(Received for publication, November 6, 1995)
From the
Oxidized low density lipoprotein (LDL), a potent atherogenic
lipoprotein, has been shown to cause the alteration of various
endothelial functions. We have examined the effect of oxidized LDL on
the cofactor activity for thrombin-dependent protein C activation and
expression of thrombomodulin (TM), a cell surface antithrombotic
glycoprotein, on cultured human umbilical vein endothelial cells.
Oxidized LDL prepared by irradiation of LDL with 254-nm ultraviolet
light did not directly affect the cofactor activity of isolated TM.
Exposure of the cells to oxidized LDL (25-200 µg/ml), but not
native LDL and acetylated LDL, reduced TM cofactor activity in parallel
with its antigen levels on the cell surface in an oxidation-,
concentration- and time-dependent manner. TM mRNA levels were reduced
prior to decrease in TM antigen levels and were 50% of the control
levels at 3.0 h after treatment of the cells with oxidized LDL. The
apparent half-life time (t = 2.8 h) of TM
mRNA in the oxidized LDL-treated cells, however, did not significantly
differ from that (t
= 2.6 h) in the
control cells when the cells were coincubated with
5,6-dichloro-1-
-D-ribofuranosylbenzimidazole, a
transcriptional inhibitor. Treatment of the cells with bafilomycin A1,
an inhibitor for the proton pump of the lysosomes, inhibited
intracellular degradation of the LDL and prevented down-regulations of
the mRNA and the cell surface TM antigen levels caused by oxidized LDL.
The inhibitor molecule in oxidized LDL was shown to be a lipid; organic
solvent extracts (300 mg/ml cholesterol, an equivalent concentration
with lipids in 200 µg/ml oxidized LDL) of oxidized LDL inhibited
expression of TM antigen to nearly the same extent as the oxidized LDL,
although water extracts did not affect TM expression on the cells.
These results suggested that down-regulation of TM on endothelial cells
exposed to oxidized LDL resulted from inhibition of its transcription
mediated by lysosomal degradation of oxidized LDL and that a lipid
component in the LDL could be an active species. A decrease in TM
expression on the surface of endothelial cells may contribute to
promote thrombosis in atherosclerotic lesions.
Vascular endothelial cells play an active role in the regulation
of blood coagulation and fibrinolysis(1) . The
thrombin-thrombomodulin (TM)()-protein C pathway is of major
physiologic significance for the antithrombotic properties of
endothelial cells(2, 3, 4) . A key cofactor
in this pathway is TM, which is a high affinity receptor for thrombin
on the endothelial cell surface(2, 3, 4) .
The TM-thrombin complex acts as a potent activator for circulating
protein C. Activated protein C functions as an anticoagulant by
proteolytically degrading the coagulation factors Va and VIIIa, which
are essential for blood
coagulation(2, 3, 4) . The physiologic
relevance of this pathway for the antithrombotic properties of
endothelial cells is emphasized by the thrombotic disorders frequently
observed in patients with protein C-resistant factor V (5, 6) or protein C deficiency(7) .
The
prothrombotic properties of endothelial cells are enhanced in response
to bacterial endotoxin (8) and inflammatory cytokines such as
interleukin 1 (9, 10, 11) or tumor necrosis
factor-(10, 11, 12, 13, 14, 15, 16, 17) .
The major components of the increase in prothrombotic properties are a
decrease in the expression of
TM(8, 9, 10, 11, 12, 13, 14, 15, 16, 17) and
induction of the expression of tissue
factor(8, 9, 10, 12, 13, 17) ,
which functions as the cellular trigger for the coagulation
cascade(18) . The down-regulation of TM activity is due to
increased internalization of surface TM(11, 14) and/or inhibition of TM transcription (15, 16) by the inflammatory cytokines. The
combination of reduced TM activity and increased tissue factor
expression on the cell surface would be expected to promote coagulation
and contribute to thrombosis in inflammatory disorders, such as septic
shock, disseminated intravascular coagulation, and
malignancy(2, 19) . Homocysteine also down-regulates
TM activity on endothelial cells(20, 21) . Patients
with hereditary homocystinuria have high levels of plasma homocysteine
and suffer from frequent arterial and venous thrombosis(22) .
The down-regulation of TM in response to homocysteine is due to direct
inhibition of TM activity on the cell surface (20, 21) as well as inhibition of TM transport between
the endoplasmic reticulum and Golgi apparatus after
transcription(20) . Down-regulation of TM has been also
reported in endothelial cells exposed to hypoxic conditions (23) or cyclosporine A(24) , which is associated with
the development of post-transplantation glomerular thrombosis and
thrombotic microangiopathy. Thus, down-regulation of TM on endothelial
cells potentiates prothrombotic properties of the vascular wall.
Thrombosis is also frequently observed in atherosclerotic lesions. Low density lipoprotein (LDL) in plasma is a major risk factor for atherosclerosis, and the LDL can be oxidized by lipid peroxidation (25, 26) and accumulate in atherosclerotic lesions(27) . Oxidized LDL induces the expression of tissue factor (28) and reduces endothelial fibrinolytic activity through increased secretion of plasminogen activator inhibitor-1 and decreased secretion of tissue-type plasminogen activator(29) . The induction of tissue factor and increased secretion of plasminogen activator inhibitor-1 were mediated by increases in transcription levels(28, 29) . It was recently suggested that the cofactor activity of TM on the surface of endothelial cells also decreases in response to stimulation with oxidized LDL(30) . The decreased mechanism is, however, unknown with respect to whether oxidized LDL directly inhibits TM cofactor activity or decreases TM antigen levels on the cell surface. The potential mechanism for reduced TM activity on the surface of cultured human endothelial cells exposed to oxidized LDL is investigated in the present work.
The cofactor activity of TM isolated from human placenta was
measured by the method described previously(33) . TM (200
ng/ml) was incubated with or without 200 µg/ml oxidized or native
LDL in PBS for 24 h at 37 °C. After incubation, the solution was
diluted 10-fold in assay buffer, which consisted of 100 µg/ml
protein C, 2 NIH units/ml thrombin, 3 mM CaCl,
0.15 M NaCl, and 5 mg/ml BSA in 20 mM Tris-HCl (pH
7.4), and then the reaction mixture was further incubated for 30 min at
37 °C. Termination of protein C activation and quantification of
activated protein C were performed as described above.
In a similar fashion, the cells were washed 3 times
with 50 mM Tris-HCl (pH 7.4) containing 0.15 M NaCl
and 0.2% BSA and followed by two washes with 50 mM Tris-HCl
(pH 7.4) containing 0.15 M NaCl. The washed cells were
solubilized with 1% Triton X-100 for 1 h at 4 °C and then
centrifuged to remove insoluble material. Proteins in the solution were
precipitated with final 10% (w/v) trichloroacetic acid, and free
[I]iodide in trichloroacetic acid-soluble
fraction was removed by the method described above. The radioactivities
of the trichloroacetic acid-insoluble or -soluble fraction were counted
as undegraded or degraded oxidized LDL, respectively, in the cells.
Specific association and degradation of the LDL in the cells was
calculated by subtracting the radioactive count of each fraction
measured in the presence of a 20-fold excess amount of the unlabeled
oxidized LDL from the radioactivity of each fraction measured in the
absence of the unlabeled oxidized LDL.
Figure 1:
Oxidation of LDL by UV irradiation and
effects on endothelial cell surface TM cofactor activity. A,
human LDL was oxidized by UV irradiation (254 nm, 0.4
mW/cm) for various times. The degree of LDL oxidation was
evaluated by formation of TBARS and is expressed as nmol of
malondialdehyde/mg of protein. B, confluent endothelial cells
were incubated with LDL (100 µg/ml) irradiated for various periods
for 24 h. After incubation, TM-dependent activation of protein C on the
cell surface was measured by the method described under
``Experimental Procedures.'' The data shown are the mean
± S.D. from four independent
experiments.
When HUVECs were incubated with 200 µg/ml of oxidized LDL produced by 12-h UV irradiation, the cell surface TM cofactor activity did not significantly change with a 1-h incubation, but a time-dependent decrease in TM cofactor activity was observed by 3 h, and the level declined to 30% of that of the control by 24 h (Fig. 2A). When the cells were incubated for 24 h with various concentrations of oxidized LDL, the cofactor activity decreased with increasing concentration of oxidized LDL, and the level was about 30% of that of the control at 200 µg/ml of the oxidized LDL (Fig. 2B). Native or acetylated LDL had no effect on surface TM activity within the 24-h incubation (Fig. 2A). When 200 ng/ml of isolated human placental TM was incubated with 200 µg/ml oxidized LDL for 24 h at 37 °C, no change in the cofactor activity (TM alone, 37.8 ± 7.2 pmol of protein C activated/min/nmol of TM; incubation with oxidized LDL, 41.8 ± 2.5 pmol of protein C activated/min/nmol of TM) was observed. These results indicated that oxidized LDL reduced surface TM activity on endothelial cells in a time- and concentration-dependent manner independent from the direct affect of the LDL on the TM molecule.
Figure 2:
Changes in TM cofactor activity on the
surface of endothelial cells exposed to oxidized LDL. A,
confluent endothelial cells were incubated for various times with 200
µg/ml native LDL (), acetylated LDL (
), or oxidized LDL
produced by UV irradiation of LDL for 12 h (
). B, cells
were incubated for 24 h with various concentrations of oxidized LDL,
which was produced by UV irradiation of LDL for 12 h. The cofactor
activity of TM on the cell surface was measured by the method described
under ``Experimental Procedures.'' The data shown is the mean
± S.D. from four independent
experiments.
Figure 3:
Effect of oxidized LDL on surface and
total TM antigen levels in endothelial cells. Oxidized LDL was produced
by UV irradiation of LDL for 12 h. A, confluent endothelial
cells were incubated with 200 µg/ml native LDL (,
) or
oxidized LDL (
,
) for various times. TM antigen levels on
the surface (
,
) and in the cell lysate (
,
) in
the cells were measured by the methods described under
``Experimental Procedures.'' B, cells were incubated
with various concentrations of oxidized LDL for 24 h, and then TM
antigen levels on the cell surface (
) and in the cell lysate
(&cjs2090;) were measured. The data represent the mean ± S.D.
from four independent experiments.
A decrease in TM antigen on endothelial cells incubated with oxidized LDL was also observed by immunocytochemistry using polyclonal rabbit anti-human TM IgG (Fig. 4A and B). In this experiment, when preimmune rabbit IgG was used instead of rabbit anti-human TM IgG in the immunocytochemical detection, no fluorescence was observed in the preparations (Fig. 4C), indicating that the immunocytochemical detection of TM was not dependent on nonspecific binding of IgG.
Figure 4: Immunofluorescence detection of TM antigen on the surface of endothelial cells. Oxidized LDL was produced by UV irradiation of LDL for 12 h. Confluent endothelial cells were treated with or without 200 mg/ml oxidized LDL for 24 h. After incubation, untreated cells (A) and oxidized LDL-treated cells (B) were fixed and then incubated with rabbit anti-human TM IgG. Antibody localization was detected with goat fluorescein isothiocyanate-conjugated anti-rabbit IgG as described under ``Experimental Procedures.'' Parallel experiments using preimmune rabbit IgG were performed to assess nonspecific immunofluorescence (C).
Figure 5: Changes in TM mRNA levels in endothelial cells exposed to oxidized LDL. Oxidized LDL was produced by UV irradiation of LDL for 12 h. Confluent endothelial cells were treated with or without 200 mg/ml oxidized LDL for various times, and total RNA was extracted from the cells (A). TM mRNA levels were analyzed by Northern blot as described under ``Experimental Procedures.'' The migration of the ribosomal RNA is indicated on the right. B, the autoradiogram of panel A was analyzed by densitometry, and the data are expressed as the percentage of the control values after normalization to the actin mRNA signal.
The half-life of TM mRNA in cells treated with or without oxidized LDL was measured using DRB, a transcription inhibitor, to investigate the effect of oxidized LDL on TM mRNA stability (Fig. 6). The levels of TM mRNA detected by Northern blotting were analyzed by densitometry (Fig. 6, D, E, and F). TM mRNA levels in the cells treated with 65 µM DRB alone decreased in a time-dependent manner, and the apparent half-life of TM mRNA was 2.6 ± 0.2 h (Fig. 6D). The half-life of TM mRNA in cells treated with oxidized LDL alone was 3.0 ± 0.3 h (Fig. 6E). The half-life in cells treated with oxidized LDL and DRB also did not differ significantly and was calculated at 2.8 ± 0.2 h (Fig. 6F).
Figure 6:
The
half-life of TM mRNA in endothelial cells treated with DRB and/or
oxidized LDL. Oxidized LDL was produced by UV irradiation of LDL for 12
h. Confluent endothelial cells were treated with 65 mM DRB
alone (A and D), 200 mg/ml oxidized LDL (B and E), or DRB plus oxidized LDL (C and F) for various times. Total RNA was isolated from the cells,
and Northern blot analysis was performed as described under
``Experimental Procedures.'' The autoradiogram was analyzed
by densitometry, and the data are expressed as percentages of control
after normalization to actin mRNA levels (D, E, and F). Each symbol (,
, and
)
represents three different experiments. The half-life (t
) for each condition was calculated by linear
regression analysis from semilog plots of the mRNA remaining versus incubation times and is reported as mean ±
S.D.
Figure 7:
The
effect of bafilomycin A1 on TM mRNA and antigen levels in endothelial
cells exposed to oxidized LDL. Oxidized LDL was produced by UV
irradiation of LDL for 12 h. Confluent endothelial cells were treated
with or without 1 mM bafilomycin A1 and/or 200 µg/ml
oxidized LDL. Bafilomycin A1 was added at 1 h before treatment of the
cells with oxidized LDL and coincubated with oxidized LDL for 6 h. A, after incubation, total RNA in the cells was isolated as
described under ``Experimental Procedures.'' RNA (10 mg) was
blotted on a nylon membrane and hybridized with P-labeled
TM or actin cDNA probes. B, after incubation, the surface TM
antigen (
) and total TM antigen in cell lysate (&cjs2090;) of
the cells were measured by the methods described under
``Experimental Procedures.'' The data represent the mean
± S.D. from four independent experiments and are analyzed by the
Student t test, *, p < 0.01 when compared with
control.
To
investigate the degradation rate of oxidized LDL, cells were incubated
with I-oxidized LDL for 2 h at 4 °C and then
incubated with or without bafilomycin A1 for 1 h at 4 °C, and the
LDL was chased with unlabeled oxidized LDL and/or bafilomycin A1 at 37
°C (Fig. 8). Culture medium and the cells were collected
after the indicated incubation time, and radioactivity recovered in
each trichloroacetic acid-soluble and -insoluble fraction was
determined. At zero time, the trichloroacetic acid-insoluble
radioactivity associated with the cells was the same for cells treated
with oxidized LDL or oxidized LDL plus bafilomycin A1. The
trichloroacetic acid-insoluble radioactivity (210 cpm; 11.7 ng of LDL
protein/mg of cellular protein) associated with the cells decreased
with time with a concomitant increase in trichloroacetic acid-soluble
radioactivity in the culture medium. An increase of 120 cpm of
trichloroacetic acid-soluble radioactivity (6.8 ng of LDL protein/mg of
cellular protein) was observed in the medium at 4-h incubation of the
cells at 37 °C. At 4 h, the increase in trichloroacetic
acid-soluble radioactivity was inhibited by 70% in cells treated with
bafilomycin A1. Trichloroacetic acid-soluble radioactivity associated
with the cells and trichloroacetic acid-insoluble radioactivity in
culture medium were negligible (data not shown). Thus, this result
indicates that about 60% of the bound
I-oxidized LDL was
degraded and released to culture medium as trichloroacetic acid-soluble
materials, and treatment of the cells with bafilomycin A1 inhibited the
degradation.
Figure 8:
The
effect of bafilomycin A1 on the degradation of I-oxidized
LDL in endothelial cells. Confluent endothelial cells were incubated
with 10 µg/ml
I-oxidized LDL at 4 °C for 2 h. The
unbound ligand was removed, and the cells were incubated in the absence
(
) or the presence of 1 µM bafilomycin A1 (
).
After incubation for 1 h at 4 °C, cells were incubated at 37 °C
for the indicated times in the absence (
) or the presence of 1
µM bafilomycin A1 (
). A, at the end of
incubation, conditioned medium was collected, and the radioactivity of
the trichloroacetic acid-soluble fraction was counted as the amount of
degraded
I-oxidized LDL as described under
``Experimental Procedures.'' B, cells were
solubilized, and the radioactivity in the trichloroacetic
acid-insoluble fraction was counted as the amount of cell associated
I-oxidized LDL as described under ``Experimental
Procedures.'' The data shown are the mean of two different
experiments.
Figure 9: The effect of organic or aqueous extraction of oxidized LDL on TM antigen levels on the surface of endothelial cells. The organic solvent-soluble and water-soluble components of native LDL or oxidized LDL were separated by the method described under ``Experimental Procedures,'' using chloroform, methanol, and water. Organic solvents in the chloroform fraction were evaporated, and the residual material was dispersed by sonication in DMEM containing 10% FCS. The aqueous extracts were concentrated by evaporation and dissolved in DMEM containing 10% FCS. All concentrations were adjusted to an equivalent concentration with lipid or protein in 200 µg protein/ml oxidized LDL. Confluent cultures of endothelial cells were incubated for 24 h in the absence or presence of native LDL (N-LDL), oxidized LDL (Ox-LDL), organic solvent extract of native (N-Lipid) or oxidized LDL (Ox-Lipid), or aqueous extract of native (N-Aq.) or oxidized LDL (Ox-Aq.). After incubation, TM antigen levels on the cell surface were measured by the method described under ``Experimental Procedures.'' Data represent the mean ± S.D. from three independent experiments.
The present study has characterized the effect of oxidized
LDL on the cofactor activity and expression of TM and TM mRNA levels in
endothelial cells. Weis et al.(30) suggested that TM
cofactor activity for thrombin-dependent protein C activation on the
surface of endothelial cells decreased in response to oxidized LDL
prepared by incubation of LDL with CuSO. The present work
used oxidized LDL prepared by UV irradiation of LDL, because the UV
irradiation method can control the extent of oxidation. We also
observed a decrease in TM cofactor activity on the surface of cells
treated with oxidized LDL prepared by UV irradiation. The present work
further demonstrates that the extent of reduction in the TM cofactor
activity progressed with increasing oxidation levels of the added LDL.
The decrease in TM cofactor activity could be a specific response in
the cells exposed to oxidized LDL, since acetylated LDL and native LDL
did not affect the activity. It was reported that Met
in
TM was oxidized by active neutrophil products and that the TM cofactor
activity subsequently decreased(50) . In our experience,
however, the cofactor activity of TM isolated from human placenta was
not directly influenced by incubation with oxidized LDL, and the
surface cofactor activity on cells treated with oxidized LDL was
reduced in parallel with TM antigen levels (Fig. 2, 3, and 4).
These results indicate that the decrease in TM cofactor activity on the
surface of cells exposed to oxidized LDL is predominantly due to
down-regulation of TM antigen levels on the cell surface irrespective
of direct oxidation of the TM molecule.
Although significant changes in TM antigen levels on the cell surface and in the cell lysate were not observed at 1 h after treatment with oxidized LDL, a time-dependent decrease in each was observed from 3 h after the treatment. In contrast, TM mRNA levels had already decreased to about 80% of control values by 1 h after treatment with oxidized LDL, and a progressive decrease in TM mRNA levels was observed earlier than decreases in TM antigen levels. There was no increase in TM antigen levels in the culture medium in oxidized LDL-treated cells. These results suggested that the down-regulation of TM on oxidized LDL-treated cells could be predominantly dependent on reduced TM biosynthesis in the cells during decline of TM mRNA. The apparent half-life of TM mRNA in the cells treated with oxidized LDL plus DRB, a transcription inhibitor, was 2.8 h and did not significantly differ from the 2.6 h observed in the control cells treated with DRB alone or the 3.0 h in the cells treated with oxidized LDL alone. These results suggest that oxidized LDL did not affect the stability of TM mRNA and repressed TM biosynthesis through inhibition of TM transcription in the cells. The apparent half-life of TM mRNA in control cells observed in the present work was comparable with that reported previously(16) . The 5`-flanking promoter region of human TM contains two regions with a DNA binding sequence (CCGCCCC) for an inhibitory transcription factor from -842 to -836 base pair and from -1269 to -1262 base pair(51) . It is not known whether oxidized LDL repressed TM transcription through activation of this mechanism or through other unidentified elements.
Oxidized LDL is internalized after binding
with the scavenger receptors on the surface of endothelial cells and
degraded by the lysosomal pathway(46) . The decreased surface
TM in endothelial cells exposed to inflammatory cytokines, such as
tumor necrosis factor-, were postulated to be due to increases in
TM degradation through the lysosomal pathway and/or decreased TM
transcription(11, 14, 15, 16) .
Therefore, the present work was designed to address the potential
contribution of lysosomal function to the down-regulation of TM in
oxidized LDL-treated cells. It is known that bafilomycin A1, a specific
inhibitor for vacuolar-type H
-ATPase, inhibits the
acidification of lysosomes(52) . Bafilomycin A1 inhibits the
degradation of oxidized LDL but does not affect on the internalization
of oxidized LDL and its transport into the lysosomes(47) .
Treatment of endothelial cells with bafilomycin A1 alone did not affect
TM mRNA levels or surface TM antigen levels but significantly increased
total TM antigen levels. Thus bafilomycin A1 did not interfere with
internalization of the surface TM, but it inhibited degradation of TM
incorporated in the lysosomes. The present result supports previous
studies showing that surface TM on endothelial cells is degraded
through the lysosomal pathway under steady state
conditions(11, 14) . Coincubation of the cells with
oxidized LDL and bafilomycin A1 prevented the decrease in surface and
total TM antigen levels. If the lysosomal degradation of surface TM was
accelerated by oxidized LDL, then the decrease in surface TM levels
could be not prevented even if the oxidized LDL-treated cells were
coincubated with bafilomycin A1, because this compound did not inhibit
internalization. This result implied that oxidized LDL did not
accelerate TM degradation through the lysosomal pathway. The present
work further indicates that bafilomycin A1 inhibited lysosomal
degradation of oxidized LDL and the decrease in TM mRNA levels induced
by oxidized LDL. Thus it was suggested that down-regulation of TM
antigen in the cells exposed to oxidized LDL was predominantly due to
inhibition of TM transcription and that the inhibition of TM
transcription was mediated by degradation of oxidized LDL through the
lysosomal function.
There is a variable distribution of scavenger
receptors for modified LDL, including acetylated and oxidized LDL, on
the surface of several cell types. Expression of the class A scavenger
receptors (type I and II) was demonstrated on the surface of
macrophages, and the receptors mediated internalization of the modified
LDL(53) . We examined the effect of dextran sulfate or
fucoidin, antagonists for the class A scavenger receptors, on
down-regulation of TM caused by oxidized LDL and found no effect (data
not shown). Therefore, internalization of oxidized LDL to induce the
down-regulation of TM may be not mediated by the class A scavenger
receptors. Naito et al.(54) recently demonstrated
that internalization of the modified LDL in endothelial cells was not
mediated by class A receptors and suggested that the cells may have
other types of scavenger receptor. Acton et al.(55) have cloned the cDNA for a scavenger receptor, which
is a new member of the CD36 family of membrane proteins (class B
scavenger receptors), and the receptors are expressed in a variety of
cells, including macrophages and endothelial cells(56) . In
addition to class B scavenger receptors, De Rijke et al.(57) recently reported that a 95-kDa oxidized
LDL-specific receptor was expressed in Kupffer and endothelial cells.
Since antagonists for these scavenger receptors are not available, we
could not examine whether the down-regulation of TM in oxidized
LDL-treated cells was mediated by the class B scavenger receptors or
the
95-kDa receptor.
Changes in the LDL particle associated
with oxidative modification include lipid peroxidation, formation of
conjugated dienes, carbonyl modification of apoB-100, and conversion of
phosphatidylcholine to lysophosphatidylcholine by phospholipase A in apoB-100 (25, 26) . It has been shown that
organic extracts of oxidized LDL interfered with endothelial cell
functions(29) . We found that TM expression on endothelial
cells is negatively regulated by the degree of LDL oxidation and that
organic extracts of oxidized LDL reduced TM antigen levels on the
surface of cells to nearly the same extent as intact oxidized LDL, but
aqueous extracts of oxidized LDL did not. Therefore, it is possible
that the down-regulation of TM was mediated by lipid components of
oxidized LDL degraded in the lysosomes. We have investigated the effect
of native LDL or acetylated LDL treated with phospholipase
A
, lysophosphatidylcholine (palmitoyl- or
stearoyl-lysophosphatidylcholine), or oxysterol (25-hydroxycholesterol
or 7-ketocholesterol) on the TM antigen levels, although no affect was
observed (data not shown). The active lipid component(s) of oxidized
LDL that causes the down-regulation of TM remains to be determined.
Oxidized LDL induces reduction of tissue-type plasminogen activator release(29) , increase in plasminogen activator inhibitor-1 release (29) , and tissue factor expression (28) in endothelial cells. Therefore, oxidized LDL disturbs the balance between anti- and prothrombotic factors in endothelial cells. Decreases in TM expression on the surface of endothelial cells caused by oxidized LDL may further contribute to promotion of thrombosis in atherosclerotic lesions.