From the Department of Clinical Pharmacology, Imperial College School of
Medicine, St Mary's Hospital (J.S.R., M.S., M.K.P., A.D.H., P.C.,
P.S.S.), and the Regional Vascular Unit (J.S.R., E.M., J.H.N.W.), St
Mary's Hospital, London, UK.
Correspondence to Dr Michael Schachter, Department of Clinical Pharmacology, St Mary's Hospital, London W2 1NY UK. E-mail m.schachter{at}ic.ac.uk
Methods and Results—In a prospective study, we correlated
antiproliferative responses to heparin in vitro with graft patency
after 1 year. Sixty-two patients with infrainguinal vein grafts were
entered into a graft surveillance program for
Conclusions—Responsiveness to heparin in cultured VSMCs is a
strong predictor of outcome for infrainguinal vein grafts, and reduced
sensitivity to heparin is correlated with decreased heparin binding.
Relative resistance to the antiproliferative action of heparin may be a
marker for aberrant regulation of VSMC growth.
Heparin was the first relatively specific antiproliferative agent
shown to inhibit the growth of VSMCs both in
vivo11 12 and in vitro.13
It remains unclear how heparin exerts its antiproliferative effect, but
similar endogenous glycosaminoglycans,
the heparan sulfates, exist in the extracellular
matrix14 and are thought to be involved in
phenotypic and growth regulation, helping to maintain VSMCs in a
contractile, nonproliferative state.15 16 This is
a property shared by heparin.17
In a previous report, we showed that VSMCs from patients with proven
stenoses are relatively resistant to the
antiproliferative effect of heparin18 and that
VSMCs obtained from a variety of patients undergoing vein bypass
procedures (cardiac or infrainguinal) exhibit a very wide range of
responses to this antiproliferative action (0% to 80% inhibition).
Among this group,
In a second study, we explored a potential mechanism underlying the
variation in responsiveness of human VSMCs to heparin. This was based
on observations that suggested that the binding of heparin to VSMCs is
required for its growth-inhibitory
action.19 20 21 In the SHR, which demonstrates
increased VSMC proliferation and vascular wall hypertrophy,
cultured VSMCs have fewer heparin binding sites and less sensitivity to
growth inhibition by heparin than normotensive WKY
controls.19 To determine whether the mechanism of
heparin resistance in human VSMCs is related to the extent of heparin
binding to the cell surface, we analyzed the binding
characteristics of radiolabeled heparin in cultured VSMCs and compared
them with their growth responses to FCS in the presence and absence of
heparin.
Cell Culture
VSMC cultures were prepared as previously
described.24 Veins were carefully dissected to
remove the adventitia, and the endothelium was scraped
away with a blade, with care taken to minimize damage to the underlying
media. The media, consisting almost exclusively of
VSMCs,25 was cut into 1-mm squares. The resulting
explants were placed in 80-cm2 tissue culture
flasks in 5 mL of nutrient medium made up of DMEM buffered with 25
mmol/L HEPES supplemented with 15% (vol/vol) FCS,
L-alanyl-L-glutamine 4 mmol/L, penicillin
100 U/mL, streptomycin 100 µg/mL, and gentamicin 25 µg/mL.
Cultures were incubated at 37°C in a humidified atmosphere of 5%
CO2 (vol/vol) in air. Over a period of 2 to 6
weeks, VSMCs migrated from the explants and proliferated to form a
confluent monolayer. At this stage, they were subcultured by
trypsinization into a fresh flask. VSMCs were then further cultured and
characterized by their typical "hill-and-valley" morphology and
indirect immunofluorescence for
Cell Proliferation Assay
Heparin Binding Assay
Statistical Analysis
Materials
To estimate the density of [3H]heparin binding
to VSMCs, at a concentration of 10-7 mol/L,
displacement curves were constructed for cell cultures from 25 patients
(examples in Figure 2
In the subgroup of 13 patients for whom binding, growth, and clinical
data were known, 6 patients had patent grafts at the end of 1 year, and
stenoses had been detected in the remainder. Both heparin
binding and growth inhibition by heparin were again significantly
correlated with clinical outcome (point biserial correlation
coefficients, r=0.56, P<0.05 and
r=0.66, P<0.02). As one would anticipate from
the overall patient data, there was also a strong correlation between
binding and growth inhibition in this subpopulation (r=0.79,
P=0.001) (Figure 4
Heparin resistance has been shown in this series to be a highly
significant predictor for the development of subsequent
stenosis. The conventional risk factors for
atherosclerosis, by contrast, have shown little or no
consistent association with clinical outcome either for
peripheral vein grafts or coronary angioplasty,
although it should be noted that our study has not been stratified for
parameters such as the size of runoff vessels or flow
velocity. Given the large difference in heparin responsiveness between
the 2 outcome groups, it is highly unlikely that these factors are a
major confounding influence. Obviously, the next step was to consider
possible mechanisms to explain this finding, and the heparin binding
study was carried out for this reason. Castellot et
al27 first described specific binding of heparin
to animal VSMCs in 1985, followed by the observation of Resink et
al19 that growth inhibition in rat VSMCs was
related to the density of heparin binding sites in the SHR model of
hypertension, in which both parameters were reduced
compared with WKY controls. Barzu et al20 showed
that it is possible to isolate heparin-resistant rat VSMCs by
growing them in culture in the presence of heparin for up to 15
passages. Here too, growth inhibition by heparin altered in parallel
with the extent of reduction in heparin binding, although this
association was not found by San Antonio et al.36
The mechanism of heparin inhibition is itself still incompletely
understood. Heparin inhibits the activation of the
mitogen-activated protein kinase in intact rat VSMCs, but the
intracellular mechanisms involved have not been
defined,37 and it is unclear whether
internalization of heparin is required for its antiproliferative
action. Because our experiments were carried out in cell monolayers,
the possibility also remains that it is the interaction of heparin with
extracellular matrix, rather than cell-associated binding sites, that
mediates its inhibitory action on these
cells.38 The same group has recently reported the
failure of heparin to inhibit intimal hyperplasia in a primate, the
baboon, while drawing attention to the existence of heparin-sensitive
and -insensitive pathways of mitogenesis.39 It
must, of course, be remembered that these concentrations of heparin are
far above therapeutic anticoagulant levels and that these results
therefore have no implications for the clinical use of heparin in
stenosis or restenosis prophylaxis.
Our results indicate a clear relationship between clinical outcome and
in vitro heparin inhibition and between inhibition and heparin binding,
and in a subgroup of patients in whom we have all 3 sets of data, a
strong correlation has been confirmed for these parameters.
Although we did not set out to answer this question, we have attempted
retrospectively to correlate heparin inhibitability with time of
detection of stenosis as an indirect measure of a
"dose-response" relationship (data not shown), but the correlation
was weak and nonsignificant: one would need a considerably larger
population to address this issue. Furthermore, one must always be
cautious about any extrapolation from in vitro cell culture to a
clinical setting, bearing in mind that cell culture inevitably implies
the loss of in vivo local and systemic regulatory mechanisms.
In conclusion, we have demonstrated for the first time a cellular
marker in vascular smooth muscle that is highly predictive of
subsequent stenosis in vein graft recipients and have also
presented evidence on which to base the formulation of a
possible mechanism. This is currently under further investigation by
our group, particularly in the context of the role of extracellular
matrix proteins as regulators of cell growth and differentiation. Our
findings have several implications for future research and ultimately
for clinical management. Altered responsiveness to heparin may itself
be a marker for a more fundamental disturbance of growth
regulation in the vascular wall, although this does not preclude the
possibility that a failure to interact appropriately with
glycosaminoglycans in the vessel wall has in itself
an important role in the disease process. Understanding these
mechanisms and how they could be modulated will contribute to better
understanding of the biology of the vascular wall and may also lead to
more rational and effective management of an important clinical
problem, with the intriguing prospect of identifying individuals at
increased risk of graft failure and ultimately reducing the likelihood
of an unfavorable outcome.
Received November 25, 1997;
revision received February 3, 1998;
accepted February 13, 1998.
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© 1998 American Heart Association, Inc.
Clinical Investigation and Reports
Vein Graft Stenosis and the Heparin Responsiveness of Human Vascular Smooth Muscle Cells
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—Vascular smooth muscle
cell (VMSC) proliferation is an essential component of myointimal
hyperplasia, which is implicated in the failure of 30% to 50% of
vascular interventions, such as coronary angioplasty and
peripheral vein grafting. We have shown that cells derived
from stenotic lesions in infrainguinal vein grafts were
significantly more resistant than controls to growth inhibition
by heparin. 
1 year. At operation,
saphenous vein segments were explanted for VSMC culture. Cell
proliferation in response to fetal calf serum was later determined in
the presence and absence of heparin. In 35 cell cultures, including 13
from the above-mentioned patients, [3H]heparin binding
was also estimated. VSMCs from patients with patent grafts were
significantly more sensitive to growth inhibition by heparin than cells
from patients with stenoses (median, 54% versus 20.9%,
P<0.001), and [3H]heparin binding was
strongly correlated with inhibition of proliferation
(r=0.81).
Key Words: veins • grafting • stenosis • heparin
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Arterial
stenosis or restenosis is one of the most important
barriers to long-tem patency after arterial interventions
such as coronary1 2 and
peripheral3 angioplasty,
percutaneous atherectomy,4 and
coronary5 grafting. It is responsible for
80% of all peripheral arterial bypass graft
failures,6 resulting in the loss of 30% of
all arterial bypass grafts. MIH comprising VSMC
proliferation (and migration) with deposition of extracellular matrix
is one major cause of the stenoses that develop between 6 weeks
and 1 year after vessel injury.7 However, it
should also be appreciated that in 50% of the affected vessels,
remodeling without hyperplasia appears to be the cause of
stenosis.8 9 10 30% are likely to develop MIH lesions severe
enough to cause clinically significant graft
stenoses.6 We therefore set up a
prospective study to investigate whether the proliferative responses of
cultured VSMCs to heparin predicted the subsequent development of
infrainguinal graft stenoses.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Patients
Between January 1991 and October 1994, 62 patients undergoing
infrainguinal (femoropopliteal) vein bypass each had a section of
redundant vein graft removed at the time of initial surgery and were
entered into a standardized graft surveillance
program.22 Relevant demographic characteristics
are summarized in the Table
, subdivided according to
clinical outcome. All patients were given heparin sodium 5000 U SC
before surgery and a further 5000 U IV during the operation. An
angiogram was performed on completion of surgery, followed by scanning
of the graft with duplex Doppler ultrasound at 6 weeks, 3 months, 6
months, 9 months, and 1 year. If a stenosis was detected by
ultrasound (in our unit, sensitivity and specificity were 100% and
98%, respectively),23 angiography was used for
confirmation. All patent grafts were examined angiographically at 1
year. For the purposes of our analysis, graft stenosis
was defined as a lesion developing between 6 weeks and 1 year after
surgery, with a V2/V1 ratio
>2 on duplex Doppler and a reduction in luminal diameter of >50%
on angiography in comparison with the immediate postoperative
measurement. All angiograms were independently reviewed at a joint
surgical and radiological conference without prior knowledge of the
results of the in vitro studies.
View this table:
[in a new window]
Table 1. Clinical Details of Patients Included in Prospective
Study
Redundant sections of vein were taken from 62 patients at the
time of initial infrainguinal bypass surgery in accordance with local
Ethics Committee guidelines and transported to the laboratory in
Hanks' balanced salt solution containing HEPES 25 mmol/L,
penicillin 200 U/mL, streptomycin 200 µg/mL, kanamycin 100 µg/mL,
and amphotericin B 1.25 µg/mL. Vein segments were explanted for cell
culture within 24 hours of operation. All the veins used in this study
were macroscopically normal, but no histological
examination was carried out because all available tissue was used for
cell culture. 
-actin, a marker
for smooth muscle cells.26
To assay the inhibitory activity of heparin, VSMCs
at second passage were trypsinized and seeded, as previously
described,24 at a density of
104 cells/mL. A total volume of 1 mL/well was put
into 24-well plates and allowed to attach overnight in standard medium
containing 15% FCS. This medium was removed, and the cells were washed
twice with PBS (without calcium and magnesium). The cells were
maintained in medium supplemented with 0.4% FCS for 72 hours to arrest
the cells in the G0 phase of the cell cycle.
Growth stimulation was obtained with growth medium containing 15%
FCS±7 µmol/L heparin. Triplicate wells were harvested by
trypsinization and counted in an electronic particle counter (Coulter)
on days 0, 3, 7, 10, and 14 after growth arrest. The growth medium in
the remaining wells was replenished at the same time points. The net
growth of VSMCs in 15% FCS containing growth medium±7 µmol/L
heparin was calculated by subtracting the starting cell number (at the
time the cells were released from G0) from the
cell number on day 14. The percentage of heparin inhibition was
determined by the following relationship: % heparin
inhibition=[1-(net growth in 15% FCS+heparin/net growth
in 15% FCS)]x100.
Displacement curves of binding of
[3H]-labeled heparin were performed as
described by Castellot et al27 to characterize
heparin binding in 25 human VSMC cultures. Cells were seeded at a
density of 105 cells/mL, 1 mL/well, in 24-well
plates and growth-arrested for 72 hours as described above. The cells
were then cooled to 0°C to 4°C and washed with ice-cold PBS.
Precooled solutions of [3H]hep-arin at
10-7 mol/L with various concentrations of
unlabeled heparin (10-9 to
10-5 mol/L) were added to triplicate wells for
30 minutes and maintained on ice during the assay. (In 13 more cell
cultures, specific binding was calculated with only
10-5 mol/L unlabeled heparin.) At the end of the
incubation period, cells were washed 3 times with ice-cold PBS and
solubilized in 1 mol/L NaOH overnight at room temperature. Cell lysates
were transferred to scintillation vials to which Ecoscint (National
Diagnostics) was added. Radioactivity was counted in a
liquid scintillation counter (Canberra, Packard) to determine the
amount of bound [3H]heparin. Proliferation and
binding assays were performed at the same time on cells from individual
cell cultures.
2 analysis was used to compare
group risk factors and the Mann-Whitney U test to compare
the median heparin inhibition values for stenosed and nonstenosed
grafts. Spearman's rank correlation coefficient was used in the
comparison between heparin inhibition and the amount of binding in
individual VSMC cultures, and the point biserial correlation
coefficient was used for analysis of dichotomous
data.28
FCS was obtained from J. Bio (Les Ulis, France), and all
other cell culture materials were obtained from GIBCO Life
Technologies/BRL. Stock [3H]heparin (1 mCi/mg,
with an average molecular weight of 14 kDa) was purchased from Du Pont.
The unlabeled heparin (Paynes & Byrne) was a kind gift of Dr B. Mulloy,
National Institute of Biological Standards and Control, South Mimms,
England, and has previously been extensively characterized for chemical
and anticoagulant properties29 as well as for its
in vitro antiproliferative effects on human
VSMCs.30 The monoclonal antibody to smooth muscle
-actin was clone 1A4 (Sigma).
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Seventy-six patients were followed up for a minimum of 1 year.
Infrainguinal grafts in 8 more patients occluded within 6 weeks of
surgery and were not analyzed in this study, because their
failure was considered to have resulted from technical mishap or
thrombosis.22 31 In 14 of the 76 patients, cell
culture was unsuccessful, leaving 62 patients suitable for
analysis. The population of patients in each outcome group was
not significantly different with respect to age, sex, diabetes, smoking
habits, or lipid profile (Table
). Given the small numbers involved, no
attempt was made to stratify for the severity of any of these
parameters. Thirty-nine patients had angiographically
confirmed unstenosed grafts after a minimum of 1 year of follow-up, and
23 (37%) had angiographically confirmed stenoses that had
developed between 6 weeks and 1 year after surgery. All of the patients
with stenoses underwent revision surgery, and in 4 cases the
intimal hyperplastic lesion was removed and examined
histologically. These lesions were all MIH. The
stenoses were first detected at the following times (figures
are cumulative): 9 by 3 months, 16 by 6 months, 21 by 9 months, and 23
by 1 year. In these 23 patients, the median VSMC inhibition in the
presence of heparin, as calculated from growth assays, was 20.9%
(range, 0% to 62%). By contrast, the median for the 39 patients whose
grafts remained patent was significantly higher at 54.0% (range, 4%
to 95%) (P<0.001, Mann Whitney) (Figure 1).
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Figure 1. Inhibition of proliferation by heparin (7
µmol/L) of VSMCs derived from saphenous vein of patients with and
without stenosis of infrainguinal vein grafts between 6 weeks
and 1 year after surgery. Inhibition was calculated as described in
"Methods." Each symbol represents a patient. ). Maximum binding
was calculated for another 13 cultures. In 13 of the 38 patients, graft
outcome data were also available, with binding and proliferation assays
performed concurrently. Analysis of the binding suggested a
single population of binding sites (data not shown) and showed a marked
variation in [3H]heparin binding between
different VSMC cultures, with the number of sites ranging from
105 to 106/cell. It
should be noted that this does not represent maximal binding,
because the concentration of labeled heparin was not saturating but
rather approximated the dissociation constant. Proliferation assays on
the same cell cultures also revealed a varied response, with a range of
values for heparin inhibition (0 to 95%; median, 42.3%). In cultures
derived from the same patient, a highly significant positive
correlation was observed between heparin inhibition of proliferation
and the density of binding of [3H]heparin
(10-7 mol/L) (r=0.81, n=38,
P<0.0001) (Figure 3). In
other experiments (data not shown), we have demonstrated that this
binding site is saturable.
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Figure 2. Representative inhibition curves
of [3H]heparin binding in a cell culture
resistant to growth inhibition by heparin (<25% inhibition,
) and a culture >75% inhibited by heparin (
).
View larger version (14K):
[in a new window]
Figure 3. Relation of inhibition of VSMC proliferation with
[3H]heparin binding. Each symbol represents a
patient. ), giving a
multiple correlation coefficient of 0.65 for the 3
parameters.
View larger version (12K):
[in a new window]
Figure 4. Relation of inhibition of VSMC proliferation by
heparin with [3H]heparin binding in patients with ( 
)
and without (
) infrainguinal vein graft stenosis.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
Vascular stenosis or restenosis has proved to be
an intractable clinical problem despite intensive attempts to devise
effective pharmacological prophylaxis. Although the basic features of
MIH are well described, it is unclear why this process, which is
essentially a reactive response to vascular
injury,31 32 becomes excessive and inappropriate
in 25% to 50% of patients6 and leads to
clinically significant luminal obstruction. Many therapeutic agents
have proved to be highly effective in attenuating MIH after vascular
injury in animal models, but this has not been predictive of success in
clinical trials, which have been consistently
negative.33 Even if one allows for weaknesses in
trial design, this suggests that such models may not be wholly
appropriate, particularly if they involve species as remote from humans
as the rat and rabbit. It is also true that remodeling, perhaps
involving the extracellular matrix, has not so far been a therapeutic
target.34 We have therefore used cultured human
VSMCs as a model system to investigate the control of cell
proliferation. In previous studies, we have shown that cells from a
given individual behave consistently over several passages,
notably in terms of response to growth inhibition by heparin.
Furthermore, this consistency is also seen in cells taken
from different vessels in the same patient, including those from
stenotic lesions themselves.35 By
contrast, in confirmation of earlier observations, we have demonstrated
that there is wide interindividual diversity in
responses.18 These findings support the
assumption that differences in cellular response are reproducible and
significant. In the present study, we have confirmed and extended
our earlier observation that VSMCs derived from patients with
stenoses are relatively resistant to growth inhibition
by heparin and report a highly significant correlation between reduced
growth inhibition by heparin in cells cultured from the grafted vessel
and the development of stenosis in the subsequent year.
Selected Abbreviations and Acronyms
MIH
=
myointimal hyperplasia
SHR
=
spontaneously hypertensive rat
VSMC
=
vascular smooth muscle cell
WKY
=
Wistar-Kyoto rat
Acknowledgments
Drs Schachter and Patel gratefully acknowledge personal
support from the Institut des Récherches Internationales Servier.
Drs Refson, Munro, and Wolfe gratefully acknowledge financial support
from Impra UK. Dr Munro was a British Heart Foundation Junior
Research Fellow.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
1.
McBride W, Lange RA, Hillis LD. Restenosis
after successful coronary angioplasty. N Engl J
Med. 1988;318:1734–1737.[Medline]
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