Circulation Research. 2001
Published online before print July 5, 2001,
doi: 10.1161/hh1401.093575
A more recent version of this article appeared on July 20, 2001
(Circulation Research. 2001;0:hh1401.093575.)
© 2001 American Heart Association, Inc.
Smooth Muscle Cell Changes During Flow-Related Remodeling of Rat Mesenteric Resistance Arteries
C. L. Buus,
F. Pourageaud,
G. E. Fazzi,
G. Janssen,
M. J. Mulvany
J. G. R. De Mey
From the Department of Pharmacology and Toxicology (C.L.B., F.P., G.E.F.,
G.J., J.G.R.D.M.), Cardiovascular Research Institute Maastricht (CARIM),
Universiteit Maastricht, and Department of Functional Developmental Biology
(C.L.B., F.P., G.E.F., G.J., J.G.R.D.M.), School of Life Sciences (tUL-SLS),
Transnational University of Limburg, Maastricht, The Netherlands; and
Department of Pharmacology (M.J.M.), University of Aarhus, Denmark. Present
address for F.P. is Department of Pharmacology, INSERM (E9937), University of
Bordeaux II, Bordeaux Cedex, France.
Correspondence to Prof Jo G.R. De Mey, Department of Pharmacology, Universiteit Maastricht, PO Box 616, 6200 MD, Maastricht, The Netherlands. E-mail j.demey{at}farmaco.unimaas.nl
Abstract
Abstract—To
obtain information on the molecular and cellular mechanisms of
flow-induced arterial remodeling, we analyzed the
morphology and smooth muscle cell (SMC) characteristics in rat
mesenteric resistance arteries after interventions that decreased and
increased flow. Juvenile male Wistar Kyoto rats were subjected to
surgery that, compared with control arteries, provided arteries with
chronic low flow and chronic high flow. Low flow resulted in a
decreased passive lumen diameter, hypotrophy of the artery wall, and
both loss and decreased size of SMCs. Time course studies, with
intervention length ranging from 2 to 32 days of altered blood flow,
showed that the narrowing of the lumen diameter in low-flow arteries
appeared within 2 days and that an early dedifferentiation of SMC
phenotype was indicated by markedly reduced levels of desmin
mRNA. High flow resulted in an increased passive lumen diameter and in
hypertrophy of the artery wall. The hypertrophy
resulted from SMC proliferation because SMC number, measured by the
3D-dissector technique, was increased and immunohistochemical
assessment of proliferating cell nuclear antigen also showed an
increase. The widening of high-flow arteries required 16 days to become
established, at which time desmin mRNA was reduced. This time was also
required to establish changed wall mass in both low-flow and high-flow
arteries. Apoptotic cells detected by TdT-mediated dUTP-biotin
nick end labeling staining were mainly located in the medial layer, and
evaluation of DNA fragmentation indicated that increased
apoptosis occurred in both low flow and high flow. This study
shows for the first time direct evidence that reduced and elevated
blood flow in resistance arteries produce, respectively, decrease and
increase in SMC number, with dedifferentiation of the SMCs in both
cases.
Key Words: rat • vascular remodeling • blood flow • small arteries
Mechanical forces
related to the velocity of the arterial blood flow are
important factors determining the caliber of
arteries.1 2
Acutely, the artery caliber responds to altered blood flow through the
change in shear stress detected by the endothelial
cells, which modulates the release of vasoactive
factors.1 These factors may
also have a role in long-term structural changes of arteries exposed to
chronically altered blood
flow,3 as may the altered
circumferential wall stress caused by the initial functional adaption
to chronic changes in blood flow (eg, Ben Driss et
al4 ).
Flow-related remodeling of resistance-sized arteries is
important for maintaining an adequate tissue perfusion to allow the
structural set point to be optimal for acute vasomotor responses.
Remodeling of skeletal muscle arteries during endurance training and
remodeling of uterine arteries during pregnancy are examples of
arterial remodeling that are due to increased blood
flow.5 6 Also, in
diseased states, hemodynamic forces are implicated in
the remodeling of, for instance, coronary collateral arteries
after ischemic injury to the
heart.7 8
Experimental analyses of flow-induced remodeling in large
arteries have primarily involved unilateral carotid artery ligation in
mice, rats, and rabbits, showing a flow-dependent decrease of lumen
diameter in the ligated artery and an increase of diameter in the
contralateral
artery.3 9 10 11
Similarly, the small arteries of the mesenteric bed of the rat have
been used for the in vivo study of flow-induced remodeling, where
ligation of feed arteries in this vascular bed acutely and chronically
results in increased blood flow through collateral pathways. Unthank et
al12 showed lumen
enlargement and medial hypertrophy in the collateral
pathway, and Pourageaud et
al13 established a model to
study both inward hypotrophic and outward hypertrophic remodeling in
response to chronically decreased and increased blood flow,
respectively.
The cellular mechanisms producing the remodeling remain
unclear. The altered morphology could be due to changes in size and
arrangement of existing SMCs or to combinations of cell proliferation
and cell death (apoptosis). Furthermore, it is not clear if the
remodeling is associated with SMC dedifferentiation. To address these
questions, we have used the ligation model of rat mesenteric arteries
during 2 to 32 days of blood flow manipulation. We found that the
outward remodeling due to high flow was a slow process compared with
the quick inward remodeling noted in chronically hypoperfused arteries.
In both cases, the flow-related arterial remodeling
involved changes in cell turnover and cell
phenotype.
Materials and Methods
Animals and Surgery
Male Wistar Kyoto rats (animal facilities,
Universiteit Maastricht) of 6 to 8 weeks of age were used for the
experiments. Blood flow–modifying surgery was performed in the animals
by distal ligation of alternate first-order mesenteric arteries to give
low flow (LF); the patent arteries between these then had a
compensatory high flow
(HF).13 14
Arteries were taken from a point proximal to the ligation
(LF artery) and from a corresponding position in the patent arteries
(HF arteries). For the stereology experiments and assessment of
phenotype markers by immunohistochemistry, control arteries
were taken from animals where a sham surgical procedure was performed
but without blood flow
modification.13 In the other
experiments, control arteries were taken from the same animal as the
flow-modified arteries as described by Ceiler et
al.14 These interventions
give, respectively, 
80% decrease and an approximate doubling of
flow in LF and HF arteries, but with similar intravascular
pressures.13 14
Stereology
Artery segments isolated at 4 weeks after surgical
intervention were mounted in an isometric
myograph.15 The artery was
exposed to a resting wall tension corresponding to a transmural
pressure of 100
mm Hg.15 Processing of the
tissue and determination of the volume and density of the cells and
nuclei in the medial layer were performed as described by Korsgaard and
Mulvany.16
Pressure-Diameter Relation
Arteries were mounted in an organ chamber (Living
Systems) filled with calcium-free Krebs-Ringer bicarbonate (KRB,
37°C, see below) aerated with 95% O2/5%
CO2. The proximal end of the artery was mounted
on a cannula and the other end was ligated as a blind
sack14 after flushing the
lumen with calcium-free KRB. The cannula was connected to a pressurized
buffer reservoir, controlled via a pressure sensor and a peristaltic
pump (Living Systems). The inner diameter of the latter set of arteries
was measured at 100 mm Hg, and afterward vessels were fixed with
4% formalin in PBS (see below).
Polymerase Chain Reaction (PCR) Amplification
of Desmin and GAPDH mRNA
Total RNA was extracted as described
previously17 and 100 ng RNA
was reverse-transcribed (RT). PCR was performed with primers for either
desmin cDNA (5'-CGAGAGCTGCGGCGCCAAGT-3', forward;
5'-CATCGTTGTTCTTATTGGCTGCC-3', reverse) or GAPDH cDNA
(5'-ACGGATTTGGCCGTATTG-3', forward; 5'-CGTCAGATCCACGACGGA-3', reverse).
The products were size-fractionated in a gel containing 1.5%
agarose, and the intensities of the bands were quantified by
densitometry.
Extraction of DNA
DNA extraction was based on the
Wizard genomic DNA purification kit from
Promega. DNA content was determined by the
fluorometric method of Labarca and
Paigen18 with calf thymus
DNA (Sigma Chemical Co) as
standard.
Ligation-Mediated PCR of Blunt-End DNA
Fragments
Ligation-mediated PCR, performed to amplify DNA
fragments characteristic for apoptotic cell
death,19 20 was
based on the ApoAlert LM-PCR ladder assay kit
(Clontech). PCR amplification was performed with
75 ng of ligated DNA. For each ligation procedure, the number of cycles
needed to detect laddering in the calibration sample, consisting of a
batch of rat thymus DNA, was determined and related to the laddering of
artery samples. A score was applied to the samples (laddering index): 2
points were given if laddering was detectable with the number of cycles
giving visible laddering for the calibration rat thymus sample, 1 point
was given if laddering was detectable with an additional 5 to 6 cycles,
and 0 points were given if laddering was not detectable after these
additional 5 to 6 cycles.
Morphometry and Immunohistochemistry
Cross sections (4 µm thick) of arteries fixed at a
transluminal pressure of 100 mm Hg were used for the
determination of medial cross-sectional area (CSA) and for assessment
of phenotypic markers by immunohistochemistry. CSA of the medial layer
was defined as the area between the internal and external elastic
laminae. Cross sections used for immunohistochemistry were incubated
overnight with different primary antibodies/antisera as indicated. The
chromogen for the color development was 3,3'-diaminobenzidine (DAB),
and sections were lightly counterstained with hematoxylin. These
stainings were evaluated blindly by two independent observers. The
sections were scored according to 4 levels of intensities: from 0 if no
stain was observed to 3 if the entire medial layer was
stained.
Longitudinal sections were used for detection of
proliferating cell nuclear antigen (PCNA, clone PC10,
DAKO). Each nucleus was determined to be either
positive (brown) or negative (blue), and at each time point the values
were normalized to the mean of the control, which was given the value
of 1. Apoptotic cells were detected by using the TdT-mediated
dUTP-biotin nick end labeling (TUNEL) method on longitudinal sections.
The sections were treated with 3% citric acid and then with proteinase
K (10 µg/mL in PBS) before incubation with TdT enzyme (300 U/mL,
Promega) and biotin-16-dUTP (0.02 nmol/L,
Boehringer Mannheim). Development of color was as described for
the above procedures. Apoptotic cells were identified when both
TUNEL staining and karyorrhexis were observed.
Solutions
The composition of the KRB buffer was (in
mmol/L) NaCl 119, KCl 4.7, CaCl2 2.5,
MgSO4 1.2, NaHCO3 25,
KH2PO4 1.2, and glucose
5.5. In calcium-free KRB buffer, CaCl2 was
omitted from the solution. The composition of the PBS solution was
(in mmol/L) NaCl 150,
Na2HPO4 11, and
KH2PO4
1.6.
Statistical Analysis
To test differences of mean values between control
arteries and arteries exposed to altered blood flow (LF and HF) we used
unpaired Student’s t test,
paired Student’s t test, or
the nonparametric tests Mann-Whitney
U test and Wilcoxon
signed-rank test, as indicated. Groups were considered significantly
different when
P<0.05.
An expanded Materials and Methods section can be found in
the online data supplement available at
http://www.circresaha.org.
Results
Flow-Induced Structural Changes
Rats (n=30) at 8 weeks of age were exposed to
flow-modifying surgery, and arteries were isolated for analysis
at days 2, 4, 8, 16, and 32 after intervention. The morphological
changes are shown in
Figure 1A
. After as little as 2 days, a significantly
decreased lumen diameter was observed for the arteries exposed to LF.
After this initial narrowing, further narrowing was especially seen
after day 16. Arteries exposed to HF showed significantly increased
lumen diameter from day 16 onward. By day 32, lumen diameters were
214±17 µm and 474±26 µm for LF and HF arteries, respectively,
compared with 403±14 µm in control arteries.
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Figure 1. Time course of the changes of lumen diameter and CSA in flow-manipulated mesenteric resistance arteries. A, Inner diameter (lumen diameter) of arteries mounted on glass cannulas at 100 mm Hg was measured by a shearing monitor (see Materials and Methods). B, CSA of the medial layer was determined histologically on cross sections of fixed arteries. In both panels A and B,  ,  , and  represent control, LF, and HF arteries, respectively. Data are mean±SEM. * and ** indicate P<0.05 and 0.01, respectively, for LF vs control or HF vs control, two-tailed Student’s t test.
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Both for LF and HF arteries, a decrease and an increase of
medial CSA, respectively, were established at day 16 after a
variable pattern for the earlier time points
(Figure 1B
). In
Table 1, stereological analysis of the SMC
number and size after 4 weeks intervention is summarized. In LF
arteries, a marked decrease (-65%) of the number of cells per
segment length was observed. Unlike for the HF arteries, LF vessels
showed a significant reduction in mean cell volume, primarily because
of a reduced mean cell length. In HF arteries, the cell number per
segment length was increased by 44% compared with control vessels. The
mean cell volume, cell length, and cell CSA were not significantly
different compared with the control.
SMC Proliferation
The 3D-dissector results presented in
Table 1 show an increased number of SMCs in the medial
layer of chronically hyperperfused arteries. To investigate if this was
associated with increased SMC proliferation, immunohistochemical
detection of PCNA (a nuclear protein abundant in cells in the S-phase
of the cell cycle21 ) was
determined. PCNA overall showed higher counts
(Figure 2) in the hyperperfused arteries, consistent
with medial hypertrophy being associated with increased SMC
proliferation. The PCNA counts were elevated (+100%) in two phases
of the time course, first at day 8 and then later at day 32
(Figure 2). There were no statistically significant
differences between LF arteries and control arteries, but the level of
PCNA immunoactivity fluctuated around the basal level with the same
biphasic appearance as seen for the HF arteries.
SMC Phenotype
In control, LF, and HF arteries, markers of SMC
phenotype were assessed by immunohistochemical stainings 4
weeks after flow manipulation
(Table 2). Smooth muscle 
-actin and smooth muscle myosin
heavy chain showed no detectable changes in response to altered blood
flow at this time point. The thin filament–associated proteins
h-caldesmon and calponin, and the intermediate cytoskeletal protein
desmin showed no change in arteries exposed to HF. However, the level
of desmin and calponin protein was reduced in the LF arteries
(Table 2), indicating a dedifferentiation of the SMCs. At
the mRNA level, desmin also showed a marked decrease at both 4 and 16
days after surgery in LF arteries
(Figure 3). At day 16, desmin mRNA expression was also
moderately suppressed in HF arteries
(Figure 3), which is in line with the nonsignificant tendency
to a decrease of the protein levels after 4-week exposure to elevated
blood flow
(Table 2).
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Figure 3. RT-PCR–based detection of desmin mRNA level in arteries at 4 days and at 16 days after modification of blood flow. mRNA from arteries was extracted, and cDNA was amplified as described in Materials and Methods. A, PCR products are separated in an agarose gel. Top, Samples isolated at 4 days after surgery. Bottom, Samples isolated at 16 days after surgery. Pairs of desmin/GAPDH PCR products from arteries at the 3 different flow conditions are indicated (control, HF, and LF). The ratio between the desmin and the GAPDH product is indicated in panel B (day 4) and panel C (day 16). Each bar indicates the mean value of 3 determinations; error bars indicate SEM. *P<0.05, **P<0.01, paired two-tailed Student’s t test. lf indicates low flow; hf, high flow.
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Apoptosis
Figure 4A shows the amplification of control tissue (rat
thymus) DNA at 22 cycles and arterial samples (control, LF,
and HF, from days 4 and 16 after intervention) amplified for an
additional 6 cycles (28 cycles). DNA fragmentation is present in
both LF and HF arteries in these two animals, which is indicative for
the general finding that apoptosis appeared higher in
flow-manipulated arteries compared with control arteries.
Semiquantitative analysis of the material (see Materials and
Methods) confirmed that apoptosis was increased in both LF and
HF arteries compared with control
(Figure 4B). Longitudinal sections from four day 16 animals
were subjected to TUNEL analysis combined with observation of
karyorrhexis, providing evaluation for apoptosis for a total of
12 110 nuclei. We detected 11 positive nuclei, of which 9 were located
in the medial layer
(Figure 5) and 2 in the adventitial layer of the
arterial wall. Of these, 5 of the 11 apoptotic
nuclei were found in the wall of LF arteries (total of 2740 nuclei), 2
in HF arteries (total of 3940 nuclei), and 4 in control arteries (total
of 5430 nuclei). Clustering of apoptotic cells was not
observed.
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Figure 4. DNA laddering as an indication of apoptosis. DNA was extracted from arteries and amplification of DNA fragments specifically generated during the apoptotic process was performed by the LM-PCR procedure and size-separated on an agarose gel as described in Materials and Methods. A, Agarose gel with samples from 2 animals. Samples 1, 2, and 3 are DNA from control, HF, and LF arteries, respectively, isolated 4 days after surgery and amplified for 28 cycles with the LM-PCR procedure. Sample 4 is DNA from a standard batch of rat thymus DNA that was amplified for 22 cycles and used as a standard (see Materials and Methods). Samples 5, 6, and 7 are DNA from control, HF, and LF arteries, respectively, isolated 16 days after surgery and amplified for 28 cycles. Sample 8 is a molecular weight standard indicating increments of 200 bp in the lower half of the gel. B, Laddering scored according to the ladder intensity (see Materials and Methods); 2 is maximum score and 0 is no laddering detected. The arteries were isolated at 2 (n=2), 4 (n=5), and 16 days (n=5) after surgery modifying blood flow through individual mesenteric arteries. Each bar indicates the mean value; error bars indicate SEM. *P<0.05, one-tailed Wilcoxon signed-rank test. lf indicates low flow; hf, high flow.
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Figure 5. Detection of apoptosis on single cell level. Longitudinal sections of artery (16 days of exposure to HF) were stained with the TUNEL method (see Materials and Methods). The adventitia is recognized on the lower part of the wall, and the small dark area in the center of the medial layer indicates an apoptotic nucleus. Original magnification x1000.
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Discussion
The results of the present study provide direct
evidence that proliferation, cell loss, and phenotypic changes of the
SMCs are involved in the remodeling response to altered blood flow in
rat mesenteric small arteries.
In line with earlier
observations,12 13 14 22 23
chronic reduction of flow ultimately resulted in a structural narrowing
of mesenteric arteries of the rat, whereas increased flow resulted in a
widening. These alterations represent remodeling rather than a
reduced or accelerated growth of the arterial wall, because
control arteries did not change their structure
(Figure 1), as also found
previously.13 23
The changes seen in LF and HF arteries have previously been
demonstrated to normalize wall shear rate and circumferential wall
stress after 4 weeks of hypoperfusion or
hyperperfusion.13 At least
two main mechanisms for these normalizations can be hypothesized: (1)
Flow-induced changes of vasomotor tone or structural lumen diameter
cause changes of the tensile forces in the artery wall, which is then
normalized by either wall hypertrophy or
hypotrophy.24 (2) Signaling
substances liberated or suppressed due to altered flow affect both
lumen caliber and artery wall mass, resulting in a tight connection
between these parameters. In the present study, both
mechanisms may be represented. Evidence for tensile force
being a signal comes from our finding in LF arteries that the
structural narrowing preceded the hypotrophy of the artery wall by
several days
(Figure 1). On the other hand, the finding in HF arteries
that final stages of widening and hypertrophy of the
arterial wall were established simultaneously
by day 16 after the intervention
(Figure 1), as also found by Tulis et
al22 in a similar system, is
more consistent with the presence of a signaling substance.
Whether this difference in inward and outward remodeling indeed results
from totally different mechanisms or is because the 80% reduction
in flow is a stronger signal to the arteries than a doubling in flow
needs to be investigated further.
Although the model used in the present study is designed
to investigate the effects of changes in flow, the vessels examined may
also have been exposed to slight changes in pressure. Thus, under
control conditions, the pressure in the vessels will be 10
mm Hg below the pressure in the superior mesenteric
artery.25 The ligation
process would be expected to cause the pressure to rise slightly in the
LF arteries as a result of the 80% decrease in flow, the rise,
however, being substantially offset by the increase in the hydrodynamic
resistance of the arteries because of the decrease in lumen diameter of
the vessels. Conversely, in the HF arteries, the ligation procedure
could cause a slight decrease in pressure, the rise being offset by the
decreased hydrodynamic resistance due to the increase in lumen
diameter. Thus, any changes in pressure in LF and HF arteries were
probably small.
The rapidity of the narrowing of the LF arteries, which
occurred within 2 days, was surprising, particularly because this
narrowing did not progress much further when the intervention period
was prolonged to up to 32 days. The lumen diameter of the arteries was
measured in vitro at 100 mm Hg in a calcium-free solution, which
suggests that the narrowing results were not due to contraction. It
cannot, however, be excluded that the narrowing is due to a prolonged
calcium-independent rigor, as seen when certain smooth muscle
preparations are exposed to protein kinase C activators
(eg, see Walsh et al26 ).
Furthermore, it has been shown that lowering shear stress causes higher
expression of endothelin 1 in endothelial cells (eg,
Malek et al27 ) and that
endothelin 1 is able to induce a stable protein kinase C–dependent
contraction of rat aorta strips in a calcium-free Krebs
buffer.28 Recently, inward
remodeling was shown for resistance arteries from rat cremaster muscle
kept in organ culture for 4 days at 75 mm Hg in the absence of
flow.29 These
authors29 suggested
fibroblast growth factor-2 (FGF-2) as a candidate mediator of the
inward remodeling, based on observations by Bryant et
al30 that anti–FGF-2
antibodies are able to inhibit the inward remodeling in the unilateral
carotid artery ligation model in
mice.30 Persistent
contraction induced by FGF-2 may be converted into a reorganization of
the cytoskeleton of arterial
SMCs.29 30 FGF-2,
endothelin 1 (see above), or a combined activity of these components
could thus be the signals for the rapid inward remodeling.
In the LF arteries, wall hypotrophy was established after 16
days, and after 4 weeks of hypoperfusion, a marked loss of SMCs was
observed by using the 3D-dissector technique
(Table 1). Our biochemical and histological
evidence indicates that apoptosis is involved. This is
consistent with the findings of Cho et
al31 showing increased
levels of nucleosomal ladders after reduction of the blood flow in the
common carotid artery of the rabbit. Furthermore, our data showed that
arteries hypoperfused for 4 weeks not only had fewer cells but the
remaining cells were considerably smaller than those in control
arteries. Cell volume regulation then has to be included as a
significant factor for the remodeling, and it would have been
interesting to see to what extent volume regulation and cell loss
contribute to the decrease of CSA at the earlier time points,
especially because cell shrinkage is one the characteristics of a cell
in apoptosis. Furthermore, with a 67% decrease of cell number
and a 40% decrease of the mean cell volume, hypotrophy of the
hypoperfused arteries is primarily a cellular phenomenon rather than as
a result of extracellular matrix degradation. A reduced proportion of
the cellular compartment of the arterial wall in LF
arteries might be responsible for this marked stiffening as indicated
by steeper stress-strain
relationships.14
The widening of the HF arteries, which reached a plateau at
day 16 after introduction of the flow modification
(Figure 1A), is in line with earlier results with the model
showing significant widening after 4
weeks13 and 2
weeks14 of intervention. It
is, however, delayed compared with the 7 days required for reaching a
significant widening in a similar ligation system of rat mesenteric
arteries analyzed by Tulis et
al.22 With regard to the
hyperperfusion-induced increase in CSA accompanying the widening of the
arteries, two lines of evidence in the present study indicate that
this is associated with proliferation. First, the 3D-dissector
analysis of arteries hyperperfused for 4 weeks showed a 44%
increase of the number of SMCs per artery length with no change in cell
volume
(Table 1). Second, the marker for SMCs in S-phase was
elevated in hyperperfused arteries, where the time course of the PCNA
levels showed a biphasic pattern with peaks at days 8 and 32
(Figure 2). The peak at day 8 preceded the increase in CSA,
consistent with the expression of PCNA preceding
hypertrophy compared with CSA also observed by Tulis et
al,22 although this appeared
at an earlier time point (days 3 through 7). The late peak of PCNA seen
at day 32 could be related to a later phase of remodeling appearing
beyond 4 weeks, as observed by Fath et
al.32
A regulation of the balance between cell proliferation and
cell death may be the most important mechanism for remodeling, as has
been suggested for embryonic development and normal tissue homeostasis
(for review, see Best et
al33 and Hamet et
al).34 This is supported by
the present study, where apoptosis determined by DNA
laddering was more frequent in both the LF and HF arteries compared
with control
(Figure 4B). The intensity of the laddering was rather
variable, which was also observed by Cho et
al31 using a similarly
sensitive laddering-detection technique in carotid arteries of rabbits.
It was also of interest that TUNEL staining
(Figure 5) did not detect clusters of cells, confirming
apoptosis as a single cell event.
An increased level of apoptosis in hypertrophic
arteries might seem inconsistent with a balance changed toward
cell proliferation. However, in spontaneously hypertensive rats treated
with
NG-nitro-L-arginine
methyl ester, an increased TUNEL count was previously observed in
hypertrophic coronary artery smooth
muscle.35 Also, in growing
coronary collateral arteries, an increased apoptosis
was observed7 and in
deoxycorticosterone acetate (DOCA)-salt hypertensive rats, an increased
CSA was associated with an increased ladder
intensity.35 As discussed by
Hamet at al34 "there is
more than a conceptual link between apoptosis and its
physiological counterbalance, cell proliferation"
(page 854). It is even suggested that apoptosis could
act as a stimulus for wall hypertrophy. Indeed evidence for
apoptosis as a normal physiological process
comes from our findings of low but detectable levels of
apoptosis in control arteries. This is consistent with
previous findings, where a significant level of DNA synthesis was
observed in 6-week-old rats, although there was no increase in DNA
content by 20 weeks of
age.36 Taken together, this
suggests that there is a basal but slow rate of cellular turnover in
intact, structurally stable, small muscular arteries, a turnover that
is increased under conditions of altered flow.
SMCs have the ability to modulate their phenotype
within a certain range from differentiated contractile type to the
synthetic phenotype (for review, see
Owens37 ). In the present
study, the level of the smooth muscle markers desmin and calponin
investigated by immunohistochemistry showed a decrease in LF arteries,
whereas the presence of smooth muscle -actin and smooth muscle
myosin heavy chain was hardly affected. Also, the levels of desmin mRNA
were markedly decreased as early as day 4, which persisted at day 16.
Thus, dedifferentiation of the contractile SMCs was pronounced and had
an early onset. This dedifferentiation could be due to the early
narrowing of the artery that at the unchanged transmural pressure
results in decreased wall stress. Such a direct role for mechanical
factors was seen in organ culture of rabbit aorta, where application of
pressure (wall stress) prevented the decreased expression of smooth
muscle differentiation markers seen in the isolated
artery.38 In the HF
arteries, when desmin mRNA is taken as a differentiation marker, a
dedifferentiation was also seen with a later onset and also the
immunohistochemical analyses showed the same tendency. The
reason for this dedifferentiation could be completely different from
the one seen in LF arteries, because proliferation of SMCs has been
linked to a conversion of the cells to more synthetic types (see
Owens37 ).
In conclusion, this work provides the first direct evidence
concerning the time course of and cellular basis for the changes in
resistance artery morphology in response to decreases and increases in
flow. The structural response to reduced blood flow consists of a rapid
reduction of lumen diameter that is followed by medial hypotrophy and
cell loss. The response to increased blood flow is a slow and parallel
increase in lumen diameter and medial mass, where at least the
increased medial mass is due to cellular proliferation as indicated by
increased PCNA counts and an increase in cell number. Both decreased
and increased blood flow lead to dedifferentiation of SMCs and
apoptosis.
Acknowledgments
This study was supported by a program
grant (902-98-291 PGN) from the Dutch Scientific Research organization
(NWO) and by the EU-sponsored initiatives EURAD and CAMMRAR (BMH1 CT
92-07777 and BMH4 CT 98-3069). Expert technical assistance of Mette
Schandorff is greatly
appreciated.
Footnotes
Original received February 14, 2001; revision received May 17, 2001; accepted May 22, 2001.
References
1.
Davies PF.
Flow-mediated endothelial mechanotransduction.
Physiol Rev. 1995;75:519–560.[Medline]
2.
Smiesko V, Lang DJ,
Johnson PC. Dilator response of rat mesenteric arcading arterioles to
increased blood flow velocity. Am J
Physiol. 1989;257:H1958–H1965.[Medline]
3.
Langille BL,
O’Donnell F. Reductions in arterial diameter produced by
chronic decreases in blood flow are
endothelium-dependent.
Science. 1986;231:405–407.[Medline]
4.
Ben Driss A,
Benessiano J, Poitevin P, Levy BI, Michel JB. Arterial
expansive remodeling induced by high flow rates.
Am J Physiol. 1997;272:H851–H858.[Medline]
5.
Huonker M, Halle M,
Keul J. Structural and functional adaptations of the
cardiovascular system by training.
Int J Sports Med. 1996;17:S164–S172.[Medline]
6.
Osol G, Cipolla M.
Pregnancy-induced changes in the three-dimensional mechanical
properties of pressurized rat uteroplacental (radial) arteries.
Am J Obstet Gynecol. 1993;168:268–274.[Medline]
7.
Wolf C, Cai WJ,
Vosschulte R, Koltai S, Mousavipour D, Scholz D, Afsah-Hedjri A,
Schaper W, Schaper J. Vascular remodeling and altered protein
expression during growth of coronary collateral arteries.
J Mol Cell Cardiol. 1998;30:2291–2305.[Medline]
8.
Schaper W, Ito WD.
Molecular mechanisms of coronary collateral vessel growth.
Circ Res. 1996;79:911–919.[Full Text]
9.
Schiffers PM,
Henrion D, Boulanger CM, Colucci-Guyon E, Langa-Vuves F, van Essen H,
Fazzi GE, Levy BI, De Mey JG. Altered flow-induced arterial
remodeling in vimentin-deficient mice.
Arterioscler Thromb Vasc Biol. 2000;20:611–616.[Abstract/Full Text]
10.
Guyton JR,
Hartley CJ. Flow restriction of one carotid artery in juvenile rats
inhibits growth of arterial diameter.
Am J Physiol. 1985;248:H540–H546.[Medline]
11.
Miyashiro JK,
Poppa V, Berk BC. Flow-induced vascular remodeling in the rat carotid
artery diminishes with age. Circ
Res. 1997;81:311–319.[Abstract/Full Text]
12.
Unthank JL, Fath
SW, Burkhart HM, Miller SC, Dalsing MC. Wall remodeling during luminal
expansion of mesenteric arterial collaterals in the rat.
Circ Res. 1996;79:1015–1023.[Abstract/Full Text]
13.
Pourageaud F, De
Mey JG. Structural properties of rat mesenteric small arteries after
4-wk exposure to elevated or reduced blood flow.
Am J Physiol. 1997;273:H1699–H1706.[Medline]
14.
Ceiler DL, De Mey
JG. Chronic
NG-nitro-L-arginine
methyl ester treatment does not prevent flow-induced remodeling in
mesenteric feed arteries and arcading arterioles.
Arterioscler Thromb Vasc Biol. 2000;20:2057–2063.[Abstract/Full Text]
15.
Mulvany MJ,
Halpern W. Contractile properties of small arterial
resistance vessels in spontaneously hypertensive and normotensive rats.
Circ Res. 1977;41:19–26.[Medline]
16.
Korsgaard N,
Mulvany MJ. Cellular hypertrophy in mesenteric
resistance vessels from renal hypertensive rats.
Hypertension. 1988;12:162–167.[Abstract]
17.
Stassen FR, Maas
RG, Schiffers PM, Janssen GM, De Mey JG. A positive and reversible
relationship between adrenergic nerves and -1A adrenoceptors in rat
arteries. J Pharmacol Exp
Ther. 1998;284:399–405.[Abstract/Full Text]
18.
Labarca C, Paigen
K. A simple, rapid, and sensitive DNA assay procedure.
Anal Biochem. 1980;102:344–352.[Medline]
19.
Wyllie AH.
Glucocorticoid-induced thymocyte apoptosis is associated with
endogenous endonuclease activation.
Nature. 1980;284:555–556.[Medline]
20.
Staley K,
Blaschke AJ, Chun J. Apoptotic DNA fragmentation is detected by
a semi-quantitative ligation-mediated PCR of blunt DNA ends.
Cell Death Differ. 1997;4:66–75.
21.
Bravo R, Celis
JE. A search for differential polypeptide synthesis throughout the cell
cycle of HeLa cells. J Cell
Biol. 1980;84:795–802.[Abstract]
22.
Tulis DA, Unthank
JL, Prewitt RL. Flow-induced arterial remodeling in rat
mesenteric vasculature. Am J
Physiol. 1998;274:H874–H882.[Medline]
23.
Pourageaud F, De
Mey JG. Vasomotor responses in chronically hyperperfused and
hypoperfused rat mesenteric arteries.
Am J Physiol. 1998;274:H1301–H1307.[Medline]
24.
Langille BL,
Bendeck MP, Keeley FW. Adaptation of carotid arteries of young and
mature rabbits to reduced carotid blood flow.
Am J Physiol. 1989;256:H931–H939.[Medline]
25.
Fenger-Gron J,
Mulvany MJ, Christensen KL. Mesenteric blood pressure profile of
conscious, freely moving rats. J
Physiol. 1995;488:753–760.[Abstract]
26.
Walsh MP,
Horowitz A, Clément-Chomienne O, Andrea JE, Allen BG, Morgan KG.
Protein kinase C mediation of
Ca2+-independent contractions of vascular
smooth muscle. Biochem Cell
Biol. 1996;74:485–502.[Medline]
27.
Malek AM, Greene
AL, Izumo S. Regulation of endothelin 1 gene by fluid shear stress is
transcriptionally mediated and independent of protein kinase C and
cAMP. Proc Natl Acad Sci
U S A. 1993;90:5999–6003.[Abstract]
28.
Shimamoto H,
Shimamoto Y, Kwan CY, Daniel EE. Participation of protein kinase C in
endothelin-1-induced contraction in rat aorta: studies with a new tool,
calphostin C. Br J
Pharmacol. 1992;107:282–287.[Medline]
29.
Bakker ENTP, van
der Meulen ET, Spaan JA, van Bavel E. Organoid culture of cannulated
rat resistance arteries: effect of serum factors on vasoactivity and
remodeling. Am J Physiol. 2000;278:H1233–H1240.
30.
Bryant SR,
Bjercke RJ, Erichsen DA, Rege A, Lindner V. Vascular remodeling in
response to altered blood flow is mediated by fibroblast growth
factor-2. Circ Res. 1999;84:323–328.[Abstract/Full Text]
31.
Cho A, Mitchell
L, Koopmans D, Langille BL. Effects of changes in blood flow rate on
cell death and cell proliferation in carotid arteries of immature
rabbits. Circ Res. 1997;81:328–337.[Abstract/Full Text]
32.
Fath SW, Burkhart
HM, Miller SC, Dalsing MC, Unthank JL. Wall remodeling after wall shear
rate normalization in rat mesenteric arterial collaterals.
J Vasc Res. 1998;35:257–264.[Medline]
33.
Best PJ, Hasdai
D, Sangiorgi G, Schwartz RS, Holmes DR Jr, Simari RD, Lerman A.
Apoptosis: basic concepts and implications in coronary
artery disease. Arterioscler Thromb Vasc
Biol. 1999;19:14–22.[Abstract/Full Text]
34.
Hamet P, DeBlois
D, Dam TV, Richard L, Teiger E, Tea BS, Orlov SN, Tremblay J.
Apoptosis and vascular wall remodeling in hypertension.
Can J Physiol Pharmacol. 1996;74:850–861.[Medline]
35.
Sharifi AM,
Schiffrin EL. Apoptosis in aorta of deoxycorticosterone
acetate-salt hypertensive rats: effect of endothelin receptor
antagonism. J Hypertens. 1997;15:1441–1448.[Medline]
36.
De Mey JG, Daemen
MJ, Boonen HC, Bosman FT, Dijkstra EH, Fazzi GE, Janssen GM, Schiffers
PM, Struyker-Boudier HA, Vrijdag MJ. In vivo DNA synthesis is not
uniformly increased in arterial smooth muscle of young
spontaneously hypertensive rats. J
Hypertens. 1991;9:695–701.[Medline]
37.
Owens GK.
Regulation of differentiation of vascular smooth muscle cells.
Physiol Rev. 1995;75:487–517.[Medline]
38.
Birukov KG,
Lehoux S, Birukova AA, Merval R, Tkachuk VA, Tedgui A. Increased
pressure induces sustained protein kinase C-independent herbimycin
A–sensitive activation of extracellular signal-related kinase 1/2 in
the rabbit aorta in organ culture. Circ
Res. 1997;81:895–903. [Abstract/Full Text]
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|
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|
|
|
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[Full Text]
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|
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23(8):
1370 - 1376.
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[Full Text]
[PDF]
|
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[Abstract]
[Full Text]
[PDF]
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[Full Text]
[PDF]
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92(8):
929 - 936.
[Abstract]
[Full Text]
[PDF]
|
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Differential structural adaptation to haemodynamics along single rat cremaster arterioles
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548(2):
549 - 555.
[Abstract]
[Full Text]
[PDF]
|
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1100 - 1105.
[Abstract]
[Full Text]
[PDF]
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June 1, 2002;
17(3):
105 - 109.
[Abstract]
[Full Text]
[PDF]
|
|
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