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Doctoral Thesis
Apolipoprotein A-I transcytosis through aortic endothelial cells
Author(s): Cavelier, Clara
Publication Date: 2006
Permanent Link: https://doi.org/10.3929/ethz-a-005296945
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ETH Library
DISS. ETH NO. 16679
APOLIPOPROTEIN A-I TRANSCYTOSIS
THROUGH AORTIC ENDOTHELIAL CELLS
A dissertation submitted to the
SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH
for the degree of Doctor of Sciences
Presented by
CLARA CAVELIER
Ingénieur de l’Institut National Agronomique Paris-Grignon (INA P-G)
born 21.10.1979
from France
Accepted on the recommendation of
Prof. Matthias PETER, examiner
Prof. Ari HELENIUS, co-examiner
Prof. Arnold von ECKARDSTEIN, co-examiner
Dr. Lucia ROHRER, co-examiner
2006
Table of contents
3
TABLE OF CONTENTS
TABLE OF CONTENTS .................................................................................... 3
ABSTRACT ....................................................................................................... 7
RESUME............................................................................................................ 9
ABBREVIATIONS ........................................................................................... 11
INTRODUCTION.............................................................................................. 13
1. Atherosclerosis and High Density Lipoproteins ................................. 13
1.1. Atherosclerosis................................................................................ 13
1.2. High Density Lipoproteins (HDL).................................................... 15
1.2.1. HDL form a Heterogeneous Class of Lipoproteins...................... 15
1.2.2. HDL Metabolism ......................................................................... 16
1.2.3. HDL and Apolipoprotein A-I are Atheroprotective ....................... 18
1.3. HDL and ApoA-I Binding Proteins.................................................. 20
1.3.1. ABCA1........................................................................................ 22
1.3.2. SR-BI .......................................................................................... 23
1.3.3. F0F1 ATPase............................................................................... 24
2. Transport of Macromolecules through Continuous Endothelia ........ 26
2.1. Transport Pathways......................................................................... 26
2.1.1. Protein Transport through Large Pores ...................................... 27
2.1.2. Interjunctional Protein Transport................................................. 27
2.1.3. Vesicular Transport..................................................................... 30
2.2. Caveolae mediated Transcytosis ................................................... 31
2.3. Lipoprotein Transport through the Endothelium.......................... 35
3. Problematic ............................................................................................ 36
Table of contents
4
MATERIALS AND METHODS.........................................................................37
RESULTS.........................................................................................................45
1. Apolipoprotein A-I Interaction with Aortic Endothelial Cells..............45
1.1. ApoA-I Binding (4°C)........................................................................45
1.2. ApoA-I Cell Association (37°C) .......................................................47
1.3. ApoA-I Internalisation and Degradation.........................................49
1.4. ApoA-I Transport through a Monolayer of Endothelial Cells .......53
2. Which Proteins mediate ApoA-I Transcytosis? ...................................57
2.1. Role of ABCA1 in ApoA-I Transcytosis..........................................57
2.1.1. Role of ABCA1 in ApoA-I Binding and Cell Association ..............57
2.1.2. Role of ABCA1 in ApoA-I Internalisation .....................................61
2.1.3. Role of ABCA1 in ApoA-I Transport ............................................62
2.2. Role of SR-BI in ApoA-I Binding and Cell Association .................64
2.3. Role of Cell Surface F0F1 ATPase in ApoA-I Transcytosis ...........66
2.3.1. Role of Cell Surface β-ATPase in ApoA-I Binding.......................66
2.3.2. Role of Cell Surface F0F1 ATPase in ApoA-I Internalisation........69
2.3.3. Role of Cell Surface F0F1 ATPase in ApoA-I Transport...............70
2.3.4. Effect of Extracellular Nucleotides on ApoA-I Internalisation ......71
2.3.5. Cell Surface F0F1 ATPase Activity...............................................72
3. Which Pathway is implicated in ApoA-I Transcytosis? ......................73
3.1. Role of Caveolin-1 in ApoA-I Transcytosis ....................................73
3.2. Role of Clathrin in ApoA-I Internalisation ......................................77
Table of contents
5
DISCUSSION................................................................................................... 79
1. ApoA-I Interaction with Aortic Endothelial Cells................................. 79
2. Which Proteins mediate ApoA-I Transcytosis?................................... 82
2.1. Role of ABCA1 in ApoA-I Transport............................................... 82
2.2. Role of SR-BI in ApoA-I Transport ................................................. 86
2.3. Role of F0F1 ATPase in ApoA-I Transport ...................................... 87
3. Which Pathway is implicated in ApoA-I Transcytosis? ...................... 90
OUTLOOK ....................................................................................................... 93
REFERENCES................................................................................................. 96
ACKNOWLEDGEMENTS.............................................................................. 115
CURRICULUM VITAE ................................................................................... 117
Abstract
7
ABSTRACT
Atherosclerosis is the major cause of death worldwide. It is a progressive
disease, characterised by the subendothelial accumulation of cholesterol-
engorged macrophages. High-density lipoproteins (HDL) are cholesterol
carriers in plasma, which major protein constituent is apolipoprotein A-I (apoA-
I). The plasma levels of both apoA-I and HDL are inversely correlated with the
risk of atherosclerotic cardiovascular diseases. Most of the atheroprotective
properties of apoA-I and HDL are exerted within the vascular wall rather than in
the plasma compartment. In deed, HDL are the most abundant lipoproteins
within the arterial intima. However, very little is known about apoA-I and HDL
transport through the endothelium. In this project, three issues were addressed:
the characterisation of apoA-I interaction with endothelial cells, the identification
of the receptors involved and the description of the pathway implicated.
First, apoA-I interaction with endothelial cells was characterised. Endothelial
cells were found to bind, internalise and transport apoA-I in a specific manner.
In immunofluorescence microscopy experiments, apoA-I was observed in
vesicles, which partially colocalised with early endosomes markers.
Furthermore, apoA-I transport was inhibited at 16°C and apoA-I was modified,
probably lipidated, in parallel to its transport. Therefore, it seems that apoA-I is
transcytosed through endothelial cells.
Second, we analysed the role of known apoA-I/HDL binding proteins (i.e.
ABCA1, SR-BI and the beta chain of F0F1 ATPase) in apoA-I interaction with
endothelial cells. Using diverse pharmacological treatments and RNA
interference, we observed that in endothelial cells ABCA1 was modulating
apoA-I binding, internalisation and transport. By contrast, reducing SR-BI
expression did not change apoA-I binding and internalisation but lowered HDL
binding. This result is consistent with the current consensus that SR-BI is a
receptor for intact HDL. Besides, F0F1 ATPase, which was found on the surface
of endothelial cells, modulated apoA-I binding, internalisation and transcytosis.
Interestingly, extracellular ADP stimulated apoA-I internalisation. In agreement
Abstract
8
with this result, F0F1 ATPase hydrolysed ATP on the surface of endothelial cells
and upon binding of apoA-I.
Third, the implication of the clathrin- and caveolin-mediated pathways in apoA-I
transcytosis was analysed. Clathrin silencing did not alter apoA-I internalisation
although it reduced LDL degradation. On the contrary, lowering caveolin-1
expression diminished apoA-I internalisation and transport. Moreover, apoA-I
bound preferentially to caveolin-1 enriched rafts transferred onto a nitrocellulose
membrane and both ABCA1 and β-ATPase were found to be expressed in
these rafts.
To conclude, three proteins were found to play a role in the transcytosis of
apoA-I: ABCA1, cell surface F0F1 ATPase and caveolin-1. It is still unclear
whether these proteins are cooperating in the same pathway to mediate apoA-I
transcytosis, but it would be a very challenging hypothesis to address.
Résumé
9
RESUME
L’athérosclérose est la première cause de mortalité dans le monde. Cette
maladie est caractérisée par l’accumulation d’éléments fibreux et de lipides
dans la paroi artérielle. Les HDL (High Density Lipoproteins) sont des
transporteurs de cholestérol dans le plasma. De faibles concentrations
plasmatiques en HDL et en apolipoprotéine A-I (apoA-I, leur principale
apolipoprotéine) sont des facteurs de risque importants de l’athérosclérose. La
majorité des effets préventifs des HDL et d’apoA-I doit être exercée dans la
paroi artérielle. Pourtant, le transport des HDL et d’apoA-I à travers
l’endothélium est un phénomène qui n’est toujours pas élucidé. Les objectifs de
ce projet sont de caractériser l’interaction d’apoA-I avec les cellules
endothéliales, d’identifier les récepteurs impliqués et de définir la voie de
transport intracellulaire.
Tout d’abord, il a été démontré qu’apoA-I s’associe à la surface des cellules
endothéliales pour être ensuite internalisée et transportée par ces cellules de
manière spécifique. ApoA-I est observée dans des vésicules intracellulaires,
dont une partie colocalise avec les marqueurs des endosomes précoces EEA1
(early endosome antigen 1) et transferrine. De plus, à 16°C le transport d’apoA-
I est significativement réduit. La majorité d’apoA-I transportée est modifiée,
probablement par association avec des lipides. Ces résultats indiquent que le
transport d’apoA-I est un phénomène de transcytose.
Par ailleurs, l’implication dans la transcytose d’apoA-I de trois protéines
importantes pour le métabolisme des HDL (ABCA1, SR-BI et F0F1 ATPase) a
été étudiée. La présente étude montre qu’ABCA1 est impliqué dans
l’association d’apoA-I avec les cellules endothéliales, son internalisation et son
transport. Au contraire, SR-BI ne semble pas être capital pour l’internalisation
d’apoA-I. Plus surprenant, la protéine mitochondriale F0F1 ATPase est
également exprimée à la surface des cellules endothéliales et est impliquée
dans l’internalisation et la transcytose d’apoA-I. Il semble, qu’à la surface des
cellules endothéliales, apoA-I stimule la production d’ADP par F0F1 ATPase.
Résumé
10
Enfin, le rôle de la clathrine et de la cavéoline-1 dans la transcytose d’apoA-I a
été étudié. Il semble que seule la cavéoline-1 soit impliquée dans la régulation
du transport d’apoA-I. Par ailleurs, il a été constaté qu’ABCA1 et F0F1 ATPase
sont exprimés dans les rafts enrichis en cavéoline-1, soulignant la possibilité
que ces protéines pourraient interagir pour réguler la transcytose d’apoA-I.
Pour conclure, cette étude montre l’implication d’ABCA1, de F0F1 ATPase et de
la cavéoline-1 dans la transcytose d’apoA-I à travers les cellules endothéliales.
Pour l’instant, rien n’a encore été démontré quant à l’interaction de ces
protéines. Néanmoins, il serait très intéressant de considérer cette hypothèse.
Abbreviations
11
ABBREVIATIONS
ABC transporter: ATP binding cassette transporter
AC: adenylate cyclase
ACAT: acyl-CoA:cholesterol acyltransferase
ADP: adenosine diphosphate
Apo: apolipoprotein
ATP: adenosine triphosphate
β-ATPase: beta-chain of the F0F1 ATPase
C: cholesterol
cAMP: cyclic adenosine monophosphate
CE: cholesterol ester
CETP: cholesteryl ester transfer protein
CsA: cyclosporin A
C-terminus: carboxy-terminus
EEA1: early endosome antigen 1
eNOS: endothelial nitric oxid synthase
GTP: guanidine triphosphate
HC: 22-R-hydroxycholesterol
HDL: high density lipoprotein
IDL: intermediate density lipoprotein
IEJ: interendothelial junctions
IP3: inositol-3 phosphate
kDa: kilo Dalton
LCAT: lecithin:cholesterol acyltransferase
LDL low density lipoprotein
LDLR: LDL receptor
LpE: apolipoprotein E containing lipoprotein
PLC: phospholipase C
NEM: N-ethyl maleimide
NSF: NEM sensitive factor
N-terminus: amino-terminus
PLTP: phospholipids transfer protein
Abbreviations
12
PM: plasma membrane
PS: phosphatidylserine
RA: 9-cis retinoic acid
RT-PCR: reverse transcription and polymerisation chain reaction
siRNA: small interfering RNA
SNAP: soluble NSF attachment protein
SNARE: soluble NSF attachment protein receptor
SR-BI: scavenger receptor type B class I
VAMP: vesicle associated membrane protein
VE-cadherin: vascular endothelial cadherin
VEGF: vascular endothelial growth factor
VLDL: very low density lipoprotein
ZO: zonula occludens
Introduction
13
INTRODUCTION
1. Atherosclerosis and High Density Lipoproteins
1.1. Atherosclerosis
Atherosclerotic cardiovascular diseases are the leading cause of death
worldwide. Atherosclerosis is a progressive disease characterised by the
accumulation of lipids and fibrous elements in the intima of large arteries. The
most common clinical complication is an occlusion of the vessel due to the
formation of a blood clot, resulting in myocardial infarction or stroke. An early
hallmark of atherosclerosis is the presence of cholesterol-loaded macrophages
(foam cells) in the intima of arteries (Fig. 1A) [10]. In contrast to most other cells
of the body, macrophages can accumulate large amounts of cholesterol by
uncontrolled scavenger receptor-mediated uptake. To circumvent the
cytotoxicity of unesterified cholesterol, they esterify cholesterol via the enzyme
acyl-CoA:cholesterol acyltransferase (ACAT). The cholesteryl esters are stored
intracellularly, mostly as cytosolic lipid droplets but also in lysosomes [11]. This
process turns macrophages into activated foam cells, which produce various
growth factors, cytokines, and proteases and thereby influence the course of
atherosclerosis [12]. These inflammatory signals stimulate the expression of cell
adhesion molecules on the surface of endothelial cells, thus facilitating the
recruitment of monocytes to the arterial wall and their subsequent differentiation
into macrophages. Progressively, the migration of smooth muscle cells and the
production of matrix proteins lead to the formation of a fibrous cap that covers
the lesion from the lumen (Fig. 1B) [4]. This cap represents a sort of healing or
fibrous response to the injury. Although advanced atherosclerotic lesions can
lead to ischemic symptoms due to progressive narrowing of the vessel lumen,
acute cardiovascular events that result in myocardial infarction and stroke are
generally caused by plaque rupture and resulting thrombosis. Exposure of lipids
and tissue factor to the blood components initiates the coagulation cascade,
causing platelets adherence and ultimately thrombosis. This generally occurs at
Introduction
14
the shoulder region of the plaque and is more likely to happen at sites of
thinning of the fibrous cap [4, 10, 12].
Figure 1: Development of an atherosclerotic lesion. An early atherosclerotic lesion
(A) consists of the accumulation of macrophages-derived foam cells. The
recruitment of monocyte is triggered by the expression of adhesion molecules by
endothelial cells. In advanced lesions (B), smooth muscle cells tent to form a fibrous
cap, which may rupture leading to thrombosis and myocardial infarction. From
reference [4].
Introduction
15
1.2. High Density Lipoproteins (HDL)
Lipoproteins are the major lipid carriers in plasma. According to their density
and diameter they can be divided in six major classes: chylomicrons,
chylomicron remnants, very low density lipoproteins (VLDL), intermediated
density lipoproteins (IDL), low density lipoprotein (LDL) and high density
lipoproteins (HDL). Important risk factors for atherosclerosis are high LDL
plasma levels and low plasma levels of both HDL and its major apolipoprotein
(apolipoprotein A-I, apoA-I). The following part focuses on HDL and their role in
atherosclerosis.
1.2.1. HDL form a Heterogeneous Class of Lipoproteins
High density lipoproteins (HDL) form a heterogeneous class of lipoproteins but
they share a high density (>1.063g/mL), small size (Stoke’s diameter 5-17nm)
and the absence of apolipoprotein B (apoB). On average, lipids constitute 50%
of total HDL mass, namely 30% phospholipids, 10-20% cholesterol and
cholesterol esters and 5% triglycerides. Phospohatidylcholine (about 80% of
phospholipids) and sphingomyelin (about 20% of phospholipids) are
indispensable structural components of HDL and are also needed to dissolve
unesterified cholesterol. Differences in the qualitative and quantitative content of
lipids and proteins result in the formation of distinct HDL subclasses (Fig. 2),
which are characterised by shape, density, size, charge and antigenicity [13].
Following agarose gel electrophoresis of plasma and anti-apoA-I-
immunoblotting, the majority of apoA-I is present in a fraction which migrates
with an α-electrophoretic mobility and is designated α-HDL. This fraction can be
further differentiated according to size and density into HDL2 and HDL3.
Approximately 5-15% of apoA-I in plasma is associated with particles which
have an electrophoretic pre-β mobility. Pre-β-HDL particles are small and
discoidal and they contain apoA-I and apoM either as lipid free apolipoproteins
or in association with a few molecules of phospholipids and free cholesterol [14-
16]. Importantly, relative to the concentration of lipid rich α-HDL, the
Introduction
16
concentration of lipid poor pre-β-HDL particles is increased in extravascular
compartments [17-19].
1.2.2. HDL Metabolism
Lipid free apoA-I or lipid poor pre-β-HDL are produced in the liver and in the
intestine, shed during lipolysis of triglyceride-rich lipoprotein by lipoprotein
lipase or formed by remodelling of HDL in plasma by cholesteryl ester transfer
protein (CETP), phospholipid transfer protein (PLTP), hepatic lipase or
endothelial lipase [13]. HDL precursors become mature lipid rich and spherical
α-HDL by acquisition of additional phospholipids and unesterified cholesterol
either from cells or from apoB-containing lipoproteins. PLTP facilitates the
transfer of phospholipids from cell onto lipoproteins and in between lipoproteins
apoA-I
Hydrophobic Coreof Cholesteryl Esters
Surface Monolayerof Phospholipids and
Free Cholesterol
pre-β-HDL α-HDL
Figure 2: Pre-β-HDL and α-HDL. HDL vary in shape, size and composition.
However, most HDL particles contain apoA-I as major apolipoprotein. Pre-β-HDL
are small, discoidal and consist of apoA-I as lipid free apolipoprotein or in
association with a few molecules of phospholipids and free cholesterol. Mature α-
HDL are large, spherical and lipid-rich particles.
Introduction
17
[20]. Free cholesterol is converted to cholesterol esters by lecithin:cholesterol
acyltransferase (LCAT) to form larger, spherical HDL particles that transport
cholesterol to the liver [21]. Moreover, in the presence of plasma CETP, a
portion of cholesterol esters is transferred to apoB containing lipoprotein
particles for clearance by the liver via the LDL receptor (LDLR) [22]. Finally,
selective uptake of cholesterol ester from circulating HDL is occurring in the
liver via the scavenger receptor BI (SR-BI) [23].
apoA-I
SR-BI
ABCA1 ABCA1
Bile
pre-β-HDLHDL3
CE C CE
LIVER PERIPHERAL TISSUE
C
LDL
LDLR
CETP
LCAT
HDL2
LCATPLTP
ABCG1
apoA-I
SR-BI
ABCA1 ABCA1
Bile
pre-β-HDLHDL3
CE C CE
LIVER PERIPHERAL TISSUE
C
LDL
LDLR
CETP
LCAT
HDL2
LCATPLTP
ABCG1
Figure 3: Schematic representation of HDL metabolism. ApoA-I (lipid-free or lipid-
poor) is secreted by the liver and acquires additional phospholipids and free
cholesterol from hepatic and peripheral tissues via ABCA1 and ABCG1 to become
spherical α-HDL (HDL2 and HDL3). These mature particle transport cholesterol to
the liver where it is used for the production of bile acids. Diverse enzymes such as
LCAT (lecithin cholesterol acyltransferase), PLTP (phospholipid transfer protein)
and CETP (cholesteryl ester transfer protein) are facilitating lipid transfers among
lipoproteins or from cells onto lipoproteins.
C: cholesterol, CE: cholesterol esters. Adapted from reference [5].
Introduction
18
1.2.3. HDL and Apolipoprotein A-I are Atheroprotective
Numerous epidemiological studies have demonstrated that plasma levels of
HDL and apoA-I are inversely correlated with the risk of atherosclerosis [24].
Moreover, rising HDL cholesterol inhibits atherogenesis in several genetic
animal models [25, 26]. HDL and apoA-I are exerting diverse potentially
atheroprotective functions. For example, they reduce oxidative damage, correct
endothelial dysfunction, inhibit inflammation and mediate lipid efflux [27]. The
most classical atheroprotective function of apoA-I and HDL, however, is the
catalysis of cholesterol efflux from the peripheral tissues including macrophages
from the vessel wall [28].
Efflux of lipids mediated by HDL and its apolipoproteins is a crucial process
regulating the cholesterol homeostasis of the organism. Efflux is the only
mechanism by which macrophages can limit or reverse the cellular cholesterol
accumulation [27]. In the absence of ATP-binding cassette (ABC) transporters
A1 or G1 macrophages accumulate massively cholesteryl esters in their
cytoplasm, highlighting the physiological importance of cholesterol efflux for
cholesterol homeostasis in macrophages [29-31]. In addition, as shown by
studies in mice with a targeted knockout of hepatic ABCA1, the lipid efflux from
liver cells mediated by ABCA1 is a rate-limiting step in the assembly of HDL and
is required for the maintenance of normal HDL cholesterol concentrations [32,
33].
Cholesterol efflux requires the conversion of intracellular cholesteryl esters into
free cholesterol as well as active transport of cholesterol to and through the
plasma membrane. This can be reached through three principal pathways (Fig.
4). First, hepatic and intestinal cells secrete lipoproteins, VLDL and
chylomicrons respectively. Macrophages and glia cells can also secrete lipids
together with endogenously produced apolipoproteins (particularly apoE) [34].
Second, some cells convert cholesterol into more hydrophilic bile acids (liver
cells) or oxysterols, notably 24S-hydroxcycholesterol (neurons) or 27-
hydroxycholesterol (macrophages), which are then secreted [35]. Third,
Introduction
19
cholesterol efflux in the proper sense is the transfer of cholesterol from or
through the plasma membrane onto extracellular acceptor particles. Only this
pathway will be further discussed.
Two major mechanisms for cholesterol efflux onto extracellular acceptors have
been described. The first process is passive aqueous diffusion of cholesterol
from the cell surface onto various extracellular acceptors including HDL, LDL,
albumin and protein-free unilamellar phospholipid vesicles. Net cholesterol
efflux by this process requires a concentration gradient between the donor cell
membrane and the various extracellular acceptor particles. Physiologically, this
is reached by extracellular cholesterol esterification through the enzyme LCAT.
LpE
cholesterylesters cholesterol
27-hydroxy-cholesterol
ApoA-I
HDL
ApoE
1
2
3
INOUT
ABCA1
ABCG1
SR-BI
LpE
cholesterylesters cholesterol
27-hydroxy-cholesterol
ApoA-I
HDL
ApoE
1
2
3
INOUT
ABCA1
ABCG1
SR-BI
Figure 4: Principal cholesterol efflux pathways in macrophages. 1 secretion of apoE
containing lipoproteins (LpE) 2 oxidation of cholesterol by CYP27 into more
watersoluble and secretable 27-hydroxycholesterol, 3 cholesterol efflux in the
proper sense onto extracellular acceptor particles.
Introduction
20
The cholesterol exchange rate between the cell membrane and lipoproteins can
be enhanced by plasma membrane receptors, for example SR-BI, which tether
lipoproteins to the cell surface and induce a redistribution of cholesterol in
lateral plasma membrane domains [36-38]. The second process requires the
interaction of apoA-I with ABCA1 or HDL with ABCG1 and may involve
retroendocytosis of apoA-I or HDL. In other words, HDL/apoA-I may be
internalised via receptor-mediated endocytosis, interact with lipid droplets for
lipidation and be resecreted without being degraded [39].
1.3. HDL and ApoA-I Binding Proteins
Several proteins have been shown to interact either with apoA-I or with HDL on
the surface of cells and thereby to play an important role in HDL metabolism:
ATP binding cassette A1 (ABCA1), scavenger receptor BI (SR-BI) and F0F1
ATPase (Fig. 5). The principal characteristics of these proteins and their role in
HDL metabolism are introduced in this section.
Introduction
21
Figure 5: Topological models of ABCA1, SR-BI and mitochondrial F0F1 ATPase.
ABCA1, SR-BI and the beta chain of F0F1 ATPase have been shown to interact with
either apoA-I or HDL and to play critical functions in HDL metabolism. ABCA1
belong to the ATP binding cassette transporter family and consist of 2 6-helix
transmembrane domains and two nucleotide binding domains (A and B design the
Walker domains A and B). SR-BI comprises two transmembrane domains, two
cytoplasmic domains and a large extracellular loop. F0F1 ATPase is a large complex
arrange in two domains: a transmembrane domain F0 (subunits a, b, c) and a
catalytic domain F1 (subunits α, β, γ, δ and ε). Adapted from [7-9]
Introduction
22
1.3.1. ABCA1
ABCA1 is a 2261 amino acid, 240 kDa protein belonging to the large ATP
binding cassette (ABC)-transporter family. ABC transporters use ATP as an
energy source that drives the transport of a wide variety of molecules. Full ABC
transporters typically consist of two six-helix transmembrane domains that make
up a pathway for the translocation of substrates across membranes and two
nucleotide binding domains that bind ATP and provide the energy for the
transport [28].
Recently, mutations in the ABCA1 gene were identified as the cause of Tangier
disease [40-43], a rare disease characterised by very low levels of plasma HDL
and accumulation of cholesterol and cholesteryl esters in macrophage foam
cells in tonsils, liver, spleen and many other tissues [44]. Furthermore,
cholesterol and phospholipid efflux to apoA-I from fibroblasts and macrophages
of Tangier disease patients is markedly reduced [44, 45]. Like Tangier disease
patients, ABCA1 knock-out mice exhibit HDL deficiency and reduced cellular
cholesterol efflux activity [33]. Both systemic and selective hepatic
overexpression of ABCA1 in mice results in an increase of HDL plasma levels
[46, 47]. Vice versa, apoA-I and HDL plasma levels are dramatically reduced in
mice with a liver specific deletion of ABCA1 [32]. Interestingly, the selective
inactivation or expression of ABCA1 in macrophages has little or no effect on
the plasma concentration of HDL [48]. Hence, hepatic ABCA1 expression is a
rate limiting factor for plasma HDL production whereas macrophages do not
contribute significantly to the formation of HDL. However, the selective knock-
out of ABCA1 in macrophages of either apoE-null or LDL receptor-null mice
significantly enhances the development of atherosclerosis [31]. Thus, although
ABCA1 in macrophages has little influence on HDL plasma levels, it crucially
prevents the excessive cholesterol accumulation in macrophages of the arterial
wall and their transformation into foam cells. However, whether ABCA1 directly
interact with apoA-I for cholesterol efflux, and hence has to be considered as a
receptor, is still ambiguous [39].
Introduction
23
Interestingly ABCA1 has been implicated in diverse endocytic processes. The
loss of ABCA1 function in Tangier fibroblasts is associated with enhanced
transferrin and dextran uptake [49]. Moreover, ABCA1, which is homologous to
the product of ced-7, one of the engulfment genes in nematode, has been
shown to promote engulfment of apoptotic cells [50]. To explain these results,
ABCA1 has been proposed to function as a phosphatidylserine translocase [50,
51], which would supply phosphatidylserine to the exofacial leaflet of the
membrane while depleting phosphatidylserine from the internal leaflet. Both of
these movements favour outward membrane bending [52, 53] and would
explain the role of ABCA1 in transferrin endocytosis, fluid phase uptake and
engulfment of apoptotic cells. However, it is still challenging to understand how
a phosphatidylserine translocase can possibly facilitate lipid efflux.
1.3.2. SR-BI
SR-BI is a 509 amino acid cell surface glycoprotein with a molecular mass of 82
kDa. Its predicted secondary structure comprises two transmembrane domains
and two cytoplasmic domains as well as a large extracellular loop containing
several N-glycosylation sites [54]. SR-BI is expressed in various mammalian
tissues and cells, including endothelial cells. The highest expression of SR-BI,
however, is in organs with critical roles in cholesterol metabolism (liver) and in
steroidogenesis (adrenal, ovary and testis) [55]. Distinct binding sites on SR-BI
have been implicated in the binding of a wide array of ligands, including anionic
phospholipids, advanced glycation end products, apoptotic cells as well as
native and modified lipoproteins (HDL, LDL acetylated, LDL, oxidized LDL and
VLDL), but not lipid free apoA-I [56].
Importantly, SR-BI mediates the selective uptake of cholesteryl esters from HDL
by cells through a process in which the cholesteryl esters are internalised
without the net uptake and degradation of the lipoprotein itself [57]. However,
the exact mechanisms for selective uptake of cholesteryl esters are largely
unknown. SR-BI reconstituted into liposomes mediates high affinity lipoprotein
binding and selective cholesterol uptake, indicating that selective uptake is an
Introduction
24
intrinsic quality of the receptor that does not require cellular structures or
compartments [58]. Alternatively, several recent studies have indicated a so-
called retroendocytosis pathway, which involves the holoparticle uptake of HDL
followed by resecretion of cholesteryl esters poor HDL leading to the net uptake
of lipids [59]. The relative contribution of each pathway is currently unknown. In
addition to its role in selective uptake of HDL cholesteryl esters, SR-BI
stimulates the bi-directional flux of free cholesterol between cells and HDL and
the rate of cholesterol efflux from various cell types correlates with the
expression of SR-BI [37, 60]. Furthermore, in endothelial cells SR-BI mediates
the activation of endothelial nitric oxide synthase (eNOS) by HDL [61]. This last
phenomenon is dependent on the presence of HDL but not lipid-free apoA-I.
Finally, Van Eck et al. showed that while hepatic cholesterol homeostasis is
maintained in SRBI-/- mice, SR-BI deficiency is associated with deregulation of
the cholesterol homeostasis in the arterial wall, resulting in increased
susceptibility to atherosclerosis [62]. Expression of SR-BI in macrophages
protects mice against atherosclerotic lesions development [63]. These data
strongly suggest a critical antiatherogenic role of SR-BI not only in the liver but
also in the arterial wall.
1.3.3. F0F1 ATPase
F0F1 ATPase is an enzymatic complex responsible for ATP synthesis in
mitochondria, prokaryote membranes and chloroplasts. The mitochondrial F0F1
ATPase (about 600 kDa) is composed of two domains: an extra-membranous
catalytic domain (F1) and a transmembrane domain (F0). Unexpectedly, it has
been found on the cell surface of endothelial cells, adipocytes, hepatocytes and
tumor cells, by immunofluorescence or after biotinylation of the cell surface [64-
68]. Although the mechanism leading to ectopic expression is unknown, F0F1
ATPase is not the only mitochondrial-matrix protein found at extramitochondrial
sites and shown to play functional roles in their unusual location [69, 70]. For
example, the cell surface fatty acid binding protein (FABP) is encoded by the
same gene as the mitochondrial aspartate aminotransferase (AspAT) [71]. It
has been shown that the mitochondrial AspAT precursor has an N-terminal
Introduction
25
mitochondrial targeting sequence, whose post-translational cleavage generates
FABP. In deed, transfection of mitochondrial AspAT in cells that do not express
it results in uptake of saturable fatty acid [72]. In addition, proteins from the
mitochondrial inner membrane (mitofilin, prohibitin, NADH-dehydrogenase,
ubiquinol-cytochrome c-reductase and F0F1 ATPase subunits α, β, γ, b, d, e, F6
and OSCP) have been found in proteomic studies performed on lipid rafts,
purified using detergent resistance or cationic silica [66, 73, 74]. Interestingly, F1
components and other mitochondrial proteins are enriched in the raft fraction
containing caveolin-1 [73]. By confocal microscopy, the α and β subunits of F0F1
ATPase were colocalised with the raft marker cholera toxin B [66].
The beta chain of F0F1 ATPase (β-ATPase) belongs to the F1 domain, which
contains the binding sites for ATP and ADP and the catalytic site for ATP
synthesis and hydrolysis [75]. In endothelial cells, angiostatin binds to and
inhibits cell surface F0F1 ATPase and anti-F0F1 ATPase antibodies reduce
endothelial cell proliferation [65, 76]. In hepatocytes, F0F1 ATPase hydrolyses
ATP upon binding of apoA-I, which triggers the uptake of HDL holoparticle [67].
It was found that the ADP produced by F0F1 ATPase stimulates P2Y13, which
ultimately regulate HDL endocytosis [67, 77]. Extracellular ADP can act through
the P2Y receptor family, which consists of 8 subtypes. P2Y1, P2Y2 and P2Y11
are the major nucleotide receptors on human vascular endothelial cells [78].
They contain seven membrane spanning domains and are coupled via G-
protein to adenylate cyclase or to phospholipase C, resulting in IP3 and Ca2+
release from intracellular stores [79, 80]. Interestingly, P2Y receptors on the
surface of endothelial cells have been involved in the regulation of cell adhesion
and permeability [81-83].
Introduction
26
2. Transport of Macromolecules through Continuous Endothelia
2.1. Transport Pathways
The endothelium forms an exchange barrier between plasma and tissues
(including the vascular wall), which is highly permeable to small molecules but
little permeable to macromolecules such as proteins. This relative
impermeability to large solutes is a prerequisite for the maintenance of fluid
equilibrium between plasma and interstitium. Still, macromolecules do cross the
endothelium to provide tissues with antibodies, protein-bound hormones, or
other macromolecules. Two mechanisms of transendothelial protein transport
have been controversially discussed. One view is that macromolecules are
shuttled by vesicles across the endothelium by “transcytosis”. The other view
favours a passive and convective transport mode across large pores located
either paracellularly or transcellularly, i.e. “porous transport” (Fig.6).
Figure 6: The different transport pathways through endothelial cells. Paracellular
transport refers to widened interendothelial junctions (IEJ). Transcellular transport
includes transport across transendothelial channels formed by fusion of caveolae
vesicles and transcytosis (vesicular transport). Adapted from reference [1].
PM: plasma membrane
Introduction
27
2.1.1. Protein Transport through Large Pores
Papenheimer described the transport of small hydrophilic molecules and
formulated the “pore theory" of capillary permeability [84]. This theory predicts
the diffusion across capillary walls of small hydrophilic solutes through water-
filled channels with a radius of 3-4 nm. It is generally accepted that small
hydrophilic molecules (such as water, sugars, amino acids and urea) are
transported paracellularly through discontinuities in the tight junctions. In 1956,
Grotte described the permeability of dextran with diverse molecular weight and
presented evidence for a two-pore barrier partitioning blood from lymph [85].
Large molecular size dextrans appeared in canine leg lymph in concentration
that decreased rapidly as a function of increasing molecular size for molecules
smaller than albumin (< 4.5 nm in radius). Larger molecules still appeared in
lymph, but at concentrations that were only slightly affected by molecular size.
This suggests that the pathway for transport of macromolecules differs from that
of small solutes. Therefore, it was proposed that a few large pores (1/30,000 of
the small pores) with a radius of 25-60 nm would account for the transport of
plasma proteins.
In an alternative hypothesis to the two-pores theory, the fibre-matrix model, the
sieving properties were attributed to the endothelial glycocalyx in series with the
interendothelial junctions [86]. The large pores theory is however not supported
by morphological studies and the structure of the large pores is still unknown. In
contrast, studies of the endothelial barrier at an ultrastructural level conclusively
showed the involvement of vesicles or vesicles-derived structures in the
transport of macromolecules through continuous endothelia [87-89].
2.1.2. Interjunctional Protein Transport
Endothelial cells adhere to one another through junctional structures formed by
transmembrane adhesive proteins. The transmembrane proteins are linked to
specific intracellular partners, which mediate anchorage to the actin
cytoskeleton and as a consequence stabilise junctions. Two major types of
Introduction
28
junctions have been described in endothelial cells: adherens junctions and tight
junctions (Fig. 7).
Adherens junctions are formed by transmembrane adhesion proteins of the
cadherin family, which mediate homophilic adhesion and are able to organise in
multimeric complexes at the cell border [90, 91]. Endothelial cells express a
specific cadherin called vascular endothelial cadherin (VE-cadherin). The
cytosolic tail of VE-cadherin is homologous to that of other classic cadherins
and through its carboxy- (C-) terminal region it binds β-catenin and
plakoglobulin. These two proteins are homologous and contain 10-13 armadillo
repeats, which are also present in many other signalling proteins. Both β-
catenin and plakoglobulin bind α-catenin, which anchors the complex to actin
[92].
Tight junctions were defined by electron microscopy as a specialisation of the
plasma membrane. In thin sections, tight junctions appear as a sequence of
fusions formed between two adjacent cells by the outer leaflets of the plasma
membrane. At higher magnification, however, it becomes clear that the
membranes are not fused but in tight contact to each other. Occludin and
claudin are transmembrane proteins at the tight junctions [93, 94]. They are
predicted to contain two extracellular loops and four membrane-spanning
regions. Both N- and C-termini are localised in the cytoplasm. The cytoplasmic
domains of occludin and claudin bind ZO-1, a cytoplasmic plaque protein from
the family of membrane-associated guanylate kinase, which plays an important
role in organising paracellular seal (Fig. 7). ZO-2 is another well-characterised
protein in the cytoplasmic plaque that links the tight junction to cytoskeletal
filaments including actin. Finally, tight junction components interact with several
signal transduction molecules, such as G proteins and protein kinases, which
ultimately regulate cell proliferation, polarity and permeability. Interestingly,
expression of occludin in the endothelium correlates with the permeability of
different segments in the vascular tree [95].
In non-absorptive epithelia (e.g. urinary bladder), tight junctions represent a
waterproof barrier. In endothelial cells, however, tight junctions only restrict but
do not block the passage of fluids. It is generally accepted that discontinuities in
the tight junctions accommodate most of the water and small solutes transport
Introduction
29
through the endothelium but would not normally allow significant passage of
macromolecules. However, in case of inflammation paracellular permeability to
plasma proteins is increased. Among extracellular stimuli acting on tight
junctions there are inflammatory cytokines. For example interferon-γ enhances
permeability of the T84 epithelial cell line, reduces ZO-1 expression, causes the
redistribution of occludin and ZO-2 and disrupts apical actin [96]. Similarly,
VEGF provokes the phosphorylation of occludin, the disassembly of tight
junctions and the increase in transport of 70 kDa dextran [97, 98].
Figure 7: The arrangement of the tight junction (ZO: zonula occludens) and the
adherens junction. The integral proteins occludin and claudin, which form the tight
junction, are displayed along with peripheral membrane proteins associated with the
tight junction, such as ZO-1 and ZO-2. The adherens junction involves homophilic
adhesion of the transmembrane protein VE-cadherin. Cadherins are linked to the
cytoskeleton via catenins and plakoglobulin. Adapted from reference [6].
Introduction
30
2.1.3. Vesicular Transport
The relative contribution of transcytosis versus large-pore transport to the
transport of macromolecules across endothelia has been controversial for the
last 50 years. Ultrastructural studies showed that electron opaque protein
tracers (albumin, insulin) in transit through the endothelium do not label
interendothelial junctions. Intravascular albumin tracers were rather detected on
the luminal endothelial membrane, in vesicles and in the interstitial space [99].
Moreover, the transport of tracers is inhibited by N-ethyl maleimide (NEM), a
reagent known to interfere with vesicular docking and fusion to target
membranes [100]. Caveolae preparations from lung vasculature contain
molecules involved in docking and fusion of vesicles such as vesicle-associated
membrane protein VAMP-2, NEM sensitive factor (NSF) and SNAP-25 as well
as in vesicle fission such as dynamin [101-104]. The use of cholesterol-binding
agents (filipin or methyl-β-cyclodextrin) which disassemble caveolae
demonstrated the implication of caveolae in albumin transcytosis [105, 106]. In
addition, the caveolin-1 (key structural component of caveolae) knockout mice
exhibit a loss of caveolae and of vesicular albumin transport. Interestingly, the
interendothelial junctions were open and capable of transporting albumin.
Although this might represent a compensatory adjustment, this result raises the
possibility that caveolae contribute also to the regulation of paracellular
endothelial permeability. Finally, as the radius of the neck region of vesicles
(~25 nm) approximates the dimension of large pores, it has been proposed that
caveolae constitute the postulated large pore system [99]. In other words, short-
lived channels might be formed by transient fusions of endothelial cell vesicles.
This last hypothesis would satisfy both the convective nature of macromolecule
transport and the ultrastructural data.
Introduction
31
2.2. Caveolae mediated Transcytosis
In endothelial cells, caveolae mediated internalisation contributes to more than
85% of the uptake process, as evaluated using cell surface biotinylated proteins
or biotinylated cholera toxin [107]. Caveolae were first identified in the 1950s by
Palade in endothelium as rounded or flask-shaped plasma membrane
invaginations of 50-80 nm in diameter (Fig. 8). They were thought to be sessile
structures but recently the highly dynamic nature of caveolae trafficking was
demonstrated [108]. In this study two subset of caveolae are described. One
subset is transport-incompetent and is found as clusters in multicaveolar
assemblies as previously described. The second subset undergoes continuous
“kiss and run” cycles in small volume below the plasma membrane and
occasionally long distance trafficking to intracellular pools.
Although caveolae do not show an electron-dense layer on their cytosolic
surface in thin-section electron microscopy, they do have a protein "coat"
composed primarily of a protein called caveolin-1 (Fig. 8). Caveolin-1 is an
integral membrane protein of 22 kDa required for the formation of caveolae. In
deed, in caveolin-1 knock-out mice caveolae are absent [109, 110]. It has an
unusual hairpin topology in that the N- and C- terminal domains are cytosolic,
connected by a hydrophobic sequence that is buried in the membrane but does
not span the bilayer. Caveolin-1 is palmitoylated in the C-terminal segment, can
be phosphorylated on tyrosine residues, binds cholesterol and forms dimers
and higher oligomers [111]. The caveolin oligomerisation is in part responsible
for the striations visualised on the cytosolic surface by electron microscopy. In
general, caveolae are highly enriched in cholesterol and sphingomyelin.
Cholesterol is required for caveolin-1 oligomerisation and recruitment at the
plasma membrane.
Introduction
32
The caveolae vesicular system is supported in endothelial cells by proteins
involved in fission, targeting and fusion (dynamin, intersectin, SNARE, etc).
Dynamin is a 100 kDa GTPase which undergoes GTP dependent self-assembly
to form higher order structures: dynamin rings and spirals [112]. The GTPase
activity generates a constricting force around the collar of vesicles undergoing
fission (Fig. 9) [113]. In endothelial cells, overexpression of a mutant dynamin
lacking normal GTPase activity not only inhibits GTP induced fission and
Figure 8: Caveolae in endothelial cells. Endothelial cells are very thin (0.2-0.5 µm) in
regions excluding nuclei (A). At a higher magnification (B and C), they present flask-
shaped invaginations attached to both the luminal and the interstitial surfaces, the
caveolae. D is a schematic representation of caveolae and its oligomeric coat
protein, caveolin-1. E is an enlarged version of D. Caveolin-1 forms hairpin
structures with both its N- and C-terminal ends facing the cytoplasm. Three
palmitoyl chains at the caveolin C-terminal tail are inserted into the bilayer.
Caveolae are enriched in cholesterol and glycosphingolipids. Adapted from [2].
Introduction
33
budding of caveolae but also prevents internalisation of cholera toxin and
albumin transcytosis [114, 115].
Intersectin is an important partner of dynamin. Two highly similar genes,
interesectin-1 and intersectin-2 have been identified, each producing two
isoforms by alternative splicing. Intersectin-1 has been localised at the caveolae
neck region and seems to recruit dynamin to generate a high local
concentration required for collar formation, caveolae fission and internalisation
[107]. Intersectin interacts also with the SNARE (soluble N-ethylmaleimide-
sensitive factor attachment protein receptor) proteins SNAP-25 and SNAP-23
[116, 117], indicating that intersectin might not only be involved in vesicle fission
but also fusion with the targeted membrane.
Normal intracellular trafficking of cholera toxin B, a caveolae marker, is impaired
when the vesicle associated membrane protein VAMP-2 is cleaved by
botulinum toxin D, suggesting that caveolar trafficking requires intact SNARE
machinery (vesicle-associated v-SNARE and target membrane-associated t-
SNARE) [104]. Diverse proteins involved in vesicle docking and fusion were
localised to endothelial caveolae: VAMP-2, syntaxin-4, SNAP-23, SNAP-25,
NSF and α-SNAP [102, 104]. Syntaxin-4 and SNAP-23 seems to cluster to
regulate caveolar fusion with the basolateral plasma membrane of endothelial
cells [117].
Introduction
34
Figure 9: Model for caveolar fission, docking and fusion mechanisms in endothelial
cells. Dynamin and proteins from the SNARE machinery (VAMP-2, syntaxin-4 and
SNAP-23) have been localised to endothelial caveolae. NSF and α-SNAP have also
been found associated with caveolae in endothelial cells. They are important
regulatory proteins which catalyse the ATP dependent disassembly of the SNARE
complex after membrane fusion. Adapted from [3].
Introduction
35
2.3. Lipoprotein Transport through the Endothelium
Within the arterial intima, HDL are the most abundant lipoproteins [15]. In
addition, relative to the amount of lipid rich mature HDL, the concentration of
lipid poor pre-β-HDL is increased in extravascular compartments, where they
are thought to exert their atheroprotective activity [118]. This suggests that HDL
and preferentially lipid poor apoA-I are transported through the aortic
endothelium. Furthermore, the flux of both pro-atherogenic LDL and anti-
atherogenic HDL into the vascular wall are considered as rate limiting steps in
atherosclerosis [12]. Studies demonstrated that the influx of lipoproteins into the
vascular wall increases with the plasma concentration and decreases with the
size of lipoproteins [119, 120]. Therefore, it is generally believed that
lipoproteins enter the vascular wall by passive leakage through damaged parts
of the endothelium [119]. Only a few experimental data on LDL transport are
available, some of them supporting transcytosis other passive filtration [121-
125]. Even less is known about the transendothelial transport of HDL [126-128].
De Vries et al. found high-affinity HDL binding sites on the surface of brain
capillary endothelial cells but observed that HDL transport is not saturable.
Thus, HDL was suggested to be transported paracellularly [128]. In another
study, HDL3 was saturably transcytosed across the blood brain barrier.
Basolateral resecreted HDL3 were partly depleted in lipid tracers and the
transcytosis was inhibited by antibodies against SR-BI, which is primarly
expressed on the apical side and colocalises with caveolin [126]. To conclude,
both paracellular and transcellular transport of HDL might occur in endothelial
cells.
Introduction
36
3. Problematic
The transport of lipoproteins into the vascular wall is considered as a rate
limiting step in the development of atherosclerosis [12]. However, little is known
about the transport of HDL and lipid poor apoA-I through endothelial cells.
Therefore, we studied the interaction of apoA-I with aortic endothelial cells and
addressed three questions:
1. How does apoA-I interact with endothelial cells and how is apoA-I
transported through a monolayer of endothelial cells?
2. Which receptor(s) or transporter(s) are involved?
3. Which pathways are involved?
The goal of the first question is to characterise apoA-I binding, cell association,
internalisation and transport in bovine aortic endothelial cells. With the second
question, we intend to study the involvement of known HDL/apoA-I binding
proteins (ABCA1, SR-BI and F0F1 ATPase) in binding, internalisation and
transport of apoA-I. The purpose of the third question is to find out whether
apoA-I transcytosis occurs via caveolae, clathrin coated pits pathway or an
alternative pathway.
Materials and Methods
37
MATERIALS AND METHODS
Isolation and Labeling of Lipoproteins and ApoA-I - Human LDL (1.019 < d <
1.063 kg/L) and HDL (1.063 < d < 1.21 kg/L) were isolated from normolipidemic
plasma of blood donors by sequential ultracentrifugation [129]. Lipid-free human
apoA-I was extracted from HDL as described previously [130] and labeled with 125I using Iodo-Beads iodination reagent (Pierce) and Na125I, according to the
manufacturer's instructions. In a typical reaction, we used 1 mCi Na125I, 1.5 mg
apoA-I, and two beads. Proteins were separated from unincorporated 125I on a
Sephadex G-25 (Amersham Biosciences) column, followed by extensive dialysis
(against 150 mM NaCl, 0.3 mM EDTA, pH 7.4) to remove residual free iodine.
The specific activity expressed as cpm/ng protein was calculated based on the
protein concentration, measured by the Dc protein assay (Bio Rad) and the 125I
counts. Specific activities of 600-1200 cpm/ng protein were obtained.
Cell Culture – Bovine aortic endothelial cells (BAEC) were isolated from bovine
aorta by collagenase digestion using standard protocols [131, 132] and cultured
in regular tissue culture dishes in Dulbecco's modified Eagle medium (DMEM)
supplemented with 5% fetal calf serum (FCS) at 37°C in a humidified 5% CO2,
95% air incubator .
125I-apoA-I 4°C Binding Assay - Cells were seeded in 24-well dishes at 100,000
cells/well and grown until confluence (2 days). On the assay day, cells were
prechilled on ice for 15 min, washed twice with DMEM and incubated in DMEM
Hepes 1% BSA containing 5 µg/mL 125I-apoA-I, in the absence (triplicate
determinations) or in the presence (at least double determinations) of a 40-fold
excess of unlabeled apoA-I (or the indicated competitor). After 2 h incubation at
4°C, cells were washed once with cold Tris/BSA wash buffer (50 mM Tris-HCl,
150 mM NaCl, pH 7.4, 2 mg/ml BSA) and twice with cold Tris wash buffer (50
mM Tris-HCl, 150 mM NaCl, pH 7.4). Cells were then solubilised in 0.5 ml of 0.1
M NaOH for 1 hour at room temperature. The amount of bound radioactivity
Materials and Methods
38
was determined using a Perkin Elmer γ-counter and the protein content was
measured with total protein urine/CSF assay, Cobas Integra, Roche.
For differential binding studies, endothelial cells were cultured for 2 days in a
two compartments system to form a tight monolayer. The label was added to
the upper (apical side) or to the lower compartment (basolateral side),
respectively with and without a 40-fold excess of unlabeled apoA-I. The
samples were analysed essentially as described before.
125I-apoA-I 37°C Cell Association Assay – Cell association of 125I-apoA-I was
performed as 4°C binding, except that the assays were conducted for 30
minutes at 37°C.
Total and Partial 125I-apoA-I Degradation Assay – Total degradation of 125I-
apoA-I was measured by quantifying the radiolabelled amino acids released in
the medium, as previously described [133]. BAEC were incubated 4 h at 37°C
with 5 µg/mL 125I-apoA-I in DMEM Hepes 1% BSA. The amount of 125I-apoA-I
degradation products in the medium was measured after TCA precipitation and
extraction (with trichlormethane) of hydrogen peroxide oxidised free iodide. The
radioactivity of the water phase containing the cellular degradation products
was measured and normalised to the protein content. Partial degradation was
assessed by loading on a SDS PAGE the cell lysate after 4 h incubation at 37°C
with 5 µg/mL apoA-I
125I-apoA-I Internalisation Assay - The assay was performed as described for
the cell association studies. After 30 min incubation with 125I-apoA-I, the cells
were washed as described earlier and chilled on ice 15 min. Cell surface
proteins were biotinylated at 4°C using 500 µg/mL EZ-link-sulfo-NHS-LC-biotin
(Pierce) in PBS containing 0.1 mM CaCl2 and 1mM MgCl2, lysed in 10 mM Tris
pH 7.4, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS. The
biotinylated cell-surface proteins were pulled down with streptavidin-conjugated
sepharose beads (Amersham Biosciences). The radioactive counts of the
supernatant containing the internal proteins were measured and normalised to
the protein contents. Alternatively, internalisation of 125I-apoA-I biotinylated with
Materials and Methods
39
EZ-link-sulfo-NHS-SS-biotin (Pierce) was studied. The biotin moiety from the
cell surface bound biotin-125I-apoA-I was cleaved at 4°C in the presence of 50
mM DTT. After lysis, internal biotinylated 125I-apoA-I was pulled down with
streptavidin sepharose.
125I-apoA-I Transport Studies – To study the transport of apoA-I, BAEC were
plated on the upper side of porous filter inserts (0.2 µm) (BD Biosciences)
coated with rat-tail collagen (BD Biosciences) (50 µg/ml in 0.05 M acetic acid) at
a density of 50’000 cells/cm2, 2 days prior the assay. Considering the
polarisation of the cells identical to the one in the vascular wall, we called the
upper compartment “apical compartment” and the lower compartment
“basolateral compartment”. A typical transport assay was conducted at 37°C for
30 min or otherwise in the indicated conditions. The tightness of the monolayer
was assessed by measuring the permeability of 3H-inulin. The medium in the
apical compartment was removed and substituted with assay medium
containing 2.5 µCi 3H-inulin/ml. Samples of 50 µl were taken in duplicate from
the basolateral compartment every 20 min (over 4 h) and replaced with 100 µl
fresh mediums. Permeability calculations were performed using equation
derived from Fick's first law, described by Youdim et al. [134].
Papp (cm/s)= VA / (A MA)*(∆MB / ∆t) (Equation 1)
Papp = apparent permeability coefficient, VA = apical volume (cm3), A =
membrane surface area (cm2), MA = apical 3H-inulin amount (cpm), ∆MB/∆t=
change in amount of 3H-inulin (cpm) in basolateral compartment over time.
For the transport studies, the growth medium was replaced with DMEM Hepes
1% BSA and 125I-apoA-I was added to the upper chamber with and without a
40-fold excess of unlabeled apoA-I or HDL at the indicated final concentrations.
During the incubation, aliquots of the medium from the basolateral compartment
were removed (100 µl) and substituted with fresh DMEM, and the radioactivity
was measured. At the end of the assay aliquots of the medium from the
basolateral compartment were collected, the radioactivity was measured and
Materials and Methods
40
the protein bound radioactivity was calculated from the difference of the total
radioactivity minus the non-trichloroacetic acid (TCA) precipitable radioactivity.
The cell permeability of 125I-apoA-I was determined using equation 2. The
equation 2 is derived from the equation 1 and takes into account mass balance
correction for 125I-apoA-I binding in the cell layer (Mcell).
Papp (cm/s)= VA / [A x (MA - Mcell)] x (∆M / ∆t) (Equation 2)
Furthermore, the medium in the basolateral compartment was analysed on a
1% agarose (50 mMol sodium barbitate, pH 8.6). Samples were loaded onto the
gels and the electrophoresis was performed at 4°C. For SDS-PAGE, the
samples were heated for 5 min at 95°C prior loading on a 10% SDS gels and
separated at room temperature. Both gels were exposed after fixation to a
phosphor imager screen.
Specific binding, cell association, internalisation, degradation and transport
were determined by subtracting the values obtained in the presence of
unlabeled apoA-I (nonspecific) from those obtained in the absence of excess
unlabeled apoA-I (total).
Pharmacological Treatments and Inhibitors – ABCA1 expression was stimulated
by a mixture of 22-R-hydroxycholesterol and 9-cis retinoic acid (Sigma), 10µM
each for 30 h. BAEC were also incubated with cyclosporin A (Sigma), 20 µM for
4 h prior the assay or with IF1 (Abnova, Taipei, Taiwan), 100 nM in DMEM
Hepes pH 6.4 containing 1% BSA for 30 minutes prior the assay. We also used
a β-ATPase blocking antibody (MS503, MitoSciences, Eugene, USA), 2 µg/mL
10 minutes prior the assay. Finally, internalisation was measured after
stimulating the cells 10 minutes before the assay with 100 nM ATP or 100 nM
ADP. Cyclosporin A, IF1, the β-ATPase blocking antibody ATP and ADP were
still present in the assay medium.
siRNA Transfection – BAEC were transfected when the monolayer were 90%
confluent. 67nM BLOCK-iTTM fluorescent oligo and 100nM Stealth siRNA
Materials and Methods
41
(Invitrogen) against SR-BI (GTCAGCAAGGTCAACTATTGGCATT), ABCA1
(GGGACTTAGTGGGACGAAATCTCTT), the beta chain of the F0F1 ATPase (G
CAGAATCCCTTCTGCTGTGGGTTA), clathrin heavy chain (GCGCTTAGTGTG
TACTTAAGGGCTA), caveolin-1 (TCTGGGCAGTTGTACCATGCATTAA) or not
coding siRNA (TCTACGTTGATGACCCGTTAGGTAA) were transfected with
Lipofectamine 2000 in OPTIMEM (Invitrogen), according to the manufacturer’s
protocol. 6 h after transfection, the medium was replaced by DMEM 5% FCS
without antibiotics. Binding, internalisation and transport assays were conducted
2-3 days after transfection. The efficiency of the silencing was evaluated by
quantitative RT-PCR and western blotting.
Quantitative RT-PCR – RNA was isolated with RNeasy mini (Qiagen) according
to the manufacturer’s protocol. Reverse transcription was performed using
Superscript II RT (Invitrogen) following the standard procedure. Quantitative
PCR was done with LightCycler FastStart DNA Master SYBR Green-I (Roche).
The primers used for the amplification were: ABCA1 (GTCATTATCATCTTCAT
CTGCTTCC, CCTCACATCTT CATCTTCATCATTC, 60°C, 5 mM MgCl2), SR-BI
(GGAATCCCCATGAACTG, CTTGGGAGCTGATGTCATC, 58°C, 5mM MgCl2),
the β-chain of the F0F1 ATPase (GGTAGCGCTGGTGTACGGTC, CGGGACAA
CACAGTGGTAGC, 64°C, 3mM), clathrin heavy chain (TGTGTAGGCCTGTAC
TTCA, CTGGACTGATACGCATAACA, 64°C, 3 mM MgCl2) and caveolin-1 (GG
AACAGGGCAACATCTACA, CAGACAGCAAGCGGTAA, 64°C, 3 mM MgCl2).
The transcription levels were normalised to GAPDH (GTCTTCACTACCATGGA
GAAGG, TCATGGATGACCT TGGCCAG, 58°C, 4mM MgCl2).
Cell Surface Biotinylation – Cell monolayers were biotinylated with 250 µg/mL
EZ-link-sulfo.NHS-LC-biotin (Pierce) in PBS containing 0.1 mM CaCl2 and 1
mM MgCl2 at 4°C for 1 hour. The reaction was terminated by a 5 min incubation
in DMEM. Cells were lysed in 10 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 1%
sodium deoxycholate, 0.1% SDS. The lysates were incubated with 25 µL
streptavidin-conjugated sepharose beads (Amersham Biosciences) at 4°C
Materials and Methods
42
overnight. The beads were washed 3 times with the lysis buffer and the pulled
down proteins were resolved on a SDS-PAGE.
Western Blotting – BAEC were lysed in RIPA buffer (10mM Tris pH 7.4, 150mM
NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, protease inhibitors
(complete EDTA, Roche)). 50 µg total protein were loaded on the gel. ABCA1
(ab18180, Abcam, Cambridge, UK), SR-BI (ab3, Abcam, Cambridge, UK), the
β-chain of the F0F1 ATPase (MS503, MitoSciences, Eugene, USA), clathrin
(ab11331, Abcam, Cambridge, UK), caveolin-1 (ab2910, Abcam, Cambridge,
UK) expression levels were normalised to actin (AC-15, Sigma).
Determination of Extracellular and Intracellular ATP Concentrations –
extracellular and intracellular ATP concentrations were measured by luciferase
driven bioluminescence (ATP bioluminescence assay kit HSII, Roche). Cells
were rinsed twice in DMEM prior incubation in DMEM Hepes 1% BSA
containing or not 5 µg/mL apoA-I. After 30 min incubation, the assay medium
was collected to measure the extracellular ATP concentration and the cells
were lysed in the provided lysis buffer to determine intracellular ATP
concentration.
Isolation of Caveolae-Enriched Membranes – BAEC were chilled on ice for 15
min and rinsed twice with ice cold PBS containing 0.1 mM Ca2+, 1 mM Mg2+.
Cells were scraped with 0.2% Triton X-100 in MBS (MES –buffered saline, 25
mM MES hydrate buffer, pH 6.5, containing 150 mM NaCl, 5mM EDTA, and
protease inhibitor (Complete, Roche)) and left on ice for 20 min. The lysate was
subjected to 10 strokes in a loose-fiting Dounce homogeniser and centrifuged
(500xg, 5 min, 4°C). The supernatant was mixed with an equal amount of 80%
(w/v) sucrose-MBS, transferred at the bottom of an ultracentrifuge tube and
overlaid with 6mL 35% sucrose-MBS and 3 mL 5% sucrose-MBS. After
centrifugation (40 000 rpm, 20 h, 4°C) in SW41 rotor (Beckman Coulter),
fractions of 1 mL were collected and analysed by western blotting or ligand
blotting [135].
Materials and Methods
43
125I-apoA-I Ligand Blot – Proteins were transferred by dot blotting onto
nitrocellulose membranes. Membranes were quickly rinsed in PBS, incubated in
PBS containing 20 mM sodium deoxycholate for 30 min, washed 3 times in PBS
and incubated with 5µg/mL 125I-apoA-I in 20mM Tris, pH 7.4, 150 mM NaCl, 1
mM EDTA at room temperature for 1hour. After washing the membrane 3 times
15 min with PBS, the membranes were exposed to a phosphor screen [136].
Immunofluorescence Confocal Microscopy – Cells were incubated 30 minutes
with 5 µg/mL apoA-I labelled with alexa-488 and 5 µg/mL alexa-594 transferrin,
washed 6 times 5 minutes in PBS, fixed in 3% paraformaldehyde for 15
minutes, permealised with 0.2% Triton X-100. The antibody used to stain the
intracellular marker EEA1 (ab15846) was purchased from Abcam and used
according to the manufacturer’s instructions. Confocal microscopy was
performed with a 63x oil-immersion lens in the sequential mode.
Results
45
RESULTS
1. Apolipoprotein A-I Interaction with Aortic Endothelial Cells
At first, the interaction of apoA-I with endothelial cells was characterised in
terms of binding at 4°C, cell association at 37°C, internalisation, degradation
and transport.
1.1. ApoA-I Binding (4°C)
The ability of apoA-I to function as a ligand for endothelial cells was tested
using lipid-free 125I-apoA-I. Nonspecific binding was measured in the presence
of a 40-fold excess of unlabeled apoA-I. At first, we verified that the
experimental data used to determine the equilibrium constant were obtained at
equilibrium. Indeed, binding of 18 nM apoA-I equilibrated in less than 2 hours
(data not shown). Using a global fitting approach, total and nonspecific binding
were fitted at once to the experimentally determined binding data (Fig 10 A).
Besides, the free apoA-I concentration was approximated to the added apoA-I
concentration, because less than 0.5 % of the apoA-I added bound to the cells
at 4°C. Bovine aortic endothelial cells (BAEC) bound 125I-apoA-I in a saturable
manner with a specific maximal binding Bmax = 1.5 ± 0.2 pmol / mg cell protein
(2.0 ± 0.3 105 binding sites per cell) and with an affinity Kd = 67 ± 20 nM. The
Scatchard plot (Fig. 10 A) gives an easier reading of the equilibrium constants
obtained after global fitting.
In addition, apoA-I binding was competable with a 40-fold excess of unlabeled
apoA-I and HDL (both over 70%) but not with an excess of BSA (Fig. 10 B).
Results
46
In the vessel wall the endothelial cells are polarised. To analyse the binding
affinity on both the apical and the basolateral side, BAEC were cultured on
collagen coated inserts to form a confluent and presumably polarised cell layer.
Figure 10: ApoA-I binding (4°C) to BAEC. (A) BAEC were incubated at 4°C with the
indicated concentration of 125I-apoA-I in the absence (total, ) or in the presence of
a 40-fold excess of unlabeled apoA-I (non-specific, ). Specific binding (-) was
obtained after fitting simultaneously total and non-specific binding. The Scatchard
plot gives an easier reading of the equilibrium constants obtained after global fitting.
(B) To study the specificity of binding, BAEC were incubated 2 h at 4°C with 5
µg/mL 125I-apoA-I in the absence or in the presence of 200 µg/mL of the indicated
competitor.
Results
47
The distribution of the apoA-I binding sites in these cells was studied by adding
the label either in the apical compartment or in the basolateral compartment.
The specific binding capacity of the apical side of the cell layer was more than
three times as high as the one of the basolateral side (Fig. 11).
1.2. ApoA-I Cell Association (37°C)
At 37°C, total and nonspecific apoA-I cell association were measured at
increased 125I-apoA-I concentrations. Specific cell association was calculated by
subtracting the nonspecific cell association values from the total cell association
values. The concentration dependence of apoA-I total, non-specific and specific
cell association is shown in Fig. 12 A. It may be noted about 5% of apoA-I
added associated with endothelial cells. In addition, apoA-I cell association was
competable with an excess of unlabeled apoA-I and HDL (both over 85%) but
not with an excess of BSA (Fig. 12 B).
Figure 11: ApoA-I binding to the apical and the basolateral side of BAEC. BAEC
were cultured on porous filter inserts and the binding of 125I-apoA-I was measured
by adding the label either into the apical compartment or into the basolateral
compartment.
Results
48
Figure 12: I-apoA-I cell association (37°C) to BAEC. (A) BAEC were incubated at
37°C with the indicated concentration of 125I-apoA-I in the absence (total, ) or in
the presence of a 40-fold excess of unlabeled apoA-I (non-specific, ). Specific cell
association (-) was obtained after fitting simultaneously total and non-specific cell
association. (B) To study the specificity of the cell association, BAEC were
incubated 2 h at 37°C with 5 µg/mL 125I-apoA-I in the absence or in the presence of
200 µg/mL of the indicated competitor.
Results
49
1.3. ApoA-I Internalisation and Degradation
The cellular distribution of labeled apoA-I was further analysed. Cell surface
biotinylation experiments clearly demonstrated that at 37°C about 20% of total
cell associated material was found internal (Fig. 13 B). Similar data were
obtained using cleavable biotinylated 125I-apoA-I for the internalisation studies
(Fig. 13 B). The uptake of 125I-apoA-I was competable with an excess of
unlabeled apoA-I and HDL and the process reached a steady-state level in
BAEC after about 2 h (Fig. 13 A).
Results
50
Figure 13: 125I-apoA-I internalisation in BAEC. (A) BAEC were incubated at 37°C
with 5 µg/mL 125I-apoA-I for the indicated time in the absence (total, ) or in the
presence of a 40-fold excess of unlabeled apoA-I (non-specific, ). Specific
internalisation ( ) was calculated by substracting the values of the non-specific
internalisation from those of the total internalisation. (B) shows the repartition of
specifically cell associated 125I-apoA-I and biotinylated 125I-apoA-I as cell surface
bound and internal.
Results
51
The internalisation of apoA-I by endothelial cells was further investigated by
confocal fluorescence microscopy. BAEC were incubated with alexa 488 apoA-I
and vesicles containing fluorescent apoA-I were partially colocalising with the
early endosome markers EEA1 (early endosome antigen 1) and alexa 594
transferrin (Fig 14). This result confirms that apoA-I is internalised by
endothelial cells.
Figure 14: Internalisation of alexa 488 apoA-I. The internalisation of apoA-I in
endothelial cells was analysed by confocal fluorescence microscopy. Confluent
BAEC were incubated for 30 min with 5 µg/ml apoA-I alexa 488 conjugates (green).
Colocalisation of vesicles containing apoA-I with the early endosome marker EEA1
and transferring (red) was assessed.
Results
52
The degradation of 125I-apoA-I after 4h was analysed by measuring the release
of radiolabelled degraded amino acids into the medium in the presence or
absence of excess unlabeled apoA-I. The specific degradation was calculated
as the difference between total degradation (without competitor) and non-
specific degradation (with competitor) and was worth about 5 ng/mg cell protein
in 4h (Fig. 15 A) which is less than 3% of the specific cell association (Fig. 12).
The SDS-PAGE analysis of the cell lysate after 4 h incubation with 125I-apoA-I
confirmed that internalised 125I-apoA-I is not degraded.
Figure 15: ApoA-I total and partial degradation. (A) Total degradation was measured
as the release of radiolabelled degraded peptides in the assay medium, after 4 h
incubation at 37°C. (B) Partial degradation was evaluated by loading the cell lysate
on a SDS PAGE after 4 h incubation with 5 µg/mL 125I-apoA-I, the starting material
(apoA-I) was used as a control.
Results
53
1.4. ApoA-I Transport through a Monolayer of Endothelial Cells
To assess the barrier function we measured the permeability coefficient (PC) of 3H-inulin, which do not cross cell membranes and hence represent a
paracellular transport marker [137]. The tracer was added to the apical chamber
and the filtered radioactivity was measured in the basolateral compartment. The
PC for 3H-inulin across the endothelial cell layer was worth 1.32 ± 0.35x10-5
cm/s, calculated over a time period of 4 h. Furthermore, the influence of apoA-I
on the permeability was determined by analysing the filtration of 3H-inulin in the
presence of 5 µg/ml apoA-I in the apical compartment. The PC of 3H-inulin in
the presence of apoA-I was worth 1.30 ± 0.35 x 10-5 cm/s and, hence, not
significantly different from the PC of 3H-inulin alone. This indicates that apoA-I
does not impair the barrier function of the monolayer. With this model we
addressed the question of whether endothelial cells transport apoA-I through
the cell layer.
The appearance of 125I-apoA-I in the basolateral chamber was measured after
adding the tracer into the apical chamber in the presence and in the absence of
excess cold apoA-I at different time points. The specific transport was
calculated by subtracting the non-specific transport from the total transport (Fig.
16). The partial competition of 125I-apoA-I transport through the cell layer points
to the occurrence of a specific transport in addition to filtration. Interestingly, in
contrast to the apical to basolateral route, no specific transport was recorded for
the opposite basolateral to apical direction whereas inulin permeability was
similar in both directions (Fig. 16 C). The radioactive proteins recovered in the
basolateral compartment were analysed by SDS-PAGE and were found to
constist of intact apoA-I (Fig. 17 B). To further analyse the transport, the
experiment was repeated at 16°C, a temperature which prevents fusion of
vesicles with the plasma membrane. The transport capacity was almost
abolished, at this reduced temperature (Fig. 16 B), which indicates that apoA-I
might be transported transcellularly.
Results
54
Figure 16: ApoA-I transport through a monolayer of endothelial cells is. (A) BAEC
were cultivated on transwell, incubated at 37°C with 5 µg/mL 125I-apoA-I (added in
the apical compartment) for the indicated time in the absence (total, ) or in the
presence of a 40-fold excess of unlabeled apoA-I (non-specific, ). Specific
internalisation ( ) was calculated by substracting the values of the non-specific
from those of the total transport. (B) ApoA-I transport from the apical to the
basolateral compartment was analysed at 37°C and 16°C. (C) apoA-I transport from
the apical to the basolateral compartment and in the opposite direction were
measured and compared to inulin permeability in both directions.
Results
55
Interestingly, charge and size fractionation on a native agarose gel of the
content of the basolateral compartment revealed two species, one with preβ-
mobility corresponding to lipid-free 125I-apoA-I and another with faster mobility,
similar to that of 125I-HDL. These α-mobile particles were absent in all samples
containing an excess of cold apoA-I indicating that it represented the fraction of
the specifically transported cargo, Fig. 17 A. Analysis of the same samples from
the basolateral compartment by SDS-PAGE revealed a single band of 28 kDa
(Fig 17 B). The mobility shift (Fig. 17 C) as well as the transport capacity (Fig.
16 B) were both almost abolished at 16°C, indicating a temperature sensitive
modification and an energy dependent transport. Thus, it is very likely that
apoA-I is transcytosed through endothelial cells.
Results
56
Figure 17: Temperature dependent change of apoA-I mobility after transport. (A)
reveals a native 1% agarose gel loaded with equal cpms of 125I-HDL (1), 125I-apoA-I
(starting material) (2). The wells 3-5 were loaded with media harvested from the
basolateral compartment: from a control insert without cells (filtrated 125I-apoA-I,
mock) (3), from inserts with BAECs incubated with 125I-apoA-I in the absence of
excess unlabeled apoA-I (4) or in the presence of excess unlabeled apoA-I (5). (B)
The media described in (A) were loaded on a SDS-PAGE. (C) Reveals an agarose
gel loaded with the identical volumes of the media isolated from the basolateral
compartment of a transport experiment performed at 37°C and 16°C.
Results
57
2. Which Proteins mediate ApoA-I Transcytosis?
To identify proteins which mediate apoA-I transcytosis, a candidate based
approach was chosen. SR-BI, ABCA1 and the β-chain of the F0F1 ATPase were
considered as candidate receptors mediating the binding, the internalisation and
ultimately the transport of apoA-I in endothelial cells.
2.1. Role of ABCA1 in ApoA-I Transcytosis
2.1.1. Role of ABCA1 in ApoA-I Binding and Cell Association
At first, we verified that ABCA1 is expressed in BAEC by RT-PCR and by
western blotting (Fig. 18). In addition ABCA1 expression and/or function were
modulated by RNA interference and pharmacological treatments. ABCA1
transcription was reduced by 90% (Fig. 18 A) and ABCA1 expression by about
75% (Fig. 18 C) in cells transfected with ABCA1 specific siRNA in comparison
to the controls. Moreover, we verified (Fig. 18 B and D) that as in macrophages
ABCA1 expression is increased after treatment with 22-R-hydroxycholesterol
(HC) and 9-cis retinoic acid (RA) [138, 139]. Finally, BAEC were treated with
cyclosporin A (CsA), a broad-spectrum multidrug resistance modulator, which
has been reported to trap ABCA1 on the cell surface [140]. We confirmed that in
BAEC as well CsA enhanced ABCA1 cell surface expression (Fig. 18 E).
Results
58
Figure 18: ABCA1 expression, silencing and stimulation in BAEC. ABCA1
expression was evaluated by quantitative RT-PCR (A, B) and western-blotting (C, D,
E). First, endothelial cells were transfected with 100 nM ABCA1 specific siRNA or
not coding siRNA (A, C). Second, 22-R hydroxycholesterol (HC) and 9-cis retinoic
acid (RA), 10 µM each, were used to stimulate the expression of ABCA1 (B, D).
Third, treating endothelial cells with 20 µM cyclosporin A (CsA) increased the
amount of ABCA1 expressed on the cell surface (E), as measured after biotinylation
of the cell surface proteins and streptavidin pull down.
Results
59
The interventions previously described were used to evaluate the role of ABCA1
in apoA-I binding and cell association. First, apoA-I binding and cell association
to BAEC were enhanced when ABCA1 expression was stimulated by a mixture
of HC and RA. (Fig. 19). Second, we used CsA, which increases the expression
of ABCA1 on the cell surface and inhibits apoA-I uptake in macrophages [140].
After 4 h of treatment with CsA, apoA-I binding and cell association to BAEC
were increased (Fig. 19), consistent with the result that CsA traps ABCA1 on
the cell surface. Third, ABCA1 expression was diminished by RNA interference.
After ABCA1 silencing, apoA-I binding and cell association were significantly
lower. These results indicate that ABCA1 critically modulates apoA-I binding
and cell association. Therefore, the implication of ABCA1 in apoA-I
internalisation and transcytosis was further studied.
Results
60
Figure 19: Role of ABCA1 in apoA-I binding (A) and cell association (B). Diverse
pharmacological treatments were used to modulate ABCA1 and evaluate its
involvement in apoA-I binding and cell association. First, ABCA1 expression was
stimulated with a mixture 22-R-hydroxycholesterol (HC) and 9-cis-retinoic acid (RA).
Second, ABCA1 was trapped on the cell surface and functionally inhibited with
cyclosporin A (CsA). Third, ABCA1 expression was reduced after transfection of
specific siRNA. All conditions were used to study apoA-I binding (A) and cell
association (B).
Results
61
2.1.2. Role of ABCA1 in ApoA-I Internalisation
Whether ABCA1 is involved in apoA-I internalisation was further investigated,
using treatments previously described. Stimulating ABCA1 expression with HC
combined with RA increased apoA-I uptake. In addition, treating the cells with
CsA reduced apoA-I internalisation in BAEC, as previously reported in
macrophages [140],. Finally, apoA-I uptake was lower in cells transfected with
ABCA1 specific siRNA than in the control cells (Fig. 20). To sum up, altering
ABCA1 expression was modulating apoA-I internalisation.
Figure 20: Role of ABCA1 in apoA-I internalisation in BAEC. ApoA-I uptake was
measured after diverse treatments. First, ABCA1 expression was stimulated with a
mixture of 22R-hydroxycholesterol (HC) and 9-cis retinoic acid (RA). Second,
ABCA1 was inhibited on the cell surface by cyclosporin A (CsA). Thrid, ABCA1
expression was reduced by RNA interference.
Results
62
2.1.3. Role of ABCA1 in ApoA-I Transport
Finally, we characterised the role of ABCA1 in apoA-I transcytosis. Treating
BAEC with CsA, added either in the apical compartment, in the basolateral
compartment or in both compartments, led to a reduced apoA-I transport, by
more than 50% (Fig. 21). Moreover, when ABCA1 expression was lowered by
RNA interference, we observed a 70% reduction in apoA-I transport (Fig. 21). In
addition, apoA-I cell association was measured in parallel to apoA-I transport
after CsA treatment. As in the previous cell association assay (Fig. 19), we
found that CsA introduced either into the apical compartment or into both
compartments increased apoA-I cell association. Interestingly, apoA-I cell
association remained unchanged when CsA was added into the basolateral
compartment, although apoA-I transport was reduced by this treatment. This
result provides a hint that ABCA1 may not only modulate apoA-I uptake but may
also regulate apoA-I trafficking and even apoA-I secretion.
Results
63
Figure 21: Role of ABCA1 in apoA-I transport through a monolayer of BAEC.
ABCA1 expression was reduced after transfecting specific siRNA. Alternatively,
ABCA1 was inhibited on the cell surface using cyclosporin A (CsA), which was
added either in the apical compartment or in the basolateral compartment or in both
compartments. (A) ApoA-I transport was evaluated after both interventions. (B)
ApoA-I cell association was also measured in the transwell system after CsA
treatments.
Results
64
2.2. Role of SR-BI in ApoA-I Binding and Cell Association
SR-BI is a well characterised HDL binding protein. We investigated if it plays a
role in apoA-I binding and apoA-I cell association in endothelial cells. At first,
SR-BI expression was studied. SR-BI was expressed in BAEC as evaluated by
RT-PCR (Fig. 22 A) and western-blotting (Fig. 22 B). In addition, it was possible
to reduce its expression after transfecting SR-BI specific siRNA. SR-BI
expression was diminished by 90% on the RNA level and by more than 50% on
the protein level. As controls, we measured the expression of SR-BI in not
transfected cells and in cells transfected with not coding siRNA.
ApoA-I binding (4°C) and cell association (37°C) were as high in cells
transfected with SR-BI specific siRNA as in cells transfected with not coding
siRNA. However, the binding of HDL (known ligand of SR-BI [141]) was
significantly reduced when SR-BI expression was diminished (Fig. 23). Thus,
Figure 22: SR-BI expression and silencing in BAEC. SR-BI expression levels were
evaluated by quantitative RT-PCR (A) and western-blotting (B). In order to reduce
SR-BI expression, specific siRNA (100 nM) were transfected with lipofectamine.
SR-BI RNA and protein levels were measured 48h after transfection.
Results
65
the role of SR-BI in apoA-I internalisation or apoA-I transport was not further
studied.
Figure 23: Role of SR-BI in apoA-I binding and cell association. ApoA-I binding (A)
and apoA-I cell association (B) were measured after reducing the expression of SR-
BI by RNA interference. As a functional control, HDL binding (C) was also evaluated
after silencing SR-BI.
Results
66
2.3. Role of Cell Surface F0F1 ATPase in ApoA-I Transcytosis
2.3.1. Role of Cell Surface β-ATPase in ApoA-I Binding
At first, the presence of the beta chain of the F0F1 ATPase (β-ATPase) on the
surface of endothelial cells was verified, after cell surface biotinylation and
streptavidin pull-down (Fig. 24). Only biotinylated β-ATPase could be pulled
down and intracellular proteins such as GAPDH were not biotinylated.
The expression of β-ATPase was reduced using RNA interference by 90% on
the RNA level and by about 50% on the protein level (Fig. 25). Intracellular ATP
levels were measured and found unchanged after transfection of siRNA specific
for β-ATPase (Fig. 25). In contrast, in cells transfected with siRNA specific for β-
ATPase the binding of apoA-I was twice as low as in not coding siRNA
transfected cells and in not transfected cells (Fig. 26).
Figure 24: Cell surface expression of β-ATPase. The cell surface expression of β-
ATPase was assessed after biotinylation of the cell surface proteins at 4°C. We
verified that GAPDH, an intracellular protein, was not biotinylated and could not be
pulled down. 10 µg from the supernatant and 60 µg of the beads were loaded on a
SDS-PAGE.
Results
67
Figure 25: Expression and silencing of β-ATPase in BAEC. β-ATPase expression
was assessed by RT-PCR (A) and western blotting (B) after tansfecting β-ATPase
specific siRNA. The intracellular concentrations of ATP (C) were also measured
after reducing the expression of β-ATPase in the presence and in the absence of 5
µg/mL apoA-I.
Results
68
Figure 26: Role of β-ATPase in apoA-I binding to BAEC. ApoA-I binding was
evaluated after transfecting BAEC with β-ATPase specific siRNA.
Results
69
2.3.2. Role of Cell Surface F0F1 ATPase in ApoA-I Internalisation
The role of β-ATPase in apoA-I internalisation was evaluated after transfection
of specific siRNA and treatment with the inhibitor IF1 or an anti β-ATPase
antibody (Fig. 27). ApoA-I internalisation in BAEC was reduced by 50% after
transfecting β-ATPase specific siRNA as compared to the controls. Moreover,
the cells were treated with IF1, which binds to the β-subunit of F1-ATPase in a
pH dependent process and inhibits the ATPase activity [142, 143]. IF1 inhibited
apoA-I internalisation by about 60%. Finally, apoA-I internalisation was reduced
in the presence of an anti β-ATPase antibody.
Figure 27: Role of β-ATPase in apoA-I internalisation in BAEC. ApoA-I
internalisation was measured in cells transfected with β-ATPase specific siRNA.
ApoA-I internalisation was also evaluated after treating the cells with the inhibitor IF1
and with an anti β-ATPase antibody.
Results
70
2.3.3. Role of Cell Surface F0F1 ATPase in ApoA-I Transport
The treatments used to study the role of β-ATPase in apoA-I internalisation
were also applied to evaluate the involvement of β-ATPase in apoA-I transport.
We found that silencing β-ATPase drastically reduces (by about 80%) apoA-I
transport through a monolayer of BAEC (Fig. 28). Moreover, when IF1 was
present in the assay medium apoA-I transport was diminished by 50%. These
results indicate that cell surface F0F1 ATPase modulates not only apoA-I binding
and internalisation but also apoA-I transcytosis.
Figure 28: Role of β-ATPase in apoA-I transport in BAEC. ApoA-I transport through
through a monolayer of BAEC was measured after silencing β-ATPase with specific
siRNA and in the presence of the inhibitor IF1.
Results
71
2.3.4. Effect of Extracellular Nucleotides on ApoA-I Internalisation
In mitonchondria, F0F1 ATPase catalysed both the synthesis and the hydrolysis
of ATP. Therefore, we tested the effect of adding extracellularly ADP or ATP on
apoA-I internalisation. Cell association and internalisation of apoA-I were both
slightly (20%) increased in the presence of 100 nM ATP and much more
augmented (50%) in the presence of 100 nM ADP (Fig. 29). This indicates that
apoA-I uptake is stimulated by extracellular ADP rather than by extracellular
ATP.
Figure 29: Effect of extracellular ADP and ATP on apoA-I cell association and
internalisation. BAEC were preincubated with 100 nM ADP or ATP for 10 min prior
apoA-I internalisation assay.
Results
72
2.3.5. Cell Surface F0F1 ATPase Activity
F0F1 ATPase activity on the cell surface was assessed by measuring
extracellular ATP concentrations in the presence and in the absence of apoA-I
and after silencing β-ATPase. First, in not transfected cells (none, Fig. 31), the
extracellular ATP concentration is reduced by 50% when the cells were
preincubated with apoA-I for 30 min. This indicates that apoA-I stimulates the
extracellular hydrolysis of ATP. Moreover, in the absence of apoA-I, ATP
accumulated extracellularly when β-ATPase was silenced (Fig. 30, black bars),
suggesting that F0F1 ATPase ihydrolyses ATP on the surface of endothelial
cells. Finally, the apoA-I specific ATP hydrolysis was reduced (by about 50%)
when β-ATPase expression was diminished by RNA interference in comparison
to the controls. In a few words, on the surface of endothelial cells F0F1 ATPase
seems to hydrolyse ATP in an apoA-I induced manner.
Figure 30: F0F1 ATPase activity with and without apoA-I. BAEC were transfected
with 100nM siRNA specific for β-ATPase. The extracellular concentration of ATP
was measured after 30 min incubation with or without 5 µg/mL apoA-I.
Results
73
3. Which Pathway is implicated in ApoA-I Transcytosis?
Finally, in order to clarify the route of apoA-I transport, we focussed on caveolin-
1 and clathrin mediated pathways.
3.1. Role of Caveolin-1 in ApoA-I Transcytosis
The expression of caveolin-1, the major structural protein of caveolae, was
verified by RT-PCR and western blotting. Moreover, it was possible to reduce
the expression of caveolin-1 by 90% on the RNA level and by more than 60%
on the protein level by transfecting specific siRNA (Fig. 31).
ApoA-I internalisation and transport were studied in BAEC transfected with
siRNA specific for caveolin-1. ApoA-I internalisation and apoA-I transport were
reduced, by 50% and by 80% respectively, when caveolin-1 was silenced (Fig.
32 A). This intervention, however, did not affect LDL degradation (Fig. 32 B).
Figure 31: Caveolin-1 expression and silencing in BAEC. After transfecting 100 nM
siRNA specific for caveolin-1 with lipofectamine, caveolin expression levels were
assessed by quantitative RT-PCR (A) and western blotting (B).
Results
74
Figure 32: Role of caveolin-1 in apoA-I internalisation and transport. ApoA-I
internalisation and transport (A) were assessed in BAEC transfected with 100 nM
caveolin-1 specific siRNA. As a control, LDL degradation (B) was also measured
after reducing the expression of caveolin-1 by RNA interference.
Results
75
Moreover, Triton X-100 insoluble fractions were isolated by flotation in a
sucrose gradient. The fractions were analysed by western blotting and probed
with antibodies against caveolin-1 and clathrin heavy chain (Fig. 33 A and B).
The Triton X-100 insoluble rafts (fractions 3 and 4) were positive for caveolin-1
but negative for clathrin heavy chain. In addition, all fractions were directly
loaded onto a nitrocellulose membrane, which was probed with 125I-apoA-I and
exposed to a phosphor screen (Fig. 33 C). ApoA-I was binding essentially to the
fractions containing the caveolin-1 enriched rafts, i.e. fractions 3 and 4.
We previously reported that ABCA1 and β-ATPase modulate apoA-I binding.
Thus, the expression of ABCA1 and β-ATPase was evaluated in Triton X-100
soluble and insoluble fractions (Fig. 34). Both proteins seemed to be expressed
in caveolin-1 enriched rafts (Fig. 35).
Figure 33: ApoA-I binding to caveolin-1 enriched rafts. Caveolae enriched
membranes were prepared after lysis in Triton X-100 at 4°C and flotation in sucrose
gradient. The fractions were analysed by western blotting for caveolin-1 and clathrin
(A and B). Moreover, the fractions were also directly loaded on a nitrocellulose
membrane, which was probed with 125I-apoA-I (C).
Results
76
Figure 34: ABCA1 and β-ATPase in caveolin-1 enriched rafts. The expression of
ABCA1 and β-ATPase was measured by western blotting in Triton X-100 soluble
(pooled fractions 11 and 12) and insoluble rafts (pooled fraction 3-4). 18 µg total
proteins were loaded in all wells.
Results
77
3.2. Role of Clathrin in ApoA-I Internalisation
Clathrin heavy chain (HC) was expressed in endothelial cells and its expression
could be reduced by 90% on the RNA level and by about 50% on the protein
level using RNA interference (Fig. 35).
Figure 35: Clathrin heavy chain (HC) expression and silencing in BAEC. 100 nM
siRNA specific for clathrin HC were transfected with lipofectamine in BAEC. Clathrin
expression levels were checked 48 h after transfection by RT-PCR (A) and western
blotting (B).
Results
78
After transfecting siRNA coding for clathrin HC, no significant reduction in apoA-
I internalisation was observed (Fig. 36). LDL are taken up via clathrin coated
pits and are degraded after internalisation [144]. Therefore, LDL degradation
was evaluated after silencing clathrin HC and was found diminished by about
50%. These results suggest that most apoA-I is not internalised in a clathrin
dependent manner.
Figure 36: Role of clathrin heavy chain (HC) in apoA-I internalisation (A) and LDL
degradation (B). Clathrin HC specific siRNA were transfected in BAEC. 48 h after
transfection apoA-I internalisation (A) and LDL degradation (B) were assessed.
Discussion
79
DISCUSSION
1. ApoA-I Interaction with Aortic Endothelial Cells
Most anti-atherogenic properties of HDL are exerted in the arterial wall.
Therefore, HDL and their main protein constituent, apoA-I, must leave the
plasma compartment and cross the endothelium [13, 145, 146]. However, only
the transcytosis of intact HDL particles through endothelial cells isolated either
from fat tissue [127] or brain [126] but not the passage of apoA-I through
endothelial cells from large arteries was previously investigated. With respect to
the pathogenesis of atherosclerosis, we believe that endothelial cells from large
arteries rather than from capillaries are the mandatory model for two main
reasons. First, the lipoproteins invade the arterial wall from the luminal side and
form the initial fatty streaks in the intima rather than in the adventitia [147].
Second, endothelial cells of different vascular origin differ qualitatively and
quantitatively by the abundance of fenestrae, clefts, junctions and receptors
which all have a strong impact on the permeability of macromolecules [148-
150]. Furthermore, the transendothelial transport of lipid free apoA-I rather than
lipidated HDL particles was evaluated because in both the intimal fluid of
arteries and the lymph the relative concentration of lipid poor HDL precursors,
pre-β−HDL, is higher than that of mature and lipidated large HDL [17-19, 151].
These lipid free or lipid poor HDL precursors are continuously generated in the
plasma compartment during lipolysis of triglyceride-rich lipoproteins by
lipoprotein lipase and interconversion of HDL subclasses by lipid transfer
proteins, hepatic lipase and endothelial lipase [13, 146, 152]. They release
phospholipids and cholesterol from hepatic and non-hepatic cells including
macrophages and smooth muscle cells from the arterial wall for the reverse
transport to the liver [5, 13, 146, 152-154].
In this study, endothelial cells bound lipid free apoA-I at 4°C with high affinity
(Kd = 67 ± 20 nM) and in a saturable manner (Bmax = 1.5 ± 0.2 pmol / mg).
This result corresponds to the presence of 2.0 ± 0.3 105 binding sites per cell.
Besides, apoA-I binding was competable with excess unlabeled apoA-I but not
Discussion
80
with excess BSA, thus considered specific (Fig. 10). It is also important to note
that the equilibrium analysis allowed us to determine the presence of only one
kind of binding sites. However, we identified two proteins modulating apoA-I
binding, ABCA1 and β-ATPase. Two major explanations for this contradiction
can be envisaged. First, if both proteins are apoA-I receptors, they might have
very similar affinities for apoA-I. Second, it is still controversially discussed
whether ABCA1 binds directly apoA-I or modify the lipid distribution at the
plasma membrane facilitating apoA-I docking [155, 156].
Savion and Gamliel [157] previously showed that lipid free apoA-I specifically
associates with BAEC at 37°C. A proportion of the associated apoA-I was
resistant to trypsinisation and hence assumed to be internalised. We verified
this assumption directly in biotinylation experiments. About 20% of apoA-I
associated with endothelial cells was internalised within 2 hours (Fig. 13 B). In
addition, less than 3% of cell associated apoA-I (or less than 15% of
internalised apoA-I) was degraded during 4 hours incubations (Fig.15 A).
Furthermore, using a Transwell culture system, we found that presumably
polarised arterial endothelial cells transport apoA-I from the apical to the
basolateral compartment (Fig. 16). In the first 30 min about 50% of apoA-I
associated with endothelial cells is transported, which suggests that apoA-I
transcytosis is a major pathway. In order to further study the nature of apoA-I
transcytosis we used vesicular transport inhibitors such as dansylcadaverine,
filipin or N-ethylenimid. Unfortunately, due to the cytotoxixity of these inhibitors
no conclusive results were obtained from these experiments.
We believe that apoA-I transcytosis is not artifactual for four reasons. First, in
cells cultured in a Transwell system, apoA-I binding was threefold stronger on
the apical side as compared to the basolateral side (Fig. 11). Our results do not
allow to conclude whether the apoA-I binding sites on the apical and basolateral
cell membranes are of different nature and function. In addition, it cannot be
ruled out that the difference in binding capacity is due, at least in part, to steric
interference of the membrane. Second, the cell surface biotinylation
experiments clearly demonstrated that apoA-I is internalised in a specific and
saturable manner (Fig. 13). It was also verified that the majority of the
Discussion
81
internalised material remained intact (Fig. 15). In addition, apoA-I internalisation
was verified by fluorescence microscopy studies of BAEC incubated with alexa
488-conjugated apoA-I (Fig. 14). ApoA-I was visualised in vesicles which
colocalised in part with the early endosome marker EEA1 and with transferrin.
We also previously reported that gold-labelled apoA-I is internalised in
intracellular vesicles [158]. These results point to a transcellular trafficking of
apoA-I. However, the nature of the vesicles containing apoA-I needs further
investigation. Third, apoA-I added to the apical compartment of a two-
compartment system was transported to the basolateral compartment as an
intact protein (Fig. 17). This transport was competable and diminished at 16°C,
a temperature which prevents vesicle fusion with the plasma membrane (Fig.
16). One may argue that the fall in temperature reduces filtration in response to
increased fluid viscosity. However, we found a greater temperature dependent
reduction in specific transport than in total transport. This supports a cell
dependent transport. Fourth, the transport changed the originally pre-β-mobile
lipid free apoA-I into an α-mobile particle without changing the integrity to apoA-
I (Fig. 17). This indicates a non-covalent modification, such as addition of acidic
phospholipids of apoA-I. In addition the charge modification was abolished by
adding excess of cold apoA-I into the donor compartment and at reduced
temperature (Fig. 17). This clearly indicates that the modification step was cell
and energy dependent. However, our experimental set-up does not permit to
determine whether the modification took place at the cell surface or
intracellularly during transport.
In conclusion, endothelial cells from bovine aorta bind and internalise lipid-free
apoA-I in a specific and saturable manner. In addition, we provided the first
evidence that apoA-I undergoes transcytosis through arterial endothelial cells
giving rise to lipidated particles. This pathway may be an important rate limiting
factor for the antiatherogenicity of HDL because apoA-I and HDL mediate many
antiatherogenic functions within the arterial wall rather than in the plasma
compartment and apoA-I must therefore cross the endothelial barrier.
Discussion
82
2. Which Proteins mediate ApoA-I Transcytosis?
Next, we aimed to unravel receptor(s) and/or transporter(s) which modulate
apoA-I binding, uptake and transport in arterial endothelial cells. A candidate
based approach was choosen: known HDL/apoA-I binding proteins (ABCA1,
SR-BI and β-ATPase) were tested for their ability to bind, internalise and
transport apoA-I in endothelial cells.
2.1. Role of ABCA1 in ApoA-I Transport
Mutations in ABCA1 have been identified as the cause of Tangier disease, a
disease characterised by extremely low HDL plasma levels and the
accumulation of lipid loaded macrophages in several organs [40-44]. In the liver,
ABCA1 mediates a rate-limiting step in the formation of HDL [46, 47]. In
macrophages, ABCA1 prevents the excessive accumulation of lipids and
thereby protects the arteries from developing atherosclerotic lesions [31].
Besides, ABCA1 activity leads, via an unknown mechanism, to the efflux of
phospholipids and cholesterol onto apoA-I [44, 45].
At first, we verified that ABCA1 is expressed in bovine aortic endothelial cells by
RT-PCR and western blotting (Fig. 18). The expression of ABCA1 in human
umbilical vein endothelial cells and human aortic endothelial cells has already
been shown [159, 160]. ABCA1 is thought to mediate phospholipid and
cholesterol efflux onto apoA-I. Endothelial cells, however, do not efflux
phospholipids and cholesterol onto apoA-I [154], which leads to the question of
the function of ABCA1 in endothelial cells.
The role of ABCA1 in apoA-I binding (4°C) and cell association (37°C) was
assessed after pharmacological treatments and siRNA mediated silencing.
First, we verified that, as in macrophages [138, 139], the expression of ABCA1
is up regulated in endothelial cells incubated with a mixture of 22-R-
hydroxycholesterol (HC) and 9-cis retinoic acid (RA) (Fig. 18 B and D). ABCA1
was also trapped on the surface of endothelial cells treated with cyclosporin A
(CsA) (Fig. 18 E), as previously reported in macrophages [140]. After both
Discussion
83
treatments, apoA-I binding and cell association were increased (Fig. 19).
Because these treatments are not specifically modulating ABCA1, the previous
results provided evidence that an ABC transporter is involved in apoA-I binding
and cell association. Furthermore, when ABCA1 expression was specifically
reduced by RNA interference, apoA-I binding and cell association were lowered.
In other words, ABCA1 is very likely the ABC transporter which was modulated
by the two first treatments, ultimately leading to increased apoA-I binding and
cell association. The result that ABCA1 modulates apoA-I binding coincides with
a previous report. In deed, overexpression of ABCA1 in macrophages increases
apoA-I binding to the cell surface [155]. However, it is still controversially
discussed whether ABCA1 binds directly apoA-I or whether it modifies the lipid
distribution at the plasma membrane facilitating apoA-I docking [155, 156].
Hence, it is still unclear whether ABCA1 can be considered as a receptor for
apoA-I.
The role of ABCA1 in apoA-I internalisation was further evaluated. Similarly to
apoA-I binding and cell association, apoA-I internalisation was increased after
stimulating ABCA1 with HC and RA (Fig. 20). In contrast, CsA inhibited apoA-I
uptake. Finally, when ABCA1 expression was reduced by RNA interference,
apoA-I uptake was also diminished. These results are consistent with data
obtained previously in macrophages and fibroblasts. In macrophages, CsA
inhibits apoA-I uptake and resecretion by trapping ABCA1 on the cell surface
[140]. In fibroblasts, apoA-I colocalises with ABCA1 in endosomes [161, 162].
Thus, it seems that ABCA1 plays a critical role in apoA-I internalisation not only
in macrophages and fibroblasts but also in endothelial cells. In order to support
these data, the colocalisation of apoA-I and ABCA1 was analysed by
fluorescence microscopy. Unfortunately, all ABCA1 antibodies tested produced
unspecific patterns. In addition, the expression of an ABCA1-GFP fusion protein
resulted in such a high background in the endoplasmatic reticulum that no
conclusion could be drawn from the experiments.
Besides, ABCA1 also critically modulated apoA-I transcytosis. Indeed,
treatment with CsA and knock-down of ABCA1 expression reduced apoA-I
transport (Fig. 21). This might be due to the role of ABCA1 in apoA-I
Discussion
84
internalisation although we cannot exclude that ABCA1 might also play a role in
apoA-I trafficking and resecretion. In deed, when added into the basolateral
compartment CsA inhibited apoA-I transport from the apical to the basolateral
compartment (Fig. 21 A) but apoA-I cell association remained unchanged (Fig.
21 B). Therefore, CsA added in the basolateral compartment did not inhibit
ABCA1 on the apical cell surface. Nevertheless, it is possible that CsA was
internalised and inhibited ABCA1 intracellular trafficking without impairing the
function of ABCA1 on the apical surface. In other words, CsA at the basolateral
side or intracellularly might inhibit apoA-I transcytosis not only by reducing
apoA-I uptake but also by affecting apoA-I intracellular trafficking or even
resecretion. However, CsA is not only modulating ABCA1 activity but inhibits
also MDR1/ABCB1 [163] and calcineurin/protein phosphatase 2B (PP2B) [164].
In order to verify that the effect of CsA on apoA-I trafficking is not due to the
inhibition of PP2B, the experiment could be repeated in the presence of PP2B
specific inhibitors such as deltamethrin or FK506. In addition, deletion of the
cytoplasmic PEST sequence in ABCA1 inhibits ABCA1 trafficking but not
cholesterol efflux from the cell surface [165]. In order to assess whether ABCA1
is implicated in apoA-I trafficking, apoA-I internalisation and transcytosis could
be studied in cells expressing this deletion mutant (ABCA-dPEST).
ABCA1 is known to mediate phospholipid and cholesterol efflux onto apoA-I but
it is also modulating transferrin and dextran uptake [49], and apoptotic cells
engulfment [50]. We found that ABCA1 modulates apoA-I internalisation and
transcytosis in endothelial cells. Taken together, these results are raising the
major question of the intrinsic activity of ABCA1. It has been suggested that
ABCA1 would control the outward translocation of phosphadidylserine in a
calcium-induced manner [166]. Thereby, ABCA1 would induce an outward
bending of the membrane which would explain its inhibitory role in transferrin
and dextran uptake [52, 53]. On the contrary, our results indicate that the
caveolin-1-mediated internalisation of apoA-I would be facilitated by the putative
phosphadidylserine translocase function of ABCA1. However, it remains
challenging to understand how the phosphatidylserine export activity of ABCA1
may improve lipid efflux. Membrane domains, which preferentially bind apoA-I,
might be created upon translocation of phosphatidylserine to the outer leaflet.
Discussion
85
Alternatively, lipid efflux onto apoA-I has been suggested to occur through a
retroendocytosis process: apoA-I would in that case be internalised, interact
with intracellular lipid pools and be resecreted as lipidated particle [167, 168].
Thus, our finding that ABCA1 modulates apoA-I transport through endothelial
cells might help understanding the still unresolved mechanism by which ABCA1
mediate lipid efflux in macrophages.
Finally, according to the current opinion, ABCA1 helps protecting against the
development of atherosclerosis by two major mechanisms. It catalyses a
limiting step in the biogenesis of HDL in the liver and it mediates cholesterol
efflux from macrophages [28]. Many of the anti-atherogenic effects of apoA-I
and HDL are to be executed within the vascular wall. Therefore, by modulating
the transport of apoA-I through the endothelium into the arterial wall, ABCA1
may exert an additional atheroprotective activity. In this context, it is important
to note that several mutations in ABCA1 were associated with cardiovascular
risk independently of HDL cholesterol [169].
Discussion
86
2.2. Role of SR-BI in ApoA-I Transport
Initially, we verified that SR-BI is expressed in bovine aortic endothelial cells by
RT-PCR and western blotting (Fig. 22). It was already known that SR-BI is
expressed in endothelial cells, where it mediates the stimulation of endothelial
nitric oxid synthase (eNOS) by HDL [61]. SR-BI expression could be diminished
by RNA interference (Fig. 22). After reducing SR-BI expression, apoA-I binding
(4°C) and cell association (37°C) to endothelial cells did not changed (Fig. 23).
By contrast, HDL binding was reduced. SR-BI has already been reported to
bind lipidated apoA-I and HDL rather than lipid free apoA-I [170, 171]. Since our
data were consistent with the literature, we did not investigate further the
implication of SR-BI in apoA-I transcytosis.
Discussion
87
2.3. Role of F0F1 ATPase in ApoA-I Transport
F0F1 ATPase, which is essentially encoded by the nuclear genome, is the
principal ATP synthesis complex in mitochondria. It consists of a catalytic
domain F1 and a transmembrane domain F0. Surprisingly, F0F1 ATPase, like
other mitochondrial proteins, has been shown to be expressed on the surface of
diverse cells [69]. It has been found on the surface of endothelial cells and
hepatocytes, and has been shown to be active in this ectopic location [65, 67].
In hepatocytes, the beta chain of F0F1 ATPase (β-ATPase), which belongs to
the F1 domain, has been characterised as an apoA-I receptor, which triggers
the internalisation of HDL [67]. For these reasons, cell surface F0F1 ATPase
was considered as a candidate receptor, which might modulate apoA-I binding
and transcytosis in endothelial cells.
At first, the presence of β-ATPase at the plasma membrane of endothelial cells
was verified after cell surface biotinylation and streptavidin pull-down (Fig. 24).
Previously, components of F0F1 ATPase (subunits α, β, γ, b, d, e, F6 and
OSCP) have been observed by immunofluorescence or detected by
biotinylation and cell fractionation on the surface of tumor cell lines [64, 68],
adipocytes [66], hepatocytes [67], keratinocytes and endothelial cells [65].
However, whether all subunits of F0F1 ATPase are present on the cell surface
and whether the complex has the same structure as in mitochondria remains to
be addressed. Although the mechanism leading to ectopic expression is still
unknown other mitochondrial-matrix proteins, including fatty acid binding protein
(FABP), HSP60 and P32, are found at extramitochondrial sites [69].
Reducing β-ATPase expression diminished significantly apoA-I binding (Fig.
26), internalisation (Fig. 27) and transport (Fig. 28). However, silencing total β-
ATPase had no detectable consequences on intracellular ATP levels (Fig. 25).
Therefore, we estimated that the reduction in β-ATPase expression did not
induce an energetic stress that could explain the previous results. In addition,
the inhibitor IF1, which binds the F1 domain of F0F1 ATPase and inhibits ATP
hydrolysis [143], also reduced apoA-I internalisation (Fig. 27) and transport (Fig
Discussion
88
28). Besides, an antibody recognising the β−chain of F0F1 ATPase inhibited
apoA-I internalisation (Fig. 27). These results are supporting the concept that
ectopic β-ATPase acts as a receptor for apoA-I on the surface of endothelial
cells. In hepatocytes, comparable results were obtained. In these cells, apoA-I
binding to the β-chain of F0F1 ATPase was shown to trigger the internalisation of
HDL [67].
Mitochondrial F0F1 ATPase efficiently catalyses both ATP synthesis and ATP
hydrolysis. Several studies demonstrated that cell surface F0F1 ATPase is also
active in both ATP synthesis [66, 76] and ATP hydrolysis [67, 172].
Interestingly, we found that ADP rather than ATP stimulated the internalisation
of apoA-I in endothelial cells (Fig. 29). This suggested that F0F1 ATPase
hydrolyses ATP and that the ADP thus produced stimulates apoA-I
internalisation. To test this hypothesis, extracellular ATP concentrations were
measured in cells transfected with β-ATPase specific siRNA, in the absence
and in the presence of apoA-I. In not transfected cells, the extracellular ATP
concentrations were lower in the presence of apoA-I than in its absence (Fig.
30). Moreover, both in the presence and in the absence of apoA-I, ATP
accumulated extracellularly (Fig. 30) after reducing β-ATPase expression.
These results indicate that apoA-I stimulates the hydrolysis of ATP by F0F1
ATPase on the surface of endothelial cells. The ATP hydrolase activity is the
main feature of the F1 domain [75]. Therefore, our data suggest that a functional
F1 domain is present on the surface of endothelial cells.
In brief, our results indicate that β-ATPase is present on the surface of
endothelial cells where it catalyses the hydrolysis of ATP upon binding of apoA-
I. This process stimulates the internalisation and transcytosis of apoA-I. All our
data converge to the conclusion that on the cell surface F0F1 ATPase modulates
apoA-I transcytosis. However, this is a very surprising result as F0F1 ATPase is
considered as a strickly mitochondrial protein. Thus, it is critical to understand
the mechanism for ectopic expression of β-ATP in order to stregthen our
functional data. However, our findings are not unprecedented. In hepatocytes,
Discussion
89
the stimulation of cell surface F0F1 ATPase by apoA-I was shown to trigger HDL
endocytosis by a mechanism strictly related to the generation of ADP.
In addition, using both a pharmacological approach and RNA interference,
P2Y13 was identified as the main partner in hepatic HDL endocytosis, in
cultured cells as well as in situ in perfused mouse liver [77]. Interestingly, P2Y
receptors on the surface of endothelial cells have been shown to regulate
vascular permeability [81-83]. Therefore, it might be that one of the endothelial
P2Y receptors is stimulated by the ADP produced by the F0F1 ATPase, leading
to the transcytosis of apoA-I via still unidentified pathways.
Finally, F0F1 ATPase subunits have been identified as cell surface receptors for
apparently unrelated ligands implicating the complex in biological events as
divers as angiogenesis, innate immunity and lipoprotein metabolism. In deed,
angiostatin, an endogenous angiogenesis inhibitor, has been shown to bind
F0F1 ATPase on the surface of endothelial cells, thus regultating cell migration
and proliferation [65]. β-ATPase has also been identified as a target for innate
cytotoxicity by natural killer and lymphokine-activated killer cells toward some
tumors [64, 68]. In hepatocytes, apoA-I is binding β-ATPase which triggers the
internalisation of HDL [67]. We found that F0F1 ATPase is modulating the
transcytosis of apoA-I. To sum up, the real function and biological significance
of ectopic F0F1 ATPase remains to be established. Besides, future work will
have to address several important issues before F0F1 ATPase can be accepted
as a cell surface apoA-I receptor: What is the structure of the complex on the
cell surface? What is the mechanism leading to cell surface expression? How
apoA-I binding to β-ATPase results in apoA-I transcytosis?
Discussion
90
3. Which Pathway is implicated in ApoA-I Transcytosis?
The role of caveolin-1 and clathrin heavy chain in apoA-I internalisation and
transport was assessed in endothelial cells. The expression of clathrin heavy
chain was reduced by RNA interference (Fig. 35). Knock-down of clathrin heavy
chain was reducing the degradation of LDL but had no effect on the uptake of
apoA-I (Fig. 36). On the contrary, reducing caveolin-1 expression diminished
apoA-I internalisation and transport but not LDL degradation (Fig. 31).
Moreover, we extracted the membrane fractions which are insoluble in TritonX-
100 at 4°C. These fractions were strongly stained by caveolin-1 antibodies and
apoA-I was preferentially binding these caveolin-1 enriched rafts transferred
onto nitrocellulose membrane (Fig. 33). Thus, it seems that most apoA-I is
internalised in a caveolin-1 dependent manner. Caveolae-internalisation
inhibitors (i.e. filipin, genistein) were used to confirm this result. Unfortunatelly,
they were cytotoxic and no specific effect could be observed. Besides, after 30
min incubation the vesicles containing apoA-I - such as the one observed by
fluorescence microscopy on Fig. 14 - were not stained with caveolin-1 (data not
shown). It is thus very likely that these vesicles were already uncoated at the
time of the observation. In addition, up to 20% of the vesicles colocalised with
the early endosomes markers transferrin and EEA1 (Fig. 14). These findings
seem to disagree with the role of caveolin-1 in apoA-I internalisation. In order to
further understand these contradictive results, it is critical to intensively
characterise the nature of the apoA-I vesicles and study apoA-I intracellular
trafficking.
Although we cannot exclude that a minor part of apoA-I might be taken up via
clathrin coated pits, most of apoA-I seems to be internalised in a caveolae-
mediated pathway. Interestingly, it is rather believed that HDL is internalised by
clathrin coated pits, at least in macrophages, CaCo2 cells and HepG2 cells,
which express low levels of caveolin-1 [168, 173-175]. On the contrary, in
endothelial cells HDL is known to interact with caveolae, thereby stimulating
endothelial nitric oxid synthase [61, 176]. Moreover, SR-BI the best known HDL
receptor is found essentially in caveolae [54]. Finally, caveolae were identified
as the major source of free cholesterol transferred onto apoA-I [177]. To
Discussion
91
conclude, it seems that HDL and apoA-I can undergo internalisation via both
clathrin-coated pits and caveolae, maybe depending on the cell type and on
their fate.
Both ABCA1 and β-ATPase were found in caveolin-1 enriched rafts (Fig. 34). It
has already been reported that F0F1 ATPase is expressed in these rafts [66, 73,
74, 178]. Moreover, in adipocytes the α and β subunits of F0F1 ATPase were
shown to colocalise with cholera toxin [66]. On the contrary, ABCA1 was shown
to be expressed in Lubrol WX insoluble rafts and not in Triton X-100 insoluble
rafts in macrophages and fibroblasts [179, 180]. We found that in endothelial
cells ABCA1 is expressed in Triton X-100 insoluble rafts. It would be neat to
confirm the localisation of ABCA1 and β-ATPase in caveolae using confocal
fluorescence microscopy. This result provides, however, the first hint that
ABCA1, F0F1 ATPase and caveolin-1 proteins might collaborate to mediate the
internalisation of apoA-I in endothelial cells.
Finally, not only HDL but also LDL [124, 181] were reported to be transcytosed
through the endothelium in a caveolae-dependent fashion. In the apoE deficient
mice, the ablation of caveolin-1 confers protection against atherosclerosis,
indicating that at least one early event involved in the development of
atherosclerosis is impaired in the caveolin-1 knockout mice [182]. Because
caveolin-1 seems to modulate the transport of both pro- and anti-atherogenic
lipoproteins, it might be a new critical target to interfere with the development of
atherosclerosis.
Outlook
93
OUTLOOK
The previous discussion revealed several issues that are to be addressed in the
future:
1. What is the intrinsic function of ABCA1? Notably, is ABCA1 directly
involved in apoA-I trafficking and resecretion?
2. Which subunits of F0F1 ATPase are present on the cell surface and how
is the complex organised?
3. Which mechanism leads to the ectopic expression of F0F1 ATPase?
4. What are the events, downstream from F0F1 ATPase, which are
modulating apoA-I transcytosis?
5. What is the nature of the vesicles containing apoA-I?
6. How are F0F1 ATPase, ABCA1 and caveolin-1 cooperating to apoA-I
transcytosis?
The last question will be now developed in more details. For this purpose, the
results discussed previously and data reported by other groups have been
integrated in a model that may be regarded as a working hypothesis (Fig. 37).
We provided evidence that, upon binding of apoA-I, F0F1 ATPase hydrolyses
ATP on the surface of endothelial cells, thus controlling apoA-I internalisation
and transcytosis. ABCA1 was also modulating the uptake and the transport of
apoA-I. Both F0F1 ATPase and ABCA1 were found in caveolae rafts, and
caveolin-1 was regulating apoA-I internalisation and transcytosis.
We propose that the ADP produced by F0F1 ATPase stimulates its cognate P2Y
receptor on the surface of endothelial cells. P2Y receptors are coupled to G-
proteins and regulate notably intracellular Ca2+ concentrations [183].
Interestingly, ABCA1 has been shown to promote Ca2+-induced exposure of
phosphatidylserine [166], which is thought to induce an outward bending of the
membrane [52, 53]. Therefore, we hypothesise that ABCA1 mediates efflux of
phosphatidylserine and creates a local change in the membrane bending, which
may promote the caveolin-1 mediated internalisation of apoA-I and ultimately
Outlook
94
apoA-I transcytosis. Besides, activation of the unidentified P2Y receptor by ADP
may also induce the Src-dependent phosphorylation of dynamin-2 and thereby
the caveolae-mediated internalisation and transport of apoA-I.
In order to validate this model (Fig. 37), several issues should be addressed.
First, simultaneous silencing of ABCA1 and β-ATPase should confirm that both
proteins contribute to the same pathway. Second, the P2Y receptor(s)
modulating apoA-I internalisation in endothelial cells must be identified and its
signalling should be studied, maybe initially focussing on PLC stimulated
release of IP3 and Ca2+ and adenyl cyclase regulation. Third, the
phosphatidylserine translocase activity of ABCA1 must be assessed and its
effect on caveolae mediated internalisation characterised.
Outlook
95
Figure 37: Model for the transcytosis of apoA-I in endothelial cells. F0F1 seems to
hydrolyse ATP upon binding of apoA-I. The ADP produced might bind one P2Y
receptor of endothelial cells, thereby stimulating the release of Ca2+ from
intracellular stores. ABCA1 was also found to modulate apoA-I internalisation and
transport. Interestingly, the phosphatidylserine transferase activity of ABCA1 is
dependent on the intracellular Ca2+ concentration. The translocation of
phosphatidylserine might induce an outward bending of the membrane which
promotes the internalisation and ultimately the transcytosis of apoA-I .
PS: phosphatidylserine, AC: adenyl cyclase, PLC: phospholipase C, cAMP: cyclic
AMP, IP3: inositol-3 phosphate
References
96
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Acknowledgements
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ACKNOWLEDGEMENTS
First of all, I would like to thank my PhD father, Matthias Peter, for following up
the development of this project over the three last years and reviewing my
thesis.
I am grateful to Ari Helenius for his constructive feedback on my project and for
agreeing to be co-examiner of this thesis.
I would also like to express my gratitude to Arnold von Eckardstein for his
continuous support and interest in my work.
I am indebted to my direct supervisor, Lucia Rohrer. She always had time to
answer my questions and to discuss work-related as well as work-unrelated
matters. Her critical feedback stimulated me to do always better.
Many thanks to Martin Hersberger for reviewing my thesis and to Thorsten
Hornemann for his always good advice.
I would like to acknowledge Jonathan D. Smith for the kind gift of the ABCA1-
GFP construct as well as the co-workers of the emz
(Elektronenmikroskopisches Zentrallabor der Universität Zürich). I would like to
thank Silvija and Yú for preparing the precious lipoproteins. I also recognise the
contribution of the technicians from the IKC routine for the many samples they
analysed for me.
Many thanks are given to the members of the lab DOPS4 for the nice working
atmosphere that developed over the years. Special thanks to Iris for her help,
her understanding and her support.
Last but not least, many thanks to my family and friends, who have been a great
source of strength all through this work.
Curriculum Vitae
117
CURRICULUM VITAE
Clara CAVELIER 21/10/1979 2003-2006 University Hospital Zurich (Switzerland) - Clinical Chemistry
Understand one critical atheroprotective property of apoA-I Study apoA-I transcytosis through aortic endothelial cells
ETH Zurich – Biochemistry Department PhD Student, 3rd year
ACADEMIC QUALIFICATIONS 2002 INA P-G (Institut National Agronomique de Paris-Grignon),
France's leading engineering school for life sciences Master’s degree, specialisation Biotechnology and Biochemistry
1997 Baccalauréat in Sciences with honours (equivalent to A-Level) WORK EXPERIENCE 2002 Feb-Sep
Aventis Pharma (France), Process Development - Biotechnology Purify, identify and characterise the unusual coenzyme of an oxidoreductase
2001 Jun-Aug
Hospital of the University of Washington (USA), Pediatrics Investigate the bridging function of hepatic lipase (a critical enzyme in the metabolism of circulating lipoproteins)
2001 Apr-Jun
Objectif Maths (France) part-time teacher Teaching of Mathematics, Physics, Chemistry and Biology
2000 Apr-Mai
French mixed family farm (France) Farm work and economic analysis of the farm
Curriculum Vitae
118
SCIENTIFIC COMMUNICATIONS 2006 Presentation at the 20th Jahrestagung der Deutschen
Gesellschaft für Arterioskleroseforschung, Germany 2005 Presentation at the 28th Annual Meeting of the European
Lipoprotein Club, Germany 2005 Presentation at the 4th Day of Clinical Research, Switzerland L. Rohrer, C. Cavelier, S. Fuchs, M.A. Schluter, W. Volker, A. von Eckardstein, Binding, internalization and transport of apolipoprotein A-I by vascular endothelial cells, Biochim Biophys Acta 1761 (2006) 186-194. C.Cavelier, I. Lorenzi, L. Rohrer, A. von Eckardstein. Lipid Efflux by the ATP Binding Cassette Transporters ABCA1 and ABCG1, Biochim Biophys Acta. 1761 (2006) 655-666. C.Cavelier, L. Rohrer, A. von Eckardstein, ABCA1 modulates apoA-I transcytosis through aortic endothelial cells, Cric Res (in press). C.Cavelier, L. Rohrer, A. von Eckardstein, Caveolin-1 modulates apoA-I transcytosis through aortic endothelial cells (submitted).
C.Cavelier, L. Rohrer, A. von Eckardstein, The β-chain of cell surface F0F1 ATPase modulates apoA-I transcytosis through aortic endothelial cells (submitted).