Clinical Science (1994) 87, 467479 (Printed in Great Britain)
467
Editorial Review
Smoothmusclecell proliferation and differentiation in
neointima formation and vascular restenosis
Paolo PAULETTO, Saverio SARTORE* and Achille Cesare PESSINA
lstituto di Medicina Clinica and *Diportimento di Scienze Biomediche. Universita di Padova,
Padova, Italy
INTRODUCTION
Revascularization procedures in significant atherosclerotic narrowings range from vascular/
endovascular surgery to interventional radiology/
cardiology (Figure 1). Percutaneous angioplasty,
endoatherectomy, vein grafts and synthetic grafts
are the most widely applied procedures today. Relatively more modern approaches of great clinical
relevance and potentiality include directional atherectomy and the use of intra-arterial stents. Biografts
and reimplantation of autologous vessels provide an
optimal management of advanced atherosclerosis. It
is through these techniques that revascularization
can be successfully achieved from the large conduit
(aorta) to muscular (coronary) arteries.
Although in most cases blood vessels can be
reconstructed either by angioplasty or by-pass surgery, they are subject to subsequent stenosis with a
reduction in blood flow and eventual thrombosis.
After carotid atherectomy, 10-20% of reconstructed
vessels develop significant luminal narrowing even
Percutaneous angioplasty
Directional atherectomy
lntra-arterial stents
Endovascular surgery
or intenentiond
radiology/cardiology
Endortherectomy
Vascular surgery
BYPW
grafting
I
Prortheses
(PTFE. Dacron)
Veins
(great saphenous,
arm, etc.)
Arteries
(mammary, arm
homologous, etc.)
Direct anastomosis
Fig. I. Revasculariution procedures
(reimplantation)
though the patients very often remain asymptomatic
[1,2]. Moreover, remodelling of coronary arteries
after balloon angioplasty is followed by haemodynamically significant restenosis with the reappearance
of angina in about 3040% of patients [3,4]. Femoropopliteal vein by-pass and superficial femoral
angioplasty or atherectomy display a similar rate of
restenosis [5,6].
Myointimal hyperplasia, thrombosis and late vascular recoil are the main causes of vascular restenosis occurring in the first six months after revascularization. In most cases, the luminal narrowing is due
to myointimal proliferation [1-3,7] and at gross
inspection the lesion appears to be fibrous and free
of overlying thrombus. At later times (more than 2
years), the intimal lesion resembles a primary atherosclerotic lesion in terms of cell composition and a
thrombus is commonly found. Hence, myointimal
proliferation seems to represent a key event, initiating a new atherogenic process leading to luminal
narrowing, and smooth muscle cells (SMCs) account
for the majority of the cells found in the restenotic
intima.
In this review, we will focus on the main mechanisms leading to myointimal proliferation and restenosis after revascularization with a special reference
to the role played by SMCs under different phenotypic status.
STRUCTURAL CHANGES AFTER
REVASCULARIZATION
Early after the different revascularization procedures, striking and specific changes in the structure of the arterial wall can be observed (Figure 2).
Angioplasty is followed by some splitting, fissuring and dissection of plaque and media, whereas
endoatherectomy involves removal of the stenosing
Key words: arterial wdl, intimal proliferation, restenoris, smooth muscle cells, smooth-muscle-celldifferentiation.
Abbreviations ACE, angiotensitxonvening enzyme; ANG II, angiotensin II; bFGF. basic fibroblast growth factor; EDRF, endotheliumderived relaxing factor; FGF, fibroblast
growth factor; IGF-I. insulin-iike growth factor I; 11-1, interleukin I; MyHC, myosin heavy chains; PCNA. anti-proliferating cell nuclear antigen; PDGF, plateletderived growth
factor; SMC, smooth muscle cell; TGF-PI, transforming growth factor PI; [PA. tissue-type plasminogen activator; uPA, urokinase-type plasminogen activator.
:Dr P. F’auletto, lrtituto di MedKina Clinka, Universiti di Padova, Via Giustiniani, 2, 35125 Padova, My.
P. Pauletto et al.
Angioplasty/endoatherectomy
(early)
Splitting, fissuring, dissection
of plaque/media
Platelet depasition
Synthetic grafts
(early)
lnsudative process
Granulation tissue
Fibrous tissue
Endothelization
neonii!tf ; e and
tion
of leukocytes
release
Chemotaxis
and mitogens
and macrophages
lntima remcdelling/formation
~
Vein grafts
//jjJJ
(early)
Increased E L . turnover
Thickening of the tunica media
Splitting of the i.e.1.
lntimal thickening
Initial accumulation of:
Proliferating/migrating SMC
Macrophage/SMCderived foam cells
Activated T lymphocytes
I
(complications)
thrombosis, aneurysms, calcifications
Fig. 2 Main structural characteristics of restenosis. Abbreviations: E.C., endothelial cells; i.e.l., internal elastic lamina.
intimal lesion along with part of the underlying
media. Both processes can be followed by platelet
deposition and, as discussed below, cellular release
of mitogens for medial SMCs such as plateletderived growth factor (PDGF), transforming growth
factor-81 (TGF-pl), basic fibroblast growth factor
(bFGF) and interleukin-1 (IL-1)[8-lo]. This kind
of injury is also liable to provoke local chemotaxis
of leucocytes and macrophages which can play an
important role in the development of subsequent
intimal lesions [8-101. On the other hand, Dacron
and poly(tetrafluoroethy1ene) (PTFE) grafts undergo
a distinct ‘repair’ process consisting of insudation of
plasma proteins, followed by formation of granulation tissue and then of fibrosis [1 1,121.This process
is completed by partial (in humans) or total (in
some experimental conditions) endothelialization
with neointima formation [12]. Vein grafts used as
arterial by-passes undergo a sort of ‘adaptive’ process to the new haemodynamic conditions, which
are very different from those reported above. Soon
after surgery, an increased turnover of endothelial
cells takes place, along with progressive thickening
of the medial layer. Splitting of the internal elastic
lamina, thickening of the intima, and extracellular
matrix deposition then occur, completing the ‘arterialization’ of the vascular wall. After 1 year, fibrin,
lipid and calcium deposits can be found [13,14].
Months or years after revascularization and the
establishment of the initial neointima, luminal narrowing can develop due to continued intimal
accumulation of proliferatindmigrating SMCs. This
process is common to all the revascularization
procedures (Figure 2) and is often complicated by
the appearance of foam cells and inflammatory cells,
so that the resulting picture may resemble that of
primary atherosclerotic lesions. Thrombosis, aneur-
ysms, and calcium deposits may represent a further
structural change (or clinical complication) of a
revascularized vessel [1 ,1 1,13,14].
On the whole, myointimal proliferation of SMCs
is considered to be the key event in restenosis [3,710). A better definition of factors initiating SMC
hyperplasia and determining the degree of its extent
seems to be of paramount importance for the
understanding of this process and for strategies
aimed at reducing the rate of arterial narrowing
after revascularization.
FACTORS INVOLVED IN MYOINTIMAL
PROLIFERATION
Some humoral and cellular factors, such as the
local expression of cytokines and growth factors
[8-lo], the renin-angiotensin system activation
[15,163, the haemodynamic characteristics of the
circulation [3,17-191, the synthetic tissue characteristics [12,20], and the phenotypic state of vascular
SMCs [21-231, are potentially involved in controlling myointimal proliferation/differentiation and
restenosis.
Interplay between SMCs, growth factors and cytokines
The balloon injury model represents an interesting tool for studying the impact of such factors. As
a consequence of balloon stripping, the endothelial
layer is removed from the injured area and the
underlying SMCs are damaged. About 24 h later,
medial SMCs start synthesizing DNA [24] and only
after several days can migrating SMCs be found in
the intima [25]. Studies by Clowes and colleagues
[24,25] have shown that after balloon injury
approximately one-half of the SMCs entering the
Restenosis after revascularization
intima are non-dividing. Non-dividing but migrating
SMCs can be identified because they are not
labelled by tritiated thymidine delivered by continuous infusion. In the rat model, dividing SMCs,
once in the intima, continue to replicate and start
synthesizing extracellular matrix proteins. These
cells accumulate in the neointima for about 2 weeks.
The further increase of neointima is mainly due to
extracellular matrix production, which accounts for
about 80% of the entire tissue volume by 3 months
after arterial injury, when the steady state of lesion
development is reached [26].
The presence in the normal vessel wall of either
media or media plus intima as well as the extent of
initial SMC damage are important factors for the
subsequent response of the arterial wall. Another
relevant factor is the injury procedure adopted. The
so-called ‘gentle filament denudation’ obtained by
Reidy and colleagues [lo] using a loop of nylon
monofilament causes endothelial cell removal without appreciable SMC damage, whereas the traditional balloon catheter denudation is accompanied by
about 25% DNA loss [lo], which is indicative of
extensive SMC death. After either procedure, intima1 SMC proliferation occurs but to a very different
extent. In fact, in filament denudation, the initial
replication rate of SMCs is 1.4% compared with
13.6% after balloon injury. This result implies that
the interaction of platelets with the exposed subendothelium does not play a crucial role in triggering
extensive SMC proliferation in uivo [24,27]. Indeed,
in rats made thrombocytopenic and subjected to
balloon injury [28], a marked replication of SMCs
was observed within 48h, but the growth of the
intimal lesion was limited. This suggested that platelets and PDGF play a role in intimal migration,
rather than in replication of SMCs. This has been
proven to be the case after experiments in which
either PDGF [29] or a PDGF-neutralizing antibody [30] was administered to animals subjected to
balloon injury. Moreover, Majesky and colleagues
[31,32] using the Northern-blotting technique found
that SMCs from rat carotid arteries express transcripts for PDGF A-chain and TGF-B1 starting
from 6 h after balloon injury. Immediate-early gene
expression such as c-myc and thrombospondin is
increased after balloon injury and precedes the
transcription of PDGF-A, TGF-PI and bFGF [33].
The precise meaning of these observations is not
fully understood as carotid neointimal SMCs are
not thought to possess the a-receptor for PDGF
[34], and TGF-Bl is secreted as an inactive precursor and has a bimodal action on SMC growth
[34,35]. These results, however, do not exclude per
se a potential for autocrine or paracrine regulation
of SMC proliferation after arterial injury. In fact,
variations in PDGF-receptor expression are part of
the arterial wall response to injury [31] and TGFB1 might be converted into the active form by the
increased levels of tissue-type plasminogen and plasmin activity usually found in injured arteries [36-
469
391. In turn, the high plasmin levels, which seem to
be induced by the increased expression of PDGF,
may represent a further stimulus for SMC migration
after injury [39].
Nevertheless, PDGF typically acts as a ‘competence factor’ in that it enables quiescent cells to
move from the GO to the G1 phase of the cell cycle
and a second ‘progression factor’, such as the
insulin-like growth factor I (IGF-I), is required for
DNA synthesis and cell replication to take place.
IGF-I gene expression markedly increases in SMCs
stimulated by PDGF itself, angiotensin I1 (ANG II),
IL-1, growth hormone and balloon injury [9,15,40].
As IGF-I is effective in stimulating elastin biosynthesis [41], the increased expression of this growth
factor after balloon injury might be part of a repair
process rather than a mechanism of SMC proliferation. In addition, synergism of action between
PDGF and IGF-I has been described in different
models of wound healing [42]. It seems likely that
PDGF released by platelets contributes to the first
wave of medial SMC proliferation soon after ballooning, whereas subsequent PDGF release by
SMCs underlies the migration into the intima
[29,30].
The bulk of SMC proliferation occurring after the
initial wave seems to be due to the action of bFGF
[10,43,44]. Acidic and basic FGF display angiogenic
properties and mitogenic activity for SMCs and
endothelial cells [45]. Although present in the basement membrane and extracellular matrix, FGFs are
largely cell-associated. As they do not possess a
signal-peptide sequence, they cannot be secreted in
the extracellular milieu like other growth factors.
Conversely, they can be released after cell death so
that the amount of FGF released after arterial
injury would reflect the extent of SMC damage.
Leucocytes and macrophages infiltrating the injured
area may also secrete heparinases and proteolytic
enzymes, which are capable of degrading extracellular matrix and releasing the FGF aliquot stored
therein. In the intact carotid artery of rat, bFGF
transcript has been found using Northern-blot
analysis, and its expression at the protein level has
been documented by immunocytochemistry and
Western blotting [43]. In situ hybridization studies
on en face preparations of injured rat arteries
showed that mRNA for bFGF and its receptor 1
was expressed by replicating SMCs and endothelial
cells only, thus suggesting a role for this growth
factor not only in maintenance of SMC replication
but also in endothelial-lining regeneration [46].
Indeed, bFGF infusion for 8 h in rats after balloon catheter injury had been produced in the
carotid artery, was followed by remarkable SMC
replication 24-48 h later, as determined by administration of C3H]thyrnidine and autoradiography [43].
The size of the intimal lesion increased twofold in
comparison with balloon-catheterized controls
receiving vehicle alone. In arteries damaged using
the filament loop, an increase in SMC replication
470
P. Pauletto et al.
similar to that found after balloon catheterization
was present. Another relevant point about the role
played by bFGF was raised by the same authors
using an antibody specific to bFGF [44]. In uitro,
this antibody inhibited the mitogenic effect of bFGF
but not that of serum or other growth factors.
When administered to rats before carotid balloon
injury, the anti-bFGF antibody was very effective in
reducing SMC replication, which turned out to be
at levels comparable with those observed after the
‘gentle filament denudation’ procedure mentioned
above (about 1.4%). It remains to be established
whether a prolonged blockade of bFGF is able to
prevent not only SMC replication during the first
hours after arterial injury but also the development
of a significant myointimal proliferation over a
longer period of time.
The potential role of monocytederived macrophages
On the whole, on the basis of the activation of
specific molecular mechanisms, massive intimal
SMC proliferation and migration occurs after experimental balloon injury, so that SMCs represent the
major cell type found in the neointima. However, as
recently pointed out by Libby and colleagues [9],
studies on myointimal hyperplasia after balloon
injury to previously normal arteries may not reflect
the response of atherosclerotic vessels. In fact, atherosclerotic lesions are rich in leucocytes (particularly
monocyte-derived macrophages) and foam cells
[47,48] producing a number of proteins, cytokines
and growth factors able to alter the physiological
response of the vascular wall. The failure of most
clinical trials to reduce restenosis despite favourable
results in the experimental setting may reflect the
fundamental biological diversity between normal
and atherosclerotic arteries. The ‘cascade model’ of
restenosis biology proposed by Libby and colleagues [9] hypothesizes that, after arterial injury,
acute local blood coagulation or thrombosis and/or
stretch or crush of the vascular wall activate
cytokine/growth factor gene expression by macrophages and/or SMCs of the plaque. Proteins such as
IL-1, PDGF, FGFs, tumour necrosis factor,
heparin-binding epidermal growth factor, transforming growth factor-a, etc. might act as mitogens for
SMCs and evoke secondary, self-sustaining expression of growth factors and cytokines by macrophages and SMCs themselves that could account for
the lag between initial injury and restenosis.
This hypothesis emphasizes the importance of
macrophages as intermediary cells between the
initial injury and SMC activation and proliferation.
Studies carried out in rabbits subjected to retrograde balloon pullback followed by cholesterol feeding [49] support this view. Under these experimental conditions, an early macrophage infiltrate was
observed along the internal elastic lamina, and a
roughly similar number of SMCs was present on the
luminal side. Moreover, the hypothesis proposed by
Libby and colleagues deserves some attention for
thrombosis and coagulation as potentially relevant
factors in the initiation and maintenance of myointima1 proliferation. Indeed, products of coagulation
and thrombosis can activate mononuclear phagocytes and elicit expression of IL-1 [SO], enhance
macrophage colony-stimulating factor-driven mitogenesis [Sl] and modify the activity of lipoprotein
receptors [52]. In addition, a-thrombin stimulates
proliferation of cultured SMCs via the activation of
a specific receptor [53] and infusion of an inhibitor
of its proteolytic activity interrupts the augmentation of PDGF-A mRNA expression induced by
arterial injury [54].
The renin-angiotensin system
The finding that angiotensin-converting enzyme
(ACE) inhibitors prevent myointimal hyperplasia
after vascular injury [SS] adds another relevant
piece of information about the mechanisms whereby
restenosis can develop, and raises the question
about the role played by the renin-angiotensin
system in the pathogenesis of myointimal hyperplasia.
ACE inhibitors might be effective in preventing
myointimal proliferation in several ways including
their effects on blood pressure and the secondary
accumulation of bradykinin which may stimulate
endothelial cells to produce growth inhibitors such
as prostacyclin (PGI,) and endothelium-derived
relaxing factor (EDRF). However, these hypotheses
are unlikely, as verapamil does not prevent development of intimal thickening after balloon injury
despite a reduction in blood pressure comparable
with that obtained with ACE inhibitors, and an
endothelium-mediated antiproliferative effect is
unlikely to occur anyhow as the endothelial cells are
completely removed at the site of injury [SS].
On the other hand, studies by Daemen and
colleagues [56] have shown that a continuous subcutaneous infusion of ANG I1 in rats is able to
induce SMC proliferation both in intact and
balloon-injured carotid arteries. Furthermore, an
overexpression of a specific ANG I1 receptor subtype, the ATl, has been described in rat aorta after
balloon angioplasty [161. Blockade of this receptor
by the specific antagonists DuP753 [lS] or TCV116 [57] prevents intimal thickening in rats after
balloon injury, with some normalization in the
relaxing properties of the arterial wall despite the
arterial trauma [57].
ANG I1 has a full potential for promoting myointimal proliferation after injury as it displays multifaceted growth properties for SMCs and is capable
of interacting in several ways with most growth
factors and cytokines involved in myointimal proliferation. ANG 11, at a minimum concentration of
100 nmol/l, was able to induce proliferation of
human SMCs from young aortas growing in
secondary cultures in the presence of 10% serum
Restenosir after revawlarkation
[58]. In secondary SMC cultures from rat mesenteric artery grown in the presence of 10% fetal
bovine serum, an increased cell proliferation was
observed in response to lower concentrations of
ANG I1 (1 nmol/l; [59]). This effect was blocked
using the antagonist saralasin [59]. On the
contrary, other authors have reported that various
concentrations of ANG I1 only have an hypertrophic effect (with polyploidy) on secondary cultures
of SMCs prepared from rat thoracic aortas [60]. It
has also been suggested that the hyperplastic response of SMCs to ANG I1 depends on the continuous exposure of these cells to factors present in
platelet-poor plasma-derived serum [6 11.
It has been hypothesized that ANG 11, as noradrenaline, has only indirect effects on SMC growth
that would depend on growth factors like PDGFAA dimers, the expression of which is stimulated by
vasoconstrictor substances [62]. ANG I1 also seems
to be able to induce an increase in SMC PDGF B
receptors making the cells more responsive to
PDGF-BB [63]. This in turn stimulates DNA synthesis and cell proliferation [63]. In contrast, other
studies have shown that ANG I1 is able to increase
the proliferative response to PDGF-BB only in
SMCs from spontaneously hypertensive rats, but
not from normotensive control rats [64]. These
experiments have also shown that TGF-B1 contributes to ANG 11-induced SMC proliferation [64].
On the other hand, studies performed by other
groups indicate that the ANG 11-induced secretion
of active TGF-Bl is involved in the inhibition of
SMC proliferation. In these latter studies, SMC
proliferation induced by ANG I1 seems to be
mediated through bFGF and PDGF-AA [65-671.
These studies showed that at least three growth
factors are involved in the modulation of SMC
growth stimulated by ANG 11. Many other variables, such as the source of SMCs and their growth
status in relation to culture conditions, may
influence the effects of these factors. Recent studies
showed the pathways by which ANG I1 stimulates
confluent and quiescent human SMCs from omental
vessels to synthesize active endothelin, reinforcing
ANG I1 stimulation on SMC contractile tone and
growth [68]. Moreover, ANG I1 increases mRNA
levels of growth-related proto-oncogenes c-fos, c-myc
and c-jun [69-71) in SMCs. This effect, at least in
the case of the increase of c-fos and phosphoinositide turnover, appears to be mediated through the
AT1 receptor [72]. In light of these findings, ANG
I1 is able to stimulate SMC growth independently
from its haemodynamic effects. It is worthwhile
noting that in our laboratory, the infusion of ANG
I1 for 2 weeks in rabbits at either subhypertensive
or hypertensive doses resulted in the development of
intimal thickening with a marked increase of
postnatal-type SMCs and the appearance of fetaltype SMCs (see the next section) both in the aortic
media and intima [73]. A similar finding was
observed in the aorta of renovascular hypertensive
47 I
rabbits [74]. Therefore, the local level of ANG I1
production seems to modulate the phenotypic
changes of aortic SMCs.
Finally, it has been recently reported that the
insertion/deletion (I/D) polymorphism of the ACE
gene may represent a new risk factor for restenosis
after coronary angioplasty. The D D genotype of ACE
is in fact associated with development of restenosis
independently from known risk factors [75].
Changes in extracellular matrix
As previously mentioned, relevant extracellular
matrix deposition takes place in the intima after
balloon injury and accounts for most of the neointima formed also in clinically relevant restenosis. In
the clinical setting, production of extracellular
matrix by SMC is a relatively long-lasting process
leading to restenosis 3-6 months after revascularization, well beyond the few weeks of SMC
proliferation/migration into the intima. The main
components of extracellular matrix in restenosis are
glycosaminoglycans and collagen [76]. The former
predominate in the early stages of arterial remodelling, whereas the latter increase in the long term.
Dermatan sulphate and chondroitin sulphate are the
main glycosaminoglycans produced by SMCs. In
rats, the glycoprotein tenascin appears in the neointima formed by proliferating SMCs 2 weeks after
balloon injury of the carotid artery and is not found
in the adjacent media [77]. I n uitro experiments
showed that tenascin expression accompanies the
shift of SMCs towards an immature (‘synthetic’)
phenotype [77]. Tenascin-containing matrix might
be important for promoting further SMC proliferation after injury because this glycoprotein has a
growth-stimulatory activity.
It is well established that the phenotypic state of
SMCs [78] is the main determinant of extracellular
matrix production. This is in fact a distinct feature
of ‘synthetic’ or undifferentiated SMCs. Other
potentially important factors are some cytokines
and growth factors such as TGF-B1 [79]. In cultured SMCs, PDGF and TGF-B1 increase the
expression of collagen types I and 111 both at the
mRNA and the protein level. This action is markedly inhibited by interferon-y, which also inhibits
SMC proliferation [SO]. ANG I1 might intervene
through secondary changes in TGF-B1 expression
[65-67).
On the other hand, heparan sulphate proteoglycan produced by regenerating endothelial cells and
the related molecule heparin are able to inhibit
SMC proliferation and migration in uitro and in uiuo
[8 1,821. Moreover, heparin interferes with the extracellular matrix composition by decreasing elastin
and interstitial collagen and increasing the amount
of proteoglycans [83]. Heparin’s molecular mechanism of action is not fully understood but it is likely
to occur through the inhibition of proto-oncogene
expression and/or synthesis and activity of various
472
P. Pauletto et al.
growth factors and their receptors [84-861. Heparin
also inhibits the expression of the tissue-type plasminogen activator (tPA) by SMCs in injured rat
carotid artery [37]. Plasminogen activators are
indirectly involved in the control of the degradation
of the matrix network surrounding SMCs and thus
may facilitate SMCs to proliferate, migrate and
produce new extracellular matrix. In fact, urokinasetype plasminogen activator (uPA) and tPA convert
plasminogen into plasmin, which not only degrades
fibrin but also a broad range of matrix molecules
and activates procollagenase to collagenase. uPA
activity, as measured in extracts of rat carotid
arteries after balloon injury, peaks 16-24h after the
procedure, whereas tPA activity is detectable at 3
days and peaks at 7 days. An increase in the related
mRNAs was also found in SMCs [36,38].
Hence, it seems that after arterial injury, medial
SMCs express uPA at the time they are proliferating
and tPA when migrating into intima [34]. The
decreased tPA activity observed in injured arteries
after heparin infusion is likely to represent one of
the mechanisms by which this molecule can inhibit
SMC migration to intima and interfere with extracellular matrix composition.
Haemodynamic factors
Several studies suggest that increased blood flow
across injured arterial segments may reduce intimal
hyperplasia. A flow-related increase in wall shear
stress has been associated with reduced intimal
thickening in balloon-injured arteries [191, experimental vein [87,88] and PTFE grafts [17,18], and
primary atherosclerotic lesions [89,90]. Under physiological conditions, as well as in atherosclerotic
vessels, arterial diameter varies in relation to
changes in blood flow in order to maintain a
relatively stable level of shear force [91-931. Moreover, media and intima thickness increase with
increasing intravascular pressure, so that a constant
wall stress is maintained in this way [94].
Adaptations of the arterial wall to both increased
and decreased blood flow are thought to be
mediated by the endothelium. A signal is likely to
start from the endothelial lining as SMCs within the
wall are unlikely to sense changes in blood-flow
characteristics. In fact, even high shear rates cause a
negligible deformation of the arterial wall [92]. On
the other hand, flow characteristics influence many
aspects of endothelium biology including cell turnover, shape, orientation and cytoskeletal composition [95-971. At least in uitro, the occurrence of a
turbulent flow increases endothelial cell proliferation
even more than the shear level per se [95]. Flow
characteristics are liable to modulate the endothelial
cell production of SMC regulatory molecules. The
endothelium is known to synthesize and secrete
some vasoactive substances which also act as
growth promoters (PDGF, TGF-1, FGFs, IL-1) or
inhibitors (EDRF, heparan sulphate). In particular,
EDRF and PDGF production by endothelial cells
are influenced by shear stress in uitro [98,99]. It is
interesting that in relatively rigid PTFE grafts
implanted in baboons [ 181, the neointimal area,
SMC proliferation and SMC content of the artificial
wall could be regulated solely by changes in the
magnitude of shear stress maintained within the
physiological range of 10-30 dyn/cm2. While most
studies agree that high magnitudes of shear are
associated with a reduced intimal thickening, it
remains to be established whether shear fluctuation
or variation in direction over time is accompanied
by a lesser or a larger intimal thickening
[17,87,100].
Synthetic tissue characteristics
In by-pass procedures, synthetic grafts represent a
good alternative to the use of vascular tissue,
especially in high-flow districts. PTFE and Dacron
represent the routine materials today. As already
mentioned, these grafts can fail because of progressive luminal narrowing due to myointimal hyperplasia, development of sudden and unexplained thrombosis, particularly under low flow conditions, and
are also subject to infection.
Some characteristics of the material from which
the graft is made potentially affect the restenosis
rate by interfering with the healing process, endothelial cell regrowth, and the extent of neointima
formation. This topic has been recently reviewed by
Clowes and Kohler [12].
Usually, complete endothelial coverage of grafts is
not achieved in humans [loll. The endothelial
lining in fact grows from the adjacent anastomoses
for the first 2 or 3 cm whereas the central portion
remains uncovered and continues to accumulate
platelets. SMCs proliferate beneath the regenerated
endothelium but not outside its edges and form a
thickened neointima mainly at the anastomotic
sides. The endothelium derives from microvessels of
the granulation tissue which are likely to penetrate
into synthetic tissue from outside under the
influence of angiogenic substances released by clotting products [102,103].
Using PTFE grafts in animal models, complete
endothelial cell coverage can be observed by 2
weeks [102,104]. Then, SMCs seemed to derive
from pericytes of microvessels growing in the synthetic tissue and proliferated to form the neointima.
This process is generally completed in 2 or 3
months. Intimal hyperplasia is found at sites of
endothelial cell regeneration and not at sites of
denudation. Large amounts of PDGF A-chain
mRNA have been found in extracts from the neointima of PTFE grafts [lOS]. Studies on perfusates of
PTFE grafts retrieved at various times showed
considerable mitogenic activity partially blocked by
a polyclonal antibody specific to PDGF [106].
Hence, growth-promoting activity seems to originate
from the graft vascular cells themselves, indicating
Ratenosis after revascularization
that platelets and their products are not the main
source of growth factors for SMC proliferation.
Experimental studies aimed at obtaining a complete luminal endothelial coverage of PTFE grafts
showed that an internodal distance of 60pm is an
optimal porosity for these grafts. Conversely, 10 and
30pm grafts do not achieve full endothelial cell
coverage and 60% of 90pm grafts exhibit focal
endothelial cell loss and platelet deposition at 3
months [20]. Dacron grafts also display an incomplete endothelial cell coverage and continue to take
up indium 111-labelled platelets for a long time
after implantation [107,108]. Moreover, exposure of
macrophages from either normal or hypercholesterolaemic rabbits to Dacron results in an increased
release of mitogenic factors from these cells which
are especially active on SMCs from hypercholesterolaemic animals [l09]. This may represent a mechanism leading to myointimal hyperplasia.
Recently, some features of bioresorbable prostheses (polyglactin 910 and polydioxanone in various proportions) have been studied in experimental
animals. Bioresorbable materials induce the formation of an inner capsule composed of myofibroblasts
and collagen beneath regenerating confluent endothelium [l lo]. In addition, macrophages which phagocytose these prostheses release growth factors
possibly involved in the formation of wall tissue
[lll]. Release of monokines from monocytes is
hampered by hypercholesterolaemia [111,112]. It is
interesting that Dacron is more effective than polyglactin 910 in stimulating DNA synthesis in cultured SMCs [112].
These studies seem to indicate that factors and
mechanisms underlying myointimal proliferation
and endothelial regeneration in synthetic grafts are
less defined than in
other
models
of
revascularization.
SMC DIFFERENTIATION IN NEOlNTlMA
FORMATION
Developmental changes in SMC phenotype
Developmental, stage-specific molecular and cellular transitions occur during vascular SMC maturation in animal and human vascular smooth muscle tissue (for review see [113,114]). Extracellular
matrix, membrane, cytoskeletal, and cytocontractile
proteins appear to be expressed differently in SMCs
from fetal and adult vascular wall. A number of
mechanical, chemical, hormonal and nervous factors
are involved in regulating the differentiation pattern
of these cells. We have recently established that,
based on the analysis of myosin isoform expression,
a three-step differentiation process takes place in
rabbit aorta during physiological remodelling occurring in the course of development, namely fetal,
postnatal, and adult [115]. In fact, the so-called
myosin heavy chains (MyHC) of non-muscle B type
(NM-B) are mainly expressed in the fetus and
473
downregulated after birth [115,1163. Another nonmuscle myosin type (NM-A) is expressed throughout development in humans [117] or downregulated
postnatally in rabbits [115]. In adult rabbit thoracic
aorta, a minor SMC population accounting for
about 4% [llS] of the entire cell population
expresses the postnatal SMC phenotype. In pathological vascular remodelling induced by endogenous
and exogenous hypercholesterolaemia, renovascular
hypertension, thyroid hormone intoxication, the size
of this SMC population markedly increased in the
media. In the intimal thickening found in these
experimental conditions, SMCs accumulating in this
tissue compartment show the fetal SMC type
[119,120].
SMC proliferation/differentiationin experimental
balloon injury
It has been established in studies performed by
the Campbells and others that SMCs involved in
experimental intimal thickening induced by balloon
denudation display unique phenotypic features compared with those of intact vessels [121-1231. Large
amounts of rough endoplasmic reticulum, free ribosomes and mitochondria, and a low volume density
of myofilaments are peculiar aspects of SMCs newly
accumulated in thickened intima after injury
[122,123]. This cell phenotype has been named
‘synthetic’ contrary to the ‘contractile’ one characterized by a high volume density of myofilaments
and poorly developed synthetic apparatus. It has
not been investigated yet whether these morphological changes can be closely correlated with modifications in MyHC expression, in analogy with the shift
in actin isoform composition observed in intimal
thickening formation. The morphological change of
medial SMCs seems to take place before proliferation and migration to the intima. This event is
likely to be a consequence of vessel damage with the
release and production of growth stimulatory and
chemotactic factors which may influence the differentiation pattern, proliferation rate and ability to
migrate [113]. In fact, SMCs in normal vessels are
in a growth-arrested state under the control of
homoeostatic mechanisms that need to be altered in
order to permit cell proliferation/migration. However, it should be pointed out that tissue-culture
experiments on vascular SMCs have failed to confirm such a strict relationship between changes in
cell phenotype and proliferation [124,1251.
As in the case of primary atherosclerotic lesions,
SMCs found in the intimal thickening display an
immature differentiative profile, as judged from the
expression of several differentiation markers
[ 1 14,1263. Experiments from Schwartz’s laboratory
on cultured SMCs isolated from adult rat carotid
neointima 2 weeks after balloon injury showed that
these cells have morphological (epithelioid) and
synthetic (PDGF-like activity, little or no PDGF a-
474
P. Pauletto et al.
receptor, expression of PDGF-B gene) properties
resembling those of the so-called rat P U P SMC
[34,113]. SMCs from neointima express the extracellular matrix genes for tropoelastin, a-1 procollagen (type I), and osteopontin. Instead, the medial
SMCs from the intact vascular wall of adult animals
show a spindle-shaped morphology, do not secrete
PDGF or express the PDGF B gene, contain high
levels of PDGF a-receptor mRNA and express
extracellular matrix genes to a lesser extent [34].
The analysis of actin and myosin isoform composition in the balloon injury model in both rat and
rabbit systems has revealed that medial SMCs
achieve an immature phenotype before migrating
into the intima [1271. Similar results have been
obtained using other markers of cell differentiation
such as desmin and vimentin. Taken together, these
data are compatible with the following hypotheses
on the origin of immature SMCs found in the
neointima: (i) phenotypic modulation, (ii) dedifferentiation, (iii) stem-like cell activation, or (iv) reexpression of a smooth muscle developmental
sequence in a unique SMC subpopulation [114].
In the first stages of neointima formation SMCs
appear to be predominantly of immature type, then
they can shift to a more differentiated phenotype as
shown by the increased content of a-actin of smooth
muscle type [1273. According to preliminary observations made in our laboratory, MyHC expression
of fetal type disappears in rabbit carotid artery 3
weeks after balloon injury. Conversely, the postnatal
type is present in all the neointimal SMCs. It is still
unclear whether the fully differentiated SMC phenotype can be achieved in the neointimal cells with
time.
Differentiation of SMCs in myointimal proliferation can be markedly affected by heparin both in
uiuo and in uitro [128]. In particular, this molecule
is able to inhibit SMC cell modulation from ‘contractile’ to ‘synthetic’ phenotype as well as actin
isoform switching after balloon injury. Although
changes in SMC differentiation seem to be causally
linked to proliferation [1211, tissue-culture experiments have shown that differentiation effects of
heparin on vascular SMCs are distinct from its
growth effects. In fact, the level of a-actin staining of
SMCs does not correlate with the sensitivity of
these cells to growth inhibition by heparin
[128,129].
SMC proliferation/differentiation in human vessels
It has been established that, under physiological
conditions, SMC proliferation in human arteries
occurs at a very low rate, as judged by labelling
with anti-proliferating cell nuclear antigen (PCNA)
[130,131] or Ki-67 antibody [132]. The same low
level of proliferative activity can be demonstrated in
the intima, although it is present at slightly higher
values [13 11. Also in primary atherosclerotic lesions,
the percentage of PCNA-positive nuclei is quite low.
For example, O’Brien and colleagues [1331 have
reported that 82% of primary specimens from
coronary atherectomy have no evidence of PCNA
labelling. A slightly higher PCNA index can be
found in restenotic specimens from the same type of
vessel. Conversely, Pickering and colleagues [134]
reported much higher PCNA-labelling in specimens
of peripheral arteries and even higher levels were
present in samples of restenotic peripheral arteries.
SMCs in primary atherosclerotic lesions can be
identified by specific ultrastructural features such as
a well developed endoplasmic reticulum, Golgi
apparatus and a less developed cytocontractile
machinery [135,136].
As for the markers of cell differentiation, in the
primary lesions, the proportion of SMCs expressing
low amounts of metavinculin, 150-kDa caldesmon,
a-actin of smooth muscle type is markedly increased
compared with subendothelial intimal cells of
normal aorta [137].
A variation in the immunostaining pattern of
SMCs mainly in the smooth muscle myosin and
desmin content can be observed in primary lesions
[138]. For example, a number of specimens proved
to be smooth-muscle myosin, a-actin and desmin
negative while still containing vimentin and nonmuscle /I-actin [138]. More recently, studies performed on myosin isoform expression indicate a
marked decrease of SM2 MyHC isoform content
and upregulation of MyHC B of non-muscle type in
primary lesions [117,139,1403. Regional differences
in the distribution of MyHC A and B myosin
isoforms of non-muscle type exist in human aorta
near the aortic arch compared with the diaphragmatic level. While in the upper aorta the thickened
intima express both isoforms at either level, in the
lower tract the media underlying the lesion contain
MyHC of A type only [117].
MyHC B mRNA is found in a greater amount
among restenotic arteries than in primary lesions in
superficial femoral and coronary arteries [1391. The
significance of such an increase, at least for restenosis in the coronary arteries, has been clarified. In
fact, in situ hybridization procedures applied to
atherectomy specimens from patients suffering from
angina have revealed that a high occurrence of
restenosis is present in patients who displayed a
high level of MyHC B mRNA expression [22]. In
particular, there is a direct relationship between the
presence of reactive cells in primary atheroma and
the degree of stenosis on follow-up angiography
c221.
In analogy with the observations on changes in
SMC phenotype and propensity to restenosis in
coronary arteries, vein graft failure might be
explained by peculiar properties of venous SMCs
[23]. The differential heparin sensitivity of SMCs
from restenotic lesions and normal vessels of the
same patients on the one hand, and that of SMCs
from control patients undergoing a primary by-pass
procedure on the other, might be attributable to
Restenoris after revarcularization
475
Table I. Potential determinants of restenoris
Lesion composition:
High prevalence of SMCs expressing NM-myosin
High expression of growth factors and cytokines
Regional flow pattern:
Low blood flow and wall shear rate
Increased wall tension
Increased intimal permeability
Synthetic tissue characteristics:
Low porosity PTFE grafts ( ClOjun internodal distance)
Dacron grafts: M w e r i v e d stimulator), factors for SMC growth
Associated risk factors:
Age, cigarette smoking, cholesterol levels, etc.
Local factors:
Small vessel size, infection, thrombus formation, vasoconstriction. etc.
altered growth regulation in all SMCs or to a
specific activation of restenosis-prone cell types [23].
Moreover, at least in the rat, an additional factor to
be considered is the tendency of SMCs from adjacent arterial tissue to colonize the subendothelial
space of vein grafts, thus contributing to the aberrant growth of the intimal layer [141].
CONCLUSIONS
The data reviewed in this paper support the view
that, in both clinical and experimental settings,
myointimal hyperplasia underlies the development
of restenosis. The main determinants of restenosis
appear to be represented by the cell pattern of the
lesion, the regional blood flow characteristics, and
the synthetic graft tissue characteristics. Although
not as clearly defined, the presence of associated
risk factors for atherosclerosis and of some local
factors can also contribute to the restenotic process
(see Table 1).
SMC proliferation and accumulation in the
intima is the fundamental process in restenosis. This
in turn depends on the intrinsic phenotypic properties of SMCs (or the degree of SMC differentiation)
and on the availability of local factors capable of
regulating SMC proliferation/migration. Some
unanswered questions remain which deserve further
investigation: (i) the precise role played by SMCs
expressing MyHC B in the restenotic lesion in terms
of proliferation and tendency to differentiate; (ii) the
reasons for the self-limiting capacity of neointima
growth; and (iii) the clues for failure of pharmacological control of myointimal proliferation/restenosis
in humans.
It is intriguing that MyHC B-expressing SMCs
progressively accumulate in secondary lesions
despite a general tendency to stop proliferating after
the regrowth of endothelial lining. It might be that
this cell type becomes unresponsive to the action of
factors which can be released by endothelial cells.
Alternatively, once in the intima, MyHC Bexpressing SMCs might be out of the paracrine
control normally exerted in the media by the other
SMC types.
The spontaneous blockade of SMC proliferation/
migration at a certain stage of neointima formation
might be due to depletion of growth factors such as
bFGF from the injured vessel. Another possibility
relates to the degree of differentiation achieved by
neointimal SMCs [21,127]. In agreement with this
hypothesis, data from our laboratory indicate that,
based on a lesser content of MyHC B, some SMC
maturation takes place in neointima. Further
investigation is needed to establish the relationship
between changes in the SMC differentiation pattern
and potential cessation of SMC proliferation/
migration. Finally, an immune mechanism might be
involved in slowing down the growth of neointimal
SMCs. Studies by Hansson and colleagues [80]
have in fact shown that interferon synthesized by a
small number of T lymphocytes present in the
injured artery causes neighbouring SMCs to express
class I1 major histocompatibility antigens (Ia) and
inhibits their proliferation. In analogy with this
observation, we can hypothesize that other
lymphocyte- or macrophage-derived cytokines act
similarly.
The pharmacological control of intimal hyperplasia in humans represents a puzzling challenge. While
the development of myointimal thickening and restenosis can be prevented by a pharmacological
approach (i.e. ACE inhibitors, calcium antagonists,
heparin, immunosuppressive agents, anti-mitotic
substances, etc.) in experimental animal models of
arterial injury/revascularization, a similar result was
not obtained in humans [142]. Some of the drugs
which failed to control restenosis in humans, such
as heparin and calcium antagonists, besides acting
as anti-proliferative agents, promote or maintain a
differentiated state in SMCs [128,143]. Thus, it is
likely that the ineffectiveness of the pharmacological
approach in humans is due to the peculiar differentiation characteristics of SMCs, i.e. the presence of
high levels of MyHC B in normal human vascular
wall [117] in comparison with rabbit vessels [74].
Alternative strategies include the use of fish oils
rich in n-3 polyunsaturated fatty acids, and some
cell or molecular biology techniques. A small to
moderate benefit in terms of the reduction in the
rate of restenosis has been obtained with fish oil
supplementation before coronary angioplasty
[144,1453. In by-pass procedures using synthetic
grafts, endothelial coverage by cell-seeding techniques [146,147] might yield a better result in terms
of reduced thrombosis and reduced susceptibility to
476
P. Pauletto et al.
infection [1481. Moreover, enhanced endothelialization of expanded PTFE grafts has been obtained in
rabbits by FGF-1 pretreatment [1493. Finally, intraluminal delivery of antisense cdc2 kinase and PCNA
antisense oligonucleotides in rats subjected to carotid balloon injury resulted in stable inhibition of
neointima formation [ 1 SO].
A further development in our knowledge of the
biology of the arterial wall will facilitate the continued development of these technologies and might
provide additional therapeutic tools in clinical
practice.
ACKNOWLEDGMENTS
We thank Lorrie Maas Fusetti for her help with
the English in this manuscript. This work was
supported by grants from the Italian National
Research Council (CNR); Targeted Project ‘Prevention and Control of Disease Factors’; Subproject 8,
by M.U.R.S.T., and by a special grant from the
Biomedical Association for Vascular Research.
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