Skip Navigation

Cardiovascular Research 2007 74(3):366-376; doi:10.1016/j.cardiores.2007.02.028
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Sawada, Y.
Right arrow Articles by Shimpo, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sawada, Y.
Right arrow Articles by Shimpo, H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2007, European Society of Cardiology

Tenascin-C synthesized in both donor grafts and recipients accelerates artery graft stenosis

Yasuhiro Sawadaa, Koji Onodaa,*, Kyoko Imanaka-Yoshidab, Junko Maruyamac, Kiyohito Yamamotoa, Toshimichi Yoshidab and Hideto Shimpoa

aDepartment of Thoracic and Cardiovascular Surgery, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan
bDepartment of Pathology and Matrix Biology, Mie University Graduate School of Medicine, Tsu, Mie, Japan
cDepartment of Physiology, Mie University Graduate School of Medicine, Tsu, Mie, Japan

* Corresponding author. Tel.: +81 59 232 1111; fax: +81 59 231 2845. Email address: k-onoda{at}clin.medic.mie-u.ac.jp

Received 24 August 2006; revised 9 February 2007; accepted 26 February 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Statistical analysis
 4. Results
 5. Discussion
 References
 
Objective: Tenascin-C, an extracellular matrix glycoprotein, is thought to play an important role in neointimal hyperplasia of artery bypass grafts. In this study, the direct contribution of tenascin-C to neointimal hyperplasia of free artery grafts and the origin of tenascin-C-producing cells were examined using tenascin-C transgenic mice.

Methods and results: Abdominal aorta-to-carotid artery interposition grafting was performed in mice. When grafts from wild-type mice were transplanted to wild-type, neointimal hyperplasia was observed in the grafts at days 14 and 28. Immunohistochemical staining showed strong expression of tenascin-C in the media and neointima of the grafts. Much less neointimal hyperplasia was seen when grafts from tenascin-C-deficient mice were transplanted to tenascin-C-deficient mice. In tenascin-C-deficient grafts transplanted to wild-type mice, tenascin-C deposition was observed only in the neointima. In the reverse combination, deposition was seen in the media and neointima. The source of the tenascin-C-producing cells was analyzed using heterozygous mice that identically express both tenascin-C and LacZ. While LacZ-positive cells were seen only in the neointima of artery grafts from wild-type transplanted to mutant mice, positive cells were detected in both the neointima and media in grafts from mutant to wild-type mice.

Conclusions: We presented direct evidence that tenascin-C is a crucial molecule in neointimal hyperplasia in free artery grafts, and that tenascin-C-producing cells are derived from both donor grafts and recipients.

KEYWORDS Cardiovascular surgery; Coronary disease; Remodeling; Transgenic animal models; Extracellular matrix

This article is referred to in the Editorial by J.W. Fischer (pages 335–336) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Statistical analysis
 4. Results
 5. Discussion
 References
 
Artery graft stenosis at the site of anastomosis induces neointimal hyperplasia and eventually results in occlusion of the graft [1]. Recruitment of smooth muscle cells (SMCs) and excess deposition of extracellular matrix (ECM) are the pathogenesis of neointimal hyperplasia [2]. Among various ECM proteins, tenascin-C (TNC) is prominent in a variety of actively remodeling cardiovascular tissues, such as in human coronary atherosclerotic plaque [3], rat balloon-injured artery [4], abdominal aortic aneurysm [5], and arterialized human vein grafts [6], as well as in the early stage after percutaneous transluminal coronary angioplasty [7]. In vitro studies have demonstrated that TNC blocks adhesion of SMCs to fibronectin [8] and promotes migration [9] and growth factor-dependent SMC proliferation [10]. In addition, platelet-derived growth factor (PDGF) and angiotensin II are stimulants for TNC synthesis in cultured SMCs [8,11].

To prevent neointimal formation after surgery, local applications of various drugs have been attempted in animal experimental models, which suppressed neointimal hyperplasia of vein grafts and injured arteries by inhibiting migration and proliferation of SMCs [12–16]. In fact, in a previous study we demonstrated that local administration of cilostazol, a specific inhibitor of cAMP phosphodiesterase III inhibitor, remarkably suppressed neointimal hyperplasia, and was associated with inhibiting TNC expression and cell accumulation in the neointima of free artery grafts of rats [17]. Cilostazol inhibited not only PDGF-induced SMC proliferation, but also TNC mRNA expression induced by PDGF in cultured aortic SMCs [17]. We also demonstrated that neointimal formation at suture sites after aortotomy in TNC-deficient mice was noticeably reduced compared to that in wild-type [18]. TNC may be a prime target for therapy in suppressing neointimal hyperplasia after vascular surgery.

The cellular origin of SMC emerging in hyperplastic neointima is still controversial. It is generally believed that SMCs in neointima of transplanted grafts are derived from the SMCs of the medial layer [19–21]. However, neointimal SMCs can be distinguished from medial SMCs by their cytoskeletal features and responses to a variety of growth factors and cytokines [22]. Recent studies have described that SMCs in neointimal lesions of transplanted grafts originate from recipients, but not donor grafts [23,24]. By analyzing animal models using immunohistochemistry and in situ hybridization, we have noticed the emergence of {alpha}-smooth muscle actin ({alpha}-SMA) positive cells, suggesting their production of TNC in hyperplastic neointima. We believe that {alpha}-SMA positive cells (SMCs and/or myofibroblast) producing TNC migrate into the neointima and proliferate, followed by neointimal formation.

In this study, using a mouse model of an abdominal aorta-to-carotid artery interposition grafting, we examined the direct contribution of TNC to neointimal hyperplasia in free artery grafts, as well as the origin of TNC-producing cells. To facilitate this, we employed gene-manipulated mice with a targeted deletion of the TNC gene by replacement with a construct with β-galactosidase (LacZ) reporter.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Statistical analysis
 4. Results
 5. Discussion
 References
 
2.1 Animals and vessel reactivity assay
Artery transplantation was performed using 8 to 10 week old male TNC-deficient (TN–), heterozygous mutant (TN+/–) and wild-type (WT) mice of the BALB/c strain. Deletion of the TNC gene was performed by targeting a construct containing the LacZ gene to the TNC locus [25]. All studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and the guidelines approved by the Mie University Animal Experiment and Care Committee.

Blood pressure measurements were performed with the BP-98A (Softron, Tokyo, Japan) tail cuff system while the animals were conscious.

The vessel reactivity of TN– mice was examined according to a method described previously [26]. Briefly, aortas removed from WT and TN– mice were carefully cleaned of fat and connective tissue in modified Krebs-Henseleit solution. Ring segments (2 mm) were cut and suspended vertically between hooks in an organ bath (20 ml) containing modified Krebs-Henseleit solution maintained at 37 °C and bubbled with a mixture of 95% air–5% CO2. The optimal resting tension for the vasocontraction and vasodilation studies was adjusted to 1 g for both WT and TN– mice. At this resting tension, the peak contraction was obtained in response to 70 mM KCl. In all experiments, changes in isometric force were measured with a force-displacement transducer (TB-651T; Nihon Kohden, Tokyo, Japan) connected to a carrier amplifier (EF601G; Nihon Kohden) and recorded on a pen recorder (WT-645G; Nihon Kohden). Concentration-response curves were obtained by cumulative addition of the studied drugs including noradrenaline (10–10–10–6 M, Sankyo, Tokyo, Japan) and phenylephrine (10–9–10–5 M, Kowa, Nogoya, Japan), respectively. Next the rings were precontracted with phenylephrine to obtain 50–80% of the maximal contraction induced by 70 mM KCl. Cumulative dose-tension curves were obtained for acetylcholine hydrochloride (10–8–10–4 M, Nacalai Tesque, Kyoto, Japan) or sodium nitroprusside (10–9–10–5 M, Nacalai Tesque). Finally, 10–4 M papaverine hydrochloride (Nacalai Tesque) was added to produce maximal relaxation. The results of each drug were expressed as a percentage of the relaxation induced by papaverine.

2.2 Gelatin zymography
The gelatinolytic activity was analyzed according to a method described previously [27]. Briefly, aortas removed from WT and TN– mice were homogenized in sample buffer (4 µl buffer/mg tissue) containing 10 mM Tris/HCl (pH 7.6), 20% glycerin, 2% SDS, and 0.1% bromphenol blue. The homogenates were clarified by centrifugation and then separated by electrophoresis on 10% polyacrylamide gel containing 0.1% SDS and 1 mg/ml gelatin as a substrate. Thereafter, gels were washed in the reaction buffer (50 mM Tris–HCl (pH 7.6), 0.15 M NaCl, 10 mM CaCl2, and 0.02% NaN3) containing 2.5% Triton X-100 for 1 h to remove SDS. The gels were then incubated for 24 h at 37 °C in the reaction buffer and stained with 0.1% Coomassie Brilliant Blue R250. The location of gelatinolytic activity was detectable as a clear band in the background of uniform staining. Pro- and activated matrix metalloproteinase (MMP) 2 bands were detected at 66 and 62 kDa, respectively. The ratio of activated MMP-2 to total MMP-2 activities (62 kDa/66 kDa+62 kDa) was calculated from their gelatinolytic activities measured by computer-assisted image analysis according to a method reported previously [28].

2.3 Free artery graft model
Donor and recipient mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg). Using a sterile technique, we exposed the aorta below the renal arteries through an abdominal incision. A 3.0-millimeter segment of infrarenal aorta was removed from the donor mouse. For the recipient mouse, the right common carotid artery was mobilized free from the bifurcation at the distal end toward the proximal end and was then cut in the middle. The donor aortic segment was transplanted into the recipient right carotid artery in an end-to-end fashion. Anastomosis was performed by interrupted 11-0 nylon sutures without injuring the intima.

Six transplant groups were assigned (Fig. 1). Four groups were used to examine the direct contribution of tenascin-C to neointimal hyperplasia of free artery grafts: WT donor graft transplanted into the WT recipient group (WT>WT), WT donor graft into the TN– recipient group (WT>TN–), TN– donor graft into the WT recipient group (TN–>WT), and TN– donor graft into the TN– recipient group (TN–>TN–). The other two groups were used to examine the origin of TNC-producing cells in the neointimal lesion: WT donor graft into the TN+/– recipient group (WT>TN+/–), and TN+/– donor graft into the WT recipient group (TN+/–>WT).


Figure 1
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Summary of interposition grafting in mice. Artery transplantation was performed using male TNC-deficient (TN–), heterozygous mutant (TN+/–) and wild-type (WT) mice of the BALB/c strain.

 
2.4 Graft harvesting and morphometric analysis
The grafts were harvested at days 14 and 28 (8 mice per group at each time point) under anesthesia. Recipient mice were perfused with 20 ml of heparin-prepared saline followed by 50 ml of 10% neutral buffered formalin solution. The grafts were exposed carefully and extracted, including proximal and distal normal right common carotid artery, and then postfixed in the same fixative at 4 °C for 1 day and embedded in paraffin. For histological analysis of the graft body, four cross-sections (4 µm-thick) were cut from the mid-portion between the distal anastomosis and the center of the graft. Four cross-sections were selected from the distal anastomotic sites and stained with elastica van Gieson to demarcate the internal elastic lamina. For each section, the ratio between the neointima and the media was calculated using an image analysis system (Adobe Photoshop version 5.0J and NIH Image version 1.61, Macintosh) as previously described [18].

2.5 Immunohistochemistry and histochemistry
Immunohistochemical staining for TNC was performed according to a previously reported method [29]. In brief, sections were first incubated with a rabbit anti-human polyclonal antibody (1 µg/ml) and then with peroxidase-conjugated anti-rabbit IgG (1:200; MBL, Nagoya, Japan), followed by color development with diaminobenzidine/H2O2 solution. Light counterstaining with hematoxylin was performed. Rabbit control IgGs were used instead of primary antibodies as negative immunostaining controls. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA), Ki67, and {alpha}-SMA was performed using mouse monoclonal antibody against PCNA (PC10, EPOS, DAKO, Tokyo, Japan), monoclonal rat anti-mouse Ki67 (DAKO, Tokyo, Japan), and peroxidase-conjugate anti-{alpha}-SMA antibody (EPOS, DAKO, Tokyo Japan), respectively [17]. The PCNA or Ki67 labeling index was determined by dividing the number of PCNA- or Ki67-positive cells, respectively, by the total number of nucleated cells in neointima and media from each section.

Collagen fibers were stained with picrosirius red as previously reported [30] and examined under a polarization microscope. Collagen content area and media area were measured in picrosirius red stained aortic tissues using a wall-tracking tool and calculated as the relative collagen content as previously reported [31].

2.6 LacZ staining
In these transgenic mice, LacZ staining reflects the endogenous expression pattern of the TNC gene [25]. LacZ staining was performed according to a previously reported method [32]. The grafts were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min at 4 °C, washed with 0.1 M phosphate buffer containing 2 mM MgCl2, 0.01% sodium deoxycholate, and 0.02% Nonidet P-40 and incubated at 37 °C overnight with X-gal solution (5.0 mM potassium ferrocyanide, 5.0 mM potassium ferricyanide, 1 mg/ml X-gal). The grafts were then further fixed in 4% paraformaldehyde, and embedded in paraffin. Thin sections (5 µm) were deparaffinized, lightly counterstained with nuclear fast red, and used for histological analysis. For double staining of LacZ and {alpha}-SMA, we performed {alpha}-SMA immunohistochemistry using the sections with LacZ staining.


   3. Statistical analysis
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Statistical analysis
4. Results
 5. Discussion
 References
 
Statistical analysis was carried out using standard software (StatView 5.0.1, SAS Institute, Inc.). Values were expressed as the mean±standard deviation (S.D.). Statistical analysis between TN– and WT mice in Table 1 was performed using the Mann–Whitney U-test. Differences between groups for repeatedly measured variables (vessel reactivity assay) were analyzed by analysis of variance (ANOVA). One-way ANOVA was used to compare differences between groups for the mean neointima/media area ratios, PCNA indices and KI67 indices. If ANOVA showed significant differences, post hoc nonparametrical testing (Fisher PLSD) was added. A p value of <0.05 was considered statistically significant.


View this table:
[in this window]
[in a new window]

  		Table 1 Characteristic profiles in WT and TN– mice

 

   4. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Statistical analysis
 4. Results
5. Discussion
 References
 
4.1 Phenotypic analysis of TN– mice
As shown in Table 1, there were no significant differences in body weight, inner diameter or lumen areas of the abdominal aorta and common carotid artery, and in blood pressure between TN– and WT mice. Vessel reactivity was examined by organ bath tension measurements using aortic rings. The aortic rings from TN– mice and those from WT mice showed similar contractions in response to noradrenaline and phenylephrine (Fig. 2A,B). Phenylephrine-precontracted aortic rings from TN– mice and those from WT mice also showed similar responses to acetylcholine and sodium nitroprusside (Fig. 2C,D). In addition, gelatin zymography revealed no significant differences in the activity of MMP-2 in aortic tissues between TN– and WT mice (Table 1). The expression of MMP-9 was also found in aortic tissues, but the band of activated MMP-9 was not detected in either TN– or WT mice. No significant differences of collagen content were recognized between TN– and WT mice (Table 1).


Figure 2
View larger version (30K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Response to noradrenaline (A) and phenylephrine (B), and relaxation response to acetylcholine (C) and sodium nitroprusside (D) of aortic rings. A and B, Contraction produced by 70 mM KCl was taken as 100%. C and D, The aortic rings were precontracted with phenylephrine to obtain 50–80% of the maximal contraction induced by 70 mM KCl. The relaxation produced by 10–4 M papaverine was taken as 100%. Values are mean±S.D.; numbers in parentheses refer to the number of rings from four mice in each group. The aortic rings show no significant differences in the response to the vasocontracting and vasodilating drugs between TN– (closed circle) and WT mice (open circle).

 
4.2 Neointimal hyperplasia in WT mice grafts
In the WT>WT group, neointimal hyperplasia was seen in both the graft body (Fig. 3A) and distal anastomotic site (Fig. 4A) at postoperative day 14 and had markedly progressed by day 28 without medial thickening. Marked neointimal hyperplasia was observed in the graft body close to the distal anastomotic site, gradually decreasing as it extended to the proximal anastomotic site. Therefore, for histological analysis we selected the mid-portion between the distal anastomosis and the center of the graft. The mean neointima/media area ratios for the graft body and the distal anastomotic site at day 28 were significantly increased compared with those at day 14 (graft body: 0.95±0.173 vs. 0.336±0.052, p<0.01, distal anastomotic site: 1.079±0.056 vs. 0.517±0.064, p<0.01) (Figs. 3B and 4BGo). Immunohistochemical staining showed {alpha}-SMA positive cells in the neointima at day 14 (data not shown) and the number of these cells increased in the neointima at day 28, paralleling the neointimal thickness (Fig. 3A).


Figure 3
View larger version (59K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Neointimal formation in the graft body in the WT>WT, WT>TN–, TN–>WT, and TN–>TN– groups. A. Elastica van Gieson-stained cross-section (a–h) and immunostaining for {alpha}-SMA (i–l). (a,e,i) WT>WT group, (b,f,j) WT>TN– group, (c,g,k) TN–>WT group, (d,h,l) TN–>TN– group. (a–d) Graft body at day 14. (e–l) Graft body at day 28. In the WT>WT group, neointimal hyperplasia is seen in the graft body at postoperative day 14 (a) and is markedly progressed by day 28 (e). The neointimas of the TN–>TN– group are distinctly smaller than those of the neointimas of the WT>WT group at days 14 (d) and 28 (h). The neointimal hyperplasia in the WT>TN– and TN–>WT groups is smaller than that in the WT>WT group and bigger than that in the TN–>TN– group at day 14 and day 28. {alpha}-SMA positive cells are seen in the neointima, especially in WT>WT group (i) and media in the four groups at day 28. Arrow heads indicate internal elastic lamina. Scale bar in (l): 50 µm. B. Neointima/media area ratio at the graft body. Four sections per animal were selected, and data are mean±S.D. of the neointima/media area ratio from eight animals per group at each time point. There is a significant difference between days 14 and 28 in the WT>WT group (*p<0.01). Significant differences are present between the WT>WT and the other groups at days 14 and 28 (day 14: **p<0.01, day 28: ***p<0.01). There are also significant differences between the TN–>TN– and WT>TN– or TN–>WT groups at days 14 and 28 (day 14: +p<0.01, day 28: ++p<0.05).

 

Figure 4
View larger version (46K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Neointimal formation at distal anastomotic sites in the WT>WT, WT>TN–, TN–>WT, and TN–>TN– groups at day 28. A. Elastica van Gieson-stained cross-section. WT>WT group (a), WT>TN– group (b), TN–>WT group (c), and TN–>TN– group (d). The neointimas of the TN–>TN– group are noticeably smaller than those of the WT>WT group. The neointimal hyperplasia in the WT>TN– and TN–>WT groups is smaller than that in the WT>WT group and bigger than that in the TN–>TN– group. Arrow heads indicate internal elastic lamina. Asterisks indicate suture thread. Scale bar in (d): 50 µm. B. Neointima/media area ratio at distal anastomotic sites. Four sections per animal were selected, and data are mean±S.D. of the neointima/media area ratio from eight animals per group at each time point. There is a significant difference between days 14 and 28 in the WT>WT group (*p<0.01). Significant differences are present between the WT>WT and TN–>TN– groups at day 14 (**p<0.01). Significant differences were present between the WT>WT and the other groups at day 28 (***p<0.01). There are also significant differences between the TN–>TN– and WT>TN– or TN–>WT groups at days 14 and 28 (day 14: +p<0.01, day 28: ++p<0.01).

 
4.3 Decrease in neointimal hyperplasia in TNC-deficient mice groups
The neointimas of the TN–>TN– group were noticeably smaller than those of the WT>WT group in both the graft body and the distal anastomotic site at days 14 and 28 (Figs. 3 and 4Go). The mean neointima/media area ratios of the graft body and distal anastomotic site in the TN–>TN– group were significantly lower than those in the WT>WT group at day 14 (graft body: 0.084±0.065 vs. 0.336±0.052, p<0.01, distal anastomotic site: 0.207±0.074 vs. 0.517±0.064, p<0.01) and day 28 (graft body: 0.120±0.082 vs. 0.95±0.173, p<0.01, distal anastomotic site: 0.31±0.112 vs. 1.079±0.056, p<0.01) (Figs. 3B and 4BGo). The mean neointima/media area ratios of the graft body and the distal anastomotic site in the TN–>TN– group were reduced to 12.6% and 28.7%, respectively, at postoperative day 28 compared to the WT>WT group. The mean neointima/media area ratios of the graft body in the WT>TN– group and TN–>WT group at days 14 and 28 were significantly lower than those in the WT>WT group (p<0.01), and but higher than those in the TN–>TN– group (day 14: p<0.01; day 28: p<0.05) (Fig. 3B). The mean neointima/media area ratios of the distal anastomotic site in the WT>TN– and TN–>WT groups at days 14 and 28 were significantly higher than those in the TN–>TN– group (p<0.01) (Fig. 4B). There were no significant differences between the WT>TN– and TN–>WT groups in mean neointima/media area ratios.

4.4 Cell proliferation in neointima and media
To assess the extent of proliferation of neointimal cells, cross-sections at days 14 and 28 were stained using monoclonal antibodies against PCNA or Ki67 and the labeling indices in the neointima and media at the graft body and distal anastomotic site were evaluated. PCNA indices at day 14 were significantly lower in the TN–>TN– group than in the WT>WT group (graft body: 14.8±2.4% vs. 31.1±6.0%, p<0.01; distal anastomotic site: 19.3±6.7% vs. 33.7±8.6%, p<0.01, Fig. 5A,B). At day 14, the PCNA indices in the WT>WT group were twice those in the TN–>TN– group. At day 28, PCNA indices at the graft body and distal anastomotic site tended to be higher in the WT>WT group than in the TN–>TN– group. In the WT>TN– and TN–>WT groups, PCNA indices at day 14 were significantly lower than those in the WT>WT group (p<0.01), and were significantly higher than those in the TN–>TN– group at the graft body (p<0.01 and p<0.05, respectively). In cell proliferation analyses using Ki67 antibody, the indices showed similar results to those for the PCNA indices (Fig. 5C,D). However, at day 28, Ki67 indices at the graft body were significantly higher in the WT>WT group than in the TN–>TN– and TN–>WT groups (p<0.01 and p<0.05, respectively), but were significantly lower in the TN–>TN– group than in the WT>TN– group (p<0.05).


Figure 5
View larger version (30K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 PCNA and Ki67 labeling indices in the neointima and media. Four sections per animal were selected, and data are mean±S.D. of the labeling index from eight animals per group at each time point. A, PCNA index at the graft body. B, PCNA index at the distal anastomotic site. C, Ki67 index at the graft body. D, Ki67 index at the distal anastomotic site. There are significant differences in PCNA indices between the WT>WT and the other groups at day 14 (*p<0.01), and significant differences in PCNA indices at the graft body are present between the TN–>TN– and WT>TN– or TN–>WT groups at day 14 (+p<0.01, ++p<0.05). There are also significant differences in Ki67 indices between the WT>WT and the other groups at day 14 (*p<0.01), and significant differences in Ki67 indices at the graft body are present between the TN–>TN– and WT>TN– or TN–>WT groups at day 14 (+p<0.05, ++p<0.01). At day 28, significant differences in Ki67 indices at the graft body are present between the WT>WT and TN–>TN– or TN–>WT groups (**p<0.01, ***p<0.05) and between the TN–>TN– and WT>TN– groups (+++p<0.05).

 
4.5 Expression of TNC in mouse artery grafts
In the WT>WT and WT>TN– groups, TNC immunolabeling was seen prominently in the neointima but also in the media at day 14 after grafting (Fig. 6a,b). At day 28, TNC staining was detected in the neointima of the graft body, whereas staining became weaker in the media (Fig. 6e,f). While TNC expression was not observed in the TN–>TN– group (Fig. 6d,h), it was clearly detected only in the neointima at days 14 and 28 in the TN–>WT group (Fig. 6c,g).


Figure 6
View larger version (69K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 TNC immunostaining of the graft body. (a,e) WT>WT, (b,f) WT>TN–, (c,g) TN–>WT, and (d,h) TN–>TN– groups. In the WT>WT and WT>TN– groups, immunostaining of TNC is seen in both the neointima and media at day 14, and TNC labeling becomes weaker in the media, but remains in the neointima at day 28. In the TN–>WT group, TNC staining is detected only in the neointima at days 14 and 28. TNC expression is not observed in the TN–>TN– group. (a–d) Postoperative day 14, (e–h) postoperative day 28. Arrow heads indicate internal elastic lamina. Scale bar in (h): 50 µm.

 
4.6 Origin of TNC-producing cells
In this experimental design (transplant of heterozygous tissues to wild mice, and vice versa), cells from both recipient and donor have the ability to synthesize TNC. The cells that originated from heterozygous tissues become positive for LacZ staining when they express TNC. Therefore, we can identify that the blue LacZ-positive cells are responsible for synthesis of TNC and originate from heterozygous tissue. In respect to the macroscopic appearance of the explanted grafts of the WT>TN+/– group (Fig. 7A), apparent blue LacZ staining was seen in the recipient vessel at the distal anastomotic site. The expression extended to the graft body across the anastomosis, which indicated that the recipient vessel synthesized TNC. In histological sections of the recipient vessel at the anastomotic site, numerous LacZ-positive cells were seen throughout the vascular wall particularly around the suture thread. LacZ-positive cells were also found in the graft body from wild-type mice, but only in the neointima, at day 14 (Fig. 7B). With careful observation of the serial sections, LacZ-positive cells were seen in a line across the distal anastomosis, suggesting that TNC-producing cells that originated from the recipient may have migrated to the transplanted graft over the anastomotic sites (Fig. 7B). In the TN+/–>WT group, intense LacZ staining was macroscopically observed in the graft body close to the distal anastomotic site, gradually decreasing as it extended to the proximal anastomotic site. In histological sections of the area, LacZ-positive cells were detected in both media and neointima of the graft body at day 14. Double staining between {alpha}-SMA and LacZ showed that some LacZ-positive cells overlapped with {alpha}-SMA positive cells in both the WT>TN+/– and TN+/–>WT groups (Fig. 7B).


Figure 7
View larger version (105K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 LacZ staining of the explanted graft at postoperative day 14. A. Macroscopic appearance of the explanted graft. (a) WT>TN+/– group, and (b) TN+/–>WT group. Blue LacZ staining is observed in the recipient vessel at the distal anastomotic site and the expression extended to the graft body across the anastomosis in the WT>TN+/– group. In the TN+/–>WT group, strong LacZ staining is seen in the graft body close to the distal anastomotic site, gradually decreasing as it extends to the proximal anastomotic site. Arrows indicate anastomotic sites. Blue staining indicates LacZ-positive cells. B. Histological sections of the explanted graft. (c,e,g) LacZ-stained semi-serial cross-section around the distal anastomotic site in the WT>TN+/– group. (c) Recipient artery just distal to the anastomotic site. (e) Donor graft of the anastomotic site. (g) Graft body. LacZ-positive cells are observed in the neointima of the WT>TN+/– group, and LacZ-positive cells in the neointima are seen in a line across the distal anastomosis, as indicated by arrows. (d) LacZ staining in the graft body in the TN+/–>WT group. (f,h) Double staining between {alpha}-SMA and LacZ in the TN+/–>WT group (f) and WT>TN+/– group (h). In the TN+/–>WT group, LacZ-positive cells are detected in both the neointima and media. Some LacZ-positive cells overlap with {alpha}-SMA positive cells in both the WT>TN+/– and TN+/–>WT groups. Arrow heads indicate internal elastic lamina. White arrows indicate examples of double-positive cells. Scale bars: 50 µm. Note that LacZ-positive cells are blue-stained.

 

   5. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Statistical analysis
 4. Results
 5. Discussion
References
 
In this study, we established an abdominal aorta-to-common carotid artery interposition grafting model by end-to-end suturing in mice based on the premise of free artery grafts in coronary artery bypass grafting. Shi et al. [33] established the first mouse model of transplant arteriosclerosis by end-to-end suturing of carotid artery segments to carotid arteries, and Koulack et al. [34] reported aortic transplantation to the infrarenal aorta by an end-to-end anastomosis. Furthermore, Dietrich et al. [35] developed a simplified mouse model of transplant arteriosclerosis of carotid artery or aortic segments to carotid arteries using a cuff technique. In this study, we modified these models by end-to-end suturing of aortic segments to carotid arteries. In the WT>WT group, neointimal hyperplasia was formed around at postoperative day 14 in both the graft body and the distal anastomotic sites with an increase in the PCNA and Ki67 indices indicating cell proliferation and was markedly progressed by day 28. It was considered that the caliber difference between the abdominal aorta and the common carotid artery yields stenosis at the distal anastomotic sites. A stenosis causes blood turbulence and/or shear stress in grafts, which results in neointimal hyperplasia through stimulated SMC migration and proliferation with deposition of ECM.

In vitro studies have demonstrated that TNC promotes migration [9] and growth factor-dependent proliferation of arterial SMCs [11]. Although marked expression of TNC has been found in various vascular lesions in active phases [3,4,6,7], its functional significance during the development of neointimal hyperplasia remains obscure. We recently demonstrated the contribution of TNC to neointimal hyperplasia in a simple aortotomy model using TNC-deficient mice [18]. In this regard, we carried out the present study to elucidate the direct contribution to stenosis of free artery graft transplantation using TNC-deficient mice. First, we confirmed that there is no difference in geometrical values of the arteries, their physiological nature, MMP expression or collagen content between WT and TN– mice. The four experimental groups in this study were a combination of donor grafts and recipients in WT and TN– mice. The mean neointima/media area ratio at day 28 in the TN–>TN– group was decreased as low as 1/6 in the graft body and as low as 1/4 at the distal anastomotic sites compared with that in the WT>WT group. PCNA labeling index at day 14 was decreased by almost half in the graft body and the stenotic site of the anastomosis. These results indicate that TNC is a crucial molecule in neointimal hyperplasia in artery grafts. Interestingly, the mean neointima/media area ratios in the WT>TN– or TN–>WT groups were significantly lower than that in the WT>WT group and higher than that in the TN–>TN– group, with no significant difference between the WT>TN– and TN–>WT groups. These results suggest that TNC produced by both donor grafts and recipient tissues may be equally involved in developing neointimal hyperplasia of artery grafts and that the severe stenotic lesion in the WT>WT group is a result of summation of the effects.

In the aortic segments donated by WT mice (WT>WT and WT>TN– groups), the changes in the distribution of TNC expression during the first 28 days after surgery, which seemed to shift from the media to the neointima, were similar to those previously observed in free artery grafts in rats [17]. By contrast, in the grafts donated by TN– mice transplanted to WT mice (TN–>WT group), the expression of TNC protein was detected only in the neointima, and was associated with the emergence of predominant {alpha}-SMA positive cells. Therefore, it seems likely that local SMCs in the media of the graft synthesize TNC and move to the neointima; simultaneously, recipient cells then migrate into the neointima from somewhere outside the graft. Traditionally, it has been believed that {alpha}-SMA positive cells in the neointima were derived from the medial SMCs of donor arteries migrating through the internal elastic lamina into subendothelial spaces [19–21]. However, recent studies have suggested that bone marrow cells are a source of smooth muscle-like cells in neointimal lesions in injured vessels [22] and transplanted grafts [23,24]. Hu and coworkers [36] demonstrated that {alpha}-SMA positive cells in neointima of atherosclerotic lesions in allografts are derived from recipients (pericytes in the microvasculature of liver and spleen), but not bone marrow progenitor cells. The authors also described that medial SMCs of recipient anastomosed arteries never migrate into the grafts, because cuff techniques were used for anastomosis of the graft to the recipient artery, in which no media SMCs contacted each other between the donor and recipient.

In the grafts from heterozygous mice, we found that LacZ-positive SMCs in the media migrated into the neointima through the internal elastic lamina, which suggest that deposition of TNC can be, at least partly, a local reaction by medial SMCs. On the other hand, we also found LacZ-positive cells in the grafts donated by wild-type mice, indicating that these TNC expressing cells evidently came from the heterozygous recipient. Careful observation also suggests that some TNC-producing cells may directly migrate from recipient vessels into the neointima of grafts across the anastomosis. In vascular lesions, several types of cells including SMCs [8,17], fibroblasts [37], myofibroblasts [38], and endothelial cells [39] have been reported to express TNC protein. The double staining of LacZ and {alpha}-SMA showed that TNC-producing cells were positive for {alpha}-SMA in both the TN+/–>WT and WT>TN+/– groups. Thus, {alpha}-SMA positive cells producing TNC in the neointima of free artery grafts can originate from not only the donor grafts but also from recipients, at least partly including recipient arteries.

Finally, this study indicates that TNC is a crucial molecule as it causes neointimal hyperplasia of artery grafts at an early stage. Therefore, we propose that TNC represents a prime target for therapy to inhibit SMC migration and proliferation in free artery grafts. On the basis of the origin of TNC-producing cells (both donor grafts and recipient arteries), we could consider that combined treatment of local and systemic drug administration may be more effective for inhibiting neointimal hyperplasia of free artery grafts.


   Acknowledgements
 
We thank Akiyo Sekimoto and Tomohiro Nishioka for technical assistant.


   Notes
 
Time for primary review 45 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Statistical analysis
 4. Results
 5. Discussion
 References
 

  1. Hosono M., Ueda M., Suehiro S., Sasaki Y., Shibata T., Hattori K., et al. Neointimal formation at the sites of anastomosis of the internal thoracic artery grafts after coronary artery bypass grafting in human subjects: an immunohistochemical analysis. J Thorac Cardiovasc Surg (2000) 120:319–328.[Abstract/Free Full Text]
  2. Davies M.G., Hagen P.O. Pathobiology of intimal hyperplasia. Br J Surg (1994) 81:1254–1269.[ISI][Medline]
  3. Wallner K., Li C., Shah P.K., Fishbein M.C., Forrester J.S., Kaul S., et al. Tenascin-C is expressed in macrophage-rich human coronary atherosclerotic plaque. Circulation (1999) 99:1284–1289.[Abstract/Free Full Text]
  4. Hedin U., Holm J., Hansson G.K. Induction of tenascin in rat arterial injury: relationship to altered smooth muscle cell phenotype. Am J Pathol (1991) 139:649–656.[Abstract]
  5. Satta J., Soini Y., Pöllänen R., Pääkkö P., Juvonen T. Tenascin expression is associated with a chronic inflammatory process in abdominal aortic aneurysms. J Vasc Surg (1997) 26:670–675.[CrossRef][ISI][Medline]
  6. Wallner K., Li C., Fishbein M.C., Shah P.K., Sharifi B.G. Arterialization of human vein grafts is associated with tenascin-C expression. J Am Coll Cardiol (1999) 34:871–875.[Abstract/Free Full Text]
  7. Imanaka-Yoshida K., Matsuura R., Isaka N., Nakano T., Sakakura T., Yoshida T. Serial extracellular matrix changes in neointimal lesions of human coronary artery after percutaneous transluminal coronary angioplasty: clinical significance of early tenascin-C expression. Virchows Arch (2001) 439:185–190.[CrossRef][ISI][Medline]
  8. LaFleur D.W., Fagin J.A., Forrester J.S., Rubin S.A., Sharifi B.G. Cloning and characterization of alternatively spliced isoforms of rat tenascin: platelet-derived growth factor-BB markedly stimulates expression of spliced variants of tenascin mRNA in arterial smooth muscle cells. J Biol Chem (1994) 269:20757–20763.[Abstract/Free Full Text]
  9. LaFleur D.W., Chiang J., Fagin J.A., Schwartz S.M., Shah P.K., Wallner K., et al. Aortic smooth muscle cells interact with tenascin-C through its fibrinogen-like domain. J Biol Chem (1997) 272:32798–32803.[Abstract/Free Full Text]
  10. Jones P.L., Rabinovitch M. Tenascin-C is induced with progressive pulmonary vascular disease in rats and is functionally related to increased smooth muscle cell proliferation. Circ Res (1996) 79:1131–1142.[Abstract/Free Full Text]
  11. Sharifi B.G., LaFleur D.W., Pirola C.J., Forrester J.S., Fagin J.A. Angiotensin II regulates tenascin gene expression in vascular smooth muscle cells. J Biol Chem (1992) 267:23910–23915.[Abstract/Free Full Text]
  12. Hu Y., Zou Y., Dietrich E., Wick G., Xu Q. Inhibition of neointima hyperplasia of mouse vein grafts by locally applied suramin. Circulation (1999) 100:861–868.[Abstract/Free Full Text]
  13. Schachner T., Zou Y., Oberhuber A., Tzankov A., Mairinger T., Laufer G., et al. Local application of rapamycin inhibits neointimal hyperplasia in experimental vein grafts. Ann Thorac Surg (2004) 77:1580–1585.[Abstract/Free Full Text]
  14. Ishizaka N., Taguchi J., Kimura Y., Ikari Y., Aizawa T., Togo M., et al. Effects of a single local administration of cilostazol on neointimal formation in balloon-injured rat carotid artery. Atherosclerosis (1999) 142:41–46.[CrossRef][ISI][Medline]
  15. Indolfi C., Avvedimento E.V., Di Lorenzo E., Esposito G., Rapacciuolo A., Giuliano P., et al. Activation of cAMP-PKA signaling in vivo inhibits smooth muscle cell proliferation induced by vascular injury. Nat Med (1997) 3:775–779.[CrossRef][ISI][Medline]
  16. Moon M.C., Molnar K., Yau L., Zahradka P. Perivascular delivery of losartan with surgical fibrin glue prevents neointimal hyperplasia after arterial injury. J Vasc Surg (2004) 40:130–137.[CrossRef][ISI][Medline]
  17. Fujinaga K., Onoda K., Yamamoto K., Imanaka-Yoshida K., Takao M., Shimono T., et al. Locally applied cilostazol suppresses neointimal hyperplasia by inhibiting tenascin-C synthesis and smooth muscle cell proliferation in free artery grafts. J Thorac Cardiovasc Surg (2004) 128:357–363.[Abstract/Free Full Text]
  18. Yamamoto K., Onoda K., Sawada Y., Fujinaga K., Imanaka-Yoshida K., Shimpo H., et al. Tenascin-C is an essential factor for neointimal hyperplasia after aortotomy in mice. Cardiovasc Res (2004) 65:737–742.[CrossRef][ISI]
  19. Kennedy L.J. Jr., Weissman I.L. Dual origin of intimal cells in cardiac-allograft arteriosclerosis. N Engl J Med (1971) 285:884–887.[ISI][Medline]
  20. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med (1999) 340:115–126.[Free Full Text]
  21. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature (1993) 362:801–809.[CrossRef][Medline]
  22. Han C., Campbell G.R., Campbell J.H. Circulating bone marrow cells can contribute to neointimal formation. J Vasc Res (2001) 38:113–119.[CrossRef][ISI][Medline]
  23. Sata M., Saiura A., Kunisato A., Tojo A., Okada S., Tokuhisa T., et al. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med (2002) 8:403–409.[CrossRef][ISI][Medline]
  24. Shimizu K., Sugiyama S., Aikawa M., Fukumoto Y., Rabkin E., Libby P., et al. Host bone-marrow cells are a source of donor intimal smooth-muscle-like cells in murine aortic transplant arteriopathy. Nat Med (2001) 7:738–741.[CrossRef][ISI][Medline]
  25. Saga Y., Yagi T., Ikawa Y., Sakakura T., Aizawa S. Mice develop normally without tenascin. Genes Dev (1992) 6:1821–1831.[Abstract/Free Full Text]
  26. Maruyama J., Yokochi A., Maruyama K., Nosaka S. Acetylcholine-induced endothelium-derived contracting factor in hypoxic pulmonary hypertensive rats. J Appl Physiol (1999) 86:1687–1695.[Abstract/Free Full Text]
  27. Brown P.D., Levy A.T., Margulies I.M.K., Liotta L.A., Stetler-Stevenson W.G. Independent expression and cellular processing of Mr 72,000 type IV collagenase and interstitial collagenase in human tumorigenic cell lines. Cancer Res (1990) 50:6184–6191.[Abstract/Free Full Text]
  28. Cai M., Onoda K., Takao M., Imanaka-Yoshida K., Shimpo H., Yoshida T., et al. Degradation of tenascin-C and activity of matrix metalloproteinase-2 are associated with tumor recurrence in early stage non-small cell lung cancer. Clin Cancer Res (2002) 8:1152–1156.[Abstract/Free Full Text]
  29. Imanaka-Yoshida K., Hiroe M., Nishikawa T., Ishiyama S., Shimojo T., Ohta Y., et al. Tenascin-C modulates adhesion of cardiomyocytes to extracellular matrix during tissue remodeling after myocardial infarction. Lab Invest (2001) 81:1015–1024.[ISI][Medline]
  30. Janqueira L., Bignolas G., Brentani R. Picrosirius staining plus polarization microscopy: a specific method for collagen detection in tissue sections. J Histochem Cytochem (1979) 11:447–455.
  31. Whittaker P., Kloner R.A., Boughner D.R., Pickering J.G. Quantitative assessment of myocardial collagen with picrosirius red staining and circularly polarized light. Basic Res Cardiol (1994) 89:1167–1171.
  32. Imanaka-Yoshida K., Matsumoto K., Hara M., Sakakura T., Yoshida T. The dynamic expression of tenascin-C and tenascin-X during early heart development in the mouse. Differentiation (2003) 71:291–298.[CrossRef][ISI][Medline]
  33. Shi C., Russell M.E., Bianchi C., Newell J.B., Haber E. Murine model of accelerated transplant arteriosclerosis. Circ Res (1994) 75:199–207.[Abstract/Free Full Text]
  34. Koulack J., McAlister V.C., Giacomantonio C.A., Bitter-Suermann H., MacDonald A.S., Lee T.D. Development of a mouse aortic transplant model of chronic rejection. Microsurgery (1995) 16:110–113.[ISI][Medline]
  35. Dietrich H., Hu Y., Zou Y., Dirnhofer S., Kleindienst R., Wick G., et al. Mouse model of transplant arteriosclerosis: role of intercellular adhesion molecule-1. Arterioscler Thromb Vasc Biol (2000) 20:343–352.[Abstract/Free Full Text]
  36. Hu Y., Davison F., Ludewig B., Erdel M., Mayr M., Url M., et al. Smooth muscle cells in transplant atherosclerotic lesions are originated from recipients, but not bone marrow progenitor cells. Circulation (2002) 106:1834–1839.[Abstract/Free Full Text]
  37. Chiquet-Ehrismann R., Tannheimer M., Koch M., Brunner A., Spring J., Martin D., et al. Tenascin-C expression by fibroblasts is elevated in stressed collagen gels. J Cell Biol (1994) 127:2093–2101.[Abstract/Free Full Text]
  38. Wallner K., Sharifi B.G., Shah P.K., Noguchi S., DeLeon H., Wilcox J.N. Adventitial remodeling after angioplasty is associated with expression of tenascin mRNA by adventitial myofibroblasts. J Am Coll Cardiol (2001) 37:655–661.[Abstract/Free Full Text]
  39. Hanamura N., Yoshida T., Matsumoto E., Kawarada Y., Sakakura T. Expression of fibronectin and tenascin-C mRNA by myofibroblasts, vascular cells and epithelial cells in human colon adenomas and carcinomas. Int J Cancer (1997) 73(1):10–15.[CrossRef][ISI][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Sawada, Y.
Right arrow Articles by Shimpo, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sawada, Y.
Right arrow Articles by Shimpo, H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?