491
Vascular Smooth Muscle Cell Hypertrophy and
Hyperploidy in the Goldblatt Hypertensive Rat
Gary K. Owens and Stephen M. Schwartz
From The Department of Pathology, University of Washington, Seattle, Washington, and the Department of Physiology, University of
Virginia, Charlottesville, Virginia
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SUMMARY. Our major objective in this study was to examine the hypothesis that the aortic
smooth muscle cell hypertrophy and hyperploidy observed in previous studies of spontaneously
hypertensive rats is not peculiar to that model, but also occurs in Sprague-Dawley rats made
hypertensive by a Goldblatt procedure (two-kidney, one-dip model). Flow microfluorometric and
microdensitometric analysis of smooth muscle cell DNA content showed a significant increase in
the frequency of tetraploid smooth muscle cells from 5.6 ± 0.9% in controls to 14.6 ± 1.94% in
hypertensives 1 month after Goldblatt surgery. Neither differences in ploidy nor elevation in
blood pressure were apparent 2 weeks after surgery. The frequency of polyploid smooth muscle
cells increased with age, duration of hypertension, and level of blood pressure. Analysis of the
interrelationship between smooth muscle cell ploidy and hypertrophy in 5-month post-surgery
Goldblatts by cytospectrophotometric measurements of the protein and DNA content of individual
smooth muscle cells showed that tetraploid and octaploid cells from Goldblatt rats had 64% and
129% greater protein mass, respectively, than diploid cells. In addition, the mean protein mass of
smooth muscle cells from Goldblatts was approximately 100% greater than that of normotensive
controls, with each of the ploidy classes in Goldblatts having a higher frequency and mass than
the corresponding cells in controls. Estimates of cell number per centimeter aortic length, based
on measurements of average DNA/cell and total aortic medial DNA, showed no difference
between hypertensives and controls. Furthermore, the rate of accumulation of polyploid cells
could account for the increased frequency of cells undergoing DNA synthesis as measured by
[3H]thymidine autoradiogTaphy. Thus, smooth muscle cell hypertrophy, not hyperplasia, was
responsible for the increased mass of smooth muscle in aortas of Goldblatt hypertensive rats
compared with normotensive controls, and this smooth muscle cell hypertrophy was accompanied
by an increase in DNA ploidy. (Circ Res 53: 491-501, 1983)
ARTERIES from hypertensive patients and animals
have a greater smooth muscle mass than those of
their normotensive counterparts (Furuyami, 1962;
Wolinsky, 1970; Jurukova et al., 1976; Wiener et al.,
1977; Warshaw et al., 1979; Olivetti et al., 1980;
Arner and Uvelius, 1982). Folkow and others (Folkow et al., 1973; Lundgren et al., 1974; Berecek and
Bohr, 1977) have suggested, based on hemodynamic
evidence, that an increase in medial wall thickness,
wall:lumen ratio, and mass of smooth muscle in
resistance vessels of hypertensive animals confers
both a functional and a structural advantage, so
that, at any given level of smooth muscle activation,
vascular resistance is greater in hypertensives than
controls. Whereas the role of structural decreases in
vessel lumenal diameter as a factor in increased
peripheral resistance in hypertension remains a
highly debated issue, there is good evidence supporting the idea that vessel hypertrophy confers a
functional advantage. Mulvany, Halpern and coworkers (Mulvany et al., 1978; Warshaw et al., 1979)
found, in studies of isolated mesenteric arteries
(150-200 fim in diameter), that vessels from spontaneously hypertensive (SH) rats developed greater
maximal contractile force than those of normoten-
sive Wistar-Kyoto (WKy) controls. The increased
force was due to an increase in the mass of smooth
muscle in SH rats rather than to a change in the
force development per unit of contractile mass. Similarly, Arner and Uvelius (1982) demonstrated that,
whereas maximum tension development of abdominal aortic-segments was greater in SH rats than in
WKy controls, no differences were found in either
force development per unit cell area or in maximum
shortening velocity. Data thus suggest that the characteristics of actomyosin interaction in SH and WKy
rats are similar, and that differences in force-generating capacity are due to differences in total contractile mass.
The studies above point out the potential significance of changes in wall mass in hypertension and
emphasize the importance of understanding the cellular basis of medial hypertrophy. In recent studies
(Owens et al., 1981; Owens and Schwartz, 1982)
we demonstrated that the increased smooth muscle
mass in aortas of SH rats relative to WKy controls
was due solely to smooth muscle cell hypertrophy
without detectable hyperplasia. Our findings were
recently confirmed in morphometric studies by Olivetti et al. (1982), who found that smooth muscle
Circulation Research/Vol. 53, No. 4, October 1983
492
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cell number did not differ, between aortas of SH
and WKy rats, and that differences in mass of
smooth muscle were due to cellular hypertrophy.
An additional finding in OUR studies was that smooth
muscle cell hypertrophy was accompanied by an
increase in DNA ploidy with up to 30% of the
smooth muscle cell population in SH rats being
polyploid. Thus, the increase in DNA synthesis and
content in an aortic coarctation model of hypertension in rabbits (Bevan, 1976; Bevan et al., 1976) in
the absence of any increase in cell number in this
same model in rats (Olivetti et al., 1980) could be
accounted for by changes in smooth muscle cell
ploidy. A critical remaining issue, however, is
whether changes in smooth muscle cell ploidy are
unique to a genetic model of hypertension such as
the SH rat. Thus, in the present study, we have
examined whether smooth muscle cell hypertrophy
and hyperploidy also occur in normotensive rats
made hypertensive by a Goldblatt procedure (Goldblatt et al., 1934). In addition we have studied the
time-course of changes in ploidy and hypertrophy
relative to changes in blood pressure, examined
interrelationships between smooth muscle cell hypertrophy, hyperploidy, and blood pressure, and
assessed the relative roles of smooth muscle cell
hypertrophy vs. hyperplasia in aortic medial thickening in Goldblatt hypertension.
Methods
Animals
Male, Sprague-Dawley rats, obtained from Tyler Laboratories, were used throughout these experiments. Rats
were maintained on normal sodium (110 mEq/^g) chow
and both food and water were administered ad libitum.
Blood pressures were measured via a tail-cuff method,
using a piezoelectric pressure transducer (Buffington Clinical Systems). Animals were lightly anesthetized with
ether, and the animal's temperature was maintained with
an electric heating pad. Although pressures measured in
this manner are slightly lower than measurements in
conscious animals, the variance between measurements is
less (unpublished observations). All animals were identified by number, and the blood pressure technician was
not aware of experimental groupings. Heart weight:body
weight ratios were determined at sacrifice, as previously
described (Owens and Schwartz, 1982).
Surgical Procedure: Induction of Goldblatt
Hypertension
Surgery was performed on rats that were 4-6 weeks of
age. Two blood pressure measurements were taken during
the week before surgery. Animals were divided randomly
into control and Goldblatt groups, and were anesthesized
with sodium pentobarbital (24 mg/kg, intraperitoneal).
The left renal artery was exposed through a midline incision and was dissected free. A 0.23-mm silver clip was
placed on the vessel. The muscle layers and skin were
closed with 000 monofilament nylon. A midline incision
was performed on control animals, so that control and
Goldblatt rats could not be identified during blood pressure measurements. Blood pressures were measured twice
weekly during the first month after surgery, and weekly
or biweekly thereafter. Operated animals were considered
hypertensive when their systolic blood pressure exceeded
140 mm Hg for three consecutive readings. The only
exception to this was with the 2-week post-surgery group
in which all operated animals, other than those showing
left kidney necrosis on necropsy, were grouped as 'hypertensive' despite the fact that their pressures were not yet
elevated (Fig. 1).
At either 2 weeks, 1 month, 3 months, or 5 months
after surgery, rats were killed by cervical dislocation following ether anesthesia. The thoracic aorta and heart were
excised, perfused with Hanks' solution (Gibco) (pH 7.4)
and placed in fresh Hanks'. Hearts were cleaned of loose
connective tissue, dried for 48 hours, and weighed. An
intima-medial preparation of the aorta was made by carefully dissecting away the adventitia and intercostal arteries
(Wolinsky and Daly, 1970). The endothelium then was
scraped off with a piece of Teflon. Resultant medial preparations were used for subsequent, isolation of smooth
muscle cells and for flow cytometric ploidy determinations
described below.
Evaluation of Aortic Hypertrophy
A quantitative assessment of the difference in mass of
smooth muscle between thoracic aortas of hypertensive
and normotensive rats was obtained by the method of
Anversa et al. (1980). This involved multiplying the wet
weight (of aortic medial preparations) per vessel length by
the fraction of aortic media occupied by smooth muscle
(i.e., volume density). Several vessel segments for transmission electron microscopy were taken from the midpoint
of the thoracic aorta, fixed in 1% paraformaldehyde, 2%
glutaraldehyde in 0.1 M phosphate buffer (pH 7.0), placed
in 1% osmium tetroxide for 2 hours, dehydrated, en bloc
stained with 3% uranyl acetate, and embedded in Epon.
Sections were cut and post-stained with uranyl acetate
and Reynold's lead citrate. Volume densities were determined on electron micrographs by standard point-counting techniques (Weibel and Bolender, 1973). A minimum
of 15 electron micrographs (approximately 1325 fim2 /
micrograph) were analyzed from each animal. By this
sampling method, the variability between electron micrographs from individual animals (expressed as the standard
['
f ,
ormedonratst
»Cwrtro)
0
O 20 S3 40 90 60TO80 90 100 IK) 120 0 0 MO BO 160
QftYS POST-CUPPING
FIGURE 1. Time-course of systolic blood pressure changes in twokidney, one-clip Goldblatt rats and control non-clipped rats. Blood
pressures were measured via a tail-cuff method under light ether
anesthesia. Each data point represents the mean value (± SE) at each
respective lime point for animals utilized in this study (Le., 34
Goldblatts and 28 controls).
Owens and Schwartz /Smooth Muscle Hypertrophy in Renal Hypertension
error divided by the mean) averaged 5.3%, which was
considered acceptable. The variability between animals,
expressed in the same manneT, was 5.4% and 3.6% for
Goldblart hypertensive and control rats, respectively.
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
Flow Cytometric Analysis of the Frequency of
Polyploid Smooth Muscle Cells
Smooth muscle cell nuclei were prepared for flow cytometric evaluation of DNA ploidy as previously described
(Owens et al., 1981). In brief, medial thoracic aortic preparations were incubated (37°C, 5% CO2, 95% air) for 1.5
to 2 hours in a collagenase and elastase solution. Fetal calf
serum (20% vol/vol) was added and the tissue centrifuged. This cell-tissue pellet then was suspended in nuclear isolation medium containing Tris-buffered isotonic
saline (pH 7.0), 10 /ig/ml diamidinophenylindole (DAPI),
0.6% Nonidet P.40 (vol/vol) 1.0 mM CaCh, 21 HIM MgCl2,
and 0.2% bovine serum albumin (wt/vol). Samples were
vortexed gently and cooled on ice. Suspensions were
filtered through 50 fim plastic mesh to remove tissue
debris. Nuclei then were syringed three times through a
26-gauge needle to ensure single nuclei. DAPI-stained
nuclei were observed in a Leitz fluorescent microscope to
determine the percentage of nuclear aggregates. If greater
than 2%, nuclei were resyringed and examined again.
Nuclear yields were about 25%, as determined by DNA
assay. As reported previously (Owens and Schwartz,
1982), the results obtained with this assay agree closely
with those obtained by Feulgen-DNA microdensitometric
analysis in tissue sections. Thus, the flow cytometric assay
provides a valid measure of the frequency of polyploid
smooth muscle cells in the intact aorta. Measurement,
acquisition, and analysis of DNA content was done on an
ICP-22 flow cytometer (Ortho Diagnostic Systems, Inc.).
Cell cycle compartments were estimated by an adaptation
of the method of Dean and Jert (1974), including fitting
and subtraction of an exponential noise background. Nonlinear least squares fitting was by the method of Marquardt (1963).
Aortic smooth muscle cells for microdensitometric evaluation of cellular hypertrophy (protein) and hyperploidy
(DNA), and for autoradiography were enzymatically dissociated into single cells using methods previously described (Owens et al., 1981; Owens and Schwartz, 1982).
Isolated cells were fixed overnight in 10% neutral-buffered
formalin. Cells were then washed with distilled water,
and cell smears made on glass slides and air-dried.
Protein: DNA Microdensitometry
To examine the relationship between smooth muscle
cell hypertrophy and hyperploidy, and to provide a quantitative measure of the degree of cellular hypertrophy, we
made simultaneous microdensitometric determinations of
the protein and DNA content of individual smooth muscle
cells, as previously described (Owens and Schwartz,
1982). In brief, smooth muscle cell smears, prepared as
described above, were stained for Fuelgen DNA determinations by the method of Fand (1970) and counterstained
for protein determinations with naphthol yellow S (Deitch,
1955; Meek, 1962). A Vickers M85 scanning-integrating
microdensitometer (Vickers Instruments) was employed
for microspectrophotometric measurements. We have previously shown that the absorption curves of the FeulgenDNA reaction product and naphthol-yellow S-protein
product are sufficiently separated to permit simultaneous
measurements of DNA and protein in the same cell (Owens and Schwartz, 1982). Protein measurements were
493
made at the napthol-yellow S absorption maximum of
445 nm, whereas DNA measures were made at 565 nm.
Standards of chick red blood cells (2.5 pg DNA) and
cultured rat aortic smooth muscle cells were processed
simultaneously. Cellular protein content of standards was
determined by the method of Lowry et al. (1951). By
comparison to standards, the absolute quantity of DNA
and protein for individual smooth muscle cells was calculated.
[3Hf 1 hymidine Autoradiography
The frequency of cells undergoing DNA synthesis was
determined by [ Hjthymidine autoradiography on smooth
muscle cell dispersions, as previously described (Owens
and Schwartz, 1982). Animals for autoradiography were
given a single intraperitoneal dose of [3H]thymidine (50
ftCi/100 g body weight; 6.7 Ci/mM; New England Nuclear) 1 hour before sacrifice. This essentially gives an
instantaneous estimate of the frequency of cells in S phase
of the cell cycle. Autoradiography was done on smooth
muscle cell dispersions prepared as described above. This
method has the advantage over conventional autoradiography on tissue sections of permitting analysis of large
numbers of cells—a necessity when dealing with very low
labeling frequencies. Slides containing cell dispersions
were coated with autoradiographic emulsion (Kodak,
NTB-2) exposed for 2 weeks (4C), developed by standard
procedures, and stained with hematoxylin. The frequency
of labeled cells was determined using counting techniques
for determining the frequency of a rare event, as described
by Schwartz and Benditt (1973). This involves counting
the total number of labeled cells (N*) per slide. An estimate
of the total number of cells per slide (N"*) was obtained
by counting the number of cells per microscopic field in
40 randomly selected fields. This then was multiplied by
the number of fields per slide to give Nto1. Thymidine
index was estimated as N*/Ntot. An average of 50,000
cells were counted per animal.
Determination of Smooth Muscle Cell Number
To assess whether smooth muscle cell hyperplasia occurred in aortas of Goldblatt hypertensive rats, an estimate
of smooth muscle cell number per unit aortic length was
obtained as previously described (Owens et al., 1981). In
brief, total DNA content of aortic tissue was determined
on medial preparations of thoracic aortas by the diphenylamine method of Burton (1955). DNA content was
expressed per unit in situ vessel length. Smooth muscle
cell number then was calculated by dividing the DNA
content per aortic length by the average DNA per smooth
muscle cell determined by flow cytometry.
Results
Blood Pressure
Figure 1 illustrates the time course of blood pressure changes after partial constriction of the renal
artery. Blood pressures were typically elevated between 16 and 20 days following surgery. Thirtyfour percent of the animals which were clipped did
not show a blood pressure increase within 30 days
(i.e., non-responders), and were discarded. It should
be noted, however, that selection of hypertensive
animals was not possible with the 2-week postsurgery group, because their pressures were not yet
Circulation Research/Vol. 53, No. 4, October 1983
494
TABLE 1
Blood Pressure, Heart Weight, and Heart WeightBody Weight Ratio of GoldblaH Hypertensive
(GB) and Control Sprague-Dawley Rats
Time*
postclipping
Heart wt
_,
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Blood pressure}
(mmHg)
Average dry
heart wt (g)
2-week, GB
n= 8
128 4 ±7.6
0.279 ± 0.010
body wt
7.88 ± 0.24
2-week, control
n=6
120.3 ± 7.7
0.269 ± 0.014
7.62 ± 0.30
1-month, GB
n= 7
154.0 ± 1.9}
0.412 ±0.017}
10.06 ± 0.74}
1-month, control
n=5
112 2 ±3.9
0.268 ± 0.004
7.34 ± 0 19
3-month, GB
n=6
163 3 ± 6.6}
0.470 ± 0.019}§
10 18 ±0.61}
3-month, control
n=6
116.7 ±5.7
0.375 ± 0.029 ||
5.94 ± 0.30 ||
5-month, GB
n=6
163.7 ±3.1}
0.477 ± 0.030}
8.83 ± 0.56}
5-month, control
n=6
114.7 ± 2 2
0.366 ± 0.015 |
5.74 ± 0 25 ||
x 10
Results are expressed as mean ± SEM.
* Time following surgical clipping of the renal artery of 4- to 6-week-old rats. Controls were agematched.
} Average of the last three measurements for the 1-month, 3-month, and 5-month groups and the
last two measurements for the 2-week group.
} Significantly greater than age-matched control (P < 0.025, analysis of variance).
§ Significantly greater than 1-month Goldblatt (P < 0.05, analysis of variance).
|| Significantly different from the 2-week and 1-month control groups (P < 0.025, analysis of
variance).
elevated over controls. Thus, for this group, all
animals except those showing gross renal necrosis
when killed were included in the hypertensive
group. As shown in Table 1, at all other time periods
examined (i.e., at 1, 3, and 5 months post-surgery),
Goldblatt rats had significantly higher blood pressures, heart weights, and heart weight:body weight
ratios than normotensive controls.
Smooth Muscle Cell Polyploidy and Rates of
DNA Synthesis
Results of flow cytometric evaluations of the frequency of polyploid cells (Figs. 2 and 3) showed that
substantial numbers of tetraploid (4C) aortic smooth
muscle cells were present in both Goldblatt hypertensive and normotensive rats. A marked increase
in the frequency of polyploid smooth muscle cells
was apparent by 1 month following renal-artery
clipping. Whereas no significant differences were
seen at 2 weeks post-surgery, it should be noted that
none of these animals were as yet hypertensive, and
that some animals included in analysis probably
would never have developed hypertension. As ob-
served previously in our studies of SH rats (Owens
and Schwartz, 1982), the frequency of polyploid
smooth muscle cells increased with age, although,
for controls, ploidy did not increase significantly
(analysis of variance) beyond 3 months following
sham operation—i.e., after animals were more than
4-5 months old. Linear regression analysis showed
a significant correlation between ploidy and animal
age in both Goldblatt hypertensive (r = 0.83, P <
0.001) and control groups (r = 0.85, P < 0.001). The
fractional increase in polyploid smooth muscle cells
with age (i.e., the slope of the linear regression line)
was more than four times greater in hypertensives
(slope = 8.27%/month) than in controls (1.96%/
month). A significant linear relationship was also
found between the frequency of polyploid smooth
muscle cells and systolic blood pressure (Fig. 4) for
combined Goldblatt and control animals (r = 0.63,
P < 0.001), for Goldblatt animals alone (r = 0.56, P
< 0.005), but not for controls alone (r = 0.02, not
significant). When the relationship between ploidy
and blood pressure was analyzed in animals of the
same age, significant relationships were observed
for the 1-month (r = 0.80, P < 0.01), 3-month (r =
Owens and Schwartz /Smooth Muscle Hypertrophy in Renal Hypertension
280
50
495
-—--O Goldblatt
*
Control
240-
40
30
20
10
0
MONTHS POST-CLIPPING
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3
4
5
6
7
8
9
O
DMA pg/nucleus
FIGURE 2. Representative DNA determinations of individual smooth
muscle cell (SMC) nuclei by flow microfluorimetry for a 5-month
post-clip Goldblatt hypertensive rat (upper) and a normotensive agematched control. Nuclei were stained for DNA with diamidmophenylindole as described by Owens et al. (1981). Distinct populations of
diploid (7 pg DNA) and tetraploid (14 pg DNA) smooth muscle cells
are apparent.
0.88, P < 0.01), and 5-month (r = 0.83, P < 0.02)
post-clip groups but not for the 2-week group (r =
0.40, not significant). Despite this apparent relationship between polyploidism and blood pressure, it is
interesting that, although ploidy increased by more
than 200% (P < 0.001, analysis of variance) in
Goldblatt rats between 1 and 5 months post-clipping
(Fig. 3), there was not a corresponding change in
blood pressure during this interval (Fig. 1). This
emphasizes the importance of an age-pressure component and shows that blood pressure appears to
peak much sooner than does the polyploid response.
To determine the possible contribution of proliferating smooth muscle cells to the tetraploid population measured by flow cytometry, the incidence of
cells in S phase of the cell cycle was measured by
[3H]thymidine autoradiography. This was done in
the 1 -month post-clip group, only since it would be
expected to be highest at this early time point (Bevan, 1976). The frequency of cells in S phase (mean
± SEM) was 3.50 ± 0.67 (x 10~4) and 1.26 ± 0.27 (X
10~4) for Goldblatt hypertensive and normotensive
controls, respectively. This difference was significant
(P < 0.025, analysis of variance). The fact that
labeling frequencies were on the order of 10~4 demonstrated that the high frequency of tetraploid cells
observed by flow cytometry did not represent the
G2 component of a rapidly proliferating population
FIGURE 3. Frequency of tetraploid aortic smooth muscle cells in
Goldblatt hypertensive and in normotensive controls as determined
by flow microfluorimetry. Goldblatts were significantly greater than
controls (P < 0.001, analysis of variance) at 1, 3, and 5 months postclipping. In addition, there was a significant increase in tetraploidy
with age at all time points examined for Goldblatt rats but only
between 1 and 3 months post-dipping (i.e., from 3 to 5 months of
age) for controls (P < 0.025, analysis of variance).
of cells. Likewise, an extremely low S phase population was evident by flow cytometry (Fig. 2) in all
age groups studied, in that cells with intermediate
DNA content between diploid and tetraploid were
not observed.
60
50
a>
O 40
I
•
O
»
'A
•
a
•
O
2
2
I
I
3
3
5
5
WK GOLDBLATT
WK CONTROL
M0 G0LD6LATT
MO CONTROL
MO GOLDBLATT
MO CONTROL
MO GOLE6LATT
M0 CONTROL
30
8.
3
20
10
u
80 90 OOIO 120130 WO 150 I6OTO180190200
Systolic Blood Pressure (mmUg)
FIGURE 4. Linear regression of the frequency of tetraploid aortic
smooth muscle cells to systolic blood pressure for both Goldblatt and
control rats. Blood pressures utilized represented the mean value of
at least two determinations taken during the week before sacrifice for
the 2-week post-clip group, during the 2 weeks before sacrifice for the
1-month post-dip group, and during the month before sacrifice from
the 3- and 5-month post-clip groups.
Circulation Research/Vol. 53, No. 4, October 1983
496
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It is interesting to note that, if the rate of DNA
synthesis measured at 1 month were to continue
until 3 months post-clipping, and assuming an S
phase duration of approximately 8 hours (Ross et
al., 1978), the calculated change in the fraction of
terraploid smooth muscle cells in Goldblatts (3.50 X
10~4 X 24 hr/8 hr X 60 days) and controls 1.26 X
10"4 X 24 hr/8 hr day X 60 days) would be 6.3%
and 2.3%, respectively. The observed change in the
fraction of tetraploids during this interval (Fig. 3)
was 9.1% for Goldblatts and 3.8% for controls.
Thus, the change in the fraction of polyploids could
account for the number of cells expected to undergo
DNA synthesis during this interval. In other words,
data suggest that the increased smooth muscle cell
DNA synthesis observed was associated with generation of polyploid cells rather than more cells. This
idea is supported by results of cell number determinations discussed elsewhere.
Interrelationship between Smooth Muscle
Hypertrophy and Hyperploidy: Simultaneous
Protein and DNA Measurements
To examine the interrelationship between cellular
hypertrophy and hyperploidy, and to provide a
quantitative measure of the degree of smooth muscle
cell hypertrophy, protein and DNA content of
freshly isolated smooth muscle cells were determined by scanning microdensitometry (Figs. 5 and
6). These measurements are extremely time-consuming and were performed only on cells from the
5-month post-clip group. Although there was considerable overlap in protein content between ploidy
classes (Fig. 5), polyploid cells from both hypertensive and normotensive rats had a greater average
protein mass than diploid cells (Fig. 6). In addition,
Gold blatt
1500
Control
8C
1300
4C
1100
4C
900
700
•g
2C
500
300
100
5.0
10.0 15.0 20.0 25.0 30.0
DNA (pg./SMC)
5X> 10.0 15.0 200 25.0 30.0
DNA(pg./SMC)
FIGURE 5. This ilustrates the interrelationship between smooth muscle cell ploidy (DNA content) and cellular mass (protein content).
Each point represents the combined cytospectrophotometric determination of protein and DNA content on individual aortic smooth
muscle cells. Data are a representative portion of the cells measured
from a single 5-month post-clip Coldblatt (left) and a normotensive
control (right). Cells were obtained by enzymatic dissociation of medial
preparations of rat aortas as described in the text. Distinct populations
of diploid (2C), tetraploid (4Q, and octaploid (8C) cells were found in
Goldblatt rats, whereas only diploid and tetraploid cells were seen in
controls. See Figure 6 for a summary of these data obtained from
several Coldblatt and control rats.
l200iI 100U
o
o.
a.
1000 -
E$$§J diploid cells
900 -
|
800 -
H£gj octaploid cells
700 -
•
| tetraploid cells
all cells
600
500
400
300
200
100
Control
Gold blatt
FIGURE 6. Summary of cytospectrophotometric determinations of
protein and DNA content on individual aortic smooth muscle cells of
5-month post-clip Coldblatt hypertensive rats (n = 4) and agematched normotensive controls (n = 5). Protein contents (mean ±
SEM) of diploid, tetraploid, and octaploid smooth muscle cells are
shown, as well as the calculated average cellular protein for all cells
combined, which takes into account both the frequency and protein
mass of each ploidy class. Approximately 100 cells were analyzed
from each rat. Note the relationship between smooth muscle cell
ploidy (DNA) and mass (protein), and that cells from Goldblatt rats
are considerably hypertrophied compared with those from normotensive controls.
diploid and tetraploid cells from Goldblatt hypertensive rats had 76% and 164% greater protein mass
respectively than the corresponding cells in normotensive controls. Thus, there was also an increase in
cellular mass which was independent of a change
in cellular ploidy. Data for octaploid cells showed a
high variance, but sample size was small (8 in a total
of 380 Goldblatt cells measured, and 0 in 487 for
control cells).
Based on the frequency and respective cellular
mass of each ploidy class, the average mass for
smooth muscle cells from Goldblatt and normotensive rats was calculated (shaded bars figure 6). As
shown, the average protein mass of smooth muscle
cells from 5-month post-clip Goldblatt rats was more
than twice that in age-matched normotensive controls.
Role of Smooth Muscle Cell Hypertrophy vs.
Hyperplasia in Vessel Hypertrophy
The relative role of smooth muscle cell hypertrophy vs. hyperplasia to aortic wall hypertrophy was
evaluated in 3-month post-clip Goldblatt hypertensive rats compared to their normotensive controls.
A quantitative estimate of the degree of vessel hypertrophy was obtained by the method of Anversa
et al., (1980), in which aortic medial wet weight (mg)
Owens and Schwartz /Smooth Muscle Hypertrophy in Renal Hypertension
497
TABLE 2
DNA Content, Cell Number, and Smooth Muscle Cell (SMQ Mass Evaluations in 3-Month Post-Clip Goldblatt Hypertensive and
Normotensive Control Rats
Aortic
medial wt (mg)
SMC
volume
density
cm length
DNA
SMC wt (mg)
cm length
aortic medial wt
(mg)
DNA Qig)
cm length
Cell number*
x 10'
cm length
Goldblatt
hypertensive
10.85 ± 0.64f
0.386 ±0.021
4.19±0.25t
1.32 +0.06
14 19 ± 0 . 7 1 |
1.63 ± 0.08
Control
8.21 ±0.19
0.366 ± 0.013
3 6 6 ±0.013
3.00 ±0.07
11.85 ±0.15
1.53 ±0.02
* Cell number was calculated by dividing the DNA/length by the average DNA/cell determined by flow microfluorimetry. Cell
number estimates for Goldblatts may be slightly high due to the fact that octaploid smooth muscle cells, which were found in small
numbers in Goldblatts only, were not included in calculations of average DNA/cell since their frequency cannot be measured accurately
by flow cytometry.
•f Significantly greater than controls (P < 0.05, analysis of variance).
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is multiplied by the fraction of the media occupied
by smooth muscle. Results (Table 2) showed a 32.2%
and 39.7% increase (P < 0.01, analysis of variance)
in aortic mass and smooth muscle mass, respectively,
in Goldblatt hypertensive rats, compared with controls. Results are in agreement with those of numerous other investigators (Wolinsky, 1970; Wiener et
al., 1977) who have demonstrated an increase in
aortic smooth muscle mass in Goldblatt hypertensive rats.
To assess the relative roles of smooth muscle cell
hypertrophy vs. hyperplasia to this increase in total
mass of smooth muscle, we made DNA and cell
number determinations on aortic medial preparations from these same animals. Whereas the DNA
content per unit in situ length was increased by 20%
(P < 0.05), analysis of variance) in Goldblatts, compared with controls, the smooth muscle cell number
per unit vessel length (calculated by dividing total
medial DNA by the average DNA/cell calculated
from flow cytometric measurements of ploidy) was
not different. Average DNA/cell was 8.7 pg for
Goldblatts, and 7.75 pg for controls. Results thus
showed that the increased mass of smooth muscle
in aortas of Goldblatt hypertensive rats compared
with controls occurred without a detectable increase
in cell number.
Discussion
As observed previously in our studies of the SH
rat (Owens et al., 1981; Owens and Schwartz, 1982),
smooth muscle cell hypertrophy and hyperploidy
also occurred in Goldblatt hypertension. The rate of
increase in polyploid cells in Goldblatt hypertensive
rats was very similar to that in SH rats, suggesting
that there is not a difference in inherent propensity
for development of smooth muscle cell polyploidy
between these models. This is important, since it
shows that changes in smooth muscle cell ploidy in
conjunction with cellular hypertrophy are not a
genetic anomaly unique to the SH rat. Although
Olivetti et al. (1980) did not measure DNA contents,
they did observe an increase in nuclear volume in
rats made hypertensive by aortic coarctatdon, suggesting that changes in ploidy occur in this hypertensive model, as well.
In previous studies we demonstrated that differences in mass of aortic smooth muscle between SH
rats and WKy controls were due solely to cellular
hypertrophy without detectable hyperplasia (Owens
et al., 1981; Owens and Schwartz, 1982). Recent
morphometric studies (Olivetti et al., 1982) likewise
showed that, whereas smooth muscle cell hyperplasia occurred in both SH and WKy rats between 21
and 45 days of age, differences in mass between SH
and WKy rats were due to cellular hypertrophy
without detectable differences in cell number. Similarly, this same group also reported (Olivetti et al.,
1980) that smooth muscle cell hypertrophy, not
hyperplasia, was responsible for aortic medial thickening in rats following subdiaphragmatic aortic stenosis. Three lines of evidence are presented in this
study to support the idea that changes in medial
smooth muscle mass in Goldblatt hypertension result from cellular hypertrophy, not hyperplasia.
First, based on biochemical measurements of total
vessel DNA and flow cytometric measurements of
cellular DNA, we were unable to detect an increase
in smooth muscle cell number in hypertrophied
aortas from Goldblatt hypertensive rats compared
with normotensive controls. Second, evaluation of
smooth muscle cell hypertrophy by cytospectrophotometric measurements of the protein content of
individual cells showed a 100% increase in average
cellular mass in Goldblatts compared with controls.
Third, the rate of accumulation of polyploid cells in
Goldblatt rats could account for the increased incidence of cells undergoing DNA synthesis in these
animals. These data thus provide very strong evidence that differences in mass of aortic smooth
muscle between Goldblatt hypertensive and normotensive controls are also due to cellular hypertrophy rather than hyperplasia. Whereas the studies
498
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above are in apparent contrast with a number of
studies that have implicated smooth muscle cell
hyperplasia in vessel thickening, the results of this
study demonstrate how the increased DNA synthesis and/or content observed by Bevan and others
(Crane and Dutta, 1963; Bevan et al., 1976; Bevan,
1976; Seidel, 1979; Rorive et al., 1980) could be
accounted for by increases in smooth muscle cell
ploidy. Additional studies implicating hyperplasia
are based on rather imprecise measurements of cell
cross-sectional areas or on assumptions that nuclear
size, shape, and orientation are the same in SH and
WKy rats (Mulvany et al., 1979; Warshaw et al.,
1979; Arner and Uvelius, 1982).
Our observation that the growth response of vascular smooth muscle cells in hypertension is limited
to cellular hypertrophy rather than hyperplasia has
important implications with regard to smooth muscle cell growth control. Cells hypertrophy in hypertension, whereas, in lipid and traumatic models of
atherogenesis, cells undergo proliferation (Imai et
al., 1970; Spaet et al., 1975; Thomas et al., 1976).
Chamley-Campbell et al. (1979, 1981) have presented evidence showing that smooth muscle cells
do not replicate in cell culture until they have lost
much of their contractile apparatus. Based on this
and other data, the Campbells have suggested that
smooth muscle cells normally exist in a nonproliferative, contractile state, and that the cells must first
dedifferentiate before proliferating. The evidence
presented in this study, as well as data of Olivetti et
al. (1982), indicate that cellular hypertrophy is the
principal mode of smooth muscle growth in both
normal and hypertensive adult rats. It is thus interesting to speculate that the hypertrophic growth
response of smooth muscle cells may represent a
mechanism whereby smooth muscle mass can increase without loss of differentiated function. There
is precedent for this idea in both cardiac and skeletal
myocytes, which respond to 'hypertrophic* stimuli
by increasing cell mass without loss of differentiation (Carlson, 1973; Baserga, 1976; Rumyantsev,
1977). Furthermore, it is interesting that, like smooth
muscle cells, significant numbers of normal adult
cardiac myocytes are polyploid and that the incidence and degree of polyploism increases in myocardial hypertrophy (Sandritter and Scomazzoni,
1964; Grove et al., 1969). Whereas our speculation
remains to be tested, it is clear from our observations
that the problem of initiation of growth of the
smooth muscle cell must be examined in a new light.
We should also point out that the cell cycle status
of the polyploid smooth muscle cell has not yet been
clearly established. We do know that tetraploid cells
do not represent the G2 plus mitotic component of
a rapidly proliferating cell population, since the frequency of smooth muscle cells undergoing DNA
synthesis was of the order of 10"4. However, there
are at least two other possibilities. First, they could
represent cells arrested in the G2 phase of the cell
cycle. Arrest of cells in G2 has been shown to occur
Circulation Research/Vol. 53, No. 4, October 1983
in a number of other cells and tissues (Gelfant, 1962;
Gordon and Lane, 1980). A G2-arrested cell would
contain 4C DNA but 2N chromosomes (where C =
haploid DNA content, N = haploid number of chromosomes) and, upon appropriate stimulation, would
be capable of undergoing mitosis without prior DNA
synthesis. The increased protein content which we
observed in polyploid cells would be quite consistent
with the increase in cell mass known to occur as
cells progress through the cell cycle (Darzynkiewicz
et al., 1979). A second possibility is that our polyploid cells are true tetraploids. A true tetraploid is a
cell which, in the resting phase of the cell cycle (Go),
contains double the number of chromosomes (4N)
and, upon appropriate mitogenic stimulation, would
initiate DNA synthesis rather than mitosis. The fact
that we observed a small number of octaploid cells
by microspectrophotomerry suggests that at least
some 4C cells are true 'tetraploids* and can reenter
S phase without undergoing mitosis.
An obvious question that needs to be addressed
deals with the mechanism responsible for initiating
the smooth muscle growth response. It has been
suggested that the medial hypertrophy that occurs
in vessels of hypertensive patients and animals represents an adaptive response to normalize vessel
wall tension. Whereas the demonstration in this
study of a significant linear relationship between the
frequency of tetraploid-hyperrrophied vascular
smooth muscle cells and blood pressure would be
consistent with this idea, our data also suggest that
polyploidy is more than just a simple response to
increased pressure. For example, we observed a
significant increase in the frequency of polyploid
smooth muscle cells in both hypertensive and normotensive rats between 1 and 5 months post-surgery
(Fig. 3), despite the fact that blood pressures did not
change during this same interval (Fig. 1). Thus, if
cellular hypertrophy is an attempt to normalize tension, then it appears to be a very slow adaptive
response. Our demonstration of a higher correlation
between polyploidism and animal age than between
polyploidism and blood pressure emphasizes the
importance of a time-pressure component. Finally,
our previous observations (Owens and Schwartz,
1982) of polyploid cells in veins, although at equal
frequencies in SH and WKy rats, also suggest that
hypertrophy and hyperploidy represent a response
to factors other than just increased pressure.
Whereas the studies reported here have focused
on a large vessel, there is reason to suspect that
similar changes occur in resistance vessels. There is
good evidence that increases in wall thickness and
smooth muscle mass occur in microvessels of hypertensive animals (Ichijma, 1969; Mulvany et al.,
1978; Warshaw et al., 1979) and man (Furuyama,
1962). Mulvany, Halpern and co-workers (Mulvany
et al., 1978; Warshaw et al., 1979, 1980) have consistently shown an increase, although not always
significant, in cross-sectional area of individual
smooth muscle cells in mesenteric arteries of SHR,
Owens and Schwartz /Smooth Muscle Hypertrophy in Renal Hypertension
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suggesting that cellular hypertrophy is present. Bevan (1976) examined rates of DNA synthesis by
[3H]thymidine autoradiography in a variety of vessels from rabbits made hypertensive by aortic stenosis. Although she did not collect quantitative data
on DNA replication in microvessels, she did suggest,
based on inspection of autoradiograms, that increases in DNA synthesis occur in small vessels as
well. Whether this reflected DNA endoreplication
or cellular hyperplasia remains to be determined. In
preliminary studies (unpublished observations), we
have observed that polyploid smooth muscle cells
are present in mesenteric arteries (approximately 150
nm in diameter) of 3-month Goldblart hypertensive
rats. However, we do not have quantitative data on
their frequency at this time.
Assuming that medial hypertrophy occurs in pressure-regulating vessels of hypertensive animals,
what role might it play in the hypertensive process?
In this study we were not able to detect a significant
increase in the frequency of polyploid cells prior to
onset of hypertension. However, it is likely that
nearly half of the 2-week Goldblart rats analyzed
would not have developed hypertension. Thus it is
not entirely clear whether changes in ploidy precede,
parallel, or follow development of hypertension. As
noted previously, an increase in mass of smooth
muscle would confer a mechanical advantage so
that, for a given level of smooth muscle cell activation, total force development would be greater in
hypertensives than controls (Folkow et al., 1973;
Berecek and Bohr, 1977; Mulvany et al., 1978; Warshaw et al., 1979). Whether this would in itself result
in an increase in vascular resistance as suggested by
Folkow et al. (1973), or whether it is simply an
adaptive response to normalize wall stress, is controversial. In either case, it would be an extremely
important component of a chronic elevation in vascular resistance.
Finally, we should comment on the significance
of a change in smooth muscle mass due to cellular
hypertrophy and hyperploidy as opposed to cellular
hyperplasia. Whereas an increase in cell number
would not be expected to result in a change in
individual cell function, there is reason to suspect
that cellular enlargement or hypertrophy may be
associated with functional and/or structural alterations. Hypertrophy of cardiac myocytes, for example, is associated with profound alterations in myosin isozymes and contractile properties (Alpert and
Mulieri, 1982, litten et al., 1982). Data of Berner et
al. (1981) suggest that smooth muscle cell hypertrophy may also be associated with major changes in
cellular protein composition. Using morphometric
techniques, they found a decrease in the relative
number of myosin filaments along with a 250%
increase in intermediate filaments in hypertrophic
venous smooth muscle of the ligated portal vein.
Consistent with this, Johansson (1976) and others
(Uvelius et al., 1981) have reported a decreased
force-generating capacity, a reduced frequency of
499
spontaneous contractions, and a lower sensitivity to
exogenous norepinephrine in this same model. Using microchemical techniques, Brayden et al. (1983)
found decreased actin and myosin content and decreased stress-generating capabilities in cerebral
(170 fim internal diameter) but not mesenteric arteries of SH rats, compared with WKy controls. Our
data (Owens et al., 1981) and those of Seidel (1979)
showed that actin and myosin content increased
proportionately with other cellular proteins in hypertrophic aortic smooth muscle cells from SH rats.
A decrease in the surface area:volume ratio as cells
enlarge (Gabella, 1979), could influence cell function
through a variety of mechanisms, including alterations in membrane receptor number and/or density.
It is interesting to speculate that development of
smooth muscle cell hypertrophy and hyperploidy
might be related to some of the functional alterations
reported for vascular smooth muscle in hypertensive
animals (Holloway and Bohr, 1973; Jones, 1973;
Hansen and Bohr, 1975; Lais and Brody, 1975). The
change in ploidy would be particularly important,
since changes in DNA, unlike changes in other
macromolecules, are irreversible except by cell
death. Although we have not as yet determined
whether hypertrophy and hyperploidy are reversible by antihypertensive therapy, Van der Heijden
and James (1975) have shown that the polyploidy
which occurs in uterine smooth muscle during pregnancy does not reverse postpartum. The ploidy
change observed in this and previous studies (Owens et al., 1981; Owens and Schwartz, 1982) may
relate to observations that treatment of animals with
chronic hypertension (Wolinsky, 1971; Hamilton,
1975; Lundgren and Weiss, 1979) is not totally effective in reversing structural or functional vascular
alterations. Thus, the development of smooth muscle cell hyperploidy may represent a fixed change
related to the establishment of a chronic hypertensive state.
Supported by Grants HL-03174, HL-0731Z and HL-19242 from
the National Institutes of Health. Dr. Schwartz is an Established
Investigator for the American Heart Association.
Address for reprints: Dr. Gary K. Owens, Department of Physiology, Box 449, University of Virginia, Charlottesville, Virginia 22908.
Received April 25, 1983; accepted for publication August 18, 1983.
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INDEX TERMS: Hypertension • Hyperploidy • Smooth muscle
hypertrophy
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Vascular smooth muscle cell hypertrophy and hyperploidy in the Goldblatt hypertensive rat.
G K Owens and S M Schwartz
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Circ Res. 1983;53:491-501
doi: 10.1161/01.RES.53.4.491
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