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Tutorial in Molecular and Cellular Biology
Pharmacology of Smooth Muscle
Cell Replication
Christopher L. Jackson and Stephen M. Schwartz
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The suggestion that smooth muscle cell proliferation contributes to hypertension, atherosclerosis, and
restenosis after angioplasty has led to a growing interest in the use of drugs to inhibit this process. This
review summarizes pharmacological studies of smooth muscle cell proliferation in vitro and in vivo and
identifies specific mediators of proliferation that are implicated by drugs binding with high affinity to
enzymes or receptors. (Hypertension 1992^0:713-736)
KEY WORDS • atherosclerosis • growth substances • hypertension, essential • muscle, smooth,
vascular
T
his review emphasizes agents that influence
smooth muscle proliferation. Four overlapping
categories of agents that could be discussed are
growth factors, drugs with known pharmacological effects, endocrine hormones with growth regulatory properties, and intracellular mediators of replication. This
review will concentrate on the last three categories. A
great deal has already been written in other reviews
about the growth factors, but the much longer list of
possible targets discussed here has not been reviewed
systematically. Moreover, a great deal is known about
the pharmacology of some of the molecules in our
review, so their therapeutic value in the treatment of
conditions arising from smooth muscle replication is
likely to be realized more quickly in a clinical sense than
treatments based on the still very poorly understood
pharmacology of growth factors.
Pathology of Smooth Muscle Proliferation
Smooth muscle proliferation has been implicated as
playing a central role in atherosclerosis and hypertension.1 The evidence that smooth muscle cell proliferation occurs in these diseases is clear. As we will briefly
review, however, evidence for a causal connection is not
well established.
Smooth Muscle Replication in Hypertension
The argument for a role of smooth muscle cell
replication in hypertension is an outgrowth of the
structural hypothesis of hypertension proposed by
Folkow.2 Folkow explained that the ability of a resistance artery to restrict blood flow depends on the
thickness of the vessel wall as well as on the size of the
vessel lumen, the responsiveness of the wall to vasoactive stimuli, and the level of stimulation by vasoconstrictors. Folkow's idea is based on the simple physical fact
that a thicker vessel wall will act as an amplifier, so that
From the Department of Pathology, University of Washington,
School of Medicine, Seattle, Wash.
Address for correspondence: Christopher L. Jackson, Department of Cardiovascular Biology, Pfizer Central Research, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK.
an equal vasoconstrictor stimulus will produce greater
narrowing of the lumen in a hypotensive vessel than in
a normotensive vessel. Studies of essential and secondary hypertension have confirmed that hypertensive vessel walls are thickened.3 Indeed, there is evidence that
such structural changes precede any elevation in blood
pressure,*-5 and, at least for the spontaneously hypertensive rat, wall thickening may depend on the increased sympathetic innervation that develops in the
spontaneously hypertensive rat vessel wall within the
first month of life.5-6 A correlation exists between the
development of sympathetic innervation and the development of arterial medial hypertrophy in normotensive
and spontaneously hypertensive rats.6 Of course, wall
thickening need not be correlated with smooth muscle
replication. There is one report, however, that DNA
synthesis precedes secondary hypertension,7 and most
studies of hypertrophied vessel walls of hypertensive
animals also show an increase in DNA content either as
an increase in cell number (hyperplasia) or in DNA
content of each cell (hyperploidy).8-9
In summary, increase in smooth muscle mass is well
established as a feature of hypertension, and the change
in mass, at least in some cases, appears to be related to
DNA synthesis. This DNA synthesis could contribute to
the chronic nature of hypertension, because DNA, once
formed, is a stable molecule that only decays after cell
death. Because the increased DNA should provide an
increased template for protein synthesis, hyperplasia or
hyperploidy might be expected to maintain increased
mass. Unfortunately, we lack evidence that increased
mass can itself be a primary event in hypertension or
that therapies directed at either mass change or replication will have any effect on blood pressure. Perhaps
more convincing evidence will become available when
we have more specific agents able to control smooth
muscle cell hypertrophy or replication.
Smooth Muscle Replication in Atherosclerosis
The role of the smooth muscle cell in atherosclerosis
can be summarized briefly: Atherosclerotic lesions occur in the intima at sites of smooth muscle proliferation.
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The smooth muscle cells contribute to the lesion by
synthesis of cell mass and extracellular matrix. Lipid
accumulates within the smooth muscle cells, in the
extracellular matrix elaborated by the smooth muscle
cells, and in macrophages that accumulate in the
plaque. Debate continues as to whether smooth muscle
proliferation is the first event in lesion formation,110 but
there is no doubt that all occlusive lesions depend on
smooth muscle cells for much of their mass. Indeed, in
sudden cardiac death, typical lesions consist largely of
smooth muscle cells and their extracellular matrix.1112
As with hypertension, the coincidence of pathology is
not clear evidence that an antiproliferative strategy will
be effective in treating atherosclerosis. Recent cell
kinetic studies show that the level of replication in the
normal human plaque is very low, perhaps one or two
divisions per year.13 It is possible that most of the
replication is an early event, occurring during vascular
development and therefore not of interest therapeutically. Alternatively, replication might occur episodically,
perhaps as a result of inflammatory events.14 Knowing
when such events might occur seems unlikely, so chronic
therapy may be necessary to ensure suppression of
replication at the critical times.
Smooth Muscle Replication After Angioplasty:
Three-Wave Model
Like atherosclerosis itself, the lesion formed after
angioplasty is a result of aberrant smooth muscle accumulation in the intima. Angioplasty is an important
topic for this review for three reasons. First, in experimental models, the response to angioplasty occurs
rapidly enough to allow various agonists or antagonists
to be readily studied. Second, the high incidence of
restenosis after angioplasty in humans makes this likely
to be the first target for clinical intervention with
inhibitors of smooth muscle proliferation. Third, animal
studies of arterial injury have begun to provide data on
the role of growth factors in the control of smooth
muscle cell proliferation in vivo.
The animal model of arterial injury that bears the
closest resemblance to angioplasty is balloon catheter
de-endothelialization. The most thoroughly investigated
system is balloon catheter injury to the rat common
carotid artery. The first phase of the response is a
dramatic increase in the replication rate of smooth
muscle cells in the media injured by the balloon dilatation. This response is massive, involving as many as 50%
of the smooth muscle cells in the wall, and probably
represents replacement of cells that die in the media as
a consequence of the injury.15-21 This repair response
does not directly contribute to the accumulation of cells
in the intima, but it is possible that this "first wave" is
necessary to provide cells that ultimately form the
intimal lesion.
The first wave is completed within 4 days. In the rat,
however, there are normally no intimal smooth muscle
cells, so formation of the neointima requires migration
of cells from the media into the intima, the "second
wave" of lesion formation. Smooth muscle cells migrate
and can be seen forming a neointima by 3-4 days after
injury.1-21 Although a discussion of potential pharmacological inhibition of smooth muscle cell migration is
beyond the scope of this review, it is possible that this
may be a legitimate target for limiting restenosis after
angioplasty. The utility of this approach clearly depends
on the contribution of migration to the accumulation of
smooth muscle cells in the human arterial intima, an
issue that is as yet unresolved. Once these cells are in
the intima, approximately 50% of the cells go on to
replicate, forming the lesion. This "third wave" involves
a much lower dairy replication rate, as low as 1-5%.
However, the response may continue for weeks or
months and presumably accounts for most of the accumulation of cells in the intima.
In any attempt to design a therapeutic regimen, it is
important to consider the probability of affecting each of
the three waves and the role that they may play in the
restenotic process. For example, it is unlikely that the first
wave itself contributes to intimal thickening. Indeed, by
inhibiting repair of the injured media, one might even
increase the possibility that the "healed" vessel will have a
narrowed lumen. On the other hand, without an adequate
first wave, too few cells may be available to migrate, and
neointimal formation may be retarded. Agents that affect
the second wave (migration) or third wave (neointimal
proliferation) would seem much more certain to have
beneficial effects. However, we also need to be aware of a
lack of data relating intimal mass to lumen size. In a recent
study of the effects of hypertension on atherosclerotic
narrowing, hypertension greatly accelerated formation of
the neointima but had no effect on narrowing of the
lumen.22
In summary, the issues in restenosis are similar to
those in hypertension. We know very little about why
the lumen becomes narrow, although we know that the
adverse effects of the lesion depend on the narrowing.
Growth Factors
Growth Factor Assays
As already mentioned, this review will not concentrate on the usual polypeptide growth factors, because
these have been comprehensively reviewed elsewhere.
Nonetheless, a brief overview is useful for completeness
and because some of the agonists and antagonists
discussed below may act by influencing growth factor
activity.
It is important to understand how growth factors are
usually defined. In theory, the assay system is quite
simple: Cultured cells are arrested by being placed in
medium deficient in growth factors. The suspected
agonist is added, and growth is determined by measuring the rate of increase in cell number or by measuring
incorporation of DNA-specific nucleotides such as thymidine or bromodeoxyuridine. However, this assay has
several problems.
1. Definition of "growth factor-deficient medium."
Cells in vivo exist in an extracellular medium very
similar to lymph. Smooth muscle cells, however, grow
quite well in lymph (Schwartz et al, unpublished data).
Quiescence is usually achieved by use of an artificial
medium such as a fully synthetic "serum-free" medium
able to sustain nongrowing cells by simply allowing the
cells to become quiescent by depleting undefined factors
in the medium. Growth factors detected under these
conditions may include molecules required for growth in
vivo but not able to stimulate growth themselves.
2. Derivation of cells used for assays. Cells must be
grown to do these assays, but cells in vivo replicate
Jackson and Schwartz Pharmacology of Smooth Muscle Replication
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rarely. Thus, the factors detected by an assay may reflect
selection of growth factor-dependent cells during the
establishment of cells in culture. For example, smooth
muscle cells obtained from rats and subcultured in
plasma-derived serum, obtained without platelet release, do not require platelet-derived growth factor
(PDGF) for growth in vitro, whereas cells initially
obtained and passaged in whole blood serum do require
PDGF. 23
3. Effects of potent agonists other than growth factors.
Because quiescence as denned in vitro is an arbitrary
and artificial state, we should not be surprised if a
number of molecules with functions other than the
initiation of growth can facilitate replication in vitro.
Referring back to the three waves discussed above, it
would not be surprising if a large number of metabolically active hormones would alter the rate of cell growth
in the third wave, i.e., when cells are already replicating.
Similarly, when in vitro assays are considered, it may be
important to distinguish factors that determine rates of
cell growth from factors that can initiate growth from
quiescence.
4. In vivo versus in vitro cell kinetics. The issue of
replication rate is very confusing when we attempt to
compare in vivo with in vitro phenomena. In vitro,
"quiescence" usually implies a daily cell replication rate
of 5% or even 10%. This compares with normal quiescent states for vascular smooth muscle of as little as
0.01%.' Indeed, replicative responses of smooth muscle
to hypertension or cholesterol feeding in vivo are usually less than the dairy replication rate seen in quiescent
cultured cells.24-26 In practice, this means that mitogenic agents with no apparent proliferative effect in vitro
may be active in vivo at levels not measurable in the
usual assays. Therefore, it would not be surprising to
discover circumstances in which a specific inhibitor
lowers replication rates in "quiescent" cultures, even
though the addition of agonists at levels beyond those
found in the basal medium has no further effect on
replication. Indeed, Emmert and Harris-Hooker27 reported just such an inhibitory effect for saralasin under
conditions in which angiotensin II did not appear to be
mitogenic.
Effects of Growth Factors in the Three-Wave Model
Recently, the availability of large amounts of growth
factors, usually as expression products of recombinant
DNA, has led to initial understanding of the possible
roles of several growth factors in the three waves of the
in vivo angioplasty response described above in the rat
carotid artery injury model. We will briefly summarize
the results.
When vessels are gently denuded of endothelium, the
first wave is greatly decreased. This is consistent with
the view expressed above that the first wave is a repair
process dependent on injury to medial smooth muscle.
However, when different growth factors were infused at
the time of gentle injury, only basic fibroblast growth
factor (bFGF) had a mitogenic effect comparable to the
usual response to balloon injury. Transforming growth
factor-^ and PDGF BB were markedly less effective.
Moreover, antibodies to bFGF greatly decreased the
response seen after balloon injury. These data, combined with evidence that bFGF is present in the uninjured vessel wall, led Lindner et al28 to propose that
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bFGF acts as a wound hormone to control the first
wave.
Similar studies by Clowes et al29 and Ross et al,30
using infused PDGF and antibodies to PDGF, imply
that its major role is stimulation of migration, i.e., the
second wave. These observations help to resolve the
paradoxical observation that antiplatelet antisera have
no effect in abolishing first wave replication but retard
the formation of the intimal lesion.16
Much less is known about controls in the third wave.
Growth factor infusion studies have suggested that
transforming growth factor-/3 and angiotensin II may be
active at this stage.24-31
In this review, we summarize current knowledge
concerning drugs that influence smooth muscle cell
proliferation. Many of these agents interact with high
affinity with receptors or enzymes and therefore implicate specific molecules in the positive or negative mediation of proliferation. With this in mind, we have
grouped drugs in terms of their interactions with potential mediators. For each of these mediators, the following questions are considered: 1) Provenance. What are
the possible in vivo sources of the mediator? Is it
present in adequate concentration? Do vascular smooth
muscle cells bear receptors that interact with it?
2) Effects of exogenous administration. Does exogenous
administration of the proposed mediator by an appropriate route significantly influence proliferation? 3) Use
of antagonists. Does administration of a suitable antagonist to the proposed mediator influence smooth muscle
cell proliferation in the predicted way?
This review is therefore limited to a discussion of
those potential mediators of smooth muscle cell proliferation that have an associated pharmacology. The
second part of the review considers drugs that influence
arterial smooth muscle cell proliferation but that do not
implicate specific mediators.
Endogenous Agents That May Control Vascular
Smooth Muscle Cell Proliferation
Adenosine
Adenosine is a nucleoside formed from 5'-adenosine
monophosphate (AMP) by the action of 5'-nucleotidase.32 The concentration of adenosine in canine arterial
blood plasma is 0.26 fiM but rises substantially in some
vascular beds after nerve stimulation.33 Cultured arterial smooth muscle cells possess both the Al and A2
adenosine receptor subtypes.34-33 Occupation of the Al
receptor by adenosine inhibits adenylate cyclase activity
and thus reduces the intracellular concentration of
cyclic AMP, whereas occupation of the A2 receptor has
the opposite effect.35 Jonzon et al36 correlated these
effects of adenosine receptor stimulation with changes
in the rate of DNA synthesis in rat aortic smooth muscle
cells. Exposure of quiescent cells to human platelet
PDGF resulted in an increase in the pHJthymidine
labeling index, which was significantly inhibited by
adenosine at concentrations greater than 10 /tM. Inhibition of PDGF-stimulated DNA synthesis was also
observed with the adenosine analogues 5'-W-ethylcarboxamidoadenosine and L-phenylisopropyladenosine.
The latter was approximately equipotent with adenosine, which had 10% of the potency of 5'-Af-ethylcarboxamidoadenosine. This adenosine analogue has
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Vol 20, No 6 December 1992
greater affinity for A2 receptors, suggesting that these
may play a role in the control of vascular smooth muscle
cell proliferation.
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Adrenocorticosteroid Hormones
The biosynthesis and release of adrenocorticosteroid
hormones from the adrenal cortex is controlled by
corticotropin. The release of this hormone from the
anterior pituitary gland is stimulated by corticotropin
releasing factor, which is present in high concentration
in neurosecretory cells in the paraventricular nucleus of
the hypothalamus. The predominantly secreted adrenocorticosteroid hormone in humans is cortisol, or hydrocortisone, which is present in plasma at a total concentration of 0.14-0.55 ^tM under normal conditions.37
Jarvelainen et al38 isolated smooth muscle cells from
human fetal aorta and grew them in secondary culture.
Addition of hydrocortisone at concentrations of 1 pM
and greater significantly inhibited fHJthymidine incorporation into DNA, and DNA content was significantly
reduced at concentrations of 100 nM and greater. Berk
et al39 found similar results using rat thoracic aortic
smooth muscle cells growing in secondary culture. Addition of hydrocortisone (2 fiM) significantly inhibited
cell replication and the incorporation of pH]thymidine
into DNA. Inhibition of replication was also seen with
the synthetic glucocorticoid dexamethasone (1 JAM) but
not with the sex hormones testosterone (2 piM) or
progesterone (2 fiM). Inhibition of cell growth by
hydrocortisone was associated with a change in cell
morphology, with the cells appearing larger, flatter, and
more polygonal. The authors suggest that this morphological change could cause density-dependent inhibition
of cell growth at lower cell densities, which is consistent
with the growth profile of hydrocortisone-treated cells.
The growth rate during the logarithmic phase was equal
to that in control cultures, but the plateau phase was
achieved at a lower cell density.
Longenecker et al 4041 also examined the effects of
glucocorticoids on smooth muscle cell growth in vitro.
Using bovine aortic smooth muscle cells, they found
significant inhibition by dexamethasone at a concentration of 1 nM. This inhibition was counteracted by
growing the cells on dishes coated with extracellular
matrix; matrix elements can bind many polypeptide
growth factors, and these may have overcome the inhibition by dexamethasone. Hydrocortisone (0.1 fiM) and
corticosterone (0.1 jiM) also inhibited growth. Interestingly, the growth of primary cultures of bovine aortic
smooth muscle cells was markedly more sensitive to
dexamethasone than the growth of secondary cultures.
In their 1984 study, Longenecker et al41 found that
dexamethasone reduced both the growth rate and the
final saturation density of smooth muscle cells, which
contradicts the lack of effect on growth rate found by
Berk et al.39 However, the earlier report from Longenecker et al40 showed a significant effect only on saturation density. Hauss et al42 examined the inhibitory
effects of the synthetic glucocorticoid prednisolone on
the proliferation of rat and porcine thoracic aortic
smooth muscle cells in secondary culture. Significant
inhibition of proliferation of porcine cells was observed
at a concentration of 56 /iM. Parenteral administration
of prednisolone in rats by an unspecified route for 4
days (2 mg per rat per day) produced a slight inhibition
of the growth of smooth muscle cells subsequently
isolated from the aortas of these animals. The development of neointimal hyperplasia in the ear artery of
rabbits in response to a crushing injury was significantly
inhibited by intravenous treatment with prednisolone
(0.2 mg per rabbit per day).43
Dexamethasone, at concentrations as low as 10 nM,
was found by Hirosumi et al44 to inhibit the proliferation
of rat thoracic smooth muscle cells growing in secondary
culture. Inhibitory effects on smooth muscle cell migration were noted at concentrations greater than 100 pM.
This study also investigated the effect of intramuscular
administration of dexamethasone on the development
of the neointima in response to investment of the rabbit
carotid artery with a polythene cuff for 21 days. Intimal
thickening was dose-dependently inhibited by dexamethasone, the lowest effective dose being 1 mg/kg/day.
Presumably, this effect was the result of inhibition both
of smooth muscle cell proliferation and of smooth
muscle cell migration to the intima. Dexamethasone
(0.05 mg/kg of body weight per day) has also been
shown to inhibit intimal thickening in the ballooninjured rabbit common carotid artery45'46 and at
0.2 mg/kg of body weight per day in a rat vasculitis
model.47 In this model, a thread soaked in bacterial
lipopofysaccharide is laid along the adventitial surface
of the femoral artery. Leukocytes adhere to the endothelium in the vicinity of the thread, and a neointima
develops that is composed primarily of smooth muscle
cells. Dexamethasone inhibits the accumulation of leukocytes in this system; it is possible that the effects of
glucocorticoids in other inflammatory models of arterial
injury, such as periarterial cuffing44 or arterial crushing,43 are related to inhibition of leukocyte function.
The main action of the glucocorticoids is induction of
lipocortin, a protein that inhibits phospholipase A2.48
This enzyme is responsible for the liberation of arachidonic acid from cellular phospholipids, and its inhibition therefore reduces eicosanoid biosynthesis. The
inhibitory effects of glucocorticoids on arterial smooth
muscle cell proliferation thus may be the consequence
of reduced availability of growth-stimulatory eicosanoids. The effects of eicosanoids on arterial smooth
muscle cell growth are reviewed below.
If glucocorticoids were an important growth-inhibitory influence on smooth muscle cells in the arterial
wall, one might expect that hypophysectomy would
cause an increase in smooth muscle cell proliferation in
response to vascular injury, because removal of the
pituitary reduces circulating levels of hydrocortisone by
approximately 90%.49 In fact, hypophysectomy significantly inhibits the development of intimal thickening in
the balloon-catheterized rat aorta.49-50 Bettmann et al50
also found that hypophysectomy accelerated the regrowth rate of endothelium in balloon-denuded rat
aorta and suggested that the inhibition of intimal thickening in hypophysectomized rats could be the consequence of suppression by the endothelium. However,
Tiell et al49 did not observe any difference in endothelial
regrowth between control and hypophysectomized animals, suggesting that the inhibition of neointimal development was not the indirect consequence of rapid
recoverage by endothelium. This leads to the conclusion
that the pituitary is necessary for the elaboration of an
essential cofactor for smooth muscle cell proliferation.
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Bettmann et a! were unable to reverse the inhibitory
effect of hypophysectomy by coadministering thyroxin,
hydrocortisone, deoxycorticosterone acetate, and
growth hormone. However, the pituitary also produces
prolactin, follicle-stimulating hormone, luteinizing hormone, thyrotropin, corticotropin, lipotropin, endorphins, enkephalins, and melanocyte-stimulating hormone.51 These hormones themselves modulate the
synthesis, secretion, and activity of a wide range of
hormones and neurotransmitters in the body. Dissection of the pathways involved in hypophysectomyinduced suppression of smooth muscle cell proliferation
will be a formidable task, but determination of the
mitogenic activity for smooth muscle cells of plasmaderived serum prepared from normal and hypophysectomized animals may provide some useful clues. This
approach was used by Stiles et al,52 who determined the
effects on the growth of the BALB/c 3T3 murine
fibroblast cell line of plasma prepared from hypophysectomized rats. This material had 5% of the potency of
normal rat plasma in promoting DNA synthesis in cells
briefly exposed to PDGF. The activity of hypophysectomized rat plasma was restored to normal by addition
of somatomedin C.
Angiotensin
Angiotensin II is a vasoactive octapeptide formed
from its inactive decapeptide precursor, angiotensin I,
by the action of a dipeptidyl carboxypeptidase known as
angiotensin converting enzyme (ACE). Angiotensin I is
derived from the cleavage of angiotensinogen by renin.
A reasonable case may be made for the presence in
vascular tissue of all of the components required for the
synthesis of angiotensin II. Canine aortic smooth muscle
cells have been shown to synthesize renin in vitro,53 and
mRNA has been detected in rat periaortic tissues for
both renin54 and angiotensinogen.53-57 Rat aorta has
been shown to be capable of generating angiotensin II
from angiotensin I, suggesting that this vessel contains
ACE activity.58 These authors suggested that this property is present in both endothelium and vascular smooth
muscle, because conversion of angiotensin I occurred in
aortic rings even after the removal of the endothelium
by rubbing. Similar results were obtained in rabbit aorta
by Saye et al.59 An alternative explanation is that the
endothelium of rat and rabbit aorta does not exhibit
ACE activity, but these results certainly indicate that
ACE activity is present in the arterial wall.
In human smooth muscle cells derived from neonatal
and juvenile aortas, growing in secondary culture in
10% fetal calf serum, exogenous angiotensin II significantly stimulates cell growth at concentrations of
100 nM and greater.60 However, this is insufficient
evidence to conclude that angiotensin II is a smooth
muscle cell mitogen. This study did not exclude the
possibilities that angiotensin II was degraded in the
culture medium to form uncharacterized mitogenic peptides or that it potentiated another mitogen present in
fetal calf serum. Also, the mitogenic concentration of
angiotensin II was at least 3,000 times higher than the
concentration in human plasma.61
These objections were answered in part by Lyall et
al62*3 in a study examining the effects of angiotensin II
on the proliferation of rat mesenteric arterial smooth
muscle cells growing in secondary culture in 10% fetal
717
calf serum. Concentrations as low as 1 nM stimulated an
increase in cell number, but this is still an order of
magnitude higher than the human plasma concentration. Stimulation of cell replication was blocked by the
angiotensin II structural analogue saralasin, which competes for the angiotensin II receptor. This suggests both
that proteoh/tic degradation of angiotensin II is not
involved in its mitogenic effect and that angiotensin II
acts directly on the smooth muscle cell, rather than
interacting with a serum component. The involvement
of angiotensin II in the growth of arterial smooth muscle
cells in vitro was highlighted by Emmett and HarrisHooker.27 They found that saralasin significantly, dosedependently, and reversibly inhibited smooth muscle
cell proliferation in the presence of fetal calf serum.
Geisterfer et al64 were unable to detect any hyperplastic effect of angiotensin II at a concentration of 1 jtM,
using rat thoracic aortic smooth muscle cells growing in
secondary culture either in 10% fetal calf serum or in a
defined serum-free medium containing partially purified
PDGF. However, at concentrations of 1 nM and greater,
significant increases in the mean cellular protein content
were observed, indicating hypertrophy. The hypertrophic
effect was blocked by saralasin. The dose-response curve
for the hypertrophic effect of angiotensin II was extremely flat, spanning five orders of magnitude between
the lowest effective concentration and the maximally
effective concentration. This may have been the consequence of breakdown of angiotensin II, which was added
in fresh culture medium every 2-3 days; Lyall et al62-63
showed by radioimmunoassay that incubation for 3.5
hours in medium containing 10% fetal calf serum degraded 99.9% of exogenous angiotensin II.
Berk et al65 also found no effect of angiotensin II on
the replication of rat thoracic aortic smooth muscle cells
growing in secondary culture, although DNA synthesis
increased significantly at concentrations of 100 nM and
greater. This suggests that polyploid cells were being
formed. Hypertrophic effects were observed at angiotensin II concentrations as low as 100 pM.
It is interesting to note that the effects of angiotensin
II on smooth muscle cell proliferation seem to be
related to exposure of the cells to serum. Angiotensin II
appears to cause hyperplasia in smooth muscle cells
maintained in growth-supporting concentrations of serum throughout the experiment60-62-6366 In contrast, in
studies in which cells are rendered quiescent by incubation in medium containing little or no serum, hypertrophic effects are observed when angiotensin II is
administered at the same time as reintroduction of
serum.6465 Hyperplastic effects of angiotensin II have
not been observed under these conditions.64-67 A possible exception is the study by Hamada et al68 in which
rat thoracic aortic smooth muscle cells in secondary
culture were incubated in serum-free medium for 1 day
before exposure to angiotensin II. The incorporation of
pHJthymidine into DNA was significantly stimulated at
concentrations of 1 nM and greater. However, cell
numbers were not determined, so it is not clear whether
angiotensin II stimulated cellular replication or simply
caused the development of polyploidy. CampbellBoswell and Robertson60 examined the effects of angiotensin II in smooth muscle cells cultured in 1% fetal calf
serum for 2 days before introduction of the experimental medium. Under these conditions, the stimulation of
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cell replication by angiotensin II was approximately
75% of that found in cells maintained in 10% fetal calf
serum throughout and exposed to the same concentration of angiotensin II. However, induction of quiescence
by culruring the cells in 5% platelet-poor plasmaderived serum for 2 days did not inhibit the subsequent
mitogenic effect of angiotensin II. Similar findings were
reported by Scott-Burden et al,66 who were able to
detect a very weak mitogenic effect of 50 nM angiotensin II in rat vascular smooth muscle cells growing in 1%
plasma-derived serum. This suggests that the hyperplastic response of smooth muscle cells is dependent on
continuous exposure to serum and that, in the presence
of low concentrations of serum, the mitogenic response
gradually disappears. It is clear that cellular quiescence
is not an issue, because quiescence induced by plateletpoor plasma-derived serum did not inhibit the mitogenic response to angiotensin II. It therefore appears that a
non-platelet-derived factor or factors in serum is critical
for the mitogenic response and that withdrawal of the
factor for 2 or more days inhibits the response. Whether
this factor operates by inducing a state of smooth
muscle cell growth in which the cells are particularly
sensitive to the mitogenic effects of angiotensin II or,
alternatively, by inducing an intracellular signal necessary for the angiotensin II mitogenic signal to be fully
transduced is open to question.
It is not clear what the cofactor for angiotensin
II-induced mitogenesis might be, Campbell-Boswell
and Robertson60 showed that removal of cationic substances from platelet-poor plasma-derived serum with
carboxymethyl Sephadex abolished its ability to support
the mitogenic effect of angiotensin II. During this
procedure, the serum was also concentrated by ultrafiltration through a filter with a 10 kd cutoff. Therefore,
the mitogenic cofactor is bound by carboxymethyl Sephadex or has a molecular weight less than 10 kd.
The mitogenic activity of angiotensin II under appropriate experimental conditions in vitro suggests that
smooth muscle cells may respond similarly in vivo. This
possibility was addressed in a study by Daemen et al24 in
which angiotensin II was administered to rats by continuous subcutaneous infusion. The labeling index of
smooth muscle cells in the carotid arterial media increased significantly both in unmanipulated vessels and
in those subjected to balloon catheter injury. In the
latter vessels, in which a neointima had been allowed to
develop, angiotensin II infusion significantly increased
the intimal smooth muscle cell labeling index also.
However, it is not clear from this study whether there
was an induction of smooth muscle cell proliferation by
angiotensin II infusion, because the data do not rule out
the possibility of an increase in cell ploidy.
The relative lack of potency of angiotensin I as a
vasoconstrictive hormone has led to the development of
synthetic ACE inhibitors as treatments for hypertension. Recently, interest has begun to focus on the use of
ACE inhibitors to restrict the development of pathological changes in arteries. Kuriyama et al69 found that the
ACE inhibitor captopril had no effect on the growth of
the A7r5 embryonic rat thoracic aortic smooth muscle
cell line in 10% fetal calf serum. This may reflect a lack
of conversion of angiotensin I to angiotensin II under
normal culture conditions. It may alternatively be the
consequence of a restricted supply of angiotensin I,
because Andre et al70 have shown that rat aortic smooth
muscle cells are able to convert exogenous angiotensin I
to angiotensin II.
The use of ACE inhibitors in studies of smooth
muscle cell replication in vivo is associated with some
serious difficulties of interpretation. In addition to
catalysis of the conversion of angiotensin I to angiotensin II, ACE also cleaves a dipeptide from bradykinin
and thus inactivates bradykinin. ACE inhibitors therefore reduce circulating levels of angiotensin II and
increase circulating levels of bradykinin.71 Bradykinin is
itself mitogenic for smooth muscle cells in vitro.72
Because angiotensin II causes release of aldosterone
from the adrenal zona glomerulosa, ACE inhibitors may
reduce circulating levels of this hormone.73 Also, angiotensin II causes increased sympathetic tone and increases vasopressin release through an effect on the
central nervous system74 as well as facilitating the
release of catecholamines from peripheral adrenergic
nerve terminals.75
Overturf et al76 administered the ACE inhibitor enalapril orally to normotensive and one-kidney, one clip
hypertensive rabbits fed a normal or cholesterolenriched diet. Drug treatment was associated with
significant reductions in aortic weight and aortic cholesterol and triglyceride content in normally fed animals,
both normotensive and hypertensive. These effects were
not observed in the corresponding cholesterol-fed
groups. Although one cannot attribute the effects of
enalapril on aortic lipid content in normally fed animals
directly to effects on smooth muscle cell proliferation, it
is possible cautiously to conclude that ACE inhibition
may have beneficial effects on arteries injured by hypertension. In contrast to the lack of effect of enalapril in
rabbits rendered hyperlipidemic by dietary manipulation, beneficial effects have been obtained by ACE
inhibition with captopril in other hypercholesterolemic
animal models. These include the cholesterol-fed cynomolgus monkey77 and the Watanabe heritable hyperlipidemic rabbit.78
Powell et al79 investigated the effect of oral administration of the ACE inhibitor cilazapril to rats in which
the left common carotid artery was injured with a
balloon catheter. This procedure induces the replication
of smooth muscle cells in the tunica media, accompanied by migration to and proliferation in the tunica
intima.80 Cilazapril significantly inhibited the accumulation of smooth muscle cells in the intima and also
decreased the proportion of the arterial circumference
adjacent to neointima. Of course, this does not prove
that cilazapril inhibits smooth muscle cell proliferation
in vivo, because this effect could equally well be caused
by inhibition of extracellular matrix accumulation or of
smooth muscle cell migration. Indeed, the reduced
circumferential extent of the neointima in cilazapriltreated animals supports the latter hypothesis. If cilazapril simply inhibited proliferation, smooth muscle cells
would still be expected to be present in the intima,
because nonproliferating smooth muscle cells do migrate to the intima in vivo.21 Because large portions of
the arterial circumference were free of neointima, it is
likely that cilazapril inhibits smooth muscle cell migration and may also inhibit smooth muscle cell proliferation. Another ACE inhibitor, benazepril, inhibits migration in this model but has no effect on smooth muscle
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cell proliferation,81 and this is also true of ramipril.82
The inhibitory effects of ACE inhibitors on intimal
thickening in the balloon-injured rat carotid artery have
prompted clinical trials of cilazapril in postangioplasty
restenosis (the Multicentre European/American Research Trial with Cilazapril After Angioplasty to Prevent Transluminal Obstruction and Restenosis). The
European trial revealed no effect of cilazapril on change
in minimal lumen diameter or on frequency of clinical
events.83 This failure may have been mechanism related;
inhibition of smooth muscle cell migration may not
prevent intimal thickening in a vessel with a preexisting
intima such as a human coronary artery. Alternatively,
it may have been dose related; on a gravimetric basis,
the dose used in the clinical trial was approximately
1.5% of that shown to be effective in rats. The former of
these two hypotheses is the more likely, because cilazapril does not prevent intimal thickening in other species
that have preexisting intimas, despite being administered at doses that powerfully inhibit ACE activity.84-85
The effects of ACE inhibitors could be the direct
result of a reduction in the availability of angiotensin II
or could be an indirect consequence of ACE inhibition.
The angiotensin II receptor antagonist losartan significantly reduces intimal thickening after balloon catheter
injury,81-86-87 so it is clear that some of the activity of
ACE inhibitors is mediated through effects on the
angiotensin system. However, effects on kinin metabolism may also be involved, because the kinin receptor
antagonist Hoe 140 potentiates the inhibition of intimal
thickening in the balloon-injured rat carotid artery
produced by ramipril.87
Catecholamines
Epinephrine and norepinephrine are synthesized ultimately from the amino acid phenylalanine. The major
sites of synthesis are postganglionic sympathetic nerve
fibers and the adrenal medulla. The plasma levels
of epinephrine and norepinephrine in rats are 1.1 and
4.0 JAM, respectively; the values in normal human
plasma are 0.2 and 2.7 ^M, respectively.88
Sympathetic agonists stimulate the growth of arterial
smooth muscle cells in vitro. Blaes and Boissel89 showed
that epinephrine dose-dependently stimulates the proliferation of rat thoracic aortic smooth muscle cells in
secondary culture, the minimum effective concentration
being 1 nM. In this study, norepinephrine and the
nonselective /3-adrenergic receptor agonist isoproterenol were also found to stimulate cellular proliferation.
At a concentration of 10 JIM, the rank order of efficacy
was epinephrine > norepinephrine > >isoproterenol.
The role of /J-adrenergic receptors in the stimulation of
proliferation was confirmed by showing that the nonselective £-adrenergic receptor antagonist propranolol
significantly inhibited the response to epinephrine.
However, there was no effect of propranolol or of the
nonselective a-adrenergic receptor antagonist phentolamine, both at a concentration of 10 /xM, on the
proliferation of control cultures growing in calf serum.
Bell and Madri90 found that norepinephrine (1 pM)
stimulates the proliferation of bovine aortic smooth
muscle cells growing in secondary culture. Epinephrine
and norepinephrine stimulate with equal potency the
proliferation of rat thoracic aortic smooth muscle cells
growing in fetal calf serum, but epinephrine is more
719
efficacious.88 Peak effects were produced at a concentration of 1 fiM. The proliferation of the A7r5 smooth
muscle cell line is stimulated by 10 fiM norepinephrine
and is inhibited by the a-adrenergic receptor antagonist
bunazocine and by the o- and 0-adrenergic receptor
antagonist labetalol.69
Similar findings were reported by Nakaki et al,91 who
investigated the effects of catecholamines on the restimulation of DNA synthesis in quiescent cloned rat aortic
smooth muscle cells in serum-free medium. In subconfluent cultures, norepinephrine stimulated DNA synthesis at concentrations at and greater than 100 pM. In
confluent cultures, the stimulatory effect was seen only
at concentrations greater than 100 nM. These effects
were inhibited by the nonselective a-adrenergic receptor antagonist phentolamine and by the a,-adrenergic
receptor antagonist prazosin. The a r adrenergic receptor agonist phenylephrine also stimulated DNA synthesis, but with lower potency than norepinephrine. Norepinephrine was found to inhibit the restimulation of
DNA synthesis in cloned cells of high passage number,
an effect apparently caused by ft-adrenergic receptor
stimulation, because it was blocked by the selective
antagonist butoxamine. This compound also augmented
the stimulatory effects of norepinephrine on DNA
synthesis in cells of lower passage number. These results
led the authors to conclude that stimulation of a,adrenergic receptors stimulates arterial smooth muscle
cell growth but that stimulation of ft-adrenergic receptors has an inhibitory effect. This runs counter to the
conclusion of Blaes and Boissel89 that isoproterenol
stimulates smooth muscle cell proliferation, but it is
possible that this discrepancy is related to the rather
different assay conditions used in the two studies. At
any rate, we may conclude that stimulation of the
a r adrenergic receptor induces or augments the proliferation of smooth muscle cells in vitro.
This hypothesis also appears to hold true in vivo.
Epinephrine stimulates DNA synthesis in the cells of
the aortic tunica media in rabbits fed a cholesterolenriched diet92; presumably, these are smooth muscle
cells. Bevan93 found that sympathetic denervation of the
ear artery by removal of the superior cervical ganglion
in young rabbits causes a significant reduction in smooth
muscle cell DNA synthesis. Chemical sympathectomy
with 6-hydroxydopamine causes a reduction in the
number of smooth muscle cells in rabbit aortic tunica
media.94 In rats sympathectomized by a combination of
immunological and chemical means, there is a significant reduction in the number of medial smooth muscle
cell layers in large and small mesenteric arteries, but not
in the superior mesenteric artery.5 Medial cross-sectional area was also reduced in large mesenteric arteries. Administration of the a r adrenergic receptor agonist methoxamine to chickens results in the formation of
thoracic aortic intimal accumulations of smooth muscle
cells.95 Another a r adrenergic receptor agonist, phenylephrine, does not stimulate DNA synthesis in the
thoracic aorta of normal rats.96 These disparate results
may be related to differences in aortic morphology
between chickens and rats; in chickens, there are occasional smooth muscle cells in the subendothelial intima,95 but these are not found in normal rats.97 Perhaps
in the uninjured vessel increased aj-adrenergic receptor
stimulation by exogenous agonist provokes the prolifer-
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Hypertension
Vol 20, No 6 December 1992
ation of intimal but not of medial smooth muscle cells.
Because sympathectomy inhibits medial smooth muscle
cell proliferation, endogenous catecholamines may be
implicated in normal cell turnover processes in the
vessel wall.
The role of a,-adrenergic receptor stimulation in the
control of the proliferative response of smooth muscle
cells to injury has been established in studies in which the
balloon catheter has been used. Prazosin inhibits DNA
synthesis in rat thoracic aortic smooth muscle cells, but not
in other proliferating tissues,98 and reduces intimal hyperplasia in the rabbit abdominal aorta."
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Eicosanoids
The eicosanoids are a family of products derived from
the oxidation of fatty acids and include the prostaglandins, prostacyclins, thromboxanes, and leukotrienes.
An appreciable amount of evidence now suggests that
arterial smooth muscle cells can synthesize eicosanoids.
Huttner et al100 showed that guinea pig aortic smooth
muscle cells secrete prostaglandins of the E and I series
into the culture medium when supplied with appropriate fatty acid precursors. Cornwell et al101 were able to
attribute this to de novo synthesis of prostaglandins
rather than release from preformed stores by showing
that the prostaglandin endoperoxide synthetase (cyclooxygenase) inhibitor indomethacin reduced the concentrations of prostaglandin (PG) E and PGI2 in conditioned medium. PGE2, PGF^, and PGI2 are synthesized
by porcine aortic smooth muscle cells supplied with
exogenous arachidonic acid, but there is no secretion of
thromboxane A2.102 Using bovine pulmonary arterial
smooth muscle cells, Menconi and colleagues103 detected synthesis of PGF^ and PGI 2 , which was augmented by exogenous arachidonic acid. There was no
detectable synthesis of PGE2 or thromboxane A2. However, punch samples of bovine pulmonary arterial tunica
media synthesized PGE 2 , PGI 2 , and thromboxane A2.
PGF^ synthesis was not determined. Larrue et al104 also
compared the prostaglandin synthetic activity of rabbit
aortic smooth muscle cells in intact artery and in
culture. PGEa, PGI 2 , and PGF^ were all synthesized in
response to addition of arachidonic acid. The major
product, both ex vivo and in vitro, was PGI2.
Thromboxane A2 production by rat thoracic aortic
smooth muscle cells was documented by Ishimitsu et
al 105.106 Thromboxane A2 was also synthesized by excised thoracic aorta, but it is not possible to attribute
this exclusively to smooth muscle cells, because endothelial cells and fibroblasts can also synthesize thromboxane A2.103
Canine and human coronary arteries synthesize leukotriene (LT) C4, LTD 4 , and LTE4 when incubated in
vivo with arachidonic acid and the calcium ionophore
A23187.107 Cultured rabbit aortic smooth muscle cells
have been shown to generate lipoxygenase products
from exogenous arachidonic acid.104 Human umbilical
arterial smooth muscle cells generate PGE^, PGI 2 ,
PGF^, 12-hydroxyheptadecatrienoic acid, and 11-hydroxy-eicosatetraenoic acid from exogenous arachidonate in vitro.108 PGE 2 is the major metabolite. All of these
are cyclooxygenase products; no evidence was found for
processing of exogenous arachidonic acid via the lipoxygenase pathway.
Prostaglandin synthesis by smooth muscle cells has
been shown to be stimulated by a variety of factors.
Interleukin-1 stimulates prostaglandin synthesis by
smooth muscle cells growing in culture.109-111 PDGF
dose-dependently stimulates PGI2 synthesis by porcine
aortic smooth muscle cells in culture, at concentrations
of 100 pg/ml and higher. Similar effects, but at higher
PDGF concentrations, were found in porcine aortic
rings denuded of endothelium.112 The same authors also
found that PDGF-stimulated PGI2 synthesis was markedly lower in human atherosclerotic femoral and carotid
arteries than in ostensibly normal vessels. Morphological classification of human atherosclerotic lesions
showed that both basal and PDGF-stimulated PGI2
production were reduced in fatty streaks, intimal thickenings, fibrous plaques, and complicated lesions. In
lesions classified as lipid deposits and fatty dots, basal
production of PGI2 was not significantly reduced, but
the response to PDGF was blunted.
A number of groups have found stimulatory effects of
vasoactive agents on smooth muscle cell prostaglandin
production. Vasopressin increases PGI2 production by
the A10 smooth muscle cell line, an effect augmented by
epidermal growth factor.113 Other vasoactive agents that
augment PGI2 production include bradykinin,114 angiotensins I and II but not angiotensin III,103 and
serotonin.114
The ability of prostanoids to modulate the proliferation of arterial smooth muscle cells in vitro was first
demonstrated by Huttner et al.100 The growth of guinea
pig aortic smooth muscle cells was dose-dependently
inhibited by PGE, and PGF^, the minimum effective
concentrations being 2 and 20 fiM, respectively.
Pietila et al115 confirmed and extended these results.
They incubated secondary cultures of rabbit aortic
smooth muscle cells with 10 fiM PGE,, PGEj, PGF la ,
or PGF^. Significant inhibition of the incorporation of
[3H]thymidine into DNA was observed with ah1 these
prostaglandins; the maximum inhibitory effect was a
40% reduction in incorporation produced by PGE!.
Although several groups subsequently have reported
the inhibitory actions of E-series prostaglandins on
arterial smooth muscle cell proliferation in vitro, it has
become clear that study conditions are critical determinants of the observed effect. In the studies of Huttner et
al,100 Pietila et al,115 Sjolund et al,116 Smith et al,117 and
Orekhov et al,118 E-series prostaglandin inhibited the
proliferation of arterial smooth muscle cells growing
continuously in serum. Loesberg et al119 rendered human aortic smooth muscle cells quiescent by culturing
them for 3 days in 0.5% human serum. Cell growth was
restimulated by addition of 1% serum supplemented
with PDGF, and PGE! was added at various time
intervals later. Addition of PGE, (25 nM) between 0
and 12 hours after restimulation inhibited pHJthymidine incorporation into DNA, but addition 14-18 hours
after restimulation was without effect. Nilsson and
Olsson120 used a similar protocol to examine the effects
of PGE, on rat thoracic aortic smooth muscle cells.
Restimulation of quiescent cells by addition of PDGF
was inhibited by PGE, (140 nM), but only if it was given
within 6 hours of restimulation. Using the A10 embryonic rat thoracic aortic smooth muscle cell line, Owen121
found that a minimal restimulation of quiescent cells by
addition of insulin was significantly and dose-depen-
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dently augmented by PGE]. The half-maximal effect was
seen at a concentration of 4 JAM. NO such stimulatory
effect was observed in quiescent cells that were restimulated with 10% fetal calf serum or with PDGF plus
insulin. Addition of PGE, to cultures growing normally
in fetal calf serum, PDGF, or insulin significantly and
dose-dependenth/ inhibited DNA synthesis, the halfmaximal effect being produced by a concentration of 20
nM. It therefore appears that the inhibitory effect of
PGEj is produced either by administration to proliferating cells or by administration to quiescent cells within
a few hours of restimulation. There is a crucial difference, however, in sensitivity between these two protocols. The concentration of PGE, required to inhibit the
proliferation of growing cells was on average 100 times
greater than that required to inhibit the restimulation of
quiescent cells.
Inhibitory effects on arterial smooth muscle cell
proliferation have also been noted with PGA,, 117
PGA2,117 P G B L 1 1 7 PGD2,117-118.120 PGEj,100.11*"7.119-120
and PGJ2.117 Effects with F-series prostaglandins have
been contradictory. Pietila et al115 found significant
inhibitory actions with PGF, a (10 /iM) and PGF^ (10
fiM), but Cornwell et al101 found significant stimulation.
No effect was found with PGF^ (0.6 fiM) by Nilsson
and Olsson120 or at unspecified doses by Smith et al117
and Loesberg et al.119
Prostacyclin (PGI2), in the form of its relatively stable
analogue carbacyclin, has been found to inhibit the
incorporation of pHlthymidine into DNA of human
aortic intimal smooth muscle cells growing in primary
culture, at micromolar concentrations, ns.122.123 Similar
effects were noted with authentic prostacyclin by
Morisaki et al,124 using rabbit aortic smooth muscle cells
growing in secondary culture. Prostacyclin was without
effect, at concentrations up to 100 fiM, on the restimulation of growth in quiescent bovine aortic smooth
muscle cells.123
Ishimitsu et al105-106 have shown that a stable thromboxane A2 analogue, 9,ll-epithio-ll,12-methanothromboxane A 2 , dose-dependenth/ stimulates the incorporation of pHJthymidine into DNA of rat thoracic
aortic smooth muscle cells, with a maximal effect at a
concentration of 10 pM. This concentration also significantly prolonged the doubling time. Similar results
were achieved by Akopov et al,123 using the stable
thromboxane A2 analogue U46619 at a concentration of
0.7 JAM to stimulate the proliferation of human aortic
intimal smooth muscle cells. However, the thromboxane
synthetase inhibitors sodium 5-(3-pyridinylmethyl)benzofuran-2-carboxylate and dazoxiben both failed to
inhibit the proliferation of canine arterial smooth muscle cells growing in fetal calf serum.126
LTB4 was initially found to inhibit rabbit aortic
smooth muscle cell growth in culture, the half-maximal
effect being produced at a concentration of 28 jiM.117
Later, Palmberg et al127-128 showed that LTB4, LTQ,
and LTD4 all significantly stimulate proliferation in rat
thoracic aortic smooth muscle cells growing in primary
culture, with maximal effects being produced at a concentration of 10 pM. LTE4 produced no effect, nor did
the biologically inactive isomer of LTD 4 , (55,125)dihydroxy-(6,8,10,14)-eicosatetraenoic acid. LTB 4 ,
LTC4, and LTD4 stimulated DNA synthesis in smooth
muscle cells in serum-free medium at concentrations as
721
low as 10 fM. These cells cannot be characterized
uniformly as quiescent, because 15% of them incorporated pHJthymidine into DNA in the absence of leukotrienes. The capacity of leukotrienes to propel quiescent cells into the cell cycle remains undetermined. The
growth stimulatory effect of LTB4, but not of LTC4, was
inhibited by indomethacin and aspirin, suggesting that
this effect is modulated by a cyclooxygenase product.127
Brinkman et al108 investigated the effects of inhibitors of
lipoxygenase on the proliferation of human umbilical
arterial smooth muscle cells in vitro. Both nordihydroguiaretic acid and caffeic acid inhibited proliferation, suggesting an involvement of lipoxygenase products. There was no evidence in these cells of metabolism
of exogenous arachidonic acid via the lipoxygenase
pathway, leading the authors to suggest that lipoxygenase metabolism of endogenous fatty acids other than
arachidonate may play a role in the control of smooth
muscle cell proliferation.
Investigation of the effects of drugs that interfere with
endogenous eicosanoid synthesis is a potentially useful
way of obtaining information regarding the role of this
process in the modulation of smooth muscle cell proliferation. The major mechanism of action of the glucocorticoids is inhibition of phospholipase A^ 48 It is
possible, at least in the rat model, that glucocorticoids
may also interfere with the release or activity of histamine and serotonin.129 Dexamethasone and cortisone
also reduce levels of cyclooxygenase mRNA in cultured
vascular smooth muscle cells, thereby reducing its activity.130 The effects of steroids on smooth muscle cell
proliferation are discussed above. It is possible that
their growth-inhibitory actions are the consequence of
the impaired biosynthesis of growth-stimulatory eicosanoids such as thromboxane A 2 , 103106 LTB4, LTC4,
LTD4,12712» and perhaps PGF^. 101 Two synthetic inhibitors of phospholipase A 2 , />-bromophenacylbromide
and mepacrine, have been shown to inhibit the proliferation of human umbilical arterial smooth muscle cells
in vitro.108 Hauss et al42 found that aspirin inhibited the
proliferation of porcine aortic smooth muscle cells in
secondary culture. The proliferation of aortic smooth
muscle cells isolated from rats dosed with aspirin was
also inhibited. However, Lindblad et al126 found no
effect of aspirin, at concentrations up to 560 fiM, on the
growth of canine carotid arterial smooth muscle cells in
fetal calf serum. Another cyclooxygenase inhibitor, ibuprofen, inhibited proliferation of these cells at a concentration of 240 ^.M. Indomethacin has been found
either not to affect smooth muscle cell proliferation in
vitro 44101108127131 or to augment it.124132 The effects of
in vivo administration of cyclooxygenase inhibitors are
discussed in detail in the section dealing with antiplatelet drugs, because these agents are potent inhibitors of
platelet aggregation.
In light of the generally negative findings with cyclooxygenase inhibitors on smooth muscle cell proliferation in vitro, it is tempting to speculate that any growthstimulatory eicosanoid or eicosanoids inhibited by
glucocorticoid treatment are lipoxygenase products.
Cicletanine is a diuretic that stimulates the synthesis
of prostacyclin in rat thoracic aortic smooth muscle
cells, both from endogenous and exogenous arachidonic
acid.133 Cicletanine inhibits the restimulation of quies-
722
Hypertension
Vol 20, No 6 December 1992
cent rat mesenteric arterial smooth muscle cells in
response to serum or PDGF.' 34
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Heparinoids
Heparin is a heterogeneous group of gh/cosaminogrycans and is particularly abundant in lung, liver, and
intestinal mucosa. It is found intracellularh/ in mast
cells, which accumulate in most organs around small
blood vessels.135 Heparan sulfate is a structurally related
compound that is present in extracellular matrix and at
cell surfaces and that has been shown to be present in
the aorta.136-137
Hoover et al138 showed that heparin dose-dependently inhibits the proliferation of rat aortic smooth
muscle cells growing in 20% fetal calf serum, with 50%
inhibition of growth occurring at a concentration of
approximately 10 jig/ml. This corresponds to 0.67 fiM,
assuming a molecular weight of 15 kd. There was no
difference in growth-inhibitory activity between anticoagulant and nonanticoagulant heparin fractions. Because the latter species has low affinity for antithrombin
III, it is unlikely that the growth-inhibitory properties of
heparin are related to inactivation of the proteoh/tic
properties of thrombin. Reilly et al139 went on to show
that the inhibition of bovine aortic smooth muscle cell
proliferation is maximized by preincubation of quiescent cells with heparin for 48 hours before reintroduction of serum.
The effects of heparin on smooth muscle cell growth
have also been investigated in animal models of arterial
injury. Clowes and Karnovsky140 administered heparin
by intravenous infusion to rats in which the common
carotid artery was injured by air drying. Infusion began
24 hours after injury. There was no difference in
neointimal thickening between control and heparintreated rats 10 days after injury. During the next 4 days,
there was considerable expansion of the intima that was
inhibited by heparin, with the result that at 14 days after
injury the neointimas of injured carotid arteries were
significantly smaller in the heparin-treated group. This
difference was attributed to inhibition of smooth muscle
cell proliferation. Similar results were reported by Guyton et al,141 who also showed a dose-related inhibitory
action of heparin on neointimal thickening. This effect
was observed with both anticoagulant and non-anticoagulant heparin fractions. Reduction of neointimal
thickening was also observed in the balloon-injured
rabbit aorta after continuous intravenous infusion of
heparin.142 Local release of heparin from a gel enclosed
in a periadventitial cuff around the rat common carotid
artery significantly inhibits balloon catheter-induced
neointimal thickening in this vessel.143 Intravenous administration of heparin to rats for 14 days significantly
inhibits the increase in the DNA content of the common
carotid artery caused by balloon catheter injury.144-'45
Further direct evidence of an effect of heparin on
arterial smooth muscle cell proliferation in vivo was
obtained by Majesky et al,146 who measured the fH]thymidine labeling index of smooth muscle cells in the rat
common carotid artery after balloon injury. Intravenous
infusion of heparin significantly reduced the labeling
index measured 33 hours after injury. Delay of commencement of heparin administration until 18 hours
after injury did not prevent the inhibitory effect, but
delay until 27 hours after injury abolished the inhibi-
tion. This is rather surprising in view of the finding by
Clowes and Karnovsky140 that heparin inhibits neointimal thickening in the air-drying model of arterial injury
through a mechanism that becomes manifest 10 days
after injury. These results suggest that heparin inhibits
both medial smooth muscle cell proliferation and
neointimal expansion. This latter effect could be the
result of inhibition of neointimal smooth muscle cell
proliferation, of synthesis of extracellular matrix in the
neointima, or of migration of smooth muscle cells from
the media to the intima. There is experimental support
for all of these alternatives.144145 The mechanism by
which heparin inhibits smooth muscle cell proliferation
has not been established, although several hypotheses
may be considered. In smooth muscle cells growing in
vitro, heparin binds via specific, high-affinity receptors147-148 and prevents progression through the G]
phase of the cell cycle.139 The specific suppression by
heparin of the elevation of the levels of the mRNAs for
c-myb and 2F1 may constitute a mechanism by which the
antiproliferative effect is exerted. Another possible
mechanism involves inhibition of intracellular Ca 2+ mobilization through an effect on the inositol 1,4,5trisphosphate receptor.149-151 A third alternative mechanism of antiproliferative action involves the capacity of
heparin to bind to specific structural domains in the
bFGF molecule.152-153 Although this mechanism may not
be important for the inhibition by heparin of smooth
muscle cells growing in serum, which contains little
bFGF, it could play a role in vivo. Lindner et al 15154
have suggested that, after balloon catheter injury to the
rat common carotid artery, bFGF is released from
wounded smooth muscle cells and stimulates replication
among their neighbors. Exogenous heparin could compete for bFGF with binding sites in the extracellular
matrix. The growth factor would therefore partition into
the systemic circulation, where clearance mechanisms
would supervene. Lastly, heparin has been shown to
disrupt the deposition of thrombospondin in the extracellular matrix of smooth muscle cells.155-156 Thrombospondin appears to be functionally essential for smooth
muscle cell proliferation in vitro.157
Unfortunately, the hypothesis that endogenous heparinoids play a role in the control of arterial smooth
muscle cell replication cannot be tested. There are no
agents currently available that can reduce or remove the
influence of these molecules, and this makes it difficult
to evaluate their biological significance. It has been
suggested that a smooth muscle cell growth-inhibitory
heparinoid is released from endothelial cells on exposure to a platelet-derived endoglycosidase.15*-160 Synthetic inhibitors of this enzyme may help to elucidate
the role of heparinoids in smooth muscle cell growth.
Serotonin
Serotonin (5-hydroxytryptamine) is found in platelets, and also in the mast cells of rodents and cattle, but
not in those of humans.161 Serotonin initiates DNA
synthesis in quiescent bovine aortic smooth muscle cells,
with a maximal effect at a concentration of 1 jtM131 or
100 fiM.i62 Serotonin (100 nM) also stimulates DNA
synthesis in continuously growing rat thoracic aortic
smooth muscle cells.72 Similar effects were noted at this
concentration by Bell and Madri90 using bovine aortic
smooth muscle cells. To put these concentrations in
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perspective, the concentration of serotonin in human
blood is approximately 40 nM, and the concentration in
human platelets is approximately 2.6 pmol per million
platelets.163
The effects of serotonin on smooth muscle cell
proliferation are blocked by serotonin-D receptor
antagonists.131 This receptor is now termed the 5-HT2
receptor.164 Stimulation of this receptor also stimulates synthesis of prostacyclin by bovine aortic smooth
muscle cells,114-131-162-165 although prostacyclin production in response to serotonin by explants of bovine
aortic tunica media is mediated by the 5-HTi receptor.166 It has been suggested that the smooth muscle
cell proliferation induced by serotonin may be mediated, in part at least, by metabolites of arachidonic
acid.131 Thus, the mitogenic concentrations studied in
vitro may represent a pharmacological artifact. It
seems unlikely that prostacyclin itself could be responsible for the mitogenic effect, because it generally
inhibits smooth muscle cell proliferation. 118,122,123 i n a
test system in which serotonin stimulates smooth
muscle cell proliferation, both prostacyclin and indomethacin have no effect.131 Because serotonin stimulates the liberation of arachidonic acid from smooth
muscle cell membrane lipids,162 it is possible that the
increased levels of prostacyclin reflect increased activity in the arachidonic acid metabolic pathway and that
growth-stimulatory leukotrienes are responsible for
the increased proliferation. This hypothesis could be
tested by examining the effects of lipoxygenase inhibitors or leukotriene receptor antagonists on the mitogenic response of smooth muscle cells to serotonin.
723
Somatostatin
Somatostatin is a tetradecapeptide hormone that
inhibits the release of growth hormone from the pituitary.167 It is found in the hypothalamus and in ganglia
of the sympathetic nervous system. Nanomolar concentrations of angiopeptin, an octapeptide analogue of
somatostatin, inhibit DNA synthesis in explants of rat
common carotid artery,168 suggesting that this material
has an effect on vascular smooth muscle cell proliferation that is not mediated by growth hormone. Subcutaneous administration of angiopeptin inhibits DNA synthesis and neointimal thickening in the rat common
carotid artery after air-drying injury,169 but this property is not shared by some other analogues of somatostatin that also inhibit the release of growth hormone.
Angiopeptin also inhibits DNA synthesis170 and neointimal thickening171 in balloon-injured rabbit arteries.
The evidence for mitogenic effects of thrombin on
vascular smooth muscle cells is controversial. Ishida and
Tanaka174 grew rabbit aortic smooth muscle cells in 10%
fetal calf serum, then added thrombin at the same time
as complete serum withdrawal. Concentrations up to
10 nM produced no effect, and cell numbers actually
decreased over a period of 7 days when exposed to a
concentration of 100 nM. Significant mitogenic effects
of 10 nM thrombin were observed in neonatal rat
vascular smooth muscle cells by Huang and Ives175 and
in bovine aortic smooth muscle cells by Graham and
Alexander.176 Berk et al177 failed to detect any effect of
a-thrombin on the proliferation of quiescent rat vascular smooth muscle cells at concentrations up to 100 nM,
despite marked increases in protein synthesis, intracellular calcium ion concentration, and intracellular pH.
Inhibition of the proteorytic activity of a-thrombin with
D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone
(PPACK) prevented all of these effects. •y-Thrombin, a
form of thrombin that exhibits enzymatic activity but
that does not convert fibrinogen to fibrin, caused increases in protein synthesis and in intracellular pH but
did not affect intracellular calcium ion concentration.
These results suggest that the proteolytic activity of
thrombin may be an important determinant of its activation of signal transduction mechanisms in vascular
smooth muscle cells. The wide spectrum of proteorytic
activity of thrombin (reviewed by Fenton178) encourages
the speculation that a proteotyticalry modified potypeptide or protein may be the proximal agonist in smooth
muscle cell cultures treated with a-thrombin. This hypothesis is supported by the studies of Walz et al,173 who
found that the contraction of strips of rabbit aorta to
different preparations of thrombin was absolutely dependent on proteolytic activity, and also by the discovery that the thrombin receptor on platelets is activated
by proteolytic modification of the receptor protein.179
However, bovine aortic smooth muscle cells have been
shown to respond mitogenically to nanomolar concentrations of both a-thrombin and diisopropytfluorophosphate-conjugated, enzymatically inactive a-thrombin. 18°
Hirudin, a leech polypeptide that specifically inhibits
thrombin, blocked its mitogenic effect. The authors
suggest that hirudin interacts with a part of the thrombin molecule, distinct from its active proteolytic site,
which is responsible for conferring mitogenic activity.
Another possibility is that there is more than one type
of thrombin receptor, with proteolytic activation being
required by some but not by others.
Thrombin
Thrombin (factor Ha) is a serine protease that catalyzes the conversion of fibrinogen to fibrin and activates
factors V, VIII, and XIII. It is derived from prothrombin (factor II) by the coordinated actions of factor V,
factor Xa, phospholipid, and calcium ions and is therefore formed at sites of vascular injury. Its physiological
inhibitors include antithrombin III, a2-macroglobulin,
and a r antitrypsin, but thrombin can be deposited on
subendothelial extracellular matrix in such a way that it
is protected from inhibition by antithrombin III.172 The
approximate maximum concentration of thrombin
achieved during blood coagulation is 140 nM.173
Hirudin and other inhibitors of thrombin such as
3,4-dihydro-3-benzyl-6-chloromethylcoumarin181 could
be used to elucidate the role of thrombin in the control
of vascular smooth muscle cell proliferation in vivo.
Warfarin is a congener of bishydroxycoumarin that
interferes with the hepatic synthesis of the vitamin
K-dependent clotting factors VIII, IX, X, and II,
prothrombin. It therefore produces a hypoprothrombinemia and reduces the capacity of the blood to
generate thrombin. Oral administration of warfarin to
rats subjected to air-drying injury to the common carotid artery significantly reduces the neointimal thickening measured 4 weeks after injury.182
724
Hypertension
Vol 20, No 6 December 1992
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Drugs That Modify Vascular Smooth
Muscle Cell Proliferation
Antiplatelet Drugs
A wide variety of agents has been used in humans to
inhibit platelet aggregability. Inhibition of cyclooxygenase results in reduced platelet aggregation. The effects
of inhibitors of this enzyme on the proliferation of
vascular smooth muscle cells in vitro are discussed in the
section dealing with eicosanoids.
Cyclooxygenase inhibitors have been used in a number of in vivo models of smooth muscle cell proliferation. In the cuffed rabbit carotid artery, indomethacin
treatment had no significant effect on intimal thickening.44 Neither aspirin nor flurbiprofen, another cyclooxygenase inhibitor, influenced the formation of a
neointima in the rat carotid artery subjected to airdrying injury.183 August and TUson182 also found aspirin
to be without effect in this model. Aspirin did not
influence the degree of intimal thickening occurring in
canine carotid arteries after endarterectomy.184 Aspirin
was without effect on smooth muscle cell proliferation
in the injured rabbit ear artery.185 However, V61ker and
Faber186 found that dietary administration of aspirin to
rats resulted in a significant reduction in balloon catheter-induced neointimal thickening in the common
carotid artery. This treatment also reduced the thickness of the media.
Dipyridamole has a number of activities, most of
which appear to be related to inhibition of cyclic AMP
phosphodiesterase.187 In addition to its effects on platelet aggregation, it is a vasodilator and it inhibits the
uptake of adenosine into erythrocytes.188 It has also
been suggested that it decreases the release of PDGF
from human platelets during blood clotting because it
inhibits the mitogenic effects of serum on human skin
fibroblasts.189 It is also an antioxidant.190 Dipyridamole
inhibits the proliferation of canine carotid arterial
smooth muscle cells in vitro, at a concentration of
20 /iM, which is 10 times greater than the plasma
concentration achieved in clinical use.126 No effect was
seen at lower concentrations. Morisaki et al190 found
that 1 JAM dipyridamole significantly increased the proliferation rate of guinea pig aortic smooth muscle cells
growing in serum. Oral administration of dipyridamole
to baboons injured by continuous infusion of homocystine resulted in a significant reduction in intimal lesion
score.191 This score was derived by counting layers of
intimal smooth muscle cells in cross sections of the
abdominal aorta and the iliac and femoral arteries.
Administration of dipyridamole to cholesterol-fed dogs
with autologous vein-to-artery grafts significantly reduced the intimal thickening measured 6 weeks later.192
There was no effect on the plasma concentrations of the
major metabolites of thromboxane A2 and prostacyclin.
Dipyridamole treatment significantly reduced the accumulation of smooth muscle cells in the intimas of rabbit
ear arteries subjected to repeated mechanical injury, as
did the structurally related compound AH-P719.185 In
contrast to these positive findings, Koster et al193 found
that dipyridamole significantly increased the number of
smooth muscle cells in the aortic intima of cholesterolfed rabbits.
A number of studies have investigated the effects of a
combination of dipyridamole with aspirin on the devel-
opment of vascular lesions. In a nonhuman vein graft
model, this therapy reduced the intimal cross-sectional
area measured 16 weeks after surgery.194 Hagen et al195
measured the degree of luminal narrowing in the region
of Gore-Tex grafts inserted in the abdominal aortas of
nonhuman primates. Aspirin/dipyridamole therapy reduced the degree of luminal narrowing in the aorta
distal to the graft. However, the vessels were not fixed in
situ under pressure, so it is not clear whether drug
treatment affected postmortem aortic contraction or
intimal hyperplasia. In a rabbit balloon injury model,
treatment with aspirin and dipyridamole caused an
increase in the intima/media ratio in the thoracic aorta
14 days after injury.196 Similar effects were noted by
Murday et al197 in a rabbit vein graft model. Radic et
al198 measured the rate of smooth muscle cell DNA
synthesis, intimal nuclei per unit vessel circumference,
and intima to intima-plus-media ratio in rabbits with
balloon injury to the abdominal aorta. Combination
therapy with aspirin and dipyridamole had no significant
effect on any of these indexes. This therapy was also
ineffective in a study reported by Rodgers et al199 in
which stents were implanted in the left anterior descending coronary arteries of hypercholesterolemic
pigs.
Ticlopidine, another inhibitor of platelet function,
had no effect on the growth of rat aortic smooth muscle
cells in serum.44 There is one preliminary report of
inhibition by ticlopidine of neointimal thickening in a
rabbit balloon injury model,200 but this compound was
ineffective in the repeated mechanical injury model
used on rabbit ear arteries by Ingerman-Wojenski and
Silver.185
U-53,059, a thiazole that inhibits platelet aggregation, reduces the extent of the pulmonary arterial
lesions caused in dogs by infection with Dirofilaria
immitis.201 The authors suggest that this is due to
reduced myointimal proliferation. However, the same
group found no effect of U-53,059 on neointimal thickening in the balloon-injured rabbit aorta.202
It must be concluded from these studies that the case
for an antiproliferative effect of antiplatelet therapy is
not proved.
Calcium Antagonists
The calcium antagonists are a group of compounds
that share the property of inhibiting the flux of Ca2+
ions into cells through a specific voltage-sensitive membrane channel, called the L-type channel. This channel
is relatively impermeable to Ca2+ ions when the membrane is normally polarized but opens when a sufficient
degree of depolarization is reached. In arterial smooth
muscle cells, this influx of Ca2+ ions and subsequent
elevation of the concentration of cytoplasmic-free Ca2+
is a critical element in the contractile process.
Many studies have focused on the influence of calcium antagonists on arterial smooth muscle cell proliferation in vitro, and these are summarized in Table 1.
This table is subdivided into four groups of compounds,
according to the scheme drawn up by the World Health
Organization.215 It is interesting to note that, in general,
the concentrations of calcium antagonists required to
inhibit the onset of proliferation in restimulated quiescent cells are considerably lower than those required to
inhibit growing cultures. For instance, the median con-
Jackson and Schwartz
Pharmacology of Smooth Muscle Replication
TABLE 1.
Effects of Calcium Antagonists on Smooth Muscle Cell Proliferation In VHro
Type
Compound
I
Veraparail
Verapamil
Verapamil
Verapamil
Verapamil
Verapamil
Verapamil
Verapamil
Verapamil
Verapamil
Verapamil
Concentration
n
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
III
Stimulus
Effect
Cell origin
033*
1
DNA synthesis
DNA synthesis
PDGF
5t
5t
DNA
DNA
DNA
DNA
DNA
Cell
DNA
DNA
Cell
DNA
Cell
DNA
DNA
DNA
DNA
Decrease
Decrease
Decrease
Decrease
Decrease
Rat aorta
Rabbit aorta
Rat aorta
Rat aorta
Rat aorta
Human artery
Human artery
Rabbit aorta
Porcine aorta
Rabbit aorta
Rat aorta
Rat aorta
Rat aorta
Rat aorta
Rat aorta
Rat aorta
Rat aorta
Rat aorta
Rat artery
Rat artery
Rat artery
Rat aorta
Rat artery
10t
10
10
10
10
50
100
Verapamil
SIM 6080
Nifedipine
Nifedipine
Nifedipine
100t
Nifedipine
Nifedipine
Nifedipine
Nifedipine
Nifedipine
Nifedipine
1-2*
2
Nifedipine
Nifedipine
Nifedipine
Nifedipine
Nifedipine
Nisoldipine
Nisoldipine
Nisoldipine
Nicardipine
Nicardipine
Nicardipine
Nimodipine
Nitvadipine
Diltiazem
Diltiazem
Diltiazem
Diltiazem
Diltiazem
Diltiazem
Diltiazem
Diltiazem
Diltiazem
rv
Measurement
G*M)
Flunarizine
5t
O.lt
0.1*
It
33t
33t
33t
5.4*
10
10
10
100
100
1-2*
1-2*
2
It
5t
5t
It
100
10
100
loot
100
0.47*
It
5t
5t
100t
2t
synthesis
synthesis
synthesis
synthesis
synthesis
number
synthesis
synthesis
number
synthesis
number
synthesis
synthesis
synthesis
synthesis
DNA synthesis
Cell number
DNA
DNA
DNA
Cell
synthesis
synthesis
synthesis
number
DNA
DNA
Cell
Cell
synthesis
synthesis
number
number
DNA synthesis
DNA synthesis
DNA synthesis
DNA synthesis
DNA synthesis
DNA synthesis
Cell number
Cell
DNA
DNA
DNA
Cell
DNA
DNA
DNA
DNA
Cell
number
synthesis
synthesis
synthesis
number
synthesis
synthesis
synthesis
synthesis
number
Cell number
Serum
PDGF
EGF
Serum
Serum
Serum
Serum
PDGF
Serum
Serum
Serum
Serum
PDGF
Serum
Serum
PDGF
Serum
Serum
Serum
Serum
PDGF
Serum
PDGF
Serum
Serum
Serum
Serum
PDGF
Serum
EGF
PDGF
Serum
Serum
Serum
Serum
Serum
Serum
Serum
PDGF
EGF
PDGF
Serum
Serum
Serum
Decrease
No effect
No effect
Decrease
Decrease
No effect
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
No effect
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
No effect
No effect
Decrease
Decrease
No effect
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Porcine aorta
Rat aorta
Rat aorta
A7r5
Rat aorta
Rat aorta
Rat aorta
Rat aorta
Rat aorta
Rat aorta
Rabbit aorta
Rat aorta
Human artery
Rat aorta
Rat aorta
Rat aorta
Rat aorta
Rat aorta
Rat aorta
Rat aorta
Rat artery
Rabbit aorta
Growth
status
Quiescent
Growing
Quiescent
Quiescent
Quiescent
Primary
Primary
Quiescent
Quiescent
Growing
Growing
Quiescent
Quiescent
Quiescent
Quiescent
Quiescent
Quiescent
Primary
Quiescent
Quiescent
Growing
Quiescent
Growing
Quiescent
Primary
Growing
Growing
Quiescent
Quiescent
Primary
Quiescent
Quiescent
Quiescent
Quiescent
Growing
Primary
Growing
Quiescent
Growing
Quiescent
Quiescent
Quiescent
Quiescent
Growing
Quiescent
725
Reference
203
204
205
205
205
206
123
207
208
209
210
211
212
211
213
211
213
213
134
134
134
203
134
208
211
210
69
213
213
213
205
205
205
207
210
123
214
211
210
203
205
205
205
134
207
PDGF, platelet-derived growth factor, EGF, epidermal growth factor.
tMinimum effective concentration.
centration of dihydropyridine (Type II) calcium antagonists required to inhibit proliferation in arterial
smooth muscle cells growing in serum is 100 fiM; the
median concentration in quiescent cultures is 1-2 fiM.
In other cell types, there is a relation between growth
state and the ability to bind dihydropyridine calcium
antagonists to L-type calcium channels. In the nonfusing muscle cell line BQH1, specific binding of pH]isradipine was undetectable in cultures growing in 20%
fetal calf serum.216 On reduction of the serum concentration to 0.5%, specific binding of [3H]isradipine began
to increase, reaching a peak 6 days after serum with-
726
Hypertension
Vol 20, No 6 December 1992
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drawal. Similar results were found in human embryonic
fibroblasts by Dudkin et al.217 The specific binding of
2,6-dimethyl-3-methoxycarbonyl-5-([2,3- 3 H 2 ]-/J-propoxycarbonyl)-4-(2'-difluoromethoxyphenyl)-l,4dihydropyridine increased to a maximum 3 days after
removal of calf serum from the culture medium. The
specific binding decreased when serum was reintroduced. These experiments indicate a loss of L-type
calcium channels from proliferating cells. If this were
also true in arterial smooth muscle cells, it might explain
the relative ineffectiveness of calcium antagonists in
proliferating cultures. The corollary of this argument is
that Ca2+ influx via L-type channels is critical for
initiating proliferation in quiescent cells but not for
maintaining proliferation in growing cultures. Because
high concentrations of calcium antagonists block other
types of ion channel, the effects reported in growing
cultures may not be related to specific blockade of
calcium influx via L-type channels.
The hypothesis that influx of Ca2+ via L-type channels is necessary for arterial smooth muscle cells to
achieve the transition from quiescence to active growth
has been addressed in vivo. Administration of nifedipine orally to balloon-catheterized rats inhibited aortic
smooth muscle cell proliferation, measured 48 hours
after injury, if the compound was given between 8 and
20 hours after injury.218 Administration more than 30
hours after injury was ineffective, suggesting that
smooth muscle cell proliferation is susceptible to the
inhibitory influence of reduced Ca 2+ ion influx via
L-type channels only during a critical period 8-20 hours
after balloon injury. These data suggest that nifedipine
inhibits a process occurring during the activation of
quiescent cells by balloon injury. Whether this is a
direct effect or a consequence of the blood pressurelowering effect of nifedipine is unknown.
In the same paper, Jackson et al218 investigated the
effects of nifedipine on balloon catheter-induced
smooth muscle cell proliferation in the rabbit abdominal
aorta. A dosage protocol that resulted in inhibition of
aortic smooth muscle cell proliferation 2 days after
balloon injury was ineffective if delayed by 7 days,
proliferation being measured in this case 9 days after
injury. This also indicates that nifedipine inhibits arterial smooth muscle cell proliferation at an early stage
and presumably does not block at a point in the cell
cycle that is traversed with every round of cell division.
The influx of calcium into smooth muscle cells can
also be blocked by lanthanum, which displaces Ca2+
ions from specific binding sites on the cell membrane,219
and by the potassium channel opener minoxidil, which
hyperpolarizes the cell membrane.220 Both of these
agents inhibit smooth muscle cell proliferation in the rat
thoracic aorta after balloon catheter injury.98
Many studies have been carried out to investigate the
antiatherogenic properties of calcium antagonists, usually in cholesterol-fed animals. These have been summarized previously.218 The results of these studies also
suggest that calcium antagonists interfere with an early
event in the development of experimental arterial lesions, an idea supported by Overturf.221 This argument
has consequences for the use of calcium antagonists to
treat human arterial disease. Administration of these
drugs to individuals in whom smooth muscle cell proliferation is already an active process would be expected
to be ineffective. Indeed, studies of their utility in
percutaneous transluminal coronary angioplasty have
yielded negative results.222-223 Strong positive support
for the hypothesis that calcium antagonists inhibit the
activation of quiescent smooth muscle cells comes from
the International Nifedipine Trial on Antiatherosclerotic Therapy (INTACT).224 In this study, patients with
angiographically proven mild coronary artery disease
were randomly assigned to treatment for 3 years with
either placebo or nifedipine. When angiograms were
measured at the end of the treatment period, nifedipine
therapy had no effect on the rate of progression of the
lesions that were present at the beginning of the trial.
However, the number of new lesions per patient was
significantly lower. It therefore seems likely that calcium
antagonists should show activity in other clinical settings in which quiescent arterial smooth muscle cells are
stimulated to proliferate, such as organ transplantation
or coronary bypass grafting. Calcium antagonists have
shown beneficial effects in experimental models of vein
bypass grafting.225-226
An important question arising from the antiproliferative actions of calcium antagonists concerns the nature
of the agonist or agonists stimulating Ca2+ ion influx via
L-type channels, as this occurs only in response to
membrane depolarization. What are the endogenous
agents capable of promoting Ca2+ influx via these
channels?
Thromboxane A2 has been shown to promote calcium
influx into rabbit coronary artery smooth muscle cells by
this route.227 Contraction of intact strips of this artery,
evoked by application of nanomolar concentrations of
the stable thromboxane A2 analogue 9,ll-epithio-ll,12methano-thromboxane A2, was inhibited by approximately 50% by nanomolar concentrations of nifedipine.
LTD4 produces contractions of strips of guinea pig
basilar artery denuded of endothelium.228 The phasic
component of these contractions is inhibited by submicromolar concentrations of nifedipine.
Endothelin is a 21-amino acid polypeptide that dosedependently stimulates DNA synthesis in quiescent rat
aortic smooth muscle cells, in the absence of serum.229-230 The peak effect is produced at a concentration of 1 nM and is completely blocked by treatment
with 1 /AM nifedipine.208
PDGF stimulates calcium influx into aortic smooth
muscle cells via L-type channels.203-208 Nifedipine and
verapamil both inhibit PDGF-stimulated DNA synthesis in aortic smooth muscle cells in vitro.203'208
a-Adrenergic receptor agonists also stimulate calcium influx into arterial smooth muscle cells via L-type
channels. The increase in intracellular Ca2+ concentration caused by application of norepinephrine to rat
aortic smooth muscle cells is inhibited by verapamil and
diltiazem.231
Oxidized low density lipoprotein causes a slight contraction of strips of rabbit femoral artery, which is
markedly potentiated by norepinephrine, phenylephrine, and serotonin. Diltiazem (10 /iM) inhibits these
contractions.232
Therefore, thromboxane A 2 , LTD«, endothelin,
PDGF, catecholamines, and oxidized lipoproteins are
all capable of inducing calcium influx via L-type channels and are also potential mitogens in vivo. However, it
is important to consider the likely provenance of each of
Jackson and Schwartz Pharmacology of Smooth Muscle Replication
these molecules, especially in the light of the report by
Fingerle et al233 that arterial smooth muscle cell replication in the balloon-catheterized rat carotid artery is
not affected by abolition of platelet adherence to the
injured vessel. This suggests that the molecule or molecules mediating proliferation are derived from the
plasma, arterial wall, or leukocytes adhering to the
injured wall.
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Cyclosporin A
Cyclosporin A is an immunosuppressive agent used
to forestall tissue rejection after organ transplantation. It acts by inhibiting both the proliferation of T
lymphocytes and their secretion of cytokines. In the
balloon-injured rat common carotid artery, cyclosporin A treatment resulted in a reduction in neointimal
volume.234 Because the drug had no effect on the
proliferation of smooth muscle cells in vitro, the
authors conclude that the inhibition of neointimal
thickening was mediated by an effect on the immune
system that resulted in impaired smooth muscle cell
proliferation. Another possibility is that cyclosporin A
treatment inhibits the migration of smooth muscle
cells from the media to the intima. However, Ferns et
al235 were able to detect an inhibitory effect of cyclosporin A on the growth of rabbit aortic smooth muscle
cells in serum. This antiproliferative effect was accompanied by vacuolation of the cells, indicating the
possibility of a mild toxicity. Cyclosporin A had no
effect on the proliferation of smooth muscle cells in
the rabbit common carotid artery after atraumatic
removal of the endothelium.
Trapidil
Trapidil is a triazolopyrimidine that was originally
developed as a coronary vasodilator and that has subsequently been found to lower elevated serum levels of
low density lipoprotein and very low density lipoprotein
in humans.236 It inhibits pITJthymidine incorporation
into human thoracic aortic intima] smooth muscle cells
growing in 10% fetal calf serum, at concentrations at
and greater than 5 /xM.236 The peak concentration
achieved in rabbit plasma after subcutaneous administration is approximately 40 /iM.237
Oral administration of trapidil at a dose of 6 mg/kg/
day to rats with air-drying injury to the common carotid
artery significantly reduced the intima-to-media ratio
measured at 14 days.238 Tiell et al239 administered
trapidil orally to rats, starting 3 days before balloon
catheter injury to the aorta. The daily dose was 180 or
360 mg/kg; the oral LDJO in rats is 235 mg/kg (Merck
Index, 1983). These treatment schedules both resulted
in significant decreases in intimal thickening in the
abdominal aorta, measured 14 days after balloon injury.
The authors attribute this effect to inhibition of myointimal hyperplasia. One must question, however, the
validity of a study in which very high doses of a
vasodilator are given before balloon catheter injury. It is
quite possible that reduced vascular tone in the aortas
of trapidil-treated rats resulted in diminished balloon
injury, and this could account for the differences in
intimal thickening seen 2 weeks later.
Liu et al237 investigated the effects of trapidil in a
rabbit model of postangjoplasty restenosis. Animals
were fed cholesterol and were subjected to focal balloon
727
injury to the iliac arteries so that stenoses developed.
These were dilated with an angioplasty balloon, and the
degree of restenosis was determined 4 weeks later.
Treated rabbits received subcutaneous trapidil, 60 mg/
kg/day, beginning 2 days before balloon dilatation.
There were significant reductions in both intimal and
medial thicknesses in trapidil-treated animals when
compared with controls. The authors tentatively attribute these beneficial effects to inhibition of smooth
muscle cell proliferation. Determinations of serum cholesterol concentrations were not made; because trapidil
has cholesterol-lowering properties, it is possible that
the observed effects were related to differences in
serum cholesterol concentration.
Drugs That Interfere With Intracellular Mitogenic
Signal Transduction Pathways
Calmodulin
Calmodulin is a protein that mediates many of the
intracellular effects of calcium ions. The complex between calmodulin and Ca2+ is disrupted by drugs such
as W-7 and trifluoperazine. W-7 inhibits induction of
DNA synthesis in quiescent rat aortic smooth muscle
cells restimulated with serum213 or with epidermal
growth factor or PDGF.205 Trifluoperazine also inhibited restimulation by these mediators.203
Cyclic Nucleotides
Nitric oxide is believed to be an endogenous vasodilator generated by the endothelium. It causes an increase in cyclic guanosine monophosphate (GMP) concentration within smooth muscle cells by inhibiting
cyclic GMP phosphodiesterase. Vasodilators that generate nitric oxide inhibit the proliferation of smooth
muscle cells in vitro. Garg and Hassid240 showed that
sodium nitroprusside, S-nitroso-N-acetylpenicillamine,
and isosorbide dinitrate inhibit DNA synthesis and
growth in rat aortic smooth muscle cells. Kariya et al241
obtained similar results with sodium nitroprusside using
rabbit aortic smooth muscle cells.
Forskolin stimulates cyclic AMP formation by adenylate cyclase and inhibits the restimulation of proliferation by serum in quiescent rat cerebral microvascular
smooth muscle cells.242 It also inhibits the growth of
human aortic smooth muscle cells in serum.118 As
already noted,169 angiopeptin, a somatostatin analogue,
has recently been studied as a smooth muscle growth
inhibitor. Although this approach was based on the
notion that somatostatin inhibits release of pituitary
hormones thought to be important in smooth muscle
proliferation, this class of compounds has more general
effects on protein secretion. These effects are believed
to be due to d protein-induced decreases in cyclic
AMP.243-243 The possibility that this pathway also directly mediates an inhibition of smooth muscle replication has not been explored.
HMGCoA Reductase
3-Hydroxy-3-methylglutaryl coenzyme A (HMGCoA)
reductase catalyzes the formation of mevalonic acid, a
precursor in the biosynthesis of cholesterol. Several
synthetic inhibitors of HMGCoA reductase have been
developed as hypolipidemic agents, and some have been
728
Hypertension
TABLE 2.
Vol 20, No 6 December 1992
Effects of Miscellaneous Compounds on Smooth Muscle Cell Proliferation In Vitro
Concentration
*
Measurement
Stimulus
Effect
Cell origin
Growth
status
Reference
DNA synthesis
Serum
Increase
Rat aorta
Growing
263
Plant poh/phenol
141 fiM
DNA synthesis
None
Increase
Quiescent
268
Laser
photosensitizer
5 /ig/mlt
Cell number
Serum
Decrease
Growing
269
106 mM
DNA synthesis
Serum
Decrease
Growing
270
Dimethyl sulfoxide
Metabolite of
dimethyl sulfoxide
Solvent
128 mM
DNA synthesis
Serum
Decrease
Growing
270
Dimethyl sulfoxide
Solvent
64mMt
Cell number
Scrum
Decrease
Growing
271
Dimethyl sulfoxide
Solvent
128 mM
DNA synthesis
No effect
Growing
272
DNA synthesis
Hyperlipidemic
serum
Serum
Bovine
aorta
Human
carotid
artery
Bovine
aorta
Bovine
aorta
Rabbit
aorta
Rabbit
aorta
Decrease
Rat aorta
Growing
273
Bovine
aorta
Rabbit
aorta
Rat aorta
Quiescent
268
Growing
274
Quiescent
Quiescent
Quiescent
Growing
275
276
276
Growing
278
Quiescent
279
Growing
280
Quiescent
268
Quiescent
268
Growing
272
Quiescent
268
Quiescent
125
Quiescent
281
268
Compound
Category
AUylamine
Industrial
chemical
Chlorogenic acid
Dihematoporphyrin
ester or ether
Dimethyl sulfone
Dinitrotoluene
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Industrial
chemical
Plant poryphenol
165/xM
DNA synthesis
None
Decrease
Etofibrate
Hypolipidemic
0.186 mMt
Cell number
Serum
Decrease
Fenofibric acid
L44
L44-0
Methyl sterculate
Hypolipidemic
Hypolipidemic
Hypolipidemic
Cyclopropcnoid
DNA synthesis
Decrease
Decrease
Decrease
Increase
Monocrotaline
pyrrole
Ellagic acid
Nicotine
Peanut lectin
25/tM§
25 A M §
2 /tg/ml
Cell number
Cell number
DNA synthesis
PDGF
Serum
Serum
Serum
Pulmonary toxin
0.5 Mg/m't
Cell number
Serum
Decrease
Tobacco
constituent
Plant lectin
1 nM
DNA synthesis
None
Increase
Rat
Rat
Rabbit
aorta
Bovine
pulmonary
artery
Rat aorta
2 jig/mlf
Cell number
Serum
Increase
Primate
200
MM§
Quercetin
Plant poh/phenol
83 fiM
DNA synthesis
None
Increase
Rutin
Tobacco
constituent
Pollen component
25/ig/mlf
DNA synthesis
None
Increase
NR
DNA synthesis
Decrease
25/ig/mlt
DNA synthesis
Terbinafine
Tobacco
constituent
Antimycotic
Hyperlipidemic
serum
None
5/Jvl§
DNA synthesis
PDGF
Decrease
TMB-8
Tobacco
gfycoprotein
Unknown
Tobacco
constituent
30jiM§
Cell number
DNA synthesis
Serum
Decrease
Increase
0-Sitosteryl
palmitate
Tar
25 jig/mlt
None
Increase
aorta
Bovine
aorta
Bovine
aorta
Rabbit
aorta
Bovine
aorta
Bovine
aorta
Rat aorta
Bovine
aorta
Quiescent
277
PDGF, platelet-derived growth factor; NR, not reported.
*Cells were isolated from animals dosed orally, 70 mg/kg of body weight per day for 20 days.
tMinimum effective concentration.
JCells were isolated from animals dosed intraperitoneally, 0.5 mg/kg of body weight per day for 8 weeks.
§100-
investigated for possible effects on vascular smooth muscle cell proliferation.
Compactin significantly inhibited DNA synthesis in
quiescent primate aortic smooth muscle cells restimulated with PDGF.2*5 This effect was reversed by addition of mevalonic acid to the cells but not by addition of
cholesterol, suggesting that some other derivative of
mevalonic acid is required for DNA synthesis in vascu-
lar smooth muscle cells. Mevinolin significantly inhibited DNA synthesis in quiescent bovine aortic smooth
muscle cells restimulated with serum.247 It should be
noted that products of mevalonic acid are incorporated
into a number of cell proteins, including nuclear
lamins.246-248-250
In cholesterol-fed rabbits, mevinolin administration
significantly reduced the neointimal cross-sectional area
Jackson and Schwartz
Pharmacology of Smooth Muscle Replication
729
TABLE 3. Effect* of Miscellaneous Componnds on Smooth Mnsde Cell Proliferation In Vivo
Compound
Route Species
Category
Dose
Defibrinogenator
100 units/kg
i.v.
Etofibrate
Hypolipidemic
200 mg/kg/day
p.o.
Etofibrate
Hypolipidemic
200 mg/kg/day
p.o.
Terbinafine
Antimycotic
200 mg/kg/day
p.o.
Arvin
Model
Duration Measurement
Air-drying
14 days
Neointimal
injury
volume
Rabbit
Electrical
4 weeks
Intimal
stimulation;
thickness
cholesterol-fed
Rabbit
Electrical
4 weeks
Intimal
stimulation;
thickness
cholesterol-fed
Rat Balloon injury 14 days Intimal area
Rat
Effect
40% decrease
Reference
33% decrease
274
46% decrease*
283
40% decrease
125
282
*No effect on serum cholesterol concentration.
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
after air-drying injury to the femoral artery.251 Possibly,
the reduced concentration of serum cholesterol in the
mevinolin-treated group was responsible for this effect.
However, in both control and treated groups, the serum
cholesterol concentration was greatly elevated above
normal, and it is conceivable that a direct effect on
cellular proliferation contributed to the reduced neointimal thickening.
Membrane Ion Transport
In many cell types, there is a close link between
mitogenesis and elevation of cytoplasmic pH via a
membrane Na7H + exchange mechanism. Amiloride
and its derivative, ethylisopropylamiloride, inhibit the
Na7H + exchange mechanism and prevent alkalinization
of the cytoplasm. Bobik et al252^253 have shown that
ethylisopropylamiloride inhibits the proliferation both
of quiescent rat aortic smooth muscle cells restimulated
with serum and of cells growing exponentially in serum.
Amiloride had similar effects but was less potent.
Monensin also inhibits cytoplasmic alkalinization by
catalyzing the expulsion of sodium ions through the
N7H + exchange mechanism. It inhibits the proliferation
of quiescent rat aortic smooth muscle cells incubated
with either PDGF or serum, with an IC*, of 5 fiM.254
Inhibition of the membrane Na+,K+-ATPase with
ouabain inhibits the proliferation of rat aortic smooth
muscle cells growing in serum252 and depresses further
the low rate of DNA synthesis in quiescent rabbit aortic
smooth muscle cells.255
Microtubules
Colchicine causes disassembly of microtubules in cultured rat aortic smooth muscle cells and stimulates the
proliferation of these cells in serum-free medium.256 When
quiescent cells were exposed to PDGF or to serum,
colchicine inhibited DNA synthesis. The authors suggest
that disassembly of microtubules is an obligatory step in
the pathway leading to DNA synthesis and that colchicine
mimics this. Because colchicine inhibits endocytosis, the
inhibitory effects on growth factor-stimulated growth may
have been the consequence of decreased growth factor/
receptor complex intemalization.
Protein Kinase C
The role of protein kinase C in vascular smooth
muscle cell mitogenesis is not clear. It has been
investigated both by using tumor-promoting phorbol
esters that activate it and by using synthetic inhibitors,
but the results are equivocal. Owen257 found that
12-O-tetradecanoylphorbol-13-acetate (TPA) stimu-
lated DNA synthesis in quiescent cultures of the A7r5
smooth muscle cell line. In quiescent cultures exposed
to epidermal growth factor, TPA significantly inhibited DNA synthesis. In quiescent neonatal rat vascular
smooth muscle cells, TPA did not stimulate DNA
synthesis, but it did inhibit the mitogenic effect of
a-thrombin.175 TPA also inhibited DNA synthesis in
quiescent rabbit aortic smooth muscle cells restimulated with serum.258.259 The inhibitory effects of TPA
in all of these reports were obtained with concentrations of approximately 5 nM. Similar concentrations
significantly stimulated the proliferation of quiescent
bovine smooth muscle cells isolated from carotid
artery260 and pulmonary artery,261 and 100 nM TPA
stimulated DNA synthesis in quiescent rat aortic
smooth muscle cells.262-264
The published results obtained with synthetic inhibitors of protein kinase C show a clearer picture. H-7
inhibited the restimulation of quiescent rat aortic
smooth muscle cells by both PDGF and epidermal
growth factor.262 Staurosporine was effective in quiescent rabbit aortic smooth muscle cells restimulated with
serum,265 and K252a had similar effects in bovine carotid arterial smooth muscle cells.260
Tyrosine Kinase
Proliferation induced by epidermal growth factor in
rat aortic smooth muscle cells was inhibited by the
tyrosine kinase inhibitor genistein, with an ICJO of
85 /iM.266 Various tyrosine kinase inhibitors of the
tyrphostin type reversibly inhibit PDGF-stimulated
growth of rabbit arterial smooth muscle cells, with IQo
values as low as 40 nM.267
Miscellaneous Agents
The effects of a variety of agents that cannot easily be
categorized are summarized in Table 2 (in vitro studies)
and Table 3 (in vivo studies).
Summary
The first point to be made in summary is that the
potential list of compounds is more extensive than the
usual limited focus on polypeptide growth factors. A
number of fairly well understood mediators seem to be
as active as growth factors in stimulating and inhibiting
smooth muscle proliferation; these pathways merit
further study. The issues are the usual ones for a
pharmacological study. Mechanisms are most easily
explored when we have well-defined antagonists as
well as agonists. For many of the mediators discussed
above, there is insufficient evidence to reach firm
730
Hypertension
Vol 20, No 6 December 1992
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conclusions because of a lack of specific antagonists.
This is a problem particularly for molecules posited to
have an inhibitory function such as steroids, heparinoids, or somatostatin. In other cases, tools are available and the experiments are feasible (angiotensin,
thrombin, eicosanoids, serotonin).
Second, the recent successful studies with growth
factors and growth factor antagonists open a new frontier. The in vivo studies suggest that these molecules
may control specific parts of the proliferative response;
for example, bFGF controls the first wave of DNA
synthesis as a repair response to cell death, whereas
PDGF seems to play a role in migration into the intima.
The greatest value of in vitro studies may be to reveal
how these mediators exert their effects on cells and thus
allow us to identify pathways that may be blocked either
with new drugs or with existing drugs that affect relevant
pathways.
Third, we must finish with a note of caution. Although
the evidence that smooth muscle proliferation occurs is
compelling in atherosclerosis, hypertension, and restenosis, the relation of smooth muscle proliferation to the
final clinical event, i.e., vascular narrowing or obstruction, is certainly not established. Perhaps the surest
benefit of being able to control smooth muscle growth
will be to determine the extent to which this pathological response is etiologic in the clinical manifestation of
vascular diseases.
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Pharmacology of smooth muscle cell replication.
C L Jackson and S M Schwartz
Hypertension. 1992;20:713-736
doi: 10.1161/01.HYP.20.6.713
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