doi: 10.1161/hq0701.092106
© 2001 American Heart Association, Inc.
Vascular Biology |
Injury Induces Dedifferentiation of Smooth Muscle Cells and Increased Matrix-Degrading Metalloproteinase Activity in Human Saphenous Vein
From the Bristol Heart Institute, University of Bristol, Bristol, UK, and the Department of Molecular Genetics (G.J.J.M.v.E.), Faculty of Medicine, University of Maastricht, Maastricht, the Netherlands.
Correspondence to Mr Jason Lee Johnson, Bristol Heart Institute, Level 7, Bristol Royal Infirmary, Upper Maudlin Street, Bristol BS2 8HW, UK. E-mail jason.l.johnson{at}bris.ac.uk
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Abstract |
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Abstract—Long-term patency of human saphenous vein bypass grafts is low because of intimal thickening and superimposed atherosclerosis. Matrix-degrading metalloproteinases (MMPs) and changes in vascular smooth muscle cell (VSMC) phenotype are thought to be essential for the VSMC migration that contributes to intimal thickening. We examined VSMC phenotype and MMP activity in saphenous veins obtained before and after surgical manipulation. Surgical preparation of the veins significantly increased pro-MMP-1 expression by 2-fold and significantly reduced tissue inhibitor of MMPs (TIMP)-2 expression, whereas MMP-3 and TIMP-1 were unaffected. Furthermore, caseinolytic and gelatinolytic activities measured by in situ zymography were dramatically elevated by injury. The expression of desmin and smoothelin was significantly decreased by injury, whereas vimentin expression was significantly increased. In addition, these changes in phenotype and MMP activity were localized to a subpopulation of VSMCs, the circumferential medial VSMCs. Our data show that surgical preparative injury induces phenotypic modulation of a subpopulation of medial VSMCs to a synthetic phenotype and increases MMP activity. This may favor matrix degradation, VSMC migration, and the subsequent intimal thickening that leads to graft failure.
Key Words: smooth muscle cell • differentiation • matrix-degrading metalloproteinases • tissue inhibitors of matrix-degrading metalloproteinases • smoothelin
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Autologous saphenous vein remains the most commonly used conduit for coronary artery bypass grafting,1 despite long-term patency rates of 50% after 10 years.2 Late vein graft failure results from intimal thickening with superimposed atheroma. Intimal thickening occurs because of migration of medial vascular smooth muscle cells (VSMCs) to the intima, where they proliferate and secrete extracellular matrix proteins.3 4 Surgical preparation of the saphenous vein injures the blood vessel and contributes to the poor long-term patency (see review by Angelini and Newby1 ).
Medial VSMCs exist in the normal blood vessel wall in the
contractile (differentiated) phenotype. These have a
"spindle-like" morphology, maintain vessel wall tone, and are rich
in contractile and intermediate filament proteins (IFPs). It has been
suggested that migrating and proliferating VSMCs have dedifferentiated
to a synthetic phenotype, characterized by a reduction in
contractile proteins and alterations in IFPs (see review by
Owens5 ). Recent studies
illustrate that the amounts of the IFPs vimentin and desmin, the
cytoskeleton-related protein smoothelin, and the contractile proteins
-smooth muscle (SM) actin, SM myosin heavy chain (SMMHC), and
tropomyosin change when VSMCs shift from the contractile to the
synthetic
phenotype.5 6 7
Mechanical injury to the blood vessel wall, particularly endothelial damage, is thought to trigger phenotypic modulation of medial VSMCs, shifting them toward the synthetic phenotype (see reviews by Thyberg and colleagues8 9 10 ). To enable VSMC migration, remodeling of the basement membrane and of the interstitial collagenous matrix that maintains VSMCs in a quiescent state must occur.11 Mechanical injury of aortic explants12 and isolated VSMCs13 stimulates the production of extracellular matrix-degrading metalloproteinases (MMPs), which are mainly associated with VSMCs of the synthetic phenotype.14 15 Injury of rat carotid arteries16 and human saphenous veins17 increases the expression of basement membrane–degrading MMP-2 and MMP-9. Furthermore, MMP inhibitors,12 18 19 20 as well as gene transfer of the endogenous tissue inhibitors of MMPs (TIMPs),21 22 23 24 25 have demonstrated the involvement of MMPs in injury-stimulated intimal thickening. Injury also affects TIMP protein levels. TIMP-2 is increased after rat carotid injury in 2 studies.26 27 Although these previous studies have demonstrated that dedifferentiation and MMP activity are required for intimal thickening, it is unknown whether these are caused by injury or whether they are colocated.
The present study aimed to examine whether injury of the
saphenous vein caused by surgical preparation affects MMP activity and
medial VSMC phenotype before its use as a coronary
artery vein graft. The expression of the cytoskeletal proteins (desmin,
vimentin, and smoothelin) and of contractile proteins (-SM actin,
SMMHC, and tropomyosin) was examined to assess VSMC phenotypic
modulation. The expressions of MMP-1, MMP-3, TIMP-1, and TIMP-2 and net
collagenolytic and gelatinolytic activity were also
determined.
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Methods |
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Materials
All reagents were of the highest grade available and were obtained from Sigma Chemical Co, unless stated differently in the text.
Vein Collection
Paired freshly isolated and surgically prepared
segments of human saphenous vein were obtained from 13 patients
undergoing coronary artery bypass graft surgery. Freshly
isolated veins (2 to 3 cm in length) were obtained immediately after
excision from the leg, with use of a no-touch
technique.28 After
completion of the last proximal anastomosis, surgically prepared veins
(up to 10 cm in length) were obtained from the same patients. Surgical
preparation involved dissection from the vascular bed, adventitial
stripping, side-branch ligation, gentle manual distension at gentle but
uncontrolled pressure (up to 600 mm Hg), and storage in
heparinized blood at room temperature (23°C) for between 50 and 180
minutes. Surgically prepared veins were obtained 60 to 180 minutes
after the freshly isolated veins. Ethical permission was obtained from
the United Bristol Healthcare Trust ethics committee (Ref.
E2847). The adventitia was removed, and the vein was cut into 5- to
10-mm segments. Segments were snap-frozen, quenched in isopentane, or
fixed in 10% (vol/vol) formalin-PBS and embedded in paraffin wax at
right angles to the original direction of blood
flow.
Immunocytochemistry
Serial 3-µm paraffin sections were dewaxed and
rehydrated (n=6 paired segments). Endogenous peroxidase
activity was inhibited with hydrogen peroxide. Antigen retrieval was
carried out by using either pressure cooking or trypsin digestion.
After the sections were blocked with 20% (vol/vol) goat serum in PBS,
sections were incubated overnight at 4°C with primary antibodies
diluted in 1% (wt/vol) BSA in PBS (mouse anti-myosin 1:75,
anti–-SM actin 1:400, anti-tropomyosin 1:400, anti-vimentin 1:200,
anti-desmin 1:50 [DAKO], and anti-smoothelin
1:5; rabbit anti–MMP-1 1:500 and anti–MMP-3 1:1500 [Dr C Long,
Pfizer, Sandwich, UK]; and sheep anti–TIMP-1 36 µg/mL and
anti-TIMP-2 35 µg/mL [Prof G. Murphy, University of Norwich,
Norwich, UK]). Sections were incubated with appropriate biotinylated
secondary antibodies (DAKO) diluted 1:200 in 1% (wt/vol) BSA in PBS
and then with either horseradish peroxidase or alkaline
phosphatase–labeled ExtrAvidin (diluted 1:400
in 1% [wt/vol] BSA in PBS). Color was developed with 0.05% (wt/vol)
3,3'-diaminobenzidine or Fast Red TR/Naphthol AS-MX (Fast Red),
respectively, and then nuclei were counterstained with Mayer’s
hematoxylin. The percentage of positive circumferential and
longitudinal cells was counted in four
0.25-mm2 fields. A negative control, for
which the primary antibody was replaced with mouse, rabbit, or sheep
IgG at the same dilution, was always included.
For dual immunocytochemical labeling (n=6 paired segments), vimentin was detected as described above with 3,3'-diaminobenzidine used as substrate. Desmin was then detected as described above except for the addition of avidin- and biotin-blocking solution (Vector Laboratories) in goat serum and primary antibody, respectively. Peroxidase-labeled ExtrAvidin was substituted with alkaline phosphatase–labeled ExtrAvidin, and Fast Red was used as substrate.
Western Blotting
Tissue levels of desmin, vimentin, and smoothelin
(n=3 paired segments) and MMP-1, MMP-3, TIMP-1, and TIMP-2 (n=6 paired
segments) were quantified by Western blot analysis, as
described previously.17
Equal concentrations of proteins were loaded on gels and detected with
rabbit anti-human MMP-1 and MMP-3 antisera diluted 1:1000, 50 µg/mL
sheep anti-human TIMP-1 and TIMP-2 antibodies, and 2 µg/mL mouse
anti-human desmin, vimentin, and smoothelin antibodies. Densitometric
scanning was performed by using a Bio-Rad model GS-690 Imaging
Densitometer.
In Situ Zymography
Caseinolytic and gelatinolytic
activities were located in frozen sections (n=4 paired segments) as
described
previously.29 30
Controls, for which the incubation buffer was supplemented with 20
mmol/L EDTA, 500 nmol/L of the nonselective MMP inhibitor
UK-231,890 (BB94) and 500 nmol/L of the gelatinase
inhibitor UK-181,587 (CT572, both generous gifts from Dr C.
Long, Pfizer Limited, Sandwich, UK), 10 µmol/L E64, 100 µmol/L
pepstatin A, and 1 µmol/L 4-amidinophenylmethanesulfonyl
fluoride (APMSF) were included.
Statistical Analysis
Values are expressed as mean±SEM. Mean values of
positive cell counts were compared by a 2-way ANOVA and
Student-Newman-Keuls post hoc test to detect differences between 2
groups. Mean values from densitometric scanning were compared by using
ANOVA and Tukey-Kramer multiple comparisons. Differences were
considered statistically significant at
P<0.05.
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Results |
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As published previously,31 a high proportion of saphenous veins used for coronary artery bypass grafting have a preexisting thickening. VSMCs in these thickenings are longitudinally orientated, in contrast to the circumferentially orientated medial VSMCs. All saphenous veins used in the present study exhibited such thickening; therefore, VSMCs are referred to as longitudinal or circumferential. Histological assessment of surgically prepared veins revealed patchy disturbance of the vessel architecture, particularly in the circumferential layer (data not shown). It has been suggested that distension of veins before implantation may induce a number of changes,32 including the loss of cell-to-cell and cell-to-matrix contacts, muscle hypertrophy, and proliferation of circumferential VSMCs.33 34
Expression of Cytoskeletal and Contractile
Proteins
The contractile proteins SMMHC, -SM actin, and
tropomyosin were detected in 78±1%, 97±2%, and 85±2% of medial
VSMCs of freshly isolated veins, respectively. Similarly, SMMHC, -SM
actin, and tropomyosin were detected in 87±2%, 96±1%, and 84±2%
of medial VSMCs of surgically prepared veins, respectively. In both
vein types, the expression was similar in longitudinal and
circumferential VSMCs.
In freshly isolated veins, a small number of longitudinal
and circumferential VSMCs, 14% and 20%, respectively, stained
vimentin positive
(Figure 1A
and Table 1). Significantly higher numbers (38%,
P<0.01)
of vimentin-positive cells were detected
in the circumferential VSMCs of surgically prepared veins
(Figure 1D and
Table 1). In contrast, the percentage of cells expressing
smoothelin and desmin was significantly higher in circumferential VSMCs
of freshly isolated veins
(Figure 1B and 1C and
Table 1) than in surgically prepared veins (75% versus
51%, respectively, for smoothelin
[P<0.01] and 79% versus
57%, respectively, for desmin
[P<0.05];
Figure 1E and 1F and
Table 1). The expression of smoothelin and desmin protein
in longitudinal VSMCs was similar in both vein types. No staining was
detected in the negative control sections (data not shown).
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Dual immunocytochemistry revealed that almost all cells
expressed desmin (red) but that less than half of these cells also
expressed vimentin (brown,
Figure 2A) in freshly isolated veins. Coexpression of these
2 proteins was restricted to circumferential VSMCs. In contrast, in
surgically prepared veins, cells expressing only vimentin were observed
(Figure 2B and 2C). Furthermore, although lower numbers of
desmin-positive cells were present, all desmin-positive cells
coexpressed vimentin
(Figure 2B). It is also noteworthy that the
vimentin-positive/desmin-negative cells in surgically prepared veins
were confined to the circumferential VSMCs.
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Therefore, our present findings have shown that vimentin is significantly increased only in circumferential VSMCs within the media of surgically prepared saphenous veins. Conversely, these cells had reduced expression of desmin and smoothelin, suggesting that injury to these veins results in activation and deactivation of proteins linked with VSMC dedifferentiation.
Western blotting for desmin, vimentin, and smoothelin
proteins confirmed the immunocytochemical results. Despite some
variation between samples, significantly less smoothelin (1.34±0.81
versus 4.82±0.73, P<0.03) and
desmin protein (4.15±0.39 versus 7.11±1.44,
P<0.05) and significantly more
vimentin protein (8.13±0.73 versus 2.73±1.35,
P<0.03) was detected in
surgically prepared veins than freshly isolated veins
(Figure 2D
and 2E). Variation between samples may be due to
differences in the degree of distension and therefore injury, because
pressure is not controlled during surgical
preparation.
Localization and Quantification of MMP-1,
MMP-3, TIMP-1, and TIMP-2
MMP-1 protein expression was detected in most medial
VSMCs in freshly isolated veins
(Figure 3A and
Table 1) and surgically prepared veins
(Figure 3E and
Table 1). However, compared with longitudinal VSMCs,
significantly greater numbers of circumferential VSMCs
(P<0.01) were positive for
MMP-1
(Table 1). A low level of MMP-3 protein expression was
detected in the longitudinal and circumferential VSMCs of freshly
isolated
(Figure 3B and
Table 1) and surgically prepared
(Figure 3F and
Table 1) veins.
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TIMP-1 protein was detected in almost all longitudinal and
circumferential cells in freshly isolated
(Figure 3C and
Table 1) and surgically prepared
(Figure 3G and
Table 1) veins. TIMP-2 protein was located in almost all
longitudinal and circumferential VSMCs of freshly isolated veins
(Figure 3D and
Table 2). However, significantly less staining was observed
in longitudinal (P<0.05) and
circumferential (P<0.001)
VSMCs in surgically prepared veins
(Figure 3H and
Table 1). Interestingly, the number of TIMP-2 positive
VSMCs in the circumferential layer was significantly less than that
detected in the longitudinal layer
(P<0.05). No signal was
detected when the antibodies were preadsorbed with the appropriate
peptides or when nonimmune IgG was used (data not shown).
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Western blotting confirmed that surgical preparation
significantly reduced TIMP-2 protein expression, whereas TIMP-1 was not
statistically affected
(Table 2). Interestingly, a significant increase in
pro-MMP-1 protein expression was detected in surgically prepared veins
compared with freshly isolated veins
(Table 2). No difference in active MMP-1 and pro-MMP-3 and
active MMP-3 protein expression was detected between the vein types
(Table 2).
Caseinolytic and
Gelatinolytic Activity
In situ zymography revealed areas of gelatin
lysis, indicating gelatinolytic activity,
throughout the media in all surgically prepared veins, whereas
caseinolytic activity was detected only in the circumferential VSMCs
(Figure 4A and 4C, n=4). Addition of the MMP
inhibitors BB94
(Figure 4E and 4G) and EDTA (data not shown) abolished
caseinolytic and gelatinolytic activity. Casein in
situ zymography supplemented with a gelatinase-specific
inhibitor (CT572) reduced but did not abolish the
caseinolytic activity
(Figure 4F and 4H) in surgically prepared veins, indicating
that MMPs other than gelatinases are responsible for the observed
substrate lysis. No caseinolytic activity
(Figure 4B) and only low levels of
gelatinolytic activity
(Figure 4D) were detected in freshly isolated veins (n=4).
Addition of other protease inhibitors (E64, pepstatin A,
and APMSF) did not affect the observed activity, demonstrating the
specificity of this method (data not
shown).
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Discussion |
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In the present study, we observed that a subset of VSMCs, the circumferential VSMCs, underwent phenotypic modulation after surgical preparation. Furthermore, this coincides with induced MMP activity. This dedifferentiation of VSMCs and MMP activity may contribute to the VSMC migration and proliferation that leads to intimal thickening and late vein graft failure.
Medial VSMC phenotypic modulation, characterized by changes in desmin and vimentin expression, in response to injury is thought to enable migration and proliferation and to result in neointimal thickening.35 36 37 In the present study, we describe decreased desmin and increased vimentin expression, which is associated with the synthetic phenotype,38 in circumferential VSMCs after surgical preparative injury of saphenous vein. In addition, we observed a significant reduction in the cytoskeleton-associated protein smoothelin, a unique protein expressed in contractile VSMCs,6 39 in circumferential VSMCs after injury. This further indicates the conversion of VSMCs from the contractile to the synthetic phenotype in this subset of VSMCs after injury. These findings are supported by the detection of less smoothelin expression in vein bypass grafts compared with saphenous vein obtained at postmortem examination.40
Focusing on the MMP/TIMP system, we also assessed the expression and proteolytic activity of these enzymes in response to injury, to determine whether an increased ability for matrix degradation coincides with VSMC phenotypic modulation. Using in situ zymography, we demonstrated that this is the case, with increased amounts of gelatinolytic and caseinolytic activity in veins after surgical preparation. The increased collagenolytic and gelatinolytic activity coincided and was colocated with induced phenotypic modulation in circumferential VSMCs. We propose that this increase in MMP activity is, in part, due to the increased expression of MMP-1 and the decreased expression of TIMP-2 observed in circumferential VSMCs. Taken with our previous findings, ie, that MMP-2 and MMP-9 levels are increased after injury,17 this suggests that the MMP/TIMP balance is shifted to favor proteolytic degradation after injury. This increase in MMP activity presumably permits matrix remodeling and the increased migration and possibly proliferation that occur in the vein graft after implantation. Although the precise role of the MMPs in inducing VSMC migration is unknown, there are several potential mechanisms.41 It may remove the physical restraint to permit migration, sever cell-matrix contacts via integrins or cell-cell contacts via cadherins, permit contacts with interstitial matrix components, expose cryptic extracellular matrix sites, or produce extracellular matrix fragments, which stimulate migration or release matrix or cell-bound growth factors.
In summary, we observed that phenotypic modulation of VSMCs and MMP activity induced by injury were located in the outer circumferential layer of the media. It could be postulated that these VSMCs are more susceptible to mechanical stretch injury as a result of vessel distension. This is of note because we have previously demonstrated that intimal thickening induced by VSMC migration and proliferation is significantly greater in surgically prepared veins than in freshly isolated veins and that VSMC proliferation occurs only in the circumferential VSMCs in surgically prepared veins and is absent in freshly isolated veins.33 42 This suggests that dedifferentiation of VSMCs and increased MMP activity induced by injury may contribute to the VSMC migration and proliferation that lead to intimal thickening. However, although we have clearly demonstrated that MMP activity is required for VSMC migration in veins,21 22 23 the direct involvement of MMP activity in VSMC proliferation is less clear. We have previously demonstrated that a synthetic MMP inhibitor reduces VSMC proliferation,43 but the overexpression of TIMP-1, TIMP-2, and TIMP-3 did not affect VSMC proliferation.21 22 23
Although we have clearly demonstrated that injury increases MMP activity and phenotypic modulation of circumferential VSMCs, we are unable to show directly that they occur in the same cells. This is due to the nature of the methods used. In situ zymography for MMP activity requires coating the section with the substrate; therefore, immunocytochemistry is impossible. However, despite this, we believe that our data provide very compelling evidence that injury induces MMP activity and dedifferentiation of a subset of VSMCs. Furthermore, the present study does not demonstrate that these changes are essential for intimal thickening, but taken together with previous studies, it suggests that injury may initiate some of the processes that occur after implantation and contribute to intimal thickening.
The present study illustrates that injury of the saphenous vein caused by surgical manipulation is sufficient to cause phenotypic modulation of a subset of VSMCs. It highlights that minimization of preparative injury may inhibit intimal thickening by reducing these changes. Furthermore, stabilizing the MMP/TIMP balance with synthetic MMP inhibitors or overexpression of recombinant TIMPs may be clinically useful in reducing intimal thickening and late vein graft failure.
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Acknowledgments |
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The British Heart Foundation and the Garfield Weston Trust supported this work.
Received March 8, 2001; accepted April 3, 2001.
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