High density lipoprotein mediated lipid efflux from retinal
pigment epithelial cells in culture
B Y Ishida, K G Duncan, K R Bailey, J P Kane, D M Schwartz
............................................................... ............................................................... .
See end of article for
Daniel M Schwartz, MD,
Box 0730, University of
California, San Francisco,
CA 94143, USA;
Accepted for publication
3 December 2005
Br J Ophthalmol 2006;90:616–620. doi: 10.1136/bjo.2005.085076
Backgound/aim: The transport of radiolabelled photoreceptor outer segments (POS) lipids was
investigated by cultured retinal pigment epithelial cells (RPE). Phagocytosis of POS by the RPE is essential
to maintain the health and function of the photoreceptors in vivo. POS are phagocytised at the apical cell
surface of RPE cells. Phagocytised POS lipids may be either recycled to the photoreceptors for
reincorporation into new POS or they may be transported to the basolateral surface for efflux into the
Results: The authors have demonstrated that high density lipoprotein (HDL) stimulates efflux of
radiolabelled lipids, of POS origin, from the basal surface of RPE cells in culture. Effluxed lipids bind
preferentially to HDL species of low and high molecular weight. Effluxed radiolabelled phosphotidyl
choline was the major phospholipid bound to HDL, with lesser amounts of phosphatidyl ethanolamine,
phosphatidyl inosotol. Effluxed radiolabelled triglycerides, cholesterol, and cholesterol esters also bound to
HDL. Lipid free apolipoprotein A-I (apoA-I) and apoA-I containing vesicles also stimulate lipid efflux.
Conclusion: The findings suggest a role for HDL and apoA-I in regulating lipid and cholesterol transport
from RPE cells that may influence the pathological lipid accumulation associated with age related macular
beneath the retina that leads to haemorrhage, accumulation
of subretinal fluid and, inevitably, replacement of macular
tissue with a scar.4Before visual loss from AMD, there is
progressive accumulation of lipids in Bruch’s membrane, a
multilayered extracellular tissue separating the retina from
its choroidal blood supply.5 6Progressive lipid deposition in
Bruch’s membrane reduces diffusional transport from the
choroid to the retina and is thought to impair retinal
function.7 8There has been considerable debate over whether
the lipid deposits in Bruch’s membrane are of circulatory or
retinal origin. Recent evidence suggests the predominant
source of this lipid is from the retina, deriving from residues
of degraded photoreceptor outer segments (POS) effluxed
from the retinal pigment epithelium (RPE) into Bruch’s
membrane.9Although cholesteryl ester and apolipoprotein B
deposition in Bruch’s membrane suggests contribution from
plasma lipids, analysis of lipids and apolipoproteins from
tissue and RPE cell cultures indicates that these cells may
account for most of the deposits.10 11Mechanisms by which
lipids efflux from the RPE across Bruch’s membrane and into
the choroidal circulation are incompletely understood. RPE
cells express apolipoprotein E (apoE),12 13scavenger receptor
BI (SR-BI),14and ATP binding cassette transporter A1
(ABCA1),15all recognised components of reverse cholesterol
transport (RCT).16Similar to macrophages, apoE expression is
regulated by nuclear hormone receptor ligands.13
AMD shares risk factors with atherosclerosis, such as
smoking, hypertension, and elevated C reactive protein (CRP)
levels.17–19The relation between AMD and hyperlipidaemia is
not consistent.20–26Investigators have speculated that since
the main source of Bruch’s membrane lipids is the retina and
RPE, and not the circulation, serum lipids levels would not
necessarily correlate with the extent of lipid deposition in
Bruch’s membrane.8 9 11Serum HDL levels have also not been
associated consistently with AMD. Several studies showed a
ge related macular degeneration (AMD) is the leading
cause of visual loss in the Western world.1–3Most visual
loss in AMD develops secondary to neovascularisation
positive correlation between serum HDL levels and advanced
stages of AMD.24–26Other studies have not confirmed these
results.22 23Recently, in a case-control study of a Veterans
Affairs Medical Center cohort, HDL levels correlated nega-
tively with the development of neovascularisation in patients
The atheroprotective properties of HDL include promotion
(reviewed by Assmann and Nofer28and Nofer et al29). The
Age-Related Eye Disease Study demonstrated that high dose
supplementation with anti-oxidant vitamins C and E, b
carotene, and zinc reduces visual loss in patients with
macular degeneration.30The importance of anti-oxidants
may be attributed to protection against lipid peroxidation
owing to the high content of oxygen, polyunsaturated fatty
acids, and light irradiation in the retina.31The anti-oxidative
and anti-inflammatory attributes of HDL may protect against
visual loss associated with AMD. The presence of CRP,
complement components, and macrophages in Bruch’s
membrane deposits is suggestive of a chronic inflammatory
response in AMD.18
To determine whether HDL may be involved in RCT from
RPE cells, we have studied human RPE cells in culture
incubated with radiolabelled POS. We demonstrate that
labelled lipids of POS origin are transported through RPE for
efflux from the cell at the basolateral surface. The effluxed
labelled lipids (primarily phospholipids) are bound preferen-
tially to HDL of both low and high molecular weight species
Abbreviations: ABCA1, ATP binding cassette transporter A1; AMD,
age related macular degeneration; apoE, apolipoprotein E; apoA-I,
apolipoprotein A-I; C, cholesterol; CE, cholesterol esters; CRP, C reactive
protein; DHA, docosahexanoic acid; HDL, high density lipoprotein;
LCAT, lecithin:cholesterol acyltransferase; LDL, low density lipoprotein;
LSC, liquid scintillation counting; PBS, phosphate buffered saline; PC, PI,
phosphatidyl choline; phosphatidyl inosotol, ; PE, phosphatidyl
ethanolamine; POS, photoreceptor outer segments; RCT, reverse
cholesterol transport; RPE, retinal pigment epithelium; SR-BI, scavenger
receptor BI; TG, triglycerides; TLC, thin layer chromatography
in a process that is stimulated by HDL and apolipoprotein A-I
MATERIALS AND METHODS
Cell culture and POS labelling
Primary cultures of normal human RPE cells from a 35 year
old male donor were grown as described.14 32RPE cells
(passage 5–10) were propagated to confluence on laminin
coated six well or 12 well Costar Transwell tissue culture
plates (Fischer Scientific, Los Angeles, CA, USA) with DMEM
H21 containing 5% FBS, 2 mM glutamine, 5 mg/ml genta-
2.5 mg/ml fungizone, 1 ng/ml bFGF, and 1 ng/ml EGF in
the top and bottom chambers. POS were prepared from
bovine retinas as described33and stored at 270˚C for use. POS
were labelled with 1,4,7,10,13,16,19-[1-14C] docosahexanoic
acid (DHA) (ICN Life Sciences, 49 Ci/mol) as described.34For
lipid efflux experiments, cell monolayers were washed three
times with Dulbecco’s phosphate buffered saline (PBS) and
lipoprotein free fetal calf serum was added to the top
chambers. Bottom chambers contained apolipoprotein and
lipoprotein acceptors in serum free medium.
14C labelled POS (50 mg/ml) and 5%
Lipoprotein purification and analyses
Low density lipoprotein (LDL) (d=1.019–1.063 g/ml) and HDL
(d=1.063–1.210 g/ml) were purified from human plasma by
KBr density gradient ultracentrifugation as described.35ApoA-I
was purified from human HDL as described.36RPE media
samples were adjusted to d= 1.25 g/ml with solid potassium
ultracentrifuged (Beckman 50.2 Ti rotor) at 45 000 rpm for
24 hours at 10˚C. The lipoprotein containing d,1.21 g/ml
fraction was transferred to a centrifugal ultrafilter (5K MCO,
Viva Sciences, Hannover, Germany), buffer exchanged to
0.15 M NaCl, 1 mM EDTA (pH 7.4), 0.025% NaN3(Sal-EN),
Lipoprotein fractions were analysed by non-denaturing
PAGE. Briefly, samples were electrophoresed in linear 0–30%
gradient PAG at 200 V at 10˚C for 3000 V hours. Gels were
calibrated to the mobilities of calibrator proteins (HMW kit,
Amersham Pharmacia, Piscataway, NJ, USA) supplemented
with LDL and ovalbumin, Stokes diameter, 25 nm and
6.0 nm, respectively. Distribution of14C label was determined
by fractionating Coomassie stained gel into 2 mm slices. Gel
samples were treated with 0.2 ml TS-1 reagent (Research
Products International, Mt. Prospect, IL, USA) at 50˚C,
overnight in a shaking waterbath, cooled; 0.04 ml glacial
acetic acid added before radioactivity was determined by
liquid scintillation counting (LSC).
Discoidal HDL composed of purified human plasma apoA-
I, DMPC, and cholesterol were produced by the sodium
cholate dialysis method37and purified by FPLC on two
tandemly connected columns (Superdex 200, Amersham
Pharmacia, Piscataway, NJ, USA).
14C cpm80 000
culture (p=0.0027, t test, n=3). Total14C cpm in basal medium (mean
(SEM)) is shown.
HDL stimulates efflux of14C labelled lipids from RPE cells in
14C labelled lipid associated with
14C cpm bound
cells fed14C labelled POS. Shown are Coomassie stained gel lanes
containing samples: control medium (lane 1), purified plasma
lipoproteins (lane 2), repurified HDL (lane 3), and repurified LDL (lane
4). Calibrator proteins of known Stokes diameter (nm) in lane labelled
Repurification of HDL and LDL following incubation with RPE
HDL + LDL
2827 26252423 222120191817161514131211 10987654321
Polyacrylamide gel lanes were fractionated from the top (fraction 1) to
the bottom (fraction 28).14C was quantified by liquid scintillation
counting. Coomassie blue stained fractions 3–5 (LDL) and fractions 9–14
Distribution of radioactivity in repurified lipoproteins.
HDL stimulates RPE lipid efflux617
Thin layer chromatography (TLC)
14C labelled lipids were extracted from HDL by the Bligh-Dyer
method38and separated by one dimensional TLC by sequen-
tial development; first in solvent 1: chloroform/methanol/
acetic acid/water (25:15:4:2) until the solvent front had
progressed half way up the plate; then in solvent 2: n-hexane/
diethylether/acetic acid (65:35:2), until the solvent front
reached the top of the plate. Lipid species were detected by
acid charring. Plates were immersed in 7.5% copper acetate,
2.5% copper sulfate, 8% phosphoric acid, and heated on a
hotplate for 1 hour. Lipid spots identified by charring were
cut out and subjected to liquid scintillation counting.
Since HDL has been demonstrated to facilitate lipid and
cholesterol efflux in macrophages, we sought to determine
whether HDL has similar effect on lipid efflux from RPE cells.
RPE cells were cultured in Transwell plates and fed14C-DHA
labelled bovine POS in the apical chambers in the presence or
absence of purified lipoproteins added to the bottom media.
(100 mg/ml), and LDL+HDL (50 mg/ml each). After 36 hours
14C in basal media was determined by liquid scintillation
counting. As shown in figure 1, total14C in basal media was
significantly increased by HDL (p=0.0027, two tailed t test).
HDL stimulated basal
compared to no lipoprotein acceptor. LDL did not signifi-
cantly increase basal efflux of14C labelled lipids (p=0.4293,
two tailed t test). When LDL and HDL were present together,
stimulation of14C labelled lipid efflux was about half that of
HDL alone (1.4-fold), although this was not significantly
different from the control (p=0.0719, two tailed t test).
In order to determine whether basally effuxed14C labelled
lipids associated with lipoproteins, like samples were
combined and lipoproteins were purified from basal media
by ultracentrifugation at a density of 1.21 g/ml. The amount
of14C in the d,1.21 g/ml density fraction for each sample
was determined by liquid scintillation and is given in table 1.
HDL bound about 14-fold more14C labelled lipids than did
LDL. When both LDL and HDL were present,
d,1.21 g/ml fraction was intermediate to the amount when
either HDL or LDL were present alone. In the absence of added
lipoproteins, control media had low, but measurable, levels of
radioactivity in the d,1.21 g/ml fraction. The ultracentrifuged
media lipoprotein fractions were resolved by non-denaturing
PAGE (fig 2). The Coomassie stained components observed in
HDL (fig 2, lane3) andLDL (fig 2, lane 4) are typical lipoprotein
profiles expected of pure LDL and HDL. For purposes of
comparison control basal medium (fig 2, lane 1) and purified
plasma lipoprotein (fig 2, lane 2) profiles are also shown.
LDL (100 mg/ml),HDL
14C labelled lipid efflux 1.9-fold
14C in the
The distribution of14C labelled lipids among the lipopro-
teins in HDL and LDL samples was determined. Gel lanes
(fig 2, lanes 3 and 4) were fractionated and counted. As
shown in figure 3, radioactivity was confined to the
lipoproteins present in each sample: HDL (1783 cpm), LDL
(266 cpm). HDL+LDL was separated on another gel (not
shown) and yielded 966 cpm in the HDL band and 380 cpm
in the LDL band. Again, HDL was a better acceptor (sixfold to
sevenfold) than LDL when tested as a pure lipoprotein and in
plasma. When purified LDL and HDL were combined, HDL
exhibited a twofold to threefold higher affinity for basally
effluxed14C labelled lipids.
Lipids were extracted from the HDL fraction, purified as
above, and partially purified by one dimensional TLC. As
shown in figure 4 several lipid spots could be identified. Most
cholesterol (C), with lesser amounts in phosphatidyl inosotol
(PI), phosphatidyl ethanolamine (PE), triglycerides (TG) and
cholesterol esters (CE) (table 2). The remaining14C label was
in a dozen other, as yet unidentified, spots.
As a first step in determining which HDL fraction was the
most potent stimulator of
fractionated plasma HDL (1.063,d,1.210) by ultracentrifu-
gation in a continuous KBr density gradient. Ten HDL
fractions ranging in density (1.07–1.18 g/ml) and particle
size (6–11 nm, Stoke’s diameter) (fig 5) were tested in
equivalent protein concentrations (100 mg/ml). All HDL
fractions stimulated basal efflux of14C labelled lipids more
than twofold (p,0.0005, t test) (fig 6). In addition, all HDL
fractions bound effluxed14C labelled lipids (not shown).
14C label was in phosphatidyl choline (PC) and
14C labelled lipid efflux, we
HDL. Following incubation with RPE cells fed14C labelled POS, HDL
bound lipids were extracted and separated by TLC (bottom of plate at the
left). Standards for pure phosphatidyl choline (PC), phosphatidyl inosotol
(PI), phosphatidyl ethanolamine (PE), and cholesterol (C) were run, as
well as triglyceride rich lipids (TRL) which contains triglycerides (TG) and
cholesterol esters (CE) were run.
TLC separation and identification of some lipids bound to
14C labelled lipid bound to HDC
ultracentrifugation, fractionated and analysed by non-denaturing PAGE.
Shown is a Coomassie stained gel. Densities of each fraction are: F1
(d=1.077), F2 (d=1.086), F3 (d=1.096), F4 (d=1.105), F5
(d=1.116), F6 (d=1.127), F7 (d=1.141), F8 (d=1.154), F9
(d=1.176), F10 (d=1.191). Calibrator proteins of known Stoke’s
diameter (nm) are in lanes labelled MW.
Isolation of HDL subspecies. HDL was separated by KBr
618Ishida, Duncan, Bailey, et al
As a first step in identifying the components of HDL
necessary and sufficient for stimulating basal efflux of14C
labelled lipids, an artificial HDL, consisting of purified apoA-
I, cholesterol, and DMPC, was synthesised as described in
Methods. Purified artificial HDL (apoA-I vesicles), average
Stoke’s diameter of 10 nm, is shown in figure 7, fractions 44–
49. The ability of purified apoA-I and apoA-I vesicles
(fractions 44–49), to stimulate basal14C labelled lipid efflux
was tested. Both purified apoA-I and apoA-I vesicles
stimulated14C labelled lipid efflux by about 1.5-fold to 2-
fold (p=0.0079, Mann-Whitney test) (fig 8).
In non-ocular cell types, where RCT and its regulation has
been studied extensively,16nascent HDL particles containing
apoA-I bind to ABCA1, promoting phospholipid and choles-
terol efflux. Binding of these lipids to HDL forms pre-beta
migrating HDL, which is then converted to larger alpha
migrating HDL through esterification of cholesterol by
lecithin:cholesterol acyltransferase (LCAT). In macrophages,
incubation with apoA-I, the major apolipoprotein component
of HDL, increases efflux, probably mediated by direct binding
of apoA-I to ABCA1.39Recent evidence suggests that apoA-I
binding to ABCA1 may reduce ABCA1 turnover, effectively
increasing overall efflux mediated by this transporter.40
Lipid efflux from RPE may be mediated, as it is in
macrophages, by SR-BI and ABCA1. We have previously
demonstrated expression of these proteins by cultured
human RPE cells and, in the case of ABCA1, have localised
expression to the basal aspect of the cell.14 15Increased lipid
efflux by RPE in the presence of HDL and apo A-I is probably
mediated by binding of the lipoproteins to ABCA1. To bind
ABCA1 in the basal RPE plasma membrane, a lipoprotein
acceptor must traverse Bruch’s membrane from the chor-
iocapillaris. With ageing, there is progressive thickening of
Bruch’s membrane. This thickening is associated with a
reduction in macromolecular permeability and hydraulic
Clover have reported a 10-fold reduction in macromolecular
permeability of Bruch’s membrane from the first to the ninth
decades of life.41They show that proteins of molecular weight
.200 kDa could traverse a young patient’s Bruch’s mem-
brane, while elderly patients had an exclusion limit of
between 100–200 kDa.
We have demonstrated that various species of HDL bind
14C labelled lipids basally effluxed by human RPE. Analyses
of these density subclasses show that they range in size of
6.0–12.2 nm Stokes diameter. Their corresponding apparent
molecular weights (Mr43–440 kDa) are consistent with the
possibility that HDL may affect lipid transport in vivo. The
potential functional differences of different HDL subspecies
or their abilities to traverse Bruch’s membrane have not been
extensively studied. Serum levels of HDL2 have been
demonstrated to be negatively correlated with risk for
coronary disease.42Gordiyenko et al have demonstrate that
rhodamine labelled LDL can traverse Bruch’s membrane in
the mouse.43However, little is known of the permeability of
human submacular Bruch’s membrane to LDL and HDL in
vivo. It is possible that, with ageing, some of the larger
molecular weight HDL species may not traverse Bruch’s
membrane efficiently. This might lead to increased lipid
accumulation inthe RPE
Furthermore, there is no known mechanism, other than
diffusion across Bruch’s membrane, for removing lipids from
Bruch’s membrane if they were effluxed by other means—for
example, SR-BI or as large apoB containing lipoproteins.10
The inability of larger molecular weight lipoprotein acceptors
to fully traverse Bruch’s membrane might contribute to
progressive lipid accumulation that occurs with ageing in
Bruch’s membrane. In the present study, apo A-I increased
lipid efflux by approximately 50% in cultured human RPE.
The molecular weight of apo A-I is 28 kDa and may be better
at traversing a thickened Bruch’s membrane in older
subjects. Thus, nascent HDL particles such as pre-beta HDL
(6.0 nm Stokes diameter, personal communication, B Ishida
2005) may be particularly important in removing lipids from
RPE and Bruch’s membrane in older individuals.
Reducing access of some HDL species to Bruch’s membrane
and the RPE may have other consequences in ageing. HDL’s
atheroprotective effects derive not only from its role in
reverse cholesterol transport, but also its anti-oxidative
properties.28HDL bound enzymes, paraoxonase, and platelet
activating factor acetylhydrolase inhibit lipid peroxidation.
RPE cells in culture (p,0.0005, t test, n=3). Total14C cpm in basal
medium (mean (SEM)) is shown.
All HDL subspecies stimulate efflux of14C labelled lipids from
MW 30 33 36 39 42 45 48 51 54 57 60 63 66 69 72 Start MW
particles from sodium cholate dispersions of DMPC, cholesterol, and apo
A-I were purified by FPLC and fractions were analysed by non-
denaturing PAGE followed by Coomassie blue staining. Starting material
(Start), and calibrator proteins of known Stoke’s diameter (nm) (MW)
Purification of synthetic HDL (apoA-I vesicles). Discrete lipid
14C cpm (% of control)
ControlApoA-I ApoA-1 Ves
stimulate efflux of14C labelled lipids from RPE cells in culture
(p=0.0079, Mann-Whitney test, n=5). Results are the combination of
two separate experiments normalised to control levels of14C cpm in
basal medium. Control is 100%.
Purified apoA-I (ApoA-I) and synthetic HDL (ApoA-I Ves)
HDL stimulates RPE lipid efflux619
Because lipid peroxidation has been implicated in both the
pathogenesis of AMD and identified as a potential therapeu-
tic target, HDL’s potent anti-oxidants may play a part in
slowing the progression of AMD. A Bruch’s membrane
barrier to HDL diffusion may effectively diminish the anti-
oxidant properties of this lipoprotein.
Since lipid accumulation in Bruch’s membrane (basal
linear deposit) is one of the best histopathological correlates
with AMD,44 45an understanding of the mechanisms of RCT
in the RPE is particularly important. The present study
demonstrates that HDL is a preferred lipoprotein acceptor for
effluxed residues derived from phagocytised POS. The
changes that occur in ageing and AMD may impair access
of HDL and apoA-I to the basal surface of the RPE and the
inner aspect of Bruch’s membrane. A resultant decrease in
RCT may contribute to the pathological deposition of lipid
and cholesterol observed in AMD. Furthermore, pharmaceu-
tical strategies to increase RCT in RPE may be useful in
treating the early stages of AMD.
This study was supported by a gift from That Man May See, Inc
(DMS); and grants from: Merit Review Grant from the Veterans
Affairs Medical Center (DMS), and National Institutes of Health
B Y Ishida, J P Kane, Cardiovascular Research Institute, University of
California, San Francisco, CA, USA
B Y Ishida, K R Bailey, D M Schwartz, Veterans Affairs Medical Center,
San Francisco, CA, USA
K G Duncan, K R Bailey, D M Schwartz, Department of Ophthalmology
University of California, San Francisco, CA, USA
Competing interests: There are no competing interests to report for any
of the authors.
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