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From the
Received for publication, May 25, 2001, and in revised form, September 21, 2001
Sub-endothelial infiltration of monocytes occurs
early in atherogenesis and is facilitated by cell adhesion molecules
that are up-regulated on activated endothelium. Apolipoprotein E (apoE) helps protect against atherosclerosis, in part, because apoE particles secreted by macrophages have local beneficial effects at lesion sites.
Here, we hypothesize that such protection includes anti-inflammatory actions and investigate whether cell-derived apoE can inhibit tumor necrosis factor- Early in atherogenesis circulating monocytes are recruited to the
arterial sub-endothelium where they differentiate into macrophages, ingest cholesterol, and develop into "foam cells" (1). Initially, monocytes adhere to activated endothelium on which up-regulated cell
adhesion molecules (CAMs)1
are displayed, a dynamic process sensitive to inflammatory cytokines, shear stress, and oxidative insults (2). Induction of vascular cell
adhesion molecule-1 (VCAM-1), a member of the immunoglobulin superfamily of CAMs, is increasingly described as the key factor in
monocyte infiltration (3, 4).
Apolipoprotein E (apoE) is a 34-kDa polypeptide synthesized mainly by
liver and helps protect against atherosclerosis, in part by mediating
hepatic clearance of remnant plasma lipoproteins (5). When apoE is
absent or dysfunctional, severe hyperlipidemia and atherosclerosis
ensue, while infusion of apoE or hepatic gene overexpression protect
(6, 7). ApoE is also abundant in atherosclerotic lesions, secreted by
resident cholesterol-loaded macrophages (6). This locally produced apoE
is atheroprotective by contributing to reverse cholesterol transport
(8), inhibiting smooth muscle cell proliferation (9), preventing
oxidation (10), and restricting platelet aggregation (11).
ApoE-deficient mice have elevated VCAM-1 in aortic lesions (3), which
enhances monocyte recruitment and adhesion (12), while apoE expression
in the artery wall reduces early foam cell lesion formation (13). These
findings imply that apoE may influence early inflammatory responses by
suppressing endothelial activation and CAM expression. However, we
found that plasma-purified apoE did not suppress VCAM-1 induction in
cultured human umbilical vein endothelial cells (HUVECs) (14).
Nevertheless, if apoE affects CAM expression and atherogenic events
in vivo, then most likely it will be released at lesion
sites by recruited macrophages. Here, we devise two models to mimic
endothelial exposure to locally synthesized, cell-derived apoE and
show, in both cases, that apoE limits cytokine-mediated VCAM-1
up-regulation in a dose-dependent manner.
Cell Culture--
HUVECs were isolated, characterized and
cultured as before (14). Cells were maintained in M199 media with
Hank's salts containing 3.6 mM glutamine, 100 IU/ml
penicillin, 100 µg/ml streptomycin, 20% (v/v) fetal bovine serum
(FBS) and 25 µg/ml endothelial growth supplement (Sigma). For
experiments, 5% (v/v) FBS was used. Plastic culture ware was precoated
with 1% (w/v) gelatin, and cells were used between passages 1-4.
Chinese hamster ovary (CHO) cells secreting human apoE2, apoE3, or
apoE4 isoforms (CHOapoE2, CHOapoE3, and
CHOapoE4) were produced by stably transfecting CHO cells
lacking the dihydrofolate reductase (DHFR) gene with apoE
expression plasmids (p7055.apoE2/3/4) encoding the selectable
DHFR gene (15). Recombinant CHOapoE cells were
maintained in selection media, Iscove's medium plus 2 mM
glutaMAX and 10% (v/v) dialyzed FBS, while medium for control CHOdhfr
Human monocytes with the apoE2/2 genotype were isolated by
leukapheresis and elutriation and then differentiated into macrophages by 12 days of cultivation in RPMI media supplemented with 20% (v/v) pooled human serum. Macrophages were then converted into foam
cells by 48 h of incubation with 100 µg of protein/ml of acetylated low-density lipoprotein as described previously (16). These
acetylated low-density lipoprotein-loaded monocyte-derived macrophages
(MDM) were incubated with serum-free RPMI for characterization of
secreted apoE.
Measurement of VCAM-1 by Enzyme-linked Immunosorbent Assay
(ELISA)--
Analysis of VCAM-1 in quadruplicate wells by ELISA used
previously described protocols and reagents (14). Briefly, HUVECs in
96-well plates were either exposed to cell-derived apoE by transient
transfection, co-culturing with CHOapoE cells or by
incubation with CHOapoE cell-conditioned media. For the
last 6 h in each experiment tumor necrosis factor- Measurement of VCAM-1 by Flow Cytometry--
Analysis of VCAM-1
by flow cytometry was carried out on confluent HUVECs in 12-well plates
as described previously (14). ApoE-enriched high-density lipoproteins
(HDL-E), from a normal subject homozygous for the Transient Transfection of HUVECs--
ApoE expression plasmids
pCMV.apoE2, pCMV.apoE3, and pCMV.apoE4 were prepared by ligating the
corresponding cDNA into pCMV.0, a mammalian expression vector
originally purchased as pCMV. Co-culture of HUVECs with CHO Cells--
Mixtures of HUVECs and
CHOapoE2 or CHOdhfr Incubation of HUVECs with CHO Cell-conditioned
Media--
CHOapoE2, CHOapoE3,
CHOapoE4, and CHOdhfr Intracellular NO Measurement--
Intracellular NO production
was measured directly in triplicate wells using the cell-permeable
fluorescent indicator 4,5-diaminofluorescein diacetate (DAF-2 DA;
Sigma) (19). Confluent HUVECs in 48 well-plates were incubated with 10 µM DAF-2 DA (0.2% Me2SO) for 1 h at
37 °C. To inhibit NOS activity, wells were preincubated for 2 h
with 100 µM ethyl-ITU prior to addition of DAF-2 DA plus
ethyl-ITU. Monolayers were washed with warm PBS and then incubated with
CHO cell-conditioned media (from either CHOapoE2 or
CHOdhfr Characterization of ApoE Particles--
To characterize apoE
particles secreted from CHOapoE cells and to compare them
with those secreted by MDM, cells were incubated for 24 h with
serum-free media. Media was then concentrated 10-fold in Vivaspin
concentrators (10,000 molecular weight cut off; Vivascience Ltd,
Lincoln, UK), and apoE particles were analyzed for electrophoretic mobility in agarose hydragels (Sebia, Issy-les-Moulineaux, France) by
apoE immunoblotting (8) or for morphology by negative staining electron
microscopy (8). Particles were also separated by FPLC gel filtration on
a Superose 6 column (Amersham Pharmacia Biotech). Fractions (0.5 ml)
were blotted onto nitrocellulose and analyzed for apoE by
immunoblotting and densitometry.
Characterization of ApoE Receptor 2 (ApoER2) in
HUVECs--
Polyadenylated RNA was extracted from HUVECs, and the
full-length open reading frame of apoER2 was amplified by reverse
transcription-polymerase chain reaction to detect splice variants as
described previously (20). To detect protein, HUVECs were labeled for
4 h with L-[35S]methionine, and
solubilized membranes were immunoprecipitated with our anti-peptide
antisera, "anti-apoER2Ins" (20). Precipitated proteins were
reduced, separated by 8% SDS-polyacrylamide gel electrophoresis, and
analyzed by fluorography. Similar analyses were carried out using CHO
cells expressing apoER2 (CHOapoER2) as positive controls.
Statistical Analysis--
This was performed for independent
experiments using GraphPad InStat version 3.01 for Windows 95, choosing
a Student's two-tailed t test, analysis of variance test,
or Pearson correlation, as appropriate. Results are shown as mean ± S.E. and p < 0.05 was considered significant.
Inability of HDL-E to Down-regulate VCAM-1--
Plasma-purified
apoE does not inhibit endothelial CAM expression (14). However, as the
purification process may attenuate apoE biological activity (21), we
studied effects of HDL-E, the minor apoE-containing subclass of bulk
plasma HDL, on VCAM-1 up-regulation. The fluorescein isothiocyanate
fluorescence profiles showed basal VCAM-1 expression was negligible,
with most cell fluorescence values under 10 units (Fig.
1A), similar to
isotype-treated controls or of cells incubated with HDL-E alone (not
shown). After TNF- VCAM-1 Is Down-regulated in HUVECs Transiently Transfected to
Secrete ApoE--
To mimic endothelial exposure to locally secreted
apoE, HUVECs were transfected with apoE expression plasmids. Although
apoE was undetectable in pCMV.0 media, it was readily detected in
pCMV.apoE2/3 transfectants with the accumulated levels of apoE2 and
apoE3 being similar (0.10 ± 0.02 versus 0.12 ± 0.02 µg/ml, respectively, n = 4). Levels of apoE4
were much lower, close to the detection limit of 10-20 ng/ml, possibly
due to enhanced re-uptake by the cells or of increased association with
cell-surface heparan sulfate proteoglycan (HSPG) (16). Variable apoE
handling and retention by HSPG, with isoform-dependence, has been
reported for various cultured cells including neurons, fibroblasts,
CHO, and HepG2 cells (22). However, while HSPG-mediated accumulation of
apoE4 occurs less readily than apoE3 in these cell types, in
macrophages apoE4 has increased surface binding to HSPG (16). Whether
there is differential apoE isoform retention by endothelial cells is unknown but merits further investigation.
HUVEC monolayers transfected with pCMV.0 responded to TNF-
One explanation for VCAM-1 suppression would be an intracellular action
of apoE trafficking in successfully transfected cells, as this
influences several processes (23). However, this possibility is
unlikely to account for down-regulation of up to 70% of VCAM-1; primary endothelial cells are notoriously difficult to transfect and we
estimated our transfection efficiency to be typically ~1% by
treating parallel wells with a plasmid encoding green fluorescent protein (pCMV.GPF; data not shown). Further support against an intracellular action of apoE was obtained by transferring media from
transfected HUVECs containing 0.12 ± 0.03 µg of apoE2/ml to
untransfected cells; induction of VCAM-1 was inhibited by 12.9 ± 2.2% (p < 0.05, Student's t test, data
not shown). Additionally, when HUVECs were co-cultured with
CHOapoE2 cells expressing apoE2, VCAM-1 induction was
down-regulated by 16.1 ± 2.4% (p < 0.05, Student's t test) when the concentration in the media was
0.14 ± 0.01 µg/ml, as compared with incubations with control
CHOdhfr Cell-derived ApoE Has an Inherent Ability to Down-regulate
VCAM-1--
To verify that apoE secreted by CHOapoE cells
inhibits VCAM-1 expression, CHOapoE2/3/4 cell-conditioned
media was added directly to HUVECs. Although VCAM-1 suppression was
less than by pCMV.apoE2/3/4 transfections, induction of VCAM-1
inversely correlated with apoE2 (r = Characterization of ApoE Particles--
Characterization of
CHOapoE cell-derived apoE revealed several similarities to
particles secreted by MDM. Both had a similar heterogeneity by agarose
gel electrophoresis (Fig. 4A).
Particles were spherical and of a similar diameter by negative staining
electron microscopy (mean diameter 14 nm; range 8-20 nm) (Fig.
4B). Additional analyses showed that, for both cells, most
apoE particles had a mean molecular mass of 400 kDa (range 150-1200
kDa; Fig. 4C) with pre- Evidence That Cell-derived ApoE Down-regulates VCAM-1 via the
ApoER2-NO Pathway--
ApoE inhibits platelet aggregation by
stimulating NOS III (endothelial NOS) (11), the NO released elevating
anti-aggregatory cGMP. The initial step was considered to be binding of
apoE by apoER2, a member of the low-density lipoprotein receptor
(LDL-R) family localized to caveolae signaling microdomains within the plasma membrane (24). Upon binding, a signal transduction cascade to
activate NOS was postulated (20, 25). As NO is an inhibitor of VCAM-1
expression (26-28), we examined whether this apoE-NOS pathway might
down-regulate VCAM-1 in endothelial cells. Suppression of VCAM-1
induction in HUVECs by CHOapoE2 cell-conditioned media was
completely blocked by the NOS inhibitor, ethyl-ITU (Fig.
5A), while intracellular cGMP
levels were increased by 46.5 ± 15% (p < 0.05, Student's t test, n = 3), indicating NO
release (Fig. 5B). However, it is unlikely that cGMP itself mediates the action of apoE because the inhibition of VCAM-1 by NO is
not a result of increases in cyclic nucleotides (27, 28). When
intracellular NO production was measured directly, using the
cell-permeable fluorescent indicator DAF-2 DA (18), NO levels increased
62% from basal levels of 100.0 ± 5.6% (in HUVECs exposed to
control-conditioned media) to 161.9 ± 24.2% (p < 0.001, Student's t test, n = 5) during a
2-h incubation with CHOapoE2 cell-conditioned media (Fig.
5C). Moreover, this apoE-induced rise in NO production was
due to activation of NOS since the effect was essentially blocked using
the NOS inhibitor (p < 0.001, Student's t
test, n = 5).
We then confirmed, that apoER2 mRNA was present in HUVECs (29) by
long-range polymerase chain reaction (Fig.
6A). As in platelets and the
megakaryocytic cell line, HEL (20), the predominant transcript (~70%
by densitometry) lacked binding repeats 4-6 (apoER2 This study provides the first evidence that cell-secreted apoE may
be anti-inflammatory by suppressing VCAM-1 induction, most probably
because endothelial cells are stimulated to release NO. By contrast, an
apoE-enriched lipoprotein, HDL-E, did not inhibit VCAM-1 expression,
endorsing the view that plasma apoE does not modulate endothelial CAMs
(14). This was important; previously we used plasma-purified apoE3
complexed to phospholipid (apoE3:DMPC), and, although active in other
systems (11, 30), this did not inhibit VCAM-1 expression (14)
presumably because the purification process (21) or lipid environment
(5) diminished biological potency. Indeed, in a direct comparison of
anti-platelet activity we found that cell-derived apoE3 was 4-fold more
active than apoE3:DMPC (data not shown). As activated endothelium is
exposed to locally secreted apoE from cholesterol-loaded macrophages
within atherosclerotic plaques (6) our findings suggest a new
atheroprotective role for apoE: the restriction of endothelial
activation by down-regulating VCAM-1 induction. This is consistent with
macrophage apoE acting early in lesion development (13).
Mimicking endothelial exposure to macrophage apoE in vitro
is complex; a simple co-culture would expose endothelial cells not only
to apoE but also to diverse macrophage secretory factors, including
inflammatory cytokines (31). To circumvent this problem, we first
transiently transfected HUVECs to expose them to locally synthesized
native apoE, albeit self-secreted; all three common apoE isoforms,
apoE2, apoE3, and apoE4, strongly inhibited VCAM-1 induction even
though media levels of apoE were <200 ng/ml. Moreover, VCAM-1
down-regulation was not solely due to intracellular actions or
autocrine functions of apoE synthesized in successfully transfected cells; cross-incubation studies, adding media from transfected HUVECs
to fresh non-transfected cells and the low efficiency of transfection
(~1%) excluded these possibilities. Rather, and in contrast to HDL-E
or plasma-purified apoE, the potency of apoE reflected its inherent
cell-derived nature. Thus, in other experiments, CHOapoE
cell-conditioned media also down-regulated VCAM-1 induction, correlating with apoE content. Significantly, this cell-derived apoE
was secreted as particles that closely resembled macrophage apoE.
Intriguingly, suppression of VCAM-1 was most marked in transfected
HUVECs when the media content of apoE was 50-75 times lower than in
CHOapoE cell-conditioned media. One explanation is that in
transfected wells entrapment of secreted apoE by endothelial HSPG (32)
may result in high local concentrations at the cell surface and
enhanced biological activity. Indeed, HSPG is abundant on the
cell-surface of cultured endothelial cells, and its production is
rapidly stimulated by apoE (33). Although our transfection studies are
not directly relevant to the situation in vivo, as
endothelium does not synthesize apoE, they do have implications for
gene therapy; transfecting endothelial cells to express apoE may limit
endothelial activation. Indeed, this approach prevents lesion
development in apoE-deficient mice (34).
ApoE activates NOS III in platelets (11) and NOS II in macrophages
(35). Because NO is a potent intracellular messenger and inhibitor of
atherogenesis, in part by suppressing cytokine-induced VCAM-1
expression and reducing monocyte adherence to endothelium (26-28), we
investigated whether apoE might stimulate endothelial NO production.
Supporting this mechanism, the NOS inhibitor, ethyl-ITU, blocked the
inhibitory effect of apoE on VCAM-1 expression, while CHOapoE cell-conditioned media not only increased cGMP, a
surrogate marker for NO release (36), but also increased intracellular
NO levels in a fluorescent assay for direct NO detection. Importantly,
E-selectin, a cytokine-induced endothelial CAM not regulated by NO in
HUVECs (26), was unaffected by apoE; rather apoE selectively inhibits VCAM-1.
Indirect evidence also implicates apoE-mediated release of NO in
down-regulating VCAM-1. In platelets, binding of apoE by its receptor,
apoER2, appears to initiate a signal transduction cascade to
up-regulate NOS (20, 25). As we found apoER2 mRNA and protein in
HUVECs, a similar apoE-apoER2-NOS pathway may function in endothelium
to limit VCAM-1 induction. Indeed, this constitutes the first report of
apoER2 protein being identified in vascular endothelial cells in
non-neuronal tissues. A role for apoER2 is also suggested by the
similar efficacy of apoE2 and apoE3; apoE2 binds poorly to the LDL-R
and LDL-R-related protein, thereby discounting them in inhibiting
VCAM-1, whereas apoER2 and its closest mammalian homolog, the
very-low-density receptor (VLDL-R), bind both isoforms efficiently (5,
20, 37). Also relevant is the proposal that both apoER2 and VLDL-R
initiate tyrosine kinase signaling to modulate neuronal positioning in
brain development (38), a process dependent on neuronal cell adhesion
molecules (39, 40). Since VLDL-R is also present in HUVECs (41), we
cannot exclude coordinate apoE signaling by the two receptors in HUVECs to activate NOS and limit VCAM-1 induction.
In summary, we have shown that cell-derived apoE inhibits
cytokine-induced VCAM-1 expression on endothelial cells and propose that this occurs through activation of NOS to release NO. However, further work will be needed to confirm an apoE-NO link in endothelium and to delineate the steps involved, not least because VCAM-1 can also
be suppressed by NO-independent mechanisms (42).
We thank J. Breslow (The Rockefeller
University, New York) for generously providing human apoE cDNAs and
D. G. Hassall (GlaxoSmithKline, Hertfordshire, UK) for help with flow cytometry.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Supported by British Heart Foundation Ph.D. Studentship
(FS/95051).
¶
Present address: Neurology Center of Excellence for Drug
Discovery, GlaxoSmithKline, New Frontiers Science Park, Third Ave., Harlow, Essex CM19 5AW, UK
**
Supported by the Deutsche Forschungsgemeinschaft (Ec116,3-3).
¶¶
To whom correspondence should be addressed: Dept. of
Medicine, Royal Free and Univ. College Medical School, Univ. College London, Royal Free Campus, Rowland Hill Street, London NW3 2PF, UK.
Tel.: 44-207-4332853; Fax: 44-207-4332852; E-mail:
j.owen@rfc.ucl.ac.uk.
Published, JBC Papers in Press, October 5, 2001, DOI 10.1074/jbc.M104812200
The abbreviations used are:
CAM, cell adhesion
molecule;
VCAM, vascular cell adhesion molecule;
apo, apolipoprotein;
HUVECs, human umbilical vein endothelial cells;
FBS, fetal bovine
serum;
CHO, Chinese hamster ovary;
MDM, monocyte-derived macrophage;
ELISA, enzyme-linked immunosorbent assay;
TNF, tumor necrosis factor;
HDL-E, apoE-rich high-density lipoproteins;
NOS, nitric oxide synthase;
ethyl-ITU, 2-ethyl-2-thiopseudourea;
vWF, von Willebrand factor;
apoER2, apolipoprotein E receptor 2;
HSPG, heparan sulfate
proteoglycan;
NO, nitric oxide;
cGMP, cyclic guanosine monophosphate;
LDL-R, low-density lipoprotein receptor.
Cell-derived Apolipoprotein E (ApoE) Particles Inhibit Vascular
Cell Adhesion Molecule-1 (VCAM-1) Expression in Human Endothelial
Cells*
§,¶,,§§,
,**,
,¶¶
Department of Medicine, Royal Free and
University College Medical School, London NW3 2PF, United Kingdom, the
Institut für Arterioskleroseforschung and Institut
für Klinische Chemie und Laboratoriumsmedizin, Westfälische
Wilhelms-Universität, 48149 Münster, Germany, and the
§§ School of Biological Sciences, Royal Holloway
University of London, Surrey TW20 0EX, United Kingdom
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mediated up-regulation of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells
(HUVECs). Two models were used to mimic endothelial exposure to
macrophage-derived apoE. In the first, HUVECs were transiently transfected to secrete apoE; VCAM-1 induction inversely correlated with
secretion of apoE into the media (r = 
0.76,
p < 0.001). In the second, incubation of HUVECs with
media from recombinant Chinese hamster ovary (CHO) cells expressing
apoE (CHOapoE) also reduced VCAM-1 in a
dose-dependent manner (r = 0.70,
p < 0.001). Characterization of CHOapoE
cell-derived apoE revealed several similarities to apoE particles secreted by human blood monocyte-derived macrophages. The suppression of endothelial activation by apoE most likely occurs via stimulation of
endothelial nitric oxide synthase; apoE increased levels of intracellular nitric oxide and its surrogate marker, cyclic
guanosine monophosphate, while the nitric oxide synthase inhibitor,
ethyl-isothiourea, blocked its effect. We propose that apoE secreted
locally at lesion sites by macrophages may be anti-inflammatory by
stimulating endothelium to release NO and suppress VCAM-1 expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells was supplemented with 0.1 mM
hypoxanthine and 16 µM thymidine.
(TNF-,
human recombinant; Sigma) was added directly into the media to a final
concentration of 10 units/ml (0.5 ng/ml) except for basal VCAM-1
controls. Any binding of monoclonal VCAM-1 antibody at 5 µg/ml (clone
BBIG-V1; R&D Systems, Abingdon, UK) was detected by StreptABComplex/HRP
Duet kit and O-phenylenediamine chromogenic substrate (both
from DAKO Ltd., High Wycombe, UK). Absorbances were measured at 492 nm
(A492) by microtitre plate spectrophotometer.
Finally, cellular protein per well was determined by solubilizing the
monolayers with 0.1 M NaOH, adding Bradford reagent
(Bio-Rad Laboratories, Hemel Hempstead, UK), and measuring the
absorbance at 620 nm (A620). VCAM-1 expression
was calculated for each well as a ratio of A492
VCAM-1 assay:A620 protein assay values for
TNF- stimulated cells. Non-cytokine-stimulated wells were subtracted
to give "VCAM-1 induction above basal", and then data was
normalized to a percentage of VCAM-1 induction in TNF- controls.
3 allele, were
prepared by our standard procedure (17) and added to cells (1.5 mg of
protein/ml) for 24 h; TNF- was added directly into the media to
a final concentration of 10 units/ml (0.5 ng/ml) for the last 6 h,
except for basal VCAM-1 controls. Primary antibody (monoclonal VCAM-1
antibody at 5 µg/ml) binding was detected using goat anti-mouse
fluorescein isothiocyanate-conjugated antibodies (DAKO Ltd), and 5 × 103 cells were analyzed per well by flow cytometry
(Coulter Epics Elite; Coulter, Hialeah, FL).
(CLONTECH, Basingstoke, UK). Subconfluent HUVECs (2 × 104
cells/well; 96-well plate) were transfected for 1 h with 0.4 µg
of DNA/well of pCMV.apoE2, pCMV.apoE3, pCMV.apoE4, or pCMV.0, using 3 µg of LipofectAMINE (Life Technologies). Cells were then incubated
for 48 h in M199 (5% FBS), adding 10 units/ml TNF- for the
last 6 h, except for basal VCAM-1 controls. ApoE from cell culture
supernatants was measured by ELISA (Apo-Tek ApoE kit; PerImmune Inc,
Rockville, MD), while HUVEC monolayers were analyzed for VCAM-1 by
cell-bound ELISA.
cells were co-cultured in
96-well plates at a ratio of 4:1 in M199 (5% FBS) for 48 h with TNF- 10 units/ml added for the last 6 h (except for basal
VCAM-1 controls). ApoE and VCAM-1 were then measured by ELISA.
cells (each ~80%
confluent) were conditioned for 24 h in M199 (5% FBS). The media
was collected, filtered (0.2 µm), and after apoE measurement was
incubated for 24 h with confluent HUVECs (3 × 104 cells/well; 96-well plate). To inhibit nitric oxide
synthase (NOS) activity, HUVECs were pre-incubated for 2 h with
100 µM ethyl-isothiourea (18) (ethyl-ITU; Calbiochem,
Nottingham, UK) prior to adding conditioned media plus 100 µM ethyl-ITU; TNF- was added 6 h before VCAM-1
analyses. In some experiments, induction of E-selectin by TNF- and
constitutive von Willebrand factor (vWF) expression were analyzed by
cell-bound ELISA using anti-E-selectin (clone BBIG-E4; R&D Systems) and
anti-vWF (DAKO Ltd) at 5 and 1.2 µg/ml, respectively. In other
experiments intracellular cyclic guanosine monophosphate (GMP) content
of confluent HUVECs in 12-well plates was analyzed by radioimmunoassay
(11) (Amersham Pharmacia Biotech).
cells) with some wells supplemented with 100 µM ethyl-ITU. Fluorescent emissions at 530 nm (bandwidth
of 25 nm) were read using a Cytofluor Series 400 plate reader
(PerSeptive Biosystems, Framingham, MA) upon excitation at 485 nm
(bandwidth of 20 nm). Results are expressed as change in relative DAF-2
DA fluorescence (arbitrary units) over a 2-h time course as a
percentage of the fluorescence signal from the control wells treated
with CHOdhfr cell-conditioned media.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
treatment, the fluorescence peak shifted right,
indicating up-regulation of cell-surface VCAM-1 (Fig. 1B).
However, pretreatment with HDL-E did not suppress this induction of
VCAM-1 expression; the fluorescence profile or mean fluorescence
intensity was not altered (Fig. 1C).
View larger version (18K):
[in a new window]
Fig. 1.
HDL-E does not suppress
TNF- -induced VCAM-1 expression in HUVECs.
Anti-VCAM-1 binding to HUVECs was detected using a secondary
fluorescein isothiocyanate-conjugated antibody before fixation in 1%
(w/v) paraformaldehyde and flow cytometric analysis. Histograms show a
three-decade log10 fluorescence scale against the number of
intact cells or "events" for a representative experiment. Basal
levels of VCAM-1 expression were negligible (A), but were
up-regulated after exposure to TNF- for 6 h (B).
Preincubation with HDL-E (1.5 mg of protein/ml) before addition of
TNF- failed to reduce VCAM-1 expression (C). Samples were
analyzed in duplicate, and findings were confirmed in independent
assays using different batches of HUVECs and HDL-E.
by a
10-fold induction of VCAM-1 as measured by cell-bound ELISA (data not
shown). Strikingly, cells secreting apoE2, apoE3, or apoE4 had
significantly reduced (all p < 0.01, Student's
t test) VCAM-1 compared with pCMV.0 transfection (67.8 ± 8.3%, 61.0 ± 12.0%, and 41.4 ± 7.4% inhibition,
respectively; Fig. 2A),
although the differences between the isoforms was not significant
(p > 0.05, analysis of variance test). Further
experiments revealed that VCAM-1 induction was inversely correlated
with concentration of secreted apoE2 (r = 0.76,
Pearson correlation, p < 0.001; n = 20, Fig. 2B) or apoE3 (r = 0.81, Pearson
correlation, p < 0.001; n = 8, data
not shown).
View larger version (25K):
[in a new window]
Fig. 2.
VCAM-1 is down-regulated in HUVECs
transiently transfected to secrete apoE. A, VCAM-1
induction by TNF- , as detected by VCAM-1 ELISA, was significantly
reduced (*, p < 0.05, Student's t test,
n = 4) when cells were transfected with pCMV.apoE2/3/4
(gray bars) compared with pCMV.0 controls
(100.0 ± 6.9% VCAM-1 induction, n = 4, black bar). B, secretion of apoE2 (
) by
transfected HUVECs inversely correlated with induction of VCAM-1
(r = 0.76, Pearson correlation, n = 20, p < 0.001). The mean VCAM-1 induction for pCMV.0
controls was 100.3 ± 3.4% (n = 22) (
),
and the graph shows experiments with five different batches of HUVECs
from different donors.
cells (data not shown).
0.70, p < 0.001; n = 10, Fig.
3A) or apoE3 and apoE4
concentration (r = 0.64, p < 0.001, n = 4 and r = 0.53, p < 0.001, n = 4, respectively; data not shown) in the
conditioned media. By contrast, CHOapoE cell-derived apoE
did not affect expression of other endothelial markers; at 5.5 ± 0.2 µg of apoE2/ml of expression of both constitutive vWF and
TNF--induced E-selectin were unchanged, even though VCAM-1 was
suppressed by 23.4 ± 2.2% (p < 0.001;
n = 3, Fig. 3B) in the same experiments.
Thus, unlike plasma-purified apoE, which was inactive (14),
CHOapoE cell-derived apoE was a selective inhibitor of
VCAM-1 expression, without affecting the constitutive levels of vWF or
the degree of E-selectin up-regulation in response to TNF-.
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Fig. 3.
Down-regulation of VCAM-1 in HUVECs by CHO
cell-derived apoE is selective. A, preincubation of
HUVECs with CHOapoE2 cell-conditioned media ( )
down-regulated TNF--stimulated VCAM-1 with the extent of VCAM-1
induction inversely correlating with the apoE2 concentration in the
media (r = 0.70, Pearson correlation,
p < 0.001). Data points represent the mean ± S.E. for each of 10 independent experiments performed in quadruplicate
using a total of six different batches of HUVECs; the mean VCAM-1
induction for CHOdhfr cell-conditioned controls was
100.2 ± 1.3%, n = 30 (). B,
although CHOapoE2 cell-conditioned media (5.5 ± 0.2 µg of apoE2/ml) suppressed TNF--stimulated VCAM-1 induction by
23.4 ± 2.2% (*p < 0.001, Student's
t test, n = 3), expression of
TNF--induced E-selectin and constitutive vWF in parallel wells were
unaffected as assessed by cell-bound ELISAs (gray bars).
Control percentage antigen expression was 100 ± 1.2% (black
bar), and data is from three independent experiments performed using
two batches of HUVECs.
mobility. A minor 90-kDa (50-150
kDa) particle with pre-mobility was also found. Additionally, MDM
media contained a very large particle (>1200 kDa) not seen in
CHOapoE cell media.
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[in a new window]
Fig. 4.
CHOapoE cell-dervived and MDM
apoE particles are similar. Secreted apoE particles in
CHOapoE2 cell-conditioned media were heterogeneous by
agarose gel electrophoresis and apoE immunoblotting (A), by
negative staining electron microscopy (B; electron
micrographs taken at ×125,000 magnification, white scale
bar represents 100 nm), and by gel filtration (C; the
bars indicate fractionation of molecular-size standards:
A, dextran blue, 2000 kDa; B, thyroglobulin, 669 kDa; C, catalase 232 kDa; and D, albumin, 67 kDa;
profiles for apoE content in media from the different cell types are
overlaid (solid line for MDM, dashed line for
CHOapoE)), but have many similarities to those in MDM
media.
View larger version (23K):
[in a new window]
Fig. 5.
Evidence that apoE down-regulates VCAM-1 via
NO release in HUVECs. A, the down-regulation of VCAM-1
induction by TNF- (23.4 ± 2.2% inhibition, *p < 0.001, Student's t test, n = 3) in
HUVECs by CHOapoE2 cell-conditioned media (5.5 ± 0.2 µg of apoE2/ml) was completely blocked by preincubation with the NOS
inhibitor, ethyl-ITU at 100 µM. B, incubation
with CHOapoE2 cell media (5.9 ± 0.94 µg of
apoE2/ml) increased intracellular cGMP levels by 46.5 ± 15%
compared with cells exposed to CHOdhfr cell-conditioned
media (*, p < 0.05, Student's t test,
n = 3). Results are expressed as a percentage of the
cGMP level in wells with control-conditioned media where cGMP varied
between 45-125 pmol/108 cells. Three different batches of
HUVECs and CHO cell-conditioned media were used in three independent
experiments. C, intracellular NO production in HUVECs was
measured directly using the fluorescent DAF-DA assay. The 61.9 ± 24.2% increase in NO from basal levels of 100.0 ± 5.6% (in
HUVECs exposed to with control-conditioned media) to 161.9 ± 24.2% (p < 0.001, Student's t test,
n = 5) during a 2-h incubation with
CHOapoE2 cell-conditioned media, was blocked by
preincubation with a NOS inhibitor (*, p < 0.001, Student's t test). Results are expressed as a percentage of
the relative fluorescence (arbitrary units) in wells treated with
control CHOdhfr cell-conditioned media. Data is from
using five different batches of HUVECs and five different preparations
of conditioned media.
4-6) but
contained the 177-base pair cytoplasmic insert, although minor transcripts of full-length apoER2±cytoplasmic insert (Ins) were also
detected. To verify protein expression we immunoprecipitated solubilized HUVEC membranes with anti-apoER2Ins (20). As the variant
lacking the cytoplasmic insert is not recognized by this anti-peptide
antibody, only two immunoreactive bands were detected, a major one at
130 kDa and a minor one at 180 kDa corresponding, respectively, to
apoER24-6 and full-length apoER2 (Fig. 6B).
View larger version (16K):
[in a new window]
Fig. 6.
ApoER2 is expressed in HUVECs.
A, long-range reverse transcription-polymerase chain
reaction identified the major apoER2 transcript in HUVECs as
apoER2 4-6, the variant with deletion of repeats 4-6 in the
ligand-binding domain, although smaller amounts of full-length
apoER2±cytoplasmic insert (Insert) were also present. B,
immunoprecipitation of 35S-labeled HUVEC membranes, with
antipeptide antisera against the cytoplasmic insert of apoER2, verified
that apoER24-6 was expressed (130 kDa) and that a smaller amount of
full-length apoER2 (180 kDa) was also present.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
Supported by the Deutsche Forschungsgemeinschaft (Cu
31/4-1).
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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