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Cell replication induces in-stent lesion growth in rabbit carotid artery with preexisting intimal hyperplasia
Atherosclerosis 162 (2002) 345– 353 www.elsevier.com/locate/atherosclerosis
Cell replication induces in-stent lesion growth in rabbit carotid artery with preexisting intimal hyperplasia Shinya Inoue, Hiroyuki Koyama *, Tetsuro Miyata, Hiroshi Shigematsu Di6ision of Vascular Surgery, Department of Surgery, Graduate School of Medicine, The Uni6ersity of Tokyo, 7 -3 -1 Hongo, Bunkyo-ku, Tokyo 113 -8655, Japan Received 24 May 2001; received in revised form 25 September 2001; accepted 8 October 2001
Abstract This study examined the responses of rabbit carotid artery with a preexisting intimal lesion, to stent implantation and balloon dilation. Rabbit carotid arteries were injured with a Fogarty catheter, and 28 days later the same arteries were subjected to implantation of a Palmaz-Schatz stent or balloon dilation angioplasty. Intimal size was significantly increased after stent implantation and balloon dilation, and no significant difference was detected between the two procedures. After stent implantation, replicating intimal cells were increased mainly in the inner intima, and the increase of cell replication was prolonged until day 28. In contrast, a significant increase of intimal cell replication was detected only at 2 days after balloon dilation. Intimal cell number after stent implantation was significantly higher than that after balloon dilation from day 7. Abundant leukocytes adhered to the luminal surface until 14 days after stent implantation, and significant infiltration of macrophages was observed in the mid-intima. Activation of proteases was prolonged, and obvious accumulation of proteoglycans was detected after stent implantation as compared with balloon dilation. These findings suggest that, an increase in cell replication is critical in the development of in-stent restenosis, and that inflammatory responses represent a unique property after stent implantation. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Angioplasty; Inflammation; Restenosis; Stents; Intimal hyperplasia
1. Introduction Intimal hyperplasia is considered to be critical in the development of in-stent restenosis after vascular stent implantation, [1] but the mechanism promoting the in-stent intimal lesion is not well understood. Previous studies of arterial injury in animal models have increased our understanding of how intimal hyperplasia develops, and showed that intimal lesion formation consists of three predominant factors; replication of arterial wall cells, cell migration and accumulation of extracellular matrix (ECM) [2,3]. However, it might be inappropriate to extrapolate the data acquired from the previous arterial injury studies to the process of in-stent lesion formation. One reason is that the arterial wall component injured in the previous vascular injury mod* Corresponding author. Tel.: +81-3-5800-8653; fax: + 81-3-38116822. E-mail address: [email protected] (H. Koyama).
els is different from that injured by stent implantation. The most widely used model of arterial injury is the balloon angioplasty model using a normal artery in small mammals. Since the normal artery of small animals possesses no or scarce intimal smooth muscle cells (SMCs), the arterial wall components injured in the balloon injury model are endothelial cells and medial SMCs. In contrast, stents are generally implanted into arteries with intimal lesions, and the injured arterial component after stent implantation is intimal lesion cells. Since previous in vivo studies noted various unique properties of intimal SMC not found in medial SMC, [4,5] there is a possibility that intimal cells respond differently to injury as compared with medial cells. To study the responses of intimal cells subjected to injury, we previously applied balloon dilation to rat carotid artery with a preexisting intimal lesion (reinjury model), and found that the increase of intimal lesion size after reinjury was associated with the accumulation of ECM, but not with an increase of intimal cell
0021-9150/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 0 1 ) 0 0 7 3 8 - 9
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number [5,6]. Another difference between stent implantation and the commonly used arterial injury model is that the stent itself possibly initiates some distinct responses in the arterial wall. Pathological studies using human specimens and animal models demonstrated inflammatory infiltration around the implanted metallic stent, and inflammatory cells secrete a variety of bioactive substances [7–9]. Thus, there is a possibility that these inflammatory substances could induce unique arterial responses not found in the balloon injury model. In the present study, we, therefore studied the responses of rabbit carotid artery with a preexisting intimal lesion, to stent implantation. We believe that this model mimics the process of in-stent restenosis formation in the clinical setting.
2. Methods
2.1. Reinjury model using stent or balloon catheter Male Japanese white rabbits (2.5– 3.0 kg, Saitama Rabbitry, Saitama, Japan) fed a normal diet were used in all experiments, and all protocols were conducted according to the Guide for Animal Experimentation, of The University of Tokyo. Rabbits were anesthetized by intramuscular injection of xylazine (2.5 mg/kg) and ketamine (50 mg/kg). A 2F Fogarty balloon catheter (Baxter Healthcare Co) was introduced through the first branch of the left external carotid artery and passed into the left common carotid artery. The balloon was inflated at 1.5 atm and passed through the common carotid artery three times with constant rotation (first injury). At 28 days after the first injury, stent implantation (stent group) or balloon angioplasty (balloon
group) was performed (Fig. 1). A midline incision in the neck was made to expose the left carotid bifurcation and external carotid artery. After intravenous administration of heparin (200 UPS units/kg), a 3-mm-diameter Palmaz-Schatz™ stent (spiral articulation type, 17.4 mm in length, Johnson and Johnson Co) mounted coaxially on a 3-mm-diameter Charger™ balloon catheter (20 mm in length, Johnson and Johnson) was passed into the left common carotid artery through the left external carotid artery. The proximal edge of the stent was positioned 15 mm from the carotid bifurcation, and the stent implantation involved two 1-min inflations of the balloon at 8 atm with a 30 s interval. In the balloon group, a 3 mm diameter Charger™ catheter was expanded with the same technique. Rabbits were killed at 2, 7, 14, and 28 days after stent implantation or balloon angioplasty. One group of rabbits was killed at 28 days after the first injury as control. Ten minutes before death by injection of an overdose of sodium pentobarbital, rabbits received an injection of Evans blue (1 ml of a 5% solution) to mark the deendothelialized area. Lactated Ringer’s solution (Otsuka Pharmaceutical Co, Tokyo, Japan) was infused for 3 min at 120 mmHg in a retrograde fashion from the abdominal aorta, and 4% phosphate-buffered paraformaldehyde was perfused for 5 min at 120 mmHg. The left carotid artery was excised and immersed in the same fixative for additional 1 h. Connective tissue around the carotid artery was removed, the artery was opened longitudinally, and five segments of artery were cut out. Segments taken 17– 19, 22–24 and 27 –29 mm from the carotid bifurcation were used for histological study after careful removal of the stent under a stereomicroscope, and segments taken 19–22 and 24–27 mm from the carotid bifurcation were analyzed by electron microscopy.
2.2. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM)
Fig. 1. Experimental protocol.
For SEM, the lumen of the artery was observed without removal of the stent struts. The segments of artery were fixed overnight in phosphate-buffered 2.5% glutalaldehyde/2% paraformaldehyde solution. The opened vessels were prepared as described previously [5] and examined with a Hitachi S-430 scanning electron microscope at 20 kV. For TEM, after removal of the stent struts, some pieces were immersed overnight in sodium acetate buffered 2.5% glutaraldehyde and 0.2% Cuprolinic blue (BDH Chemicals) with 0.3 mol/l magnesium chloride, washed three times in 1% aqueous sodium tungstate, dehydrated through graded ethanol containing 1% sodium tungstate, and embedded in EPON 812 (TAAB Laboratories) [10]. Other pieces were fixed overnight in phosphate-buffered 2.5% glutalaldehyde/2% paraformaldehyde solution, and embed-
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ded. Thin sections were mounted on copper grids, stained with uranyl acetate and lead citrate, and examined with a Hitachi H-7000 transmission electron microscope at 75 kV.
2.3. Morphometry and histology Fixed segments of the resected carotid artery (three segments per animal) were embedded in paraffin, and 4 mm cross-sections were cut and stained with hematoxylin/eosin. The area of the intimal layer was measured, total number of nuclei in the intima was counted as intimal cell number, and intimal cell density was calculated as described previously, for each slide [5]. At 1, 9, and 17 h before the rabbits were killed, each rabbit was injected subcutaneously with 5-bromo-2%-deoxyuridine (BrdU) (25 mg/kg, Boehringer-Mannheim). Replicating cells were identified by means of a monoclonal antibody against BrdU, and BrdU index was calculated as previously described [5]. Monoclonal antibody against rabbit macrophage (1:100, RAM11, DACO) was applied after enzyme digestion with 0.125% trypsin. Subsequent incubation with biotinylated goat anti-mouse IgG (1:100, Vector Laboratories) and an ABC Elite kit (Vector) was performed. The percentage of macrophage number to total cell number was calculated for each slide (macrophage index).
2.4. Western blot and zymography Other rabbits were killed at 2, 7, 14, and 28 days after stent implantation or balloon dilation (3– 4 animals for each time point in each group) as described above, and four rabbits were killed at 28 days after the first injury as control. The lysates of carotid arteries were prepared, and equal amounts (10 mg of total protein) of each lysate were subjected to western blot analysis using mouse monoclonal antibody against human interleukin-1b (IL-1b) (1:500, Genzyme) according to the protocol described previously [5,11]. The lysates of carotid arteries were also subjected to plasminogen activator (PA) zymography and gelatin zymography, as described previously [6]. To determine urokinase PA (UPA) activity, amiloride (1 mmol/l) or monoclonal antibody against human tissue PA (TPA) (250 mg/ml, American Diagnostica) was incorporated into the underlay gel of some PA zymograms [12]. For identification of matrix metalloproteinase (MMP)-2 and -9, some carotid lysates were analyzed by western blot using mouse monoclonal antibodies against human MMP-2 (1:500, ICN) or human MMP-9 (1:500, ICN) under non-reducing condition [13]. Further, some lysate samples were subjected to immunoprecipitation with monoclonal antibody against MMP-2 (ICN) or normal mouse IgG, and the resulting supernatants were analyzed by means of gelatin zymography [14].
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2.5. Statistics To evaluate morphometric data, BrdU index and macrophage index, the mean of each value from three carotid segments per animal was calculated for statistical analysis. Analysis of variance (ANOVA) followed by Fisher’s protected least significant difference test was used for the time course of morphometric data. The Kruskal–Wallis test followed by Scheffe’s F test was used for the time course of BrdU index and macrophage index. Differences between the stent group and the balloon group were evaluated by unpaired Student’s t-test, and all data were considered significant at PB 0.05.
3. Results
3.1. Luminal surface SEM showed that the carotid lumen was predominantly covered with platelets and leukocytes after stent implantation (Fig. 2A and B). Platelet adhesion and thrombus formations were observed at 2 days after stent implantation, but had decreased on day 7. In contrast, abundant leukocytes were attached to the luminal surface at 2, 7, and 14 days after stent implantation. In the balloon group, platelet adhesion was seen only on day 2, and few leukocytes were attached to the surface at all the time points (Fig. 2C and D).
3.2. Extracellular matrix of arterial wall TEM identified elastin and collagen fibrils as pale amorphous islands and fibers with a distinctive banding pattern, and proteoglycans was stained with Cuprolinic blue. In the ECM of stent-implanted intima, proteoglycans was abundantly expressed as compared with neointima in the balloon group and control, while no change of other matrix components was observed after stent implantation and balloon dilation (Fig. 2E–G).
3.3. Histological morphometry The intimal area was significantly increased from 7 days after stent implantation and from 14 days after balloon angioplasty, and continued to widen until day 28 (Fig. 3A). No difference was observed between the stent and balloon groups. Intimal cell number was also increased from 7 days after stent placement and balloon angioplasty, though the increase of intimal cells in the stent group was significantly larger than that in the balloon group (Fig. 3B). Intimal cell density was calculated as the ratio of intimal cell number to intimal area, and the density was significantly increased at 14 and 28 days after stent implantation (Fig. 3C). In contrast, no
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significant change in density was detected after balloon angioplasty.
3.4. Cell replication in intima and media Both stent implantation and balloon angioplasty promoted cell replication in the intima, which was maximal on day 2 (Fig. 3D and Fig. 4). In comparison with the control, a significant increase in intimal BrdU index was observed at 2 and 7 days after stent implantation, while the index was significantly increased only at 2 days after balloon angioplasty. The intimal BrdU index in the stent group was significantly higher than that in the balloon group from day 7 to 28 (Fig. 3D and Fig. 4). Especially in the stent group, BrdU-positive cells in the intima were mainly distributed in the inner part of the intima as compared with the middle and outer intima (Fig. 4B and C). Medial BrdU index was significantly increased on day 2 and 7 in the stent group and only on day 2 in the balloon group (Fig. 3E and Fig. 4).
3.5. Macrophage distribution in intima Immunostaining using anti-macrophage antibody (RAM11) revealed the distribution of macrophages in the intima. Macrophage index showed a significant increase of macrophage number in the intima after stent implantation, and the index continued to increase until day 28 (Fig. 3F). RAM11-positive cells were predominantly located around the stent struts and in the mid-intima between the stent struts (Fig. 5B and C). In the balloon group, no significant increase of macrophage index was detected in the intima (Fig. 3F).
3.6. Expression of IL-1i Western blot analysis using anti-IL-1b antibody demonstrated that protein expression of IL-1b was increased in the intima after stent implantation, while low level expression was detected in the intima after balloon dilation (Fig. 6).
3.7. Plasminogen acti6ator acti6ity Fig. 2. Scanning electron microscopic appearance of carotid lumen after stent implantation (A and B; bar = 100 mm) and balloon dilation (C and D; bar = 100 mm) and transmission electron microscopic appearance of arterial intima (E, F, and G; bar = 1 mm). Panel A is from a rabbit at 2 days after stent implantation, and panel B is on day 14. Abundant leukocytes adhere to the luminal surface. Panel C is from a rabbit at 2 days after balloon dilation, and Panel D is on day 7. A few leukocytes are observed on the luminal surface. At 2 days after stent implantation and balloon dilation, the luminal surface is covered with platelets and thrombus. Panel E is from a control rabbit, panel F is from a rabbit at 28 days after stent implantation, and panel G is at 28 days after balloon dilation. Samples of all panels were prepared with 0.2% Cuprolinic blue with 0.3 mol/l magnesium chloride. Note that ECM after stent implantation became proteoglycans-rich. Arrowhead indicates proteoglycans; E, elastin; C, collagen.
The PA zymogram showed an increase of PA activity with molecular mass of 54 kDa in both the intima and media at 2 days after stent implantation and balloon angioplasty (Fig. 7A and B). In the stent group, the 54 kDa activity in the intima and media on day 14 was approximately equal to that on day 2 and 7. However, in the balloon group, the 54 kDa activity on day 14 was lower than that on day 2 and 7 in both the intima and media. The PA activity with 54 kDa molecular mass represented UPA activity, since amiloride completely blocked the activity, while anti-TPA antibody did not inhibit the activity (Fig. 7C) [12]. Other PA activity was not detected in the PA zymograms.
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3.8. Gelatinase acti6ity In the control vessel, two prominent bands of gelatinolytic activity were observed in both the intima and media, with molecular masses of 62 and 72 kDa (Fig. 8A and B). At 2 days after stent implantation or balloon dilation, an extra band of 95 kDa appeared in both the intima and media. The 95 kDa band was detected in the intima at 7, 14 and 28 days after stent implantation, though the band disappeared by day 7 in
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the balloon group and in the media in the stent group. Western blot analysis with anti-MMP-2 antibody recognized two bands at 62 and 72 kDa, and western blot probed with anti-MMP-9 antibody showed 95 kDa band, suggesting that 62 and 72 kDa activities represented MMP-2 activities and 95 kDa activity represented MMP-9 activity (Fig. 8C). The supernatant after immunoprecipitation with anti-MMP-2 antibody showed lower activities at 62 and 72 kDa than that with normal mouse IgG (Fig. 8D).
Fig. 3. Time course of intimal area (A); intimal cell number (B); intimal cell density (C); intimal BrdU index (D); medial BrdU index (E); and macrophage index in intima (F) after stent implantation (solid bars) and balloon dilation (open bars). Control values (hatched bars) are from rabbits 28 days after first injury. Values are shown as mean 9S.D. (*PB 0.01 vs. control, **PB0.05 vs. control, c PB 0.01 stent group vs. balloon group, §P B0.05 stent group vs. balloon group). Cont, control; d, days after stent implantation or balloon dilation.
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Fig. 4. Photomicrographs of carotid arteries after stent implantation or balloon dilation. (A) Control artery; (B) 2 days after stent implantation; (C) 28 days after stent implantation; (D) 2 days after balloon dilation; (E) 28 days after balloon dilation. Sections were immunostained with anti-BrdU antibody and counterstained with hematoxylin. BrdU-positive cells stain brown. L, lumen; I, intima; M, media; *, strut hole; arrowhead, internal elastic lamina. Bar = 100 mm. Fig. 5. Macrophage infiltration after stent implantation or balloon dilation. (A) Control artery; (B) 2 days after stent implantation; (C) 28 days after stent implantation; (D) 28 days after balloon dilation. All sections were stained with anti-macrophage antibody (RAM11) and counterstained with hematoxylin. Macrophages stain brown, and are located around stent struts and in the mid-intima between struts. L, lumen; I, intima; M, media; *, strut hole; arrowhead, internal elastic lamina. Bar =100 mm.
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Fig. 6. Western blot probed with antibody against IL-1b shows time course of IL-1b expression in the intima after stent implantation or balloon dilation. Cont, control; d, days after stent implantation or balloon dilation.
4. Discussion In the present study, we evaluated the responses of rabbit carotid artery with a preexisting intimal lesion after stent implantation and balloon dilation. Although, analysis of lesion size showed no significant difference between the stent group and balloon group, several interesting differences between the two groups were observed in the other responses of arterial wall. One obvious difference was that the increase of intimal cells replication after stent implantation was prolonged as compared with that after balloon dilation. For the prolonged cell replication, the intimal cell number in the stent group was significantly higher than that in the balloon group from day 7 to 28. Since, no difference was detected in the intimal lesion size between the two groups, as mentioned previously, intimal cell density in the stent group was also significantly higher than that in the balloon group on day 14 and 28. These findings indicate that intimal cell replication played an important role in the lesion increase after stent implantation. Indeed, a histological study using human atherectomy
Fig. 7. PA zymograms show time course of PA activity in intima (A) and media (B). PA activity at 54 kDa was blocked by amiloride, but not inhibited by anti-TPA antibody, suggesting that the 54 kDa band represented UPA (C). Day 2 sample of the balloon group was used to identify UPA. Cont, control; d, days after stent implantation or balloon dilation.
Fig. 8. Gelatin zymograms show time course of gelatinolytic activities in intima (A) and media (B). Western blot analysis using antibodies against MMP-2 or -9 suggested the gelatinolytic activities at 62 and 72 kDa represented MMP-2 activities and 95 kDa activity represented MMP-9 activity. To identify MMP-2 activity, lysate samples were immunoprecipitated with anti-MMP-2 antibody or normal mouse IgG, and supernatants were subjected to gelatin zymography. The supernatant treated with anti-MMP-2 antibody shows low level activities at 62 and 72 kDa as compared with that with normal mouse IgG. Day 2 sample of the balloon group was used to identify MMP-2 and -9. Cont, control; d, days after stent implantation or balloon dilation.
specimens with in-stent restenosis showed ongoing cell replication and activation of the cell cycle, [7] while a lack of cell replication was seen in human lesions after balloon angioplasty [15]. Recently, Sousa et al. presented a clinical study using rapamycin-coated stents for coronary artery stenosis, and reported favorable results [16]. We think that rapamycin is a reasonable drug as coating material, since, it blocks cell replication by several mechanisms. If cell replication is important also in human in-stent lesion formation, stent-coating with anti-mitogenic drug(s) might be a promising strategy for preventing in-stent restenosis. In contrast, Kollum et al. implanted a Palmaz-Schatz stent in the rabbit iliac artery, and showed that intimal cell density after stent implantation was significantly lower than that after balloon angioplasty at 4 weeks, [9] which was
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contrary to the result of the present study. One explanation for the discrepancy in the results was difference in responses between the iliac artery and carotid artery. Additionally, the above study used normal healthy arteries, and as mentioned previously, medial cells have quite different properties as compared with intimal cells. Then, there was also a possibility that the discrepancy in the results reflected the difference between normal artery and artery with an intimal lesion as the subject for stent implantation. Inflammatory responses are also an interesting feature after stent implantation. In the present study, we showed prolonged adhesion of leukocytes to the luminal surface and an increase of macrophages with IL-1b expression in the intima after stent implantation. Leukocyte adhesion was observed immediately after stent implantation, and abundant leukocytes were detected until day 14. Since the time course of leukocyte adhesion was similar to that of intimal cell replication after stent implantation, we presumed that leukocytes on the luminal surface play a role in stimulating intimal cells. Indeed, histological findings showed that replicating cells in the intima were predominantly located in the inner intima after stent implantation, suggesting the presence of mitogenic factor(s) near the lumen. It is of interest that macrophage infiltration was mainly located around stent struts and in the mid-intima between stent struts. This distribution of macrophages was different from that of replicating cells in the intima. Further, macrophage index increased gradually until 28 days after stent implantation, while intimal BrdU index decreased after day 2. The discrepancy in the findings suggests that macrophages are not critical for intimal cell replication after stent implantation. However, previous studies using arterial injury models showed that infiltrating macrophages in the intima possibly promote an increase of intimal lesion size [17,18]. One possible explanation for these findings is that macrophages in intimal lesions promoted cell migration and/or accumulation of ECM to increase the lesion size after stent implantation. To develop an intimal lesion inside the stent, cell movement through the stent mesh might be required, as well as lesion expansion by cell replication and ECM accumulation. Cell movement in the arterial wall is linked to activation of some proteases, [19,20] and macrophages potentially secrete various proteases, including MMPs [21,22]. Further, a previous study showed that UPA acts as a physiological activator of MMPs [23]. In the present study, both MMP-9 and UPA activation were observed until 28 and 14 days after stent implantation, respectively, whereas these activities were detected only at 2 days after balloon dilation. Another interesting finding of the present study is that ECM of intimal lesions changed to become proteoglycans-rich after stent implantation as compared with
balloon dilation. Although it is poorly understood how the expression and accumulation of ECM molecules are regulated after stent implantation and balloon dilation, various studies showed that the contents of ECM such as collagen, elastin and proteoglycans were upregulated after arterial injury [10,24,25]. Strauss et al. demonstrated a significant increase of ECM synthesis after balloon dilation in their study using a rabbit reinjury model, and the synthesis of collagen and elastin continued to be significantly high until 12 weeks after balloon dilation, whereas proteoglycans synthesis decreased to below the control level by 4 weeks [24,26]. These data suggest that balloon dilation of intimal lesions promotes low-level accumulation of proteoglycans in the vessel wall, which is similar to the findings of the present study. In addition to this, macrophage accumulation in the intima was possibly linked to upregulation of proteoglycans synthesis. A previous in vitro study showed that some humoral factors released from macrophages in certain conditions could stimulate other cells to secrete proteoglycans, and further, macrophages themselves could secrete some proteoglycans [21,27]. Finally, we have to be aware of unsolved issues and limitations in the present study. One issue could be the limited period of observation after stent implantation and balloon dilation. Previous clinical studies reported that symptoms of in-stent restenosis were commonly developed from several months after stent implantation [28]. The present study, however, analyzed arterial responses for only 28 days after stent implantation. A number of studies using the rat carotid artery balloon injury model showed that an increase of SMC number promoted intimal formation in the early phase after injury, whereas intimal thickening was due to accumulation of ECM in the late phase after injury [2,29]. Thus, there is a possibility that longer observation would provide more crucial evidence for understanding the mechanisms of in-stent lesion formation. Meanwhile, a limitation of the present study is that the rabbit carotid artery with a preexisting intimal lesion mimics a diseased artery in the clinical setting, but is not exactly same. In the present study, since balloon injury induced the preexisting intimal lesion under a normal diet, the dominant cell component of the intimal lesion was SMC [29]. However, the human atherosclerotic lesion is composed of other cell components as well as SMC [30]. In particular, foam cells play an important role in the progression of human atherosclerosis [30]. We are now considering implanting a stent in a rabbit artery with an atherosclerotic lesion induced by a cholesterolrich diet, and investigating the responses of the arterial wall. In summary, the present study showed that stent implantation into a rabbit artery with preexisting intimal hyperplasia induced a significant increase in size of
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the in-stent intimal lesion. Intimal cell replication after stent implantation was significantly greater than that after balloon dilation, even at day 28, and prolonged cell replication was suggested to be a main reason for the increase of intimal lesion size. Meanwhile, stent implantation promoted arterial inflammatory responses not found after balloon dilation. Abundant leukocytes adhered to the luminal surface after stent implantation, and significant infiltration of macrophages and accumulation of proteoglycans were observed in the intima. Further, in the stent group, activation of proteases was detected for a longer period than in the balloon group. These data demonstrate unique properties after stent implantation, and might provide insights into understanding the pathophysiology of in-stent restenosis.
Acknowledgements This study was supported by a Grant-in Aid for Scientific Research (B) from the Ministry of Education, Science, and Culture of Japan (12470236).
References [1] Dussaillant GR, Mintz GS, Pichard AD, et al. Small stent size and intimal hyperplasia contribute to restenosis: a volumetric intravascular ultrasound analysis. J Am Coll Cardiol 1995;26:720 – 4. [2] Clowes AW, Reidy MA, Clowes MM. Mechanisms of stenosis after arterial injury. Lab Invest 1983;49:208 – 15. [3] Schwartz SM, deBlois D, O’Brien ER. The intima. Soil for atherosclerosis and restenosis. Circ Res 1995;77:445 – 65. [4] Koyama H, Olson NE, Dastvan FF, Reidy MA. Cell replication in the arterial wall: activation of signaling pathway following in vivo injury. Circ Res 1998;82:713 – 21. [5] 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 – 17. [6] Koyama H, Reidy MA. Expression of extracellular matrix proteins accompanies lesion growth in a model of intimal reinjury. Circ Res 1998;82:988 –95. [7] Kearney M, Pieczek A, Haley L, et al. Histopathology of in-stent restenosis in patients with peripheral artery disease. Circulation 1997;95:1998 – 2002. [8] Komatsu R, Ueda M, Naruko T, Kojima A, Becker AE. Neointimal tissue response at sites of coronary stenting in humans: macroscopic, histological, and immunohistochemical analyses. Circulation 1998;98:224 –33. [9] Kollum M, Kaiser S, Kinscherf R, Metz J, Kubler W, Hehrlein C. Apoptosis after stent implantation compared with balloon angioplasty in rabbits. Role of macrophages. Arterioscler Thromb Vasc Biol 1997;17:2383 –8. [10] Snow AD, Bolender RP, Wight TN, Clowes AW. Heparin modulates the composition of the extracellular matrix domain surrounding arterial smooth muscle cells. Am J Pathol 1990;137:313 – 30. [11] Tanaka H, Sukhova G, Schwartz D, Libby P. Proliferating arterial smooth muscle cells after balloon injury express TNF-alpha but not interleukin-1 or basic fibroblast growth factor. Arterioscler Thromb Vasc Biol 1996;16:12 – 8.
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[12] Pepper MS, Vassalli JD, Montesano R, Orci L. Urokinase-type plasminogen activator is induced in migrating capillary endothelial cells. J Cell Biol 1987;105:2535 – 41. [13] Bayer IM, Adamson SL, Langille BL. Atrophic remodeling of the artery-cuffed artery. Arterioscler Thromb Vasc Biol 1999;19:1499 – 505. [14] Shan L, Nishimura Y, Kotani Y, Yokoyama M. Platelet-activating factor increases the expression of metalloproteinase-9 in human bronchial epithelial cells. Eur J Pharmacol 1999;374:147 – 56. [15] O’Brien ER, Alpers CE, Stewart DK, et al. Proliferation in primary and restenotic coronary atherectomy tissue. Implications for antiproliferative therapy. Circ Res 1993;73:223 – 31. [16] Sousa JE, Costa MA, Abizaid A, et al. Lack of neointimal proliferation after implantation of sirolimus-coated stents in human coronary arteries: a quantitative coronary angiography and three-dimensional intravascular ultrasound study. Circulation 2001;103:192 – 5. [17] Weidinger FF, McLenachan JM, Cybulsky MI, Fallon JT, Hollenberg NK, Cooke JP, Ganz P. Hypercholesterolemia enhances macrophage recruitment and dysfunction of regenerated endothelium after balloon injury of the rabbit iliac artery. Circulation 1991;84:755 – 67. [18] Stadius ML, Rowan R, Fleischhauer JF, Kernoff R, Billingham M, Gown AM. Time course and cellular characteristics of the iliac artery response to acute balloon injury. An angiographic, morphometric, and immunocytochemical analysis in the cholesterolfed New Zealand white rabbit. Arterioscler Thromb 1992;12:1267 – 73. [19] Bendeck MP, Zempo N, Clowes AW, Galardy RE, Reidy MA. Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat. Circ Res 1994;75:539 –45. [20] Clowes AW, Clowes MM, Au YP, Reidy MA, Belin D. Smooth muscle cells express urokinase during mitogenesis and tissue- type plasminogen activator during migration in injured rat carotid artery. Circ Res 1990;67:61 – 7. [21] Edwards IJ, Wagner WD, Owens RT. Macrophage secretory products selectively stimulate dermatan sulfate proteoglycan production in cultured arterial smooth muscle cells. Am J Pathol 1990;136:609 – 21. [22] Galis ZS, Sukhova GK, Kranzhofer R, Clark S, Libby P. Macrophage foam cells from experimental atheroma constitutively produce matrix-degrading proteinases. Proc Natl Acad Sci USA 1995;92:402 – 6. [23] Woessner JF Jr. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J 1991;5:2145 – 54. [24] Strauss BH, Chisholm RJ, Keeley FW, Gotlieb AI, Logan RA, Armstrong PW. Extracellular matrix remodeling after balloon angioplasty injury in a rabbit model of restenosis. Circ Res 1994;75:650 – 8. [25] Nikkari ST, Jarvelainen HT, Wight TN, Ferguson M, Clowes AW. Smooth muscle cell expression of extracellular matrix genes after arterial injury. Am J Pathol 1994;144:1348 – 56. [26] Strauss BH, Robinson R, Batchelor WB, et al. In vivo collagen turnover following experimental balloon angioplasty injury and the role of matrix metalloproteinases. Circ Res 1996;79:541 –50. [27] Nathan CF. Secretory products of macrophages. J Clin Invest 1987;79:319 – 26. [28] George CJ, Baim DS, Brinker JA, et al. One-year follow-up of the Stent Restenosis (STRESS I) Study. Am J Cardiol 1998;81:860 –5. [29] Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium. Lab Invest 1983;49:327 – 33. [30] Stary HC, Chandler AB, Dinsmore RE, et al. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation 1995;92:1355 – 74.
Cell replication induces in-stent lesion growth in rabbit carotid artery with preexisting intimal hyperplasia Shinya Inoue, Hiroyuki Koyama *, Tetsuro Miyata, Hiroshi Shigematsu Di6ision of Vascular Surgery, Department of Surgery, Graduate School of Medicine, The Uni6ersity of Tokyo, 7 -3 -1 Hongo, Bunkyo-ku, Tokyo 113 -8655, Japan Received 24 May 2001; received in revised form 25 September 2001; accepted 8 October 2001
Abstract This study examined the responses of rabbit carotid artery with a preexisting intimal lesion, to stent implantation and balloon dilation. Rabbit carotid arteries were injured with a Fogarty catheter, and 28 days later the same arteries were subjected to implantation of a Palmaz-Schatz stent or balloon dilation angioplasty. Intimal size was significantly increased after stent implantation and balloon dilation, and no significant difference was detected between the two procedures. After stent implantation, replicating intimal cells were increased mainly in the inner intima, and the increase of cell replication was prolonged until day 28. In contrast, a significant increase of intimal cell replication was detected only at 2 days after balloon dilation. Intimal cell number after stent implantation was significantly higher than that after balloon dilation from day 7. Abundant leukocytes adhered to the luminal surface until 14 days after stent implantation, and significant infiltration of macrophages was observed in the mid-intima. Activation of proteases was prolonged, and obvious accumulation of proteoglycans was detected after stent implantation as compared with balloon dilation. These findings suggest that, an increase in cell replication is critical in the development of in-stent restenosis, and that inflammatory responses represent a unique property after stent implantation. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Angioplasty; Inflammation; Restenosis; Stents; Intimal hyperplasia
1. Introduction Intimal hyperplasia is considered to be critical in the development of in-stent restenosis after vascular stent implantation, [1] but the mechanism promoting the in-stent intimal lesion is not well understood. Previous studies of arterial injury in animal models have increased our understanding of how intimal hyperplasia develops, and showed that intimal lesion formation consists of three predominant factors; replication of arterial wall cells, cell migration and accumulation of extracellular matrix (ECM) [2,3]. However, it might be inappropriate to extrapolate the data acquired from the previous arterial injury studies to the process of in-stent lesion formation. One reason is that the arterial wall component injured in the previous vascular injury mod* Corresponding author. Tel.: +81-3-5800-8653; fax: + 81-3-38116822. E-mail address: [email protected] (H. Koyama).
els is different from that injured by stent implantation. The most widely used model of arterial injury is the balloon angioplasty model using a normal artery in small mammals. Since the normal artery of small animals possesses no or scarce intimal smooth muscle cells (SMCs), the arterial wall components injured in the balloon injury model are endothelial cells and medial SMCs. In contrast, stents are generally implanted into arteries with intimal lesions, and the injured arterial component after stent implantation is intimal lesion cells. Since previous in vivo studies noted various unique properties of intimal SMC not found in medial SMC, [4,5] there is a possibility that intimal cells respond differently to injury as compared with medial cells. To study the responses of intimal cells subjected to injury, we previously applied balloon dilation to rat carotid artery with a preexisting intimal lesion (reinjury model), and found that the increase of intimal lesion size after reinjury was associated with the accumulation of ECM, but not with an increase of intimal cell
0021-9150/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 0 1 ) 0 0 7 3 8 - 9
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number [5,6]. Another difference between stent implantation and the commonly used arterial injury model is that the stent itself possibly initiates some distinct responses in the arterial wall. Pathological studies using human specimens and animal models demonstrated inflammatory infiltration around the implanted metallic stent, and inflammatory cells secrete a variety of bioactive substances [7–9]. Thus, there is a possibility that these inflammatory substances could induce unique arterial responses not found in the balloon injury model. In the present study, we, therefore studied the responses of rabbit carotid artery with a preexisting intimal lesion, to stent implantation. We believe that this model mimics the process of in-stent restenosis formation in the clinical setting.
2. Methods
2.1. Reinjury model using stent or balloon catheter Male Japanese white rabbits (2.5– 3.0 kg, Saitama Rabbitry, Saitama, Japan) fed a normal diet were used in all experiments, and all protocols were conducted according to the Guide for Animal Experimentation, of The University of Tokyo. Rabbits were anesthetized by intramuscular injection of xylazine (2.5 mg/kg) and ketamine (50 mg/kg). A 2F Fogarty balloon catheter (Baxter Healthcare Co) was introduced through the first branch of the left external carotid artery and passed into the left common carotid artery. The balloon was inflated at 1.5 atm and passed through the common carotid artery three times with constant rotation (first injury). At 28 days after the first injury, stent implantation (stent group) or balloon angioplasty (balloon
group) was performed (Fig. 1). A midline incision in the neck was made to expose the left carotid bifurcation and external carotid artery. After intravenous administration of heparin (200 UPS units/kg), a 3-mm-diameter Palmaz-Schatz™ stent (spiral articulation type, 17.4 mm in length, Johnson and Johnson Co) mounted coaxially on a 3-mm-diameter Charger™ balloon catheter (20 mm in length, Johnson and Johnson) was passed into the left common carotid artery through the left external carotid artery. The proximal edge of the stent was positioned 15 mm from the carotid bifurcation, and the stent implantation involved two 1-min inflations of the balloon at 8 atm with a 30 s interval. In the balloon group, a 3 mm diameter Charger™ catheter was expanded with the same technique. Rabbits were killed at 2, 7, 14, and 28 days after stent implantation or balloon angioplasty. One group of rabbits was killed at 28 days after the first injury as control. Ten minutes before death by injection of an overdose of sodium pentobarbital, rabbits received an injection of Evans blue (1 ml of a 5% solution) to mark the deendothelialized area. Lactated Ringer’s solution (Otsuka Pharmaceutical Co, Tokyo, Japan) was infused for 3 min at 120 mmHg in a retrograde fashion from the abdominal aorta, and 4% phosphate-buffered paraformaldehyde was perfused for 5 min at 120 mmHg. The left carotid artery was excised and immersed in the same fixative for additional 1 h. Connective tissue around the carotid artery was removed, the artery was opened longitudinally, and five segments of artery were cut out. Segments taken 17– 19, 22–24 and 27 –29 mm from the carotid bifurcation were used for histological study after careful removal of the stent under a stereomicroscope, and segments taken 19–22 and 24–27 mm from the carotid bifurcation were analyzed by electron microscopy.
2.2. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM)
Fig. 1. Experimental protocol.
For SEM, the lumen of the artery was observed without removal of the stent struts. The segments of artery were fixed overnight in phosphate-buffered 2.5% glutalaldehyde/2% paraformaldehyde solution. The opened vessels were prepared as described previously [5] and examined with a Hitachi S-430 scanning electron microscope at 20 kV. For TEM, after removal of the stent struts, some pieces were immersed overnight in sodium acetate buffered 2.5% glutaraldehyde and 0.2% Cuprolinic blue (BDH Chemicals) with 0.3 mol/l magnesium chloride, washed three times in 1% aqueous sodium tungstate, dehydrated through graded ethanol containing 1% sodium tungstate, and embedded in EPON 812 (TAAB Laboratories) [10]. Other pieces were fixed overnight in phosphate-buffered 2.5% glutalaldehyde/2% paraformaldehyde solution, and embed-
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ded. Thin sections were mounted on copper grids, stained with uranyl acetate and lead citrate, and examined with a Hitachi H-7000 transmission electron microscope at 75 kV.
2.3. Morphometry and histology Fixed segments of the resected carotid artery (three segments per animal) were embedded in paraffin, and 4 mm cross-sections were cut and stained with hematoxylin/eosin. The area of the intimal layer was measured, total number of nuclei in the intima was counted as intimal cell number, and intimal cell density was calculated as described previously, for each slide [5]. At 1, 9, and 17 h before the rabbits were killed, each rabbit was injected subcutaneously with 5-bromo-2%-deoxyuridine (BrdU) (25 mg/kg, Boehringer-Mannheim). Replicating cells were identified by means of a monoclonal antibody against BrdU, and BrdU index was calculated as previously described [5]. Monoclonal antibody against rabbit macrophage (1:100, RAM11, DACO) was applied after enzyme digestion with 0.125% trypsin. Subsequent incubation with biotinylated goat anti-mouse IgG (1:100, Vector Laboratories) and an ABC Elite kit (Vector) was performed. The percentage of macrophage number to total cell number was calculated for each slide (macrophage index).
2.4. Western blot and zymography Other rabbits were killed at 2, 7, 14, and 28 days after stent implantation or balloon dilation (3– 4 animals for each time point in each group) as described above, and four rabbits were killed at 28 days after the first injury as control. The lysates of carotid arteries were prepared, and equal amounts (10 mg of total protein) of each lysate were subjected to western blot analysis using mouse monoclonal antibody against human interleukin-1b (IL-1b) (1:500, Genzyme) according to the protocol described previously [5,11]. The lysates of carotid arteries were also subjected to plasminogen activator (PA) zymography and gelatin zymography, as described previously [6]. To determine urokinase PA (UPA) activity, amiloride (1 mmol/l) or monoclonal antibody against human tissue PA (TPA) (250 mg/ml, American Diagnostica) was incorporated into the underlay gel of some PA zymograms [12]. For identification of matrix metalloproteinase (MMP)-2 and -9, some carotid lysates were analyzed by western blot using mouse monoclonal antibodies against human MMP-2 (1:500, ICN) or human MMP-9 (1:500, ICN) under non-reducing condition [13]. Further, some lysate samples were subjected to immunoprecipitation with monoclonal antibody against MMP-2 (ICN) or normal mouse IgG, and the resulting supernatants were analyzed by means of gelatin zymography [14].
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2.5. Statistics To evaluate morphometric data, BrdU index and macrophage index, the mean of each value from three carotid segments per animal was calculated for statistical analysis. Analysis of variance (ANOVA) followed by Fisher’s protected least significant difference test was used for the time course of morphometric data. The Kruskal–Wallis test followed by Scheffe’s F test was used for the time course of BrdU index and macrophage index. Differences between the stent group and the balloon group were evaluated by unpaired Student’s t-test, and all data were considered significant at PB 0.05.
3. Results
3.1. Luminal surface SEM showed that the carotid lumen was predominantly covered with platelets and leukocytes after stent implantation (Fig. 2A and B). Platelet adhesion and thrombus formations were observed at 2 days after stent implantation, but had decreased on day 7. In contrast, abundant leukocytes were attached to the luminal surface at 2, 7, and 14 days after stent implantation. In the balloon group, platelet adhesion was seen only on day 2, and few leukocytes were attached to the surface at all the time points (Fig. 2C and D).
3.2. Extracellular matrix of arterial wall TEM identified elastin and collagen fibrils as pale amorphous islands and fibers with a distinctive banding pattern, and proteoglycans was stained with Cuprolinic blue. In the ECM of stent-implanted intima, proteoglycans was abundantly expressed as compared with neointima in the balloon group and control, while no change of other matrix components was observed after stent implantation and balloon dilation (Fig. 2E–G).
3.3. Histological morphometry The intimal area was significantly increased from 7 days after stent implantation and from 14 days after balloon angioplasty, and continued to widen until day 28 (Fig. 3A). No difference was observed between the stent and balloon groups. Intimal cell number was also increased from 7 days after stent placement and balloon angioplasty, though the increase of intimal cells in the stent group was significantly larger than that in the balloon group (Fig. 3B). Intimal cell density was calculated as the ratio of intimal cell number to intimal area, and the density was significantly increased at 14 and 28 days after stent implantation (Fig. 3C). In contrast, no
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significant change in density was detected after balloon angioplasty.
3.4. Cell replication in intima and media Both stent implantation and balloon angioplasty promoted cell replication in the intima, which was maximal on day 2 (Fig. 3D and Fig. 4). In comparison with the control, a significant increase in intimal BrdU index was observed at 2 and 7 days after stent implantation, while the index was significantly increased only at 2 days after balloon angioplasty. The intimal BrdU index in the stent group was significantly higher than that in the balloon group from day 7 to 28 (Fig. 3D and Fig. 4). Especially in the stent group, BrdU-positive cells in the intima were mainly distributed in the inner part of the intima as compared with the middle and outer intima (Fig. 4B and C). Medial BrdU index was significantly increased on day 2 and 7 in the stent group and only on day 2 in the balloon group (Fig. 3E and Fig. 4).
3.5. Macrophage distribution in intima Immunostaining using anti-macrophage antibody (RAM11) revealed the distribution of macrophages in the intima. Macrophage index showed a significant increase of macrophage number in the intima after stent implantation, and the index continued to increase until day 28 (Fig. 3F). RAM11-positive cells were predominantly located around the stent struts and in the mid-intima between the stent struts (Fig. 5B and C). In the balloon group, no significant increase of macrophage index was detected in the intima (Fig. 3F).
3.6. Expression of IL-1i Western blot analysis using anti-IL-1b antibody demonstrated that protein expression of IL-1b was increased in the intima after stent implantation, while low level expression was detected in the intima after balloon dilation (Fig. 6).
3.7. Plasminogen acti6ator acti6ity Fig. 2. Scanning electron microscopic appearance of carotid lumen after stent implantation (A and B; bar = 100 mm) and balloon dilation (C and D; bar = 100 mm) and transmission electron microscopic appearance of arterial intima (E, F, and G; bar = 1 mm). Panel A is from a rabbit at 2 days after stent implantation, and panel B is on day 14. Abundant leukocytes adhere to the luminal surface. Panel C is from a rabbit at 2 days after balloon dilation, and Panel D is on day 7. A few leukocytes are observed on the luminal surface. At 2 days after stent implantation and balloon dilation, the luminal surface is covered with platelets and thrombus. Panel E is from a control rabbit, panel F is from a rabbit at 28 days after stent implantation, and panel G is at 28 days after balloon dilation. Samples of all panels were prepared with 0.2% Cuprolinic blue with 0.3 mol/l magnesium chloride. Note that ECM after stent implantation became proteoglycans-rich. Arrowhead indicates proteoglycans; E, elastin; C, collagen.
The PA zymogram showed an increase of PA activity with molecular mass of 54 kDa in both the intima and media at 2 days after stent implantation and balloon angioplasty (Fig. 7A and B). In the stent group, the 54 kDa activity in the intima and media on day 14 was approximately equal to that on day 2 and 7. However, in the balloon group, the 54 kDa activity on day 14 was lower than that on day 2 and 7 in both the intima and media. The PA activity with 54 kDa molecular mass represented UPA activity, since amiloride completely blocked the activity, while anti-TPA antibody did not inhibit the activity (Fig. 7C) [12]. Other PA activity was not detected in the PA zymograms.
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3.8. Gelatinase acti6ity In the control vessel, two prominent bands of gelatinolytic activity were observed in both the intima and media, with molecular masses of 62 and 72 kDa (Fig. 8A and B). At 2 days after stent implantation or balloon dilation, an extra band of 95 kDa appeared in both the intima and media. The 95 kDa band was detected in the intima at 7, 14 and 28 days after stent implantation, though the band disappeared by day 7 in
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the balloon group and in the media in the stent group. Western blot analysis with anti-MMP-2 antibody recognized two bands at 62 and 72 kDa, and western blot probed with anti-MMP-9 antibody showed 95 kDa band, suggesting that 62 and 72 kDa activities represented MMP-2 activities and 95 kDa activity represented MMP-9 activity (Fig. 8C). The supernatant after immunoprecipitation with anti-MMP-2 antibody showed lower activities at 62 and 72 kDa than that with normal mouse IgG (Fig. 8D).
Fig. 3. Time course of intimal area (A); intimal cell number (B); intimal cell density (C); intimal BrdU index (D); medial BrdU index (E); and macrophage index in intima (F) after stent implantation (solid bars) and balloon dilation (open bars). Control values (hatched bars) are from rabbits 28 days after first injury. Values are shown as mean 9S.D. (*PB 0.01 vs. control, **PB0.05 vs. control, c PB 0.01 stent group vs. balloon group, §P B0.05 stent group vs. balloon group). Cont, control; d, days after stent implantation or balloon dilation.
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Fig. 4. Photomicrographs of carotid arteries after stent implantation or balloon dilation. (A) Control artery; (B) 2 days after stent implantation; (C) 28 days after stent implantation; (D) 2 days after balloon dilation; (E) 28 days after balloon dilation. Sections were immunostained with anti-BrdU antibody and counterstained with hematoxylin. BrdU-positive cells stain brown. L, lumen; I, intima; M, media; *, strut hole; arrowhead, internal elastic lamina. Bar = 100 mm. Fig. 5. Macrophage infiltration after stent implantation or balloon dilation. (A) Control artery; (B) 2 days after stent implantation; (C) 28 days after stent implantation; (D) 28 days after balloon dilation. All sections were stained with anti-macrophage antibody (RAM11) and counterstained with hematoxylin. Macrophages stain brown, and are located around stent struts and in the mid-intima between struts. L, lumen; I, intima; M, media; *, strut hole; arrowhead, internal elastic lamina. Bar =100 mm.
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Fig. 6. Western blot probed with antibody against IL-1b shows time course of IL-1b expression in the intima after stent implantation or balloon dilation. Cont, control; d, days after stent implantation or balloon dilation.
4. Discussion In the present study, we evaluated the responses of rabbit carotid artery with a preexisting intimal lesion after stent implantation and balloon dilation. Although, analysis of lesion size showed no significant difference between the stent group and balloon group, several interesting differences between the two groups were observed in the other responses of arterial wall. One obvious difference was that the increase of intimal cells replication after stent implantation was prolonged as compared with that after balloon dilation. For the prolonged cell replication, the intimal cell number in the stent group was significantly higher than that in the balloon group from day 7 to 28. Since, no difference was detected in the intimal lesion size between the two groups, as mentioned previously, intimal cell density in the stent group was also significantly higher than that in the balloon group on day 14 and 28. These findings indicate that intimal cell replication played an important role in the lesion increase after stent implantation. Indeed, a histological study using human atherectomy
Fig. 7. PA zymograms show time course of PA activity in intima (A) and media (B). PA activity at 54 kDa was blocked by amiloride, but not inhibited by anti-TPA antibody, suggesting that the 54 kDa band represented UPA (C). Day 2 sample of the balloon group was used to identify UPA. Cont, control; d, days after stent implantation or balloon dilation.
Fig. 8. Gelatin zymograms show time course of gelatinolytic activities in intima (A) and media (B). Western blot analysis using antibodies against MMP-2 or -9 suggested the gelatinolytic activities at 62 and 72 kDa represented MMP-2 activities and 95 kDa activity represented MMP-9 activity. To identify MMP-2 activity, lysate samples were immunoprecipitated with anti-MMP-2 antibody or normal mouse IgG, and supernatants were subjected to gelatin zymography. The supernatant treated with anti-MMP-2 antibody shows low level activities at 62 and 72 kDa as compared with that with normal mouse IgG. Day 2 sample of the balloon group was used to identify MMP-2 and -9. Cont, control; d, days after stent implantation or balloon dilation.
specimens with in-stent restenosis showed ongoing cell replication and activation of the cell cycle, [7] while a lack of cell replication was seen in human lesions after balloon angioplasty [15]. Recently, Sousa et al. presented a clinical study using rapamycin-coated stents for coronary artery stenosis, and reported favorable results [16]. We think that rapamycin is a reasonable drug as coating material, since, it blocks cell replication by several mechanisms. If cell replication is important also in human in-stent lesion formation, stent-coating with anti-mitogenic drug(s) might be a promising strategy for preventing in-stent restenosis. In contrast, Kollum et al. implanted a Palmaz-Schatz stent in the rabbit iliac artery, and showed that intimal cell density after stent implantation was significantly lower than that after balloon angioplasty at 4 weeks, [9] which was
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contrary to the result of the present study. One explanation for the discrepancy in the results was difference in responses between the iliac artery and carotid artery. Additionally, the above study used normal healthy arteries, and as mentioned previously, medial cells have quite different properties as compared with intimal cells. Then, there was also a possibility that the discrepancy in the results reflected the difference between normal artery and artery with an intimal lesion as the subject for stent implantation. Inflammatory responses are also an interesting feature after stent implantation. In the present study, we showed prolonged adhesion of leukocytes to the luminal surface and an increase of macrophages with IL-1b expression in the intima after stent implantation. Leukocyte adhesion was observed immediately after stent implantation, and abundant leukocytes were detected until day 14. Since the time course of leukocyte adhesion was similar to that of intimal cell replication after stent implantation, we presumed that leukocytes on the luminal surface play a role in stimulating intimal cells. Indeed, histological findings showed that replicating cells in the intima were predominantly located in the inner intima after stent implantation, suggesting the presence of mitogenic factor(s) near the lumen. It is of interest that macrophage infiltration was mainly located around stent struts and in the mid-intima between stent struts. This distribution of macrophages was different from that of replicating cells in the intima. Further, macrophage index increased gradually until 28 days after stent implantation, while intimal BrdU index decreased after day 2. The discrepancy in the findings suggests that macrophages are not critical for intimal cell replication after stent implantation. However, previous studies using arterial injury models showed that infiltrating macrophages in the intima possibly promote an increase of intimal lesion size [17,18]. One possible explanation for these findings is that macrophages in intimal lesions promoted cell migration and/or accumulation of ECM to increase the lesion size after stent implantation. To develop an intimal lesion inside the stent, cell movement through the stent mesh might be required, as well as lesion expansion by cell replication and ECM accumulation. Cell movement in the arterial wall is linked to activation of some proteases, [19,20] and macrophages potentially secrete various proteases, including MMPs [21,22]. Further, a previous study showed that UPA acts as a physiological activator of MMPs [23]. In the present study, both MMP-9 and UPA activation were observed until 28 and 14 days after stent implantation, respectively, whereas these activities were detected only at 2 days after balloon dilation. Another interesting finding of the present study is that ECM of intimal lesions changed to become proteoglycans-rich after stent implantation as compared with
balloon dilation. Although it is poorly understood how the expression and accumulation of ECM molecules are regulated after stent implantation and balloon dilation, various studies showed that the contents of ECM such as collagen, elastin and proteoglycans were upregulated after arterial injury [10,24,25]. Strauss et al. demonstrated a significant increase of ECM synthesis after balloon dilation in their study using a rabbit reinjury model, and the synthesis of collagen and elastin continued to be significantly high until 12 weeks after balloon dilation, whereas proteoglycans synthesis decreased to below the control level by 4 weeks [24,26]. These data suggest that balloon dilation of intimal lesions promotes low-level accumulation of proteoglycans in the vessel wall, which is similar to the findings of the present study. In addition to this, macrophage accumulation in the intima was possibly linked to upregulation of proteoglycans synthesis. A previous in vitro study showed that some humoral factors released from macrophages in certain conditions could stimulate other cells to secrete proteoglycans, and further, macrophages themselves could secrete some proteoglycans [21,27]. Finally, we have to be aware of unsolved issues and limitations in the present study. One issue could be the limited period of observation after stent implantation and balloon dilation. Previous clinical studies reported that symptoms of in-stent restenosis were commonly developed from several months after stent implantation [28]. The present study, however, analyzed arterial responses for only 28 days after stent implantation. A number of studies using the rat carotid artery balloon injury model showed that an increase of SMC number promoted intimal formation in the early phase after injury, whereas intimal thickening was due to accumulation of ECM in the late phase after injury [2,29]. Thus, there is a possibility that longer observation would provide more crucial evidence for understanding the mechanisms of in-stent lesion formation. Meanwhile, a limitation of the present study is that the rabbit carotid artery with a preexisting intimal lesion mimics a diseased artery in the clinical setting, but is not exactly same. In the present study, since balloon injury induced the preexisting intimal lesion under a normal diet, the dominant cell component of the intimal lesion was SMC [29]. However, the human atherosclerotic lesion is composed of other cell components as well as SMC [30]. In particular, foam cells play an important role in the progression of human atherosclerosis [30]. We are now considering implanting a stent in a rabbit artery with an atherosclerotic lesion induced by a cholesterolrich diet, and investigating the responses of the arterial wall. In summary, the present study showed that stent implantation into a rabbit artery with preexisting intimal hyperplasia induced a significant increase in size of
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the in-stent intimal lesion. Intimal cell replication after stent implantation was significantly greater than that after balloon dilation, even at day 28, and prolonged cell replication was suggested to be a main reason for the increase of intimal lesion size. Meanwhile, stent implantation promoted arterial inflammatory responses not found after balloon dilation. Abundant leukocytes adhered to the luminal surface after stent implantation, and significant infiltration of macrophages and accumulation of proteoglycans were observed in the intima. Further, in the stent group, activation of proteases was detected for a longer period than in the balloon group. These data demonstrate unique properties after stent implantation, and might provide insights into understanding the pathophysiology of in-stent restenosis.
Acknowledgements This study was supported by a Grant-in Aid for Scientific Research (B) from the Ministry of Education, Science, and Culture of Japan (12470236).
References [1] Dussaillant GR, Mintz GS, Pichard AD, et al. Small stent size and intimal hyperplasia contribute to restenosis: a volumetric intravascular ultrasound analysis. J Am Coll Cardiol 1995;26:720 – 4. [2] Clowes AW, Reidy MA, Clowes MM. Mechanisms of stenosis after arterial injury. Lab Invest 1983;49:208 – 15. [3] Schwartz SM, deBlois D, O’Brien ER. The intima. Soil for atherosclerosis and restenosis. Circ Res 1995;77:445 – 65. [4] Koyama H, Olson NE, Dastvan FF, Reidy MA. Cell replication in the arterial wall: activation of signaling pathway following in vivo injury. Circ Res 1998;82:713 – 21. [5] 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 – 17. [6] Koyama H, Reidy MA. Expression of extracellular matrix proteins accompanies lesion growth in a model of intimal reinjury. Circ Res 1998;82:988 –95. [7] Kearney M, Pieczek A, Haley L, et al. Histopathology of in-stent restenosis in patients with peripheral artery disease. Circulation 1997;95:1998 – 2002. [8] Komatsu R, Ueda M, Naruko T, Kojima A, Becker AE. Neointimal tissue response at sites of coronary stenting in humans: macroscopic, histological, and immunohistochemical analyses. Circulation 1998;98:224 –33. [9] Kollum M, Kaiser S, Kinscherf R, Metz J, Kubler W, Hehrlein C. Apoptosis after stent implantation compared with balloon angioplasty in rabbits. Role of macrophages. Arterioscler Thromb Vasc Biol 1997;17:2383 –8. [10] Snow AD, Bolender RP, Wight TN, Clowes AW. Heparin modulates the composition of the extracellular matrix domain surrounding arterial smooth muscle cells. Am J Pathol 1990;137:313 – 30. [11] Tanaka H, Sukhova G, Schwartz D, Libby P. Proliferating arterial smooth muscle cells after balloon injury express TNF-alpha but not interleukin-1 or basic fibroblast growth factor. Arterioscler Thromb Vasc Biol 1996;16:12 – 8.
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[12] Pepper MS, Vassalli JD, Montesano R, Orci L. Urokinase-type plasminogen activator is induced in migrating capillary endothelial cells. J Cell Biol 1987;105:2535 – 41. [13] Bayer IM, Adamson SL, Langille BL. Atrophic remodeling of the artery-cuffed artery. Arterioscler Thromb Vasc Biol 1999;19:1499 – 505. [14] Shan L, Nishimura Y, Kotani Y, Yokoyama M. Platelet-activating factor increases the expression of metalloproteinase-9 in human bronchial epithelial cells. Eur J Pharmacol 1999;374:147 – 56. [15] O’Brien ER, Alpers CE, Stewart DK, et al. Proliferation in primary and restenotic coronary atherectomy tissue. Implications for antiproliferative therapy. Circ Res 1993;73:223 – 31. [16] Sousa JE, Costa MA, Abizaid A, et al. Lack of neointimal proliferation after implantation of sirolimus-coated stents in human coronary arteries: a quantitative coronary angiography and three-dimensional intravascular ultrasound study. Circulation 2001;103:192 – 5. [17] Weidinger FF, McLenachan JM, Cybulsky MI, Fallon JT, Hollenberg NK, Cooke JP, Ganz P. Hypercholesterolemia enhances macrophage recruitment and dysfunction of regenerated endothelium after balloon injury of the rabbit iliac artery. Circulation 1991;84:755 – 67. [18] Stadius ML, Rowan R, Fleischhauer JF, Kernoff R, Billingham M, Gown AM. Time course and cellular characteristics of the iliac artery response to acute balloon injury. An angiographic, morphometric, and immunocytochemical analysis in the cholesterolfed New Zealand white rabbit. Arterioscler Thromb 1992;12:1267 – 73. [19] Bendeck MP, Zempo N, Clowes AW, Galardy RE, Reidy MA. Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat. Circ Res 1994;75:539 –45. [20] Clowes AW, Clowes MM, Au YP, Reidy MA, Belin D. Smooth muscle cells express urokinase during mitogenesis and tissue- type plasminogen activator during migration in injured rat carotid artery. Circ Res 1990;67:61 – 7. [21] Edwards IJ, Wagner WD, Owens RT. Macrophage secretory products selectively stimulate dermatan sulfate proteoglycan production in cultured arterial smooth muscle cells. Am J Pathol 1990;136:609 – 21. [22] Galis ZS, Sukhova GK, Kranzhofer R, Clark S, Libby P. Macrophage foam cells from experimental atheroma constitutively produce matrix-degrading proteinases. Proc Natl Acad Sci USA 1995;92:402 – 6. [23] Woessner JF Jr. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J 1991;5:2145 – 54. [24] Strauss BH, Chisholm RJ, Keeley FW, Gotlieb AI, Logan RA, Armstrong PW. Extracellular matrix remodeling after balloon angioplasty injury in a rabbit model of restenosis. Circ Res 1994;75:650 – 8. [25] Nikkari ST, Jarvelainen HT, Wight TN, Ferguson M, Clowes AW. Smooth muscle cell expression of extracellular matrix genes after arterial injury. Am J Pathol 1994;144:1348 – 56. [26] Strauss BH, Robinson R, Batchelor WB, et al. In vivo collagen turnover following experimental balloon angioplasty injury and the role of matrix metalloproteinases. Circ Res 1996;79:541 –50. [27] Nathan CF. Secretory products of macrophages. J Clin Invest 1987;79:319 – 26. [28] George CJ, Baim DS, Brinker JA, et al. One-year follow-up of the Stent Restenosis (STRESS I) Study. Am J Cardiol 1998;81:860 –5. [29] Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium. Lab Invest 1983;49:327 – 33. [30] Stary HC, Chandler AB, Dinsmore RE, et al. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation 1995;92:1355 – 74.