(Circulation. 1998;97:2486-2490.)
© 1998 American Heart Association, Inc.
Heparin Responsiveness In Vitro as a Prognostic Tool for Vascular Graft Stenosis
A Tale of Two Cell Types?
Jürgen R. Sindermann, MD;
; Keith L. March, MD, PhD
From the Krannert Institute of Cardiology, R.L. Roudebush VA Medical
Center, and Indiana University Medical Center, Indianapolis, Ind.
Correspondence to Keith L. March, MD, PhD, Krannert Institute of Cardiology, Indiana University Medical Center, 1111 W 10th St, Indianapolis, IN 46254. E-mail march{at}kimail.dmed.iupui.edu
Key Words: Editorials • heparin • stenosis • muscle, smooth
Vascular
graft stenosis after infrainguinal vein grafting or
coronary artery bypass graft surgery is a significant cause of
morbidity and suboptimal long-term clinical outcome of patients with
vascular disease. Numerous animal and clinical studies have been
undertaken to reveal the pathophysiological
mechanisms accounting for this detrimental process. Stenotic
lesions are dominated by neointima formation with migration
and proliferation of SMCs and deposition of extracellular matrix. This
appears to be a conserved response in the vasculature not only after
vein grafting but also after various injurious stimuli such as
angioplasty, endarterectomy, embolectomy, and
arterial catheterization. However, the
mechanisms of the process of stenosis are still not fully
understood, and there is neither an effective treatment for prevention
nor a diagnostic test for reliable prediction of patients
at risk for developing graft stenosis. Most clinical and
experimental studies have focused on the control of SMC proliferation
with the intention of developing strategies for the prophylaxis of
restenosis, whereas relatively little has been achieved in the
area of early tests to define the prognosis and need for clinical
monitoring of particular patients.
The anticoagulant heparin has been used for many years in the
therapy and prophylaxis of thrombotic conditions. It is also known for
its antiproliferative effects on SMCs when applied in higher doses and
has been studied extensively in animal models and clinical studies for
the inhibition of neointima formation after vascular
injury. As for many other compounds, encouraging results obtained in
animal models have not been matched by successful clinical studies,
perhaps because of dissimilar dosing levels or strategies in the
patients or different contributions of SMC proliferation in
stenotic lesions of animals and humans. Notwithstanding the
failure of heparin to prevent graft stenosis, recent
investigations, including that by Refson et al in this issue of
Circulation,1 focus attention on a new
potential utility of heparin in the management of patients with bypass
graft surgery. This application of heparin is as a biological test
reagent for stratifying patient populations with respect to their risk
for chronic graft occlusion. The ability of heparin to contribute to
such an assay is based on the observations that SMCs from given
individuals may be categorized on the basis of their diversity of
response in culture to the antiproliferative activity of heparin and
that the heparin-resistant phenotype is associated with
an increased risk for subsequent graft stenosis. Underlying
this approach is a growing recognition of the phenotypic diversity of
vascular SMCs among subjects as well as among distinct sites in
particular individuals.
The findings of this and the other related studies, as with many
emerging experimental observations, raise many more questions than they
answer. These provocative questions have a number of
implications for either vascular biology, clinical management, or both.
Some of these issues may be summarized as follows.
What are the molecular transducers of the heparin response that are
altered in heparin-resistant cells? Are the key abnormalities
to be found at surface-receptor or intracellular levels?
It is known that the effects of heparin on cell proliferation are
dissociable from the anticoagulant function of heparin, because
nonanticoagulant fractions that retain antiproliferative activity have
been described. It is also clear from clinical experience that patients
differ widely in their responsiveness to heparin, even when the extent
of thrombus and weight-adjusted dosing are considered. This raises the
question, not addressed by the study of Refson et al, as to whether the
observed heparin resistance correlates with an alteration in heparin
anticoagulant function.
Are the interindividual differences in SMCs based at the genetic level,
and thus heritable, or rather epigenetic, with a causal basis in the
environment, diet, or other factors? Currently, no clearly described
genetic syndromes of altered heparin sensitivity are known.
If the molecular mechanism for the variability in heparin response is
due to an epigenetic alteration, is this an alteration that occurs
throughout the vascular tree, or does it instead reflect local
selection of a population characterized by heparin resistance and a
concurrent "hyperstenotic" phenotype? In either
case, what are the key factors that play a role in this selection? Are
they classic risk factors of atherosclerosis or local
hydraulic or mechanical forces that affect the vessels later to be used
as grafts?
If the molecular basis of the variability in heparin response occurs at
the genomic level, can molecular genetic testing be used to stratify
patients with respect to their vascular smooth muscle responsiveness to
heparin? In this case, it would be particularly important to pursue
research to define the gene(s) whose allelic variants underlie heparin
resistance. Successful identification of these genes would permit the
development of comparatively straightforward diagnostic
testing. Such tests might be based on polymerase chain reaction for the
underlying altered allele, which could then be performed on any
cellular material (such as leukocytes in standard blood samples) rather
than requiring vascular tissue sampling at the time of surgical
intervention, or could be based on immunoassay for an altered protein
in tissue exhibiting its expression.
Will the information gained from molecular or cellular analysis
truly find a place in clinical decision-making and management for
patients with peripheral vascular disease? The widespread
adoption of any such analysis in general patient care settings
will be strongly influenced by the practicality of the specific test
for physicians as well as patients in a nonresearch setting.
Accordingly, the work necessary to translate these cellular findings
into tests not based on tissue culture analyses will take
priority, provided that the current data can be replicated in more
extensive studies.
Does the described interindividual variability in smooth muscle
behavior also predict the behavior of synthetic grafts with respect to
their frequency of anastomotic stenosis, which is similarly
dependent on smooth muscle proliferation? Also, does this phenomenon
hold true for venous and arterial grafts in
coronary positions? Furthermore, if relative heparin resistance
is a characteristic of the entire vascular tree in particular
individuals, does it form a basis for prognostic information with
regard to angioplasty or stent restenosis?
What would be the optimum use of information concerning the
heparin-related vascular cell genotype or phenotype?
Possible effects on patient care might include (1) directing selection
of endovascular versus surgical bypass therapy; (2) influencing
the aggressiveness/timing of bypass surgery; (3) influencing the type
of bypass to be used, ie, venous, arterial, or synthetic
grafts; (4) helping to determine the frequency of postprocedural
follow-up of patients; and (5) altering the heparin regimen according
to heparin sensitivity. This latter might involve addition/substitution
of alternative perioperative and postoperative
anticoagulant medications or intensification of the level of
heparinization in heparin-resistant individuals. This would be
conceptually similar to the use of exogenous insulin in
insulin-resistant states.
Might patients known to be heparin-resistant be treated
systemically in ways that facilitate restoration of heparin
sensitivity? This would be directly analogous to the use in diabetes of
agents that promote peripheral insulin sensitivity.
Would information concerning the heparin sensitivity of SMCs in
given individuals be useful to direct selection of nonsurgical
management options, such as the choice of heparin-coated versus
standard stents or the decision to use local delivery of heparin in the
context of angioplasty or stenting?
A clear identification of the mechanism(s) underlying the range of
responses to heparin may be challenging because of complexity at
several levels: (1) heparin preparations are themselves polydisperse
polymers with subfractions possessing distinctive properties, so that
antiproliferative and anticoagulant properties are clearly
distinguishable; (2) mechanisms of heparin action are multiple, even
when only proliferation effects are considered; and (3) SMC populations
possess significant phenotypic diversity when viewed with respect to
any of several parameters, not limited to heparin
sensitivity.
We will now examine literature relevant to some of these issues and
questions.
Molecular Bases of Heparin Effects on Proliferation
Among the spectrum of the effects of heparin on SMCs, a potential
for differential influences on SMC proliferation, depending on the
applied dose, environmental conditions, and treatment duration, has
been noted in vitro and in vivo. Heparin in high concentrations
(µg/mL) is typically an inhibitor of arterial
SMC replication,2 whereas heparin in lesser
quantities (ng/mL) may facilitate binding of bFGF to its receptor as
well as expression of the elements of this system, thereby mediating a
potential for growth stimulation.2 3 Further
evidence for a paradoxical growth-stimulatory effect of heparin is
provided by recent investigations on human saphenous veins, in which it
has been shown that heparin can displace bFGF from binding sites at the
luminal surface, with released bFGF available to stimulate SMC
proliferation.4 Studies of rat SMCs showed that
heparin inhibits MAPK in the presence of FCS but not in the presence of
epidermal growth factor, thus suggesting heparin-sensitive and
-insensitive pathways of MAPK activation.5 A
study performed on baboon SMCs similarly revealed heparin inhibition of
MAPK activity when stimulated by serum but not platelet-derived
growth factor. When bFGF was used, heparin had a stimulatory effect on
MAPK activity.6 Although it has been shown in the
balloon injury model of the rat carotid artery that heparin inhibits
neointima formation and that the injury causes the
activation of MAPK, it could not be demonstrated that the
antiproliferative effect of heparin is exerted by the inhibition of
MAPK.7 Accordingly, the ability of heparin to
block cell-cycle entry at the comparatively early stage of MAPK
inhibition may reflect differences in growth factor sensitivity among
discrete cell populations.
In addition to inducing arrest before G1 entry,
presumably related to MAPK effects, heparin exhibits activity at
several subsequent points of the cell cycle. Activity is also noted
during the G1 phase, with reduction of cell-cycle
regulatory factors such as cyclin D1 mRNA and
protein, cdk2 mRNA and cdc2 protein,8 and
traverse inhibited through the G2/M
phase.3 9 It has also been described as blocking
the expression of c-myb, an event that occurs in
mid-G1 phase.10 At
present, the extent to which such specific cell-cycle effects might
vary with the SMC site of origin is not known. Evidence for species- or
location-specific effects of heparin has been provided by
investigations showing that the growth-inhibitory effect of
heparin on rat, bovine, and human SMCs required the presence of a
biologically active transforming growth factor-ß only in the case of
rat and bovine arterial SMCs but not for SMCs of human
saphenous veins.11
The molecular results of heparin exposure also extend beyond
interactions with cell cycle–regulatory factors, to effects on
proteases required for cell movement, and on matrix formation as it
inhibits collagenase gene
expression,12 the transcription and expression of
tissue-type plasminogen
activator,13 14 and the induction of
matrix metalloproteinases such as stromelysin and 92-kDa
gelatinase.15 Some of these effects, as well as
cell-cycle effects, may be modulated by activation of one or more PKC
isoforms.12 16 The relative significance of these
particular heparin activities remains to be clarified.
Phenotypic Diversity Among Vascular SMCs
Several studies have demonstrated the existence of relatively
heparin-sensitive and heparin-resistant populations of SMCs.
Some of these investigations have involved prolonged in vitro exposure
to heparin, in which case the finding either may reflect selection of a
preexistent phenotype from among an inhomogeneous
population or might represent the induction of a novel
phenotype in culture under selection pressure. The phenomenon
of cell clones developing a resistance to compounds is not
heparin-specific, because similar effects are well known to occur on
exposure to chemotherapeutic agents. For example, rat thoracic aortic
SMCs exposed to long-term culture with 200 µg/mL heparin have
exhibited a significant loss of sensitivity to growth inhibition by
heparin. This heparin resistance was stable even after cells were grown
for two passages in heparin-free medium, suggesting effective selection
for this SMC phenotype.17
Characterization of the heparin-resistant SMCs compared with
their nontreated controls revealed the treated cells to be smaller, to
possess less smooth muscle 
-actin and one half of the PKC activity,
and to show a paradoxically greater contact inhibition. A marked
decrease in heparin binding capacity was seen in the resistant
cells. However, another group has shown that similar
heparin-resistant and heparin-sensitive SMCs derived from rat
abdominal aorta bound and internalized comparable amounts of
heparin.18 The ability of heparin treatment to
increase the percentage of cells that express smooth muscle -actin
was preserved in the heparin-resistant cells, suggesting that
the antiproliferative and the differentiation-promoting effects of
heparin were independent.18 With regard to
extracellular matrix proteins, it has been shown that
heparin-resistant SMCs differ from control cultures by low
production of fibronectin, prevalent expression of laminin, and
decreased cell-associated
glycosaminoglycans.19 With
each of these observations, the question remains whether the
alterations represent primary characteristics of cells with the
heparin-resistant phenotype or are secondary to a
selection-induced SMC phenotypic shift.
These data have been extended by experiments not involving prolonged in
vitro heparin exposure. SMCs derived from SHRs have been found to be
less sensitive to the growth-inhibitory effect of heparin
than controls from Wistar-Kyoto rats, in conjunction with a reduced
capacity but comparable affinity for heparin binding to extracellular
surface receptors. In addition, SHR-derived SMCs exhibited a greater
capacity, but not affinity, for epidermal growth factor
binding.20 Recent investigations of the rat
carotid artery demonstrated that the typical inhibition of SMC
replication by heparin acutely after a balloon injury was not
present after a reinjury performed 28 days
later.21 These results may be viewed in the
context of an altered phenotype of SMCs to be found in vivo in
neointima after vascular injury; it would be interesting to
see whether the heparin resistance would return after a longer time
interval subsequent to the initial injury, which might permit the SMCs
to return to a contractile phenotype more similar to their
baseline. A basis for variable heparin sensitivity in the clinical
setting could thus reside in the intensity and timing of mechanical or
biochemical injury, with resultant phenotypic shift.
Findings in Human Smooth Muscle
In some studies performed on human cells, aortic and saphenous
vein SMCs have shown different sensitivities to heparin, with DNA
synthesis in venous SMCs manifesting heparin dependence different from
that found in corresponding aortic SMCs. This phenomenon could be
demonstrated with paired aortic and venous samples obtained from the
same individual as well as with pairs mixed from different
patients.2 Another study design comparing heparin
sensitivity among human aortic and saphenous vein SMCs in culture did
not yield significant differences based on the site of origin but did
show interindividual differences, supporting systemic rather than
regional variation. Despite these differences in heparin sensitivity,
baseline proliferation was consistent among SMCs derived from
the various individuals.22 The question arises
whether such conflicting results may be explained by different sites of
vessel origin or different patient populations.
Such work characterizing the diversity of SMCs with respect to heparin
sensitivity has provided an impetus to determine whether this
phenomenon might correlate with the clinical problem of vascular graft
stenosis. A retrospective study was thus performed on the
effect of heparin on proliferation of cultured human SMCs derived from
stenotic lesions (at the time of reoperation) and from
apparently normal vessels of the same patient as well as on SMCs grown
from vessels of patients undergoing primary bypass
surgery.23 Although the vascular tissues in that
study were derived from a range of different vessels, including
internal mammary artery, common femoral artery, popliteal artery, iliac
artery, and saphenous veins, this previous investigation yielded the
provocative initial result that SMCs derived from patients
with early stenosis showed much lower sensitivity to growth
inhibition by heparin than the controls. This effect was apparent
whether SMCs were grown from stenotic lesions or undiseased
veins. In light of the prognostic potential, the group proceeded with
the current prospective study, in which SMCs were cultured by outgrowth
from redundant sections of veins at the time of initial infrainguinal
bypass surgery and the resulting SMC cultures incubated with heparin. A
diminished growth inhibition by heparin (median of 20.9%, compared
with 54% in control cells) was found with SMCs derived from vessels of
patients who subsequently developed graft stenosis over a
follow-up period of at least 1 year. In an attempt to explain this
phenomenon, the group investigated a subset of the SMC cultures for
heparin-binding parameters, which appeared to be correlated
with growth inhibition as well as the clinical outcome.
Future Implications
The study by Refson et al1 provides a basis
for expanded investigation of the laboratory assessment of vascular
patients by evaluation of heparin effects on SMC growth as a prognostic
approach for the development of graft stenosis. In the design
and execution of such future studies, certain methodological cautions
warrant consideration. The authors point out that more information is
needed about the relation of environmental and hereditary risk factors
to in vitro properties of SMCs cultured from patients. This is
particularly true given the substantial overlap in heparin
sensitivities found between the groups of patients with and without
stenoses. Such overlap will confound attempts to use this
approach for individuals (rather than populations), and it will thus be
important to control for other variables and optimize the culture
conditions to provide maximally predictive separation between patients.
As part of an approach to understanding effects that might be reflected
by early pathological changes in the vascular site, it would be helpful
to collect tissue samples for histological or
physiological evaluation as well as culture.
Another area for specific consideration in trial planning would be the
choice of consistent heparin formulations, given the well-known
heterogeneity of heparin activities, depending on
source, processing, etc.
Experiments designed to determine whether the heparin-resistant
phenotype is inherited or based on somatic diversity will be
important. The finding that the heparin-resistant
phenotype persists despite passaging in vitro does indeed
support a genetic difference but does not necessarily verify a
germline-level genotype, because it is possible that particular
environmental factors favor either mutation or selection for particular
smooth muscle populations from among a group with preexisting
functional heterogeneity based on somatic diversity in
gene expression. Regional vascular genetic
heterogeneity reflected by X-chromosome inactivation
patterns has indeed been demonstrated within atherosclerotic plaques as
well as normal coronary vessels.24 It may
be speculated that distinct types or intensities of chronic vascular
injury result in cell-cycle reentry of specific types of SMCs, or SMCs
that develop distinct phenotypes and growth properties once
cell proliferation and migration has started. The distinction between
systemic and regional genetic variability is significant for future
clinical application, because an inherited genetic basis would provide
for the possibility of molecular phenotyping for heparin-resistance
status based on blood or other peripheral sampling. Local
clonal expansion of particular vascular cell types based on selection
pressures might also support molecular assignment of the
phenotype, but this approach would presumably require screening
of vascular tissue(s) in which the selection and clonal expansion was
thought to have occurred. Each of these issues will require
consideration as the development of a new test for the clinical
management of vascular disease is contemplated.
Another intriguing clinical implication of the identification of
heparin insensitivity in cultured cells is the suggestion of a state of
in vivo functional heparin insensitivity. This implies that an increase
in heparin sensitivity of SMCs might be a novel therapeutic target. The
discovery of pharmacological interventions capable of inducing such an
increase could lead to enhanced in vivo activity of heparin to prevent
graft stenosis or might conceivably be manifested as an
alteration in SMC proliferative activity without the need for modified
heparin administration. Research exploring these as well as the other
intriguing questions raised by the study of Refson and colleagues may
be expected to yield interesting results in both basic and clinical
arenas of vascular medicine in the near future.
Selected Abbreviations and Acronyms
| bFGF |
= |
basic fibroblast growth factor |
| MAPK |
= |
mitogen-activated protein kinase |
| PKC |
= |
protein kinase C |
| SHR |
= |
spontaneously hypertensive rat |
| SMC |
= |
smooth muscle cell |
|
Acknowledgments
This work was supported in part by a Merit Review from the
Veterans Administration (Dr March) and grant R01-HL57411 from the
NIH/US Public Health Service (Dr March) as well as the Cryptic Masons
Medical Research Foundation (Dr Sindermann). The authors wish to thank
Drs Hans-Peter Stoll and Denzil D'Souza for helpful comments and
discussion.
Footnotes
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
References
1.
Refson JS, Schachter M, Patel MK, Hughes AD, Munro
E, Chan P, Wolfe JHN, Sever PS. Vein graft stenosis and the
heparin responsiveness of human vascular smooth muscle cells.
Circulation. 1998;97:2506–2510.[Abstract/Free Full Text]
2.
Stöhr S, Meyer T, Smolenski A, Kreuzer H,
Buchwald AB. Effects of heparin on aortic versus venous smooth muscle
cells: similar binding with different rates of
[3H]thymidine incorporation. J
Cardiovasc Pharmacol. 1995;25:782–788.[Medline]
[Order article via Infotrieve]
3.
Skaletz-Rorowski A, Schmidt A, Breithardt G, Buddecke
E. Heparin-induced overexpression of basic fibroblast growth factor,
basic fibroblast growth factor receptor, and cell-associated
proteoheparan sulfate in cultured coronary smooth muscle cells.
Arterioscler Thromb Vasc Biol. 1996;16:1063–1069.[Abstract/Free Full Text]
4.
Medalion B, Merin G, Aingorn H, Miao HQ, Nagler A,
Elami A, Ishai-Michaeli R, Vlodavsky I. Endogenous basic
fibroblast growth factor displaced by heparin from the lumenal surface
of human blood vessels is preferentially sequestered by injured regions
of the vessel wall. Circulation. 1997;95:1853–1862.[Abstract/Free Full Text]
5.
Ottlinger ME, Pukac LA, Karnovsky MJ. Heparin
inhibits mitogen-activated protein kinase activation in intact
rat vascular smooth muscle cells. J Biol Chem. 1993;268:19173–19176.[Abstract/Free Full Text]
6.
Daum G, Hedin U, Wang Y, Wang T, Clowes AW. Diverse
effects of heparin on mitogen-activated protein
kinase-dependent signal transduction in vascular smooth muscle cells.
Circ Res. 1997;81:17–23.[Abstract/Free Full Text]
7.
Lille S, Daum G, Clowes MM, Clowes AW. The regulation
of p42/p44 mitogen-activated protein kinases in the injured rat
carotid artery. J Surg Res. 1997;70:178–186.[Medline]
[Order article via Infotrieve]
8.
Vadiveloo PK, Filonzi EL, Stanton HR, Hamilton JA. G1
phase arrest of human smooth muscle cells by heparin, IL-4 and cAMP is
linked to repression of cyclin D1 and cdk2.
Atherosclerosis. 1997;133:61–69.[Medline]
[Order article via Infotrieve]
9.
Grainger DJ, Witchell CM, Watson JV, Metcalfe JC,
Weissberg PL. Heparin decreases the rate of proliferation of rat
vascular smooth muscle cells by releasing transforming growth factor
beta-like activity from serum. Cardiovasc Res. 1993;27:2238–2247.[Abstract/Free Full Text]
10.
Reilly CF, Kindy MS, Brown KE, Rosenberg RD, Sonenshein
G. Heparin prevents vascular smooth muscle cell progression through the
G1 phase of the cell cycle. J Biol
Chem. 1989;264:6990–6995.[Abstract/Free Full Text]
11.
McCaffrey TA, Falcone DJ, Brayton CF, Agarwal LA, Welt
FGP, Weksler BB. Transforming growth factor-ß activity is
potentiated by heparin via dissociation of the transforming growth
factor-ß/2-macroglobulin inactive complex.
J Cell Biol. 1989;109:441–448.[Abstract/Free Full Text]
12.
Au YPT, Montgomery KF, Clowes AW. Heparin inhibits
collagenase gene expression mediated by phorbol
ester–responsive element in primate arterial smooth muscle
cells. Circ Res. 1992;70:1062–1069.[Abstract/Free Full Text]
13.
Au YPT, Kenagy RD, Clowes AW. Heparin selectively
inhibits the transcription of tissue-type plasminogen
activator in primate arterial smooth muscle
cells during mitogenesis. J Biol Chem. 1992;267:3438–3444.[Abstract/Free Full Text]
14.
Clowes AW, Clowes MM, Kirkman TR, Jackson CL, Au YPT,
Kenagy R. Heparin inhibits the expression of tissue-type
plasminogen activator by smooth muscle cells in
injured rat carotid artery. Circ Res. 1992;70:1128–1136.[Abstract/Free Full Text]
15.
Kenagy RD, Nikkari ST, Welgus HG, Clowes AW. Heparin
inhibits the induction of three matrix metalloproteinases (stromelysin,
92-kD gelatinase, and collagenase) in primate
arterial smooth muscle cells. J Clin
Invest. 1994;93:1987–1993.
16.
Herbert JM, Clowes M, Lea HJ, Pascal M, Clowes AW.
Protein kinase C alpha expression is required for heparin inhibition of
rat smooth muscle cell proliferation in vitro and in
vivo. J Biol Chem. 1996;271:25928–25935.[Abstract/Free Full Text]
17.
Bârzu T, Herbert JM, Desmoulière A, Carayon
P, Pascal M. Characterization of rat aortic smooth muscle cells
resistant to the antiproliferative activity of heparin
following long-term heparin treatment. J Cell Physiol. 1994;160:239–248.[Medline]
[Order article via Infotrieve]
18.
San Antonio JD, Karnovsky MJ, Ottlinger ME, Schilling
R, Pukac LA. Isolation of heparin-insensitive aortic smooth muscle
cells: growth and differentiation. Arterioscler Thromb. 1993;13:748–757.[Abstract/Free Full Text]
19.
Bârzu T, Pascal M, Maman M, Roque C, Lafont F,
Rousselet A. Entry and distribution of fluorescent
antiproliferative heparin derivatives into rat vascular smooth muscle
cells: comparison between heparin-sensitive and
heparin-resistant cultures. J Cell Physiol. 1996;167:8–21.[Medline]
[Order article via Infotrieve]
20.
Resink TJ, Scott-Burden T, Baur U, Bürgin M,
Bühler FR. Decreased susceptibility of cultured smooth muscle
cells from SHR rats to growth inhibition by heparin. J Cell
Physiol. 1989;138:137–144.[Medline]
[Order article via Infotrieve]
21.
Koyama H, Reidy MA. Reinjury of arterial
lesions induces intimal smooth muscle cell replication that is not
controlled by fibroblast growth factor 2. Circ Res. 1997;80:408–417.
22.
Munro E, Chan P, Patel M, Betteridge L, Gallagher K,
Schachter M, Sever P, Wolfe J. Consistent responses of the
human vascular smooth muscle cell in culture: implications for
restenosis. J Vasc Surg. 1994;20:482–487.[Medline]
[Order article via Infotrieve]
23.
Chan P, Patel M, Betteridge L, Munro E, Schachter M,
Wolfe J, Sever P. Abnormal growth regulation of vascular smooth muscle
cells by heparin in patients with restenosis.
Lancet. 1993;341:341–342.[Medline]
[Order article via Infotrieve]
24.
Murry C, Gipaya C, Bartosek T, Benditt EP, Schwartz SM.
Monoclonality of smooth muscle cells in human
atherosclerosis. Am J Pathol. 1997;151:697–706.[Abstract]
