© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Upregulation of Endothelial Receptor for Oxidized LDL (LOX-1) by Oxidized LDL and Implications in Apoptosis of Human Coronary Artery Endothelial Cells
Evidence From Use of Antisense LOX-1 mRNA and Chemical Inhibitors
From the Departments of Medicine and Physiology, University of Florida and VA Medical Center, Gainesville.
Correspondence to J.L. Mehta, MD, PhD, Department of Medicine, University of Florida College of Medicine, 1600 Archer Rd, PO Box 100277 JHMHC, Gainesville, FL 32610. E-mail mehta{at}medmac.ufl.edu
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Abstract |
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Abstract—A specific lectinlike endothelial receptor for oxidized low density lipoprotein (LOX-1), distinct from the scavenger receptor in monocytes/macrophages, has been identified and cloned. In this study, we examined the regulation of LOX-1 by oxidized low density lipoprotein (ox-LDL) and determined the role of LOX-1 in ox-LDL–induced apoptosis of cultured human coronary artery endothelial cells (HCAECs). Incubation of HCAECs with ox-LDL (40 µg/mL), but not native LDL, for 24 hours markedly increased LOX-1 expression (mRNA and protein). After 48 hours of preincubation of HCAECs with a specific antisense to LOX-1 mRNA (antisense LOX-1), ox-LDL–mediated upregulation of LOX-1 was suppressed (P<0.01). In contrast, treatment of HCAECs with sense LOX-1 had no effect. Ox-LDL also induced apoptosis (determined by terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling and DNA laddering) of HCAECs in a concentration- and time-dependent fashion. LOX-1 played an important role in ox-LDL–mediated apoptosis of HCAECs because antisense LOX-1 inhibited this effect of ox-LDL. Polyinosinic acid and carrageenan, 2 different chemical inhibitors of LOX-1, also decreased ox-LDL–mediated apoptosis of HCAECs. Nuclear factor (NF)-
B was markedly activated in ox-LDL–treated HCAECs. The
critical role of NF-B activation became evident in experiments with
antisense LOX-1, which abolished ox-LDL–mediated NF-B activation.
In this process, an NF-B inhibitor, caffeic acid
phenethyl ester, also inhibited ox-LDL–mediated apoptosis of
HCAECs. These findings indicate that ox-LDL upregulates its own
endothelial receptor. Ox-LDL–induced apoptosis
is mediated by the action of LOX-1. In this process, NF-B activation
may play an important role as a signal transduction mechanism.
Key Words: apoptosis • endothelial cells • LOX-1 • nuclear factor-B • oxidized LDL
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Introduction |
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Vascular endothelial cells in culture1 and in vivo2 internalize and degrade oxidized LDL (ox-LDL) through a putative receptor-mediated pathway that does not seem to involve the classic macrophage scavenger receptor. The endothelial receptor for ox-LDL (LOX-1) is a membrane protein that belongs structurally to the C-type selectin family and is expressed in vivo in vascular endothelium and in vascular-rich organs.3 Because endothelial uptake of ox-LDL is important in the genesis of atherosclerosis, understanding of the role and regulation of LOX-1 may be of immense clinical significance.
Recent studies show that cytokine tumor necrosis factor
(TNF)-
4 and fluid shear stress5 markedly
upregulate LOX-1 gene expression in endothelial cells.
Another study6 has shown that LOX-1 expression is
dramatically increased in hypertensive rats. A more recent study from
our laboratory7 has demonstrated that
angiotensin II upregulates LOX-1 expression as well as the
uptake of ox-LDL in human coronary artery
endothelial cells (HCAECs).
It is widely appreciated that LDL, especially its oxidatively modified form (ox-LDL), is a critical factor in atherogenesis. Endothelial dysfunction elicited by ox-LDL has been identified in the course of atherogenesis and its complications.8 LDL is oxidized in vascular endothelial cells to a highly injurious product that results in characteristic cell dysfunction in large arteries and resistant vessels. Endothelial dysfunction (ie, loss of vasodilation, vasoconstriction, thrombosis, and inflammation) occurs before and throughout the development of atherosclerosis and particularly during plaque rupture. Ox-LDL appears to induce this cellular dysfunction in a time- and concentration-dependent manner.9 Recent studies show that apoptosis, which is a common accompaniment of atherosclerosis, is induced by ox-LDL in cultured vascular smooth muscle cells,10 monocytes/macrophages,11 and human endothelial cells.12 The mechanisms of ox-LDL–mediated apoptosis, particularly in endothelial cells, and of its relation with LOX-1 have not been defined.
Nuclear factor (NF)-B, a transcription factor, regulates the
transcription of a variety of cellular genes, including injury response
and growth control.13 Activation of NF-B is inhibited
by antioxidant compounds, such as
N-acetyl-L-cysteine, pyrrolidine
dithiocarbamate,14 and vitamin E.15 It
has thus been proposed that NF-B is primarily an oxidative
stress–responsive transcription factor.14 15 Previous
studies have demonstrated that NF-B is activated in
accelerated and in advanced
atherosclerosis.16 Recent studies show
that NF-B activation plays a critical role in
apoptosis17 18 and that ox-LDL induces the
activation of NF-B in fibroblasts and in endothelial
and smooth muscle cells.19
Accordingly, we hypothesized that (1) ox-LDL upregulates its own
receptor, LOX-1; (2) ox-LDL–mediated apoptosis of cultured
HCAECs is associated with the action of LOX-1; and (3) NF-B
activation plays an important role in this process. The present
study was conducted to examine these hypotheses.
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Methods |
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Cell Culture
The initial batch of HCAECs was purchased from Clonetics Corp. The endothelial cells were pure, on the basis of morphology and staining for factor VIII and acetylated LDL. These cells were 100% negative for
-actin smooth muscle expression.
Microvascular endothelial growth medium consisted of
500 mL of endothelial cell basal medium, 5 ng of human
recombinant epidermal growth factor, 5 mg of hydrocortisone, 25 mg of
gentamycin, 25 µg of amphotericin B, 6 mg of bovine brain extract,
and 25 mL of FBS.
Fifth generation HCAECs7 12 were incubated with ox-LDL (40
µg/mL) for 24 hours to study the expression of LOX-1 as well as
NF-B activity. Several groups of HCAECs were incubated with
different concentrations of ox-LDL (10 to 100 µg/mL) for 24 hours to
observe apoptosis. HCAECs were preincubated with antisense to
LOX-1 mRNA (antisense LOX-1) or sense LOX-1 (0.5 µmol/L) for 48
hours. HCAECs were then incubated with ox-LDL (40 µg/mL) to determine
the expression of LOX-1. HCAECs incubated with antisense LOX-1 or sense
LOX-1 were also used to examine ox-LDL–mediated apoptosis and
NF-B activation. Parallel groups of HCAECs were incubated with 2
different chemical inhibitors of LOX-1 (250 µg/mL
polyinosinic acid or 250 µg/mL carrageenan) or NF-B
inhibitor (25 µg/mL caffeic acid phenethyl ester
[CAPE]) for 1 hour and then exposed to ox-LDL for 24 hours to observe
NF-B activity and apoptosis of HCAECs. The concentration of
these inhibitors was based on previous
studies.20 21
Preparation of Antisense LOX-1
Antisense phosphorothioate oligonucleotides
(ODNs) and sense phosphorothioate ODNs (as controls) directed to the
5'-coding sequence of the human LOX-1 mRNA were designed and
manufactured by Biognostik GmbH. The antisense LOX-1 was synthesized as
a 16-mer product (8 bases) targeted at 5'-CAG TTA AAT GAG GCC G-3'
of the LOX-1 sequence. The corresponding control (sense) was 16-mer (8
bases) targeted at 5'-ACC TAC GTG ACT ACG T-3'. Hereafter, the
antisense and sense to LOX-1 mRNA will be referred to as antisense
LOX-1 and sense LOX-1, respectively. Logarithmically growing
endothelial cells were transfected by adding ODNs into
culture medium according to the instructions of the manufacturer.
Recent experiments from our laboratory demonstrated that the uptake of
antisense LOX-1 was maximal, with 0.5 µmol/L after incubation
for 48 hours. Hence, the present study describes the use of a
0.5 µmol/L concentration of antisense ODNs.
Preparation of Lipoproteins
Native LDL and ox-LDL were prepared as described
earlier.12 In brief, human native LDL was isolated from
human blood plasma by discontinuous centrifugation. LDL
was oxidized by exposure to CuSO4 (5
µmol/L free Cu2+ concentration) in PBS at
37°C for 24 hours. The thiobarbituric acid–reactive substance
content of ox-LDL was 18.2±0.28 versus 0.56±0.16 nmol per 100 µg
protein in the native LDL preparation (P<0.01). LDL and
ox-LDL were kept in 50 mmol/L Tris-HCl, 0.15 mol/L NaCl, and
2 mmol/L EDTA at pH 7.4 and were used within 10 days of
preparation.
RT-PCR for LOX-1 mRNA Expression in HCAECs
Total RNA (1 µg) extracted from cultured HCAECs was
reverse-transcribed with oligo dT (Promega) and M-MLV reverse
transcriptase (Promega) at 37°C for 1 hour. Two microliters of the
reverse-transcribed material was amplified with Taq DNA polymerase
(Promega) by using a primer pair specific to human
endothelial receptor (sense primer,
5'-TTACTCTCCATGGTGGTGCC-3'; antisense primer,
5'-AGCTTCTTCTGCTTGTTGCC-3'). The polymerase chain reaction (PCR)
product was 193 bp. For PCR, 35 cycles were used at 94°C for 40
seconds, 55°C for 1 minute, and 72°C for 1 minute. The reverse
transcription (RT)-PCR–amplified samples were visualized on 1.5%
agarose gels by using ethidium bromide. ß-Actin was amplified as a
reference for quantification of LOX-1 mRNA. The signal intensity of
each LOX-1 mRNA band was normalized by that of ß-actin. The RT-PCR
product for LOX-1 was sequenced. The sequence of RT-PCR product
for LOX-1 in HCAECs was the same as the previously published sequence
for LOX-1.3 7
Western Blot Analysis for LOX-1 in HCAECs
HCAEC lysates from each experiment (30 µg per lane) were
separated by 10% SDS-PAGE and transferred to nitrocellulose membranes.
After incubation in blocking solution (4% nonfat milk, Sigma Chemical
Co), membranes were incubated with 1:100 dilution primary antibody
(monoclonal antibody to human LOX-1 was a gift of Dr Sawamura, National
Cardiovascular Center Research Unit, Osaka, Japan) overnight at
4°C. Membranes were washed and then incubated with secondary antibody
(1:2000 dilution, Amersham Life Sciences) for 1 hour, the membranes
were detected with the ECL system (Amersham Life Sciences), and
relative intensities of protein bands were analyzed by an
MSF-300G Scanner (Microtek Laboratory).7
Determination of Apoptosis
In Situ TUNEL and Propidium Iodide Staining
To detect DNA fragmentation in situ, terminal
deoxynucleotidyl transferase–mediated dUTP nick
end-labeling (TUNEL) was performed as previously
described.22 Briefly, the cells plated on slides were
fixed with 4% methanol-free formaldehyde, pH 7.4, for 25 minutes at
4°C and washed with PBS. The slides were incubated with 0.2% Triton
X-100 for 5 minutes on ice to increase cell permeability and were
equilibrated with terminal deoxynucleotidyl
transferase (TDT) buffer (including 30 mmol/L Tris-HCl, pH 7.2,
140 mmol/L sodium cacodylate, and 1 mmol/L cobalt chloride)
for 10 minutes at room temperature. The slides were covered with 0.3
U/µL TDT and 0.04 nmol/µL fluorescein-12-dUTP (Promega)
in TDT buffer for 60 minutes at 37°C. The slides were immersed in 2x
SSC buffer for 15 minutes at room temperature and then washed with PBS
to remove unincorporated fluorescein-dUTP. The slides were
immersed in 1 µg/mL of propidium iodide in PBS for 15 minutes at room
temperature and washed with deionized water. The slides were viewed
under a fluorescence microscope with green fluorescence
set at 520 nm and red fluorescence (of propidium iodide) set at
>620 nm. The negative controls were cells without TDT enzyme. The
positive controls were samples pretreated with DNase I.
DNA Fragmentation Gel Electrophoresis (DNA Laddering)
HCAECs were removed from culture dishes, washed twice with PBS,
and pelleted by centrifugation. Cell pellets were then
treated for 10 minutes with lysis buffer (1% NP-40 in 20 mmol/L
EDTA and 50 mmol/L Tris-HCl, pH 7.5). After
centrifugation for 5 minutes at 1600g, the
supernatant was collected, and the extraction was repeated with the
same amount of lysis buffer. The supernatants were brought to 1% SDS
and treated for 2 hours with RNase A (final concentration 5 µg/µL)
at 56°C, followed by digestion with proteinase K (final concentration
2.5 µg/µL) for 2 hours at 37°C. After addition of 1/2 vol of 10
mol/L ammonium acetate, the DNA was precipitated with 2.5 vol of
absolute ethanol. DNA was recovered by centrifugation
at 12 000g for 10 minutes and dissolved in gel loading
buffer. DNA was separated by electrophoresis in 1.6% agarose gel with
ethidium bromide.22
Preparation of Nuclear Extracts
Nuclear extracts were prepared as described
previously.22 Briefly, the cells were washed with 1 mL PBS
and resuspended in 100 µL hypotonic buffer (mmol/L: HEPES 10, pH 7.3,
KCl 10, MgCl2 1.5, dithiothreitol [DTT]
1, and phenylmethylsulfonyl fluoride [PMSF] 1). After
centrifugation, cells were lysed by resuspension in 300
µL lysis buffer (10 mmol/L HEPES, pH 7.3, 10 mmol/L KCl,
1.5 mmol/L MgCl2, 0.4% Nonidet P-40, 1
mmol/L DTT, 1 mmol/L PMSF, 1 µg/mL leupeptin, and 15 µg/mL
aprotinin). After a 10-minute incubation at 4°C, nuclei were
collected by centrifugation for 1 minute at
8000g, and the pellets were washed once in 1 mL of 20
mmol/L KCl buffer (20 mmol/L HEPES, pH 7.3, 22% glycerol, 20
mmol/L KCl, 1.5 mmol/L MgCl2, 0.2
mmol/L EDTA, 1 mmol/L DTT, 1 mmol/L PMSF, 1 µg/mL
leupeptin, and 15 µg/mL aprotinin). The isolated nuclei were
resuspended in 100 µL of 484 mmol/L KCl buffer (20 mmol/L
HEPES, pH 7.3, 22% glycerol, 484 mmol/L KCl, 1.5 mmol/L
MgCl2, 0.2 mmol/L EDTA, 1 mmol/L DTT,
1 mmol/L PMSF, 1 µg/mL leupeptin, and 15 µg/mL aprotinin).
Nuclear proteins were extracted by incubation on ice for 30 minutes.
After centrifugation for 15 minutes at
8000g, the supernatant containing nuclear proteins was
transferred to a precooled microcentrifuge tube, and nuclear
protein concentration was quantified.
Western Blot for NF-B
Monoclonal antibody to the P65 subunit of NF-B from mouse to
mouse hybrid cells was purchased from Boehringer-Mannheim. The
antibody recognizes an epitope overlapping the nuclear location signal
of the P65 subunit and, therefore, selectively binds the
activated form of NF-B. Equal amounts of protein (15 µg)
from the nuclear extract of each group were separated by 7.5% SDS-PAGE
and transferred to nitrocellulose filters (Sigma). The membrane was
blocked by incubating the membrane for 1 hour in Tris-saline buffer (pH
7.4) containing 3% nonfat milk (Sigma) and incubating the membrane in
the same buffer containing 7.5 µg/mL of monoclonal antibody to the
P65 subunit of NF-B. Anti-mouse alkaline phosphatase–conjugated
antibody was used as a secondary antibody at 1: 3000 dilution. The blot
was used for color development on the membrane. Sites of antigen
localization turn a dark purple color as a result of alkaline
phosphatase activity. Relative intensities of bands of interest were
analyzed by use of an MSF-300G Scanner (Microtek
Laboratory).7 12 22
Data Analysis
All data represent the mean of duplicate samples from 6
independently performed experiments. Data are presented as
mean±SD. Statistical significance was determined in multiple
comparisons among independent groups of data in which ANOVA and the F
test indicated the presence of significant differences. A value of
P<0.05 was considered significant.
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Results |
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Regulation of LOX-1 by Ox-LDL
A previous study from our laboratory showed that LOX-1 expression is upregulated by ox-LDL in a concentration-dependent manner, with maximal effect at 40-µg/mL.20 Therefore, a concentration of only 40 µg/mL ox-LDL was used in the present study. In keeping with the previous report, incubation of HCAECs with ox-LDL (40 µg/mL) for 24 hours increased LOX-1 mRNA (RT-PCR) and protein expression (Western analysis), whereas native LDL had no effect on the regulation of LOX-1. This upregulation of LOX-1 by ox-LDL was blocked by pretreatment of HCAECs with antisense LOX-1, but not with sense LOX-1. Notably, basal LOX-1 mRNA and protein were also suppressed by antisense LOX-1, but not by sense LOX-1 (Figure 1
).
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Ox-LDL and Apoptosis in HCAECs
Because a small number of cells normally die during culture
or are damaged during processing, 1% to 5% (3.8±1.6%) of control
cells stained positive on TUNEL staining. Cultured HCAECs incubated
with ox-LDL (10 to 100 µg/mL) for 24 hours showed a
concentration-dependent increase in the number of apoptotic
cells (from 6.8±3.0% to 48.4±4.2%). Ox-LDL also caused a
time-dependent (6- to 24-hour) increase in the number of
apoptotic cells (Figure 2). The
proapoptotic effect of ox-LDL was also confirmed by DNA
fragmentation on gel electrophoresis (see below).
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LOX-1 Inhibition and Apoptosis in HCAECs
Pretreatment of cells with antisense LOX-1 markedly decreased the
number of apoptotic cells in response to ox-LDL
(P<0.01). In contrast, sense LOX-1 had no effect on the
degree of apoptosis. These effects of antisense LOX-1 were
confirmed by TUNEL staining (Figure 3) and gel electrophoresis (Figure 4).
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The presence of 2 different chemical inhibitors of LOX-1,
polyinosinic acid and carrageenan, in the HCAEC culture medium before
the cells were exposed to ox-LDL also reduced the number of
apoptotic cells in response to ox-LDL (P<0.01).
These observations were confirmed by TUNEL staining as well as by DNA
laddering (Figure 4
).
LOX-1 Inhibition and NF-B Activation and Apoptosis
in HCAECs
Cultured HCAECs in normal conditions did not show NF-B
activation. Treatment of cells with ox-LDL, on the other hand, markedly
enhanced the activation of NF-B. Pretreatment of cells with
antisense LOX-1 for 48 hours before the cells were exposed to ox-LDL
significantly inhibited ox-LDL–mediated activation of NF-B (Figure 5). The chemical inhibitors
of LOX-1, polyinosinic acid and carrageenan, also inhibited the
ox-LDL–mediated activation of NF-B (P<0.05, Figure 5).
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The presence of CAPE, an inhibitor of NF-B, in the cell
culture medium before the cells were exposed to ox-LDL markedly reduced
the number of apoptotic cells in response to ox-LDL
(P<0.01), but alone, it had no effect on the magnitude of
apoptosis (Figure 6).
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Discussion |
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We have recently identified high-affinity LOX-1 in cultured HCAECs by RT-PCR, Western blot, and radioligand binding.7 20 Native LDL does not bind to this receptor. We have also demonstrated that angiotensin II enhances LOX-1 expression, uptake of ox-LDL, and injury to HCAECs.7 In the present study, we confirm that ox-LDL upregulates the expression of its own endothelial receptor, LOX-1. Additionally, we show that ox-LDL induces apoptosis of HCAECs via action of LOX-1. The critical role of LOX-1 in the genesis of apoptosis became evident in that a highly specific antisense to LOX-1 mRNA as well as 2 different chemical LOX-1 inhibitors attenuated the proapoptotic effect of ox-LDL. Last, these studies indicate that NF-
B activation is an important signal
transduction mechanism in LOX-1–mediated apoptosis, because
the antisense as well as the chemical inhibitors of LOX-1
prevented the ox-LDL–mediated activation of NF-B. The NF-B
inhibitor, CAPE, also significantly attenuated the
ox-LDL–induced apoptosis of HCAECs.
Regulation of LOX-1
Inflammatory stimuli, such as TNF- and phorbol
12-myristate 13-acetate, increase LOX-1 expression in a time-
and dose-dependent fashion in cultured bovine aortic
endothelial cells. Upregulation of the expression of
LOX-1 by TNF- and phorbol 12-myristate 13-acetate in bovine
endothelial cells is associated with an increase in the
uptake of ox-LDL.4 Fluid shear stress, which
activates endothelium, also upregulates LOX-1
expression by enhancing intracellular calcium
mobilization.5 In an in vivo study, Nagase et
al6 found that LOX-1 mRNA is markedly upregulated in the
aorta and vein of hypertensive rats, which may be the basis of impaired
endothelium-dependent vasodilation in these rats. In a
recent study,7 we demonstrated that LOX-1 expression is
upregulated by angiotensin II via activation of the Ang II
type 1 receptor and that the upregulation of LOX-1 is associated with
an increased uptake of ox-LDL by HCAECs and induction of cell injury.
In the present study, we confirm that ox-LDL upregulates its own
receptor LOX-1 mRNA and protein expression. These observations may be
the basis of the increased uptake of ox-LDL and
endothelial activation and dysfunction as plasma LDL
levels rise.
LOX-1 and Apoptosis
Nishio and Watanabe10 have suggested that ox-LDL, but
not native LDL, induces apoptosis in cultured rabbit vascular
smooth muscle cells. Yang et al11 have also shown that
ox-LDL induces apoptosis of human monocytes and
macrophages. Recent work from our laboratory12 has
shown that ox-LDL markedly augments
hypoxia/reoxygenation-mediated
apoptosis of cultured HCAECs. Ox-LDL decreases
endogenous superoxide dismutase activity and constitutive
nitric oxide synthase activity and increases free radical generation
beyond that caused by hypoxia/reoxygenation. In
the present study, we demonstrate that ox-LDL, per se, induces
apoptosis of cultured HCAECs in a concentration- and
time-dependent fashion. The importance of LOX-1 expression became
obvious in our experiments in which a specific antisense to LOX-1 mRNA
decreased ox-LDL–mediated apoptosis of HCAECs by 75%. In
other experiments, we found that 2 different nonspecific chemical
inhibitors of LOX-1, polyinosinic acid and carrageenan,
also decreased ox-LDL–mediated apoptosis of HCAECs by 62% and
72%, respectively. Notably these 2 LOX-1 inhibitors
prevent [125I]ox-LDL binding to LOX-1 by 60%
to 90%.4 20
Apoptosis in the present study was documented by 2 independent methods, TUNEL stating and gel electrophoresis. The results of the 2 methods were similar and complementary. We have shown previously that apoptosis in HCAECs in response to ox-LDL, as measured by TUNEL staining and DNA laddering, is associated with characteristic changes in markers of apoptosis, such as bcl-2 and Fas proteins.12
LOX-1 and NF-B Signal Transduction
The mechanism of ox-LDL–induced apoptosis continues
to be discussed. Nishio and Watanabe10 have shown that
oxysterols induce apoptosis in cultured smooth muscle cells
through CPP32 protease activation and bcl-2 protein downregulation.
Dimmeler et al23 have shown that ox-LDL induces
apoptosis of endothelial cells through
activation of CPP32-like proteases. Another study24 has
shown that ox-LDL increases cellular calcium to trigger
apoptosis. Recent work12 from our laboratory has
shown that ox-LDL changes the expression of certain genes associated
with apoptosis in endothelial cells, such as
bcl-2 and Fas. Concurrently, ox-LDL activates protein kinase C
and protein tyrosine K, and inhibitors of protein kinase C
and protein tyrosine K markedly reduce ox-LDL–mediated
apoptosis.
NF-B is an oncogene protein that regulates transcription of a
variety of cellular genes, including immune and inflammatory response
and growth control.13 NF-B is present in cytosol as
a heterodimer composed of NF-B1 (P50) and Rel (P65) subunits bound
to an inhibitor protein, I-B. After activation, NF-B
translocates from the cytosol to the nucleus of the cell, binds to
specific DNA sequences, and initiates transcription. Maziere et
al19 have shown that ox-LDL activates NF-B in
fibroblasts and endothelial and smooth muscle cells and
causes cell injury. Collins25 has suggested oxidative
activation of endothelial cell transcription factors,
especially NF-B, as a mechanism for changing
endothelial cell phenotype and for initiating
atherosclerotic lesions. Hernan dez-Presa et al16 have
also demonstrated NF-B activation in early
atherosclerosis. Other studies have shown a critical
role of NF-B activation in myocardial26 and
endothelial cell17 apoptosis. Some
studies have also shown NF-B as a proapoptotic signal in
human endothelial cells.27 It is possible
that NF-B activation plays different roles depending on the stimulus
and conditions for cell injury. In the present study, we
demonstrate that ox-LDL induces NF-B activation. The role of LOX-1
in this process appears to be important, because antisense LOX-1 and
chemical LOX-1 inhibitors significantly reduced NF-B
activation in cultured HCAECs. Importantly, we found that CAPE, an
inhibitor of NF-B,21 markedly attenuated
ox-LDL–induced apoptosis of HCAECs. Natarajan et
al21 demonstrated that CAPE completely blocks the
activation of NF-B induced by TNF- but that it has no effect on
other transcription factors, such as activator protein-1,
Oct-1, and TFIID. Although a few studies show that CAPE also
induces apoptosis in fibroblast cells,28 we did
not find evidence for a proapoptotic effect of CAPE. We propose
that ox-LDL via LOX-1 triggers a suicide pathway leading to
apoptosis in HCAECs and that this pathway is closely linked
with the activation of NF-B. It is possible that ox-LDL–induced
apoptosis may involve other signal transduction pathways, such
as the activation of CPP32-like protease and protein kinase C, because
the NF-B inhibitor did not completely block the effect
of ox-LDL.
In summary, ox-LDL upregulates the expression of its own receptor in
HCAECs. Ox-LDL also induces apoptosis in HCAECs via LOX-1 in
concert with upregulation of its receptor, and last, NF-B activation
plays an important role as a signal transduction mechanism in this
process. The confirmation of these concepts was obtained with the use
of a specific antisense to LOX-1 mRNA and 2 different chemical blockers
of LOX-1, polyinosinic acid and carrageenan, as well as CAPE, a
powerful NF-B inhibitor. These observations may have
important implications with regard to the propagation of
endothelial cell injury in the presence of ox-LDL.
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Acknowledgments |
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This study was supported by a Merit Review Award from the VA Central Office and an award from the Department of Defense, Washington, DC.
Received July 13, 1999; accepted December 1, 1999.
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