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Cardiovascular Research 2005 68(2):249-258; doi:10.1016/j.cardiores.2005.06.016
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Copyright © 2005, European Society of Cardiology

Xenogenic smooth muscle cell immunization reduces neointimal formation in balloon-injured rabbit carotid arteries

Masakazu Shinohara, Seinosuke Kawashima*, Tomoya Yamashita, Tomofumi Takaya, Ryuji Toh, Tatsuro Ishida, Tomomi Ueyama, Nobutaka Inoue, Ken-ichi Hirata and Mitsuhiro Yokoyama

Division of Cardiovascular and Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan

* Corresponding author. Tel.: +81 78 382 5846; fax: +81 78 382 5859. Email address: kawashim{at}med.kobe-u.ac.jp

Received 6 October 2004; revised 20 June 2005; accepted 22 June 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 
Objective: Intimal hyperplasia plays an important role in a variety of types of vascular remodeling, particularly luminal narrowing after vascular injury. The vascular smooth muscle cells (VSMCs) in the neointimal area are a synthetic phenotype and have different epitopes from VSMCs in the normal media. The synthetic VSMCs in the neointima contain various possible antigens that can be targeted by the immune system. In this study, we tried to develop a new immunotherapy, which targets the synthetic VSMCs, for prevention of neointimal formation after angioplasty.

Method and results: Rabbits were repeatedly immunized with fixed xenogenic rat cultured VSMCs suspended in adjuvant as immunogens or injected with adjuvant and phosphate-buffered saline (PBS) or rat hepatocytes as controls every 2 weeks for 3 times. One week after the last immunization/injection, balloon injury of the left common carotid artery was performed. Four weeks after the injury, rabbits were euthanized and the neointimal lesion formation was assessed. The mean neointimal area of the PBS-injected, non-immunized group and the rat hepatocyte-immunized, control group was not statistically different (0.339 ± 0.036 and 0.350 ± 0.041 mm2, P = NS). However, immunization with rat VSMCs significantly reduced the intimal lesion area (0.219 ± 0.0286 mm2; P<0.05 vs. PBS-injected, non-immunized group and rat hepatocyte-immunized group.) PCNA-immunopositive proliferating VSMCs in the neointima were suppressed by the rat VSMC immunization (1.34 ± 0.49% vs. 5.78 ± 0.47%; P<0.05 vs. PBS-injected, non-immunized group). Rat VSMC immunization induced antibodies which had strong cross-reactivity against rabbit synthetic VSMCs. In experiments in vitro, proliferation and migration of rabbit VSMCs that were stimulated by serum, angiotensin (AT) II, platelet-derived growth factor (PDGF)-BB, fibroblast growth factor (FGF), and the phorbol ester PMA were significantly suppressed by treatment with immunoglobulin extracted from the VSMC-immunized rabbit plasma, implying that the immunoglobulin had some global effects on VSMCs. The rat VSMC-immunized rabbit immunoglobulin bound the rabbit AT1a receptor protein, which was expressed in COS7 cells by transfection of rabbit AT1a receptor pcDNA3. This binding to AT1a receptor may be one of mechanisms of the effects of VSMC-immunized immunoglobulin.

Conclusion: Xenogenic, synthetic rat VSMC immunization in rabbits induced auto-antibodies against synthetic rabbit VSMCs in a cross-reaction. The induced auto-antibodies against synthetic VSMCs may provide a possibility of new immunotherapy for vascular remodeling that forms neointimal lesions.

KEYWORDS Neointimal formation; Vascular remodeling; Vascular smooth muscle; Antibody; Immunotherpy

This article is referred to in the Editorial by Thaunat et al. (pages 183–185) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 
Intimal hyperplasia plays an important role in a variety of types of vascular remodeling, particularly luminal narrowing after vascular injury such as that seen in restenosis following percutaneous coronary intervention. Proliferation and migration of vascular smooth muscle cells (VSMCs) from the media play a central role in neointimal formation, and various strategies have been developed to inhibit it, including inhibition of cell cycle of VSMCs, induction of apoptosis of VSMCs, and inhibition of intracellular signal transduction in VSMCs [1–4].

Recently immunological modulation has attracted attention as a possible therapeutic strategy of atherosclerosis [5–22]. Although only limited information is available, several studies have shown the possibility of immunotherapy against neointimal lesion formation after balloon injury. It seems that adequate induction of B-lymphocyte-related immunity has potential beneficial effects on neointimal formation. Nilsson et al. showed that immunization with homologous oxidized low density lipoprotein reduced neointimal formation after balloon injury in hypercholesterolemic rabbits [23]. In balloon-injured rat carotid arteries, it was demonstrated that treatment with an anti-P selectin monoclonal antibody reduced inflammation, in neointimal formation, and vascular remodeling [24]. However, until now, only few epitopes have been reported as appropriate targets of immunotherapy against neointimal formation.

It is possible that synthetic VSMCs in the neointima contain various possible antigens that can be targeted by the immune system. Since VSMCs play a central role in neointimal formation, we hypothesized that immune modulation targeting the synthetic VSMCs-associated antigens may inhibit neointimal formation after balloon injury. To prove this hypothesis, rabbits were immunized with the synthetic xenogenic rat VSMCs and then balloon-injured to induce neointimal formation.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgment
 References
 
2.1 Materials and animals
All drugs and culture media used in this study were purchased from Sigma Chemical Co. (MO) and WAKO (Japan). Male Japanese White Rabbits were purchased from a breeder (SLC, Hamamatsu, Japan) and kept under conventional conditions in our animal facility. Rabbits were fed normal chow (Oriental Yeast, Japan) and water ad libitum and maintained on a 12 h light/dark cycle. All animal experiments were conducted according to the "Guidelines for Animal Experiments at Kobe University Graduate School of Medicine", which complies NIH Guidelines.

2.2 Preparation for rat vascular smooth muscle cells and rat hepatocytes as immunogens
Rat VSMCs were prepared from thoracic aortas of male Sprague–Dawley rats by the collagenase digestion method and cultured as described [25]. For all experiments, rat aortic VSMCs from passage 5 to 8 were used. Rat hepatocytes (RLC-18, Cell No. RCB1484) were purchased from Rikken Cell Bank (Japan).

Rat VSMCs and rat hepatocytes were cultured in DMEM with 10% fetal bovine serum (FBS), and approximately 1 x 108 cells were collected and washed three times with phosphate buffered saline (PBS). The cells were fixed with 10% neutralized formaldehyde (WAKO, Japan) for 24 h at 4 °C. After being washed three times and incubated at 37 °C for 2 h to remove residual formaldehyde, the cells were washed again and re-suspended in PBS for use as immunogens.

2.3 Experimental protocol
Male Japanese White Rabbits (body weights were 2.0 kg) were divided into three groups. Injection of either rat VSMCs as immunogens (1 x 108 cells per rabbit), vehicle PBS for control non-immunized group or rat hepatocytes (1 x 108 cells per rabbit) for control immunized group was started. Those injections were subcutaneously performed in the rabbits' back with an equal volume of adjuvant every two weeks for three times. We used the Freund complete adjuvant for the first immunization, and the Freund incomplete adjuvant for the second and the third immunization.

One week after the last injection, rabbits were anesthetized with sodium pentothal. Blood sample was collected from the ear vein. Plasma immunoglobulin (IgG) levels were assessed using a commercially available kit (Bethyl Laboratories Inc. TX). A 2F Fogarty embolectomy catheter (Baxter, USA) was introduced through an aseptic neck incision produced in the facial branch of the external left carotid artery and positioned approximately at the origin of the common carotid artery. An acute balloon injury was performed by inflating the balloon with 0.1 mL saline solution and then gently pulling it back along the entire length of the common carotid artery with constant rotation as described before [26]. The catheter was then removed, the artery branch was ligated, and the surgical wound was closed. Two and four weeks after balloon injury, rabbits were euthanized, and the neointimal lesion formation was assessed.

2.4 Histological and immunohistochemical analysis of neointimal lesions
Serial equally spaced cross sections (5 µm thick) were obtained throughout the entire length of the carotid artery for histological analysis (average of six sections per animal). All samples were routinely stained with hematoxyline and eosin or subjected to immunostaining with the anti-proliferating cell nuclear antigen (PCNA). For immunohistochemistry, slides were preincubated with 1% bovine serum to decrease nonspecific binding. Sections were incubated overnight at 4 °C with the mouse anti-PCNA antibody (DAKO, Denmark).

2.5 Quantification of neointimal lesions in sections of carotid arteries
Six equally spaced cross sections of the entire length of carotid arteries were used in all rabbits to quantify neointimal lesions. Using NIH imaging software, total cross-sectional neointimal area was measured between the endothelial cell monolayer and the internal elastic lamina. Total cross-sectional medial area was measured between the external and internal elastic lamina.

2.6 Quantification of proliferating neointimal cells
Serial cross sections (5 µm thickness) of carotid arteries were made. One section was used for HE-staining to calculate the total cell number in the neointimal area, and the next section was used for PCNA immunohistochemistry. The total PCNA-immunopositive neointimal cells and total neointimal cells in HE-staining in each serial section were counted. Then, the percentage of PCNA-immunopositive cells per total number of neointimal cells in each section was calculated, and the average of the six sections per animal was obtained for each animal.

2.7 Detection of apoptosis in the neointimal cells
Apoptosis in the neointimal cells was detected by TUNEL technique. Modified TUNEL assay was performed by use of DeadEnd colorimetric apoptosis detection system (Promega) according to the manufacturer's instructions. Briefly, tissue sections from the neointima of PBS injected non-immunized group and rat VSMC-immunized group were washed in PBS, and endogenous peroxidase was blocked by 0.3% hydrogen peroxide. Biotinylated nucleotide was incorporated at the 3'-OH DNA ends using terminal deoxynucleotidyl transferase (TdT). Streptavidin-HRP was then bound to these biotinylated nucleotides, which were detected using hydrogen peroxide and diaminobenzidine (DAB). Some of slides were treated with Dnase I for positive controls. Stained cells were counted under a light microscope.

2.8 Immunoglobulin purification from rabbit plasma
The immunoglobulin from the pooled plasma derived from the rabbits on day 7 after the third immunization or from control rabbits was purified with the use of a Protein G column (Sigma, MO).

2.9 Immunoblotting using rabbit plasma and protein analysis
Rabbit VSMCs were primarily cultured by the explant method and cultured in DMEM with 10% fetal bovine serum (FBS). Rabbit VSMCs were homogenized in a homogenizing buffer containing 50 mM Tris–HCl (pH 7.4), 1 mM EDTA, 1 mM phenylmethyosulfonylfluoride, 10% glycerol, and 20 mM CHAPS. The whole protein (50 µg per lane) was resolved by SDS-PAGE (10% polyacrylamide), transferred to a PVDF membrane and probed with rabbit plasma obtained from PBS injected non-immunized control rabbits, rat hepatocyte-immunized control rabbits, or rat VSMC-immunized rabbits. Immunoreactive bands were visualized with horseradish peroxidase-conjugated anti-rabbit IgG (Amersham, England; 1:3000 dilution) and an ECL detection kit (Amersham, England).

2.10 Counting of VSMC number in vitro
Cell proliferation was quantified by total cell number as previously described [27]. Rabbit VSMCs (5000 per well) were seeded in 96-well microtiter plates in 0.1 mL of DMEM–10% FBS. After 12 h incubation, the medium was replaced by DMEM with purified immunoglobulin from rat hepatocyte-immunized group and that from rat VSMC-immunized group (20 µg/mL, respectively). At 48 h after stimulation with agonists (10% FBS, 1 µM ATII, 10 ng/ml PDGF-BB, 10 ng/ml FGF, 1 µM PMA), cells were fixed by addition of 10 µL of glutaraldehyde and shaken for 15 min. After being washed 3 times with deionized water, plates were air-dried and stained for 20 min with 0.1% crystal violet solution in 200 mM MES, pH 6.0. After being washed 3 times with deionized water to remove excess dye, plates were air-dried before solubilization of the bound dye in 10% acetic acid. The optical density of dye extracts was measured at 595 nm by using a microplate reader (Bio-Rad). Values are means ± SEM of 8 separate experiments in each group.

2.11 VSMC migration
Rabbit VSMCs were grown to confluence in 6-well culture plates. The monolayer-wounding cell migration assay was performed as described previously [28,29]. Cell layers were scraped with a sterile single edged razor blade and re-incubated in DMEM containing 5 mM hydroxyurea with purified immunoglobulin from rat hepatocyte-immunized rabbits or rat VSMC-immunized rabbits (20 µg/mL, respectively). Hydroxyurea was added to eliminate any confounding effects of cell proliferation. At 24 h after stimulation with agonists (10% FBS, 1 µM ATII, 10 ng/ml PDGF-BB, 10 ng/ml FGF, 1 µM PMA), cells were fixed and the maximum migration distance across the wound edge was analyzed.

2.12 VSMC viability assay
Rabbit VSMCs (5000 per well) were incubated in 96-well plastic plates with purified immunoglobulin from rat VSMC-immunized rabbits or rat hepatocyte-immunized rabbits (20 µg/mL, respectively). After 24 h, cell viability was assessed by measuring mitochondrial NADH-dependent dehydrogenase activity with a Cell Counting Kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) using sulfonated tetrazolium salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) [30]. Each measurement was done in triplicate and the results were presented as a percentage of the value for rat hepatocyte-immunized and control-immunized groups.

2.13 Identification of a target protein which rat VSMC-immunized immunoglobulin recognizes
Rabbit AT1a receptor cDNA was a generous gift from Dr. Raymond C. Harris (Vanderbilt Medical Center, Vanderbilt University). The complete coding site was excited and cloned into the expression vector pcDNA3 (Invitrogen), and pcDNA3 without any expression insert was used as the control vector. COS7 cells were purchased from the American Type Culture Collection (Manassas, VA) and cultured according to the manufacture's recommendations. COS7 cells were transfected by control pcDNA3 and rabbit AT1a receptor pcDNA3, respectively. The whole protein of these two groups of COS7 cells were resolved by SDS-PAGE, transferred to a PVDF membrane and probed with rabbit plasma obtained from PBS injected non-immunized control rabbits or rat hepatocyte-immunized control rabbits or rat VSMC-immunized rabbits respectively. Immunoreactive bands were visualized with horseradish peroxidase-conjugated anti-rabbit IgG (Amersham, England; 1:3000 dilution) and an ECL detection kit (Amersham, England).

2.14 Statistical analysis
Values represent means ± SEM. Differences between groups were compared using a one-way analysis of variance test followed by Fisher protected least significant difference. P<0.05 was accepted as statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgment
 References
 
3.1 Blood analysis and general appearance of rabbits
Rat VSMC-immunization induced no significant changes in liver function, renal function and lipid profiles. Mean body weight and behavior of rabbits did not change by the immunization. In comparison with the PBS injected non-immunized control group, rat VSMC-immunization did not affect plasma IgG levels (284 ± 61 mg/dl in PBS injected group vs. 268 ± 44 mg/dl in rat VSMC-immunized group; P = NS).

3.2 Effect of rat VSMC immunization on the neointimal lesion formation
All animals developed concentric intimal lesions in response to the balloon injury. At 4 weeks after balloon injury, the mean neointimal area of PBS injected non-immunized group and rat hepatocyte-immunized control group were 0.339 ± 0.036 and 0.350 ± 0.041 mm2, respectively (P = NS). The intimal lesion area was not significantly reduced by the rat hepatocytes control immunization. However, immunization with rat VSMCs significantly reduced the intimal lesion area (0.219 ± 0.0286 mm2; P<0.05 vs. PBS injected non-immunized group and rat hepatocyte-immunized control group, Fig. 1A and C). The intimal/medial ratio was also significantly reduced in rat VSMC-immunized group (0.349 ± 0.049) compared with PBS injected non-immunized group (0.662 ± 0.114; P<0.01). There was no sign of infiltration of inflammatory cells at the contralateral common carotid artery (Fig. 1B). Immunization with rat VSMCs did not induce any pro-inflammatory effects on normal contractile VSMCs.


Figure 1
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  		Fig. 1 (A) Representative light photomicrograph images showing injured carotid arteries from PBS-injected non-immunized control group, rat hepatocyte-immunized control group, and rat VSMC-immunized group at 4 weeks after balloon injury. Arrowhead indicates internal elastic lamina (IEL). Original magnifications are x 40 (upper) and x 200 (lower panels). Bars indicate 100 µm. (B) Representative light photomicrograph images showing the untouched contralateral common carotid artery from rat VSMC-immunized group. Arrowhead indicates internal elastic lamina (IEL). Original magnifications are x 40 (upper) and x 200 (lower panels). Bars indicate 100 µm. (C) Quantitative analysis of neointimal area and I/M ratio in rabbit carotid arteries at 4 weeks after injury in rat VSMC-immunized group (white bars), PBS-injected non-immunized control group (black bars), and rat hepatocyte-immunized control group (striped bars). Values are means ± SEM in each group. *P<0.05 vs. PBS-injected non-immunized control group and rat hepatocyte-immunized control group. **P<0.05 vs. PBS-injected non-immunized control group.

 
3.3 Rat VSMC immunization induced rabbit VSMC-reactive antibodies
To detect the antibodies, which were induced by xenogenic rat VSMC immunization, rabbit VSMCs proteins were resolved by SDS-PAGE, and immunoblotted with plasma obtained from the PBS injected non-immunized control rabbits, rat hepatocyte-immunized control rabbits, or the VSMC-immunized rabbits. Only a few non-specific bands were detected when the plasma from the PBS injected non-immunized control rabbits was applied. Some bands were detected when the plasma from rat hepatocyte-immunized control rabbits was applied. In contrast, several new bands were clearly recognized in the immunoblot when we applied the plasma from rat VSMC-immunized rabbits (Fig. 2). It implies that the rat VSMC-immunized immunoglobulin recognized some new proteins of rabbit VSMCs that reacted with neither immunoglobulin from PBS injected non-immunized rabbits nor that from rat hepatocyte-immunized rabbits.


Figure 2
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  		Fig. 2 Immunoblottings of proteins extracted from rabbit VSMCs using the plasma obtained from PBS injected non-immunized control rabbits (left), rat hepatocyte-immunized control rabbits (middle), or rat VSMC-immunized rabbits (right) as primary antibodies, respectively.

 
3.4 Effect of rat VSMC immunization on the proliferation and apoptosis of neointimal cells
To investigate whether the reduced neointimal lesion formation was due to the reduced proliferation of VSMCs in the neointimal lesion, the proliferation of neointimal VSMCs was investigated by the PCNA immunostaining on the day 14 after the injury (Fig. 3A). The percentage of PCNA-immunopositive cells was significantly reduced in VSMC-immunized group compared with in PBS injected control group (1.34% ± 0.49 vs. 5.78% ± 0.47, P<0.01, Fig. 3B).


Figure 3
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  		Fig. 3 (A) Photomicrographs of HE stainings (left panels) and PCNA immunostainings (right panels) in the neointima of injured carotid arteries at 2 weeks after balloon injury. Original magnification is x 200. A bar indicates 100 µm. (B) Quantitative analysis of PCNA positive proliferating cells in the neointima at 2 weeks after injury. Values are means ± SEM of at least six rabbits in each group. {dagger}P<0.01 vs. PBS injected non-immunized control group.

 
Then to clarify the role of apoptosis in the reduction of neointimal formation, the TUNEL assay was carried out. As shown in Fig. 4, rat VSMC-immunization did not increase the number of TUNEL positive neointimal cells.


Figure 4
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  		Fig. 4 Representative light photomicrograph images showing apoptosis in the neointimal cells using TUNEL technique.

 
3.5 Effect of VSMC immunized immunoglobulins on rabbit VSMC proliferation and migration in vitro
The immunoglobulin (20 µg/ml) obtained from VSMC-immunized rabbits significantly suppressed rabbit VSMC cell numbers stimulated by FBS, ATII, PDGF, FGF, and PMA (Fig. 5) and rabbit VSMC migration stimulated by FBS, ATII, PDGF, and PMA (Fig. 6), compared with that from rat hepatocyte-immunized control rabbits.


Figure 5
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  		Fig. 5 Quantitative analysis of proliferation of cultured rabbit VSMCs treated with immunoglobulin obtained from rat hepatocyte-immunized control rabbits (striped bars) or rat VSMC-immunized rabbits (white bars). Values are means ± SEM of separate six experiments in each group. {dagger}P<0.01 vs. the rat hepatocyte-immunized control group. *P<0.05 vs. PBS-injected non-immunized control group.

 

Figure 6
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  		Fig. 6 Quantitative analysis of the migration distance evaluated by the maximally migrated point from the wounded edge in rat hepatocyte-immunized control group (striped bars) and in rat VSMC-immunized group (white bars), respectively. Values are means ± SEM of five separate experiments in each group. {dagger}P<0.01 vs. the rat hepatocyte-immunized control group. *P<0.05 vs. PBS-injected non-immunized control group.

 
3.6 Effect of immunized immunoglobulin on VSMC viability
The rat VSMC-immunized immunoglobulin used in the proliferation and the migration assays did not alter rabbit VSMC viability assessed by WST-1 assay (rat hepartocyte-immunized control group: 100% vs. rat VSMC-immunized group: 106 ± 8.6%, P = NS).

3.7 AT1a receptor may be one of a target protein which rat VSMC-immunized immunoglobulin recognizes
As shown in Fig. 7, only rat VSMC-immunized rabbit plasma recognized the rabbit AT1a receptor protein which was expressed in COS7 cells by transfection of rabbit AT1a receptor pcDNA3 (molecular weight is about 50 kDa). No bands were detected by immunoglobulin from PBS-injected rabbits or that from hepatocyte-immunized rabbits.


Figure 7
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  		Fig. 7 The rabbit AT1a receptor protein expressed in COS7 cells by transfection of the rabbit AT1a receptor pcDNA3 was immunoblotted with PBS injected non-immunized control rabbit plasma (left panel), rat hapatocyte-immunized rabbit plasma (middle panel), or rat VSMC-immunized rabbit plasma (right panel), respectively. pcDNA3 without any expression inserts was used as the control transfection.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgment
 References
 
In the present study, we demonstrated for the first time that, immunization with xenogenic rat synthetic VSMCs reduced neointimal formation after balloon injury, in association with induction of auto-antibodies against rabbit VSMCs. The VSMCs which contribute to form neointimal lesions are different in phenotype from ones in the normal media [31]. VSMCs in the normal media consisted of a contractile phenotype, whereas VSMCs which contribute to form neointimal lesions show a synthetic phenotype [32]. And the epitopes of VSMCs also may be different between the contractile phenotype and the synthetic phenotype. We used cultured rat VSMCs as the xenogenic immunogens against rabbits. These rat VSMCs are the synthetic phenotype, and therefore immunization with rat VSMCs was likely to induce various antibodies against the synthetic VSMCs. And importantly, it seemed that certain antibodies cross-reacted between rat and rabbit VSMCs.

As demonstrated in Fig. 2, when we applied rat VSMC-immunized plasma as the primary antibody, several new proteins of rabbit VSMCs were detected by the Western blot analysis, which were not detected when PBS injected non-immunized control plasma or rat hepatocyte-immunized control plasma was applied It implies that the rabbit plasma obtained from rat VSMC-immunized rabbits had an antibody response distinct from the one induced by PBS or rat hepatocytes. Importantly, as demonstrated in Fig. 1, only rat VSMC-immunization reduced the neointimal lesion formation after balloon injury. These results might implicate that the induced antibodies reacting with the synthetic VSMCs but not others might play critical roles in the reduction of neointimal area in this study.

Apoptosis of VSMCs is implicated in the formation of neointimal lesions [33]. In the aortic allograft model in rats, Plissonnier et al. reported the induction of apoptosis by alloantisera [34]. Therefore, we investigated apoptosis of VSMCs in both the neointimal lesion and the normal media by TUNEL staining, but we did not find significant differences between rat VSMC-immunized group and PBS injected non-immunized group. On the other hand, the proliferative capability of VSMCs estimated by PCNA staining in neointimal lesions was significantly suppressed by rat VSMC-immunization (Fig. 3A and B), whereas no differences were detected in the media, at 2 weeks after balloon injury. Thus, proliferation of VSMCs in the neointima was suppressed by rat VSMC-immunization. This may be related to that neointimal lesions themselves may be more antigenic than the normal media. Plissonnier et al. reported that normal media was a "privileged" immunological site as compared to neointimal lesions in the vascular chronic rejection process in allotransplantation/immunization [35].

To further clarify the effects of the immunization on VSMC function, we performed in vitro assay. For in vitro assay, we purified immunoglobulin from plasma using a protein G column, because we needed to avoid the effects of many cytokines and growth factors which were contained in the immunized plasma. Therefore, in the study in vitro, we could investigate the direct effect of induced immunoglobulins. At first we compared viability of rabbit VSMCs between rat hepatocyte-immunized immunoglobulin treated control group and rat VSMC-immunized immunoglobulin treated group by use of WST-1 assay, and confirmed that viability of VSMC did not differ between two groups. Therefore VSMC-immunized immunoglobulin didn't have severe cell toxicity against VSMCs. The VSMC-immunized rabbits immunoglobulin significantly suppressed rabbit VSMC cell numbers stimulated by FBS, ATII, PDGF, FGF, and PMA (Fig. 5) and rabbit VSMC migration stimulated by FBS, ATII, PDGF, and PMA (Fig. 6). We applied immunoglobulins to the culture media, which bind only to the cell surface proteins. Therefore we speculate that rat VSMC-immunized immunoglobulin might bind to some surface proteins which globally regulate the proliferation and migration of rabbit VSMCs. As one of candidates for such surface proteins, we focused on AT1a receptor, since the inhibitory effects of VSMC-immunized rabbits immunoglobulin were prominent in ATII-induced proliferation and migration. As shown in Fig. 7, rat VSMC-immunized immunoglobulin was immunoreactive against the AT1a receptor protein which was expressed in COS7 cells by the rabbit AT1a receptor pcDNA3 transfection, whereas no bands were detected by immunoglobulin from PBS-injected rabbits or that from hepatocyte-immunized rabbits. Therefore, it is possible that rabbit AT1a receptor protein is one of target proteins which rat VSMC-immunized immunoglobulin recognizes and that binding of rat VSMC-immunized immunoglobulin to rabbit AT1a receptors might have inhibited the ATII signaling. Not only ATII signaling but almost all stimuli tested, however, were affected by the adjunction of rat VSMC-immunized immunoglobulin. Therefore it is likely that a much more global effect than the blocking effect on these receptors was involved. At the present time, we cannot show more detailed molecular mechanisms of the inhibitory effects of xenogenic VSMC immunization, and further investigation is needed.

This study demonstrated for the first time that xenogenic VSMC immunization significantly reduced neointimal formation in the balloon-injured carotid arteries. Our data indicate the possibility of immunotherapy for neointimal formation with xenogenic VSMCs by breaking immune tolerance against autologous VSMCs in a cross-reaction between the xenogenic homologs and self molecules, similar to the case of immunotherapy for tumor angiogenesis [36].

In the present study, rat VSMC-immunization didn't cause any serious side effects. The body weight and behavior of rabbits did not change by the immunization, and also blood analysis didn't show any significant changes in liver functions, renal functions, and lipid profiles. However, at the present time we cannot show the target protein(s) of VSMC-immunized immunoglobulin that regulates the functions of rabbit VSMC globally. Although further investigation on the target protein(s) and the possibility of immunological regulation is needed, immunological regulation shown in our study may be a new immunotherapy for vascular remodeling which forms neointimal lesion.


   Acknowledgment
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgment
References
 
We appreciate Kiyoko Matsui for her secretarial assistance and technical support (cell culture, blood cell count, and animal care).


   Notes
 
Time for primary review 20 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 

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