Enhancement Effect of Ethanol on Lipopolysaccharide-induced Procoagulant Status in Human Umbilical Endothelial Cells
In spite of the inhibitory effects of ethanol (EtOH) on platelet function, soft blood clots are often observed in cadaveric blood in cases of sudden death after alcohol ingestion. In order to resolve this discrepancy, we have focused on the role of vascular endothelial cells. We tried to investigate the effects of EtOH and LPS on endothelial cells from various perspectives; thrombogenic factor (Von Willebrand factor, VWF), fibrinolytic factor (tissue plasminogen activator, tPA) and inflammatory factor (Interleukin-6, IL-6). Human umbilical vein endothelial cells (HUVECs) were incubated with various concentrations of EtOH (0~160 mM) with or without LPS. Treatment with EtOH and LPS increased VWF release from HUVECs without enhancement mRNA expression. Treatment with 40 mM of EtOH also increased IL-6 release from HUVECs without enhancement mRNA expression. Although EtOH inhibited LPS-induced IL-6 mRNA expression, 20 mM of EtOH still had an increasing effect on the release of IL-6. These doses of EtOH are consistent with a moderate drunkenness level in a normal person. On the other hand, mRNA expression and release reaction of tPA were not affected by EtOH and LPS addition. In conclusion, EtOH enhances procoagulant status via VWF release and IL-6 production cooperation with LPS and may contribute to soft blood clot formation in cadaveric blood.
*; Minori Nishiguchia
; Shie Yoshidaa
; Nao Ohtsua
; Nobuyuki Adachia
; Yoshihiko Sakuraic
; Midori Shimac
; Motonari Takahashia
; Katsuhiko Hatakeb
; Hiroshi Kinoshitaa
aDepartment of Legal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo, 663-8501, Japan
bDepartment of Legal Medicine, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara, 634-8521, Japan
cDepartment of Pedia
; Present address: Department of Legal Medicine, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara, 634-8521, Japan
Vyšlo v časopise:
Soud Lék., 54, 2009, No. 4, p. 44-48
Navzdory inhibičnímu účinku ethanolu na funkci destiček, měkká krevní koagula jsou často pozorována v kadaverosní krvi v případech náhlého úmrtí po požití alkoholu. Ve snaze vysvětlit tuto diskrepanci, zaměřili jsme se na roli vaskulárních endotheliálních buněk. Snažili jsme se prozkoumat účinek ethanolu a lipopolysacharidů (LPS) na endotheliální buňky z různých hledisek: trombogenního faktoru (von Willebrandův faktor, vWF), fibrinolytického faktoru (tkáňový aktivátor plasminogenu, tPA) a zánětlivého faktoru (interleukin 6, IL-6). Endotheliální buňky lidské pupečníkové žíly byly inkubovány při různých koncentracích ethanolu (0 – 160 mmol) s nebo bez LPS. Působení ethanolu a LPS zvýšilo uvolňování vWF z endotheliálních buněk lidské pupečníkové žíly bez zvýšení exprese mRNA. Ačkoliv ethanol inhiboval expresi LPS indukovanou IL-6 mRNA, 20 mmol roztok ethanolu ještě měl potencující účinek na uvolňování IL-6. Tyto koncentrace ethanolu odpovídají mírné hladině opilosti u normální osoby. Z druhé strany, exprese mRNA a uvolňující reakce tPA nebyly ovlivněny přidáním ethanolu a LPS. V souhrnu, ethanol podporuje prokoagulační stav cestou uvolňování vWF a tvorbou IL-6 podpořenou LPS a může přispívat ke tvorbě měkkých krevních koagul v kadaverosní krvi.
Fluidity of the cadaveric blood
is one of the most important characteristics of a sudden death.
The prevailing view of this phenomenon attributes it to the release
of tissue plasminogen activator (tPA) from endothelial cells (23,
24). Yet it is also empirically known that soft blood clots have been
observed in cases of sudden death after alcohol ingestion. A number
of studies have stated that ethanol attenuates platelet activation
induced by several agonists (14, 19, 20). Moreover, it has been
reported that expression of tPA and urokinase-type PA (uPA) are
up-regulated in endothelial cells after ethanol (EtOH) treatment (4,
22). The underlying mechanism of the discrepancy between fluidity of
cadaveric blood and soft-clot formation in sudden death after alcohol
ingestion remains to be fully elucidated.
In order to resolve this
discrepancy, we focused on the role of vascular endothelial cells. It
has long been understood that blood clot formation can be associated
with the following factors known as Virchow’s triad; reduction in
blood flow, endothelial injury and alterations in the constitution of
blood (9). Endothelial cells in normal condition provide various
anticoagulants such as or nitric oxide,
whereas they express thrombogenic activity when they are injured. The
most common injury to endothelial cells is caused by inflammation due
Lipopolysaccharide (LPS) is
a major component of the outer membrane of Gram-negative
bacteria and is the prototypical example of an endotoxin. LPS-induced
inflammation causes various types of reaction, and the relationship
between coagulation and inflammation have been well-discussed (7).
Since absorption of LPS in the intestine is enhanced by alcohol
ingestion (25), it is just conceivable that an interaction between
EtOH and LPS affects the endothelial function.
In the present study, we tried to
investigate the effects of EtOH and LPS on endothelial cells from
various perspectives; thrombogenic factor, fibrinolytic factor and
inflammatory factor. Von Willebrand factor (VWF) is one of the most
important thrombogenic factor produced and released from endothelial
cells. tPA is the most potent fibrinolytic factor which is also
produced and released from endothelial cells. Interleukin-6 (IL-6) is
the most important proinflammatory cytokine, which correlates with
various aspects of hemostasis (15). In this paper, in order to
explore potential mechanism of soft-clot formation after alcohol
drinking, we determined mRNA expression and secretion of VWF, tPA and
IL-6 by using human umbilical vein endothelial cells (HUVECs).
Materials and Methods
HUVECs and culture medium were
purchased from Cell Applications (San Diego, CA, USA). Absolute EtOH
and LPS from Escherichia
coli serotype O55 were
purchased from Wako Pure Chemical Industries (Osaka, Japan).
HUVECs were grown for 2-3 days
until reaching 80% confluence in a humidified chamber with a 5%
mixture at 37 °C. For experiments, HUVECs were passaged using
trypsin-EDTA. They were seeded into 24-well or 96-well tissue culture
plates. The cells were confluent after 2 days. HUVECs were incubated
with EtOH alone or EtOH with LPS. We employed 10 experimental groups;
Group A (EtOH 0 mM), Group B (EtOH 20mM), Group C (EtOH 40 mM),
Group D (EtOH 80 mM), Group E (EtOH160 mM), Group F (EtOH 0mM, LPS
100ng/ml), Group G (EtOH 20 mM, LPS 100 ng/ml), Group H (EtOH 40
mM, LPS 100ng/ml), Group I (EtOH 80 mM, LPS 100 ng/ml) and Group
J (EtOH 160 mM, LPS 100 ng/ml). EtOH alone treatment groups (A~E)
were incubated with various concentrations of EtOH for indicated
hours, while EtOH and LPS treatment groups (F~J) were first incubated
for 30 min at 37 °C in culture media containing various
concentrations of EtOH and incubated for indicated hours with
addition of LPS. All experiments were performed on second passage
The cytotoxicity of EtOH and LPS
were evaluated with a WST-8 colorimetric assay (Kishida
Chemical, Osaka, Japan). HUVECs seeded into a 96-well plate were
treated for 20 hours in 100 l of culture media containing
various concentrations of EtOH with or without LPS. Next, 10 μl
of WST-8 reagent were added and incubation conducted for 2 hours.
Cell viability was determined according to the manufacturer’s
instructions. The data are presented as a ratio to the response
in Group A and are expressed as mean Ī SEM.
After 4 hours of treatment, cell
monolayers were washed twice in PBS, after which total RNA was
extracted using RNeasy micro kit and treated with RNase free DNase
(Qiagen, Valencia, CA, USA). Reverse transcription-polymerase chain
reaction (RT-PCR) was performed using 1 μg
of total RNA, and Superscript RT kit (Invitrogen) with random
hexamers. Quantitative real time RT-PCR analysis wasperformed with an
Applied Biosystems StepOne Plus Real time PCRSystem using TaqMan®
universal PCR master mix accordingto the
manufacturer’s specifications for human VWF, tPA and IL-6 genes
(Applied Biosystems,Foster City, CA,
USA). The TaqManprobes and primers
for human VWF, tPA and IL-6 gene (assay identification number,
Hs00169795_m1, Hs00938315_m1 and Hs99999032_m1, respectively) were
assay-on-demand gene expression products (Applied Biosystems), while
human GAPDH gene (assay identification number Hs99999905_m1) was used
as an endogenous control. The data were analyzed by the ∆∆Ct
method (17) and were presented as normalized gene amount relative to
of VWF, tPA and IL-6
Culture supernatants (400 μl)
from 4 hour-treated HUVECs were centrifuged at 15,000 g to
remove cell debris and frozen at -80 °C until use. VWF was measured
by ELISA as described elsewhere (3). The VWF values were presented as
a percentage of pooled normal human plasma. tPA (Assaypro, St
Charles, MO, USA) and IL-6 (Biosource International, Camarillo, CA,
USA) were obtained with ELISA kits according to the manufacturer’s
instructions. The data are presented as a ratio to the response
in Group A and are expressed as mean Ī SEM.
According to the results of WST
assay and ELISA, statistical analysis was performed using Statview
software (SAS Institute, Cary, NC, USA). Differences between the
groups were assessed by a Kruscal-Wallis test with post-hoc
testing (Tukey-Kramer test). A value of
p < 0.05 was
considered statistically significant.
At first, to rule out the
possibility that a high concentration of EtOH and LPS had
a lethal effect on endothelial cells, we assessed cell viability
following EtOH and LPS treatment. We found that incubation of HUVECs
for 20h with EtOH with or without LPS did not significantly affect
cell viability, as determined by WST-8 assay (Figure 1). These
results suggested that EtOH and LPS did not induce protein release
due to necrotic cell collapse.
The results of real time
quantitative PCR are summarized in Table 1. EtOH alone did not show
remarkable change in mRNA expression of VWF, tPA and IL-6 (Table1.
a). Co-stimulation with LPS and EtOH (Table 1. b) also did not show
significant change in mRNA expression of VWF and tPA. mRNA expression
of IL-6 was up-regulated by LPS stimulation (Group F). This is
because LPS stimulates the synthesis of inflammatory cytokines by
activating nuclear factor kappaB(NF-μB)-dependent
signaling mechanisms. However, addition of EtOH inhibited LPS-induced
mRNA expression (Group G-J).
In view of protein secretion, the
mean value of concentrations of VWF, tPA and IL-6 in group A were
5.73%, 0.34 ng/ml and 41.3 pg/ml, respectively. EtOH alone did not
significantly affect the release of VWF and tPA in conditioned media
(Figure. 2 a, c).
However, by addition of LPS, VWF
release was significantly increased (Figure.2 b). On the other hand,
tPA release was not affected at all by the addition of LPS (Figure. 2
d). IL-6 secretion in conditioned media was significantly increased
by addition of 40mM EtOH (Figure.2 e, Group C). Naturally, LPS
treatment increased IL-6 secretion (Figure.2 f, Group F). Although
real time PCR analysis demonstrated that addition of EtOH inhibited
LPS-induced mRNA expression of IL-6, co-stimulation with 20 mM
of EtOH and LPS (Group G) still increased IL-6 secretion compared to
Group A. There were no significant difference between group F
and Group G in IL-6 secretion.
We have investigated the role of
endothelial cells in EtOH administration. We demonstrated that
moderate dose of EtOH enhanced IL-6 release and co-stimulation with
EtOH and LPS increased VWF release without an enhancement of tPA
Since LPS is a potent
substance that causes inflammation, while a high dose of EtOH
also may denature the cell membrane, there was a possibility
that the release reactions were due to cell collapse. We therefore
confirmed that EtOH and LPS did not induce cell death even after 20h
incubation and consider that the increase of release reactions were
not due to cell collapse.
VWF release from HUVECs would
promote soft clot formation, since VWF plays a crucial role in
hemostasis as a mediator for platelet aggregation and carrier
protein for coagulation factor VIII. Although expression of VWF mRNA
was not affected by EtOH with or without LPS, co-stimulation with
EtOH and LPS increased release of VWF from HUVECs. These results
suggest that co-stimulation with EtOH and LPS induces VWF release
from internal store without de novo synthesis.
Inflammatory cytokines contribute
to coagulation activation by increasing fibrinogen concentration and
inducing tissue factor expression on the cell surface (7).
Conversely, coagulation activation also stimulates production of
inflammatory cytokines (10-12). There exist many reports using
various kinds of cells, such as osteoblast-like cells, hepatocytes,
monocytes, and dermal microvascular endothelial cells. However, it is
still controversial whether EtOH activates NF-kB (16, 18, 21, 27). It
may be that the various cell lines give rise to the differences seen
in the effect of EtOH on NF-μB
activation. However, Jonsson et al. reported that EtOH inhibited
activation in HUVECs (13). In our experiments, HUVECs significantly
increased IL-6 release at 40 mM EtOH treatment (Group C).
Although EtOH inhibited LPS-induced IL-6 mRNA expression, we have
demonstrated that 20 mM of EtOH has an increasing effect on the
release of IL-6. These concentrations of EtOH are often seen in
autopsy cases. A recent report has revealed that soft clot
formation in cadaveric blood is associated with a blood ethanol
level having a mean value of 0.237 mg/ml (around 40 mM) (8).
This is consistent with our results; i.e., that moderate EtOH
concentration supports soft blood clot formation. Taken together, LPS
acts synergistically with EtOH to release VWF and IL-6 from HUVECs.
Although further investigation should be required to clarify the
underlying mechanism of this phenomenon, it is possible that EtOH and
LPS would change the membrane permeability.
On the other hand, mRNA
expression and secretion of tPA remained unchanged, even under
co-stimulation with EtOH and LPS. Since tPA is the most important
fibrinolytic factor, unchanged tPA release would contribute to soft
blood clot formation. According to a previous report, tPA mRNA
was up-regulated by EtOH stimulation (4). However, in that study,
relatively low levels of EtOH were used (< 20 mM). In our present
study, we used relatively high levels of EtOH (> 20 mM, moderate
ebrious ~ non physiological levels). As EtOH affects many kinds of
proteinkinase as well as NF-kB (1, 2, 5, 26), tPA production
mechanism are likely to be modified intricately by the difference in
the concentration of EtOH. Unlike VWF, EtOH and LPS did not induce
tPA secretion. We considered that the stimuli for their secretion may
be different because tPA and VWF are stored in different vesicles
each other (6).
In conclusion, a moderate
dose of EtOH enhances procaogulant status via VWF and IL-6 release
cooperation with LPS. These actions do not involve an enhancement of
tPA production. Enhancement of LPS absorption due to alcohol
ingestion would contribute to soft blood clot formation in cadaveric
We would like to thank Ms. Junko
Kato for her excellent technical assistance.
to: Shogo Kasuda, M.D., Ph.D.
of Legal Medicine, Nara Medical University School of Medicine, 840
Shijo-cho, Kashihara, Nara, 634-8521, Japan
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