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* Center for Molecular and Vascular Biology and
Department of Pharmacology, KU Leuven, B-3000 Leuven, Belgium;
Laboratoire de Biochimie et Biologie Cellulaire, FUNDP;
§ INSERM Unité 321, Hôpital de la Pitié, Paris, France; and
¶ Department of Physiology, KU Leuven, B-3000 Leuven, Belgium
1Correspondence: Center for Experimental Surgery and Anesthesiology-CEHA, Onderwijs en Navorsing, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium. E-mail: paul.holvoet{at}med.kuleuven.ac.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: atherosclerosis • lipoproteins • leukocytes • cell adhesion molecules • signal transduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. It
correlates inversely with restenosis after coronary angioplasty
(2)
. Even small increments of HDL-C protect against
cardiovascular disease (3)
. Transgenic expression of
apolipoprotein AI (apo AI) increases HDL and delays atherosclerosis
(4
5
6)
.
HDL may exert its protective effect by functioning as a cholesterol
acceptor in reverse cholesterol transport (7)
or by
reducing leukocyte/endothelial cell interactions. The latter effect has
been attributed to suppression of endothelial cytokine-induced vascular
cell adhesion molecule 1 (VCAM-1) expression (8)
or to
protection of low density lipoprotein (LDL) from oxidative
modification, possibly mediated by HDL-associated paraoxonase
(9)
and platelet-activating factor acetylhydrolase
(PAF-AH) (10)
. We have recently shown that
oxidized LDL (oxLDL) predicts the risk for cardiovascular disease in
humans. OxLDL levels were inversely related to HDL cholesterol levels
(11)
. Oxidation of the phospholipid content of LDL
generates compounds with PAF-like bioactivity (10)
. These
substances, in vitro, induce expression of the adhesion
molecules VCAM-1 and intercellular adhesion molecule 1 (ICAM-1) on the
endothelial cell surface. The transcriptional activation is partially
due to rises of cytosolic Ca2+ (12)
.
Adhesion molecule expression depends on NF
B and can be prevented
in vitro by antioxidants (13)
, HDL and
PAF-AH (14
, 15)
.
In the present study the effect of HDL on endothelial cell/leukocyte
interaction was evaluated in apoE-/- mice using
the macrophage homing assay of Patel et al. (16)
. The
beneficial effect of high levels of HDL is explained by an attenuation
of oxidative stress and decreased endothelial adhesiveness in
vivo. Human PAF-AH gene transfer studies reveal the
antioxidative capacity of PAF-AH as an active principle for
the antiatherogenic effect of HDL and suggest a potential gene therapy
approach for atherosclerosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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were
backcrossed for 10 generations into the C57BL/6 background. Human apo
AI transgenic mice (4)
in the C57BL/6
apoE-/- background have been described
elsewhere (6)
. Groups were of mixed but matched gender and
fed regular mouse chow; macrophage donors were 8 wk old C57BL/6 mice
(Jackson Laboratories, Bar Harbor, Maine). The Institutional Research
Animal Care Committee approved all animal procedures.
Macrophage homing assay
Macrophages were isolated, labeled, and injected as described
(16)
. ICAM-1 and VCAM-1 blocking studies in
vivo were carried out with monoclonal antibodies (mAb) 3E21
(18)
and 429 (19)
. Hamster immunoglobulin G
(IgG) mAb G235–2356 or R35–95 rat IgG2a
served as corresponding isotype controls (PharMingen, San Diego,
Calif.). The antibodies (100 µg) were injected intraperitoneally
(i.p.) 6 h prior to macrophages.
After 4 days, blood was drawn from barbiturate anesthetized mice in 0.1 vol of 0.1 M citrate/1 mM EDTA/10 µM butylated hydroxytoluene/20 µM vitamin E. Mice were perfused with 0.9% saline/1 U/ml heparin. Thoracic aortas were dissected and snap frozen for RNA isolation. Heart base and ascending aorta were OCT embedded, stored at -80°C, and cryosectioned at 7 µm. Macrophages phagocytosed between 1 and 10 microspheres and were counted in fluorescent light as single or groups of microspheres that were associated with a macrophage adherent to the aortic wall. Macrophages were counted in 140 serial sections per mouse, spanning the proximal 1 mm of the aortic valve region. After intravenous (i.v.) injection in apoE-/- mice, numbers of homing macrophages were 353 ± 85 at 24 h, 259 ± 159 at 48 h, 235 ± 37 after 96 h, and 316 ± 132 after 8 days (n=6, 8, 13, 3, P=ns). Aortic lesion areas were assessed on oil red O-stained sections. The macrophage specific antibody MAC3 (PharMingen) was used to stain and detect areas occupied by macrophages with the Quantimet 600 image analyzer (Leica, Brussels, Belgium).
Flow chamber experiments
Monolayers of fEnd.5 (20)
grown on collagen-coated
glass coverslips were mounted in a parallel plate flow chamber and
superfused with 2 x 105/ml
bis-carboxyethyl-carboxyfluorescein-acetoxymethyl ester (BCECF-AM,
Molecular Probes, Eugene, Oreg.) -labeled macrophages. Adhesion was
determined in 15 high-power fields of 1.5 mm2
each of the coverslip as described elsewhere (21)
. fEnd.5
were treated for 6 h with 20% plasma in DMEM (0.1 U heparin/ml)
or ßVLDL fractions (50 µg/ml in HBSS/1% bovine serum albumin/1.2
mmol/l Ca2 +) at 37°C.
Ca2+ measurements
Cytosolic Ca2+ was assayed as described
previously (22)
. Cells were loaded with Fluo-3-AM and
baseline fluorescence was recorded after washing. Inhibitors [WEB
2086, 10 µM (Boehringer, Mannheim, Germany) and HDL 100 µg/ml apo
AI] were added, followed by ßVLDL. Fluorescence was measured
confocally using a Meridian Insight microscope (Meridian, Okemos,
Mich.) and an S-plan APO 60x (NA1.4) oil immersion lens (Olympus,
Tokyo, Japan). An oscillating slit-shaped 488 nm light beam was used
for excitation. Emission was measured at 530 nm, recorded on videotape,
and normalized to baseline fluorescence after background correction.
Real time rtPCR for ICAM-1, VCAM-1, and HPRT
First strand cDNA generated from 10 ng total thoracic
aorta RNA was subjected to quantitative real time reverse
transcriptase-polymerase chain reaction (rtPCR) according to the
suppliers protocol (Perkin-Elmer, Zaventem, Belgium). Oligonucleotides
used as forward primer (F), reverse primer (R), and probes (P) labeled
with the fluorescent quencher TAMRA (3') and the indicator dye FAM (5')
were: for ICAM-1: F: 5'-TGATCCCTGGGCCTGGT-3'; R:
5'-TTTCAGCCACTGAGTCTCCAAG-3'; P:
5'-FAM-CTCATGCAAGGAGGACCTCAGCCTG-TAMRA-3'; for VCAM-1: F:
5'-GTATCACGTGG ACATCTACTCTTTC C-3'; R: 5'-CTGTCTGTTCATGAGCTGGTCAC-3';
P: 5'-FAM-TGACCGTGACCGGCTTCCCAAAC-TAMRA-3'. The copy numbers
were calculated from plasmid cDNA standards containing the rtPCR
amplicon. ICAM-1 and VCAM-1 levels were expressed as copy number per
1000 copies of hypoxanthine transferase (HPRT). HPRT oligonucleotides
were: F: 5'-TTATCAGACTGAAGAGCTACTGTAATGATC-3'; R: 5'-TTACC
AGTGTCAATTATATCTTCAACAATC-3'; P: 5'-JOE-TGAGAGATCATCTCCACCAATAACT
TTTATGTCCC-TAMRA-3'
Determination of MDA-LDL auto-antibodies
Auto-antibodies against oxidized LDL in mice were determined as
described earlier (23
, 24)
. Ninety six-well ELISA plates
were coated with in vitro MDA-modified human LDL or native
LDL overnight and blocked with 1% bovine serum albumin. Plasma samples
were added at serial dilutions from 1:10 until 1:100 and incubated for
2 h. Plates were washed and a horseradish peroxidase-conjugated
rabbit anti-mouse IgG was added. After developing, the absorbance was
read at 492 nm. Auto-antibody levels are expressed as MDA-LDL/native
LDL ratio to account for unspecific binding to unmodified human LDL.
Adenoviral gene transfer of human PAF-acetylhydrolase
Recombinant adenovirus (AdPAF-AH) containing the cytomegalovirus
promoter/enhancer and the human PAF-AH cDNA was generated by
cotransfecting rescue plasmid pJM17 and shuttle plasmid pLpA containing
the human PAF-AH cDNA into 293 cells (25)
.
PAF-AH cDNA was a gift from Dr P. Kolkhof (Bayer Pharma,
Wuppertal, Germany). 5 x 108 plaque-forming
units (pfu) of either AdPAF-AH or AdRR5 control virus were injected
i.v. in apoE-/- mice. Citrated blood was drawn
from the retrobulbar plexus on indicated days. PAF-AH
activity was assessed as described (26)
.
Statistical methods
Nonparametric Kruskal-Wallis testing compared results of
macrophage homing, ex vivo adhesion and
Ca2+ amplitudes. Individual differences were
identified by Mann-Whitney-U or, where required, by Dunnett‘s test to
correct for multiple testing. Responding cell fractions in the
Ca2+ experiments were compared by
2 test, followed by Fisher’s Exact test for
individual differences. A P value < 0.05 was
considered significant. InStat V2.05 was used (Graphpad Software, San
Diego, Calif.).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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on normal chow were not different with respect
to non-HDL cholesterol levels (580±25 mg/dl and 610±30 mg/dl,
n=6/10, P=ns). Circulating human apo AI (240±19
mg/dl) led to an increase of HDL cholesterol to 105 ± 3.5 mg/dl
in apoE-/-/AI+/+ compared
to 37 ± 2.3 mg/dl in apoE-/- mice
(P<0.01, n=6/10).
HDL effect on macrophage homing
Peritoneal macrophages displayed typical leukocyte homing
patterns as described previously (16)
. Macrophages
injected i.p. or subcutaneously into C57BL/6 mice displayed active
trafficking. Cells migrated along the lymphatic vessels, homed to the
lungs, and subsequently to liver and spleen (Fig. 1a
, b
). When injected i.v., 235 ± 37 (n=13)
macrophages were found in the aortic root of 6-month-old
apoE-/- mice (Fig. 1f
). In
3-month-old apoE-/-, similar numbers of
macrophages were counted (n=8, P=ns). In C57BL/6
mice, 37-fold fewer macrophages homed (n=6,
P<0.0001, Fig. 1f
). Lesion areas in the
aortic roots of apoE-/- were 72,400 ± 996
µm2 at 3 months and fourfold larger at 6 months
(P<0.001, Fig. 1g
). These data indicated that
macrophage homing is independent of lesion size. In 6 month
apoE-/-/AI+/+, lesion
areas were similar to those in 3 month apoE-/-
mice (Fig. 1g
), but macrophage homing was threefold reduced
in comparison to both 3 month and 6 month
apoE-/- mice (n=8,
P<0.01, Fig. 1f
). The fraction of
lesion surface area immunoreactive for macrophages was 13 ± 1.7%
in apoE-/-/AI+/+
(n=4) and 2.0- and 1.7-fold larger in 3 month and 6 month
apoE-/- mice (n=6/7,
P<0.01, Fig. 1h
), respectively.
|
Ex vivo macrophage adhesion is induced by
apoE-/- plasma and ßVLDL
To elucidate the mechanism of the reduction of macrophage homing
in apoE-/-/AI+/+ mice,
adhesion of peritoneal macrophages to fEND.5 cells was studied in a
flow chamber at a shear rate of 400s-1.
Macrophages rolled on the endothelium before adhering firmly
(Fig. 2a
). When fEnd.5 cells were exposed to
apoE-/- plasma prior to superfusion, 3.7-fold
more adhesion (n=5, P<0.05) was observed
compared to C57BL/6 plasma whereas
apoE-/-/AI+/+ plasma
induced a nonsignificant 1.7-fold increase (n=5, Fig. 2b
). ßVLDL from apoE-/- plasma
increased adhesion 4.9-fold over C57BL/6 ßVLDL (n=5,
P<0.001, Fig. 2c
).
Palmitoyl-lysophosphatidylcholine (p-LPC) increased macrophage adhesion
5.5-fold compared to vehicle control or C57BL/6 plasma (n=5,
P<0.001, Fig. 2d
).
ApoE-/- ßVLDL or p-LPC-induced adhesion was
reduced 3-fold or 3.4-fold by
apoE-/-/AI+/+ HDL
(n=5, P<0.01, Fig. 2c
, d
) whereas
apoE-/- HDL fractions had no inhibitory effect
(Fig. 2c
). PAF receptor antagonist WEB2086 blocked ßVLDL
or p-LPC-induced adhesion (n=5, P<0.01, Fig. 2c
, d
). In contrast, human rTNF
-induced adhesion was not
affected by WEB2086, suggesting that both ßVLDL and p-LPC, in
contrast to TNF-, induced macrophage adhesion through a
PAF-sensitive pathway (Fig. 2d
).
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HDL reduces ßVLDL triggered cytosolic Ca2+ signaling
In fEND.5 monolayers, ßVLDL elicited biphasic cytosolic
Ca2+ transients consisting of a rapid early peak
within 150 s after stimulation and a sustained component within 10
to 15 min (Fig. 3a
, b
, c
). The initial phase of the Ca2+
transient was unaffected in Ca2+-free buffer (2
mM EGTA/HBSS) or with 1 mM Ni2+ [100% and 88%
responding cells; relative peak fluorescence (rpf) 3.2 ± 0.6 and
3.7 ± 0.8 arbitrary units, n=20/40]; it was therefore
due to Ca2+ release from intracellular stores. In
contrast, the second Ca2+ rise was abolished and
reflected Ca2+ influx. In responders, the
amplitude of the Ca2+ signal was similar with 50
µg/ml apoE-/- ßVLDL or
apoE-/-/AI+/+ ßVLDL
(rpf 2.46±0.14 vs. 2.49±0.44, n=81/82). However, 7.3% of
the cells responded to 50 µg/ml
apoE-/-/AI+/+ ßVLDL
compared to 62% for apoE-/-ßVLDL
(n=81/82 cells, P<0.0001, Fig. 3d
). The concentration required to induce
Ca2+ signaling in 50% of the cells was 87
µg/ml for apoE-/-/AI+/+
ßVLDL and 46 µg/ml for apoE-/- ßVLDL
(n=5, P<0.01). In the presence of
apoE-/-/AI+/+ HDL only
5.8% (n=82, P<0.01) responded to 50 µg/ml
apoE-/- ßVLDL. In contrast
apoE-/- HDL had no inhibitory effect.
ApoE-/-/AI+/+ HDL but not
apoE-/- HDL (n=7/37) reduced
(P<0.05) peak fluorescence intensities of the
Ca2+ signal. In the presence of WEB 2086 only
16% of the cells responded to apoE-/- ßVLDL
(16%, n=60, P<0.0001, Fig. 3d
).
Thus, PAF-like activity in oxidatively modified ßVLDL elicits
cytosolic Ca2+ signaling.
|
HDL reduces adhesion molecule expression ex vivo and
in vivo
Expression of VCAM-1 and ICAM-1 was studied at the transcriptional
and functional level ex vivo and in vivo. rtPCR
on total RNA purified from the thoracic aorta detected 2.68 ±
1.26 copies of ICAM-1 per 1000 copies of HPRT in
apoE-/- mice, but only 0.38 ± 0.2 copies
in apoE-/-/AI+/+ mice
(n=8, P<0.05). VCAM-1 expression was also
2.7-fold lower in
apoE-/-/AI+/+ compared to
apoE-/- mice (n=8,
P<0.05, Fig. 3e
). As an estimate for expression
of functional ICAM-1 and VCAM-1 protein, in vivo blocking
studies were carried out in the two strains of mice. VCAM-1 inhibition
reduced macrophage homing to apoE-/- aortas by
48% (n=5, P<0.05), whereas blocking of both
VCAM-1 and ICAM-1 reduced homing by 75% (n=5,
P<0.05, Fig. 3f
). In contrast, blocking
of VCAM-1 and ICAM-1 did not reduce in vivo macrophage
homing in apoE-/-/AI+/+
mice (Fig. 3f
).
To verify that
apoE-/-/AI+/+ ßVLDL was
less potent to induce ICAM-1 and VCAM-1 expression, ex vivo
blocking studies were carried out. In the flow chamber, anti-ICAM-1 and
-VCAM-1 mAbs elicited a dose-dependent inhibition of macrophage
adhesion induced by apoE-/- plasma (not shown).
At plateau concentrations of the combined antibodies, a 3.4-fold
inhibition of macrophage adhesion was obtained with
apoE-/- plasma-stimulated fEnd.5, whereas the
3-fold reduced adhesion in response to
apoE-/-/AI+/+ was further
reduced 4.7-fold (n=4, P<0.05, Fig. 3g
). Separately, ICAM-1 and VCAM-1 accounted for 48% and
39% of ex vivo adhesion in response to
apoE-/- plasma (n=4,
P<0.01) and for 8% and 53% of the reduced adhesion
observed in response to
apoE-/-/AI+/+ plasma
(n=4, P<0.05, VCAM-1 vs. control, Fig. 3g
). Thus, reduced endothelial adhesiveness in
apoE-/-/AI+/+mice is
attributable to a greater reduction of ICAM-1 than VCAM-1 expression.
HDL decreases oxidative stress in apoE-/- mice
The ratio of antibodies against oxidatively modified LDL over
antibodies recognizing human native LDL were 7.7 ± 3.4 in
apoE-/- plasma compared to 4.9 ± 1.4 in
apoE-/-/AI+/+ plasma
(n=12, P<0.01), indicating a decrease of
immunogenic neoepitopes in ßVLDL when high levels of HDL were
present.
Breakdown of PAF-like compounds in vivo reduces
macrophage adhesion ex vivo and in vivo
In apoE-/- mice, liver-directed
PAF-AH gene transfer induced a 1.5-fold increase in
PAF-AH plasma activity (n=13,
P<0.0001, day 0 vs. 7, Fig. 4a
). AdRR5 did not induce an increase in PAF-AH
plasma activity (23.5±1.6 vs. 25.2±2 AU, day 0 vs. 7,
n=4). AdRR5-ßVLDL induced 3.5-fold more macrophage
adhesion (n=5, P<0.01, Fig. 4b
) than
AdPAF-AH-ßVLDL. Compared to AdRR5, AdPAF-AH reduced macrophage homing
in apoE-/- mice 2.6-fold (P<0.02,
n=6/13, Fig. 4c
).
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previously, the antiadhesive effect of HDL has been well established
in vitro (8)
. However, using the same mouse
model in the prelesion state, Dansky et al. did not observe reduced
VCAM-1 expression or recruitment of mononuclear cells by
immunohistochemistry (27)
. We find in slightly more
advanced apoE-/- lesions than studied by Dansky
and colleagues that less endogenous macrophages accumulate in lesions
of mice with high levels of human-like HDL. These data agree with
previous reports on apoE-/- mice
(28)
. In our study, reduced macrophage content as
established in the functional in vivo homing assay is due to
reduced endothelial adhesiveness. This conclusion is supported by
quantitative rtPCR data at the mRNA and by functional blocking studies
for adhesion molecules at the protein level. Differences in the outcome
of the studies are potentially due to the different time points chosen
for the investigation since subtle differences in the dynamic state of
a lesion will be harder to detect in younger mice.
Our study demonstrates that in vivo oxidative modification
of atherogenic lipoproteins (in the mouse, predominantly ßVLDL) is
responsible for increased leukocyte homing in
apoE-/- mice. ApoE-/-
ßVLDL-associated compounds with PAF-like bioactivity
(29)
induced cytosolic Ca2+
signaling through a PAF-sensitive pathway leading to VCAM-1 and ICAM-1
expression. Overexpression of human apo AI (4)
in
apoE-/- mice results in an increase of HDL
causing breakdown of ßVLDL-associated PAF-like bioactivity.
Autoantibodies against modified lipoproteins in
apoE-/- plasma, as a measure for the in
vivo oxidation of ßVLDL, were reduced by human-like HDL reducing
its potency to induce ex vivo cytosolic
Ca2+ signals. This blunted in vivo and
ex vivo expression of ICAM-1 and VCAM-1. These findings
suggest that HDL may exert its antiatherogenic effect by antagonizing
oxidation of lipoproteins that are associated with cardiovascular
disease and inversely correlated to HDL-C in humans (11)
.
In vitro, accumulation of proadhesive PAF-AH
substrates in atherogenic lipoproteins and the protective effect of HDL
have been demonstrated (14
, 29)
. The antioxidative effect
of HDL has been attributed to paraoxonase and PAF-AH
circulating with the mature lipoprotein particle (9
, 10)
.
ApoE-/--HDL-associated PAF-AH
activity indeed is markedly increased in apo AI transgenics (B. De
Geest et al., unpublished results), suggesting that increasing HDL
concomitantly increases the antioxidative capacity of
PAF-AH.
Ex vivo, the effect of apo
E-/--ßVLDL was essentially entirely due to
PAF-like bioactivity, as indicated by the nearly complete inhibition of
Ca2+ signals and macrophage adhesion in the
presence of the PAF receptor antagonist WEB2086. This suggests that the
loss of macrophage adhesion in response to ßVLDL isolated from
AdPAF-AH-treated mice compared to the control virus treated mice is due
to reduced accumulation of PAF-like bioactivity in ßVLDL. Beyond
that, increases of PAF-AH also diminished macrophage homing in
vivo. Additional studies are needed to establish reduction of
lesion formation by PAF-AH gene transfer in vivo.
Compared to strategies directed at elevating circulating apo AI, which
requires much higher viral doses (25
, 30)
, a 1.5-fold
increase in PAF-AH activity as achieved in this study using
5 x 108 pfu of AdPAF-AH was
sufficient to significantly reduce macrophage homing.
Mutations in the PAF-AH gene are independent risk factors
for coronary artery disease (31)
, and low levels of
PAF-AH are found in myocardial infarction (32)
,
suggesting that our findings in apoE-/- mice
are reflecting human pathophysiology.
Taken together, our results suggest that if HDL levels are low PAF-like
bioactivity accumulates in proatherogenic lipoprotein particles. It is
this bioactivity that ultimately leads to endothelial activation and
monocyte recruitment (Fig. 5
). Increasing HDL levels allows breakdown of these PAF-like compounds by
the associated increase of PAF-AH activity (Fig. 5)
.
Increasing PAF-AH activity without affecting HDL-levels
significantly reduced endothelial adhesiveness and macrophage
recruitment to lesion prone sites, suggesting that in
apoE-/- mice raising HDL may be antiatherogenic
through the concomitant increase of antioxidative capacity conveyed by
PAF-AH (Fig. 5)
.
|
In conclusion, our study establishes the relation between HDL-associated PAF-AH, prevention of oxidation of atherogenic lipoproteins, and macrophage homing, which is the initial step in atherogenesis. PAF-AH gene transfer is a very promising therapeutic strategy to abolish the oxidation of atherogenic lipoproteins in vivo. These data suggest that even if means to increase HDL levels are not available, the quality of the HDL particles can be improved dramatically. Further studies to evaluate the clinical effect of reducing oxidative stress by modulating the antioxidative potential of HDL are warranted.
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ACKNOWLEDGMENTS |
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Received for publication December 9, 1999.
Revision received April 18, 2000.
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T missense in exon 9 of the plasma platelet-activating factor acetylhydrolase gene as an independent risk factor for coronary artery disease in Japanese men. Metabolism 47,177-181[Medline]
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L. Calabresi, M. Gomaraschi, and G. Franceschini Endothelial Protection by High-Density Lipoproteins: From Bench to Bedside Arterioscler. Thromb. Vasc. Biol., October 1, 2003; 23(10): 1724 - 1731. [Abstract] [Full Text] [PDF]
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S. Blankenberg, D. Stengel, H. J. Rupprecht, C. Bickel, J. Meyer, F. Cambien, L. Tiret, and E. Ninio Plasma PAF-acetylhydrolase in patients with coronary artery disease: results of a cross-sectional analysis J. Lipid Res., July 1, 2003; 44(7): 1381 - 1386. [Abstract] [Full Text] [PDF]
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V. Tsimihodimos, A. Kakafika, A. P. Tambaki, E. Bairaktari, M. J. Chapman, M. Elisaf, and A. D. Tselepis Fenofibrate induces HDL-associated PAF-AH but attenuates enzyme activity associated with apoB-containing lipoproteins J. Lipid Res., May 1, 2003; 44(5): 927 - 934. [Abstract] [Full Text] [PDF]
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H. Noto, M. Hara, K. Karasawa, N. Iso-O, H. Satoh, M. Togo, Y. Hashimoto, Y. Yamada, T. Kosaka, M. Kawamura, et al. Human Plasma Platelet-Activating Factor Acetylhydrolase Binds to All the Murine Lipoproteins, Conferring Protection Against Oxidative Stress Arterioscler. Thromb. Vasc. Biol., May 1, 2003; 23(5): 829 - 835. [Abstract] [Full Text] [PDF]
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A. Mertens, P. Verhamme, J. K. Bielicki, M. C. Phillips, R. Quarck, W. Verreth, D. Stengel, E. Ninio, M. Navab, B. Mackness, et al. Increased Low-Density Lipoprotein Oxidation and Impaired High-Density Lipoprotein Antioxidant Defense Are Associated With Increased Macrophage Homing and Atherosclerosis in Dyslipidemic Obese Mice: LCAT Gene Transfer Decreases Atherosclerosis Circulation, April 1, 2003; 107(12): 1640 - 1646. [Abstract] [Full Text] [PDF]
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 I. GOUNI-BERTHOLD and A. SACHINIDIS Does the coronary risk factor low density lipoprotein alter growth and signaling in vascular smooth muscle cells? FASEB J, October 1, 2002; 16(12): 1477 - 1487. [Abstract] [Full Text] [PDF]
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 G. Theilmeier, C. Michiels, E. Spaepen, I. Vreys, D. Collen, J. Vermylen, and M. F. Hoylaerts Endothelial von Willebrand factor recruits platelets to atherosclerosis-prone sites in response to hypercholesterolemia Blood, May 29, 2002; 99(12): 4486 - 4493. [Abstract] [Full Text] [PDF]
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E. Boisfer, D. Stengel, D. Pastier, P. M. Laplaud, N. Dousset, E. Ninio, and A.-D. Kalopissis Antioxidant properties of HDL in transgenic mice overexpressing human apolipoprotein A-II J. Lipid Res., May 1, 2002; 43(5): 732 - 741. [Abstract] [Full Text] [PDF]
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T. M. Forte, G. Subbanagounder, J. A. Berliner, P. J. Blanche, A. O. Clermont, Z. Jia, M. N. Oda, R. M. Krauss, and J. K. Bielicki Altered activities of anti-atherogenic enzymes LCAT, paraoxonase, and platelet-activating factor acetylhydrolase in atherosclerosis-susceptible mice J. Lipid Res., March 1, 2002; 43(3): 477 - 485. [Abstract] [Full Text] [PDF]
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V. Tsimihodimos, S.-A. P. Karabina, A. P. Tambaki, E. Bairaktari, G. Miltiadous, J. A. Goudevenos, M. A. Cariolou, M. J. Chapman, A. D. Tselepis, and M. Elisaf Altered distribution of platelet-activating factor- acetylhydrolase activity between LDL and HDL as a function of the severity of hypercholesterolemia J. Lipid Res., February 1, 2002; 43(2): 256 - 263. [Abstract] [Full Text] [PDF]
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