; 44(4): 1753 - 1759. [Abstract] [Full Text] [PDF]
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G. Malek, C.-M. Li, C. Guidry, N. E. Medeiros, and C. A. Curcio Apolipoprotein B in Cholesterol-Containing Drusen and Basal Deposits of Human Eyes with Age-Related Maculopathy Am. J. Pathol., February 1, 2003; 162(2): 413 - 425. [Abstract] [Full Text] [PDF]
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D. Bok New insights and new approaches toward the study of age-related macular degeneration PNAS, November 12, 2002; 99(23): 14619 - 14621. [Full Text] [PDF]
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L. V. Johnson, W. P. Leitner, A. J. Rivest, M. K. Staples, M. J. Radeke, and D. H. Anderson The Alzheimer's Abeta -peptide is deposited at sites of complement activation in pathologic deposits associated with aging and age-related macular degeneration PNAS, September 3, 2002; 99(18): 11830 - 11835. [Abstract] [Full Text] [PDF]
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S. Dithmar, N. A. Sharara, C. A. Curcio, N.-A. Le, Y. Zhang, S. Brown, and H. E. Grossniklaus Murine High-Fat Diet and Laser Photochemical Model of Basal Deposits in Bruch Membrane Arch Ophthalmol, November 1, 2001; 119(11): 1643 - 1649. [Abstract] [Full Text] [PDF]
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G. Hoppe, A. D. Marmorstein, E. A. Pennock, and H. F. Hoff Oxidized Low Density Lipoprotein-Induced Inhibition of Processing of Photoreceptor Outer Segments by RPE Invest. Ophthalmol. Vis. Sci., October 1, 2001; 42(11): 2714 - 2720. [Abstract] [Full Text] [PDF]
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E. F. Moreira, C. Jaworski, A. Li, and I. R. Rodriguez Molecular and Biochemical Characterization of a Novel Oxysterol-binding Protein (OSBP2) Highly Expressed in Retina J. Biol. Chem., May 18, 2001; 276(21): 18570 - 18578. [Abstract] [Full Text] [PDF]
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