CLINICAL STUDY
Enhanced extracellular matrix accumulation in restenosis of coronary arteries after stent deployment
Ick-M. o Chung, MD*,
Herman K. Gold, MD
,
Stephen M. Schwartz, MD, PhD*,
Yuji Ikari, MD
,
Michael A. Reidy, PhD* and
Thomas N. Wight, PhD*
,*
* Department of Pathology, University of Washington, Seattle, Washington, USA
Hope Heart Institute, Seattle, Washington, USA
Division of Cardiology, Massachusetts General Hospital, Harvard University, Boston, Massachusetts, USA
Division of Cardiology, Mitsui Memorial Hospital, Tokyo, Japan
Manuscript received January 9, 2002;
revised manuscript received August 22, 2002,
accepted August 29, 2002.
* Reprint requests and correspondence: Dr. Thomas N. Wight, Vascular Biology, The Hope Heart Institute, 1124 Columbia Street, Suite 783, Seattle, Washington 98104, USA.
twight{at}hopeheart.org
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Abstract
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OBJECTIVES: The goal of this study was to evaluate the cellular and extracellular composition of human coronary arterial in-stent restenosis after various periods of time following stent deployment.
BACKGROUND: Neointimal in-growth rather than stent recoil is thought to be important for coronary arterial in-stent restenosis. There is only limited data on the cellular and extracellular composition changes with time after stent deployment.
METHODS: We analyzed 29 coronary arterial in-stent restenotic tissue samples (14 left anterior descending coronary artery, 10 right coronary artery, and 5 left circumflex artery) retrieved by using directional coronary atherectomy from 25 patients at 0.5 to 23 (mean, 5.7) months after deployment of Palmaz-Schatz stents employing histochemical and immunocytochemical techniques.
RESULTS: Cell proliferation was low (0% to 4%). Myxoid tissue containing extracellular matrix (ECM) enriched with proteoglycans was found in 69% of cases and decreased over time after stenting. Cell-depleted areas were found in 57% of cases and increased with time after stenting. Versican, biglycan, perlecan, and hyaluronan were present with varying individual distributions in all samples. Positive transforming growth factor-ß1 staining was found in 80% of cases. Immunostaining with alpha-smooth muscle actin identified the majority of cells as smooth muscle cells with occasional macrophages present (
12 cells per section).
CONCLUSIONS: These data suggest that enhanced ECM accumulation rather than cell proliferation contribute to later stages of in-stent restenosis. Balloon angioplasty of in-stent restenosis may, therefore, fail due to ECM changes during: 1) additional stent expansion, 2) tissue extrusion out of the stent, or 3) tissue compression.
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Abbreviations and Acronyms
| | ECM | | extracellular matrix | | HABP | | hyaluronan binding protein | | PBS | | phosphate buffered saline | | PCNA | | proliferating cell nuclear antigen | | SMC | | smooth muscle cell | | TGF-ß1 | | transforming growth factor-ß1 |
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Although intracoronary stent implantation has been shown to reduce the rate of restenosis and to improve clinical outcome compared with standard balloon angioplasty (1), in-stent restenosis rate is still 15% to 30% in six months (1–3). The mechanism of stent stenosis is not well understood. Neointimal in-growth, rather than tissue remodeling or stent recoil, is thought to be an important mechanism for in-stent restenosis because stents subject the artery to permanent dilation. These ideas are supported by clinical intravascular ultrasound studies (2,3) and animal studies (4).
Only a few histopathologic studies of stent restenosis have been performed in patients with coronary or peripheral artery disease (5–7). Myxoid tissue characterized by stellate-shaped smooth muscle cells (SMC) in a loose extracellular matrix (ECM) is a frequent observation in restenotic tissue (5,6). Similar tissue has been found in coronary restenotic lesions and, to a lesser extent, in primary atherosclerotic lesions (8). Cell replication rates as measured by proliferating cell nuclear antigen (PCNA) vary according to the study. For example, no PCNA-positive cells were found in 10 coronary stented restenotic samples (5), whereas approximately 25% of the SMC of peripheral arterial stented restenotic samples were PCNA-positive (6). Data on human coronary restenotic lesions after balloon angioplasty show cell replication rates varying from mostly <1% (9) to as much as 15.2% (10). The reasons for these differences are not clear.
In the present study, we analyzed histopathologic sections of human coronary arterial stent neointima retrieved by directional atherectomy from 25 patients who underwent previous Palmaz-Schatz stent deployment with the specific aim of assessing cellularity, cell replication rate, and ECM composition over time after stenting.
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Method
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Atherectomy specimens.
Twenty-nine Palmaz-Schatz coronary stent restenotic independent specimens (14 left anterior descending coronary artery, 10 right coronary artery, and 5 left circumflex) were obtained from different anatomical locations from 25 patients (male/female: 18/7; age: 59 ± 13 years) and analyzed separately. In four patients, two different specimens were retrieved at different sites within the stent. Specimens were retrieved percutaneously by directional coronary atherectomy using a Simpson Coronary Atherocath (Devices for Vascular Intervention, Redwood City, California) in the Massachusetts General Hospital (Boston, Massachusetts) and Mitsui Memorial Hospital (Tokyo, Japan) between August 1995 and November 1996. Indications for atherectomy were angiographically documented stent restenosis with one of the following: 1) positive exercise tolerance test with or without stable angina (n = 5: numbers 2, 6, 16, 20, 21 in Table 1), 2) recurrent unstable angina pectoris (n = 21), and 3) acute myocardial infarction (n = 3: numbers 1, 4, 12 in Table 1). Time interval between initial stent deployment and later atherectomy was 5.7 ± 5.4 (range: 0.5 to 23) months (Table 1, Fig. 1). During the time intervals studied, all patients that had in-stent restenosis where the stent was approachable were used in the study. Tissue was taken from within the stent. Great care was taken not to go outside the stent margins. All stents were postdilated with a high-pressure balloon from 14 to 18 atms. All patients had >75% stenosis for the initial stent placements. Reference diameter of the vessel was 
3.0 mm. Fifteen tissue specimens were immediately immersed in methyl Carnoy’s fixative for two to three days. Fourteen specimens were immersed in 4% paraformaldehyde and divided for immunocytochemical staining and transmission electron microscopy.
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Figure 1 Bar graph showing the distribution of age of specimens after stent deployment. DCA = directional coronary angioplasty.
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Histology
Histochemistry was performed on serial sections from each specimen. Each tissue section ranged in area between 0.3 to 3.0 mm. Hematoxylin and eosin staining was done to determine the cellularity and general morphologic patterns of the specimens. The distribution of myxoid tissue was graded semiquantitively: 1+ for small foci and 2+ for large microscopic areas. Modified Movat’s staining was performed to identify different ECM topological patterns (11). For example, ECM was graded semiquantitatively for the relative distribution of proteoglycans and collagen by analyzing modified Movat’s staining: proteoglycans as a dominant component (grade 1), proteoglycans > collagen (grade 2), proteoglycans approximately equal to collagen (grade 3), proteoglycans < collagen (grade 4), and collagen as a dominant component (grade 5). The distribution of transforming growth factor (TGF)-ß1 labeling was semiquantitatively graded: 1+ for positivity of <5% of cells; grade 2+ for positivity 5% of cells per microscopic field. The total number of Ki-67-positive cells per slide was counted visually at x 400 magnification by two pathologists (M.R. and I.C.), and the percentage of positive Ki-67 cells in each tissue fragment was calculated. A cell-depleted area is defined as a microscopic field area in which fewer than 320 cells/mm2 are found.
Antibodies
To identify SMCs, tissue sections were stained with mouse monoclonal anti-human 
-smooth muscle actin antibody (IgG2a, DAKO, Carpinteria, California). Macrophages were identified by staining the specimens with mouse monoclonal CD68 antibody (IgG3, DAKO). To identify proliferating cells, tissue sections were stained with monoclonal MIB-1 antibody (IgG1, Immunotech Inc., Westbrook, Maine), which detects Ki-67 nuclear antigen. Biotinylated hyaluronan binding protein (HABP) was kindly provided by Dr. Charles B. Underhill (Georgetown University Medical Center, Washington, DC) and used as a probe for hyaluronan (12). Three different ECM proteoglycans were analyzed. Versican was detected by rabbit polyclonal antibody (versican anti-poly E, IgG) raised against a fusion protein encoding for amino acid residues 387 to 692 of versican, which was kindly provided by Dr. Richard LeBaron (University of Texas, San Antonio, Texas). Perlecan was detected by rabbit antisera EY9 to purified murine perlecan produced by Engelbreth-Holm-Swarm tumor, which was kindly provided by Dr. John R. Hassell (Shriners Hospital, Tampa, Florida) (13). Biglycan was detected by antisera LF51 to human biglycan synthetic peptide that was kindly provided by Dr. Larry W. Fisher (National Institute of Dental Research, Bethesda, Maryland). Rabbit polyclonal affinity purified anti-human TGF-ß1 antibody (IgG, 1.25 µgm/ml) was generously provided by Dr. Leslie Gold (New York University Medical Center, New York, New York). Monoclonal antibody T2G1 was used for staining of fibrin. Monoclonal antibody T2G1 (6.7 µg/ml, Centocor, Malvern, Pennsylvania) reacts with Bß 15 to 42 peptides as well as fibrin II and cross-linked fibrin II.
Immunocytochemical staining
Immunocytochemistry was done on serial sections from each tissue specimen. After deparaffinization, endogenous peroxidase activity was blocked with 0.3% H2O2 in methanol. Nonspecific binding of the polyclonal antibodies was blocked by incubation with 10% normal goat serum or 10% normal horse serum in phosphate buffered saline (PBS), 1% body surface area depending on the source of secondary antibody. Slides were incubated with primary antibody for 1 h at room temperature. After washing with PBS, slides were incubated with secondary antibody for 1 h at room temperature. Two kinds of secondary antibodies were used. A biotinylated horse anti-mouse IgG (Vector Laboratories, Inc., Burlingame, California) was used with mouse primary antibody, whereas a biotinylated goat anti-rabbit IgG (Vector Laboratories, Inc.) was used with rabbit primary antibody. Further staining was performed using the avidin-biotin-peroxidase technique (Vectastain, Vector Laboratories, Inc.). DAB (3,3'-Diaminobenzidine Tetrahydrochloide, Sigma, St. Louis, Missouri) was used as a chromogen. Tissues were then counterstained with either methylgreen or hematoxylin. To confirm activity of the antisera, tissues positive for the various antisera such as human tonsil (anti-human -smooth muscle actin, CD 68, and MIB-1), human bladder (HABP), monkey pulmonary artery (TGF-ß1), and human organizing thrombi (ß-fibrin) were used. Negative control sections were incubated with same concentration of either normal mouse serum or normal rabbit serum depending upon the source of primary antibody instead of primary antiserum.
Antigen retrieval technique (14) was used for 4% paraformaldehyde-fixed tissue. Briefly, after deparaffinization, hydration, and blocking of endogenous peroxidase with 0.3% H2O2, slides were placed in a plastic container that was filled with 10 mM sodium citrate, pH 6.0. Slides were heated in a microwave oven (800 W) for 8 min. After heating, the container was removed from the oven and allowed to cool for 20 min at room temperature. After slides were rinsed in PBS, further immunohistochemical staining was performed on the slides.
Hyaluronan labeling was done with biotinylated HABP. After deparaffinization, tissue specimens were dehydrated with graded ethanol. Endogenous peroxidase activity was blocked with 0.3% H2O2 in 100% methanol. For negative control, human bladder tissue was digested with hyaluronidase (20 U/ml, 0.2 M sodium acetate, pH 5.5) for 1 h at 37°C. Nonspecific binding was blocked by incubation with 10% normal goat serum in PBS, 1% BSA for 1 h. Slides were incubated with biotinylated HABP (2 to 4 µg/ml) in 5% normal goat serum/PBS overnight at 4°C. Slides were rinsed three times in PBS, and further staining was performed using the avidin-biotin peroxidase technique.
Transmission electron microscopy
After initial fixation and transport in 4% paraformaldehyde/PBS, the tissue was rinsed for 5 min with 0.1 M cacodylate, pH 7.3, and treated for 2 h with a fixative that includes 2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.1 M cacodylate and 0.2% ruthenium red (Johnson-Matthey Co., West Hill, Massachusetts) to stabilize the proteoglycan component of the ECM, as described (15). After multiple rinses with 0.1 M cacodylate containing 0.1% ruthenium red, the tissue was postfixed with 1% osmium tetroxide and 0.05% ruthenium red in 0.1 M cacodylate for 2 h. After rinsing with water, the tissue was processed routinely for plastic embedment with dehydration through graded ethanol and infiltration with the epoxy resin Medcast (Pelco, Redding, California). Ultrathin sections (80 to 100 nm) were stained with 6% aqueous uranyl acetate and lead citrate.
Statistics
All statistics were calculated by use of SPSS 7.5 for Windows, and data are expressed as mean ± SD. To compare the frequency of specimens containing either cell-depleted areas or myxoid tissue between different stent age groups, we used Fisher exact test. A value of p < 0.05 indicates statistical significance.
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Results
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To facilitate any recognition of changes over time, these specimens were grouped into three categories: stented for under 3 months (n = 9), between 3 and 6 months (n = 13), and more than 6 until 23 months (n = 7).
Cellularity of lesion.
The cellularity of stent neointima, in general, decreases in time after stenting. The early lesion is hypercellular, whereas the late lesion becomes hypocellular (Fig. 2). The majority (59%) of lesions contain cell-depleted areas (Table 1). Interestingly, the frequency of specimens containing cell-depleted areas increases over time after stent deployment (Fig. 3) (1 of 9 in <3 months vs. 10 of 13 in 3 to 6 months after stenting, p < 0.01; 1 of 9 in <3 months vs. 6 of 7 in >6 months after stenting, p < 0.01). At other sites in the stent neointima, myxoid tissue characterized by stellate-shaped cells in a disorganized pattern was observed (Fig. 2). As can be seen in Figure 4, the frequency of specimens containing myxoid tissue diminishes in time after stenting (8 of 9 in <3 months vs. 2 of 7 in >6 months, p < 0.05). The majority of cells in these specimens were labeled with -smooth muscle actin, confirming that they are SMC (Fig. 2). Specimens were also stained with antibodies (CD68), which recognize macrophages. Nine of 15 samples showed no CD68-positive cells, while the remaining samples contained 1 to 12 macrophages per section (data not shown). Thus, stent neointima is primarily composed of SMC with minimal inflammatory cells.
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Figure 3 Bar graph showing the frequency of specimens containing foci of cell-depleted areas. Note that specimens with foci of cell depleted areas tend to increase over time after stenting. *p < 0.01 for both 3 to 6 months and <6 months vs. <3 months. DCA = directional coronary angioplasty.
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Figure 4 Bar graph showing the frequency of specimens containing myxoid tissue. Note that specimens with myxoid tissue tend to decrease over time after stenting. *p < 0.05 for <6 months vs. <3 months. DCA = directional coronary angioplasty.
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Cell proliferation
Overall, very few replicating cells (range, 0% to 4%) as assessed by Ki-67 staining were noted, and 16 of 26 analyzed specimens exhibited no replicating cells (Table 1). Ten specimens had more than one Ki-67-positive cell, and two specimens had more than 1% Ki-67-positive cells. The maximum number of Ki-67-positive cells were observed in a specimen that was taken 23 months after stent deployment (Fig. 5). When these data were organized into three groups with respect to time after stenting, the same pattern was observed (Fig. 6). Low cell replication rate of all specimens was also confirmed by PCNA immunolabeling (data not shown).
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Figure 5 Immunostaining for Ki-67 shows one positive nucleus (brown color, arrow) in the myxoid tissue of human coronary stent neointima at 2.5 months after stenting (A). In tissue of stent neointima at 23 months after stenting, several Ki-67-positive nuclei were noted (B). Tissue of human tonsil as a positive control shows many positive Ki-67-stained nuclei of lymphocytes in the follicular structure (C). Counterstaining with either hematocylin (A, B) or methylgreen (C). Bar = 100 µm.
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Figure 6 Numbers of in-stent restenotic specimens grouped according to various degrees of Ki-67 nuclear antigen labeling. Note that, regardless of stent age, majority of specimens had no Ki-67 labeling. Solid bar = <3 months; hatched bar = 3 to 6 months; diamond bar = >6 months.
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Identification of ECM
The majority of specimens after three months after stenting demonstrated cell-depleted areas. Two kinds of cell-depleted areas were noted: collagen-rich or collagen-poor as determined by histochemical staining. There was little staining for proteoglycans in the collagen-rich, cell-depleted areas. Immunocytochemistry revealed that the proteoglycan biglycan was commonly associated with collagen-rich areas in approximately 60% of cases. The collagen-poor, cell-depleted areas immunostained for a variety of proteoglycans in which versican was most prominent (Fig. 7). Hyaluronan, and to a lesser degree perlecan, was also noted at these collagen-poor sites. There was a general tendency for collagen to accumulate in the ECM over time after stenting, whereas proteoglycans were more frequently observed in the early period after stenting (Table 1). In myxoid tissue, proteoglycans were dispersed diffusely (Fig. 8), and versican was, in general, dominant although hyaluronan and biglycan were also identified in these zones. Perlecan staining was minimal and was usually localized in the matrix surrounding SMC (Fig. 8). Most of myxoid tissue examined by electron microscopy exhibited ECM enriched in ruthenium red positive granules depicting proteoglycans with occasional immature segments of elastic fibers present (Fig. 9).
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Figure 7 Immunohistochemical analysis of extracellular matrix (ECM) of cell-depleted areas in human coronary stent neointimal tissue sections adjacent to the section of Figure 2C. In modified Movat staining, two kinds of cell-depleted areas were characterized. The collagen-rich ECM (pinkish yellow, thick arrow) is colocalized with the dense matrix seen in Figure 2C, and the proteoglycan-rich ECM (blue, thin arrow) with the loose matrix. Fibrin and/or fibrinogen are labeled as red (asterisk). Versican is abundant in the proteoglycan-rich area (brown, thin arrow), but is spared in the collagen-rich ECM (thick arrow). The distribution of hyaluronan is similar to that of versican. The biglycan-stained area is mostly colocalized with the dense collagen-rich matrix (brown, thick arrow). A few fibrin-labeled areas (dark brown) assessed by T2G1 antibodies are shown, counterstaining with methylgreen. Bar = 100 µm.
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Figure 8 Immunohistochemical analysis of myxoid tissue in atherectomized human coronary stent neointima at two months after stenting. In modified Movat’s staining, this specimen is composed of two different kinds of tissue: one is the myxoid tissue (labeled as M) enriched in blue-colored proteoglycans, and the other is collagen-rich cell-depleted area (labeled as C). Both versican and hyaluronan are abundant in this myxoid tissue, whereas both molecules are spared in the collagen-rich cell-depleted area. Perlecan staining shows labeling around the stellate-shaped smooth muscle cells. Transforming growth factor-ß1 staining shows cytoplasmic labeling in the stellate cells. Counterstaining with either methylgreen or hematoxylin. Bar = 100 µm.
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Figure 9 Transmission electron micrograph of an atherectomized human coronary stent neointima specimen containing the loose proteoglycan-rich matrix. Extracellular matrix contained numerous ruthenuium red positive proteoglycan granules, often linked together by fine filamentous threads, and occasional segments of immature elastin (labeled as E). Bar = 500 nm.
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These samples were also examined for the presence of fibrin. Fibrin was present in only 6 of 21 specimens (Table 1). In five specimens the fibrin staining was mostly found in cell-depleted areas (Fig. 7). There was no correlation between the presence of fibrin and time after stenting.
The ECM-enriched regions contained TGF-ß1-positive cells throughout the stent neointima (Fig. 4). Sixteen of 20 specimens stained positive with anti-TGF-ß1 antibody. Transforming growth factor-ß1 immunostaining tended to be abundant in myxoid tissue.
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Discussion
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Conventional wisdom is that the critical lesion of in-stent neointima is an excess of proliferative SMCs. Our data indicate that proliferation is not a factor contributing to in-stent restenosis at times greater than two months after interventions. Instead, the in-stent neointima in human specimens consists primarily of large amounts of a proteoglycan-rich ECM with a relatively small number of proliferative SMCs. A major finding in this study is the variability of cellularity in stent neointima. Cell-depleted areas are identified in 59% of cases and tended to increase with time after stent deployment. The cell density observed in cell-depleted areas (mean: 194; range: 48 to 320 cells/mm2) is as low as that detected in primary atherosclerosis (10). Other data (5) show a trend toward reduction in the cell density of the coronary arterial neointima formed after stenting with time. These data are consistent with the cell replication data in our study. Both Ki-67 nuclear antigen and PCNA labeling indexes are low, in most cases below 1%. This is true regardless of the period of time after stenting. Furthermore, the myxoid tissue observed in this study did not exhibit enhanced proliferative activity as others have observed (6,8). Thus, in-stent neointima may be a stable end-stage tissue similar to the advanced atherosclerotic plaque where both cell replication and death reach a minimal level. The low replication rate is also observed in primary lesions and in restenotic lesions after either stent implantation or balloon angioplasty (5,9,16). Furthermore, there are several studies in experimental animals to indicate that reinjury to existing intimal lesions stimulates low and transitory proliferative response by SMC (17,18). Smooth muscle cells cultured from human lesions also exhibit a marked decrease in the ability to replicate (19). Differences in detecting replication rates may reflect use of different reagents to detect proliferation. Ki-67 detects replicative cells more accurately than PCNA (20,21). However, it is possible that overestimates of cell proliferation can be made because both Ki-67 and PCNA are positive in proliferating cells not only during S phase, but also during the G1 and G2 phase of the cell cycle (16,20). One shortcoming of our study is that we are unable to rule out the possibility of transient increases in cell replication during the period immediately after stent insertion due to lack of specimens at this time period.
There are at least two possible mechanisms to explain the occurrence of cell-depleted areas in the stent restenotic specimens. One is cell death, and the other is ECM expansion in the absence of cell proliferation. As it may well be that both are operative, the recent observation that reduction in vascular tensile stress induced programmed cell death suggests that cell death may play a role (22). For example, the expansion of the stent in the artery should lead to a decrease in tension in the neointima formed inside of stent. Therefore, cell loss might be an expected outcome.
On the other hand, it is quite clear that the bulk of the lesion mass after stenting is due to accumulation of specific components of the ECM. Cell-depleted areas are characterized by both collagen-rich as well as proteoglycan-rich ECMs. Similar reciprocal ECM patterns of collagen and proteoglycan are observed in human restenotic peripheral arteries (23). However, in the present study, collagen-rich ECMs tended to be more common in cell-depleted areas suggesting that this ECM component may play a role in cell death (24,25).
The accumulation of ECM in these specimens may be influenced by the presence of TGF-ß1. Extracellular matrix accumulation in lesions of atherosclerosis and restenosis occurs in TGF-ß1-positive areas (26,27). Transforming growth factor-ß1 may be derived from resident vascular cells (28) or from platelet degranulation (29). Transforming growth factor-ß1 is elevated in vascular SMC of human coronary and peripheral arteries, and was significantly higher in restenotic lesions compared with primary lesions (27). Transforming growth factor-ß1 is known to regulate the synthesis and turnover of a variety of ECM components such as proteoglycans, hyaluronan, fibronectin, and collagen (30–34). Interestingly, blocking TGF-ß1 activity after vascular injury dramatically decreases ECM accumulation and suppressed intimal hyperplasia in the rat model of arterial injury (35).
Hyaluronan is abundant, especially in myxoid tissue, and is scarce in collagen-rich cell-depleted areas in our specimens. Similar inverse relation between hyaluronan and collagen was previously observed in human restenotic peripheral arteries (36). Hyaluronan accumulation around PCNA-positive SMCs is observed in human restenotic arteries and in injured rat carotid arteries, suggesting that hyaluronan significantly contributes to expansion of the neointima (36,37), possibly through the trapping of water and tissue swelling. In experimental animal studies, an increase of ECM hyaluronan is found to be responsible for the closing ductus arteriosus during fetal development (38). Hyaluronan may also influence in-stent restenosis by promoting the migration of SMCs (39–41).
On the other hand, hyaluronan may effect the constrictive remodeling that occurs in this form of restenosis. For example, hyaluronan added to collagen gels promotes collagen gel contraction by SMC (42), suggesting that hyaluronan could also promote vessel shrinkage if bound to collagen. However, in most cases hyaluronan was not present in collagen-rich regions in the stent restenotic samples.
Surprisingly, we observed little fibrin labeling (29% of cases) in these samples, which is lower than observed by others (6). Low frequency of fibrin deposits suggests that acute thrombosis does not play a significant role in the restenosis of stented arteries as shown in a recent clinical study in which acute thrombosis occurred in only 1.4% of cases (43).
Our study shows that ECM accumulation, rather than cell proliferation, in the developing in-stent lesion at two months or greater contributes to lesion severity. This concept is supported by the recent study showing that high activity 32P radioactive stent promotes the formation of neointima after six months in porcine coronary stent models (44). We cannot rule out the involvement of cell proliferation as a contributing factor in the early time points after stenting, nor can we rule out an involvement of cell proliferation outside the stent such as in the adventitia media and deep plaque. Cell proliferation is accompanied by changes in the ECM, and such changes may, in part, regulate the proliferative and survival properties of the cells. The hygroscopic property of the ECM in in-stent restenotic tissue may limit the effectiveness of balloon angioplasty due to transient tissue compression. This idea is supported by a recent clinical study showing that balloon angioplasty for treating stent restenosis led to less acute gain than at time of stenting and carried a particularly high recurrent restenosis rate (45). Our human aortic clonality study suggests that intima probably originates from underlying media (46). Therefore, on the basis of our study results, SMCs comprising the stent neointima may originate mostly from the underlying atherosclerotic plaque outside of stent or media, and subsequent ECM accumulation seems to play a key role in stent restenosis. Further study to identify the role of myoxoid tissue may be crucial to understand the mechanisms.
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Acknowledgments
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The authors thank Dr. Charles B. Underhill for providing biotinylated HABP, Dr. Richard LeBaron for providing versican antisera, Dr. John R. Hassell for EY9, Dr. Larry W. Fisher for LF51, and Dr. Leslie Gold for transforming growth factor-ß1 antibodies. The authors also thank Stephanie Lara for hyaluronan labeling and tissue preparation for electron microscopy. The authors are grateful for valuable discussions with Dr. Charles E. Murry, Department of Pathology, University of Washington, and Dr. Michael Kinsella at The Hope Heart Institute in Seattle, Washington. The authors are also grateful to Ms. Ellen Briggs for the typing of this manuscript.
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Footnotes
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Supported by NIH grant HL-41103 (M.R.) and HL-18645 (T.W.).
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