© 2001 American Heart Association, Inc.
Thrombosis |
Shear Stress Decreases Endothelial Cell Tissue Factor Activity by Augmenting Secretion of Tissue Factor Pathway Inhibitor
From the Cardiovascular Thrombosis Laboratory (E.F.G., A.J.R., P.G.T.) and the Vascular Surgery Research Laboratory (O.T., R.W.O.), Massachusetts General Hospital, Boston, Mass.
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Abstract |
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Abstract—Monolayers of human umbilical vein endothelial cells were activated with 50 U/mL interleukin-1
(IL-1) for 3 hours
and simultaneously conditioned with shear stresses of 0,
0.68, or 13.2 dyne/cm2 in a parallel-plate
flow chamber. In the presence of an inflow buffer containing 100 nmol/L
factor X and 10 nmol/L factor VII, production of factor Xa, a
measure of functional tissue factor (TF), was determined as the
product of outflow concentration of factor Xa
(chromogenic assay performed under quasi-static flow
conditions after the shear period) and flow rate. Similarly,
production of TF pathway inhibitor (TFPI) was
estimated as the product of antigenic TFPI (by enzyme-linked
immunosorbent assay) in the supernatant and flow rate. In parallel
experiments, total RNA was isolated for determination of amplification
products of TF mRNA by reverse transcription–polymerase chain
reaction. We found that shear stress reduced factor Xa
production (mean±SE; n=number of experiments) from
13.33±1.14 (n=16) fmol/minxcm2 at 0 shear
stress to 5.70±2.51 (n=5) and 0.54±0.54 (n=4)
fmol/minxcm2 at shear stresses of 0.68 and
13.2 dyne/cm2, respectively. At the same
time, immunogold labeling showed that TF antigen on the
endothelial surface increased >5-fold with shear
stress, whereas TFPI antigen on the surface increased 2-fold. The
secretion of TFPI (appearance of new supernatant TFPI) rose from
7.4±2.4 (n=12)
x10-3
fmol/minxcm2 at 0 shear stress to 23.7±7.3
(n=9) and 50.2±14.3 (n=4)
x10-3
fmol/minxcm2 at 0.68 and 13.2
dyne/cm2, respectively. TF mRNA
amplification products were not markedly changed by shear stress.
We conclude that acute application of shear stress reduces functional,
but not antigenic, expression of TF by intact, activated
endothelial cell monolayers in a manner associated with
shear stress–augmented endothelial cell secretion of
TFPI.
Key Words: tissue factor • endothelial cells • shear stress • tissue factor pathway inhibitor
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Introduction |
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Tissue factor (TF) is the membrane-bound glycoprotein that serves as the nonenzymatic cofactor for factor VII/VIIa in the initiation of blood coagulation, whereas TF pathway inhibitor (TFPI), its principal inhibitor, binds to both activated factor X and the ternary complex formed by TF, factor VIIa, and factor Xa. Both TF and TFPI are synthesized by endothelial cells (ECs), the former usually only after cell activation.1 With regard to increasing shear stress, functional TF has been shown to increase over a wide range of shear stress for a noncellular system of lipid bilayers, to which has been added exogenous TF that has been immobilized on the inner surface of a glass tube.2 A similar increase has been demonstrated for monolayers of human fibroblasts,3 which express TF in the absence of cytokine activation. With respect to monolayers of activated human ECs, however, the dependence on shear stress is more complex, passing through a maximum with respect to shear stress under 1 set of conditions3 or decreasing monotonically with shear stress under another.4 Prior work has not established whether this complex dependence is due predominantly to a reduction in TF protein and TF mRNA, augmentation of cell-associated TFPI, or other factors.
This flow dependence itself, moreover, may explain the fact that TF activity has not normally been found on human endothelium in vivo, whereas TF activity has long been identified on the surface of activated, cultured endothelial monolayers under static conditions in vitro.5 6 TF is not found on normal coronary endothelium or on saphenous veins and internal mammary arteries obtained during coronary bypass surgery, although TF mRNA and protein are expressed in macrophages, foam cells, monocytes adjacent to cholesterol clefts, and smooth muscle cells.7 8 More recently, on the other hand, digoxigenin-labeled factors VIIa and X have been used to demonstrate the presence of TF not only in atherosclerotic plaques but also in a subset of ECs overlying these plaques.9
In the present work, therefore, we explored the effects
of fluid shear stress (none, venous, and arterial) on the
expression of TF by interleukin-1 (IL-1)–activated EC
monolayers. We found that for acute changes in shear stress
simultaneous with EC activation with IL-1 and of 3
hours’ duration or less, dependence of the expression of TF on shear
stress is regulated largely by levels and secretion rates of TFPI and
not by a reduction in TF antigen (nonfunctional plus functional TF) or
TF mRNA.
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Methods |
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EC Monolayers
Human umbilical vein ECs were harvested as described elsewhere10 and seeded on Permanox chamber slides (21x45 mm, Nalge Nunc, Inc) or on glass slides (38x75 mm, Fisher Scientific Co) precoated with fibronectin (10 µg/mL). The medium consisted of medium 199 (Whittaker Bioproducts), 20% fetal calf serum, 150 µg/mL heparin (from porcine intestinal mucosa, Sigma Chemical Co), 50 µg/mL EC growth supplement (Biomedical Technologies), 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (both from Sigma). Monolayers were used when fully confluent, generally at 3 to 7 days of age, as assessed by means of phase-contrast microscopy.
Shear Preconditioning
Monolayers were mounted in specially designed
parallel-plate flow chambers, 1 of
which11 accommodated the
21x45-mm Permanox slides, while the
other12 accepted the larger
38x75-mm glass slides. Both chambers were placed within an incubator
and perfused with complete culture medium containing 50 U/mL IL-1
(Genzyme, Inc) by interposing a given chamber between a sterile
reservoir and a withdrawal pump (shear stress of 0.68
dyne/cm2) or by placing the chamber in a
recirculating-flow device driven by a gravity head, in turn maintained
by a roller pump (shear stress of 13.2
dyne/cm2). The second chamber required
larger flow rates for the same shear stresses, given its larger
cross-sectional area. While the first chamber
(endothelial surface area of 1.9
cm2) was used for measurements of TF
antigen, TFPI antigen, TFPI protein in the supernatant, and factor Xa
production (see below), the second chamber (surface area of
13.2 cm2) was necessary for both accurate
measurements of TFPI secretion and harvest of large numbers (0.5 to
1x106) of shear-exposed ECs for studies of
TF mRNA. Control (static) monolayers, both large and small, were placed
in an incubator in a tissue culture dish containing complete medium and
the same concentration of IL-1. Duration of flow (or stasis) was
always 3 hours. Unless otherwise stated, IL-1 exposure was
simultaneous with flow.
In further experiments with the first chamber type
concerning factor Xa production, monolayers were (1) exposed to
a shear stress of 0.68 dyne/cm2 for 3 hours
in the absence of IL-1 and then exposed to IL-1 in the absence of
shear for an additional 3 hours or (2) exposed to IL-1 in the
absence of shear for 3 hours and then exposed to a shear stress of 0.68
dyne/cm2 for 3 more hours in the absence of
IL-1.
Immunogold Labeling for TF and TFPI
Antigens
TF and TFPI antigens were identified by
immunocytochemistry for light microscopy. After exposure to shear
stress, monolayers were fixed in 4% paraformaldehyde
solution, washed 3 times in PBS, coated with 10% normal goat serum (as
the blocking agent) in PBS for 20 minutes, and then incubated for 1
hour at room temperature with either 30 nmol/L of a polyclonal
anti-TF antibody (raised in rabbits to the extracellular domain of
recombinant soluble TF, residues 1 to 218; courtesy of Dr Yale
Nemerson, Mt Sinai Medical Center, New York, NY) or 30 nmol/L of a
monoclonal antibody against TFPI (mouse anti-human; American
Diagnostica). The primary antibodies were detected with a
secondary gold-conjugated antibody (goat anti-rabbit with 10-nm gold
particles, or goat anti-mouse with 5-nm gold particles; Amersham Life
Science). As a control for nonspecific binding of the secondary
antibody, the primary antibody was omitted from a portion of every
monolayer that nonetheless received the secondary antibody. Monolayers
were then postfixed with 2.5% glutaraldehyde in
cacodylate buffer. For light microscopy (Optiphot, Nikon), the bound
gold particles were subsequently enhanced with silver (IntenSE M silver
enhancement kit RPN491, Amersham Life Science) and visualized with
dark-field and interference reflection contrast microscopy.
Quantification of bound antibody against TF or TFPI was performed on
light microscopy specimens by interference reflection contrast
microscopy with a x20 objective. Interference reflection contrast
microscopy was favored over dark-field microscopy because
epi-illumination prevented scattered light from the ECs and revealed
only the silver-enhanced gold particles. By means of a CCD camera
(model 300-RC, Dage-MRI, Inc) and a frame grabber with Inspector
software (Matrox Electronics Systems, Ltd), 5 random images of each
immunolabeled EC monolayer were digitized, recorded onto hard disk
(Dimension XPS P120C, Dell Computer Corp), and analyzed for the
percentage of image pixels occupied by silver-enhanced gold particles.
Because immunolabeling may have varied on different days, we compared
EC labeling performed on the same day only.
Chromogenic Assay for Factor Xa
Under Nearly Static Flow Conditions
As a measure of functional TF, we determined levels
of activated clotting factor X (factor Xa) that appeared in the
supernatant of intact endothelium or
subendothelial matrix by using an amidolytic
technique.3 For this purpose,
EC monolayers (on Permanox slides) after exposure to shear stress and
IL-1 were incubated at 37°C under nearly static conditions (see
below) with a "reaction complex" consisting of 0.01 mol/L HEPES,
0.14 mol/L NaCl, 10 nmol/L factor VII, 100 nmol/L factor X (both from
American Diagnostica), 5.0 mmol/L
CaCl2, and 1 mg/mL bovine serum albumin.
Aliquots of postincubation reaction complex were mixed with an equal
volume of 75 mmol/L EDTA to inhibit further activation of factor X
and then incubated with Spectrozyme Xa (0.5 mmol/L final
concentration, American Diagnostica) at 37°C for 30
minutes. To block further action of factor Xa on the
chromogenic substrate, we then added 30% acetic acid (1
part to 5 parts reaction complex). Samples were read in a microplate
reader at 405 nm for the increase in absorbance of free chromophore; a
calibration chart had been constructed previously by using known
concentrations of purified factor Xa (A. Guha, Mt Sinai Medical Center,
New York, NY).
Factor Xa production was assayed under nearly static conditions at 0.054 dyne/cm2 in the same flow chamber used for shear preconditioning or, when true static conditions were used, monolayers were mounted for assay purposes in an identical chamber (control static monolayers) at 0.054 dyne/cm2. The low shear stress (0.054 dyne/cm2) during the factor Xa assay, which characterized the nearly static conditions, was sufficient to allow chamber outflow sampling (over a 40-minute period) without disassembly of the flow chamber yet approximated well the true static conditions when compared with either of the shear preconditioning stresses (0.68 or 13.2 dyne/cm2). Factor Xa production was estimated as the product of the nearly static flow rate and the steady-state outflow concentration of factor Xa (achieved after 15 minutes).
The specific TF origin of factor Xa activity was confirmed by additional experiments in which activated monolayers under "true" static conditions were incubated for 30 minutes with 100 nmol/L of the above polyclonal anti-human TF antibody, versus 100 nmol/L of a nonspecific rabbit IgG. Factor Xa production was calculated from the rate of increase of factor Xa over time.
Anti-TFPI Assay
In view of the reported
equilibrium13 14
that is believed to exist between supernatant "free" TFPI and
EC-associated TFPI, we
previously15 characterized
the affinity in solution of a monoclonal antibody directed against
Kunitz domain 1 of human TFPI (American Diagnostica) for
EC-associated TFPI in terms of Michaelis-Menten kinetics. For human
umbilical vein ECs,15 we
estimated an apparent rate constant,
k, of 8.68±3.08 µg/mL
(mean±SE) and a maximum production rate of factor Xa,
Fmax, of 1.385±0.191
fmol/cm2 per 100 000 cells (mean±SE).
Therefore, some monolayers were washed with HEPES buffer with 0.1%
bovine serum albumin and incubated with 100 nmol/L anti-TFPI
for 30 minutes at 37°C. After a single wash with the same HEPES
buffer, the monolayers were assayed for factor Xa production as
described above. Control experiments were carried out in which TF
activity was assessed in the absence of cytokine activation
("baseline" activity), without and with
anti-TFPI.
Assay for TFPI
Antigenic levels of TFPI in cell culture supernatants
were determined by using an ELISA for total (full-length plus
truncated) TFPI (product 849, American Diagnostica).
TFPI secretion after IL-1 activation with shear preconditioning at
0.68 dyne/cm2 or IL-1 activation during a
period of true stasis was measured from chamber outflow sampling in a
manner similar to that described above for factor Xa
production; ie, nearly static conditions were used. TFPI
secretion after 3 hours at 0.68 dyne/cm2 or
after 3 hours under static conditions was calculated as the product
of the nearly static flow rate and the steady-state outflow
concentration of TFPI (achieved after 30 minutes). For 13.2
dyne/cm2, samples after 3 hours were taken
directly from the upper reservoir of the recirculation device, which
had been filled with medium containing or not containing IL-1. TFPI
secretion was then calculated as the product of the rate of rise of
TFPI concentration and the total volume (15 to 20 mL) of the
recirculation device. Preliminary experiments had shown that TFPI
levels in complete media were <0.1 ng/mL and were augmented to only a
minor extent by cytokine activation without or with shear
preconditioning.
Quantitative RT-PCR Amplification Products
of TF mRNA
Total RNA from guanidine isothiocyanate extracts of
cell cultures (isolated as indicated for Northern blot
analysis) was used for reverse transcription–polymerase chain
reaction (RT-PCR). First-strand cDNA synthesis (RT reaction) used 4
µg of total RNA and 0.2 A260 U of
random-hexamer primers (Boehringer-Mannheim) and was performed
for 1 hour at 37°C with 400 U of M-MLV reverse transcriptase
(Promega) in Promega reaction buffer and 40 U of RNase
inhibitor (Boehringer-Mannheim). PCR was performed
in a Mini Cycler (MJ Research). In each reaction we used 5% of the RT
reaction (2 µL of a 40-µL RT total volume), 2 pmol of primer
oligonucleotides for TF, 1 pmol of primer
nucleotide for glyceraldehyde 3-phosphate
dehydrogenase (GAPDH), and 2 U of
Taq polymerase (Fisher
Scientific), in PCR reaction buffer B (Fisher Scientific) supplemented
with 4 µL of 25 mmol/L MgCl2 to a final
concentration of 4 mmol/L in a final reaction volume of 25 µL.
Oligonucleotide primers for TF were synthesized from
published primer sequences4
and yielded a product of 204 bp. Those for GAPDH were constructed
complementary to the known sequence for GAPDH. The primer sequences for
GAPDH were AAG GTG AAG GTC GGA GTC AA for the 5' end and TGA GTC CTT
CCA CGA TAC CA for the 3' end, resulting in a 511-bp product.
Thermal cycling parameters were as follows: initial
denaturation at 95°C for 1 minute followed by 94°C for 30 seconds,
annealing at 50°C for 30 seconds, and extension at 72°C for 2
minutes per cycle, with a final extension at 72°C for 5 minutes.
Linear amplification (log optical density of RT-PCR amplification
products) was established with respect to cycle number for both TF
and GAPDH primer pairs. Quantification of the products was
performed by measuring optical density in the range of 20 to 22
cycles.
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Results |
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Endothelial Monolayer Morphology
No loss of EC monolayer integrity was observed at 4 hours and over the range of shear stress used (0 to 13.2 dyne/cm2). ECs also did not undergo elongation and alignment with flow direction, a phenomenon observed by others16 after longer periods of shear stress.
Immunogold Labeling for TF and TFPI
Antigens
Because unstimulated ECs do not express TF antigen at
their surfaces, we considered the weak immunolabeling (0.5%) found at
those cell surfaces to be nonspecific (Figure
I; published online at
http://atvb.ahajournals.org), an impression confirmed in every 1 of our
experiments by a similar degree of weak labeling observed in the
absence of primary antibody but in the presence of the secondary
antibody (Figure II; published online at http://atvb.ahajournals.org).
Cytokine activation of the ECs increased TF labeling from this
background level to 5% to 8% (n=3,
Figure 1a versus Figure I). Subjecting ECs to venous
and arterial shear stress while simultaneously
being activated with IL-1 further increased surface TF
antigen expression by 2-fold and 5-fold, respectively (n=3 in each
case,
Figures 1b and 1c). As for TFPI antigen, a baseline level of
detectable antigen was present in the absence of IL-1, which was
not detectably increased after cytokine activation, in accord
with only a modest 25% increase observed by
others17 after stimulation
with thrombin. However, this baseline level in the presence of
cytokine activation was increased 
2-fold by shear stress
over the range of shear stress investigated
(Figures 2a and 2b). In the absence of primary antibody but in
the presence of secondary antibody, only weak labeling was observed
(Figure III; published online at
http://atvb.ahajournals.org).
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Factor Xa Production
Preconditioning of the ECs with a shear stress of 0.68
dyne/cm2 for 3 hours, with
simultaneous activation with IL-1, reduced functional TF
to 43% of baseline, while detectable factor Xa was not significantly
different from 0 at 13.2 dyne/cm2
(Figure 3). When shear stress preconditioning for 3 hours in
the absence of IL-1 preceded cytokine activation under
static conditions for 3 hours, functional TF was reduced to 12% of
control. However, when cytokine activation preceded shear
preconditioning, functional TF declined to only 71% of baseline
(Figure 4). Control experiments under static conditions with
100 nmol/L of the polyclonal antibody to human TF reduced functional TF
by 92±6.8% (n=4).
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Anti-TFPI Studies
Under static conditions, the presence of 100 nmol/L
anti-TFPI augmented (P<0.01)
measurable factor Xa production after IL-1 stimulation by
2.1-fold, from a mean±SE (n=number of experiments) of
1.27±0.25 (n=6) to 2.61±0.19 (n=6). In the absence of IL-1
activation, factor Xa production was similar with and without
anti-TFPI: 0.11±0.016 (n=4) and 0.08±0.020 (n=4)
fmol/minxcm2,
respectively.
TFPI Secretion
TFPI secretion after IL-1 activation increased
significantly with simultaneous shear stress
(P<0.01), attaining 3-fold and
nearly 7-fold increments at 0.68 and 13.2
dyne/cm2, respectively, over that under no
shear stress
(Figure 5). Results did not differ significantly in the
presence versus absence of IL-1 stimulation.
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Quantitative RT-PCR
In 3 separate experiments, the RT-PCR
amplification products for both TF mRNA and control GAPDH mRNA
neither increased nor decreased significantly with shear stress
(Figure 6).
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Discussion |
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We report here that shear stress reduces functional, but not antigenic (nonfunctional plus functional) expression of TF by intact, activated EC monolayers in a time- and shear stress level–dependent manner. Indeed, the expression of surface TF antigen was markedly enhanced by shear stress, whereas for acute changes in shear stress investigated in this study, there was no significant change in TF mRNA. The decrease in functional TF instead may be due to secretion into the medium of sufficient TFPI to offset any increased production of factor Xa. The endothelium accomplishes this by ensuring that its rate of secretion of TFPI (and any component of "secretion" that may be new TFPI synthesis) is both shear rate augmented and always just large enough so that the rate-limiting step for transport into the culture medium (or, in vivo, into the blood) is convective diffusion, not the rate of secretion itself. The net result is the maintenance, despite increasing shear rates, of a relatively constant concentration of TFPI in a "boundary layer" immediately adjacent to the endothelium. (See the Appendix.) This boundary layer concentration is believed to be in equilibrium with cell surface–associated TFPI.13 14 Therefore, cell surface–associated TFPI need not increase markedly with shear stress (see below). We also have previously reported no significant change in TFPI secretion after cytokine activation,15 an observation consistent with that of Ameri et al,18 who found increases in TFPI levels only of the order of 25%.
In parallel with this mechanism, shear stress may also favor, to a modest extent, the redistribution and increased exposure of TFPI on the EC plasma membrane, such as has been reported for thrombin stimulation under static conditions.17 Such TFPI is believed to "encrypt" TF by forming TF/factor VIIa/factor Xa/TFPI assemblies that inhibit TF/factor VIIa in glycosphingolipid-rich microdomains and translocate into low-density, detergent-insoluble cellular fractions enriched in caveolin (caveolae19 20 ). In this regard, we note that surface TFPI antigen in the present study, as assessed by immunogold labeling, was maintained—even doubled—over the range of shear stress from 0 to 13.2 dyne/cm2. A third mechanism for the effects of shear stress on functional TF may be a shift in the partitioning of TF between apical and basolateral surfaces, possibly by bringing more TF to the apical surface where it can be inhibited by the formation of TF/factor VIIa/factor Xa/TFPI assemblies.
Whatever the mechanism, a potent role for TFPI is supported
by our findings under static conditions of a 2.1-fold increase in
functional TF with 100 nmol/L anti-TFPI and by our earlier observation
under static conditions15 of
a 3.7-fold increase with a molar excess of anti-TFPI. Under flow
conditions with a lower concentration of IL-1, we demonstrated
increases in functional TF of 15-fold and 25-fold at shear stresses of
0.68 and 2.7 dyne/cm2, respectively, with
the use of 300 nmol/L of a polyclonal anti-TFPI (calculations from data
of Figure 3
of Reference 33 ). TFPI, therefore, may have an even more
potent role under shear stress conditions.
Matsumoto et al4
have reported a decrease with respect to shear stress in functional TF,
surface TF antigen, and TF mRNA by tumor necrosis
factor-–activated ECs, the magnitude of the decrease in
functional TF being dependent on both shear intensity and duration. In
most of their experiments, the ECs were preconditioned with shear
stress for 15 hours before the onset of stimulation with tumor necrosis
factor-. Shorter periods of preconditioning led to lower degrees of
reduction in TF mRNA (Y. Ikeda, unpublished data, August 1999). These
workers interpreted their findings to be indicative of shear
attenuation of TF mRNA, a conclusion that may apply to longer (eg,
15-hour) periods of preconditioning, but not to shorter (eg, 3-hour)
periods.
In the "closed" cone-in-plate system used by Matsumoto et al,4 it is furthermore probable that TFPI accumulated over time and contributed to the observed reduction in functional TF for intact endothelium. In contrast, the reduction in functional TF of the present study was observed at 0.68 dyne/cm2 in a single-pass system, which eliminates the possibility of such TFPI accumulation. This is not a trivial matter, because the presence of an antibody directed against Kunitz domain I of human TFPI was found in the present work and in previous work15 to more than double measurable production of factor Xa under static conditions, indicating a marked downregulation by TFPI of EC functional TF even under static circumstances. We hypothesize that the downregulation of functional TF under flow conditions is due chiefly to TFPI when changes in shear stress are acute (eg, nearly simultaneous with the onset of cytokine exposure) but may be due as well to downregulation of TF mRNA (and possible upregulation of TFPI mRNA) when changes in shear stress are more chronic (eg, lasting several hours or more).
Lin et al21 and
Houston et al22 both
reported that shear stress alone (no cytokine exposure)
transiently induced TF mRNA over periods of 1 to 3 hours. Transcription
factors likely involved in these shear stress–induced changes in TF
mRNA include Sp1, whose increased activity is associated with
hyperphosphorylation,21
and/or
Egr-1,22 23 24
but not nuclear factor-
B.4
In those studies, either human umbilical vein ECs were subjected to 12
dyne/cm2 for 1 to 2
hours21 or human aortic ECs
were exposed to 15 dyne/cm2 for 1 to 3
hours.22 Lin et al found
that TF mRNA by Northern blotting was essentially no longer present
after 6 hours; Houston et al, who performed RT-PCR, did not look beyond
3 hours. Although the present work does not address this issue, we
did find that TF antigen at 3 hours increased with increasing shear
stress. Although we also found no significant effect of shear stress on
TF mRNA at 3 hours, it is possible that shear stress may have shortened
the time required for TF mRNA to attain a "ceiling" level, thereby
still having an effect on the area under the curve for TF mRNA. A
further point is that both Matsumoto et
al4 and the present work
used cytokine stimulation, whereas Houston et al and Lin et al
did not: stimulation with tumor necrosis factor- or IL-1 may have
quantitatively masked any response due to shear stress itself. In
support of this is our earlier observation with a 10-fold
lower concentration of IL-1:
factor Xa production, with or without anti-TFPI, was actually
enhanced at a shear stress of 0.68 dyne/cm2
compared with no shear
stress.3
These findings suggest that flow has a direct effect on diminishing the ability of activated ECs to generate functional TF. One may speculate that stasis in vivo may therefore promote coagulation by permitting the relative upregulation of TF and TF mRNA and downregulation of TFPI secretion. Such a process may be operative in deep venous thrombosis or in coronary artery thrombosis as a compounding event after acute near-closure due to platelet thrombi and/or vessel spasm. This remains to be clarified in future work.
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Appendix 1 |
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TopAbstractIntroductionMethodsResultsDiscussionAppendix 1References |
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Convective Diffusion of TFPI
The convective diffusion of a substance from a surface can in general be considered a 2-step process. Step 1 is the local "production and/or release rate" of the substance at the surface itself, which for TFPI in the current study is its rate of secretion. Step 2 is convective diffusion of the substance into the bulk of the adjacent flow and downstream. When the first step is rate limiting, the gradient in the concentration of the substance normal to the surface is fixed, while the surface concentration diminishes with increasing flow rate. Convective diffusion is then said to be surface reaction–rate limited. When the second step is rate limiting, the surface concentration of the substance is fixed, while the surface concentration gradient increases with increasing flow rate. Convective diffusion is then said to be diffusion limited. Because the secretion rate of TFPI in the current work was found to increase with increasing shear stress (or shear rate), it is likely that the latter condition well approximates the convective diffusion of TFPI. Mathematically, such a diffusion-limited process for a concentration boundary layer confined to a fraction of the thickness of the chamber flow path and for isotropic diffusion (according to the Fick equation) in a homogeneous, incompressible fluid25 is described by the Leveque26 equation:
 |
where -D(dc/dy)y=h is the diffusion (production) rate of TFPI, in mol/cm2xs, from the EC surface; D is the Brownian diffusion coefficient for TFPI, in cm2/s; c is the local concentration of TFPI, in mol/cm3; y is the distance from the plane bisecting the chamber flow path, in cm; C0 is the concentration of TFPI at the EC surface, in mol/cm3; h is half of the height of the flow chamber, in cm; U is the mean flow velocity in the flow chamber, in cm/s; and x is the axial (downstream) position from the chamber inlet, in cm.
For an increase in shear stress (or mean flow velocity) from 0.68 to 13.2 dyne/cm2, -D(dc/dy)y=h should increase by a factor equal to the one-third power of (13.2/0.68), or by a factor of 2.7. The observed increase is a factor of 2.4. The present TFPI data are therefore consistent with diffusion-limited convective diffusion, which predicts a surface concentration of TFPI that changes little with shear stress. This is also in accordance with the present experimental observations involving immunogold labeling of surface TFPI, which showed that the surface antigen concentration of TFPI changes at most by a factor of 2 over the range of shear stress studied.
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Acknowledgments |
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This work was supported by grant No. HL33095 from the National Heart, Lung, and Blood Institute, Bethesda, Md. Dr Reininger’s salary was supported by DFG grant No. RE-1293/3–1.
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Footnotes |
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Reprint requests to Eric F. Grabowski, MD, Dr Eng Sci, Pediatric Hematology/Oncology, Blake 255, Massachusetts General Hospital, Fruit St, Boston, MA 02114.
May 22, 2000; revision accepted November 1, 2000.
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References |
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