© 2000 American Heart Association, Inc.
Vascular Biology |
Increased Flow and Shear Stress Enhance In Vivo Transforming Growth Factor-ß1 After Experimental Arterial Injury
From the Departments of Surgery and Pathology (S.G.), The University of Chicago, Chicago, Ill.
Correspondence to Hisham S. Bassiouny, MD, Department of Surgery, The University of Chicago, 5841 South Maryland Ave, MC 5028, Chicago, IL 60637. E-mail hbassiou{at}surgery.bsd.uchicago.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—We have previously demonstrated that high-flow (HF) conditions inhibit experimental intimal hyperplasia. We hypothesized that such flow conditions may alter transforming growth factor-ß1 (TGF-ß1) after mural injury. The right common carotid artery (CCA) was balloon-injured in 54 New Zealand White male rabbits. Flow was thereafter preserved (normal flow [NF]), reduced by partial outflow occlusion (low flow [LF]), or increased by ligation of the left CCA (HF). Four sham-operated animals served as uninjured controls. Mean blood flow and pressure in the right CCA were measured before and after flow modulation and before euthanasia (3, 7, and 14 days). TGF-ß1 mRNA and protein levels in the right CCA were determined by Northern and ELISA analyses at each time point. At 7 and 14 days, intimal hyperplasia was quantified, and the transmural localization of TGF-ß1 was determined by immunohistochemical analysis. Mean flow was reduced from 22±1 to 10±3 mL/min in the LF group and increased to 34±2 mL/min in the HF group (P<0.001). Blood pressure was not different among the flow groups for all time points. Wall shear stress was markedly decreased in the LF group to 14±4 dyne/cm2 and increased in the HF group to 63±6 dyne/cm2 at 7 days compared with values in uninjured controls (39±2 dyne/cm2, P<0.001) and the NF group (44±7 dyne/cm2, P<0.001). At 14 days, wall shear stress was similar among the flow groups. The intima-to-media ratio was 5- and 2-fold greater in the LF group than in the HF and NF groups at 14 days. mRNA levels for TGF-ß1 and its active ligand were increased in the HF group by at least 2- and 3-fold, respectively, at 3 and 7 days compared with levels in uninjured controls and the LF group (P<0.05) but were not different among the flow groups at 14 days. TGF-ß1 preferentially localized in the abluminal vascular smooth muscle cells of the HF arterial segments. Flow- and shear-mediated release of TGF-ß1 may therefore play a role in abrogating the proliferative and migratory response of vascular smooth muscle cells in the early stages after mural injury.
Key Words: hemodynamics • hyperplasia • transforming growth factor-ß1
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Hemodynamic shear forces have been implicated in the induction and localization of intimal thickening.1 2 3 More specifically, reduced wall shear stress (WSS) has been shown to be associated with enhanced intimal lesion formation in atherosclerosis,4 vascular grafts,5 and balloon-injured vessels.6 Conversely, relative elevations of blood flow and shear stress appear to have an atheroprotective role and inhibit intimal thickening after vascular injury.7 8 9 Hemodynamic forces have been shown to modulate endothelial and vascular smooth muscle cell (VSMC) responses, which involve the induction of cytokines, such as platelet-derived growth factor (PDGF), basic fibroblast growth factor, and transforming growth factor-ß1 (TGF-ß1),10 11 in addition to other mediators, such as nitric oxide,7 tissue plasminogen activator,12 13 and matrix metalloproteinases (MMPs),14 involved in vascular remodeling.
TGF-ß1 has been described as a multifunctional regulator; its actions are dependent on species, cell phenotype, growth conditions, and interaction with other growth factors.15 Several studies have shown that TGF-ß1 inhibits VSMC growth and migration in various species, including human, rat, bovine, and rabbit,11 16 17 18 19 by inhibiting DNA synthesis18 20 and causing cell-cycle arrest at the late G1 phase.21 The latter is similar to the manner in which cell growth is inhibited by shear stress.22 Although in vivo studies have demonstrated that overexpression of TGF-ß1 by direct transfer of TGF-ß1 gene into porcine arteries23 or systemic administration of TGF-ß1 after balloon injury in rabbits24 induces extracellular matrix (ECM) synthesis and deposition in arterial wall, the role of flow and related shear-mediated forces in modulating wild-type TGF-ß1 in the arterial wall has not been investigated. In the present study, we determined TGF-ß1 mRNA and protein levels in the injured New Zealand White male rabbit carotid arteries in relation to varying flow and wall shear conditions at 3, 7, and 14 days. Immunohistochemical staining for TGF-ß1 was also examined in the involved arterial segments.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Animal Model
Adult New Zealand White male rabbits, weighing 3.0 to 3.5 kg and fed normal rabbit chow (n=58), were used for the study. Anesthesia was induced with intramuscular injection of ketamine hydrochloride (40 mg/kg) and xylazine (5 mg/kg) and was maintained with 1% halothane via endotracheal intubation. A 2F balloon catheter was introduced twice via the facial branch of the right common carotid artery (CCA) for 10 cm to induce mural endothelial denudation and the usually associated subjacent medial injury of the entire length of the right CCA. The balloon was consistently inflated with 0.2 mL of normal saline. Injury was induced in 54 animals. Housing and handling of animals were in compliance with Principles of Laboratory Animal Care and Guide for the Care and Use of Laboratory Animals (NIH publication No. 80-23, revised 1985).
Flow Modulation and Hemodynamic Measurements
After balloon injury, blood flow in the right CCA was reduced by
ligation of 3 of the 4 terminal internal and external branches (n=18,
low-flow [LF] group) or increased by ligation of the contralateral
left CCA (n=18, high-flow [HF] group). In another group of animals
with right CCA injury, flow was preserved without
hemodynamic manipulation (n=18, normal flow [NF]
group). Four other animals served as sham-operated controls
(U-controls) in which neither balloon injury nor flow modulation was
performed.
Mean blood flow was measured by use of transit time ultrasound
(Transonics Inc) in the right CCA before and after flow modulation.
Mean arterial pressure was also monitored before and after
flow modulation with an intra-arterial 22-gauge catheter
introduced via the facial artery into the distal CCA. Flow and pressure
were simultaneously recorded at 200 Hz for 10 seconds
with a digital acquisition system (Laboratory Master DMA, Scientific
Solutions, Inc) at day 0 and at days 3, 7, and 14. WSS was calculated
using the Hagen-Poiseuille formula:
=4
Q/
(
r)3, where is WSS
(dyne/cm2), is the blood viscosity (0.03
poise), Q is volume flow (mL/s), is 3.14, is 1.25 (the
shrinkage index,25 which is the ratio of artery diameter
before and after imbedding in paraffin for histological
sections), and r is the arterial radius (cm).
The right CCA was harvested at days 3, 7, and 14 immediately after the flow and pressure measurements. In each of the experimental groups, 3 to 4 cm of the right CCA was prepared for RNA and protein extraction (n=4 per time point). Histomorphometric and immunohistochemical studies were performed on 7 and 14 days (n=3 per time point).
Histomorphometry
At 7 and 14 days, the rabbits were killed with an
intravenous overdose of pentobarbital, and the carotid
arteries were fixed in situ via the facial branch by perfusion with 4%
paraformaldehyde in PBS, pH 7.0, at 100 mm Hg
pressure for 20 minutes. The right CCA was excised and sectioned into
four 5-mm segments. Each segment was embedded in paraffin,
cross-sectioned at 5-µm intervals, and stained with hematoxylin and
eosin and Gomori’s trichrome aldehyde fuchsin preparation for
connective tissue elements. By use of digitized computer-assisted
morphometry (Nikon Co), the intimal, medial, and luminal areas were
measured with Scion Image software. Luminal radius was calculated from
luminal area S (r2), where
r=(
S/)x.
Mural TGF-ß1 Analyses
TGF-ß1 mRNA
cDNA Probe
A 1.0-kb cDNA of human TGF-ß1 was obtained from American Type
Culture Collection. The cDNA probe was digested with
XbaI/HindIII restriction enzyme and isolated by
electrophoresis. The probe was labeled with
[
-32P]CTP by use of the random hexamer
priming method (Promega).
Total RNA Isolation and Northern Blotting
Total RNA was isolated from carotid arteries by
homogenization in 4 mol/L guanidinium
thiocyanate–containing mercaptoethanol and extraction with
phenol/chloroform. RNA was precipitated at -70°C with isopropanol
and redissolved in distilled water containing 0.1%
diethylpyrocarbonate. After denaturation with formamide and
formaldehyde, 15 µg RNA (as measured at 260 nm) from each
sample was electrophoresed in a 1.1% agarose gel. After
electrophoresis, RNA was transferred to a nylon filter by capillary
blotting for 16 hours and cross-linked by UV light. The filter was then
prehybridized in 50% formamide, 1 mol/L NaCl, 10 mmol/L
NaH2PO4, 5x Denhardt’s
solution, 1% SDS, and 250 µg/mL salmon sperm DNA at 42°C for 4
hours, followed by hybridization in the same buffer with
1x106 cpm/mL of the
32P-labeled probe. Incubation was continued
overnight at 42°C. The filter was washed with 2x SSC sodium
chloride 0.15 mol/L, sodium citrate (0.015 mol/L), and 0.1% SDS three
times for 5 minutes each at room temperature, followed by 2 washes in
0.2x SSC and 0.1% SDS for 20 minutes each at 60°C. The filter was
subsequently exposed to x-ray film at -70°C for 72 hours.
Densitometric Analysis
Autoradiographic bands after Northern blotting were
quantified by scanning densitometry (Bio-Rad 620 scanner). To correct
for differences in RNA loading, the density of the 18S rRNA band on the
photographic negative of the ethidium bromide–stained gel was also
determined, and the relative densities of the probe and the rRNA band
were compared.
Active and Total (Latent Plus Active) TGF-ß1
Protein levels of TGF-ß1 in the uninjured control and injured
right CCA segments were measured by ELISA. Samples were extracted by
using chilled glass homogenizers in ice-cold PBS
(15 mmol phosphate and 135 mmol NaCl, pH 7.2) containing 1%
Triton X-100 and 0.1% SDS. A 96-well plate (Costar) was coated
with 100 µL monoclonal mouse anti-human TGF-ß1 antibody (capture
antibody, R & D Systems) in PBS (2 µg/mL, pH 7.4) for 16 hours at
room temperature. The plate was then washed 3 times with PBS containing
0.05% Tween 20, followed by blocking the plate with PBS containing 5%
Tween 20, 5% sucrose, and 0.05% sodium azide for 1 hour at room
temperature. Because the monoclonal antibody recognizes only
activated TGF-ß1, to quantify total TGF-ß1, its latent form
was converted to the active form by acidification with 1.0N HCl for 15
minutes at room temperature and then neutralized with 1.2N NaOH and 0.5
mol/L HEPES. For the active TGF-ß1 assay, equal amounts of samples
(20 µg) were diluted in 100 µL PBS containing 0.1% BSA without
acidification. The plate was subsequently incubated with 100 µL of
sample or standard for 60 minutes at 37°C. After a wash, 100 µL
polyclonal anti-human TGF-ß1 antibody (detection antibody, R & D
Systems) was added into each well, and the plate was incubated for 60
minutes at 37°C. Subsequently, it was washed and incubated with 100
µL streptavidin horseradish peroxidase (Zymed) for 30 minutes at room
temperature. After another wash, substrate solution containing
tetramethylbenzidine (Genzyme) and
H2O2 was added for 30
minutes, and then the reaction was stopped by adding an equal volume of
0.5N sulfuric acid. Absorbance at 450 nm was read with use of an ELISA
microtiter plate reader, and active TGF-ß1 concentrations were
determined from a standard curve, with human recombinant TGF-ß1 used
as the standard (R & D Systems). All samples were assayed before and
after acidification to determine active and total (active plus latent)
TGF-ß1, respectively.
Immunohistochemical Localization of TGF-ß1
Sections were deparaffinized, rehydrated in graded ethanol
solution, and incubated with 3%
H2O2 in methanol for 10
minutes. Nonspecific binding was blocked with 10% BSA in PBS for 1
hour at room temperature. After a wash in Tris-buffered saline (TBS),
the sections were incubated with a polyclonal chicken anti-human
TGF-ß1 antibody (R & D Systems) at a concentration of 20 µg/mL in
PBS overnight at 4°C in a humid chamber. The slides were washed twice
in TBS and once in TBS containing 3% BSA and 0.05% Triton X-100 (TBT)
and then incubated with biotinylated goat anti-chicken IgG (Jackson
Immuno Research) diluted 1/200 in TBT for 30 minutes at room
temperature. After a wash in TBS and TBT, peroxidase-conjugated avidin
diluted to 20 µg/mL (Zymed) in TBS with 0.5% (wt/vol) dried skimmed
milk was applied to the sections for 30 minutes at room temperature.
Peroxidase activity was developed by incubating sections in 0.05%
mol/L Tris-HCl buffer, pH 7.0, containing 0.05% of
3,3'-diaminobenzidine and 0.03%
H2O2 for 10 minutes. The
slides were counterstained with Harris’ hematoxylin, dehydrated in
ethanol and xylene, and mounted with Permount (Fischer
Scientific). Positive control for TGF-ß1
immunostaining was performed on the sections of human
carotid plaque. For a negative control, the sections were incubated in
PBS without the primary antibody or were incubated with nonimmune
rabbit serum.
Data Analysis
Data were expressed as mean±SEM. The group means were compared
by ANOVA, followed by the Scheffé multiple comparison test. A
value of P<0.05 was considered to be significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Hemodynamic Parameters
Mean Blood Flow
Mean baseline blood flow ranged from 17 to 25 mL/min and was similar in all flow groups (22±1 mL/min). At day 0, in the LF animals, outflow ligation reduced baseline flow by 55%, to 10±3 mL/min (P<0.001), whereas in the HF animals, ligation of the contralateral left CCA increased baseline flow by 50%, to 33±2 mL/min (P<0.001). In the NF animals, blood flow was unchanged. At 3, 7, and 14 days, the blood flow measurements in each group were not significantly different from the initial values recorded on day 0 (Figure 1
).
|
Mean Arterial Pressure
Mean arterial pressure in the right CCA ranged from 40
to 62 mm Hg and was not different among the NF (53±2
mm Hg), LF (55±4 mm Hg), and HF (51±5 mm Hg) groups at
all time points.
Wall Shear Stress
WSS values were not significantly different between U-controls
(39±2 dyne/cm2) and the NF group at 7 days
(44±7 dyne/cm2) and 14 days (46±11
dyne/cm2). There was a 64% reduction in WSS
values in the LF group to 14±4 dyne/cm2 and a
62% increase in the HF group to 63±6 dyne/cm2
at 7 days compared with values in U-controls and the NF group
(P<0.001). At 14 days, WSS values were elevated to 63±11
dyne/cm2 in the LF group and declined to 48±7
dyne/cm2 in the HF group compared with the values
at 7 days. These values were not significantly different among the flow
groups at this time point (Figure 2).
|
Histomorphometric Analysis
Histomorphometric analysis of arterial cross
sections at 7 and 14 days are shown in the
Table. At 7 days, intimal
thickening was present in only the LF group but not in the HF and
NF groups. At 14 days, the intimal area and intima-to-media ratio were
4- and 5-fold greater in the LF group (0.25±0.05
mm2 and 0.59±0.03, respectively) compared with
the HF group (0.06±0.03 mm2 and 0.12±0.04,
respectively) and were 3- and 2-fold greater in the LF group compared
with the NF group (0.09±0.02 mm2 and
0.25±0.05, respectively); values were significantly different in the
LF group compared with the NF and HF groups (P<0.03
[intimal area] and P<0.005 [intima-to-media ratio],
respectively). Medial area was not different among the flow groups at
either 7 or 14 days. The luminal radius of the injured arteries was not
influenced by flow conditions at 7 days compared with the value in
uninjured carotid arteries (0.7 mm). At 14 days, there was a 30%
decrease in luminal radius (from 0.7 to 0.5 mm) in the LF group
and a 7% increase in luminal radius (from 0.7 to 0.75 mm) in the
HF group compared with the 7-day measurements. Luminal radius in the NF
group was unchanged at 7 and 14 days (0.7 mm).
|
TGF-ß1 mRNA Levels
Wild-type rabbit TGF-ß1 mRNA was detected in the uninjured
carotid arterial segments (Figure 3A, U-controls), suggesting a
constitutive expression of TGF-ß1. At 3 and 7 days, TGF-ß1 mRNA
expression in the injured arterial segments was increased
in the NF and HF groups compared with the U-control and LF groups, as
represented in Figure 3A. Relative mRNA levels were
determined by densitometry and expressed as a fold of U-control values
(Figure 3B). There were 1.5- and 2-fold increases in TGF-ß1
mRNA levels in the NF and HF groups, respectively, compared with
U-controls (P<0.05) at 3 and 7 days. Conversely, TGF-ß1
mRNA levels in the LF group were similar to levels in U-controls at
corresponding time points. Within the injured arterial
segments, TGF-ß1 mRNA levels were 50% and 30% less in the LF group
and 20% more in the HF group compared with levels in the NF group at 3
and 7 days, respectively. There was also a 2-fold increase in TGF-ß1
mRNA levels in the HF group compared with the LF group
(P<0.05). At 14 days, TGF-ß1 mRNA levels in the HF group
declined and approached those levels in the NF and LF groups.
|
P<0.05 compared with
LF group.
TGF-ß1 Protein Levels
Active TGF-ß1 levels in the U-controls were 0.35±0.03 ng/mg
protein. At 3 and 7 days after injury, active TGF-ß1 levels were
increased by 3-fold in the NF group (1.0±0.02 and 1.0±0.3 ng/mg
protein, respectively) and by 3- and 5-fold in the HF group (1.0±0.1
and 1.8±0.2 ng/mg protein, respectively) compared with U-controls
(P<0.05). The increased values in the HF group were also
2.5-fold greater compared with those in the LF group (0.4±0.07 and
0.7±0.1 ng/mg protein, respectively; P<0.05). Active
TGF-ß1 levels in the HF group were increased by 
2-fold compared
with the levels in the NF group at 7 days (1.8±0.2 versus 1.0±0.3
ng/mg protein), yet the differences were not statistically significant.
At 14 days, active TGF-ß1 increased in the LF group to 1.3±0.1 ng/mg
protein and declined in the HF group to 1.2±0.2 ng/mg protein,
approaching the NF group values (1.1±0.2 ng/mg protein). The values of
all flow groups remained significantly greater than U-control values
(P<0.05); however, no significant differences were observed
among the various flow groups at this time point (Figure 4A).
|
A similar trend was also found in the total TGF-ß1 levels (active
plus latent), with a 2-fold increase in the HF group compared with the
LF group at 3 and 7 days. However, these changes were not statistically
significant compared with U-control and NF group values (Figure 4B
).
Immunolocalization of TGF-ß1
Immunostaining of TGF-ß1 is shown in Figure 5. In the uninjured CCA, TGF-ß1 was
mainly localized in the endothelial layer, with minimal
medial and adventitial immunostaining (Figure 5A). Seven days after injury, TGF-ß1 staining was present
in the CCA throughout the media and adventitia (Figure 5C). In
the CCA subjected to LF conditions, TGF-ß1 was evident within the
neointimal VSMC cytoplasm and surrounding ECM, whereas
staining was minimal throughout the media and adventitia (Figure 5E). In contrast, heavy immunostaining of
TGF-ß1 was present in the CCA subjected to HF conditions and was
preferentially localized in the abluminal VSMCs and throughout the
media and adventitia (Figure 5G). At 14 days, TGF-ß1 staining
was present in all flow groups, with no discernible differences in
localization patterns (Figure 5D [NF], 5F [LF], and 5H
[HF]). Sections incubated with PBS (negative controls) did not
exhibit any immunoreaction (Figure 5B).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The results of the present study indicate that at 3 and 7 days after experimental mural injury, TGF-ß1 mRNA and protein levels are markedly increased in those arterial segments subjected to HF but not in injured arterial segments subjected to LF. At 14 days, TGF-ß1 mRNA and protein levels were not different between the LF and HF groups, yet they remained elevated above U-control values. Early increase in TGF-ß1 may retard the migration of medial VSMCs and their subsequent proliferation in the intima; critical cellular events in the development of injury induced intimal thickening. Such findings additionally substantiate the tenet that relative increases in flow and shear are inhibitory to the intimal hyperplastic response after mural injury.
HF and elevated shear stress have been previously shown to modulate TGF-ß1 synthesis and activation. Cucina et al11 reported that increasing shear stress induces release of TGF-ß1 by arterial endothelial cells in a concentration inhibitory to VSMC proliferation. Ohno et al26 also demonstrated that exposure of cultured endothelial cells to increased steady laminar shear stress (20 dyne/cm2) induced biologically active TGF-ß1. This increase in active TGF-ß1 was associated with a sustained increase in TGF-ß1 mRNA expression. Ueba et al27 also found identical results in cultured bovine VSMCs. In their study, VSMCs exposed to shear stress levels of 28 dyne/cm2 for 24 hours demonstrated a 2-fold increase in TGF-ß1 gene expression. Additionally, the levels of both latent and active forms of TGF-ß1 in conditioned media of VSMCs were elevated. Furthermore, an anti–TGF-ß1 antibody reversed shear-induced inhibition of VSMC growth. Increased in vivo TGF-ß1 by HF in the present study is consistent with the above reported in vitro investigations.
Migration of VSMCs from the media to the intima and remodeling of ECM are principal events in arterial wall intimal thickening. VSMC proliferation occurs in 1 to 3 days and is greatest between 2 and 7 days after injury.28 Proliferating and nonproliferating VSMCs migrate across the intimal elastic lamina into the intima during this time. After 2 weeks, the smooth muscle cell content of the arterial wall does not appear to change, but matrix production continues, resulting in intimal thickening.29 Hemodynamic forces have been shown to modulate intimal hyperplasia (IH) at regions prone to atherogenesis30 and to restenosis after percutaneous transluminal coronary angioplasty31 by regulation of VSMC proliferation32 and migration.6 Kohler and Jawien6 showed that early IH is increased when flow is reduced after balloon injury of the rat carotid artery. Their study suggests that enhanced flow attenuated VSMC migration more than proliferation, suggesting that VSMC migration affected more than smooth muscle cell growth by flow. Chen et al33 reported similar findings indicating that reduced blood flow accelerates IH in endarterectomized canine arteries. In their experiment, this advanced IH response was associated with a relatively increased ECM component.
VSMCs in vivo are surrounded by and embedded in ECM; therefore, ECM degradation and remodeling are essential to VSMC migration to subendothelial space. We have previously studied flow regulation of MMP-2, a critical MMP known to degrade the subendothelial basement membrane.14 We found that LF upregulated and HF inhibited injury-induced MMP-2 mRNA and MMP-2 activity. The role of flow in these experiments appeared to override the MMP response to injury. Such increased MMP-2 activity may facilitate migration of the VSMCs and the subsequent development of intimal thickening. TGF-ß1 is an important modulator of MMPs and thus plays a key role in matrix degradation.34 It has been recently reported that a TGF-ß inhibitory element is found in the promoters of several genes that are downregulated by TGF-ß, including urokinase, elastase, and collagenase.35 36 Thus, HF-enhanced and elevated shear stress–enhanced TGF-ß1 would favor ECM synthesis by inhibiting MMPs. Conceivably, matrix stabilization would impede the migration of medial SMCs into the intima, thereby attenuating early intimal thickening.
Another fundamental observation is that alteration in blood flow and shear may regulate vessel wall dimension. Zarins et al37 demonstrated that an increase in blood flow produced by creating an arteriovenous fistula in an atherogenic model resulted in arterial dilatation and normalization of WSS. Langille and O’Donnell38 documented a 21% decrease in the diameter of the rabbit CCA 2 weeks after flow was reduced by 70%. These data implicate an adaptive response that tends to maintain a constant shear stress at the vessel wall. In the present study, we found a 4-fold increase in cross-sectional intimal area in the LF group compared with the HF group at 14 days after injury. Increased intimal area resulted in a reduction of luminal area and radius in the LF group and a concomitant increase in WSS. This was associated with elevated TGF-ß1, which may contribute to the expansion of the ECM in late intimal thickening. On the other hand, WSS was reduced by 25% to near baseline levels in the HF group as a result of arterial dilation. It is our contention that the convergence of TGF-ß1 mRNA and protein levels in the LF and HF groups at 2 weeks is related to such adaptive dimensional responses.
Increased activity of TGF-ß1 may be related to flow modulation of regulatory proteins involved in release of the active growth factor from its latent complex. Such mechanisms of activation include plasmin or thrombospondin-1 plus other nonspecific stimuli, such as extremes of pH, heat, or chaotropic agents. It has been reported that elevated shear stress enhances tissue plasminogen activator gene expression, which converts plasminogen to plasmin, in cultured VSMCs and that it is concomitant with increased activation of TGF-ß1.27 Thrombospondin-1 has been found to be a major activator of TGF-ß1 in vivo.39 Histological abnormalities in young TGF-ß1 null and thrombospondin-1 null mice were strikingly similar in 9 organ systems. Lung and pancreas pathologies were similarly induced in wild-type pups by systemic treatment with a peptide that blocked the activation of TGF-ß1 by thrombospondin-1. To date, however, the relation between flow and vascular thrombospondin-1 activity is unknown. The mechanisms underlying flow-induced and shear stress–induced activation of TGF-ß1 in vivo deserve further study.
Recently, a shear stress–responsive element (SSRE) was proposed in several pathophysiologically relevant genes. For example, SSRE (core binding sequence GAGACC) has been found in the promoter of the human PDGF-B chain gene, which interacts with DNA binding proteins in the nuclei of shear-stressed endothelial cells to upregulate transcriptional activity.40 A sequence that is complementary to the putative SSRE described within the PDGF-B chain promoter is present at position -1219 in the human TGF-ß1 promoter.41 Ohno et al26 have further postulated the existence of different novel SSRE cis and trans elements within the TGF-ß1 promoter, which are modulated by K+ channel activity. These shear-responsive genetic regulatory elements and their trans activity may suggest a potential mechanism by which the HF-induced and related shear stress–induced TGF-ß1 is regulated at the transcriptional level. A recent study by Topper et al42 reported that 2 genes, Smad6 and Smad7, encoding members of the MAD-related family of molecules, were selectively induced in cultured human vascular endothelial cells by steady laminar shear stress. MAD-related proteins are intracellular proteins that are thought to be essential components in the signaling pathways of the serine/threonine kinase receptors of the TGF-ß superfamily.
In summary, we have demonstrated that increased blood flow and shear stress enhance in vivo TGF-ß1 expression and activation at 3 and 7 days after experimental arterial injury. At 14 days, shear stress declined to near baseline values, resulting in downregulation of TGF-ß1 transcription to levels similar to U-control levels. Elevations of TGF-ß1 in the present study appear to be relatively lower compared with previous observations by Majesky et al.43 This is likely related to deferring techniques; in the control groups of Majesky et al, the endothelium, which is rich in TGF-ß1, was stripped, in contrast to the present study, in which it was preserved. Nonetheless, it is our contention that because TGF-ß1 is a potent cytokine, modest alterations in its tissue concentration can greatly modulate the VSMC proliferative and matrix-remodeling responses. Our results indicate that flow- and shear-mediated release of TGF-ß1 is a critical mechanism in abrogating the proliferative and migratory response of VSMCs in the early stages after mural injury and thereby attenuates the mural healing response to injury. Mechanical force transduction of TGF-ß1 may therefore constitute an important pathway for the adaptive and remodeling response of the injured vessel wall to altered shear forces.
|
Acknowledgments |
|---|
This study was supported by American Heart Association Grant-in-Aid 10843-01-01 and National Institutes of Health grant R01-HL-555296-01. Dr Fortunato was supported by Cardiovascular Pathophysiology and Biochemistry Training Program (University of Chicago) grant 5t32 HL-07237. The authors would like to thank James Vosicky for his excellent assistance in operative and postoperative animal care.
Received May 14, 1999; accepted November 3, 1999.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Bassiouny HS, White S, Glagov S, Choi E, Giddens DP, Zarins CK. Anastomotic intimal hyperplasia: mechanical injury or flow induced. J Vasc Surg. 1992;5:708–717.
2.
Zarins CK, Giddens DP, Bjaradaj BK, Sottiurai
VS, Mabon RF, Glagov S. Carotid bifurcation
atherosclerosis: quantitative correlation of plaque
localization with flow velocity profiles and wall shear stress.
Circ Res. 1983;53:502–514.
3.
Ku DN, Giddens DP, Zarins CK, Glagov S.
Pulsatile flow and atherosclerosis in the human carotid
bifurcation: positive correlation between plaque localization and low
and oscillating shear stress.
Arteriosclerosis. 1985;5:293–302.
4. Glagov S, Zarins C, Giddens DP, Ku DN. Hemodynamics and atherosclerosis: insights and perspectives gained from studies of human arteries. Arch Pathol Lab Med. 1988;112:1018–1031.[Medline] [Order article via Infotrieve]
5.
Geary RL, Kohler TR, Vergel S, Kirkman TR,
Clowes AW. Time course of flow-induced smooth muscle cell proliferation
and intimal thickening in endothelialized baboon
vascular grafts. Circ Res. 1994;74:14–23.
6.
Kohler TR, Jawien A. Flow affects development of
intimal hyperplasia after arterial injury in rats.
Arterioscler Thromb. 1992;12:963–971.
7.
Mattsson EJR, Kohler TR, Vergel SM, Clowes AW.
Increased blood flow induces regression of intimal hyperplasia.
Arterioscler Thromb Vasc Biol. 1997;17:2245–2249.
8. Bassiouny SH, Krievins D, Zarins CK, Abu-Hamad G, Glagov S. Distal arteriovenous fistula inhibits experimental anastomotic intimal thickening. Surg Forum. 1993;44:345–346.
9.
Kohler TR, Kirkman TR, Kraiss LW, Zierler BK,
Clowes AW. Increased blood flow inhibits neointimal
hyperplasia in endothelialized vascular grafts.
Circ Res. 1991;69:1557–1565.
10. Sterpetti AV, Cucina A, Fragale A, Lepidi S, Cavallaro A, D’Angelo LS. Shear stress influences the release of platelet derived growth factor and basic fibroblast growth factor by arterial smooth muscle cells. Eur J Vasc Surg. 1994;8:138–142.[Medline] [Order article via Infotrieve]
11. Cucina A, Sterpetti AV, Borrelli V, Pagliei S, Cavallaro A, D’Angelo LS. Shear stress induces transforming growth factor-beta1 release by arterial endothelial cells. Surgery. 1998;123:212–217.[Medline] [Order article via Infotrieve]
12.
Diamond SL, Eskin SG, McIntire LV. Fluid flow
stimulates tissue plasminogen activator
secretion by cultured human endothelial cells.
Science. 1989;243:1483–1485.
13. Diamond SL, Sharefkin JB, Dieffenbach C, Frasier-Scott K, McIntire LV, Eskin SG. Tissue plasminogen activator messenger RNA levels increase in cultured human endothelial cells exposed to laminar shear stress. J Cell Physiol. 1990;143:364–371.[Medline] [Order article via Infotrieve]
14.
Bassiouuny HS, Song RH, Hong XF, Singh A,
Kocharyan H, Glagov S. Flow regulation of 72-kD collagenase
IV (MMP-2) after experimental arterial injury.
Circulation. 1998;98:157–163.
15. Koyama N, Koshikawa T, Morisaki N, Saito Y, Yoshida S. Bifunctional effects of transforming growth factor-ß on migration of cultured rat aortic smooth muscle cells. Biochem Biophys Res Commun. 1990;169:725–729.[Medline] [Order article via Infotrieve]
16. Halloran BG, Prorok GD, So BJ, Baxter BT. Transforming growth factor-ß1 inhibits human arterial smooth muscle cell proliferation in a growth rate dependent manner. Am J Surg. 1995;170:193–197.[Medline] [Order article via Infotrieve]
17. Mii S, Ware AJ, Kent CK. Transforming growth factor-beta inhibits human vascular smooth muscle cell growth and migration. Surgery. 1993;114:464–470.[Medline] [Order article via Infotrieve]
18.
Owens GK, Geisterfer AAT, Yang YW, Komoriya
A. Transforming growth factor-ß-induced growth inhibition and
cellular hypertrophy in cultured vascular smooth muscle
cells. J Cell Biol. 1988;107:771–780.
19. Morisaki N, Kawano M, Koyama N, Koshikawa T, Umemiya K, Saito Y, Yoshida S. Effects of transforming growth factor-ß1 on growth of aortic smooth muscle cells: influence of interaction with growth factors, cell state, cell phenotype, and cycle. Atherosclerosis. 1991;88:227–234.[Medline] [Order article via Infotrieve]
20. McCaffrey TA, Consigli S, Du B, Falcone DJ, Sanbom TA, Spokolny AM, Bush HL Jr. Decreased type II/type I TGF-ß receptor ratio in cells derived from human atherosclerotic lesions. J Clin Invest. 1995;96:2667–2675.
21. Reddy KB, Howe PH. Transforming growth factor-ß1-mediated inhibition of smooth muscle cell proliferation is associated with a late G1 cell cycle arrest. J Cell Physiol. 1993;156:48–55.[Medline] [Order article via Infotrieve]
22. Sterpetti AV, Cucina A, Santoro L, Cardillo B, Cavallaro A. Modulation of arterial smooth muscle cell growth by hemodynamic forces. Eur J Vasc Surg. 1992;6:16–20.[Medline] [Order article via Infotrieve]
23.
Nabel EG, Shum L, Pompili VJ, Yang ZY, San H, Shu
HB, Liptay S, Gold L, Gordon D, Derynck R, Nabel GJ. Direct transfer of
transforming growth factor ß1 gene into arteries stimulates
fibrocellular hyperplasia. Proc Natl Acad Sci U S A. 1993;90:10759–10763.
24.
Kanzaki T, Tamura K, Takahashi K, Saito Y,
Akikusa B, Oohashi H, Kasayuki N, Ueba M, Morisaki N. In vivo effect of
TGF-ß1: enhanced intimal thickening by administration of
TGF-ß1 in rabbit arteries injured with a balloon catheter.
Arterioscler Thromb Vasc Biol. 1995;15:1951–1957.
25. Zarins CK, Zatina MA, Glagov S. Correlation of postmortem angiography with pathologic anatomy: quantitation of atherosclerotic lesions. In: Bond MG, ed. Clinical Diagnosis of Atherosclerosis: Quantitative Methods of Evaluation. New York, NY: Springer-Verlag; 1986:283–306.
26. Ohno M, Cooke JP, Dzau VJ, Gibbons GH. Fluid shear stress induces endothelial transforming growth factor beta-1 transcription and production: modulation by potassium channel blockade. J Clin Invest. 1995;95:1363–1369.
27.
Ueba H, Kawakami M, Yaginuma T. Shear stress as
an inhibitor of vascular smooth muscle cell proliferation:
role of transforming growth factor-ß1 and tissue-type
plasminogen activator. Arterioscler
Thromb Vasc Biol. 1997;17:1512–1516.
28. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury, I: smooth muscle growth in the absence of endothelium. Lab Invest. 1983;49:327–333.[Medline] [Order article via Infotrieve]
29.
Clowes AW, Schwartz SM. Significance of quiescent
smooth muscle migration in the injured rat carotid artery. Circ
Res. 1985;56:139–145.
30. Friedman MH, Hutchins GM, Bargeron CB, Deters OJ, Mark FF. Correlation between intimal thickness and fluid shear in human arteries. Atherosclerosis. 1981;39:425–436.[Medline] [Order article via Infotrieve]
31. Binns RL, Ku DN, Stewart MT, Ansley JP, Coyle KA. Optimal graft diameter: effect of wall shear stress on vascular healing. J Vasc Surg. 1989;10:326–337.[Medline] [Order article via Infotrieve]
32. Tohda K, Masuda H, Kawamura K, Shozawa T. Difference in dilatation between endothelium-preserved and -desquamated segments in the flow-loaded rat common carotid artery. Arterioscler Thromb. 1992;2:519–528.
33. Chen C, Coyle KA, Hughes JD, Lumsden AB, Ku DN. Reduced blood flow accelerates intimal hyperplasia in endarterectomized canine arteries. Cardiovasc Surg. 1997;5:161–168.[Medline] [Order article via Infotrieve]
34. Roberts AB, Hline UI, Flanders KC, Sporn MB. Transforming growth factor-ß: major role in regulation of extracellular matrix. Ann N Y Acad Sci. 1990;580:225–230.[Medline] [Order article via Infotrieve]
35. Kerr LD, Miller DB, Matrisian LM. TGF-beta1 inhibition of transin/stromelysin gene expression is mediated though a Fos binding sequence. Cell. 1990;61:267–278.[Medline] [Order article via Infotrieve]
36. Matrisian LM, Ganser GL, Kerr LD, Pelton RW, Wood LD. Negative regulation of gene expression by TGF-beta. Mol Reprod Dev. 1992;32:111–120.[Medline] [Order article via Infotrieve]
37. Zarins CK, Zatina MA, Giddens DP, Ku DN, Glagov S. Shear stress regulation of artery diameter in experimental atherogenesis. J Vasc Surg. 1987;5:413–420.[Medline] [Order article via Infotrieve]
38.
Langille BL, O’Donnell F. Reductions in
arterial diameter produced by chronic decreases in blood
flow are endothelium-dependent. Science. 1986;231:405–407.
39. Crawford SE, Stellmach V, Murphy-Ullrich JE, Ribeiro SMF, Lawler J, Hynes RO, Boivin GP, Bouck N. Thrombospondin-1 is a major activator of TGF-ß1 In vivo. Cell. 1998;93:1159–1170.[Medline] [Order article via Infotrieve]
40.
Resnick N, Collins T, Atkinson W, Bonthron DT,
Dewey CF Jr, Gimbrone MA Jr. Platelet-derived growth factor B chain
promoter contains a cis-acting fluid shear-stress-responsive element.
Proc Natl Acad Sci U S A. 1993;90:4591–4595.
41. Resnick N, Gimbrone MA Jr. Hemodynamic forces are complex regulators of endothelial gene expression. FASEB J. 1995;9:874–882.[Abstract]
42.
Topper JN, Cai J, Qiu Y, Anderson KR, Xu YY,
Deeds JD, Feeley R, Gimeno CJ, Woolf EA, Tayber O, Mays GG, Sampson BA,
Schoen FJ, Gimbrone MA Jr, Falb D. Vascular MADs: two novel MAD-related
genes selectively inducible by flow in human vascular
endothelium. Proc Natl Acad Sci U S A. 1997;94:9314–9319.
43. Majesky MW, Lindner V, Twardzik DR, Schwartz SM, Reidy MA. Production of transforming growth factor ß1 during repair of arterial injury. J Clin Invest. 1991;88:904–910.
This article has been cited by other articles:
 |
|
 |
|
  P. J. Mack, Y. Zhang, S. Chung, V. Vickerman, R. D. Kamm, and G. Garcia-Cardena Biomechanical Regulation of Endothelium-dependent Events Critical for Adaptive Remodeling J. Biol. Chem., March 27, 2009; 284(13): 8412 - 8420. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. P. Ng, B. Hinz, and M. A. Swartz Interstitial fluid flow induces myofibroblast differentiation and collagen alignment in vitro J. Cell Sci., October 15, 2005; 118(20): 4731 - 4739. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Friedl, J. Li, B. Schumacher, H. Hanke, J. Waltenberger, A. Hannekum, and S. Stracke Intimal Hyperplasia and Expression of Transforming Growth Factor-{beta}1 in Saphenous Veins and Internal Mammary Arteries Before Coronary Artery Surgery Ann. Thorac. Surg., October 1, 2004; 78(4): 1312 - 1318. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. L. Schott, R. P. Wildman, S. Brockwell, L. R. Simkin-Silverman, L. H. Kuller, and K. Sutton-Tyrrell Segment-Specific Effects of Cardiovascular Risk Factors on Carotid Artery Intima-Medial Thickness in Women at Midlife Arterioscler Thromb Vasc Biol, October 1, 2004; 24(10): 1951 - 1956. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. L. Myers, K. J. Harmon, V. Lindner, and L. Liaw Alterations of Arterial Physiology in Osteopontin-Null Mice Arterioscler Thromb Vasc Biol, June 1, 2003; 23(6): 1021 - 1028. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Tateshima, Y. Murayama, J. P. Villablanca, T. Morino, K. Nomura, K. Tanishita, and F. Vinuela In Vitro Measurement of Fluid-Induced Wall Shear Stress in Unruptured Cerebral Aneurysms Harboring Blebs Stroke, January 1, 2003; 34(1): 187 - 192. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. S. Jackson, A. I. Gotlieb, and B. L. Langille Wall Tissue Remodeling Regulates Longitudinal Tension in Arteries Circ. Res., May 3, 2002; 90(8): 918 - 925. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Negishi, D. Lu, Y.-Q. Zhang, Y. Sawada, T. Sasaki, T. Kayo, J. Ando, T. Izumi, M. Kurabayashi, I. Kojima, et al. Upregulatory Expression of Furin and Transforming Growth Factor-{beta} by Fluid Shear Stress in Vascular Endothelial Cells Arterioscler Thromb Vasc Biol, May 1, 2001; 21(5): 785 - 790. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. van Royen, J. J. Piek, I. Buschmann, I. Hoefer, M. Voskuil, and W. Schaper Stimulation of arteriogenesis; a new concept for the treatment of arterial occlusive disease Cardiovasc Res, February 16, 2001; 49(3): 543 - 553. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. S. Jackson, A. I. Gotlieb, and B. L. Langille Wall Tissue Remodeling Regulates Longitudinal Tension in Arteries Circ. Res., May 3, 2002; 90(8): 918 - 925. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||