© 1999 American Heart Association, Inc.
Vascular Biology |
Focal Expression of Angiotensin II Type 1 Receptor and Smooth Muscle Cell Proliferation in the Neointima of Experimental Vein Grafts
Relation to Eddy Blood Flow
From the Biomedical Engineering Department, Northwestern University, Evanston, Ill.
Correspondence to S.Q. Liu, PhD, Biomedical Engineering Department, Northwestern University, 2145 Sheridan Rd, Evanston, IL 60208-3107. E-mail sliu{at}nwu.edu
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Abstract—Eddy flow has been shown to promote focal smooth muscle cell (SMC) proliferation and neointimal formation in experimental vein grafts. This study focuses on whether the angiotensin II type 1 (AT1) receptor mediates these events. Experimental vein grafts with and without eddy flow were created in the rat. Losartan was used to assess the influence of the AT1 receptor on SMC proliferation. In vein grafts with eddy flow, apparent focal expression of AT1 mRNA and protein was found in the leading region of the proximal focal neointima, where eddy flow occurred, but not in the trailing region, where eddy flow diminished, at days 5, 10, 20, and 30. The rate of SMC proliferation in the leading region (10.9±1.4%, 19.5±2.2%, 12.2±2.0%, and 6.9±1.3% at these times, respectively) was significantly higher than that in the trailing region (9.5±1.8%, 15.3±2.0%, 8.2±1.9%, and 3.2±0.7%) in these vein grafts. When eddy flow was prevented in engineered vein grafts, no apparent location difference was found in the distribution of AT1 receptor mRNA and protein in the neointima, and the rate of SMC proliferation (5.3±1.0%, 5.8±0.9%, 3.4±1.0%, and 3.7±0.9% at days 5, 10, 20, and 30, respectively) was reduced significantly. In vein grafts with losartan, the rate of SMC proliferation in the leading region of the neointima (9.4±1.8%, 10.1±1.3%, 8.3±0.9%, and 4.2±0.5% at days 5, 10, 20, and 30, respectively) was significantly lower than that in vein grafts without losartan. These results suggested that eddy flow upregulated the AT1 receptor, which in turn mediated focal SMC proliferation in the neointima of experimental vein grafts.
Key Words: fluid shear stress • tensile stress • intimal hyperplasia • vascular tissue engineering
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Introduction |
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Blood vessels develop and remodel under the influence of mechanical factors, including blood pressure and flow. It has long been hypothesized that these mechanical factors contribute to the development of vascular diseases such as atherosclerosis. Previous clinical and experimental investigations have provided convincing evidence for this hypothesis. These studies have demonstrated that atherosclerotic lesions in human and animal arteries develop mainly in curved and bifurcation regions where eddy or secondary blood flow occurs.1 2 3 4 The coincidence of eddy blood flow with atheroma suggests that eddy blood flow may play a role in the initiation and development of vascular atherosclerosis.
Vein grafts, which are commonly used to replace malfunctioned arteries, are subject to eddy blood flow. Experimental studies have demonstrated that the location and pattern of eddy blood flow vary in different vein graft models, depending on the vessel geometry for a given blood flow rate and blood viscosity.5 6 7 8 In an end-to-end anastomosed vein graft model, eddy blood flow occurs in the proximal region due to graft-host diameter mismatch, whereas in an end-to-side or side-to-side anastomosed vein graft, eddy blood flow occurs in the distal toe and heel regions due to geometric distortions and curvatures. It has been known that eddy blood flow in these regions is often associated with focal intimal hyperplasia, a pathological event causing vein graft restenosis.5 6 7 8 Thus, these vein graft models have been used to study the influence of blood flow on vascular remodeling. Recently, the author demonstrated, by using an end-to-end anastomosed vein graft model, that focal intimal hyperplasia and smooth muscle cell (SMC) proliferation were initiated in the proximal region where eddy flow was found. When eddy flow was eliminated by matching the graft-host diameters by using a biomechanical engineering approach, focal intimal hyperplasia and SMC proliferation were significantly prevented.7 These results further support the role of eddy blood flow in the regulation of intimal hyperplasia and SMC proliferation. However, the mechanisms that link fluid dynamics to focal SMC proliferation remain unclear.
Studies using molecular and cellular approaches have demonstrated that growth-related factors regulate cell proliferation. In blood vessels, endothelial cells and SMCs are able to produce and release a number of growth-related factors, including acidic and basic fibroblast growth factors, platelet-derived growth factor (PDGF), vascular endothelial cell growth factor, insulin-like growth factor, monocyte chemotactic protein-1, and endothelin. These growth factors have been shown to interact with their receptors in the cell membrane and to promote SMC proliferation and atherogenesis in human and animal arteries9 10 as well as in vein grafts.11 12 13 Further studies have demonstrated that growth factors may mediate mechanical stress–induced biological activities such as cell proliferation and hypertrophy. A typical example is the involvement of angiotensin II and the angiotensin II type 1 (AT1) receptor in the regulation of cardiomyocyte hypertrophy. As shown in several studies, mechanical stretch enhanced expression of the AT1 receptor,14 which together with angiotensin II possibly mediated tensile stress–induced cardiac myocyte hypertrophy.15 16
Several recent studies have demonstrated that the AT1 receptor is upregulated in the neointima of experimental vein grafts,17 and an AT1 receptor antagonist, L158809, significantly suppresses the rate of intimal hyperplasia.18 These studies suggest that the AT1 receptor possibly plays a role in the regulation of intimal hyperplasia in vein grafts. On the basis of these investigations, it can be hypothesized that the AT1 receptor may mediate eddy flow–related SMC proliferation in the neointima of experimental vein grafts. This study was designed to verify this hypothesis and to achieve these goals: (1) to demonstrate whether eddy flow influences the expression of AT1 receptor mRNA and protein and (2) to investigate whether the AT1 receptor influences the rate of SMC proliferation in the vein graft neointima that is subject to eddy flow.
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Animals and Observation Schedules
Eighty male, 3-month-old Sprague-Dawley rats (Harlan, Indianapolis) were randomly separated into 16 groups with 5 rats in each group. Four groups were used for each of the following experimental series: (1) nonengineered vein grafts with eddy flow and increased tensile stress; (2) engineered vein grafts without eddy flow and without increased tensile stress; (3) engineered vein grafts with eddy flow but without increased tensile stress; and (4) nonengineered vein grafts with administration of losartan. For each animal, the right jugular vein was used as a graft, and the left jugular vein was used as a normal vein control. Observations were carried out at 5, 10, 20, and 30 days after surgery. The experimental procedures were approved by the Animal Care and Use Committee of Northwestern University.
Experimental Models
Experimental Series 1: Nonengineered Vein Grafts With Eddy Flow and
Increased Tensile Stress
Vein grafts were created by using conventional
procedures.7 In brief, a rat was anesthetized by
an intraperitoneal injection of sodium
pentobarbital (50 mg/kg). The right jugular vein was isolated, treated
with heparinized host blood (100 U/mL) and papaverine/saline (1 mg/mL),
and grafted into the abdominal aorta below the renal arteries by using
an end-to-end anastomotic technique with 8 to 10 interrupted stitches
(10-0 nylon sutures) at each end. After surgery, arterial
blood flow was initiated, the external diameters of the vein graft and
the host artery were measured, the abdominal cavity was closed, and the
rat was allowed to recover.
In this model, the diameter of the vein graft was larger than that of
the host aorta, with a graft-to-host diameter ratio of 
1.6. Eddy
blood flow developed at the proximal region of the vein grafts due to
graft-host diameter mismatch, and tensile stress in the vein graft wall
increased owing to exposure to arterial blood pressure and
enlargement of the vessel diameter.
Experiment Series 2: Engineered Vein Grafts Without Eddy Flow and
Without Increased Tensile Stress
In this series, vein grafts were engineered to match the
graft-host diameters and to increase the wall thickness by using an
engineering approach as described previously.7 In brief, a
flat piece of fixative-treated small intestine, with its stiffness
similar to that of the aorta, was cut into a submucosa/SMC sheet with a
thickness similar to that of the aorta by using a cryomicrotome. The
width of the intestinal sheet was determined on the basis of the aortic
diameter, and the length of the sheet was determined on the basis of
the in vivo length of the jugular vein. After vein graft surgery, the
intestinal sheet with desired dimensions was placed around the vein
graft and sutured into a cylindrical sheath, with the 2 axial ends
anchored to the adventitia of the host aorta by suture stitches. In all
cases observed, the diameter of the vein graft was similar to that of
the host aorta, and thus, no apparent eddy flow developed in this
model. In addition, because the vein graft was restricted in a smaller
and more rigid engineered sheath, tensile stress in the vessel wall was
reduced.
Experimental Series 3: Engineered Vein Grafts With Eddy Flow but
Without Increased Tensile Stress
In addition to eddy flow, increased tensile stress in the vessel
wall influences vein graft remodeling. To distinguish the effect of
eddy flow from that of tensile stress, vein grafts were engineered to
reduce the graft diameter by 25% with respect to the diameter of
nonengineered vein grafts, leading to a graft-host diameter ratio of
1.3. In such a case, eddy flow developed due to graft-host diameter
mismatch, but tensile stress in the vein graft wall was reduced because
the vein graft was restricted in a smaller and more rigid sheath, which
carried the tensile load in the vessel wall. Thus, the influence of
eddy flow could be examined independent of tensile stress. Methods
described in the preceding section on experimental series 2 were used
in this series of experiments as well, except for the difference in the
graft-to-host diameter ratio.
Experimental Series 4: Nonengineered Vein Grafts With
Losartan
Losartan, an AT1 receptor
antagonist, was used to examine the effect of the
AT1 receptor on SMC proliferation and intimal
hyperplasia. Rats were administrated losartan (30 mg ·
d-1 · kg-1) in their
drinking water for a period from 2 days before vein graft surgery to
the end of observation.19 Nonengineered vein grafts were
created by using the method described for experimental series 1.
Observations were carried out at the same times as for the other
experimental series described above.
Formation of Eddy Flow
The formation and characteristics of eddy flow in a vein graft
were studied with the aid of a glass model. In a divergent blood vessel
such as a vein graft, eddy flow develops under the influence of an
adverse blood pressure gradient along the endothelial
surface. The formation of an adverse pressure gradient in a vein graft
depends on the wall slope at the proximal anastomosis or on the
leading-edge slope of the proximal focal neointima. (Note
that the definitions of these slopes are shown in Figure I, which can
be found online at
http://atvb.ahajournals.org/cgi/content/full/19/11/2630/DC1.)
In this study, a relationship between wall slope and eddy flow
formation was determined by using a glass model with actual vein graft
geometry and size under a physiological Reynolds
number, because it is difficult to characterize eddy flow formation in
an actual rat vein graft.
To determine such a relationship, 23 glass models were constructed with
various proximal anastomotic wall slopes ranging from 0 to 0.5. A
Harvard rodent-blood pump (model 1407) was used to introduce pulsatile
flow (200 beats/min) to the model, with flow and pressure magnitudes
and waveform similar to those of the rat. The system was perfused with
a mixture of glycerine, water, and 0.03 g/100 mL hollow glass beads of
30-µm diameter (Potters Industries, and a gift from Dr R.M. Lueptow
of Northwestern University), with a fluid viscosity similar to that of
rat blood (0.06 poise). A slit-light beam was applied to the graft
model along the centerline in the axial direction. Images of glass bead
streak lines were recorded in the direction perpendicular to the
slit-light plane by using a Sony Betacam SP video recorder
(UVW-1400 A) and a CCD camera (kindly provided by Dr M.R. Glucksberg,
Northwestern University). The recorded images were
analyzed, and a causal relationship between anastomotic wall
slope and eddy flow was established.
Expression of AT1 Receptor mRNA
At scheduled times, vein graft specimens were fixed under in
vivo arterial blood pressure by using 4% formaldehyde in
PBS. The proximal portion (50%) of each vein graft was selected,
cut into several axial strips, and embedded in 5% gelatin in PBS at
37°C to prevent specimen distortion during preparation. After gelatin
solidification, the gelatin-embedded specimens were fixed for 15
minutes and cut into histological sections along the
vessel axis, with each section containing the graft-host junction, by
using a cryomicrotome. Several sections with the thickest intimal
hyperplasia were selected from each graft for in situ hybridization of
AT1 receptor mRNA. All solutions used for in situ
hybridization were treated with diethyl pyrocarbonate (0.02%), and all
tools and glassware were autoclaved.
A rat vascular AT1 receptor cDNA (Ca18b) sample,
kindly provided by Dr T.J. Murphy of Emory University, Atlanta,
Ga,20 was used to produce digoxigenin-conjugated sense and
antisense cDNA probes by using a polymerase chain reaction (PCR)
method.21 In brief, 0.1 ng of the template
AT1 receptor cDNA was mixed on ice with
AT1 receptor oligonucleotide
primers 1 and 2 (5'-GTCATGA-TCCCTACCCTCTACAGC-3' and
5'-CCGTAGAACAGAGGG TTCAGGCAG-3',22 respectively; 1
µmol/L each), 1x PCR buffer with 2.5 mmol/L
MgCl2, dNTPs with digoxigenin-11-dUTP (200
µmol/L of each dNTP), and 2.5 U Taq DNA polymerase
(Boehringer Mannheim). The mixture was placed in a PCR
tube and subjected to 30 cycles of denaturation (94°C, 1 minute),
annealing (60°C, 2 minutes), and primer extension (72°C, 3 minutes)
in a Perkin-Elmer 2400 thermocycler. The PCR product was verified
by restriction enzyme digestion and agarose gel electrophoresis in
accordance with the restriction map of the AT1
receptor gene.20 The AT1
receptor cDNA probe was heat denatured at 95°C for 2 minutes and
mixed with a hybridization solution (50% deionized formamide, 0.3
mol/L NaCl, 10 mmol/L Tris-Cl, 1 mmol/L EDTA, 1x Denhardt’s
solution, 500 µg/mL sonicated salmon sperm DNA, 50 mmol/L
DTT, and 10% polyethylene glycol of molecular weight 6000) for
specimen labeling.
Selected specimens were mounted onto microscopic slides; acetylated in a mixture of 0.1 mol/L triethanolamine hydrochloride, 0.25% acetic anhydride, and 0.9% NaCl; washed in 2x NaCl–trisodium citrate · 2H2O solution (SSC); dehydrated; delipidated in a graded series of ethanol; rehydrated; and washed in 2x SSC. Specimens were then incubated with the hybridization mix containing the AT1 receptor cDNA probe for 16 hours at 37°C, washed in 2x SSC (2 changes) and 1x SSC (2 changes) at 37°C for 2 hours and in 0.1x SSC (2 changes) at 37°C for 10 minutes, and incubated with a blocking solution (2% sheep serum and 0.3% Triton X-100 in 100 mmol/L Tris-Cl and 150 mmol/L NaCl) for 30 minutes, and washed in PBS. The specimens were then incubated with a mixture of 20 µg/mL rhodamine-conjugated anti-digoxigenin antibody (Boehringer Mannheim) and 1% BSA in PBS at 37°C for 1 hour, washed in PBS, and examined and photographed with an Olympus BX40 fluorescence microscope. In the analysis of fluorescent images, because not all specimens were prepared at the same time, comparisons were limited to the same specimen between different locations only.
A sense AT1 RNA probe was synthesized23 and used as a control probe for the examination of AT1 mRNA in vein grafts. In brief, a KS+ pBluescript vector, which contained the Ca18b AT1 cDNA20 (a gift from Dr T.J. Murphy), was linearized and used as a template to produce "runoff" transcripts. Approximately 1 µg of the template was mixed with 2 µL of NTP labeling solution (containing 10 mmol/L ATP, 10 mmol/L CTP, 10 mmol/L GTP, 6.5 mmol/L UTP, and 3.5 mmol/L digoxigenin-11-UTP), 40 U of T3 RNA polymerase, 10 U of RNase inhibitor, and transcription buffer, with a final volume of 20 µL (note that all reagents were from Boehringer Mannheim). The mixture was incubated for 2 hours at 37°C. The runoff transcripts were used to react with selected vein graft specimens by the method described above.
Expression of AT1 Receptor Protein
Axial cryosections of vein grafts were prepared by using a
method described above, incubated with a blocking solution (10% goat
serum in PBS) for 30 minutes, reacted with a rabbit anti-rat
AT1 receptor antibody (AB1525, Chemicon) in 1%
BSA–PBS (1:100) at 37°C for 1 hour, washed in PBS, incubated with a
rhodamine-conjugated goat anti-rabbit IgG antibody (Chemicon) at 37°C
for 1 hour, washed in PBS, and examined with an Olympus BX40
fluorescence microscope. Specimens incubated with only the
secondary antibody were used as controls.
SMC Identification
Specimens used for AT1 mRNA and protein
labeling were also used to identify SMCs by using an antibody against
the SMC isoform of 
-actin. In brief, selected specimens were
incubated with a mixture of 5 µg/mL anti–-actin antibody
(Boehringer Mannheim),13 24 1% BSA, and PBS at
37°C for 1 hour; washed with PBS; incubated with 1:20
fluorescein-conjugated anti-IgG2a
antibody (Boehringer Mannheim) at 37°C for 1 hour; washed in
PBS; and examined with an Olympus BX40 fluorescence microscope.
Specimens incubated with only the secondary antibody were used as
controls.
SMC Proliferation
A 5-bromo-2'-deoxyuridine (BrdU) labeling method was used to
determine the rate of SMC proliferation in the neointima of
vein grafts. At scheduled times, a rat was injected intramuscularly
with 30 mg/kg BrdU (Boehringer Mannheim) 3 times at 17, 9, and
1 hour before the rat was killed.25 Vein graft specimens
were prepared by using methods described above. Selected vein graft
sections were digested in 0.5% pepsin in 0.1N HCl for 30 minutes at
37°C, incubated in 1.5N HCl for 30 minutes at 37°C, washed in 0.1
mol/L borax buffer (pH 8.5) and then in Tris-buffered saline (pH
7.6),26 and blocked with 10% goat serum in PBS. The
sections were incubated with an anti-BrdU antibody (Boehringer
Mannheim) at a dilution of 1:20 in 1% BSA–PBS at 37°C for 30
minutes, washed in PBS, incubated with a secondary anti-IgG antibody
conjugated with fluorescein (Boehringer Mannheim)
at a dilution of 1:20 at 37°C for 30 minutes, and washed in PBS
again. The specimens were examined with an Olympus BX40
fluorescence microscope. Two types of specimen were used as
BrdU labeling controls: (1) specimens without BrdU injection, which
were processed by the method described above, and (2) specimens with
BrdU injection and incubated with only the secondary antibody. SMCs
were identified by positive anti–-actin antibody labeling in the
same specimens.
Hoechst 33258 was used to label the cell nuclei.27 The
numbers of BrdU-labeled and Hoechst 33258–labeled SMCs, which were
identified by positive anti–-actin antibody labeling, were measured
in the leading and trailing regions of the area with proximal focal
neointima. The proximal focal neointima was
defined as that found in the proximal region of the vein graft. The
leading and trailing regions were separated by a line perpendicular to
the vein graft wall and passing through the maximal convex curvature of
the focal neointima. (Note that the definitions of these
regions are shown in Figure I, which can be found online at
http://atvb.ahajournals.org/cgi/content/full/19/11/2630/DC1.)
In engineered vein grafts, no apparent proximal focal
neointima was found. In such a case, the proximal region,
with the axial length equivalent to the average axial length of the
proximal focal neointima in the nonengineered vein grafts
at the same observation time, was measured and divided into 2
subregions of equal length. The 1 region adjacent to the anastomosis
was defined as the trailing region, and the distal 1 was defined as the
leading region. The percentage of BrdU-labeled cells in each region was
calculated on the basis of the number of BrdU-labeled cells and the
number of Hoechst 33258–labeled cell nuclei.
Average Thickness of the Proximal Focal Neointima
Axial histological sections of engineered and
nonengineered vein grafts were used to measure the average thickness of
the proximal focal neointima. The proximal focal
neointima was identified by using the following criteria:
(1) the geometry of focal intimal hyperplasia and vein grafts; ie, a
focal neointima is located on top of the venous wall, with
a distinct boundary between the 2 structures in specimens prepared by
using histological and immunological methods; and (2)
the pattern of cell nucleus distribution; ie, there exists a narrow
zone of low cell density between the focal neointima and
the venous wall in Hoechst 33258–labeled specimens. The area of the
proximal focal neointima was measured by using a
point-counting method, and the average thickness was calculated as the
ratio of the area to the length.
Statistics
Means and SDs were calculated for all measured
parameters. The method of Student’s t test was
used to determine the significance of difference in each
parameter between any 2 selected groups. The method of
1-way ANOVA for multiple comparisons was used to determine the
significance of difference in each parameter between data
collected at different times. A difference was considered statistically
significant at P<0.05.
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Results |
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Formation of Eddy Flow
Flow visualization with the use of a glass vein graft model with anastomotic wall slopes ranging from 0 to 0.5 demonstrated that under pulsatile flow at aortic blood pressure, flow rate, and blood viscosity, a slope of
0.2 was sufficient to induce visible flow
separation and eddy flow in the proximal region of the model. Under
pulsatile flow at aortic blood pressure, flow rate, and blood
viscosity, eddy flow appeared during systole when the flow velocity
accelerated to a critical level, and eddy flow disappeared during
diastole when flow velocity decelerated to a critical
level. (Note that the flow patterns in the glass model at selected time
points during the pulsatile cycle are demonstrated in Figure II, which
can be found online at
http://atvb.ahajournals.org/cgi/content/full/19/11/2630/DC2.)
Results from actual vein grafts showed that the anastomotic wall slope
and the leading-edge slope of the proximal focal neointima
of nonengineered vein grafts were larger than the critical value of 0.2
for eddy flow formation at days 5, 10, 20, and 30 (see Figure 1
). Flow visualization in actual vein
grafts showed that eddy blood flow appeared at the proximal region or
in front of the leading edge of proximal focal intimal hyperplasia of
nonengineered vein grafts, but not in engineered vein grafts, in all
observed cases. This result was similar to that reported in a previous
study.7
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Influence of Eddy Flow on AT1 Receptor
Expression
Figure 2 shows the distribution of
AT1 receptor mRNA in the proximal focal
neointima of nonengineered vein grafts with eddy flow. At
days 10, 20, and 30 after surgery, focal expression of
AT1 receptor mRNA was found in the leading region
of the proximal focal neointima, whereas the level of
AT1 mRNA in the trailing region was apparently
lower than that in the leading region. Similar results were found in
engineered vein grafts with eddy flow. Note that
AT1 receptor mRNA was also highly expressed in
the host aorta. In specimens labeled with a sense
AT1 receptor RNA probe, little positive labeling
of AT1 receptor mRNA was found in the leading
region of proximal focal intimal hyperplasia of nonengineered vein
grafts (see Figure III, which can be found online at
http://atvb.ahajournals.org/cgi/content/full/19/11/2630/DC3).
Simultaneous examination of AT1
receptor mRNA and SMC -actin localization showed that the major cell
type that expressed AT1 receptor mRNA was the SMC
in the neointima of vein grafts (photomicrographs available
on request). In engineered vein grafts without eddy blood flow, no
apparent difference in the distribution of AT1
receptor mRNA was found in the neointima at all observation
times (see Figure 3).
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Further examination with the use of an immunohistological method demonstrated that the distribution pattern of AT1 receptor protein was similar to that of AT1 receptor mRNA in nonengineered and engineered vein grafts, although the location of expression between AT1 receptor protein and AT1 receptor mRNA did not match exactly at each observation time. Photomicrographs of AT1 receptor protein distribution in the neointima of nonengineered vein grafts are presented in Figure IV, which can be found online at http://atvb.ahajournals. org/cgi/content/full/19/11/2630/DC4.
Influence of Eddy Flow on SMC Proliferation
Figure 8 shows the distribution of
BrdU-labeled cells in a normal jugular vein and in nonengineered vein
grafts with eddy flow at days 5, 10, 20, and 30 after surgery. In
normal jugular veins, BrdU-labeled SMCs were rarely seen. In
nonengineered vein grafts with eddy flow, the density of BrdU-labeled
SMCs in the proximal focal neointima increased
significantly from days 0 to 10 and then decreased toward the normal
level (P<0.001, ANOVA). Furthermore, SMC density in the
leading region was significantly higher than that in the trailing
region of the proximal focal neointima of the same
specimens of nonengineered vein grafts at all observation times except
on day 5 (see Figures 4 and 5). In
specimens with control labeling, no BrdU-labeled cells were found
(photomicrographs available on request).
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P<0.05,
In engineered vein grafts without eddy flow, as shown in Figure 5
, the density of BrdU-labeled SMCs was significantly higher
than that in normal veins but significantly lower than that in the
nonengineered vein grafts in the leading and trailing regions of the
proximal focal neointima. No difference was found in the
density of BrdU-labeled SMCs between the leading and trailing regions
of engineered vein grafts at all observation times (see Figure 5). An immunohistological examination with the
use of an anti–-actin antibody showed that the major type of
proliferating cell was the SMC in the neointima in both
nonengineered and engineered vein grafts (photomicrographs available on
request).
Influence of Losartan on SMC Proliferation and Intimal
Hyperplasia
As shown in Figure 5, administration of losartan
significantly lowered the density of BrdU-labeled SMCs in the proximal
focal neointima of nonengineered vein grafts with eddy flow
at all observation times except for day 5, despite the locally
increased expression level of AT1 receptor mRNA
and protein in the leading region. The distribution pattern of
AT1 mRNA and protein in these specimens was
similar to that in the neointima of nonengineered vein
grafts without losartan. In specimens with losartan,
the density of BrdU-labeled SMCs in the leading region of the proximal
focal neointima was significantly higher than that in the
trailing region of the same specimens at days 10, 20, and 30 after
surgery.
Figure 6 shows the influence of
losartan on the average thickness of the proximal focal
neointima in nonengineered vein grafts with eddy flow. The
average thickness was significantly reduced in nonengineered vein
grafts with losartan compared with vein grafts without
losartan at all observation times except day 5.
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Discussion |
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Eddy Flow and Focal Intimal Hyperplasia
The formation of eddy blood flow in a blood vessel depends on several factors, including vessel geometry, flow velocity, and blood viscosity. For a given flow velocity and blood viscosity, eddy blood flow occurs because of adverse pressure gradients in blood vessels with divergent geometry, such as those distal to bifurcations and bypass grafts with a graft-host diameter mismatch. Previous studies showed that eddy blood flow often coincided with focal intimal hyperplasia in experimental vein grafts.5 6 7 8 To understand the role of eddy blood flow in the regulation of this pathological event, a number of investigators have studied the fluid dynamic features of eddy flow and demonstrated that it is associated with local oscillatory, low, fluid shear stress and large shear gradients in experimental vein grafts.28 29 30 Thus, it has been hypothesized that oscillatory, low, fluid shear stress and/or large shear gradients28 29 30 31 may play a role in the regulation of vein graft intimal hyperplasia. Although no direct evidence is available for this hypothesis, supporting data have been obtained from related experimental and clinical observations. For instance, lowered fluid shear stress, with reference to physiological blood shear stress, promoted SMC proliferation and intimal hyperplasia, whereas increased fluid shear stress prevented these evens in experimental polymer vascular grafts in nonhuman primates.26 32 33 Investigations on human and animal arterial atherosclerosis have supported these observations.1 2 3 4 However, how oscillatory, low, fluid shear stress and increased shear stress gradients differentially regulate vein graft remodeling remains to be investigated.
Role of Signaling Molecules and Growth Factors in Mechanical
Stress–Induced Vascular Remodeling
For the last decade, numbers of studies have demonstrated that
fluid shear stress and strain rate influence the structure and regulate
a variety of activities of vascular endothelial cells
and SMCs.34 35 36 37 38 39 40 41 42 For instance, altered fluid shear stress
influences the activities of mitogen-activated protein
kinases43 44 and G proteins,45 the
production rate of prostacyclin,46 the activities
of growth factors,26 33 35 47 48 and the
morphology49 50 and cytoskeletal structure51
of endothelial cells. These changes have been
implicated in the mediation of mechanical stress–related vascular
atherogenesis. Several studies have provided experimental evidence for
this mechanism. In these studies, a cis-acting, shear
stress–response element has been identified in the promotor region of
the PDGF B-chain gene by a gene-deletion method.48
Fluid-shear stress was shown to activate nuclear factor-
B, a
transcription regulatory factor activated through signal
transduction pathways. Activated nuclear factor-B further
interacted with the shear stress–responsive element of the PDGF
B-chain gene, leading to transcription of this gene.52 The
PDGF B chain is a well-known growth factor that promotes cell
proliferation and vascular hypertrophy. Thus, it becomes
clear that a regulatory cascade, which involves fluid shear stress,
signaling transduction pathways, transcription regulatory factors, and
growth factors, may transduce fluid shear signals from the cell
membrane to the cell nucleus and induce DNA synthesis and cell
proliferation. It is expected that further studies may reveal the roles
of other signaling molecules and growth factors in the mediation of
mechanical stress–related vascular SMC proliferation.
Influence of Eddy Blood Flow on AT1 Receptor
Expression
The AT1 receptor is a potent promoter of
vascular SMC proliferation53 54 and has been implicated in
the mediation of tensile stress–induced biological events in cardiac
myocytes.14 15 16 However, the role of this factor in the
mediation of blood flow–related SMC proliferation has not been well
studied. Thus, this study was designed to investigate this issue. By
using experimental vein graft models with and without eddy flow, the
present study demonstrated that eddy flow was associated with the
focal upregulation of AT1 receptor mRNA and
protein in the proximal focal neointima. When eddy flow was
prevented by restricting the vein graft into an engineered sheath whose
diameter was identical to that of the host artery, no difference in the
distribution of AT1 receptor mRNA and protein was
found. These results suggest that eddy flow possibly promotes
AT1 receptor expression in the
neointima of vein grafts. The influence of blood flow on
the expression of growth factors has been studied by using different
experimental models. These studies showed that reduced blood flow
upregulated the PDGF gene, whereas increased blood flow exerted an
opposite effect in nonhuman primate polymer vascular
grafts.26 33 These results clearly demonstrated that blood
flow played a role in the regulation of mitogen expression in blood
vessels.
The present study showed that AT1 receptor mRNA and protein were highly expressed in SMCs that were not directly exposed to altered fluid shear stress. This result suggests that certain local mediators may be involved in the transduction of the shear stress signal from endothelial cells to SMCs. It is possible that signaling molecules and mitogenic factors may be involved in the transduction of a shear stress signal from endothelial cells to SMCs in vein grafts. However, the exact mechanisms remain to be determined.
Tensile stress is another mechanical factor that is increased in the wall of nonengineered vein grafts and that promotes leukocyte activation and intimal hyperplasia.55 56 57 58 59 60 Previous studies showed that relief from increased tensile stress in the vein graft wall by use of a rigid, external support could partially prevent intimal hyperplasia and medial thickening, indicating a role for tensile stress in the regulation of vein graft remodeling.56 57 58 59 60 Several recent studies further showed that increased tensile stress induced SMC actin filament degradation and SMC death,24 influenced the orientation of newly generated SMCs, and might contribute to medial and adventitial hypertrophy in experimental vein grafts.27 Thus, tensile stress is another potential factor that regulates expression of the AT1 receptor.
To distinguish the influence of eddy flow from that of increased tensile stress, an experimental model with eddy flow but without increased tensile stress was also created by using an engineering technique in the present study. In this model, the vein graft was restricted in a cylindrical engineered sheath that was larger than the host artery in diameter, leading to the formation of eddy flow in the proximal region of the vein graft. However, tensile stress in the vessel wall was reduced because the vein graft was restricted in a smaller and more rigid sheath that carried the tensile load due to arterial blood pressure. In such a model, focal expression of the AT1 receptor was found in the leading edge of the proximal focal intimal hyperplasia, where eddy flow occurred. This result provided further evidence for the role of eddy flow in the regulation of focal AT1 receptor expression in the neointima of experimental vein grafts. Tensile stress, which is relatively uniformly distributed in the vessel wall, may not be directly related to the focal AT1 receptor expression in the neointima of experimental vein grafts. Whether and how tensile stress influences the activities of the AT1 receptor in experimental vein grafts remain to be investigated.
It should be noted that fluid shear stress in regions with and without eddy blood flow was not measured and analyzed in this study because of the lack of available techniques for small blood vessels with complex geometry. Several experimental methods, including particle tracing and photochromic tracing methods, have been used to assess fluid shear stress in large blood vessels.61 62 However, for a rat vein graft of 2 to 3 mm in diameter, the accuracy of fluid shear stress measurements is always an issue to be considered. Thus, further studies are necessary for the development of experimental techniques that will allow fluid shear stress measurements in small blood vessels with complex geometry. Although fluid shear stress was not analyzed, this study provides information into the role of eddy blood flow in the regulation of vascular SMC proliferation and atherogenesis.
Influence of the AT1 Receptor on SMC Proliferation and
Intimal Hyperplasia
The present study demonstrated that focal expression of
the AT1 receptor was associated with an increase
in the rate of SMC proliferation in the leading region of the proximal
neointima, whereas the activity of the
AT1 receptor and the rate of SMC proliferation
were both reduced in the trailing region of the same
neointima in vein grafts. These results suggest that the
AT1 receptor possibly regulates SMC
proliferation. As observed in a previous study7 as well as
in the present study, the proximal focal neointima
elongated gradually toward the distal anastomosis in experimental vein
grafts after surgery. Dynamic expression of the
AT1 receptor and SMC proliferation in the leading
region likely facilitate the elongation process of the focal
neointima.
The role of the AT1 receptor in the regulation of SMC proliferation in vein grafts was further verified by using losartan, an AT1 receptor antagonist. In nonengineered vein grafts with losartan, the rate of SMC proliferation and the degree of intimal hyperplasia were significantly reduced compared with those in nonengineered vein grafts without losartan. However, losartan did not completely prevent SMC proliferation and intimal hyperplasia in nonengineered vein grafts. This observation suggested that, in addition to angiotensin II and its AT1 receptor, other local mediators are possibly involved in the regulation of blood flow–related focal SMC proliferation and intimal hyperplasia in vein grafts.
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Acknowledgments |
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This project was supported by the American Heart Association (AHA) of the Metropolitan Chicago Affiliate and the AHA National Center. The author is an Established Investigator of the AHA.
Received March 18, 1999; accepted June 21, 1999.
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References |
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