749
Angiotensin II Induces Hypertrophy, not
Hyperplasia, of Cultured Rat Aortic Smooth
Muscle Cells
Anja A.T. Geisterfer, Michael J. Peach, and Gary K. Owens
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We have explored the hypothesis that contractile agonists are important regulators of smooth muscle
cell growth by examining the effects of one potent contractile agonist, angiotensin II (All), on both
cell proliferation and cellular hypertrophy. All neither stimulated proliferation of cells made
quiescent in a defined serum-free media nor augmented cell proliferation induced by serum or
platelet-derived growth factor. However, All did induce cellular hypertrophy of postconfluent
quiescent cultures following 4 days of treatment, increasing smooth muscle cell protein content by 20%
as compared with vehicle-treated controls. All-induced hypertrophy was maximal at 1 |xM, had an
EDn of 5 nM, and was blocked by the specific All receptor antagonist Sar^Ile' All. The cellular
hypertrophy was due to an increase in protein synthesis, which was elevated within 6-9 hours following
All treatment, while no changes in protein degradation were apparent. ATI was even more effective
in inducing hypertrophy of subconfluent cultures, causing a 38% increase in protein content after 4
days of treatment (1 |AM) and showing a maximal response at concentrations as low as 0.1 nM.
Interestingly, in subconfluent cultures, ATI treatment (1 |xM, 4 days) was associated with a 50%
increase in the fraction of cells with 4C DNA content with the virtual absence of cells in S-phase of
the cell cycle, consistent with either arrest of cells in the G, phase of the cell cycle or development of
tetraploidy. These studies show that All is a potent hypertrophic agent but has no detectable mitogenic
activity in cultured rat aortic smooth muscle cells and describe an in vitro model that should be
extremely valuable in exploring the cellular controls of smooth muscle cell hypertrophy. (Circulation
Research 1988;62:749-756)
here is considerable interest in the cellular
mechanisms involved in the control of vascular
smooth muscle growth and its role in the
etiology of hypertension and atherosclerosis. It has
been demonstrated that aortic smooth muscle cells
(SMC) are capable of two distinct cellular growth
responses in vivo, depending on the mode of growth
stimulation.1"7 Coarctation models and experimental
injury models of atherosclerosis are characterized by
extensive proliferation and intimal migration of aortic
SMC.'"3 In contrast, in Goldblatt and spontaneously
hypertensive rats, the increase in aortic smooth muscle
mass can be accounted for primarily by enlargement of
existing cells, or cellular hypertrophy, rather than
cellular proliferation.4"7 Interestingly, SMC hypertrophy is often accompanied by increases in ploidy.
However, SMC hypertrophy is not dependent on
development of poryploidy because diploid cells from
hypertensive animals are also hypertrophied with
respect to diploid cells from normotensive controls.7
There is indirect evidence that SMC hypertrophy
may represent a response to increased blood pressure.
We consistently have observed a high degree of
correlation between blood pressure and SMC hypertrophy and hyperploidy.6-9 Furthermore, pharmacolog-
T
ical lowering of blood pressure can prevent development of SMC hypertrophy and hyperploidy and can
reverse cellular hypertrophy that already has occurred
in spontaneously hypertensive rats.8"10 However, these
data must be interpreted with caution because there is
no direct evidence that these effects on growth are due
to lowering of blood pressure as opposed to some other
direct or indirect effect of the drugs on cellular DNA
and protein synthesis. In fact, recent drug intervention
studies in this and other laboratories have demonstrated
dissociation of blood pressure changes and SMC
hypertrophy and hyperploidy in spontaneously hypertensive rats treated with different antihypertensive
drugs. 910 Results suggested that medial SMC hypertrophy was not simply a response to increased blood
pressure and implicated a direct role of specific
contractile agonists in control of SMC growth. For
example, we observed that captopril, a converting
enzyme inhibitor, was more effective than hydralazine
or propranolol in preventing hypertrophy of aortic
SMC in spontaneously hypertensive rats than was
predicted based on the magnitude of the decrease in
blood pressure, implicating a role for angiotensin
(All). Similarly, Yamori and coworkers10 have presented indirect evidence that catecholamines might be
involved in regulation of SMC growth.
From the Department of Physiology (A.A.T.G. and G.K.O.) and
the Department of Pharmacology (M.J.P.), University of Virginia,
School of Medicine, Charlottesville, Virginia.
Supported by National Institutes of Health grant PO1 HL-19242.
Address for reprints: G.K. Owens, Department of Physiology,
Box 449, University of Virginia, Charlottesville, VA 22908.
Received Jury 27, 1987; accepted October 23, 1987.
The hypothesis that contractile agonists might play
some direct role in mediation of SMC growth is of
interest because it may explain how an SMC might alter
its mass in response to an induced change in its work
load. It is worth noting that contractile agonists share
many common intracellular signaling mechanisms
750
Circulation Research
Vol 62, No 4, April 1988
with growth factors.""24 However, very few studies
have been done to explore the possible growth properties of contractile agonists in SMC, and no studies
have specifically explored the potential role of contractile agonists in mediation of cellular hypertrophy.
Thus, in the present studies, we have addressed the
possible role of one contractile agonist, All, in
regulation of SMC growth by studying the effect of All
on both proliferation and hypertrophy in cultured rat
aortic SMC.
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Materials and Methods
Cell Culture
Rat thoracic aortic smooth muscle cells were isolated
and cultured by the procedure described previously25
with the following changes: 150-175-g rats were used
instead of 200-225-g rats, and the following enzymes
in Hanks' Balanced Salt solution were used for cell
isolations: 1 mg/ml collagenase (type II, 158 M-/mg,
Cooper Biomedical, Freehold, New Jersey), 0.25 mg/
ml elastase (type I, 3 p-/mg, Cooper Biomedical), 1
mg/ml soybean trypsin inhibitor (Cooper Biomedical).
Cells were harvested for passaging at confluency
(approximate 5-day intervals) with trypsin-EDTA
(0.05% trypsin, 0.02% EDTA, GIBCO, Grand Island,
New York) solution and plated at 2-5 x 103 cells/cm2.
Passaged cells were grown in a one-to-one mixture of
Dulbecco's Modified Eagle Medium (GIBCO) and
Ham's F12 medium (GIBCO), containing either 10%
fetal bovine serum (FBS, Hyclone, Logan, Utah),
L-glutamine (0.68 mM, Sigma Chemical, St. Louis,
Missouri), penicillin (100 U/ml) and streptomycin (100
(xg/ml) (designated DF10) or insulin (5xlO~ 7 M,
Sigma Chemical), transferrin (5 M-g/ml, Sigma Chemical), ascorbate (0.2 mM, Sigma Chemical),
L-glutamine, and antibiotics (designated serum-free
medium, SFM). This SFM has been shown to maintain
SMC in a quiescent, noncatabolic state for extended
periods of time26 and to promote expression of smooth
muscle specific contractile proteins.a-" Cell cultures
were incubated at 37° C in a humidified atmosphere of
5% CO2-95% air with media changes every 2-3 days.
Growth Curves
Cultures were washed with a calcium-magnesium
free phosphate-buffered saline (PBS) containing (mM)
NaCl 137, N a ^ P O ^ . l , KC12.7, and KH2PO41.5, pH
7.4 harvested with trypsin-EDTA, diluted with 0.85%
NaCl (Columbia Diagnostics, Springfield, Virginia),
and counted on a Particle Data Electrozone Celloscope
(Elmhurst, Illinois) with orifice size of 95 |xm and
sample volume of 500 p.1. Triplicate wells were counted
for each group at every time point.
[3H]Thymidine Autoradiography
Cultures were pulsed with [3H]thymidine (1 jiCi/ml,
6.7 Ci/mmol, New England Nuclear, Boston, Massachusetts) in culture medium. Two hours later, cultures
were washed twice with PBS and fixed in 2% glutaraldehyde for 5 minutes, dehydrated, and coated with
Kodak NTB2 emulsion (Rochester, New York) (diluted
1:1 with distilled water). Dishes were exposed for 4
days at 4° C and then developed in D-19 (Eastman
Kodak), fixed with Rapid-Fix (Eastman Kodak), and
stained with hematoxylin. The percentage of cells
synthesizing DNA was determined by counting the
number of labeled cells (nuclei) in a random sample of
at least 1,000 cells from each dish. Triplicate dishes
were analyzed for each sample point.
Flow Microfluorimetric Analysis of Cellular DNA and
Protein Content
Cultures were washed with PBS and harvested using
either trypsin-EDTA or 1 mg/ml collagenase, 0.25
mg/ml elastase, and trypsin (0.25%, GIBCO) in
Hanks' Balanced Salt solution. Samples were divided
for protein and DNA analysis and then centrifuged
(113g, 6 minutes). For the protein staining, cells were
fixed in 75% methanol/25% PBS on ice for 30 minutes,
centrifuged (113g, 6 minutes), stained with FITC (75
ng/ml fluorescein isothiocyanate, Sigma Chemical; 0.5
M NaHCOj, pH 8) on ice for 30 minutes, and then
centrifuged (113g, 6 minutes) and resuspended in
0.85% NaCl. For measurement of DNA, cells were
stained with either propidium iodide (Sigma Chemical)
or Hoechst 33258 (Sigma Chemical). For propidium
iodide staining, cells were resuspended in a propidium
iodide staining solution (propidium iodide 50 ng/ml,
NaCl 150 mM, Tris base 100 mM, CaCl21 mM, MgSO4
0.54 mM, MgCl2 103 mM, bovine serum albumin
[BSA, Fraction V, Sigma Chemical] 2 mg/ml, concentrated HC10.7% vol/vol, NP40 0.6% vol/vol), kept
on ice 5 minutes, centrifuged (113g, 6 minutes), and
resuspended in the propidium iodide staining solution.
For the Hoechst staining, cells were fixed for 30
minutes on ice in 75% methanol/25% PBS, centrifuged
(113g, 6 minutes), and resuspended in a Hoechst 33258
staining solution (Hoechst 33258 0.58 (xg/ml, NaCl
145 mM, Tris 100 mM). All samples for protein and
DNA measurements were kept on ice until analyzed on
a Coulter Epics V fluorescence activated cell sorter
(Hialeah, Florida). Cell clumping was less than
1% based on analysis of peak versus integrated
fluorescence.
Protein Synthesis
Cultures were treated with either All or vehicle
(control) in SFM. Beginning at time of treatment and
then every 3 hours, cultures were pulsed with
["SJmethionine (2.0 n,Ci/ml, 1,100 Ci/mmol, New
England Nuclear). Cold L-methionine (1 mM, Sigma
Chemical) was added to reduce possible pool effects on
protein determination, as recommended by Rannels et
al.28 At the end of the 3-hour interval, the culture was
washed with PBS, and the cells were enzymaticalry
harvested into tubes containing 10 n-g BSA in solution.
Trichloroacetic acid (TCA) was added to each tube for
afinalconcentration of 10%, and samples were kept on
ice for a minimum of 20 minutes. TCA-precipitable
counts were collected on Whatman GF/F filters (Maidstone, England) and counted in 5 ml Scintiverse II
(Fisher Scientific, Springfield, New Jersey) in a
Geisterfer el al
Smooth Muscle Cell Growth
751
FIGURE 1. Influence of angiotensin 11 (All) on smooth
muscle cell cultures fed either serum free medium (circles),
10% fetal bovine serum (squares), or serum free medium +
partially purified platelet-derived growth factor (2.5 nglmL,
triangles) and treated with either All (1 \xM, solid symbols)
or vehicle (open symbols). Cell counts were performed at
time zero and at 2-day intervals thereafter using an Electrozone Celloscope. Each sample was counted at least three
times, and triplicate wells were evaluated for each group at
each time point. Values are expressed as mean ± SEM, n = 3.
All-treated groups were not significantly different from
respective control (p > 0.05). Results similar to these were
obtained in four independent experiments.
I.0E5-
«I0E4-
Days following plating
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Beckman LS8100 liquid scintillation counter (Irvine,
California).
obtained from the acid-insoluble material collected at
the end of the experiment.
Protein Degradation
Protein degradation assays were performed as described by Libby and O'Brien.29 Briefly, cells were
loaded with [14C]tyrosine (0.2 ^Ci/ml, 500mCi/mmol,
New England Nuclear) in SFM for 3 days. Cultures
were then washed and incubated twice for 1-hour
intervals with SFM plus 5 mg/ml BSA to remove
short-lived proteins. Dishes were then fed with SFM
plus 1 mM tyrosine and either All, vehicle (control),
or 10% FBS. At 12 hours, the medium was removed
and replaced with fresh medium. At 24 hours, the
medium was removed, and perchloric acid-insoluble
material was collected from the dish. Medium removed
at 12 and 24 hours was treated with perchloric acid
(final concentration 0.2 M) and BSA (final concentration 2 mg/ml) and centrifuged in a table-top microfuge,
and the supernatant was counted. Acid-insoluble material from each dish was collected in 1% sodium
dodecyl sulfate in 5 mM NaOH after a 0.2 M perchloric
acid treatment and two washes with Hanks' Balanced
Salt solution.
Statistics
All results were analyzed by either Student's t test
or an analysis of variance combined with NewmanKeuls multiple range test for intergroup comparisons.
Probabilities of 0.05 or less were interpreted as being
statistically significant. Values reported in the text are
mean ± SEM.
The fraction of cell protein degraded during the time
interval was calculated by dividing the counts obtained
during a specific time interval by the total counts
obtained by summing all time intervals plus the counts
Results
Effects of Angiotensin II on Smooth Muscle Cell
Proliferation
To determine whether All would induce proliferation of rat aortic SMC, cell counts were obtained from
cultures that were maintained in SFM for 3 days to
induce quiescence and were then treated with All (1
^M) or vehicle in either 10% FBS, partially purified
platelet-derived growth factor (PDGF, 2.5 ng/ml; a gift
from R. Ross) in SFM, or SFM alone (Figure 1).
Results demonstrated that All was not mitogenic by
itself nor did it augment the growth response to serum
(p>0.05). The failure to see an enhanced growth
response in the presence of serum was unlikely due to
degradation of All because no response was seen with
Sar1 All, a more stable analogue of All (data not
shown). In addition, All did not enhance the growth
Effects of Angiotensin II on the Frequency of Cells Undergoing DNA Replication in Postconflluent and
Subconfluent
it Rat Aortic Smooth Muscle Cells as Determined by [*H]Thymidine Autoradiography
TABLE 1.
Labeled cells (%)
Day 0
Day 2
Day 4
Postconfluent cells
1.5±0.2
Serum free medium
0.2±0.1
0.6±0.1
0.4±0.1
0.4±0.1
Angiotensin II (1 /xM)
Subconfluent cells
0.7±0.4
2.3±0.1
Serum free medium
1.9 + 0.3
0.3 + 0.2
3.8 + 0.3
Angiotensin II (1 /xM)
ND
31.7±1.1*
Fetal bovine serum (10%)
Cells were pulsed with [:)H]thymidine for 2 hours (1 jiCi/ml, 6.7 Ci/mMol). [3H]Thymidine labeling indexes were
determined by randomly counting 1,000 cells per well. Triplicate wells were analyzed per group.
*Significantly greater than serum free medium control or angiotensin II groups (p<O.OS, analysis of variance,
Newman-Keuls). Values are mean + SEM.
752
Circulation Research
Vol 62, No 4, April 1988
2C
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Relative DNA Content
FIGURE 2. Cell cycle histogram obtained from postconfluent
smooth muscle cells maintained in serum free medium for 3 days
to induce quiescence and then treated with angiotensin II (1 \iM)
for 4 days. Cells were harvested using trypsin, stained with
propidium iodide, and cellular DNA measured on a Coulter
Epics V fluorescence activated cell sorter.
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response to a submaximal dose of a defined competence
factor, PDGF (/?>0.05). Consistent with cell number
data, All had no effect on the fraction of cells
undergoing DNA replication in either postconfluent or
subconfluent SMC as assessed by [3H]thymidine autoradiography (Table 1). In fact, both SFM control and
All-treated cultures had extremely low [3H]thymidine
labeling indexes throughout the treatment period.
Likewise, flow microfluorimetric analysis of cell cycle
of postconfluent cultures demonstrated that by far the
majority of cells were in Go/G, with few cells in S +
G2, and no differences were observed between the
All-treated and control groups (Figure 2).
Effects of Angiotensin II on Cellular Protein Content
Although All was not mitogenic for rat aortic SMC,
it did induce cellular hypertrophy (Figure 3). We chose
to assess cellular hypertrophy, defined as an increase
in mass or size of a cell, by flow microfluorimetric
analysis of eel lular protein content because greater than
50% of the dry mass of the cell is made up of protein
and nucleic acids and the remaining components of the
cell are largely dependent on them." The mean protein
content of All-treated cells (1 u,M All in SFM, 4 days)
was increased by approximately 20% in postconfluent
cultures as compared with the vehicle-treated control
cultures (Figure 4). Cultures in 10% FBS treated with
either All or Sar' All showed no increase in protein
content as compared with the control cultures (data not
shown). The All-induced increase in protein content in
quiescent cultures (i.e., those maintained in SFM) was
concentration-dependent with the maximal response
seen at 1 u.M in postconfluent cultures (Figure 5).
Receptor Dependence of the Angiotensin II
Hypertrophic Response
Cultures were treated daily for 4 days with equimolar concentrations of All and either Sar1,He8 All
(an All receptor antagonist) or Des-Phe8 All (a
biologically inactive All analogue). Cellular hypertrophy was then analyzed by flow microfluorimetric
analysis of cellular protein content. Sar1,He8 All
completely blocked the All-induced hypertrophy,
Relative Protein Content
FIGURE 3. Histogram of relative protein content of control
and angiotensin II-treated postconfluent cells as measured by
flow microfluorimetric analysis offluoresceinisothiocyanate—
stained methanol fixed smooth muscle cells. Cells were plated
and fed DF10 until confluent. Cultures were maintained in
serum free medium for 3 days to induce quiescence and then
) or vehicle
treated with either angiotensin II (1 \\M,
(control,
) for 4 days.
while Des-Phe8 All had no effect (Figure 6). When
used alone, neither analogue exerted any effect on
protein content. While the observation that Allinduced hypertrophy in postconfluent cultures occurred over several log orders of concentration is of
some concern, the fact that hypertrophy was blocked
by the highly specific All antagonist, Sar1,He8 All,
strongly indicates that the hypertrophic response is
receptor mediated. Likewise, the observation that
Des-Phe8 All did not effect the All-induced hypertrophy indicates that the response to All did not
involve some non-receptor-mediated effect of AIL It
is likely that the gradual slope of the concentrationdependence curve in postconfluent cultures is due to
degradation of All in these high cell density cultures
and that the true concentration of AH is less than that
calculated based on what was added.
Effects of Angiotensin II on Protein Synthesis
and Degradation
The increase in protein content observed in Alltreated cultures could reflect either an increase in
protein synthesis or a decrease in protein degradation
Control
All
FIGURE 4. Summary of mean protein content determinations
of control and angiotensin II-treated cultures as determined by
flow microfluorimetry (see Figure 3). Results are mean ± SEM
of six independent experiments. Angiotensin II-treated group is
significantly greater than control (p < 0.05).
Geisterfer et al
753
FIGURE 5. Concentration dependence of angiotensin
ll-induced hypertrophy. Quiescent postconfiuent cultures of smooth muscle cells were treated daily for 4 days
with various concentrations of angiotensin II ranging
from 0.1 nM to 10 \LM, and relative cellular protein
content was measured by flow microfluorimetry
Mean ± SEM, n = 3. Angiotensin II at concentrations of
I nM or greater are significantly different from control
fp < 0.05). Similar results to these were obtained in three
independent experiments. Note that the vertical axis is
truncated.
120-
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Smooth Muscle Cell Growth
100-
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Concentration of Angiotensin H ( M)
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or some combination of the two. To test the first
possibility, cultures of quiescent SMC were treated
with All (1 u.M) or vehicle (control) in SFM, and
relative rates of protein synthesis were determined at
3-hour intervals for a total of 75 hours following
initiation of treatment. Results indicated that protein
synthesis was significantly elevated in All-treated cells
by 6-9 hours following initial treatment (Figure 7) and
remained elevated for the 75-hour duration of the
experiment. In contrast, All-treatment had no net effect
on protein degradation rates (Figure 8). The Allinduced increase in [35S]methionine incorporation into
protein was blocked by Sar'Jle 8 All (data not shown).
Additional studies were done in which cultures were
pulsed with All for varying periods of time ranging
from 5 minutes to 24 hours and protein synthesis
assessed between 12 and 24 hours. Results demonstrated that a minimum 3-hour exposure to All was
necessary to increase the rate of protein synthesis. In
contrast to the effects of All, addition of 10% FBS to
cells in SFM both increased protein synthesis and
decreased the rate of protein degradation, although in
this case, there was also a significant mitogenic
response (Figure 1, Table 1).
Response of Subconfluent Cultures to Angiotensin II
To determine whether cell density or contact inhibition of growth might be an important component of
All-induced SMC hypertrophy, whether All could
induce cellular hypertrophy in subconfluent cells made
quiescent by maintenance in SFM was explored. All (1
u,M) induced a 38% increase in cellular protein content
in subconfluent cultures treated continuously with the
peptide for 4 days (Figure 9). All was extremely potent
in inducing hypertrophy under these conditions in that
a maximum response was obtained at a concentration
of 0.1 nM (data not shown).
An interesting observation in studies of subconfluent
SMC was that a large increase in the fraction of cells
in G2 occurred in All-treated cultures as compared with
control (Figure 10), despite an extremely low fraction
of cells in the S-phase based on either flow cytograms
(Figure 10) or [3H]thymidine autoradiography (Table
1). The mean fraction of cells in G2 in the All-treated
(1 ^M) and control cultures was 25.1 ± 0 . 6 % and
16.7% ± 1 . 1 , respectively, following
treatment.
4 days of
Discussion
In the present study, we have explored the hypothesis
that contractile agonists are involved in the mediation
of growth of cultured vascular SMC by examining the
effects of one potent contractile agonist, All, on SMC
hypertrophy and cell proliferation. Under the conditions examined, All neither was mitogenic nor potentiated the proliferative responses to FBS or PDGF. The
failure of All to stimulate proliferation of rat aortic
SMC in the present study contrasts with results of
Campbell-Boswell and Robertson,30 who found that
All increased growth rates of subconfluent human
aortic SMC in the presence of 10% FBS. The differences in the results of our study and that of CampbellBoswell and Robertson may reflect either a species
difference or differences in serum. The failure of All
to enhance growth of rat aortic SMC in 10% FBS
probably was not because the SMC were already
maximally stimulated since All also did not augment
the replication induced by submaximal doses of PDGF.
Alternately, the differences may relate to differences in
the differentiational status of the rat SMC used in the
present study as compared with the human SMC used
by Campbell-Boswell and Robertson. We have previously demonstrated that under the conditions used in
these studies, our cells express high levels of smooth
muscle-specific contractile proteins,23-27 while the
explant-derived SMC used by Campbell-Boswell and
Robertson were most likely highly modulated
and expressed little, if any, of these differentiated
proteins.31
Although All was not mitogenic for rat aortic SMC
in these studies, we found that it was extremely potent
in inducing receptor-dependent hypertrophy of both
subconfluent and postconfluent quiescent SMC. It is of
interest that the degree of hypertrophy observed in
these cultured SMC (i.e., 20% and 38% increases in
protein content in postconfluent and subconfluent cells,
respectively) is comparable to that observed in aortic
SMC in vivo in the spontaneously hypertensive rat
following development of chronic hypertension, where
the protein content of diploid aortic SMC is increased
754
Circulation Research
Vol 62, No 4, April 1988
12510075u
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All
Sar1 He 8
Sar1 H e 8
+ AII
Des Ptie8
Des Phe8
+AII
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FIGURE 6. Receptor dependence of angiotensin II (AII)-induced hypertrophy of postconfluent smooth muscle cells treated daily for
4 days with AH and equimolar concentrations of either Sar', lie'All (a specific All receptor antagonist) or Des-Phe'AlI (a biologically
inactive All analogue). Mean (protein contents) ±SEM, n=3. Only the All and Des-Phe" All + All groups are significantly different
from control (p < 0.05). Results similar to these were obtained in an independent experiment.
between 20% and 50%. 6 An extremely interesting
observation in subconfluent cells was that All induced
a 50% increase in the fraction of cells in G2 without a
detectable increase in the fraction of cells in S-phase
of the cell cycle. Whether this represents induction of
true tetraploidy (i.e., doubling of chromosome number
in quiescent cells) as has been shown to occur in vivo
in conjunction with SMC hypertrophy6 or whether it
represents arrest of cells in the G2 phase of the cell cycle
will require extensive further investigation. Nevertheless, results indicate that the SMC hypertrophy induced
in vitro resembles hypertrophy that occurs in vivo in
many respects and that this model will be very useful
in exploring the underlying mechanisms involved in
SMC hypertrophy. Indeed, these studies lend support
for a role of All in the in vivo hypertrophic response,
as initially suggested by our drug intervention studies.9
However, we caution that these studies provide no
direct evidence as to the factors that mediate SMC
hypertrophy in vivo in models such as the spontaneously hypertensive rat.
The mechanism whereby All induces SMC hypertrophy is not known. All may increase protein synthesis
by stimulation of ribosomal protein S6 phosphorylation, which is believed to play a role in control of
protein synthesis. This could occur through AII-
stimulated Na + -H + exchange,l6-17 which has been
implicated in control of S6 phosphorylation.21-32 Alternatively, All may have a direct effect on ribosomal
genes. Robertson and Khairallah33 have shown that
All, or a rapidly formed degradation product, is found
in the perinuclear space in coronary smooth muscles
and cardiac myocytes within 45 seconds after injection
of All into the vena cava. Also, Re et al34 have shown
that All binds with high affinity to nuclear membranes.
Another possibility is that All could increase protein
synthesis via increased intracellular Ca 2+ , which has
been implicated in skeletal muscle hypertrophy.35 One
other possibility that must be considered is that the
increased protein synthesis may be induced by mechanical forces, although it is unclear whether All
causes active stress development in these cells and how
this might result in increased protein synthesis.
It is interesting to note that contractile agonists and
growth factors have many properties in common. Both
types of agents have been reported to mobilize intracellular calcium,'1"14 increase inositol trisphosphate
turnover,1516 activate the sodium-proton antiport,17"21
and have overlapping protein phosphorylation
profiles.22 Also, PDGF and other growth factors cause
contraction of aortic rings.23-24 Thus, the response of a
cell to a contractile agonist involves similar intracel-
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6
9
12
15
18 21
24
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Time at end of 3 hr pulse (hrs.)
FIGURE 7. Influence of angiotensin II on protein synthesis
rates. Cells were plated and grown to confluency in DF10
and then switched to serum free medium for 3 days to induce
quiescence. At time zero, all cultures were refed with serum
free medium and treated with either vehicle or AH (1 \iM).
Every 3 hours, cultures were pulsed with {"Sjmethionine
(2.0 \i.Cilml), and trichloroacetic acid-precipitable counts
determined for each interval. Values are mean ± SEM, n = J.
Protein synthesis values after 6 hours are significantly
differentfrom control fp < 0.05). Results similar to these were
obtained in five independent experiments. Note that the
vertical axis is truncated.
Geisterfer et al
Smooth Muscle Cell Growth
755
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FIGURE 8. Influence of angiotensin II on the rate of protein
degradation. Cells were plated and fed DF10 until confluency.
Cells were then maintained in serum free medium for 3 days to
induce quiescence. Cultures were prelabeled with ["CJtyrosine
in serum free medium for 2 days, washed with serum free
medium, and then treated with either angiotensin II (1 \iM,
) , vehicle (control,
) , or 10% fetal bovine serum
(—•—). Percentage of total protein degraded was calculated by
dividing trichloroacetic acid-soluble counts released into media at 0-12 hours or 12-24 hours by total of trichloroacetic
acid-soluble counts and trichloroacetic acid-insoluble counts
(collected from dish at 24 hours). Values are mean ± SEM,
n=3. At both 12and24hours, the 10% fetal bovine serum group
is significantly different from control (p < 0.05). Results similar
to these were obtained in one other independent experiment.
Note that the vertical axis is truncated.
lular signals as growth factors but does not stimulate
cell division. In light of the current concept of cellular
growth control as requiring multiple signals (or factors)
in a finite temporal sequence (see reviews by Baserga36
and Rozengurt37), it is interesting to speculate that All
might induce cellular hypertrophy and hyperploidy by
acting as a partial growth factor, activating only a
subset of the intracellular messengers necessary for
complete cell replication. For example, All stimulation might increase cellular size and protein content as
is known to occur during normal cell cycle
progression36 and, in very limited cases, DNA replication but not those signals requisite for cytokinesis.
This hypothesis obviously remains to be tested but is
consistent with the suggestion by Baserga38 that there
may be separate growth factors for cell hypertrophy
versus DNA replication. We should note that the
accumulation of significant numbers of cells with
tetraploid DNA content could occur with very small
increases in the fraction of cells undergoing DNA
replication if it occurred over a relatively long time
period. This might explain why we failed to see a
statistically significant increase in the fraction of cells
in S-phase using a 2-hour [3H]thymidine pulse (see
Table 1) but did see an increase in the fraction of cells
with tetraploid DNA with chronic All treatment. One
must also consider the alternative possibilities that the
accumulation of cells with 4C DNA occurs as a result
All
FIGURE 9. Influence of angiotensin II (All) on subconfluent
quiescent cultures of smooth muscle cells. Cells were plated in
DF10 at 3X103 cells/cm2. Medium was changed to serum free
medium 24—48 hours later. After 3-5 days, cultures were treated
with All for 4 days. Protein content was determined as shown
in Figure 3. Results are mean ± SEM of three independent
experiments. The All-treated group is significantly greater than
control (p<0.05).
of cell fusion39 or by selective replication of a preexisting tetraploid population.
In summary, results of the present study demonstrate
that All is a potent hypertrophic agent for cultured rat
aortic SMC. Findings are consistent with the hypothesis that contractile agonists may modulate SMC
hypertrophy and thereby provide a mechanism for
SMC to regulate its mass in response to its work load.
Further studies are required to determine the cellular
mechanisms responsible for All-induced hypertrophy,
whether the hypertrophy observed with All is also a
property of other contractile agonists, and whether
such mechanisms are important in mediation of smooth
muscle hypertrophy in vivo.
Acknowledgments
The authors thank Chris Eichman for technical
assistance in the performance of flow cytometric
analysis, Leslie Cox and the Vascular Smooth Muscle
Program Project (HL-19242) Cell Culture Core Laboratory for assistance with the various cell culture
aspects of these studies, and Dr. Russell Ross, University of Washington School of Medicine, for his
generous gift of platelet-derived growth factor.
= JOOO-
1000Rtlotiv* ONA Conttnt
FIGURE 10. Cell cycle histogram obtained from subconfluent
cultures using Hoechst 33258 stain. Staining cultures were
maintained in serum free medium for 3 days and then treated
with either angiotensin II (1 \iM,
) or vehicle (control,
) for 4 days.
756
Circulation Research
Vol 62, No 4, April 1988
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KEY WORDS • vascular smooth muscle cells • cellular
hypertrophy • protein content • angiotensin II • tetraploidy
Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle
cells.
A A Geisterfer, M J Peach and G K Owens
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Circ Res. 1988;62:749-756
doi: 10.1161/01.RES.62.4.749
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