From the University of Minnesota School of Dentistry, Department of Oral Sciences, Minneapolis, Minnesota 55455
Received for publication, November 12, 2002, and in revised form, January 21, 2003
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ABSTRACT |
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Tissue factor (TF) initiates the
extrinsic coagulation cascade on the surface of macrophages and
endothelial cells. In septic patients, the extrinsic coagulation
cascade is activated. When septic patients are febrile, mortality is
decreased. The purpose of this study was to investigate the role of
elevated temperatures on TF expression by endothelial cells during a
sepsis-like challenge. Human endothelial vein cells (HUVECs) were
incubated with lipopolysaccharide (LPS) or interleukin-1 Disseminated intravascular coagulation
(DIC)1 is a pathological
condition precipitated by sepsis, trauma, or certain cancers (1-4). In
DIC the coagulation system activates, promoting fibrin deposition in
the microvasculature leading to thrombosis, organ failure, depletion of
coagulation factors, and uncontrolled bleeding (5). Tissue factor (TF)
is involved in the development and progression of DIC during sepsis;
however, its precise role is unknown (3, 6-9).
Fever is a physiological response that benefits the host during
experimental infections and is correlated with improved patient survival (10, 11). Fever is characterized by the generation of acute
phase proteins, activation of the immune response, and cytokine-mediated core temperature rise (12). The effects of elevated
core temperature were modeled in rodents given a lethal dose of LPS
(14). Heat-stressed rodents challenged with endotoxin express heat
shock proteins, which positively correlate with survival (13, 14). The
data suggest that heat stress and/or heat shock protein induction are
important in survival during sepsis. Little is understood, however,
about the molecular mechanism(s) that may explain protection.
Heat shock inhibits cytokine- and endotoxin-mediated NF- Cell Culture, Heat Shock, and LPS--
HUVECs were isolated as
described previously (18, 19). HUVECs, which were provided by Dr.
Gregory Vercellotti (Dept. of Medicine, University of Minnesota), were
grown in modified Eagle's media 199 containing 10% fetal bovine serum
(Invitrogen), 4.7 mM L-glutamine, 1 mM sodium pyruvate, 100 µg/ml penicillin/streptomycin, 25 µg/ml ampicillin (Invitrogen), 5 units/ml heparin sulfate
(Sigma), and 50 µg/ml ENDOGROTM (VecTechnologies, Rensselaer, NY) at
37 °C in 5% CO2. HUVECs from passages 1 to 4 were used
in all experiments. To induce heat shock, HUVEC-containing flasks were
immersed in a water bath equilibrated in a 43, 41.5, or 40 °C
incubator. Some HUVEC cultures were stimulated with Escherichia
coli LPS serotype 55:B5 (Sigma) or IL-1 RNA Preparation--
Total RNA was isolated from HUVEC
monolayers by the guanidium-phenol extraction method with TRIzolTM
reagent (Invitrogen) according to the manufacturer's protocol (20).
Cell suspension/TRIzol was stored at Generation of Radioactive Antisense cRNA
Probes--
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), GAPDH
short product (GAPDHs), heat shock protein 72 (HSP72), and TF-purified polymerase chain reaction products were ligated into linearized plasmids that contained T3 and T7 RNA polymerase promoters
(PCR-ScriptTM; Amp Cloning Kit, Stratagene). The primers used to
generate PCR products were as follows: GAPDH
(5'-CGGAGTCAACGGATTTGGTCGTAT-3', 5'-AGCCTTCTCCATGGTGGTGAAGAC-3',
PCR product length 307 bp); GAPDHs (5'-GACCCCTTCATTGACCTCAACTAC-3',
5'-AGCCTTCTCCATGGTGGTGAAGAC-3', PCR product length 222 bp); TF
(5'-GACAATTTTGGAGTGGGAACCC-3', 5'-CACTTTTGTTCCCACCTG-3', PCR product
length 310 bp); HSP72 (5'-CTCCAGCATCCGACAAGAAGC-3', 5'-ACGGTGTTGTGGGGGTTCAG-3', PCR product length 234 bp).
Authenticity and orientation were confirmed by nucleotide
sequencing. Using a combination of primers specific for each gene
product and phage promoter flanking region, PCR products were generated
containing T7 or T3 promoters. The flanking region primers were as
follows: T7F, 5'-GGTAACGCCAGGGTTTTCCCAG-3'; and T3R,
5'-TCCGGCTCGTATGTTGTGTGGA-3'. Amplified PCR product size and
purity were confirmed by gel electrophoresis. [ Ribonuclease Protection Assay--
RPA was performed using the
Ambion RPA II kit (Ambion) according to the manufacturer's
instructions. Total RNA was mixed with 32P-labeled
antisense TF or HSP72 and GAPDH or GAPDHs probes. The RNA/probe mixture
was hybridized overnight at 42 to 45 °C and then incubated with 1:50
RNase solution (T1/A) for 30 min at 37 °C. Protected RNA products
were precipitated and then separated by electrophoresis on 5%
acrylamide gel containing 8 M urea (60 min at 200 V). Gels
were exposed to a PhosphorImager (Amersham Biosciences) for
20-24 h.
Analysis of mRNA--
To quantify mRNA, the
phosphorimage of an exposed gel was scanned with a Storm System 840 scanner; ImageQuaNTTM analysis software (version 4.2a) was used to
circumscribe the "protected" mRNA fragment and analyze the
density of each pixel within each "box." After correction for
background, relative abundance of TF- and HSP72-specific mRNAs was
quantified as the ratio to GAPDH or GAPDHs (internal controls) within
each sample.
Flow Cytometry--
HUVECs were analyzed for surface TF
expression by flow cytometry (BD Biosciences). Trypsin/EDTA solution
was used to disrupt cell monolayers. Preliminary experiments were
preformed to assess the effect of trypsin on the detection of surface
TF. Two monolayer disruption protocols were compared. The first used
EDTA to disrupt HUVEC monolayers, and the second protocol used a
trypsin/EDTA combination. The trypsin/EDTA protocol produced no
detectable differences in TF surface expression compared with EDTA
treatment when assessed by flow cytometry (data not shown). Because TF
was unaffected and the protocol disrupted HUVEC monolayers more
efficiently, the trypsin/EDTA protocol was used for the flow cytometry
experiments. HUVEC monolayers were washed with PBS containing 0.5 mM EDTA for 2 min. The supernatant wash was decanted, and
HUVEC monolayers were dispersed by incubation with 0.5 mM
EDTA/0.017% trypsin for 1 min. Dispersed HUVECs were washed with cold
PBS containing 10% (v/v) FBS, centrifuged at 400 rpm for 5 min at
4 °C, and counted in a hemocytometer after resuspension in 250 µl
of PBS containing 2% (v/v) FBS. HUVECs (5 × 105)
were incubated with 8 µg/ml anti-TF or IgG isotype control antibodies for 30 min at 4 °C, washed with 25× the incubation volume with PBS
containing 2% FBS, and incubated with an anti-isotype secondary antibody labeled with fluorescein isothiocyanate (FITC, Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min at 4 °C. To
assess nonspecific binding, some HUVECs were washed and resuspended in
300 µl of PBS containing 2% FBS and incubated with the secondary antibody only. To assess HUVEC viability, propidium iodine (1 µg/ml
final concentration) was added to each cell suspension prior to flow cytometry.
Two-stage TF Activity Assay--
HUVEC monolayers were washed
with PBS containing 0.5 mM EDTA for 2 min. Supernatants
were removed, and the monolayers were disrupted by incubation with 0.5 mM EDTA/0.017% trypsin for 1 min. To inhibit trypsin
activity, HUVEC suspensions were washed with cold Tris-HCl, pH 7.4, containing 10% (v/v) FBS. HUVECs (6 × 105) were
pelleted, washed with buffer, and then incubated with 15 nM
factor VIIa (Clinical Enzyme Laboratories, South Bend, IN) and
transferred to 96-well microtiter plates. Factor X (Clinical Enzyme
Laboratories, South Bend, IN) and S-2222 (DiaPharma Group Inc,
West Chester, OH) were added to each cell suspension at final concentrations of 300 µM and 1.4 µg/ml, respectively.
Cleavage of S-2222 by factor Xa was detected by a change in absorbance at Statistical Methods--
Repeated measures analysis of variance
(ANOVAs) was applied in which HUVECs were "subjects," and the
within-subject fixed effects were heat shock (present or absent), time
(treated as categories), and ionomycin (present or absent). The one
exception is the analysis supporting Fig. 3I, which included
the between-subject factors heat shock and temperature. All
post hoc tests used the Bonferroni correction to
maintain an alpha (type I error rate) of 0.05.
LPS-stimulated HUVECs Express Detectable TF-specific but not
HSP72-specific mRNAs--
Confluent HUVEC monolayers were
incubated with 0.1 µg/ml LPS for various times. At each time point,
cells were harvested, and total RNA was isolated and analyzed for TF-,
HSP72-, and GAPDH-specific mRNA expression by an RPA. Fig.
1A is a representative
phosphorimage from a time course experiment. The right
panel shows ribonuclease digestion of the unbound RNA target
probe. In the left panel, TF-specific mRNA
expression by HUVECs appeared to maximize at 2 h of
LPS-stimulation and return to near baseline levels at 6 h. The
increase in the TF-specific message was significant at 1, 2, and 4 h when compared with non-stimulated HUVECs and maximized at 2 h
(Fig. 1B). To determine the LPS dose that would maximally stimulate TF-specific mRNA expression, confluent HUVEC monolayers were incubated for 2 h with each LPS concentration. LPS-stimulated HUVECs did not express detectable quantities of HSP72-specific mRNA
at any time or concentration tested (data not shown). LPS concentrations from 0.01 to 10 µg/ml induced TF-specific mRNA expression, which was maximal at 0.1 µg/ml (Fig. 1, C and
D).
Heat-shocked HUVECs Induce Expression of HPS72 but Not TF-specific
mRNAs--
HUVEC cultures were heat shocked at 43 °C over time.
At the times indicated, total RNA was isolated and analyzed for
expression of HSP72-, TF-, and GAPDH-specific mRNA. HUVEC cells
expressed HSP72-specific mRNA, which appeared to peak at 2 h
(Fig. 2A). Heat-shocked
HUVECs did not express detectable quantities of TF-specific mRNA (data not shown).
Heat-shocked, LPS-stimulated HUVECs Express Reduced TF-specific
mRNA--
Based upon the optimization experiments, a standard
protocol was used unless noted (Fig. 2B). LPS (0.1 µg/ml)
was added, and HUVEC cultures were heat shocked at 43 °C for 2 h and then re-equilibrated at 37 °C for up to 4 h (Fig.
2B). To determine the effect of heat shock on TF mRNA
expression, LPS was incubated with HUVEC monolayers in the presence or
absence of heat shock. LPS induced TF-specific mRNA in heat-shocked
and non-heat-shocked HUVECs (Fig. 2C). Heat-shocked HUVECs
express HSP72-specific mRNA (Fig. 2C, right
panel). At 2 h, heat shock significantly reduced
expression of TF-specific mRNA by LPS-stimulated HUVECs when
compared with non-heat shocked, LPS-stimulated HUVECs (Fig.
2D). After 2 h of incubation with LPS, cells were
allowed to re-equilibrate at 37 °C. The reduction in the expression
of TF-specific mRNA caused by heat shock was not apparent (Fig.
2D).
Immunoreactive TF (iTF) Surface Protein Expression Reduced by Heat
Shock--
To determine whether the surface iTF expression of heat
shocked, LPS-stimulated HUVECs paralleled the decrease in TF-specific mRNA expression, monolayers were stimulated with LPS or IL-1
Because reduced HUVEC viability caused by heat shock could explain the
reduction of surface iTF, monolayers were assessed by propidium iodine
dye exclusion. Heat-shocked and non-heat-shocked LPS-stimulated HUVECs
maintained similar viability over time (Fig. 3H). To
determine whether a temperature between 37 and 43 °C depressed TF
expression, LPS-stimulated HUVECs were heat shocked at 40 and 41.5 °C. At temperatures between 37 and 43 °C, smaller reductions in iTF surface expression were detected (Fig. 3I).
TF Surface Activity Is Reduced by Heat Shock--
To determine
whether the reduction in iTF surface expression by heat shock was
functional, TF/factor VIIa activity was estimated as factor Xa
generation by a serum-free, two-stage clotting assay. LPS-stimulated
HUVECs were either maintained at 37 °C or heat shocked for 2 h
and then allowed to recover for 2 h at 37 °C. Other cultures
were heat shocked without LPS. Equal numbers of HUVECs were analyzed
for TF activity. Factor Xa generation was largely inhibited by heat
shock (Fig. 4A). To show that
the generation of factor Xa required iTF, anti-TF antibodies or isotype
controls were added to the LPS-stimulated HUVECs before the addition of factor X and S-2222 (Fig. 4B). Inhibition by anti-TF
antibodies confirmed that factor Xa generation was
TF-dependent. To determine whether the heat shock-induced
reduction in surface activity might be due to the encryption of
TF, ionomycin was added to LPS-stimulated heat-shocked and
non-heat-shocked HUVECs. TF surface activity decreased significantly in
heat-shocked, LPS-stimulated HUVECs independent of the treatment with
ionomycin (Fig. 4C). Although total TF activity increased,
ionomycin did not substantially affect the ratio of TF surface activity
in heat-shocked and non-heat-shocked LPS-stimulated HUVECs.
In this study, we hypothesized that LPS-stimulated endothelial
cells modulate TF when heat shocked. During LPS stimulation at
37 °C, TF-specific mRNA expression maximized in HUVECs at 2 h and returned to near baseline by 6 h (Fig. 1, A and
B). For the first time, we showed that heat shock
significantly reduced TF-specific mRNA expression during 2 h
of LPS stimulation when compared with non-heat-shocked, LPS-stimulated
cells (Fig. 2, C and D). To determine whether TF
surface protein levels paralleled the decrease TF mRNA, we
performed flow cytometry experiments. The largest number of surface iTF
(+) HUVECs were detected at 4 h of LPS-stimulation (Fig.
3F). Heat shock significantly reduces surface iTF (+) HUVECs
after stimulation with LPS for 2, 4, 6, or 8 h (Fig.
3F). Regulation of the expression of TF by heat shock may be
under the control of the nuclear transcription factor NF- Heat shock regulation of TF expression was not specific to LPS. Flow
cytometry experiments were repeated using IL-1 TF surface expression was reduced on heat-shocked LPS- or
IL-1 (IL-1
)
for 0, 2, 4, 6, or 8 h. At the 0-h time point, some HUVECs were
heat shocked at 43 °C for 2 h and then recovered at 37 °C
for 0, 2, 4, or 6 h. Heat-shocked and non-heat-shocked
LPS-stimulated HUVECs were analyzed for TF-specific mRNA
expression by ribonuclease protection assay (RPA), surface TF
expression by flow cytometry, and TF activity by a two-stage clotting
assay. Heat shocked LPS-stimulated HUVECs expressed significantly reduced TF-specific mRNA, TF surface protein levels, and TF surface activity when compared with non-heat-shocked, LPS-stimulated HUVECs (p < 0.0125, p < 0.0125, and
p < 0.0001, respectively; repeated measures analysis of
variance, ANOVA). If heat shock models elevated core temperature, these
results suggest that fever may protect the host during sepsis by
reducing TF activity on the surface of endothelial cells.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B nuclear
translocation and I-
B degradation in cultured cells (15, 16) and
in vivo (17). Thus, the heat shock response may protect the
host during infection by modulation of proinflammatory genes during
sepsis. TF expression in response to LPS and cytokines is also
partially regulated by NF-
B. We hypothesized, therefore, that heat
shock modulates tissue factor expression by endothelial cells during
LPS challenge. In this study we show that heat shock significantly
reduces expression of TF-specific mRNA, surface protein, and
activity by LPS-stimulated endothelial cells.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(R & D Systems,
St. Paul, MN).
20 °C until RNA purification.
The total amount of RNA isolated from each sample was quantified by
absorbance at 260 nm. Purified RNA was stored at
20 or
80 °C
until analyzed by ribonuclease protection assay (RPA).
-32P]UTP-labeled single-stranded RNA target gene
probes were transcribed with T3 or T7 RNA polymerase (Stratagene).
Transcription products were purified by 5% acrylamide and 8 M urea gel electrophoresis (45 min at constant 200 V) and
exposed to radiographic film. Full-length, radioactively labeled RNA
probes were excised and eluted from acrylamide by overnight incubation
in elution buffer from Ambion RPA II kit (Ambion, Austin, TX) at
37 °C. To determine target probe specific activity, a liquid
scintillation counter (LKB Wallac 1214 Rackbeta) was used to obtain
counts per minute from aliquots of the transcription reaction mixture
and target probe eluent. Probes were stored at
20 °C until use.
Probe sizes were as follows: GAPDH (unprotected 393 bp, protected 307 bp); GAPDHs (unprotected 308 bp, protected 222 bp); TF (unprotected 396 bp, protected 310 bp); and HSP72 (unprotected 280 bp, protected 234 bp).
= 405 nm (Bio-Rad microplate reader model 3550).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Optimizing LPS and heat shock conditions for
the expression of TF- and HSP72-specific mRNA by HUVECs.
HUVECs were stimulated with 0.1 µg/ml LPS for 0, 1, 2, 4, or 6 h
at 37 °C (A) or with 0, 0.01, 0.1, 1, or 10 µg/ml of
LPS for 2 h at 37 °C (C) and then analyzed for TF-
and HSP72-specific mRNAs by RPA. RNase-protected fragments of TF
and GAPDH were separated according to size by gel electrophoresis (TF,
310 bp; GAPDHs, 222 bp) and analyzed from the phosphorimage. Ratios of
TF/GAPDHs were calculated to determine relative levels of mRNA
expression. A and C, digestion of unprotected
probe is shown in the right panels. B
and D, relative abundance of TF-specific message in
LPS-stimulated HUVECs. The results are reported as mean ± S.E.
(n = 7, n = 3, respectively). *,
p < 0.0125, repeated measures ANOVA.
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Fig. 2.
Heat shock reduces LPS-stimulated HUVEC
TF-specific mRNA expression. A, HUVECs were heat
shocked for 0, 1, 2, 4, 6, or 8 h at 43 °C and then analyzed
for HSP72-specific messages by RPA. RNase-protected fragments of HSP72
and GAPDH were separated according to size by gel electrophoresis
(HSP72, 234 bp; GAPDH, 307 bp). B, protocol for heat shock
experiments. After LPS addition, HUVEC cultures were heat shocked for
2 h and then re-equilibrated at 37 °C for another 0, 2, 4, or
6 h. Some HUVEC monolayers were stimulated with LPS without heat
shock or with heat shock without LPS. Untreated cells were compared and
shown to be negative for TF- and HSP72-specific mRNA at each
condition (data not shown). C, induction of TF-specific
mRNA by heat-shocked and non-heat-shocked HUVECs at 2, 4, and
6 h of LPS stimulation. In a representative phosphorimage
(left panel), TF- and GAPDHs-specific mRNA
bands migrate at 310 bp and 222 bp, respectively. To confirm heat
shock, HUVEC expression of HSP72 was analyzed after 2 h of heat
shock (right panel). HUVECs expressed
HSP72-specific mRNA (234-bp band). HUVECs treated with heat
shock but without LPS did not express detectable levels of TF message
(data not shown). D, relative abundance of TF-specific
mRNA expressed by LPS-stimulated HUVECs. The results are reported
as mean ± S.E. (n = 7). *, p < 0.01 for heat shock versus no heat shock, repeated measures
ANOVA).
and
heat shocked. Monolayers were dispersed and analyzed by flow cytometry
for iTF expression. Non-heat shocked, LPS-stimulated HUVECs were also
analyzed for comparison. After 4 h of LPS stimulation, 31.7% of
HUVECs were surface iTF-positive in a representative histogram (Fig.
3A, shaded area).
After heat shock, only 22.2% of LPS-stimulated HUVECs were surface iTF
positive (Fig. 3B, shaded area) (reactions with
isotype control antibodies are shown as unshaded histograms). Virtually
no iTF was detected on HUVECs that were unstimulated (Fig.
3C) and heat shocked (2 h), followed by recovery for 2 h at 37 °C (Fig. 3D), or stimulated with LPS for 4 h
and incubated with secondary antibody only (Fig. 3E). Surface expression of iTF by HUVECs stimulated with LPS (Fig. 3F) or IL-1
(Fig. 3G) was significantly
reduced by heat shock at all time points.
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Fig. 3.
Heat shock reduces iTF surface expression by
stimulated HUVECs. A, a representative histogram from a
flow cytometry experiment demonstrating the surface iTF-positive
population of HUVECs after stimulation with LPS for 4 h
(shaded curve). The unshaded curve
represents the fluorescence of LPS-stimulated HUVECs stained with
nonspecific isotype control primary antibodies. B, surface
iTF positive HUVECs heat shocked for 2 h and stimulated with LPS
for 4 h (shaded curve). C, the histogram
generated by an unstimulated HUVEC population (shaded
curve). Within the unstimulated HUVECs the unshaded
curve represents the iTF positive HUVECs after incubation
with a primary nonspecific IgG. Histograms are also shown for HUVECs
incubated with isotype control antibody (unshaded curve) and
anti-TF (shaded curve) after (D) heat
shock (2 h), followed by 2 h at 37 °C, or (E)
LPS-stimulation incubated with secondary antibody only. Immunoreactive
TF expression by HUVECs incubated with LPS (F) or IL-1
(G) with or without heat shock. The results are expressed as
mean ± S.E. (n = 7 and n = 3, respectively); statistically significant results are indicated with an
asterisk (*, p < 0.01 for heat shock
versus no heat shock, repeated measures ANOVA).
H, viability of heat-shocked and non-heat-shocked
LPS-stimulated HUVECs by propidium iodine exclusion staining.
I, LPS-stimulated HUVECs were heat shocked for 2 h at
40, 41.5, or 43 °C and then returned to 37 °C for 0, 2, 4, or
6 h. In parallel, non-heat-shocked, LPS-stimulated HUVECs were
harvested at the same time points. HUVECs were harvested at the times
indicated and analyzed for iTF surface expression by flow cytometry.
The graph represents the average percent reduction of surface iTF ± S.E. at each heat shock temperature over the time course.
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Fig. 4.
Heat shock reduces surface TF activity by
LPS-stimulated HUVECs. HUVECs were stimulated with LPS for 4 h. During the initial 2 h, some HUVECs were heat shocked at
43 °C and then returned to 37 °C for the final 2 h.
A, a representative two-stage clotting assay demonstrates a
severe reduction in surface TF activity by heat-shocked, LPS-stimulated
HUVECs assessed by colorimetric cleavage of a factor Xa-specific
substrate. B, anti-TF-specific antibody control two-stage
clotting assay demonstrating a TF-specific reaction. C, TF
activity from heat-shocked and non-heat shocked LPS-stimulated HUVECs
with and without 12 µM inomycin (final concentration).
Mean slope values were calculated from the linear portion of each curve
generated from S-2222 cleavage (OD versus time). The results
are expressed as mean slope values ± S.E. (n = 7). Statistically significant (*, p < 0.0001 for heat
shock versus no heat shock, repeated measures ANOVA).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B. NF-
B
regulates cytokine- and LPS-mediated TF expression (21, 22). Heat shock
attenuates NF-
B nuclear translocation and the induction of
NF-
B-dependent nitric oxide synthase in murine
epithelial cells (15). Future studies will determine whether
NF-
B nuclear translocation is reduced in heat shocked,
LPS-stimulated HUVECs.2
to stimulate TF
expression. In response to IL-1
, heat shock significantly reduced
surface iTF (+) HUVECs at all times (Fig. 3G). Although the
magnitude of the heat shock inhibition of iTF expression was temperature dependent (Fig. 3I), the extent of reduction
appeared to be independent of the strength of the procoagulant signal. Interleukin-1
or LPS stimulation induced similar surface iTF expression, with peak expression appearing to occur at 4 h. At 4 h, however, IL-1
induced expression on more HUVECs (
60%) compared with LPS (
36%) (Fig. 3, F and G).
Heat shock also caused a greater reduction in the iTF (+) HUVEC
population when IL-1
was used as the stimulating agent. In contrast,
heat shock of LPS- and IL-1
-stimulated HUVECs reduced iTF (+) to
19.5 and 18% of the population, respectively. Heat shock appeared to
down-regulate TF expression to this minimum level despite differences
in stimulus potency, which may be an important control of coagulation
if the anti-coagulant effect of heat shock occurs in
vivo.
-stimulated HUVECs, but it was expressed. Therefore, we assessed surface TF procoagulant activity by a two-stage clotting assay. Heat
shock significantly reduced surface TF activity by LPS-stimulated HUVECs (Fig. 4, A and C). Because TF-specific
antibodies inhibited the two-stage clotting assay (Fig. 4B),
the procoagulant activity expressed on HUVECs was produced by TF. The
reduction in TF surface activity was proportional to decreases in TF
protein and TF-specific mRNA expression (Fig.
5), which strongly suggests that heat
shock mediated the modulation of transcription without detectable
post-translational modification.
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Fig. 5.
Heat shock proportionally reduces the
expression of TF-specific mRNA, iTF, and activity on HUVECs.
Heat shock reduces the expression of TF-specific mRNA, iTF, and
activity on LPS-stimulated HUVECs. The data from all experiments are
expressed as the mean percent reduction ± S.E.
Encrypted TF is the non-functional proportion of the total quantity of surface TF (23, 24). Heat shock may have altered the proportion of encrypted TF and, therefore, could account for the decreased TF activity. To assess the total functional TF activity, we added ionomycin to HUVECs prior to incubation with factor VIIa. Ionomycin treatment increased surface TF activity, suggesting that a fraction of the TF was encrypted. Heat-shocked, LPS-stimulated HUVECs treated with ionomycin, however, showed significantly reduced surface TF activity (Fig. 4C). Sixty-one percent of TF activity was encrypted on LPS-stimulated HUVECS compared with 83% on heat shocked cells. The data suggest that heat shock reduced the absolute quantity and functional activity of surface TF.
We considered several other explanations for the results. The reduction
in TF expression may have reflected the loss of LPS or IL-1 activity
due to heat denaturation during heat shock. Therefore, LPS and IL-1
were pre-heated at 43 °C for 2 h and then added to HUVEC
cultures. The surface iTF (+) HUVEC population was virtually identical
when stimulated with pre-heated LPS or IL-1
(data not shown),
indicating that heat denaturation or degradation did not alter
activity. Perhaps heat shock itself contributed to cell injury or death
and thus reduced TF expression. To test this possibility, cell
viability was compared in heat stressed and unstressed LPS-stimulated
HUVECs. Although HSP72-specific mRNA was up-regulated by heat shock
and served as a positive control, viability was unaffected by heat
shock when assessed by propidium iodine exclusion staining (Figs.
2C and 3H). To rule out cellular injury as a
cause of reduced TF expression, cellular respiration and gross
morphological changes were compared in heat-stressed and -unstressed
LPS-stimulated HUVECs. Cellular respiration and morphologic changes
were evaluated by AlamarBlueTM reduction and light microscopy,
respectively. Heat shock did not produce detectable changes in HUVEC
respiration or morphology (data not shown). Heat shock modulates the
expression of certain genes without apparent effect on other cell
functions. For example, heat shock reduced the expression of TNF-
mRNA and protein by LPS-stimulated macrophages, which retained the
ability to ingest antibody-coated erythrocytes like non-heat-shocked
macrophages (25). Collectively, these experiments suggest that heat
shock can modulate TF gene expression without an effect on specific
cell functions, injury, or death. Reduction of TF expression by heat
shock in LPS-stimulated HUVECs was therefore not due to cellular injury
or death.
Based on our study, we hypothesize that fever may protect septic hosts
because of the reduced activation of extrinsic coagulation. Attenuated
expression of TF would be expected to decrease development of DIC and
improve the clinical prognosis. Fever may also play a role in other
disease processes such as atherosclerosis. Viral and bacterial
infections have been suggested as being implicated in the pathogenesis
of atherosclerosis (26-28). For example, specific pathogen-free
chickens infected with Marek's disease virus and fed
cholesterol-supplemented diets develop arterial fatty-fibro lesions
similar to atheromas (29). In humans, the presence of cytomegalovirus
antibodies is an independent risk factor for the development of
atherosclerosis (30). Chlamydia antigens are detected in ~80% of
coronary atherectomy sites compared with 4% in non-diseased vessels
(31). In view of our data, it is noteworthy that the host responds to
chlamydia and cytomegalovirus infections with antibody production, but
clinical symptoms such as fever are usually absent. We hypothesize that
the absence of fever generation during acute chronic vascular
infections may increase the risk of thrombosis, contributing to the
development of atheromas and acute coronary events such as myocardial
ischemia or infarction.
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ACKNOWLEDGEMENT |
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We thank Julia Nguyen for isolation and characterization of the HUVECs used in our study.
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FOOTNOTES |
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* This work was supported by NIDCR, National Institutes of Health Grants K16 DE00270 and RO1 DE11831.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.
To whom correspondence should be addressed: University of
Minnesota School of Dentistry, Dept. of Oral Sciences, 17-164 Moos Tower, 515 Delaware St. S. E., Minneapolis, MN 55455. Tel.:
612-625-8404; Fax: 612-626-2651; E-mail: mcherzb@umn.edu.
Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.M211540200
2 D. L. Basi, K. F. Ross, J. S. Hodges, and M. C. Herzberg, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
DIC, disseminated
intravascular coagulation;
TF, tissue factor;
iTF, immunoreactive TF;
LPS, lipopolysaccharide;
HUVEC, human endothelial vein cell;
IL-1, interleukin-1
;
RPA, ribonuclease protection assay;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
GAPDHs, GAPDH short product;
HSP72, heat shock protein 72;
PBS, phosphate-buffered saline;
FBS, fetal bovine serum;
ANOVA, analysis of variance.
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