This Article Abstract Full Text (PDF) All Versions of this Article: 93/8/783    most recent 01.RES.0000096651.13001.B4v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tanaka, K. Articles by Nagai, R. Search for Related Content PubMed PubMed Citation Articles by Tanaka, K. Articles by Nagai, R. Pubmed/NCBI databases Substance via MeSH Related Collections Restenosis Apoptosis Pathophysiology

(Circulation Research. 2003;93:783.)
© 2003 American Heart Association, Inc.

Integrative Physiology

Diverse Contribution of Bone Marrow Cells to Neointimal Hyperplasia After Mechanical Vascular Injuries

Kimie Tanaka, Masataka Sata, Yasunobu Hirata, Ryozo Nagai

From the Department of Cardiovascular Medicine (K.T., M.S., Y.H., R.N.), University of Tokyo Graduate School of Medicine, Tokyo, Japan; PRESTO (M.S.), Japan Science and Technology Agency, Kawaguchi, Saitama, Japan.

Correspondence to Dr Masataka Sata, Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail sata-2im{at}h.u-tokyo.ac.jp

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We and others have suggested that bone marrow-derived progenitor cells may contribute to the pathogenesis of vascular diseases. On the other hand, it was reported that bone marrow cells do not participate substantially in vascular remodeling in other experimental systems. In this study, three distinct types of mechanical vascular injuries were induced in the same mouse whose bone marrow had been reconstituted with that of GFP or LacZ mice. All injuries are known to cause smooth muscle cell (SMC) hyperplasia. At 4 weeks after wire-mediated endovascular injury, a significant number of the neointimal and medial cells derived from bone marrow. In contrast, marker-positive cells were seldom detected in the lesion induced by perivascular cuff replacement. There were only a few bone marrow-derived cells in the neointima after ligation of the common carotid artery. These results indicate that the origin of intimal cells is diverse and that contribution of bone marrow-derived cells to neointimal hyperplasia depends on the type of model.

Key Words: bone marrow • smooth muscle cells • atherosclerosis • restenosis • inflammation

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
There is accumulating evidence that adult tissues may contain multipotent stem cells that can differentiate into various lineages, beyond their differentiative and regenerative potential for the tissues in which they reside. Many studies documented that somatic stem cells contributed to regeneration and remodeling of remote organs.³ On the other hand, recent reports have cast doubt on the pluripotential of adult stem cells in vivo under physiological conditions.⁴ Similarly, it was reported that bone marrow does not contribute substantially to vascular remodeling in some experimental systems,^5,6 whereas we and others have suggested that bone marrow cells can participate in the pathogenesis of vascular diseases in models of graft vasculopathy, postangioplasty restenosis, and hyperlipidemia-induced atherosclerosis.^7–11 We are concerned that such opposite conclusions have been drawn using different experimental systems to test hypotheses.

The present study was undertaken to determine whether bone marrow contribution to neointimal hyperplasia depends on the type of model. After three distinct types of mechanical injuries were induced in a single mouse, the proportion of bone marrow-derived cells in the vascular lesions was compared. Results demonstrate that the origin of neointimal cells is diverse and that the mode of injury is important for the recruitment of bone marrow cells to vascular remodeling.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals
ROSA26 mice, which are knock-in mice expressing the LacZ gene in essentially all tissues (C57BL/6x129S background),¹² were originally purchased from Jackson Laboratory (B6; 129S-Gtrosa26, Stock Number 002073, Bar Harbor, Maine). ROSA26 mice were maintained in our animal facility and intercrossed with C57BL/6J mice. The resulting littermates were used for this study.⁷ All wild-type mice were purchased from SLC (Shizuoka, Japan). Transgenic mice (C57BL/6 background) that ubiquitously express enhanced green fluorescent protein (GFP mouse) were a generous gift from Dr Masaru Okabe (Osaka University, Osaka, Japan).¹³ ApoE-deficient mice (ApoE^-/- mice) were purchased from Jackson Laboratory.¹⁴ All procedures involving experimental animals were performed in accordance with protocols approved by the institutional committee for animal research of The University of Tokyo and complied with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 86-23, revised 1985).

Bone Marrow Reconstitution
Bone marrow transplantation (BMT) was performed as described previously.^7,15 Bone marrow cells were harvested from femora and tibias of donor mice as already described.¹⁵ Eight- to 10-week-old male wild-type mice or ApoE-deficient mice were lethally X-irradiated with a total dose of 9.5 or 8.8 Gy (MBR-1520RB, Hitachi), respectively. One day later, the recipient mice received unfractionated bone marrow cells (3x10⁶) suspended in 0.3 mL PBS by tail vein injection. Twenty-seven to 30 weeks after BMT, three distinct types of mechanical injuries were induced in the recipient mice. Peripheral leukocytes (80% to 90%) had been reconstituted as determined by flow cytometry (BMT^GFPApoE mice or BMT^GFPWild mice) or FISH (fluorescence in situ hybridization) for Y-chromosome (BMT^MaleFemale mice).

Wire-Mediated Endovascular Injury
Transluminal arterial injury was induced by inserting a straight spring wire (0.38 mm in diameter, No. C-SF-15-15, COOK) into the left femoral artery as already described.¹⁶ The wire was left in place for 1 minute to denude and dilate the artery. A copy of the tutorial video of the surgical procedure can be sent on request; alternatively, the video can be viewed at http://plaza.umin.ac.jp/msata./

Cuff-Mediated Perivascular Injury
A cuff-mediated vascular injury was induced by placing a polyethylene tube around the right femoral artery.¹⁷ After isolating the right femoral artery from the surrounding tissues, a tube (2-mm PE-50; Becton-Dickinson) was opened longitudinally, loosely placed around the artery and then closed with sutures.

Flow-Restriction Vascular Injury
A flow-restriction vascular injury was caused by ligating the bifurcation of the left common carotid artery.^18,19 The distal left common carotid artery and its bifurcation into the external and internal carotid arteries were exposed using minimal dissection. The common carotid artery was completely ligated just proximal to the bifurcation with a 6-0 silk suture. All animals recovered and showed no symptom of a stroke.

Plastic Embedding to Detect GFP Signal
Four weeks after surgery, mice were euthanized with overdose of pentobarbital and perfused at a constant pressure via the left ventricle with 0.9% sodium chloride solution, followed by perfusion fixation with 4% paraformaldehyde in PBS. The injured arteries were further fixed in 4% paraformaldehyde overnight at 4°C. The arteries were illuminated with a GFP-lighting system (Illumatool Tunable Lighting System, LT-9800, Lightools Research) and observed using a cooled CCD camera (VB-6010, Keyence). To preserve GFP signal for histological analyses, the arteries were embedded in plastic resin (Technovit 8100, Heraeus Kulzer) according to the manufacturer’s instructions. Briefly, the arteries were washed overnight in PBS containing 6.8% sucrose at 4°C, dehydrated in 100% acetone, and embedded using a polyethylene capsule (Capsule 414 to 2, Energy Beam Sciences, Inc) as a mold. The polymerized block was fixed onto a block (Histobloc, Heraeus Kulzer) with an adhesive agent (EP001, Semedain) and cut using a rotary microtome (HM335E, MICROM International GmbH) with a disposable knife (Histoknife, Heraeus Kulzer). Thin sections (3 to 4 µm) were stretched in a water bath, mounted on silanized slides (Matsunami), and dried for 2 hours at 37°C. The sections were washed in PBS and used for immunofluorescence studies.

Detection of LacZ
LacZ was detected as described.⁷ After perfused with 0.9% sodium chloride solution, the arteries were excised and stained with X-gal solution containing 1 mg/mL X-gal, 2 mmol/L MgCl₂, 5 mmol/L K₃Fe(CN)₆, 5 mmol/L K₄Fe(CN)_6, 0.01% sodium deoxycholate, and 0.02% NP40 in PBS at 37°C overnight, further fixed in 4% paraformaldehyde overnight at 4°C, and embedded in paraffin. Thin sections (5 µm) were deparaffinized and used for histological analyses. To detect LacZ by immunofluorescence staining, the injured arteries were snap-frozen in OCT compound (TissueTek, Tokyo). Frozen sections (4 µm) were stained with anti-LacZ polyclonal (ICN) or monoclonal (clone GAL-13, Sigma) antibodies.

Double-Immunofluorescence Study
Immunofluorescence double staining was performed as described elsewhere.⁷ After blocking in 0.5% horse serum, frozen sections or plastic-embedded sections were incubated with first antibodies (Cy3-conjugated anti--smooth muscle actin (-SMA), Sigma; anti-macrophage, clone F4/80, Serotec; anti-CD31, clone MEC13.3, BD Biosciences, San Jose, Calif; anti-panendothelial cell antigen, clone MECA-32, BD Biosciences; anti-CD45, clone 30-F11, BD Biosciences; anti-von Willebrand factor (VWF), clone F8/86, DAKO; anti-smooth muscle myosin heavy chain, clone HSM-V, Sigma; anti-smooth and skeletal myosin polyclonal antibody, Sigma) followed by incubation with FITC, Rhodamine, or Cy3-conjugated secondary antibodies (Jackson ImmunoResearch). When mouse monoclonal antibody was used, the M.O.M immunodetection kit (Vector) was used. Endothelial cells were identified using the biotinylated Bandeiraea simplicifolia Lectin 1 (BS-Lectin 1, Vector Laboratory),²⁰ followed by incubation with Rhodamine-conjugated streptavidin (Immunotech, Cedex 9). Nuclei were counterstained with Hoechst 33258 (Sigma). The sections were mounted with the ProLong Antifade Kit (Molecular Probes) and observed under a confocal microscope (FLUOVIEW FV300, Olympus).

TUNEL Staining
To detect apoptotic cell death, TUNEL staining was performed as descrebed.^7,16 The cross sections (5 µm) were deparaffinized and permeabilized with 20 µg/mL proteinase K for 15 minutes. Terminal deoxynucleotidyl transferase enzyme and fluorescein-conjugated dUTP were added to the tissue sections according to the manufacturer’s specifications (Roche Molecular Biochemicals, in situ death detection kit). Nuclei were counterstained with propidium iodide.

Immunohistochemistry
Paraffin-embedded sections (5 µm thick) were deparaffinized and blocked with 0.5% horse serum. Endogenous biotin and biotin-binding proteins were blocked with a blocking kit (Vector Laboratories). The sections were incubated with anti-MCP-1 (R&D System), anti-SDF-1 (clone 79014, R&D System), anti-VEGF (sc-507, Santa Cruz) antibodies, or an alkaline phosphatase-conjugated antibody to -SMA (clone 1A4, Sigma), followed by the avidin-biotin complex technique and Vector Red substrate (Vector Laboratories). Sections were counterstained with hematoxylin.

Transmission Electron Microscopy
The femoral arteries were excised, stained with X-gal solution overnight, and fixed in 2% glutaraldehyde and 2.5% paraformaldehyde. Samples were postfixed in 1% osmium tetroxide, dehydrated, and embedded in epoxy resin (Epon 812). Thin sections were stained with 3% uranyl acetate and examined with an electron microscope (H-7000, Hitachi, Tokyo). No lead citrate was used for counterstaining.²¹

Statistics
All data are expressed as mean±SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Diverse Contribution of Bone Marrow Cells to Neointimal Hyperplasia
BMT was performed from GFP mice to wild-type (BMT^GFPWild mice) or ApoE-deficient mice (BMT^GFPApoE mice). After 12 to 32 weeks, three models of vascular injuries were induced in a single BMT mouse. At 4 weeks after surgery, GFP-positive (GFP⁺) cells accumulated in the injured arteries of BMT^GFPWild mouse under a fluorescence illuminator (Figure 1A). No GFP⁺ cell was observed in uninjured artery of BMT^GFPApoE mice (Figure 1B). In the left femoral artery where the wire had been inserted, GFP⁺ cells were detected in the neointima (38.9±5.8%) and media (61.4±5.8%) (n=4, Table 1). A significant percentage of -smooth muscle actin-positive (-SMA⁺) cells expressed GFP in neointima (26.6±6.4%) and media (35.4±9.6%) (Figure 1C, Table 1). Cuff-replacement around the right femoral artery induced neointimal hyperplasia that was exclusively composed of SMCs. Notably, most of the medial cells were negative for -SMA. A smaller number of GFP⁺ was found in the neointima (7.0±2.1%) and media (15.1±2.2%). Few GFP⁺ cells expressed -SMA. In the common carotid artery, neointimal hyperplasia was caused by proliferation of -SMA-positive cells. GFP⁺ cells were found in the neointima (24.1±5.3%) and media (33.1±8.2%). A few GFP⁺ cells expressed -SMA. Consistent with the results obtained in BMT^GFPApoE mice, there were no LacZ⁺ cells in uninjured artery of BMT^LacZWild mice. A significant percentage of LacZ⁺ cells were found in the neointima (56.3±7.8%) and media (54.3±8.0%) after the wire injury (Figure 1C) (n=5). We could readily detect LacZ⁺ cells that expressed -SMA. In the right femoral artery, around which a polyethylene cuff had been placed, bone marrow-derived cells were seldom detected in the neointima, whereas many inflammatory cells in the adventitia expressed LacZ. There were only a few LacZ⁺ cells in the neointima of the common carotid artery after ligation.

View larger version (67K): [in this window] [in a new window]   Figure 1. Marked diversity in the contribution of bone marrow-derived cells to vascular remodeling after three distinct types of injuries. A, Endovascular injury was induced in a BMTGFPWild mouse by inserting a large wire into the left femoral artery (Wire). Perivascular injury was induced by placing a polyethylene tube around the right femoral artery of the same mouse (Cuff). Flow-restriction vascular injury was induced by ligating the left common carotid artery (Ligation). Injured arteries and the right common carotid artery (Uninjured) were harvested at 4 weeks and observed with a GFP-lighting system and a cooled CCD camera. Bar=1 mm. B, Vascular injuries were induced in BMTGFPApoE mice (n=4). Arteries were fixed in 4% paraformaldehyde and embedded in plastic resin. Thin sections (3 to 4 µm) were stained with Cy3-conjugated anti--SMA and observed under a confocal microscope (FLUOVIEW FV300, Olympus). Arrows indicate the internal elastic lamina. NI indicates neointima; M, media; -SMA, -smooth muscle actin; and DIC, differential interference contrast. Bar=20 µm. C, Vascular injuries were induced in BMTLacZWild mice (n=5). After 4 weeks, the injured arteries were stained with X-gal to detect LacZ. Paraffin-embedded sections were rehydrated and stained with hematoxylin. Adjacent sections were stained with alkaline phosphatase-conjugated anti--SMA. Arrowheads indicate the internal elastic lamina; arrows, LacZ+ cells. Scale bar=20 µm.

View this table: [in this window] [in a new window]   Table 1. Frequency of GFP-Positive Cells in Neointima and Media per Cross Section 4 Weeks After Vascular Injuries in BMTGFPApoE

Differentiation of Bone Marrow-Derived Cells to Vascular Cells After Wire Injury
Next, we characterized the bone marrow-derived cells observed in the femoral arteries after wire injury. In BMT^GFPApoE mice, GFP⁺ cells on the luminal side were positive for endothelial markers (BS-lectin, CD31, or VWF) (Figure 2A). 42.9±8.5% of VWF⁺ cells were GFP⁺ (Table 2). In neointima, abundant GFP⁺ cells expressed -SMA (Figure 2B). In BMT^LacZApoE mice, LacZ⁺ cells were readily detected to express endothelial markers on the luminal side (Figure 2C) or -SMA in the neointima. The ultrastructure of bone marrow-derived neointimal cells was examined in BMT^LacZWild mice. Without lead staining after X-gal staining, electron-dense crystalloid precipitates could be observed in cytoplasm of the cells from LacZ mice (Figure 2E), but not from wild-type mice (Figure 2D).²¹ In the injured artery of BMT^LacZWild mice, LacZ⁺ cells were readily identified (Figure 2G), whose morphology was quite distinct from that of peripheral leukocytes (Figure 2F). These results suggest that bone marrow-derived cells differentiate into certain cell-types that contribute to vascular remodeling after wire injury.

View larger version (50K): [in this window] [in a new window]   Figure 2. Contribution of bone marrow-derived cells to endothelial regeneration and neointimal formation after wire-mediated vascular injury. A and B, Cross sections of the vascular lesion of BMTGFPApoE mice 4 weeks after the wire-mediated endovascular injury. GFP-signal (green) was well preserved in plastic-embedded sections. Thin sections were stained with BS-lectin and an anti-CD31 antibody (red). Bar=10 µm (A). Vascular lesion of BMTGFPApoE mice was stained for -smooth muscle actin (-SMA). Bar=10 µm (B). C, Left femoral arteries of BMTLacZApoE mice were snap-frozen at 32 weeks and stained for LacZ (Green) and endothelial cell-specific antigen (Clone MECA-32, Red). Bar=10 µm. Arrows point to double-positive cells. Arrowheads indicate the luminal side of the artery. D through G, Femoral arteries of C57/BL6 (D), LacZ (E), and BMTLacZWild (F and G) mice were stained with X-gal and observed with an electron microscope. Arrowheads indicate electron-dense crystalloid precipitates. Bar=2 µm. L indicates lumen; NI, neointima; and M, media.

View this table: [in this window] [in a new window]   Table 2. Number of GFP-Positive Cells Among von Willebrand Factor-Positive Cells per Cross Section 4 Weeks After Vascular Injuries in BMTGFPApoE Mice

Characterization of Bone Marrow-Derived -SMA-Positive Cells
Bone marrow-derived -SMA-positive cells were further characterized. Cross sections of BMT^LacZWild mice were stained for LacZ and various markers of SMCs. An anti--SMA antibody (clone 1A4) recognized more than half of the LacZ⁺ cells in the neointima (Figure 3A). On the other hand, a few LacZ-positive cells were stained for myosin (clone HSM-V, or polyclonal antibody) (Figure 3A).¹ Double-immunofluorescence study revealed that most of the medial cells expressed both -SMA and myosin in the uninjured artery, whereas a few -SMA⁺ cells were positive for smooth muscle myosin in injured arteries (Figure 3B). Furthermore, immunoreactive -SMA was expressed by some CD45-positive cells in the neointima (Figure 3C).

View larger version (55K): [in this window] [in a new window]   Figure 3. Differentiation of bone marrow-derived cells in vascular lesions. A, Left femoral arteries of BMTLacZWild mice were snap-frozen at 8 weeks after the wire injury and stained for LacZ (green) and markers of smooth muscle cells (red). Bar=10 µm. Anti-LacZ polyclonal antibody was used with monoclonal anti-myosin antibodies (clone 1A4, -SMA; clone HSM-V, myosin heavy chain). An anti-LacZ monoclonal antibody (clone GAL-13) was used with anti-myosin polyclonal antibody. Arrowheads indicate double-positive cells. B, Uninjured and wire-injured femoral arteries of BMTLacZWild mice were snap-frozen and stained for -SMA (red) and myosin (green). Arrowheads indicate double-positive cells. Bar=20 µm. C, Wire-injured femoral arteries were stained for -actin (red) and CD45 (D; green). Bar=5 µm. Arrow indicates a double-positive cell.

Vascular Changes Induced by the Vascular Injury
To understand the mechanism by which contribution of bone marrow cells to vascular remodeling depends on type of injury, the vascular changes induced by the injuries were investigated in C57BL/6 mice (n=3). At 6 hours, wire injury caused complete endothelial denudation, marked enlargement of the lumen, and thinning of the media (Figure 4). TUNEL staining revealed massive apoptosis of the medial cells. Most of the medial SMCs were eliminated by the injury as determined by immunostaining for -SMA.¹⁶ The cellularity of the media remained low until 1 or 2 weeks after injury. In the opposite femoral artery, we found some apoptotic cells in the media (39.1±9.9%) at 6 hours after cuff-replacement. The medial smooth muscle layer remained thick. The luminal side was coated with a monolayer of endothelial cells. Six hours after flow-restriction vascular injury, the endothelium and media remained almost intact with few TUNEL-positive cells (0.3±0.3%).

View larger version (87K): [in this window] [in a new window]   Figure 4. Tissue damage induced by the vascular injury. Cross sections of the arteries of C57BL/6J mice 6 hours after the surgery were analyzed. Total nuclei and apoptotic nuclei were stained with propidium iodide (PI, red) and TUNEL (green), respectively. Smooth muscle cells and endothelial cells were identified by immunostaining for -SMA and CD31, respectively.16 Arrows and arrowheads indicate the internal and external elastic laminas, respectively. Scale bar=20 µm. Endothelial layer was preserved 6 hours after cuff injury and ligation (insets).

One week after wire injury, MCP-1, SDF-1, and VEGF were highly expressed in the left femoral artery (Figure 5). After cuff-replacement, chemokines were abundantly expressed in adventitia, but not in the vessel wall. After ligation of carotid artery, those factors were slightly expressed on the luminal side.

View larger version (85K): [in this window] [in a new window]   Figure 5. Chemokine and cytokine expression 1 week after the vascular injury. Injured arteries were harvested at 1 week and snap-frozen in OCT compound. Frozen sections were stained for SDF-1, MCP-1, and VEGF. Arrowheads indicate the external elastic lamina; arrows point to the positive cells. Scale bar=30 µm.

Inflammatory Response Induced by Vascular Injuries
At 4 weeks, inflammatory cell accumulation were identified by immunostaining for CD3 (T cells) or F4/80 (macrophages) antigen in BMT^LacZWild mice (Table 3) (n=4). Inflammatory cells were predominantly macrophages in all models.^16,18 Robust infiltration of macrophages was observed in neointima and adventitia after cuff-replacement. On the other hand, a few macrophages were detected in the vascular lesions induced by wire or ligation.

View this table: [in this window] [in a new window]   Table 3. Number of Macrophages per Cross Section of Injured Arteries 4 Weeks After Vascular Injuries in BMTLacZWild Mice

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, three distinct types of mechanical injuries were induced in the same mouse whose bone marrow had been reconstituted with that of GFP or LacZ mice. After wire-mediated endovascular injury, a significant number of the neointimal and medial cells derived from bone marrow. In contrast, marker-positive cells were seldom detected in the lesion induced by perivascular cuff replacement. There were only a few bone marrow-derived cells in the neointima after ligation of the common carotid artery. The results of the present study suggest that the mode of injury is crucial for the recruitment of bone marrow-derived cells to tissue remodeling and that the potential participation of bone marrow cells in organ regeneration may be underestimated in certain types of experiments.

It remains unknown why contribution of bone marrow-derived cells to neointimal hyperplasia depends on the type of model. There was no correlation between the number of inflammatory cells in vessel wall and the degree of bone marrow cell contribution. Vascular inflammation should be essential, but may not be sufficient to recruit bone marrow-derived cells to lesions. After perivascular cuff-replacement or flow-restriction by ligation, endothelial cells and medial cells remained relatively intact with mild expression of MCP-1, SDF-1, and VEGF. Those minimal changes in vessel wall were associated with little contribution of bone marrow cells to neointimal hyperplasia. In contrast, wire injury induced complete endothelial denudation and medial cell loss due to apoptosis.⁷ In this model, the cellularity of the injured media remains very low until 1 or 2 weeks after the injury.¹⁶ The injury induces expression of MCP-1, SDF-1, and VEGF that may be important for homing of bone marrow-derived cells. It was observed that neointimal hyperplasia developed when the media remained acellular.¹⁶ It is most likely that bone marrow-derived cells must be recruited to repair the injured artery, when there are not enough local mesenchymal cells for the process.

Recent advances in gene-manipulating techniques have produced various genetically modified mice to determine the role of specific molecules in vascular remodeling, such as post-PCI restenosis. However, mouse arteries, unlike those of larger animals, are too small for transluminal injury with a balloon. Alternatively, several models of vascular injury^17,22,23 have been shown to produce neointima-like hyperplasia and are used to evaluate the susceptibility of transgenic/knock-out mice to vascular lesion formation. Our findings suggest that we should be cautious about the difference in the mechanisms of neointimal hyperplasia when we compare findings obtained in different experimental systems.

Given the complexity of human atherosclerotic lesions, none of the vascular injury models would represent the exact pathogenesis of human vascular diseases. Previous reports suggested that SMCs in human vascular lesions are composed of cells of diverse origin^24,25 and that the cellular constituents should differ depending on the type of vascular injury. Our findings suggest that bone marrow cells would substantially contribute to lesion formation when arteries are subjected to severe injuries. Advanced atherosclerotic lesions exhibit a higher incidence of internal elastic rupture and intimomedial interface damage,²⁶ which are associated with focal intraplaque microhemorrhage.²⁷ PCI denudes endothelium completely and mechanically dilates atherosclerotic lesions with a tear in the luminal surface.²⁸ Circulating progenitors would be recruited to those severely injured human vessels. Consistent with this notion, an analysis of sex-mismatched bone marrow transplant subjects revealed that SMCs throughout the atherosclerotic vessel wall can derive from donor bone marrow² and that these cells are extensively recruited in diseased compared with undiseased segments.

Most of the bone marrow-derived cells expressed -SMA, but not markers for highly differentiated SMCs. Some CD45-positive cells also expressed -SMA. These results indicate that bone marrow-derived cells present in the neointima easily express -SMA even when they remain positive for hematopoietic markers. In contrast, it seems a rare event, if not at all, for bone marrow-derived cells to express markers of highly differentiated SMCs, at least within a few months after a wire injury. Consistently, it was reported that most of the bone marrow-derived cells detected in human atherosclerotic plaques expressed -SMA-positive cells, but not calponin, a marker for differentiated SMCs.²

In summary, our data clearly demonstrate that the contribution of bone marrow cells to vascular remodeling highly depends on the type of arterial injury. When we extrapolate data obtained in animal experiments to the pathogenesis of human diseases, we should be aware that the origin of vascular lesions is diverse and that distinct mechanisms may regulate neointimal formation in different models of vascular injury.

	Acknowledgments

 
This study was supported in part by grant-in-aid from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (M.S.).

	Footnotes

 
Original received December 20, 2002; first resubmission received February 28, 2003; second resubmission received August 4, 2003; revised second resubmission received September 8, 2003; accepted September 10, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Groschel-Stewart U, Schreiber J, Mahlmeister C. Production of specific antibodies to contractile proteins, and their use in immunofluorescence microscopy, I: antibodies to smooth and striated chicken muscle myosins. Histochemistry. 1976; 46: 229–236.[CrossRef][Medline] [Order article via Infotrieve]
2. Caplice NM, Bunch TJ, Stalboerger PG, Wang S, Simper D, Miller DV, Russell SJ, Litzow MR, Edwards WD. Smooth muscle cells in human coronary atherosclerosis can originate from cells administered at marrow transplantation. Proc Natl Acad Sci U S A. 2003; 100: 4754–4759.[Abstract/Free Full Text]
3. Korbling M, Katz RL, Khanna A, Ruifrok AC, Rondon G, Albitar M, Champlin RE, Estrov Z. Hepatocytes and epithelial cells of donor origin in recipients of peripheral-blood stem cells. N Engl J Med. 2002; 346: 738–746.[Abstract/Free Full Text]
4. Wagers AJ, Sherwood RI, Christensen JL, Weissman IL. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science. 2002; 297: 2256–2259.[Abstract/Free Full Text]
5. Hillebrands JL, Klatter FA, van Dijk WD, Rozing J. Bone marrow does not contribute substantially to endothelial-cell replacement in transplant arteriosclerosis. Nat Med. 2002; 8: 194–195.[CrossRef][Medline] [Order article via Infotrieve]
6. Hu Y, Davison F, Ludewig B, Erdel M, Mayr M, Url M, Dietrich H, Xu Q. 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]
7. Sata M, Saiura A, Kunisato A, Tojo A, Okada S, Tokuhisa T, Hirai H, Makuuchi M, Hirata Y, Nagai R. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med. 2002; 8: 403–409.[CrossRef][Medline] [Order article via Infotrieve]
8. Shimizu K, Sugiyama S, Aikawa M, Fukumoto Y, Rabkin E, Libby P, Mitchell RN. 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][Medline] [Order article via Infotrieve]
9. Campbell JH, Han CL, Campbell GR. Neointimal formation by circulating bone marrow cells. Ann N Y Acad Sci. 2001; 947: 18–24.[Abstract/Free Full Text]
10. Kashiwakura Y, Katoh Y, Tamayose K, Konishi H, Takaya N, Yuhara S, Yamada M, Sugimoto K, Daida H. Isolation of bone marrow stromal cell-derived smooth muscle cells by a human SM22 promoter: in vitro differentiation of putative smooth muscle progenitor cells of bone marrow. Circulation. 2003; 107: 2078–2081.[Abstract/Free Full Text]
11. Kaushal S, Amiel GE, Guleserian KJ, Shapira OM, Perry T, Sutherland FW, Rabkin E, Moran AM, Schoen FJ, Atala A, Soker S, Bischoff J, Mayer JE Jr. Functional small-diameter neovessels created using endothelial progenitor cells expanded ex vivo. Nat Med. 2001; 7: 1035–1040.[CrossRef][Medline] [Order article via Infotrieve]
12. Zambrowicz BP, Imamoto A, Fiering S, Herzenberg LA, Kerr WG, Soriano P. Disruption of overlapping transcripts in the ROSA ßgeo 26 gene trap strain leads to widespread expression of ß-galactosidase in mouse embryos and hematopoietic cells. Proc Natl Acad Sci U S A. 1997; 94: 3789–3794.[Abstract/Free Full Text]
13. Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. Green mice as a source of ubiquitous green cells. FEBS Lett. 1997; 407: 313–319.[CrossRef][Medline] [Order article via Infotrieve]
14. Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science. 1992; 258: 468–471.[Abstract/Free Full Text]
15. Okada S, Yoshida T, Hong Z, Ishii G, Hatano M, Kuro-O M, Nabeshima Y, Nabeshima Y, Tokuhisa T. Impairment of B lymphopoiesis in precocious aging (klotho) mice. Int Immunol. 2000; 12: 861–871.[Abstract/Free Full Text]
16. Sata M, Maejima Y, Adachi F, Fukino K, Saiura A, Sugiura S, Aoyagi T, Imai Y, Kurihara H, Kimura K, Omata M, Makuuchi M, Hirata Y, Nagai R. A mouse model of vascular injury that induces rapid onset of medial cell apoptosis followed by reproducible neointimal hyperplasia. J Mol Cell Cardiol. 2000; 32: 2097–2104.[CrossRef][Medline] [Order article via Infotrieve]
17. Moroi M, Zhang L, Yasuda T, Virmani R, Gold HK, Fishman MC, Huang PL. Interaction of genetic deficiency of endothelial nitric oxide, gender, and pregnancy in vascular response to injury in mice. J Clin Invest. 1998; 101: 1225–1232.[Medline] [Order article via Infotrieve]
18. Sata M, Walsh K. Fas ligand-deficient mice display enhanced leukocyte infiltration and intima hyperplasia in flow restricted vessels. J Mol Cell Cardiol. 2000; 32: 1395–1400.[CrossRef][Medline] [Order article via Infotrieve]
19. Kumar A, Hoover JL, Simmons CA, Lindner V, Shebuski RJ. Remodeling and neointima formation in the carotid artery of normal and P-selectin-deficient mice. Circulation. 1997; 96: 4333–4342.[Abstract/Free Full Text]
20. Meeson AP, Radford N, Shelton JM, Mammen PP, DiMaio JM, Hutcheson K, Kong Y, Elterman J, Williams RS, Garry DJ. Adaptive mechanisms that preserve cardiac function in mice without myoglobin. Circ Res. 2001; 88: 713–720.[Abstract/Free Full Text]
21. Malouf NN, Coleman WB, Grisham JW, Lininger RA, Madden VJ, Sproul M, Anderson PA. Adult-derived stem cells from the liver become myocytes in the heart in vivo. Am J Pathol. 2001; 158: 1929–1935.[Abstract/Free Full Text]
22. Carmeliet P, Moons L, Stassen J-M, Mol MD, Bouche A, van den Oord JJ, Kockx M, Collen D. Vascular wound healing and neointima formation induced by perivascular electric injury in mice. Am J Pathol. 1997; 150: 761–776.[Abstract]
23. Kumar A, Lindner V. Remodeling with neointima formation in the mouse carotid artery after cessation of blood flow. Arterioscler Thromb Vasc Biol. 1997; 17: 2238–2244.[Abstract/Free Full Text]
24. Li S, Fan YS, Chow LH, Van Den Diepstraten C, van Der Veer E, Sims SM, Pickering JG. Innate diversity of adult human arterial smooth muscle cells: cloning of distinct subtypes from the internal thoracic artery. Circ Res. 2001; 89: 517–525.[Abstract/Free Full Text]
25. Zalewski A, Shi Y, Johnson AG. Diverse origin of intimal cells: smooth muscle cells, myofibroblasts, fibroblasts, and beyond? Circ Res. 2002; 91: 652–655.[Free Full Text]
26. Moreno PR, Purushothaman KR, Fuster V, O’Connor WN. Intimomedial interface damage and adventitial inflammation is increased beneath disrupted atherosclerosis in the aorta: implications for plaque vulnerability. Circulation. 2002; 105: 2504–2511.[Abstract/Free Full Text]
27. Kockx MM, Cromheeke KM, Knaapen MW, Bosmans JM, De Meyer GR, Herman AG, Bult H. Phagocytosis and macrophage activation associated with hemorrhagic microvessels in human atherosclerosis. Arterioscler Thromb Vasc Biol. 2003; 23: 440–446.[Abstract/Free Full Text]
28. 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: 224–233.[Abstract/Free Full Text]

This article has been cited by other articles:

				J. Kim, L. Zhang, K. Peppel, J.-H. Wu, D. A. Zidar, L. Brian, S. M. DeWire, S. T. Exum, R. J. Lefkowitz, and N. J. Freedman {beta}-Arrestins Regulate Atherosclerosis and Neointimal Hyperplasia by Controlling Smooth Muscle Cell Proliferation and Migration Circ. Res., July 3, 2008; 103(1): 70 - 79. [Abstract] [Full Text] [PDF]

				H. Iwata and M. Sata Origin of Cells That Contribute to Neointima Growth Circulation, June 17, 2008; 117(24): 3060 - 3061. [Full Text] [PDF]

				N. Yajima, M. Takahashi, H. Morimoto, Y. Shiba, Y. Takahashi, J. Masumoto, H. Ise, J. Sagara, J. Nakayama, S. Taniguchi, et al. Critical Role of Bone Marrow Apoptosis-Associated Speck-Like Protein, an Inflammasome Adaptor Molecule, in Neointimal Formation After Vascular Injury in Mice Circulation, June 17, 2008; 117(24): 3079 - 3087. [Abstract] [Full Text] [PDF]

				K. Satoh, T. Matoba, J. Suzuki, M. R. O'Dell, P. Nigro, Z. Cui, A. Mohan, S. Pan, L. Li, Z.-G. Jin, et al. Cyclophilin A Mediates Vascular Remodeling by Promoting Inflammation and Vascular Smooth Muscle Cell Proliferation Circulation, June 17, 2008; 117(24): 3088 - 3098. [Abstract] [Full Text] [PDF]

				H-J Kang, Y-S Kim, B-K Koo, K W Park, H-Y Lee, D-W Sohn, B-H Oh, Y-B Park, and H-S Kim Effects of stem cell therapy with G-CSF on coronary artery after drug-eluting stent implantation in patients with acute myocardial infarction Heart, May 1, 2008; 94(5): 604 - 609. [Abstract] [Full Text] [PDF]

				D. Chen, J. M. Abrahams, L. M. Smith, J. H. McVey, R. I. Lechler, and A. Dorling Regenerative repair after endoluminal injury in mice with specific antagonism of protease activated receptors on CD34+ vascular progenitors Blood, April 15, 2008; 111(8): 4155 - 4164. [Abstract] [Full Text] [PDF]

				Y. Diao, S. Guthrie, S.-L. Xia, X. Ouyang, L. Zhang, J. Xue, P. Lee, M. Grant, E. Scott, and M. S. Segal Long-Term Engraftment of Bone Marrow-Derived Cells in the Intimal Hyperplasia Lesion of Autologous Vein Grafts Am. J. Pathol., March 1, 2008; 172(3): 839 - 848. [Abstract] [Full Text] [PDF]

				W.-C. Shyu, S.-Z. Lin, P.-S. Yen, C.-Y. Su, D.-C. Chen, H.-J. Wang, and H. Li Stromal Cell-Derived Factor-1{alpha} Promotes Neuroprotection, Angiogenesis, and Mobilization/Homing of Bone Marrow-Derived Cells in Stroke Rats J. Pharmacol. Exp. Ther., February 1, 2008; 324(2): 834 - 849. [Abstract] [Full Text] [PDF]

				K. Tanaka, M. Sata, T. Natori, J.-r. Kim-Kaneyama, K. Nose, M. Shibanuma, Y. Hirata, and R. Nagai Circulating progenitor cells contribute to neointimal formation in nonirradiated chimeric mice FASEB J, February 1, 2008; 22(2): 428 - 436. [Abstract] [Full Text] [PDF]

				T. Shimizu, T. Nakazawa, A. Cho, F. Dastvan, D. Shilling, G. Daum, and M. A. Reidy Sphingosine 1-Phosphate Receptor 2 Negatively Regulates Neointimal Formation in Mouse Arteries Circ. Res., November 9, 2007; 101(10): 995 - 1000. [Abstract] [Full Text] [PDF]

				T. Yamada, T. Kondo, Y. Numaguchi, M. Tsuzuki, T. Matsubara, I. Manabe, M. Sata, R. Nagai, and T. Murohara Angiotensin II Receptor Blocker Inhibits Neointimal Hyperplasia Through Regulation of Smooth Muscle Like Progenitor Cells Arterioscler. Thromb. Vasc. Biol., November 1, 2007; 27(11): 2363 - 2369. [Abstract] [Full Text] [PDF]

				J. F. Bentzon, C. S. Sondergaard, M. Kassem, and E. Falk Smooth Muscle Cells Healing Atherosclerotic Plaque Disruptions Are of Local, Not Blood, Origin in Apolipoprotein E Knockout Mice Circulation, October 30, 2007; 116(18): 2053 - 2061. [Abstract] [Full Text] [PDF]

				S. Koide, M. Okazaki, M. Tamura, K. Ozumi, H. Takatsu, F. Kamezaki, A. Tanimoto, H. Tasaki, Y. Sasaguri, Y. Nakashima, et al. PTEN reduces cuff-induced neointima formation and proinflammatory cytokines Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H2824 - H2831. [Abstract] [Full Text] [PDF]

				B. H. Walpoth, P. Zammaretti, M. Cikirikcioglu, E. Khabiri, M. K. Djebaili, J.-C. Pache, J.-C. Tille, Y. Aggoun, D. Morel, A. Kalangos, et al. Enhanced intimal thickening of expanded polytetrafluoroethylene grafts coated with fibrin or fibrin-releasing vascular endothelial growth factor in the pig carotid artery interposition model J. Thorac. Cardiovasc. Surg., May 1, 2007; 133(5): 1163 - 1170. [Abstract] [Full Text] [PDF]

				M. Sumi, M. Sata, S.-i. Miura, K.-A. Rye, N. Toya, Y. Kanaoka, K. Yanaga, T. Ohki, K. Saku, and R. Nagai Reconstituted High-Density Lipoprotein Stimulates Differentiation of Endothelial Progenitor Cells and Enhances Ischemia-Induced Angiogenesis Arterioscler. Thromb. Vasc. Biol., April 1, 2007; 27(4): 813 - 818. [Abstract] [Full Text] [PDF]

				J. Sainz and M. Sata CXCR4, a Key Modulator of Vascular Progenitor Cells Arterioscler. Thromb. Vasc. Biol., February 1, 2007; 27(2): 263 - 265. [Full Text] [PDF]

				Y. Shiba, M. Takahashi, T. Yoshioka, N. Yajima, H. Morimoto, A. Izawa, H. Ise, K. Hatake, K. Motoyoshi, and U. Ikeda M-CSF Accelerates Neointimal Formation in the Early Phase After Vascular Injury in Mice: The Critical Role of the SDF-1-CXCR4 System Arterioscler. Thromb. Vasc. Biol., February 1, 2007; 27(2): 283 - 289. [Abstract] [Full Text] [PDF]

				M. Sahara, M. Sata, T. Morita, K. Nakamura, Y. Hirata, and R. Nagai Diverse Contribution of Bone Marrow Derived Cells to Vascular Remodeling Associated With Pulmonary Arterial Hypertension and Arterial Neointimal Formation Circulation, January 30, 2007; 115(4): 509 - 517. [Abstract] [Full Text] [PDF]

				K. Kawai-Kowase and G. K. Owens Multiple repressor pathways contribute to phenotypic switching of vascular smooth muscle cells Am J Physiol Cell Physiol, January 1, 2007; 292(1): C59 - C69. [Abstract] [Full Text] [PDF]

D. L. Basi, N. Adhikari, A. Mariash, Q. Li, E. Kao, S. V. Mullegama, and J. L. H