SupportNational Eye InstituteResearch to Prevent Blindness,
Inc.International Retinal Research FoundationEyeSight Foundation of
AlabamaRoger Johnson Prize in Macular Degeneration ResearchMacula
Vision Research Foundation
20051028, Curcio-2
For images on the next slide:
Holz, Sheraidah, Pauleikhoff, and Bird (1994) Arch. Ophthalmol.
112: 402.Fig. 3: composition of lipids extracted from human macular
and peripheral Bruchs membrane; mean SD, 32 eyes.
Sheraidah, Steinmetz, Maguire, Pauleikhoff, Marshall, & Bird
(1993). Ophthalmology 100: 47. Fig. 1: Correlateion between age and
total lipid extracted from Bruchs membrane in the first series of
specimens. Curve is exponential.
20051028, Curcio-220051028, Curcio-2Curcio, Presley, Medeiros,
Malek, Avery, Kruth Exp Eye Res 2005, in press20051028,
Curcio-2ARM, like coronary artery disease, involves the
Response-to-Retention of apoB-containing lipoprotein particles in a
vascular intima, with the twist that the apoB-containing
lipoprotein comes largely from the retinal pigment epithelium
rather than plasma.
Each step of this process is a testable hypothesis. I am going
to focus on the development of the extracellular lesions, drusen
and basal linear deposits.
20051028, Curcio-2This story begins with important observations
made by Dr. Bird and his fellows, Drs. Pauliekhoff and Holz. As
described by Pauliekhoff, macular BrM from older adults contains
abundant oil red O stained neutral lipid that is largely absent in
younger eyes. This lipid was quantified in extracts of BrM and
choroid using thin layer chromatography. These studies established
that BrM lipids were present in normal eyes, that lipids increased
with age, and that they were more prominent in the macula than in
the periphery. A 4th study demonstrated oil red O stained lipids in
drusen and basal deposits. This is one of the largest age-effects
in the human outer retina. Yet for 15 years these findings have
been like the elephant in the room-- something very big that no one
talks about, except to express the fervent belief that the retinal
pigment epithelium is somehow responsible for this. I would submit
that this subject has been neglected because up to now there was
neither a plausible mechanism to account for the arrival of lipids
in Bruchs membrane nor a plausible role for extracellular lipid
deposition in the progression of ARM, once they arrived. 20051028,
Curcio-250 years into an era of intensive biomedical research, we
should be able to find testable hypothesis of pathogenesis that
accounts for the overall trajectory of ARM.Because the principal
lesions (drusen and basal linear deposit) are in a vessel wall
outside the blood-retina barrier, and extracellular lipids
accumulate in BrM with age, our thinking should be guided by
research on coronary artery disease, another disease featuring
extracellular lipid deposition in a vessel wall.
20051028, Curcio-2Starting with the basics, lipoproteins are
multimolecular complexes that solubilize a neutral lipid core
(essentially an oil droplet) containing triglyceride and EC, within
a 2 nm thick surface of protein, UC, and phospholipid. The major
classes are based on flotation properties in density gradient
ultracentrifugation. Diameters range from 600 nm for CM to 7 nm for
HDL. Outer shell (2 nm) consists of apolipoproteins, UC, PL;
spherical neutral lipid core is TG and EC. CM & VLDL have most
TG and least protein (1-10%); LDL and HDL have most EC in core and
30-50% proteins. Low density (LDL, 22 nm) Core: EC; Surface: Apo
BVery low density (VLDL, 75 nm) Core: TG, EC; Surface: Apo B, E,
CIIIChylomicrons (>75 nm) Core: TG, EC; Surface: Apo B, E, CIII,
ATG-rich lipoproteins are large, produced by the liver (VLDL) and
intestine (chylomicrons), and contain apolipoprotein
B.Apolipoproteins act as co-factors for enzymes & ligands for
cellular receptors. The lipoproteins that initiate atherosclerotic
cardiovascular disease contain a single molecule of apoB, one of
the largest of plasma proteins.ApoB-100 from liver and apoB-48 from
intestine are products of the same gene, and apoB48 is produced by
editing of the full-length mRNA.Top Left: Vance JE: Assembly and
secretion of lipoproteins. Biochemistry of Lipids, Lipoproteins and
Membranes. Edited by Vance DE, Vance JE. Amsterdam, Elsevier, 2002,
pp 505-526, Figure 1Main Figure: Jonas, A. (2002). Lipoprotein
structure. Biochemistry of Lipids, Lipoproteins and Membranes. D.
E. Vance and J. E. Vance. Amsterdam, Elsevier. 36: 483-504.
Lipoproteins are separable by their density, size, charge, and the
composition of their core and surface components.20051028,
Curcio-2Cholesterol, exists in two chemical formsunesterified
(free), which I will call UC, and is present in all eukaryotic
membranesOr bound by an ester linkage to a fatty acid at the
3-beta-hydroxy group to produce esterified cholesterol, which I
will call EC. Esterified cholesterol is the storage and transport
form and forms part of the neutral lipid core.20051028,
Curcio-2Lusis AJ, Fogelman AM, Fonarow GC. Genetic basis of
atherosclerosis: part I: new genes and pathways. Circulation.
2004;110:1868-73.Lipids are transported through the circulation as
complexes with various apolipoproteins that package lipids and act
as cofactors for enzymes or ligands for uptake by cellular
receptors. Dietary lipids are absorbed in intestine, packaged into
chylomicrons, and secreted into lymph. On entering circulation,
triglycerides are hydrolyzed through action of LPL and the
resulting remnants taken up by interaction of apoE with LDL
receptor (apoB, E receptor) or LRP. During lipolysis, surface
phospholipids and chylomicron proteins slough off to give rise to
HDL precursors. Liver cells package triglycerides and cholesterol
esters into VLDL particles. LPL acts on them, hydrolyzing TG to
release fatty acids to produce intermediate-density lipoproteins
(IDL), which can be taken up by B, E-receptor, or further
lipolyzed, partly through hepatic lipase (HL) action, to produce
LDL. LDL retains a single major protein, apoB100, and is removed
from circulation by the apoB, E receptor. Because of slow kinetics
of LDL uptake, LDL particles constitute major cholesterol-carrying
particle in most individuals. LDL can complex with apo(a) protein
to form Lp(a) particles, which appear par-ticularly atherogenic.
Mature, spherical HDLs are formed largely in the circulation from
apoAI and AII secreted by liver and intestine and from chylomicrons
surface and VLDL during their lipolysis. HDL precursors take up
cholesterol from various tissues through interaction with ABCA1
transporter, and cholesterol is esterified by lecithin:cholesterol
acyl transferase (LCAT) action. Lipids can be transferred between
lipoproteins through the actions of cholesteryl ester transfer
protein (CETP) and phospholipid transfer protein (PLTP).20051028,
Curcio-2Glycoprotein, very large, prone to degradation, insoluble
when delipidated, sedimentation velocity and molecular weight
determinations very dependent on concentration of solution.
Segrest JP, Jones MK, De Loof H, Dashti N: Structure of
apolipoprotein B-100 in low density lipoproteins. J Lipid Res 2001,
42:1346-1367.Top Fig. 2. Schematic diagram of the pentapartite
structural model, NH3- ba1- b1- a2- b2- a3-COOH, for
apoB-100.Upper: the five domains defined previously (35, 44) have
been modified as follows: first, the N-terminal a1 (globular)
domain, based upon homology to lipovitellin (59), has been expanded
to encompass residues 1 1,000 and, because of the presence of both
amphipathic a-helical and b-strands in the sequence, renamed the a1
domain. Thus, the b1 domain has been shortened to encompass
residues 1,0012,000. Bottom Fig. 1. Schematic diagram of the
structure of apoB-100 on the surface of LDL. Trypsin-releasable and
trypsin-nonreleasable regions are shown on the inside and outside
of the LDL surface, respectively. The five proposed domains are
demarcated by dashed lines. The two thrombin-cleavable sites are
marked at residues 1,297 and 3,249. Open circles represent
N-glycosylated carbohydrates, and shaded circles represent cysteine
residues. Apo B-100 (4536 residues) is produced in the liver as
VLDL. Apo B-100 has 5 domains with globular, -helical amphipathic,
and -sheets. Apo B-48 (2152 residues) is produced in the small
intestine as dietary chylomicrons. Apo B-100 and apo B-48 are
products of the same gene, with apo B48 arising from
post-transcriptional editing of apo B-100 mRNA. Successful transfer
of lipids to nascent apo B regulates whether apo B is secreted in a
mature lipoprotein particle or degraded via the
ubiquitin-proteasome system.20051028, Curcio-2Segrest JP, Jones MK,
Dashti N: J Lipid Res 1999, 40:1401-1416. Fig. 7. Lipid pocket
model for assembly of apoB-containing lipoprotein particles.
Lipoproteins are assembled not synthesized. An incomplete lipid
pocket is formed by amphipathic b strands (blue dashes) located in
the a1 domain of hAPOB. MTP is required for this pocket to fill
with lipid (yellow, neutral lipid; light blue, phospholipid head
groups; black,fatty acyl chains), perhaps acting as a co-structural
element to complete the pocket (see C). Once the pocket is filled,
additional amphipathic b strands from the b1 domain of hAPOB are
co-translationally added, allowing the lipid pocket to expand until
defined lipoprotein particles with HDL, then VLDL density
result.
20051028, Curcio-2The Response to Retention hypothesis of
atherosclerotic states that coronary artery disease is a
Response-to-Retention of apoB-containing lipoproteins from plasma
in the arterial intima. This hypothesis summarized 8 decades of
research, culminating in the late 1980s and early 1990s
establishing that the earliest and largest cholesterol component in
the lipid-rich core derives from plasma apo B-containing
lipoproteins that directly infuse into intima. These are apoB-100
containing VLDL from liver, and its metabolite LDL, and apoB-48
containing dietary chylomicrons from intestine and their partly
hydrolyzed remnants. Lipoprotein particles enter intima via
endothelial cell trancytosis and bind to specific proteoglycan
molecules(such as biglycan and versican). Retained lipoproteins are
modified by oxidative and non-oxidative processes, evoking a
cascade of downstream events. These include deposition of
cholesterol, foam cell formation, proliferation of smooth muscle
cells, endothelial cell injury, cytokine release,
neovascularization, thrombosis, and inflammation. Williams KJ,
Tabas I: Arterioscler Thromb Vasc Biol 1995, 15:551-561; Born J et
al J Clin Invest 1998, 101:2658-2664.; Williams KJ, Tabas I: Curr
Opin Lipidol 1998, 9:471-474; Sklen K et al: Nat Med 2002,
417:750-754. Figure modeled after Proctor et al. Curr Opin Lipidol
2002, 13:461-470.20051028, Curcio-2Curcio, C.A., C.L. Millican, T.
Bailey, and H.S. Kruth (2001) Accumulation of cholesterol with age
in human Bruch's membrane. Invest. Ophthalmol. Vis. Sci. 42:
265-274, Figure 3.
We used the cholesterol-specific fluorescent marker filipin and
digital fluorescence microscopy to determine whether the oil red O
staining material in human Bruchs membrane includes EC. Filipin
binds to UC but in tissue that is extracted and hydrolyzed with
cholesterol esterase it binds to UC that is newly released. We
found that normal human Bruchs membrane markedly accumulates EC in
the macula throughout adulthood. Accumulation occurs in the
periphery but about 7-fold less. Similar results were found for UC.
20051028, Curcio-2Curcio, C.A., C.L. Millican, T. Bailey, and H.S.
Kruth (2001) Accumulation of cholesterol with age in human Bruch's
membrane. Invest. Ophthalmol. Vis. Sci. 42: 265-274.
In order to determine the ratio of EC and UC, we assayed
cholesterol by enzymatic fluorimetry in extracts of partially
isolated BrM preparations. From 8 mm diameter punches for preserved
maculas >70 yr, we found that 60% of the cholesterol recovered
was esterified. Less than 6% of it could be accounted for by plasma
lipoproteins remaining in choriocapillaries.BrM EC is therefore
17-40-fold enriched relative to plasma.20051028, Curcio-2Since the
1960s eye researchers have noted electron-lucent profiles in BrM
and called them vesicles, that is, liposomes with aqueous
interiors.15 years ago it was shown that normal and atherosclerotic
arterial intima accumulates 100 nm diameter particles rich in EC.
Using the same lipid-preserving ultrastructural methods used in
intima, we determined that the electron-lucent droplets visible by
conventional EM {shown here} were not vesicles but rather solid
lipid containing particles {shown here}.We further determined that
these particles are extractable with lipid solvents. In foveal BrM
of older adults, they occupied 30% of the volume of inner BrM and
formed a dense band 3-4 droplets thick external to the RPE basal
lamina. 20051028, Curcio-2Ruberti JW, Curcio CA, Millican CL, Menco
BPM, Huang J-D, Johnson M: Quick-freeze/deep-etch visualization of
age-related lipid accumulation in Bruchs membrane. Invest
Ophthalmol Vis Sci 2003, fig. 5
In a collaboration with Jeff Ruberti in Mark Johnsons lab at
Northwestern University, we compared transmission EM and
quick-freeze deep etch preparations of the same maculas. We called
this band the lipid wall, shown here dramatically by conventional
and QFDE methods in an eye from a 41 year old donor and a 76 year
old donor. The older eye contains a tightly-packed layer of
space-filling particles (C) that displace the structural collagen
fibers present at the same location in the younger eye (A). The
electron lucent particles seen in thin-section transmission EM (C)
correspond to solid particles seen in QFDE (D).
20051028, Curcio-2QFDE also revealed detail of of individual
lipid particles in Bruchs membrane. These are collagen fibrils
viewed end-on. The lipid rich particles have a surface and core
substructure, resembling lipoproteins.
Ruberti JW, Curcio CA, Millican CL, Menco BPM, Huang J-D,
Johnson M: Quick-freeze/deep-etch visualization of age-related
lipid accumulation in Bruchs membrane. Invest Ophthalmol Vis Sci
2003, Fig. 5. 20051028, Curcio-2We further examined lesions in ARM
eyes processed with the OTAP post-fixation method. The question was
whether membranous debris, described by Shirley Sarks as the
principal component of basal linear deposit, was membranes, or
another form of lipid, poorly preserved. Here we addressed whether
membranous debris resembles plasma membranes delimiting local
cellular processes.
Figure 3: membranous debris differs from cellular processes.
Bars = 0.5 m. A. Distorted electron-dense web-like material (single
arrow) contrasts with basal infoldings (double arrowhead) of RPE
(R), which have a less dense exterior and a more homogeneous
interior; glutaraldehyde/paraformaldehyde, OTAP. Black arrowheads
indicate RPE basal lamina. B. An electron-dense web-like material
(single arrow) extends throughout a thick basal deposit but
contrasts in density and form from basal infoldings (double
arrowheads) of RPE (R); paraformaldehyde, OTAP. C. In processes of
presumed Mller cells (double arrowheads) within BlamD (asterisk) in
eyes with severe RPE atrophy and photoreceptor loss, plasma
membranes are not electron-dense,; glutaraldehyde/paraformaldehyde,
2% osmium. Black arrowheads indicate RPE basal lamina. D.
Individual profiles in a sub-RPE aggregate of membranous debris
(arrowheads) are enlarged and partially extracted by processing;
glutaraldehyde/paraformaldehyde, 2% osmium. E. Individual profiles
in a similar sub-RPE aggregate of the same eye as D are solid and
surrounded by an electron-dense band;
glutaraldehyde/paraformaldehyde, OTAP. 20051028, Curcio-2In fellow
eyes with ARM, shown here post-fixed by 2% osmium (left) and OTAP
(right) ultrastructural methods, electron-lucent particles in basal
laminar deposits are actually solid lipid-containing particles that
are also abundant in the layer of basal linear deposits.
Figure 6: Droplets in basal laminar and BlinD are solid
particles, in eyes with late Ex-ARM. A. Lipid particles appear as
electron-lucent spaces (arrows) of varying diameter.
glutaraldehyde/paraformaldehyde, 2% osmium. B. In the fellow eye,
lipid particles are solid (arrows). Arrowheads denote RPE basal
lamina, which overlies BlinD. paraformaldehyde, OTAP. Bar = 0.5
m.
Other eyes showed pools of neutral lipid rather membranous coils
in a layer of basal linear deposits. 20051028, Curcio-2Figure 2.
Cholesterol-containing macular drusen in eyes with ARM. Bright
field (A,C,E,G,I) and filipin fluorescence images (B,D,F,H, and J)
of the same drusen in hematoxylin-counterstained cryosections. UC,
unesterified cholesterol; EC, esterified cholesterol; wide-field
epi-fluorescence and 35 mm film; bar in J = 20 m. A,C show
confluent drusen containing abundant UC (B) and EC (D) in Case 15
(see caption for Figure 1). UC in cellular membranes of the
neurosensory retina, RPE, and choroid also binds filipin (B).
Arrowheads in A indicate Bruchs membrane. E,G show two confluent
hard drusen, partly detached from BrM, in Case 1. The left druse
has more UC (F) and EC (H) than the right druse. I shows a detached
soft druse with putative membranous debris, corresponding to loops
of UC-containing material (J) in Case 3. Panel J is focused to
reveal druse contents. Out-of-focus RPE membranes form a haze
around the druse interior.
20051028, Curcio-2Malek, G., C.-M. Li, C. Guidry, N.E. Medeiros,
and C.A. Curcio (2003) Apolipoprotein B in cholesterol-containing
drusen and basal deposits in eyes with age-related maculopathy. Am.
J. Pathol. 162: 413-425, Figure 4.In addition to cholesterol we
also localized apoB immunoreactivity in inner BrM and in drusen. No
label appeared in control sections, and the pattern of specific
immunoreactivity distinctly differed from that of autofluorescence
in the same section. In peripheral retina of normal eyes where we
could get a large enough sample, as many drusen contained apoB as
apoE, previously established as a druse constituent by Anderson,
Hageman, and colleagues. Fig. 4: Apo B immunofluorescence and
autofluorescence in cryosections of normal and ARM eyes. Sections
were probed with polyclonal anti-apo B (A,B,D,E) or non-immune
serum (C,F). Images were obtained with a 40X plan fluor oil
objective and either a rhodamine filter set (A,C,D,E) or an
autofluorescence filter set (B,E). A-C, normal , macula,
87-year-old man. D-F, ARM, periphery. A. Apo B immunoreactivity is
present in Bruchs membrane (BrM), especially on the inner aspect
(arrowhead). RPE is autofluorescent (*). B. In the same section as
A, autofluorescence in BrM is distributed differently from the
specific fluorescence in A. C. No fluorescence is detected in BrM
of a control sections at the same exposure time as A. D. Apo B
immunoreactivity is present in a druse. E. In the same section as
D, autofluorescence is present in RPE and BrM and within the druse,
in a different pattern from D. F. No fluorescence in a druse in a
control section.20051028, Curcio-2Is there evidence for local
expression of apoB, and more importantly, MTP?
These data show evidence for mRNA for apoB, MTP, and PDI in
neurosensory retina, native and cultured RPE, and HepG2, a human
hepatoma cell line. No evidence for expression of APOBEC-1, which
edits mRNA in the process of apoB-48 synthesis.
Accordingly, the sequence of the mRNA for RPE apoB was the
full-length hepatic form, not the shortened intestinal form.
Finally apoB protein was found in native and cultured RPE, as
was the 97KD large subunit of MTP.
Cells that express MTP, which are found in liver, intestine, and
heart, do so for one reason only -- because they are in the
business of assembling and secreting apoB containing lipoproteins.
20051028, Curcio-2A standard paradigm for achieving secretion of
apoB lp in culture has been used for HepG2 hepatoma cells for
almost 20 yr. This entails supplementing serum-starved cells with
radiolabled oleic acid bounds to fatty acid-free bovine serum
albumin. Following this stimulation, at 24 or 48 hr intervals,
lipids were extracted, separated by thin layer chromatography and
quantified by liquid scintillography. These experiments showed that
ARPE-19 cells was capable of synthesizing and secreting TG, EC, and
PL. Negative stain EM shows plasma VLDL and particles isolated by
centrifugation (d
