© 2000 American Heart Association, Inc.
Thrombosis |
Fluid Flow Induces Upregulation of Synthesis and Release of Tissue Factor Pathway Inhibitor In Vitro
From the Vascular Biology Laboratory, Weston Centre for Experimental Research, Thrombosis Research Institute, London, and the Department of Histopathology (T.K.), Imperial College School of Medicine, Hammersmith Hospital, London, UK.
Correspondence to Dr Florea Lupu, Vascular Biology Laboratory, Thrombosis Research Institute, Emmanuel Kaye Building, Manresa Road, Chelsea, London SW3 6LR, UK. E-mail flupu{at}tri-london.ac.uk
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Abstract—Fluid flow modulates the synthesis and secretion by endothelial cells (ECs) of several proteins that control the hemostatic properties of the vessel wall. Tissue factor pathway inhibitor (TFPI), also synthesized by ECs, is the main downregulator of tissue factor–dependent procoagulant activity. In the present study, we investigated the effect of physiological shear stress on the expression, distribution, and release of TFPI in cultured ECs. The EA.hy926 cell line was grown in a hollow-fiber perfusion system and exposed for variable times to different shear values: 0.27 dyne/cm2 (minimal flow), 4.1 dyne/cm2 (venous flow), and 19 dyne/cm2 (moderate arterial flow). Step increase of the shear stress from 0.27 to 19 dyne/cm2 induced a sharp increase of TFPI released into the medium and a parallel decrease and redistribution of cell-associated TFPI, which suggests that an acute release of TFPI occurred from the cellular pools. During 24 hours of high shear stress, cell-associated TFPI antigen and mRNA increased time-dependently. Subjecting ECs to steady shear stress for 72 hours also upregulated the expression and production of TFPI, in direct correlation with the degree of the shear. The secretion of TFPI was enhanced 1.9-fold under venous flow and 2.4-fold under arterial flow compared with minimal flow. Equally, cell-associated TFPI antigen and cell surface TFPI activity increased proportionally with the shear stress. The expression of TFPI mRNA, as determined by Northern blotting, increased up to 2-fold in ECs under venous flow and up to 3-fold under arterial flow. These results suggest that shear forces regulate TFPI by modulating its release and gene expression in ECs in vitro.
Key Words: tissue factor pathway inhibitor • shear stress • flow • endothelial cells
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Endothelial cells (ECs) serve as a functional barrier between blood and the vessel wall and control the fluidity of the blood by distinct mechanisms. Accordingly, ECs produce antiplatelet aggregants and vasoactive compounds such as prostacyclin (PGI2) and NO; antithrombotic proteins such as thrombomodulin, tissue factor (TF) pathway inhibitor (TFPI), and heparan sulfate proteoglycans; and fibrinolytic proteins such as tissue plasminogen activator (tPA) (for a review, see Reference 1 ). Because of their strategic location, ECs interface with fluid shear forces, which in turn affect their function and gene expression. Examples include the reorganization of the cytoskeletal components,2 regulation of gene expression for proteins that play key roles in the maintenance of homeostasis,3 4 cell migration,5 and cell growth.6 Hemodynamic forces also regulate several of the hemostatic proteins produced by ECs. Fluid shear stress induces significant increases in the release of PGI27 and NO,8 9 as well as enhanced synthesis of regulators that inactivate certain clotting enzymes or cofactors. Recent studies showed that shear stress regulates the generation of thrombomodulin,10 11 which interacts with protein C and protein S to inactivate factor (F)V and FVIII. Shear stress also stimulates the production and release of tPA,3 4 the major activator of vascular fibrinolysis.
The effect of shear stress on the expression of TFPI, however, has not been addressed so far. TFPI is the most physiologically significant inhibitor of the TF-FVIIa complex (for a review, see Reference 1212 ). TFPI uses the tandem Kunitz type domains in its structure to form a quaternary complex with FXa bound on TF-FVIIa and thus prevents further production of FXa and FIXa through the TF-dependent pathway.
The main purpose of the present study was to investigate the effect of physiological levels of fluid shear stress on the acute release of TFPI as well as the long-term effect on the gene expression and synthesis of TFPI in human ECs in culture. Using an in vitro hollow-fiber perfusion system,13 we demonstrated both acute and chronic alterations in TFPI secretion, storage, and expression in ECs in response to shear stress.
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Reagents
The polyclonal anti–recombinant TFPI1-161 IgG developed in our laboratory was immunoaffinity-purified and tested for specificity (50 µg/mL produced 95% inhibition of TFPI activity in normal human plasma; by immunofluorescence, the competition experiments, eg, staining in the presence of recombinant TFPI, were negative). Mouse monoclonal antibodies against caveolin were from Transduction Laboratories. FITC and Texas red–conjugated secondary antibodies and Vectashield mounting medium were from Vector Laboratories Inc. Secondary antibodies conjugated to 10-nm colloidal gold were from BioCell Research Labs, and all the reagents used for electron microscopy were from TAAB Laboratory Equipment Ltd. Octyl ß-D-glucopyranoside (OGP) was from Calbiochem-Novabiochem Ltd. Human coagulation FVIIa, FX, and FXa were purchased from Enzyme Research Labs Ltd, and N-benzoyl-L-isoleucyl-L-glutamyl-(piperidyl)-glycyl-L-arginine-p-nitroanilide hydrochloride (chromogenic substrate S-2337) was from Quadratech. TRIzol was from Life Technologies Ltd, Rapid-Hyb hybridization buffer and [
-32P]dCTP were
from Amersham Life Science Ltd, and Gene Screen transfer membrane was
from NEN Research Products. Cell culture media and supplements were
from GIBCO unless stated otherwise. Phenylmethylsulfonyl
fluoride, HEPES, Tris, BSA, sodium orthovanadate, Triton X-100,
the glucose concentration assay kit, and all other reagents were
purchased from Sigma Chemical Co unless stated otherwise.
Cell Culture and the Flow System
We used the immortalized endothelial cell line
EA.hy926, kindly provided by C.J.S. Edgell (University of North
Carolina, Chapel Hill). This cell line has been previously
characterized with regard to the expression and release of
TFPI14 15 16 and other endothelium-dependent
parameters, such as von Willebrand factor,
thrombomodulin, tPA, and plasminogen activator
inhibitor-1,17 18 and has been found to behave
very much like human umbilical vein endothelial cells,
from which it was initially derived. The cells were grown on T-75
flasks in DMEM containing 4 mmol/L glutamine, 15 mmol/L
HEPES, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 1:10 (vol/vol)
heat-inactivated FCS (Harlan Seralab). After reaching
confluence under static conditions, ECs were trypsinized from the
flasks and seeded at a density of 4x106 cells
per cartridge into the CELMAX QUAD artificial capillary system (Cellco
Inc),13 which was then maintained in a standard
CO2 incubator. The capillary system consisted of
50 semipermeable ProNectin-F–coated polypropylene capillaries (length
13 cm, internal diameter 330 µm, pore size, 0.5 µm)
(Protein Polymer Technology Inc), supported within a Perspex cylinder
(ICI Inc). The culture medium was directed through the
reservoir, pump, and capillaries via silicone tubing that allowed for
the exchange of oxygen and carbon dioxide. By regulating the flow rate
at the pump, the shear stress (
) to which ECs were exposed could be
calculated according to the equation
=
4Q/
r3, where Q is the flow rate (in
milliliters per second), is the viscosity (0.01 for medium plus
FCS), and r is the capillary radius (in centimeters).
Design of the Study
The parameters of the intraluminal flow used in the
present study are summarized in the
Table
. ECs were allowed to adhere
to the capillaries for 3 hours, after which time the minimal flow was
established. ECs reached confluence (determined by glucose
depletion)19 over a period of 5 days under the minimal
flow rate.
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The response of ECs to flow modulation was investigated in 2 sets of experiments: (1) transient shear stress and (2) steady shear stress.
Transient Shear Stress
Fresh medium was added to the cartridges after ECs had reached
confluence and the minimal flow had been maintained for 2 hours, during
which time 100-µL samples were withdrawn at different time intervals
from the flow path immediately after the capillaries. The shear stress
was then increased to the arterial level for a further 2
hours, during which time another series of 100-µL aliquots were
removed, as described above, at defined time intervals (at 30 seconds,
5, at 10, 15, and 30 minutes, and at 1 and 2 hours). In another
experiment, the period of time during which the ECs were kept under
arterial flow was extended up to 24 hours.
Steady Shear Stress
After reaching confluence under minimal flow rate, ECs were
either maintained at minimal flow or subjected to a gradual increase of
the flow over 2 days, up to shear stress levels of 4.1 or 19
dyne/cm2, after which these flow rates were kept
constant for 72 hours. The medium was then discarded and replaced with
a fresh medium for a further 6 hours under the same flow rates, at
which time the medium was collected for further assay. Finally, ECs
were harvested by mechanical backflushing with 5 mmol/L EDTA in
PBS.
Total Cell Lysate Preparation
Cells were centrifuged at 3000 rpm for 10 minutes, and
the pellets were washed twice in ice-cold Tris-buffered saline (TBS,
consisting of 0.1 mol/L Tris-HCl and 0.15 mol/L NaCl), pH 7.8,
containing a cocktail of inhibitors (1 mmol/L
phenylmethylsulfonyl fluoride, 1 mmol/L aprotinin, 1
mmol/L sodium orthovanadate, and 10 mmol/L EDTA). After
centrifugation, cell pellets were resuspended in
ice-cold lysis buffer (TBS containing 10 g/L Triton X-100, 60
mmol/L OGP, and the inhibitors mentioned above) and
incubated for 30 minutes at 37°C with vigorous vortexing, after which
the insoluble proteins were removed by centrifugation,
and the supernatants representing the total cellular
lysates were stored at -70°C until the assay.
Differential Detergent Extraction
Differential detergent extraction of ECs with Triton X-100 and
OGP was performed as previously described.15
TFPI Antigen Assay
The measurement of TFPI antigen in supernatants and cell lysates
was performed by ELISA as described.14 15 Estimation of
the total protein concentration was performed by use of the
bicinchoninic acid assay kit (Pierce & Warriner Ltd).
TFPI Activity Assay
The quantification of the inhibitory activity of
TFPI against TF-FVIIa was performed as previously
described,14 16 by using the 2-stage amidolytic
chromogenic assay. In brief, the cells were fixed in
suspension with 1% (wt/vol) paraformaldehyde in PBS
for 10 minutes, washed with PBS, and incubated for 30 minutes at 37°C
with 600 µL of a combined reagent containing (all final
concentrations) 2.5 mg/L FVIIa, 5 U/L FXa, 1:80 diluted rabbit brain
thromboplastin (from one vial reconstituted with 2 mL distilled water),
and 15 mmol/L CaCl2. After
centrifugation, the supernatants were transferred into
the wells of a 96-well microtiter plate, to which a mixture of 0.4 U/mL
FX and 0.3 mmol/L chromogenic substrate S-2337 was
added. The rate of substrate cleavage was monitored over 25 minutes at
37°C in a microplate reader (Molecular Devices THERMOmax, Alpha Labs,
Ltd) by use of the dual kinetic mode
(L405–L650, where L
is the excitation wavelength [nm]).
TFPI activity was extrapolated from a standard curve constructed with serial dilutions of normal human plasma, which was assigned a TFPI functional potency of 1 U/mL.
Northern Blotting
Total RNA was extracted from ECs by an acid phenol method with
the use of TRIzol reagent according to the manufacturer’s
instructions. Electrophoresis and transfer of the total RNA,
prehybridization, and hybridization were performed as
described.16 The probes used were a 601-bp fragment from
EcoRI/ClaI digestion of human full-length TFPI
cDNA (gift from Dr G. Broze, Washington University, St. Louis, Mo) and
a S26 full-length cDNA housekeeping gene,20 both
radiolabeled by random priming with
[-32P]dCTP. Densitometry and comparisons
between the intensity of the bands were performed by using the public
domain NIH Image software.
Immunofluorescence
Randomly selected fibers were removed from cartridges, gently
flushed with PBS at 37°C, fixed with 4% (wt/vol)
paraformaldehyde in PBS at room temperature,
cryoprotected with sucrose, embedded in Tissue-Tek OCT compound
(Raymond A. Lamb Ltd), and frozen in liquid nitrogen-cooled
isopentane. The cellular localization of TFPI was studied on
cryosections (6-µm thickness) by indirect
immunofluorescence as previously
described.14 15 For double-labeling procedures, the
sections were incubated with cocktails containing a polyclonal IgG
anti-TFPI and monoclonal IgG anti-caveolin. As detection antibodies, a
mixture of goat anti-rabbit Texas red and goat anti-mouse FITC was
applied. Samples were examined with a Bio-Rad MRC 600 confocal laser
scanning unit attached to a Nikon Diaphot inverted microscope
(Bio-Rad Microscience Ltd). To allow a semiquantitative comparison
between different shear stress conditions, the parameters
of image collection (neutral density filter, confocal diaphragm
opening, gain, and black levels) were kept constant.
Images were processed by computer-assisted pseudo–color banding with the use of Confocal Assistant software (Bio-Rad).
Immunoelectron Microscopy
The ultrastructural localization of TFPI in the ECs subjected to
flow was studied by a postembedding immunogold labeling
procedure.15 16 After fixation in situ, the capillaries
were removed and cut into 
1-mm-length pieces and embedded in
Lowicryl K4M (TAAB Laboratories Ltd). Thin sections (50 to 80
nm) were cut on a Reichert Ultracut microtome (Reichert-Jung, Optishche
Werke) and placed on Formvar-coated 200-mesh nickel grids (TAAB
Laboratories Ltd). Sections were immunogold-labeled for TFPI
essentially as described.16
Statistical Analysis
The experiments involving acute exposure to shear stress were
repeated 3 times, and those involving steady (chronic) shear were
repeated 5 times. Assays were performed on 3 aliquots for each
experimental condition. Optical readings were also made in triplicate.
Northern blot analysis was repeated twice for each condition.
Data within groups were statistically compared between each other by
the unpaired t test and expressed as mean±SD. The
differences were considered significant at P
0.05. The
correlation analysis was performed with the use of StatView for
Macintosh (Abacus Concepts Inc).
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Results |
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Characterization of ECs Grown in Artificial Capillary System
Cells growing in the capillaries cannot be directly observed. Therefore, the depletion of the carbon source in the medium, which is directly related to the cell growth, was assessed by monitoring the glucose consumption as an index of growth. Fresh medium was added to the reservoir when the glucose level of the perfusing medium approximated 50% of the starting glucose concentration. The experiments described were carried out when the consumption of glucose was constant for 3 consecutive days.
ECs subjected to flow grew to confluence within the capillary lumen,
forming a typical confluent monolayer (Figure 1a and 1b) separated from the capillary
wall by a basement membrane (Figure 1f). The plasma
membrane exhibited specific flask-shaped invaginations (caveolae) at
luminal and basolateral fronts (Figure 1d and 1f). ECs showed
elongated cytoplasm projections that either formed overlapping
structures with adjacent cells or penetrated deep into the capillary
matrix (Figure 1c [round inset] and 1f). This feature was much
more prominent with the cells grown under arterial levels
of flow. The adjacent cells were always closely adherent to one another
(Figure 1c), connected at the lateral borders through cell
junctions, often with the characteristics of tight junctions (Figure 1c [oval inset]). ECs exhibited abundant organelles, vesicles,
and microfilaments. A well-developed protein synthesis machinery
consisting of rough endoplasmic reticulum (Figure 1c) and Golgi
apparatus (Figure 1e) represented common
features of the cells subjected to the arterial level of
flow.
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Flow-Mediated Acute Release of TFPI
To test the effect of the arterial flow on TFPI in
ECs, confluent monolayers grown in the capillaries at 0.27
dyne/cm2 were subjected to a step increase of the
stress to 19 dyne/cm2. The time course of the
secretion of TFPI in the cell medium over the following 2 hours (Figure 2a) showed a sharp increase in the first
30 seconds after the onset of the arterial flow. The amount
of released TFPI decreased after 60 minutes but maintained an 60%
increase over minimal flow values during the following 60 minutes.
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The amount of cell-associated TFPI was also determined at different
time points during 24 hours of high shear stress. The partition of TFPI
between the Triton extract and the OGP extract was determined in ECs
kept under minimal flow and compared with that in cells subjected to
arterial levels of flow for 2, 6, 12, and 24 hours (Figure 2b
). A significant decrease of cellular TFPI was observed after
2 hours, which occurred equivalently in both fractions
(P<0.01). After 6 hours of subsequent arterial
flow, the OGP-soluble pool of TFPI was restored and remained at
approximately the same level for 24 hours. In contrast, the amount of
TFPI in the Triton-soluble fraction was still very small after 6 hours
(P=0.008 for the difference between time points); however,
this pool was also replenished after 12 hours and slightly increased
after 24 hours compared with the starting point (P=0.1).
We also studied the expression of TFPI mRNA in ECs subjected to
arterial flow for up to 6 hours. By Northern blotting
(Figure 2c), we identified the 2 transcripts of 4 kb and 1.4 kb,
originally described by Girard et al.21 Subjecting the
cells to a sudden increase of shear stress led to an increase of TFPI
expression, which was manifested by both transcripts and apparently
time dependent (Figure 2c). The densitometry of the blots
confirmed that the intensity of both TFPI bands was strongly enhanced
after 6 hours of arterial flow (Figure 2d).
The quantitative data were paralleled by the immunocytochemical
localization of TFPI in ECs subjected to different shear stress values.
The cell surface–located TFPI was visualized in double labeling with
caveolin, the major protein resident of the caveolae (Figure 3a through 3c). ECs under minimal flow
displayed the characteristic immunostaining for TFPI
that was previously observed in human umbilical vein
endothelial cells and EA.hy926 grown under static
conditions,14 with uniform distribution of TFPI over the
cell surface (Figure 3b and 3d). Pseudo–color-banding
analysis revealed that after 1 hour of exposure to
arterial flow, ECs exhibited a decrease in the intensity of
fluorescence (Figure 3e) compared with that in ECs grown
under minimal flow (Figure 3d).
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Effects of Steady Fluid Flow
ECs grown in capillary cartridges were subjected to different flow
rates for 3 days, after which time the medium was changed and collected
after 6 hours. The amount of TFPI released into the medium and that of
cellular TFPI were determined at the end of this incubation time and
compared for different shear values. As illustrated in Figure 4a, the production of TFPI by ECs
was positively correlated with the level of shear stress (correlation
coefficient 0.968). Cells under venous or arterial flow
secreted significantly more TFPI than did the cells maintained under
minimal flow (1.9-fold increase for the venous flow and 2.4-fold
increase for the arterial flow, P<0.0001 for
both). The difference between the arterial and venous flow
with regard to the TFPI secretion was also significant
(P=0.01). The distribution of TFPI in total cell lysates
matched the TFPI released into the medium. Accordingly, total lysates
prepared from ECs kept under venous or arterial flow
contained more TFPI (50% increase for venous shear stress and 100%
increase for arterial shear stress, P<0.05 and
P<0.001, respectively) than did the equivalent lysates of
cells maintained under minimal levels of flow (Figure 4b).
Interestingly, the cell surface–exposed TFPI displayed considerably
higher inhibitory activity toward TF-FVIIa after exposure
to shear stress (Figure 4c, 3 times for the venous flow and
4 times for the arterial flow).
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Northern blotting indicated considerable differences in the expression
of TFPI by cells under different shear rates. As shown by densitometry
in Figure 4d, the intensity of both TFPI transcript bands was
stronger in cells subjected to chronic venous or arterial
flow than in cells under minimal shear stress. However, the
arterial flow induced the highest level of TFPI mRNA
expression. The 1.4-kb band showed the strongest increase of the
intensity (2 times for venous flow and 3 times for
arterial flow), whereas the 4-kb transcript increased 2
times for both shear values.
By immunofluorescence, ECs subjected to chronic
venous flow (Figure 3i) displayed less staining for TFPI than
did cells under arterial flow (Figure 3j). The
latter showed clusters of TFPI distributed all over the plasma membrane
(Figure 3j), also colocalizing with caveolin (Figure 3f
through 3h). A strong accumulation of TFPI occurred at the apical and
lateral front of the cells and on the cell projections entering the
capillary wall (arrowheads, Figure 3f through 3j).
The negative controls, which consisted of the omission of the primary
antibody, did not show notable staining (Figure 3k and 3l).
The immunogold labeling of TFPI in ECs subjected to minimal flow showed
mainly single gold particles that uniformly decorate the plasma
membrane (Figure 3m), whereas the cells that were chronically
subjected to arterial flow also exhibited clusters of gold
particles located in the caveolae (Figure 3p). Moreover, these
cells displayed a higher intracellular labeling for TFPI in the
endoplasmic reticulum (Figure 3q) compared with that in the
cells exposed to minimal flow (Figure 3n), suggesting an
increase synthesis of this protein. The negative controls, which
consisted of secondary antibodies or protein A conjugated to colloidal
gold particles, showed a low background staining (Figure 3o).
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Discussion |
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We have used the perfusion system developed by Redmond et al19 and commercialized by Cellco Inc to study the effect of hemodynamic forces on the TFPI production and release by ECs in vitro. The model consisted of an enclosed perfused bundle of semipermeable polypropylene capillaries that permitted acute or chronic exposure of ECs to pulsatile flow within the range of physiological shear stress. To date, most of the experimental studies on the effects of flow on ECs have used simple systems, such as the conic plate viscometer or the parallel-plate flow chamber, to generate uniform laminar shear. In these cases, the shear force was being applied to static cultures and therefore represented an abrupt transition in the biomechanical loading of the system.22 In contrast, the system used by us offered the possibility to evaluate the response of ECs subjected continuously to flow. The system permitted the generation of temporal fluctuations of the flow parameters as well as the combination of shear stress and hydrostatic pressure in the same in vitro system, which generated a more physiological biomechanical environment for the ECs. The levels of shear used in the present study were within the physiological range of the vascular system.23
The electron microscopic data in the present study showed that cells grown under flow displayed typical ultrastructural characteristics of endothelium (eg, caveolae, tight junctions, and basement membrane). The cells chronically exposed to flow exhibited pseudopodia-like projections that penetrated into the pores of the polypropylene tubes. These may represent adaptive changes to increased mechanical forces, inasmuch as similar cell protrusions were described in an in vivo model of flow-induced arterial remodeling.24 The increased presence of TFPI on these projections may indicate a role in cell adhesion to the matrix, as was suggested by Ruf’s group (Fischer et al25 ).
Our main findings were that a step increase of the shear stress induces the acute release of TFPI from ECs, whereas steady fluid flow upregulates the production of TFPI at the transcriptional level. The acute release occurred rapidly within minutes after the step increase of the shear stress from 0.27 to 19 dyne/cm2 and probably involved the redistribution of preexisting TFPI molecules. We have shown that in resting ECs in culture, TFPI is located as uniform clusters on the cell surface and within an intracellular apical pool14 and that stimulation with thrombin or the Ca2+ ionophore A23187 led to a rapid (within 1 minute) increase of the TFPI exposed on the plasma membrane and to the acute release of the inhibitor.16 The ECs that were acutely challenged with arterial shear levels showed redistribution of TFPI in a manner similarly described for thrombin.14 Because vascular endothelium responds to changes in shear stress by elevation of cytosolic Ca2+26 and because of our observations that TFPI was rapidly released when [Ca2+]i was increased by treatment with Ca2+ ionophore,16 we speculate that a similar mechanism may be responsible for the acute release of TFPI induced by the onset of arterial flow.
Apart from inducing the acute release of TFPI, the high shear stress
also enhanced TFPI gene expression. A variety of proteins are rapidly
"activated" by shear stress in ECs, including certain cell
surface potassium channels, members of the mitogen-activated
and stress-activated protein kinase cascades, several
transcription factors such as nuclear factor-
B, and subsets of
receptor-associated G proteins.27 28 29 The mechanism(s)
through which shear stress regulates TFPI expression remains to be
explained. Recent data suggest that caveolae play a role in
mechanotransduction.30 Caveolae are small
plasmalemma invaginations rich in cholesterol
and glycosphingolipids that can function as cell surface–organized
signal transduction centers.31 32 It has been shown that
the number of cell surface–opened caveolae is increased after EC
exposure to shear stress.33 TFPI settles in caveolae in
resting ECs15 and associates with glycosphingolipid
microdomains, a feature that confers insolubility in Triton
X-100.34 By using the differential detergent extraction
technique, we established that TFPI is decreased in Triton-soluble and
-insoluble fractions in ECs subjected to an acute increase in shear
stress. The enhanced synthesis of TFPI probably led afterward to the
replenishment of the Triton-insoluble pool, because the ultrastructural
images confirmed that TFPI consistently immunolabeled the
caveolae. After 6 hours of arterial flow, only the fraction
of cellular TFPI soluble in Triton, which was probably responsible for
the long-term sustained secretion of TFPI, was still variable.
Inasmuch as the caveolar fraction of TFPI was depleted after 2 hours of
arterial flow, we speculate that this process may have been
mediated through shear stress signals transduced in caveolae (possibly
Ca2+ transients). The events downstream from
early signal transduction that may affect TFPI expression are not known
either. For several of the shear-responsive promoters identified so
far, the ability to respond to shear stress can be directly linked to
the presence of specific cis-acting sequence elements, which
have been named shear stress response elements.5 6 35
The promoter region of the TFPI gene contains the consensus sequence of
the shear stress response element (104-GAGACC-109) that is responsible
for shear regulation of the B chain of platelet-derived growth
factor.6 We suggest that this element may control the
shear dependence of TFPI expression as well.
Our results demonstrate for the first time that shear stress modulates the release and expression of TFPI, which may have significant pathophysiological roles in the regulation of hemostatic balance at the EC surface. Increasing shear levels from minimal to venous or arterial values of flow led to the enhancement of cellular TFPI antigen, cell surface activity against TF-FVIIa, and TFPI mRNA levels. Our results correlate well with previous reports showing that human umbilical vein endothelial cells stimulated with lipopolysaccharide and interleukin-1ß developed a significantly greater TF-dependent procoagulant activity when perfused at a venous shear stress than at an arterial shear stress.36 This suggests that the cell-associated TF activity was inhibited to a greater extent under arterial shear stress. Likewise, Grabowski et al37 showed that ECs exposed to shear forces >0.68 dyne/cm2 exhibited a considerable increase of TF-dependent FXa generation when TFPI was neutralized with anti-TFPI antibodies. We found that the inhibitory potency of cell surface TFPI against TF-FVIIa activity increased to a higher extent than that of the cell-associated TFPI antigen in ECs exposed to flow. By correlating these data with the fact that flow increases the number of caveolae30 and with our findings that flow induces an increase of caveolar fraction of TFPI, one could speculate that the TFPI located in caveolae is more active than the TFPI located on the plasmalemma proper. These observations would endorse the data suggesting that the formation of the TF-FVIIa-FXa-TFPI quaternary complex takes place in glycosphingolipid-rich microdomains (caveolae).38
More work remains to be carried out to establish whether the flow-induced changes in the release and expression of TFPI in vitro also reflect a heterogeneous in vivo expression of the inhibitor in ECs located in vascular beds exposed to different hydrodynamic forces. A comparative analysis of the expression of TFPI in vivo in the vascular system is currently under way in our laboratory. On the basis of previous results that showed that the responses induced by flow in vitro appeared to recapitulate many of the morphological features of the EC in vivo,2 39 we expect our in vitro data to mirror the in vivo situation.
The modulation of TFPI expression by shear stress may have a role in the pathogenesis of vascular disorders. Atherosclerotic lesions distribute focally at the bifurcations and curvatures of the arterial tree, where disturbed flow with low or fluctuating shear stress occurs,40 41 suggesting that such hemodynamic stimuli are critical regulators of the initiation and progression of the disease. Currently, there is clear evidence that areas of low shear stress are prone to atherosclerosis42 and that low shear stress promotes intimal hyperplasia.43
Several lines of evidence indicate that the endothelial genes encoding products that can exert potent antithrombotic,10 antiadhesive,44 and anti-inflammatory45 46 effects in ECs are upregulated by high shear flow. These findings suggest that coordinated induction of these genes in ECs by the uniform shear stress may explain why the nonbranched linear regions of the vasculature are protected from atherosclerosis.47 We assume that the shear stress–enhanced production of TFPI in ECs may also contribute to the maintenance of the anticoagulant and nonatherogenic properties of the quiescent endothelium in vivo.
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Acknowledgments |
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This work was supported by stipends from the British Heart Foundation (C.L., grant PG/97045) and Rovi SA (A.D.W.). F.L. is a Leopold Muller Trust Senior Investigator. We are grateful to Dr Paul Cahill (School of Biotechnology, Dublin City University, Dublin, Ireland) for his assistance with hydrostatic pressure measurements and to Sally Mill and Emma Goulding for technical assistance.
Received August 12, 1999; accepted March 5, 2000.
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