Articles |
Correspondence to Berislav V. Zlokovic, MD, 2025 Zonal Ave, RMR 506, Los Angeles, Calif 90033. E-mail zlokovic{at}hsc.usc.edu
| Abstract |
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(10 U/mL), produced a time-dependent decrease in brain
capillary TM mRNA (t1/2 of 2.1 and 3.9 hours, respectively)
and reduced endothelial TM activity. Incubation of
brain capillaries with retinoic acid (10 µmol/L) and dibutyryl
cAMP (3 mmol/L) resulted in a 4-fold increase in TM mRNA at 4 and
8 hours, respectively, followed by an increase in protein C activation.
We conclude that TM at the blood-brain barrier is likely to be an
important physiologic anticoagulant in brain microcirculation. Its
downregulation by cytokines may contribute to ischemic
brain damage and potentially could be counteracted by retinoic acid
and cAMP.
Key Words: thrombomodulin blood-brain barrier cytokines retinoic acid cAMP
| Introduction |
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TM offers major protection against a variety of thrombotic events. Anti-TM antibodies potentiate thrombin-induced thromboembolism in mice,5 while purified or recombinant TM protects against thromboembolism.5,6 The APC provides protection against procoagulant and lethal effects in a sepsis model.7 Resistance to the effects of APC is closely linked to venous thrombosis in humans,8 while blockade of APC in a coronary occlusion model results in impaired cardiac outcome.9 Recently, lower levels of circulating APC have been suggested as an important risk factor in infection-associated stroke.10 Although thrombosis is of paramount importance in the pathophysiology of ischemic stroke, the mechanisms that govern anticoagulant activity in brain endothelium, ie, at the site of the BBB, are still poorly defined.1113
There is uncertainty in the literature whether TM is in fact expressed
in brain. Some studies reported that TM protein is present in human
brain exclusively in microvessels,14,15 whereas
others claimed its absence or very limited
expression.1618 Recently it has been shown that
TM is expressed in cultured bovine brain endothelial
cells,13 and the presence of TM mRNA has been
demonstrated in cerebral microvessels in adult bovine and rat
brain.19,20 The factors involved in TM regulation
in cerebral vasculature, however, remain largely speculative. It is
well documented that pathogenic mediators of stroke, IL-1ß, and
TNF-
21,22 downregulate TM expression in
peripheral vascular endothelial
cells,2326 but it is not known whether these
cytokines exert similar effects on brain
endothelial TM, which in turn could contribute to
ischemic brain damage. RA2729 and
cAMP,3034 on the other hand, stimulate TM
expression in systemic vascular endothelium and
counteract the effects of IL-1ß and TNF-
. Whether RA and increased
intracellular cAMP levels may enhance the anticoagulant function of the
BBB is unclear, but if yes, these strategies may have the potential to
ameliorate focal ischemic brain injury. In this study, we used
isolated bovine BCs as an in vitro model of the
BBB35,36 to address these questions.
| Methods |
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(2.7x108
U/mg) and IL-1ß (6.7x107 U/mg) were purchased
from Genzyme Corporation. All-trans-RA, RA, and db-cAMP were
obtained from Sigma Chemical Co.
-Glutamyl transpeptidase activity
was determined using a Sigma diagnostic assay. All other
chemical reagents were purchased from Sigma Chemical Co, except
where indicated.
Isolation of BCs
BCs were isolated from bovine cerebral cortical mantles,
cerebellum, and pons by a modified mechanical
homogenization technique.37
Briefly, bovine brains were rinsed in ice-cold buffer B (mmol/L:
103 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2
KH2PO4, 1.2
MgSO4, 15 HEPES; pH, 7.4). The cerebral cortical
mantles, cerebellum, and pons were rapidly freed of meninges (ie, pial
vessels) under sterile conditions, and the arteries of the circle of
Willis, veins, and choroid plexi were discarded. Cerebral and
cerebellar cortices and pons were used to isolate the capillaries.
Homogenization of brain tissue in buffer A (buffer
B + the following in mmol/L: 25 HCO3,
10 glucose, 1 sodium pyruvate, and 0.156 dextran,
Mr=64,000) with a hand-held Teflon
homogenizer was followed by dextran density
centrifugation at 5800g at 4°C. The pellet
was resuspended in buffer A and passed over an 85-µm nylon mesh.
Arterioles and venules remained on top of the mesh, while the
capillaries, red cells, nuclei, and other debris were collected in the
filtrate passing through the mesh. This filtrate was then passed over a
3x4-cm glass bead column (0.45-mm glass beads) with 44-µm nylon mesh
at the bottom, and the column was washed with buffer B. The BCs adhere
to the glass beads while the other contaminants pass unimpeded.
Capillaries were recovered by repeated gentle agitation of the glass
beads in buffer A, and the supernatant with capillaries was decanted
and spun at 500g for 5 minutes to obtain the final pellet.
The purity of the cerebral capillary preparation was checked by light
and phase microscopy. The cerebral capillaries were free of adjoining
brain tissue, and preparations consisted primarily of capillaries, but
also contained minor amounts (5% to 10%) of small arterioles, as
described.36,37 The presence of no more than
minimal detectable activity of a specific cerebrovascular marker,
-glutamyl transpeptidase, was used to confirm the absence of
contamination of capillary-depleted brain parenchyma with microvessels,
as reported.38
Incubation of BCs
Freshly isolated cortical capillaries were incubated in
medium-199/Ham's F-12 media with 10% fetal calf serum, 1 mol/L
Na pyruvate, 2 mg/L NaHCO3, 2
mmol/L L-glutamine, 100 U/mL penicillin, 100
µg/mL streptomycin, and 2.5 µg/mL Fungizone in
six-well tissue culture plates in a humified 5%
CO2/95% air incubator at 37°C. Capillaries
were incubated for 2, 4, 8, 12, 16, and 24 hours with 10 U/mL IL-1 ß,
or 10 U/mL TNF-
, or 10 µmol/L RA, and/or 3
mmol/L db-cAMP. Each experiment had its own control group
incubated with the vehicle. Experiments were performed repeatedly (n=3)
in triplicate.
RT-PCR Analysis
Total RNA was extracted from bovine capillaries and
capillary-depleted brains with TRIZOL reagent (GIBCO BRL).
cDNA was synthesized by RT using 4 µg of total RNA in 20 µL of
reaction buffer containing 1 mmol/L each dNTP, 25
mmol/L Tris-HCl (pH 8.3), 1 µg of oligo(dT), 25
mmol/L KCl, 5 mmol/L MgCl2,
5 mmol/L dithiothreitol, 0.25 mmol/L
spermidine, 10 U of RNase inhibitor (Boehringer
Mannheim), and 10 U of AMV reverse transcriptase (Promega). The
reaction was carried out at 42°C for 1 hour and terminated at 52°C
for 40 minutes. The cDNA was stored at -20°C until use. PCR was
performed with 2 µL of the RT reaction mixture, 50 mmol/L
KCl, 10 mmol/L Tris-HCl (pH 8.3), 1 mmol/L
MgCl2, 0.5 mmol/L of each TM primer,
200 µmol/L each dNTP, and 1.0 U of Taq
polymerase (Boehringer Mannheim) in a final volume of 50 µL.
Amplification was carried out in a DNA thermal cycler (Perkin Elmer
Corp). After an initial denaturation at 95°C for 5 minutes, samples
were subjected to 30 cycles (95°C, 1 minute; 58°C, 1 minute;
72°C, 2 minutes). PCR products (10 µL) were analyzed by
electrophoresis on a 1% agarose gel. The bovine thrombomodulin primers
(National Biosciences Inc) correspond to nucleotides 228 to
256 (sense primer, 5'-CTCGGCAACTACACGTGCATCTGCGAG-3') and 907 to 935
(antisense primer, 5'-GCCACCACCAGAGACAGGCTTGCAATGG-3'), of the
published sequence for bovine TM.39 ß-Actin
primers (forward primer, 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3'; reverse
primer, 5'-CTAGAAGCATTTGCGGTGGACGATGGAGG3'; Stratagene) were used to
amplify ß-actin mRNA as a housekeeping nonchanging control for gene
expression.
The RT-PCR analysis was performed by comparing the relative change in TM mRNA expression to ß-actin. The number of cycles used30 was within the linear range of the amplification response for both genes. The amplification reaction for each cDNA prepared from BCs and/or no RT blank was monitored by varying cycle numbers (cycle titration, 20, 25, 30, 35, 40, and 45), and 10 µL of the PCR product was analyzed by electrophoresis on 1% agarose gel. Gels were visualized with ethidium bromide staining and ultraviolet transillumination. Photographs were taken with positive/negative type 55 film, (Polaroid Corp), and the negatives were scanned with a Hoefer GS 300 scanning densitometer interfaced to an IBM PC computer with a DT 2805 analog and digital system (Data Translation).
QC-PCR
To validate whether the RT-PCR method as described above could
be used for quantitative comparisons, the QC-PCR, as we
described,13 was used in parallel in some
experiments, and the results were compared with densitometric readings
obtained with RT-PCR in which the change in TM mRNA level was expressed
relative to ß-actin. QC-PCR tubes contained all the amplification
reagents (described in the above section), a constant amount of target
TM cDNA from each preparation, and serial dilutions of known
concentrations of a competitor TM cDNA template. The reaction mixture
was coamplified, as described above. The competitor cDNA template was
prepared by site-directed mutagenesis.40 A single
base change of A to G at base pair 346 created a unique Sal
I restriction site.
After coamplification, the PCR products were digested with Sal I: 10 µL of the PCR product, 1 U of Sal I restriction enzyme, and 2 µL of enzyme buffer incubated at 37°C for 2 hours. The digested competitor (591 bp) and target (707 bp) cDNAs were separated by electrophoresis on a 2% agarose gel, and visualized by ethidium bromide and ultraviolet transillumination. Negatives were prepared with a Polaroid (Polaroid Corp) and scanned by optical densitometry as above. Density readings of the target cDNA were multiplied by 591/707 to correct for differences in molecular weight. The ratio of amplified versus competitor cDNA optical densities was plotted as a function of competitor template concentration.13 The initial concentration of target cDNA was derived from the point at which the ratio of target and competitor cDNA optical density equaled 1.
Thrombomodulin Cofactor Assay
Endothelial cell TM activity was assayed from
capillary cellular homogenates by measuring the increase in
protein C activation as reported.26,41,42 After
incubation with cytokines, RA, and/or db-cAMP, capillaries were
washed with phosphate-buffered saline three times
(centrifugation at 10 000 rpm for 5 minutes), the
microvascular pellet was sonicated, resuspended in phosphate-buffered
saline and spun three times at 10 000 rpm for 5 minutes. Microvascular
homogenate samples were assayed for protein content by the
method of Lowry et al.43 A standard amount of
microvascular proteins from each sample (35 µg) was incubated in 100
µL of assay buffer containing 50 mmol/L Tris-HCl (pH
8.0), 2 mmol/L CaCl2, 0.1
mol/L NaCl, 0.1% bovine serum albumin, 0.6% Triton
X-100, and 2.2 nmol/L of bovine thrombin (Enzyme Research
Laboratory), and 65 nmol/L bovine protein C (Enzyme Research
Laboratory) for 60 minutes at 37°C. The reaction was terminated by
adding antithrombin III (5 µg) and heparin (5 U), and the reaction
mixture was centrifuged at 10 000 rpm for 5 minutes. Equal
volumes of the reaction mixture and a chromogenic substrate
(S-2366, Kabi Pharmacia) were added to a 96-well microtiter plate.
Optical densities were measured with a spectrophotometer (EL-311SX,
BioTek Instruments) at 405 nm.
Statistical Analysis
Results were compared by analysis of variance.
P<.05 was taken to be statistically significant.
| Results |
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-glutamyl transpeptidase activity, the contamination of
capillary-depleted cortical brain parenchyma by brain microvessels was
minimal and <0.5%, as we reported.38 The
relative amounts of mRNA for ß-actin gene were comparable in the
vasculature and capillary-depleted cortical brain (Fig 1B
|
We then performed QC -PCR (Fig 2
)
to determine TM mRNA expression from brain regional capillary
preparations. Cortical capillaries expressed the highest concentration
of TM mRNA (mean±SD), 24.3±1.2 pg; compared with cerebellar,
19.8±5.7 pg, or pontine, 5.5±0.5 pg. The QC-PCR findings were
consistent with the RT-PCR analysis performed on the
same BC samples demonstrating reduced TM mRNA expression in the BCs
from cerebellum and pons (Fig 3
). When
values in cortical BCs were arbitrarily set as 1, the RT-PCR and QC-PCR
analysis of the regional abundance of BC TM mRNA revealed, in
fact, similar results (Figs 2
and 3
). For example, in the cerebellum
the level of TM mRNA in BCs was 0.85 (RT-PCR) and 0.82 (QC-PCR) of the
cortical level, and in the pons the level of TM mRNA in BCs was
0.24 (RT-PCR) and 0.23 (QC-PCR) of the cortical level. As there were no
significant differences between the levels of TM mRNA determined by
RT-PCR and QC-PCR, in experiments described below the RT-PCR
analysis was used as a simpler, less time-consuming. and
less-expensive method.
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The regional differences in BC TM mRNA level correlated well with differences in TM BC activity determined in BC cellular homogenates by measuring the increase in protein C activation. For example, in BCs from the cortex and pons, the protein C activities expressed as optical density determined at 405 nm were 2.54±0.26 and 0.72±0.07 A405/min (mean±SD, n=3), respectively.
Effects of Immunomodulators on BC TM mRNA and Activity
Addition of IL-1ß to freshly isolated cortical BCs produced
significant downregulation of TM mRNA with a t1/2
of 2.1 hours compared with control values obtained when BCs were
incubated for the same periods of time with vehicle only. Incubation
with vehicle did not result in significant changes in either TM or
ß-actin mRNA levels within 24 hours of incubation (data not shown).
The TM mRNA signal was undetectable from 12 to 24 hours of incubation
with IL-1ß (Fig 4A
). The levels of
ß-actin mRNA were not altered during the 24-hour period of incubation
with IL-1ß (data not shown). A decrease in TM mRNA was accompanied by
reduced BC TM activity (Fig 4B
), but the TM activity was still
measurable and about 20% of control values after 12 hours of
incubation with IL-1ß.
|
Addition of TNF-
produced a similar time-dependent decrease of TM
mRNA, but with a somewhat longer t1/2 of 3.9
hours compared with control values (Fig 5A
). As with IL-1ß, the levels of
ß-actin mRNA were not altered during a 24-hour incubation with
TNF-
(data not shown). There was progressive reduction in TM
activity, and both TM mRNA and TM activities were undetectable after 16
hours of incubation (Fig 5B
). Changes in the levels of TM activity
followed decreases in TM mRNA, but some minor discrepancies were also
noted; for example, between 4 and 8 hours there was a significant drop
in TM mRNA level, but no significant difference was found in TM
activity (Fig 5B
).
|
Effects of RA and db-cAMP on BC TM mRNA and Activity
A time course evaluation indicated that RA amplifies TM mRNA by
4.2-fold after 4 hours (Fig 6A
), followed
by a decline in TM mRNA; after 24 hours a 2-fold increase was still
observed in comparison with a control incubated with vehicle only (Fig 6B
). The TM activity followed a trend of TM mRNA increase, and a
2.6-fold maximal increase was found at 4 hours (Fig 6B
). This effect
was sustained through 12 hours, but declined at 24 hours toward the
values at the beginning of incubation. As with IL-1ß and TNF-
, RA
did not produce significant changes in the levels of ß-actin mRNA
within 24 hours of incubation (data not shown).
|
Exposure to the cAMP analog db-cAMP resulted in a maximal 4-fold
increase in TM mRNA by 8 hours (Fig 7A
),
followed by a decline in TM mRNA level, and about a 2-fold increase
relative to control values between 16 and 24 hours (Fig 7B
). The peak
2.3-fold increase in TM activity occurred at 4 hours. The effect was
sustained through 4 hours, but began to decline toward control values
between 12 and 16 hours (Fig 7B
). At 24 hours with db-cAMP, the TM
activity dropped by about 50% below control in spite of the 2-fold
increase in TM mRNA. No changes in the levels of ß-actin mRNA were
observed within 24 hours of incubation with db-cAMP (data not
shown).
|
| Discussion |
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BCs may be contaminated by astrocytes,35,36 but
it is unlikely that the TM expression observed in this study is on
astrocytes, as the TM signal or activity was not found in
astrocyte-rich brain parenchyma. Regional amounts of BC TM mRNA have
been demonstrated for the first time (Fig 3
), and both the RT-PCR and
quantitative QC-PCR analysis have revealed similar differences
in TM mRNA regional brain BC levels (Figs 2
and 3
). This correlated
well with regional BC differences in the level of protein C activation
determined in cellular homogenates of these capillaries. A
previous immunocytochemical study in human brain has suggested that the
abundance of TM antigen was reduced in several subcortical regions
where brain infraction is common.14 It is
possible that decreased level of BC TM mRNA and reduced
endothelial TM activity in certain brain regions may
reflect reduced anticoagulant capacity in those local brain
microcirculations that could favor thrombosis as in other
organs.8,9 Low circulating levels of APC may
predispose to larger strokes and arterial or venous
cerebral thrombosis,9,44 while downregulation of
BC TM by stroke risk factors in rats was shown to be associated with
enhanced ischemic brain injury.20
Proinflammatory cytokines, IL-1ß and TNF-
, are
produced by stimulated astrocytes45,46 and during
an ischemic brain insult,47,48 when they
act as key pathogenic mediators of ischemic brain
damage.21,22,49 Both cytokines suppress
endothelial cell surface anticoagulant TM activity in
the systemic circulation,2327 which leads to
the formation of fibrin strands on the endothelial cell
surface.23,50 It has also been shown that
microvascular fibrin deposition and obstruction during early focal
cerebral ischemia and reperfusion contribute to brain
parenchymal damage.50 This study demonstrates for
the first time that IL-1ß and TNF-
downregulate TM mRNA and reduce
the rate of protein C activation in BCs (Figs 4
and 5
). These effects
of cytokines are not unexpected since qualitatively similar
results were obtained in vascular systemic endothelial
cells, ie, in HUVECs.27,51 Whether brain
endothelial cells are more sensitive to
cytokine-mediated downregulation than HUVECs remains to be
determined. It is likely that these two cytokines may elicit
comparable effects on the BBB in vivo under pathologic conditions
associated with activation of astrocytes and during cerebral
ischemia. Thus, suppression of BBB anticoagulant activity along
with tissue factor-initiated coagulation,50 may
facilitate fibrin formation in brain during ischemia and
contribute to ischemic brain damage.
The present results suggest that the effects of both
cytokines are mediated at the transcriptional level, which is
followed by decreases in TM cofactor activity. Previous reports in
HUVECs suggested that TNF-
reduces TM transcription, but does not
appear to alter TM mRNA stability or protein
degradation.24,52 Other factors may contribute to
TM antigen brain endothelial depletion in vivo as shown
for neutrophils.26 This study does not suggest a
rapid release of TM antigen that would precede TM mRNA downregulation
(Figs 4
and 5
). It is also unlikely that some minor discrepancies
between TM mRNA and TM activity reductions (Fig 5
) are due to
post-transcriptional mechanisms, such as changes in internalization and
endothelial degradation of TM
antigen.53,54 It is possible that these
cytokines (eg, TNF-
) decrease TM brain
endothelial expression by reducing intracellular cAMP
content,25 via a cAMP-responsive element in the
TM gene,55 as in other types of
endothelial cells.
RA and cAMP enhance TM biosynthesis in peripheral vascular
endothelial cells via elevation of TM
mRNA.2734 Similar effects were observed in this
study in BCs (Figs 6
and 7
). RA upregulates TM mRNA transcription via
an RA-responsive element located in the 5'-flanking region of the human
TM gene.56 A cAMP-responsive element in the TM
gene has been identified in the 3'-untranslated
region.55 Thus, the accessibility of the
RA-responsive and cAMP-responsive elements to the binding proteins
induced in endothelial cells and in different types of
blood cells may control TM expression.28 RA at a
plasma concentration of 10 nmol/L57 may
antagonize TM downregulation in HUVECs exposed to
TNF-
.27 Our preliminary results confirm
similar activity of RA in BCs (data not shown), raising the possibility
that RA may normally stimulate cerebrovascular TM expression. At
pharmacologic concentrations (Fig 6
) RA exhibited a more rapid
effect on BCs than on HUVECs.29 As circulating RA
is taken up by the retinol-binding protein at the
BBB,58 BCs in vivo are likely to respond to RA
treatment by elevating TM activity that may counteract downregulation
by cytokines.
Drugs that increase intracellular endothelial cAMP content, such as isoproterenol and forskolin (ie, stimulators of adenylate cyclase activity) or rolipram (ie, inhibitor of cAMP phosphodiesterase activity)59,60 have a potential to enhance anticoagulant mechanisms in brain. This could be important under pathologic conditions when cytokines and stroke risk factors,61,62 transform the BBB from an actively anticoagulant into a procoagulant membrane.11 Strategies to increase cAMP levels at the BBB may not only improve the TM anticoagulant function, but also could suppress the procoagulant tissue factor and plasminogen-activator inhibitor-1, while stimulating the production of tissue plasminogen activator, a key fibrinolytic enzyme.63,64 All these events in turn could restore the antithrombotic properties of the BBB and ameliorate ischemic brain damage.
| Selected Abbreviations and Acronyms |
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| Acknowledgments |
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| Footnotes |
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Received June 2, 1997; accepted August 26, 1997.
| References |
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2.
Dittman WA, Majerus PW. Structure and function of
thrombomodulin: a natural anticoagulant. Blood. 1990;75:329336.
3.
Esmon CT. The roles of protein C and thrombomodulin in
the regulation of blood coagulation. J Biol Chem. 1989;264:47434746.
4. Pindon A, Hantai D, Jandrot-Perrus M, Festoff BW. Novel expression and localization of active thrombomodulin on the surface of mouse brain astrocytes. Glia. 1997;19:259268.[Medline] [Order article via Infotrieve]
5.
Kumada J, Dittman WA, Majerus PW. A role for
thrombomodulin in the pathogenesis of thrombin-induced thromboembolism
in mice. Blood. 1987;71:728733.
6.
Gomi K, Zushi M, Honda G, Kawahara S, Matsuzaki
O, Kanabayashi T, Yamamato S, Maruyama I, Suzuki K. Antithrombotic
effect of recombinant human thrombomodulin on thrombin-induced
thromboembolism in mice. Blood. 1990;75:13961399.
7. Taylor FB, Chang A, Esmon CT, D'Angelo A, Vigano-D'Angelo S, Blick KE. Protein C prevents coagulopathic and lethal effects of Escherichia coli injection in the baboon. J Clin Invest. 1987;79:918925.
8.
Svensson PJ, Dahlback B. Resistance to
activated protein C as a basis for venous thrombosis.
N Engl J Med. 1994;330:517522.
9.
Snow TR, Deal MT, Dickey DT, Esmon CT. Protein C
activation following coronary artery occlusion in the in situ
porcine heart. Circulation. 1991;84:293299.
10.
Macko RF, Ameriso SF, Gruber A, Griffin JH, Fernandez
JA, Barndt R, Quismorio FP, Weiner JM, Fisher M. Impairments of the
protein C system and fibrinolysis in
infection-associated stroke. Stroke. 1996;27:20052011.
11. Zlokovic BV, Wang L, Schreiber SS, Fisher M. Hemostatic functions of the blood-brain barrier: possible implications in the pathogenesis of stroke. In: Greenwood J, Begley DJ, Segal MB, eds. New Concepts of the Blood-Brain Barrier. New York, NY: Plenum Press; 1995:141145.
12. Fisher M. Immunohematologic mechanisms in stroke. In: Caplan LR, ed. Brain Ischemia: Basic Concepts and Clinical Relevance. New York, NY: Springer-Verlag; 1995:7103.
13.
Tran ND, Wong VLY, Schreiber SS, Bready JV, Fisher M.
Regulation of brain capillary endothelial
thrombomodulin mRNA expression. Stroke. 1996;27:23042310.
14. Wong VLY, Hofman FM, Ishii H, Fisher M. Regional distribution of thrombomodulin in the human brain. Brain Res. 1991;556:15.[Medline] [Order article via Infotrieve]
15. Boffa MC, Jackman RW, Peyri N, Boffa JF, George B. Thrombomodulin in the central nervous system. Nouv Rev Fr Hematol. 1991;33:423429.
16.
Ishii H, Salem HH, Bell CE, Laporata EA, Majerus PW.
Thrombomodulin, an anticoagulant protein, is absent in the human brain.
Blood. 1986;67:362365.
17. Isaka T, Yoshimine T, Motokiko M, Kurode R, Ishii H, Hayakawa T. Altered expression of antithrombotic molecules in human glioma vessels. Acta Neuropathol (Berl). 1994;87:8185.[Medline] [Order article via Infotrieve]
18. Maruno M, Yoshimine T, Isaka T, Kurode R, Ishii H, Mayakawa T. Expression of thrombomodulin in astrocytomas of various malignancy and in gliotic and normal brain. J Neurooncol. 1994;19:155160.[Medline] [Order article via Infotrieve]
19. Wang L, Kittaka M, Schreiber SS, Zlokovic BV. Regulation of thrombomodulin expression in bovine brain capillaries. Stroke. 1996;27:187. Abstract.
20. Wang L, Tran ND, Schreiber SS, Zlokovic BV. Thrombomodulin expression in rat brain capillaries: down-regulation in diabetic and chronic nicotine intoxication models. Stroke. 1997;28:259. Abstract.
21.
Yamasaki Y, Mansura N, Shozuhara H, Onodera H, Itoyama
Y, Kogure K. Interleukin-1 as a pathogenetic mediator of
ischemic brain damage in rats. Stroke. 1995;26:676681.
22. Meistrell M III, Cockroft KM, Botchkina GI, Di Santo E, Bloom O, Vishnubhakat J, Ulrich P, Ghezzi P, Tracey KJ. TNF mediates brain damage in stroke. Stroke. 1997;28:255. Abstract.
23.
Nawroth PP, Handley DA, Esmon CT, Stern DM.
Interleukin 1 induces endothelial cell procoagulant
while suppressing cell-surface anticoagulant activity. Proc Natl
Acad Sci U S A.. 1986;83:34603464.
24.
Lentz SR, Tsiang M, Sadler E. Regulation of
thrombomodulin by tumor necrosis factor-
: comparison of
transcriptional and posttranscriptional mechanisms. Blood. 1991;77:542550.
25.
Koga S, Morris S, Ogawa S, Liao H, Bilezikian JP, Chen
G, Thompson WJ, Ashikaga T, Brett J, Stern DM, Pinsky DJ. TNF modulates
endothelial properties by decreasing cAMP.
Am J Physiol. 1995;268:C1104C1113.
26.
Boehme MWJ, Deng Y, Raeth U, Bierhaus A, Ziegler R,
Stremmel W, Nawroth PP. Release of thrombomodulin from
endothelial cells by concerted action of TNF-
and neutrophils: in vivo and in vitro studies. Immunology. 1996;87:134140.[Medline]
[Order article via Infotrieve]
27.
Ishii H, Horie S, Kizaki K, Kazama M. Retinoic acid
counteracts both the downregulation of thrombomodulin and the induction
of tissue factor in cultured human endothelial cells
exposed to tumor necrosis factor. Blood. 1992;80:25562562.
28.
Koyama T, Hirosawa S, Kawamata N, Tohda S, Aoki N.
All-trans retinoic acid upregulates thrombomodulin and downregulates
tissue-factor expression in acute promyelocytic leukemia cells:
distinct expression of thrombomodulin and tissue factor in human
leukemic cells. Blood. 1994;84:30013009.
29. Horie S, Kizaki K, Ishii H, Kazama M. Retinoic acid stimulates expression of thrombomodulin, a cell surface anticoagulant glycoprotein, on human endothelial cells. Biochem J. 1992;281:149154.
30.
Hirokawa K, Aoki N. Up-regulation of
thrombomodulin in human umbilical vein endothelial
cells in vitro. J Biochem. 1990;108:839845.
31. Hirokawa K, Aoki N. Regulatory mechanisms for thrombomodulin expression in human umbilical vein endothelial cells in vitro. J Cell Physiol. 1991;147:157165.[Medline] [Order article via Infotrieve]
32. Archipoff G, Beretz A, Bartha K, Brisson C, de la Salle C, Froget-Leon C, Klien-Soyer C, Cazenave J-P. Role of cyclic AMP in promoting the thromboresistance of human endothelial cells by enhancing thrombomodulin and decreasing tissue factor activities. Br J Pharmacol. 1993;109:1828.[Medline] [Order article via Infotrieve]
33. Shirayoshi Y, Imada S, Katayanagi S, Uyeno M, Imada M. Cyclic AMP-mediated augmentation of thrombomodulin gene expression: cell type-dependent usage of control regions. Exp Cell Res. 1993;208:7583.[Medline] [Order article via Infotrieve]
34. Traynor AE, Cundiff DL, Soff GA. cAMP influence on transcription of thrombomodulin is dependent on de novo synthesis of a protein intermediate: evidence for cohesive regulation of myogenic proteins in vascular smooth muscle. J Lab Clin Med. 1995;126:316323.[Medline] [Order article via Infotrieve]
35. Joo F. The blood-brain barrier in vitro: recent progress. Neurochem Int. 1993;23:499521.[Medline] [Order article via Infotrieve]
36. Zlokovic BV. Critique of the blood-brain barrier in vitro: recent progress. Neurochem Int. 1993;23:523.[Medline] [Order article via Infotrieve]
37.
Zlokovic BV, Mackic JB, Wang L, McComb JG, McDonough A.
Differential expression of Na, K-ATPase
and ß subunit
isoforms at the blood-brain barrier and the choroid plexus.
J Biol Chem.. 1993;268:80198025.
38. Zlokovic BV, Wang L, Sun N, Haffke S, Verrall S, Seeds NW, Fisher MJ, Schreiber SS. Expression of tissue plasminogen activator in cerebral capillaries: possible fibrinolytic function of the blood-brain barrier. Neurosurgery. 1995;37:955961.[Medline] [Order article via Infotrieve]
39.
Jackman RW, Beeler DL, DeWaters L, Rosenberg RD.
Characterization of a thrombomodulin cDNA reveals structural similarity
to the low density lipoprotein receptor. Proc Natl Acad Sci
U S A.. 1986;83:88348838.
40.
Higuchi R, Krummel B, Saiki K. A general method of in
vitro preparation and mutagenesis of DNA fragments: study of protein
and DNA interactions. Nucleic Acids Res. 1988;16:73517367.
41. Tsiang M, Lentz SR, Dittman WA, Wen D, Scarpati EM, Sadler JE. Equilibrium binding of thrombin to recombinant human thrombomodulin: effect of hirudin, fibrinogen, factor Va, and peptide analogs. Biochemistry. 1990;29:1060210612.[Medline] [Order article via Infotrieve]
42. Suzuki K, Kusumoto H, Deyashiki Y, Maruyama I, Zushi M, Kawahara S, Honda G, Yamamoto S, Horiguchi S. Structure and expression of human thrombomodulin, a thrombin receptor on endothelium acting as a cofactor for protein C activation. EMBO J. 1987;6:18911897.[Medline] [Order article via Infotrieve]
43.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ.
Protein measurement with the Folin phenol reagent. J
Biol Chem. 1951;193:265275.
44.
Martinez HR, Rangel-Guerra RA, Marfil LJ.
Ischemic stroke due to deficiency of coagulation
inhibitors. Report of 10 young adults. Stroke. 1993;24:1925.
45.
Chung I, Norris G, Benveniste E. Tumor necrosis factor
alpha production by astrocytes: induction by
lipopolysaccharide, IFN-
, and IL-1ß. J
Immunol. 1990;144:29993007.[Abstract]
46. Fontana A, Kristensen F, Dubs R, Gemsa D, Weber E. Production of prostaglandin E and an interleukin-1 like factor by cultured astrocytes and C6 glioma cells. J Immunol. 1982;129:24132419.[Abstract]
47.
Liu T, McDonnell PC, Young PR, White RF, Siren AL,
Hallenbeck JM, Barone FC, Feuerstein GZ. Interleukin-1ß mRNA
expression in ischemic rat cortex. Stroke. 1993;24:17461751.
48.
Liu T, Clark RK, McDonnell PC, Young PR, White RF,
Barone FC, Feuerstein GZ. Tumor necrosis factor-
expression in
ischemic neurons. Stroke. 1994;25:14811488.[Abstract]
49.
Lavine S, Hofman F, Zlokovic BV. Circulating antibody
against tumor necrosis factor-
protects rat brain from
reperfusion injury. J Cereb Blood Flow Metab (In press).
50. Okada Y, Copeland BR, Fitridge R, Koziol JA, del Zoppo GJ. Fibrin contributes to microvascular obstructions and parenchymal changes during early focal cerebral ischemia and reperfusion. Stroke. 1994;25:18471854.[Abstract]
51.
Kapiotis S, Besemer J, Bevec D, Valent P, Bettelheim P,
Lechner K, Speiser W. Interleukin-4 counteracts pyrogen-induced
downregulation of thrombomodulin in cultured human vascular
endothelial cells. Blood. 1991;78:410415.
52.
Conway E. M, Rosenberg R. D. Tumor necrosis factor
suppresses transcription of the thrombomodulin gene in
endothelial cells. Mol Cell Biol. 1988;8:55885592.
53.
Moore KL, Esmon CT, Esmon NL. Tumor necrosis
factor leads to the internalization and degradation of thrombomodulin
from the surface of bovine aortic endothelial cells in
culture. Blood. 1989;73:159165.
54.
Scarpati E. M, Sadler J. E. Regulation of
endothelial cell coagulation properties: modulation of
tissue factor, plasminogen activator
inhibitors, and thrombomodulin by phorbol
12-myristate 13-acetate and tumor necrosis factor. J
Biol Chem.. 1989;264:2070520713.
55. Tazawa R, Yamamoto K, Suzuki K, Hirokawa K, Hirosawa S, Aoki N. Presence of functional cyclic AMP responsive element in the 3'-untranslated region of the human thrombomodulin gene. Biochem Biophys Res Commun. 1994;200:13911397.[Medline] [Order article via Infotrieve]
56. Nelson S, Palomba ML, Thames E, Greer P, McCachren SS, Dittman WA. Identification and characterization of a retinoic acid responsive element in the gene for human thrombomodulin. Blood. 1993;82:276a. Abstract.
57. De Leenheer AP, Lambert WE, Claeys I. All-trans-retinoic acid: measurement of reference values in human serum by high performance liquid chromatography. J Lipid Res. 1982;23:13621367.[Abstract]
58.
MacDonald PN, Bok D, Ong DE. Localization of cellular
retinol-binding protein and retinol-binding protein in cells comprising
the blood-brain barrier of rat and human. Proc Natl Acad Sci
U S A.. 1990;87:42654269.
59.
Barnard JW, Seibert AF, Prasad VR, Smart DA, Strada SJ,
Taylor AE, Thompson WJ. Reversal of pulmonary capillary
ischemia-reperfusion injury by rolipram, a cAMP
phosphodiesterase inhibitor. J Appl
Physiol. 1994;77:774781.
60.
Seibert A, Thompson W, Taylor A, Wilborn W,
Barnard J, Haynes J. Reversal of increased microvascular permeability
associated with ischemia-reperfusion: role of cAMP.
J Appl Physiol. 1992;72:389395.
61.
Kittaka M, Wang L, Sun N, Schreiber SS, Seeds NW,
Fisher M, Zlokovic BV. Brain capillary tissue plasminogen
activator in a diabetes stroke model. Stroke. 1996;27:712719.
62. Wang L, Kittaka M, Sun N, Schreiber SS, Zlokovic BV. Chronic nicotine treatment enhances focal ischemic brain injury and depletes free pool of brain microvascular tissue plasminogen activator in rats. J Cereb Blood Flow Metab. 1997;17:136146.[Medline] [Order article via Infotrieve]
63. Lyberg T. Effect of cyclic AMP and cyclic GMP on thromboplastin (factor III) synthesis in human monocytes in vitro. Thromb Haemost. 1983;50:804809.[Medline] [Order article via Infotrieve]
64.
Santell L, Levin EG. Cyclic AMP potentiates phorbol
ester stimulation of tissue plasminogen
activator release and inhibits secretion of
plasminogen activator inhibitor-1
from human endothelial cells. J Biol
Chem.. 1988;263:1680216808.
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