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Published as doi: 10.1096/fj.06-6884com.
 (The FASEB Journal. 2008;22:428-436.)
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(The FASEB Journal. 2008;22:428-436.)
© 2008 FASEB

Circulating progenitor cells contribute to neointimal formation in nonirradiated chimeric mice

Kimie Tanaka*,||, Masataka Sata*,{ddagger},1, Takeshi Natori{ddagger}, Joo-ri Kim-Kaneyama§, Kiyoshi Nose§, Motoko Shibanuma§, Yasunobu Hirata* and Ryozo Nagai*

* Department of Cardiovascular Medicine,

{dagger} Department of Advanced Clinical Science and Therapeutics, and

{ddagger} Department of Surgery, University of Tokyo Graduate School of Medicine, Tokyo, Japan;

§ Department of Microbiology, Showa University School of Pharmaceutical Sciences, Tokyo, Japan; and

|| Japan Health Sciences Foundation, Tokyo, Japan

1 Correspondence: Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: msata-circ{at}umin.net


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent evidence suggests that bone marrow-derived cells may contribute to repair and lesion formation following vascular injury. In most studies, bone marrow-derived cells were tracked by transplanting exogenous cells into bone marrow that had been compromised by irradiation. It remains to be determined whether endogenous circulating progenitors actually contribute to arterial remodeling under physiological conditions. Here, we established a parabiotic model in which two mice were conjoined subcutaneously without any vascular anastomosis. When wild-type mice were joined with transgenic mice that expressed green fluorescent protein (GFP) in all tissues, GFP-positive cells were detected not only in the peripheral blood but also in the bone marrow of the wild-type mice. The femoral arteries of the wild-type mice were mechanically injured by insertion of a large wire. At 4 wk, there was neointima hyperplasia that mainly consisted of {alpha}-smooth muscle actin-positive cells. GFP-positive cells were readily detected in the neointima (14.8±4.5%) and media (31.1±8.8%) of the injured artery. Some GFP-positive cells expressed {alpha}-smooth muscle actin or an endothelial cell marker. These results indicate that circulating progenitors contribute to re-endothelialization and neointimal formation after mechanical vascular injury even in nonirradiated mice.—Tanaka, K., Sata, M., Natori, T., Kim-Kaneyama, J., Nose, K., Shibanuma, M., Hirata, Y., and Nagai, R. Circulating progenitor cells contribute to neointimal formation in nonirradiated chimeric mice.


Key Words: smooth muscle cell • irradiation • stem cells • vascular repair • parabiosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
EXUBERANT ACCUMULATION OF smooth muscle cells (SMCs) plays a principal role in the pathogenesis of vascular diseases (1) . In atherosclerotic lesions, SMCs proliferate and synthesize an extracellular matrix, contributing to lesion mass. SMC hyperplasia is not only a major cause of reocclusion after successful percutaneous coronary interventions (PCIs) (2 , 3) and coronary bypass surgery (4) but also the primary cause of graft vasculopathy (5) . Therefore, much effort has been devoted to understanding the molecular pathways that regulate SMC behavior (6) . Recent studies indicate that putative progenitors of smooth muscle-like cells and endothelial-like cells may significantly contribute to vascular repair, new vessel formation, and lesion formation (7 8 9 10 11 12 13 14) .

Most of these studies analyzed chimeric animal and human cells that had experienced lethal irradiation followed by exogenous bone-marrow cell transplantation. However, there is a concern that irradiation may have deleterious effects on recipient animals and affect the process of neointimal formation. Thus, it remains to be determined whether circulating progenitors actually contribute to vascular remodeling in nonirradiated animals.

In this study, chimerism and lesion formation were investigated using a mouse parabiosis model in which two mice were conjoined subcutaneously without any surgical anastomosis of vasculatures. Observation revealed that circulating cells were exchanged between the two joined mice. Partner-derived circulating cells contributed to re-endothelialization and neointima formation after wire-mediated vascular injury in nonirradiated chimeric mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Wild-type C57BL/6 mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). Transgenic mice (C57BL/6 background) that ubiquitously express enhanced green fluorescent protein (GFP mice) were a generous gift from Dr. Masaru Okabe (Osaka University, Osaka, Japan) (15) . Adult, male, 8- to 12-wk-old mice were used throughout the study. All mice were kept in microisolator cages on a 12-h day/night cycle and fed regular chow. All experimental procedures and protocols were approved by the Animal Care and Use Committee 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) (16) .

Parabiosis
Pairs of sex- and weight-matched, wild-type and GFP-transgenic mice were parabiosed using the method described previously (17 , 18) . The mice were anesthetized by intraperitoneal injection of 50 mg/kg pentobarbital (Abbott Laboratories, North Chicago, IL, USA) diluted in 0.9% sodium chloride solution. The corresponding lateral aspects of each mouse were shaved and sterilized with 70% ethanol. A longitudinal skin incision was made from the olecranon to the knee joint of each mouse, taking care not to open the peritoneal cavity. After dissection of the subcutaneous connective tissue along the incision, the corresponding skin edges were approximated symmetrically, slightly converted, and joined with a 4-0 silk suture.

Bone marrow transplantation with irradiation
Bone marrow transplantation (BMT) was performed as described previously (7 , 19) . Eight- to nine-week-old male wild-type mice were lethally X-irradiated with a total dose of 9.5 Gy (MBR-1520RB, Hitachi, Tokyo, Japan). Bone marrow cells were harvested from the femora and tibiae of the donor mice (19) . One day later, the recipient mice received unfractionated bone marrow cells from GFP mice (3x106) suspended in 0.3 ml PBS by tail vein injection.

Determination of chimerism in peripheral blood and bone marrow
The chimerism of peripheral blood as well as bone marrow was measured at the time points indicated (n=2 or 3 for each group). Peripheral blood samples were collected from the retro-orbital venous plexus. Total bone marrow cells were harvested from the femora (19) . Erythrocytes were lysed with ACK lysing buffer (0.155 mol/L ammonium chloride, 0.1 mol/L disodium EDTA, and 0.01 mol/L potassium bicarbonate) (19) . Cell suspensions were analyzed by flow cytometry to measure the GFP signal (XL, Beckman-Coulter, Miami, FL, USA).

Wire-mediated endovascular injury
At the indicated time points after BMT or parabiosis operation, transluminal arterial injury was induced by inserting a straight spring wire (0.38 mm in diameter, No. C-SF-15-15, Cook, Bloomington, IN, USA) into the femoral arteries as already described (20) . The wire was left in place for 1 min to denude and dilate the artery (untreated group, n=6; parabiosis group, n=5; BMT group, n=5). A copy of the tutorial video of the surgical procedure can be sent on request, or the video can be viewed at http://plaza.umin.ac.jp/~msata/.

Plastic embedding to detect GFP signal
Four weeks after surgery, the mice were sacrificed with an overdose of pentobarbital and perfused at a constant pressure via the left ventricle with 0.9% sodium chloride solution. The injured arteries were further fixed in 4% paraformaldehyde overnight at 4°C. To preserve GFP signal for histological analyses, the arteries were embedded in plastic resin (Technovit 8100, Heraeus Kulzer, Wehrheim, Germany) 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 414–2, Energy Beam Sciences, Inc., Agawam, MA, USA) as a mold. The polymerized block was fixed onto a block (Histobloc, Heraeus Kulzer) with an adhesive agent (EP001, Semedain, Tokyo, Japan) and cut using a rotary microtome (HM335E, Microm International GmbH, Walldorf, Germany) with a disposable knife (Histoknife, Heraeus Kulzer). Thin sections (3–4 µm) were stretched in a water bath, mounted on silanized slides (Matsunami, Tokyo, Japan) and dried for 2 h at 37°C. The sections were washed in PBS and used for immunofluorescence studies. Immunofluorescence double staining on the plastic-embedded sections was performed as described elsewhere (7 , 12 , 21) . After blocking in 0.5% horse serum, plastic-embedded sections were incubated with a primary antibody (anti-CD31, clone MEC13.3, BD Biosciences, San Jose, CA, USA), followed by incubation with a Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA). Smooth muscle-like cells were identified by a Cy3-conjugated anti-{alpha} smooth muscle actin ({alpha}-SMA) antibody (Sigma Chemical Company, St. Louis, MO, USA). Nuclei were counterstained with Hoechst 33258 (Sigma) or propidium iodide (Sigma).

Triple immunofluorescence staining
At 8 wk after BMT, the femoral artery was injured with a wire (n=5). The artery was harvested at 6 wk after the injury and snap-frozen in OCT compound (Sakura, Tokyo, Japan). Immunofluorescence double or triple staining was performed as described elsewhere (7 , 12 , 20) . After blocking in 0.5% horse serum, frozen sections were incubated with primary antibodies (an anti-GFP rabbit polyclonal antibody, Molecular Probes, Eugene, OR, USA; an anti-CD45 monoclonal antibody, BD Biosciences; and a Cy3-conjugated anti-{alpha}-SMA antibody, Sigma), followed by incubation with secondary antibodies (Alexa Fluor® 488-conjugated anti-rabbit Ig; Alexa Fluor® 647-conjugated anti-rat Ig).

Confocal z-axis analysis of the colocalizing signals
After immunofluorescence staining, the sections were mounted with the ProLong Antifade Kit (Molecular Probes) and observed under a confocal microscope (Fluoview FV300, Olympus, Tokyo, Japan). The series of confocal optical XY images was acquired through the thickness of the double-positive cells. The images were reconstructed in the z axis (0.5 µm pit, 11 slices). Vertical sections of the x–z and y–z planes were generated.

Immunoelectron microscopy
Wire-mediated endovascular injury was induced to the femoral artery at 8 wk after the BMT. The injured femoral artery was harvested at 6 wk and fixed in 4% paraformaldehyde solution including 0.1% glutalaldehyde and 0.05% TritonX-100, then embedded in glycol methacrylate. Ultrathin (80 to 100 nm) sections were incubated with 1% BSA in 0.01 M PBS for 1 h and rinsed with 0.01 M PBS for 15 min. The sections were incubated overnight with 1:200 diluted anti-{alpha}-SMA mouse monoclonal antibody (Sigma) and 1:100 diluted anti-GFP rabbit polyclonal antibody (Molecular Probes) at 4°C. After the sections were washed with 0.01 M PBS, 15-nm gold-labeled goat anti-rabbit IgG (BBinternational, Cardiff, UK) and 10-nm gold-labeled goat anti-mouse IgG (BBinternational) were applied as secondary antibodies for 2 h. The sections were counterstained with uranyl acetate and Reynold’s lead citrate, then examined under an electron microscope (Hitachi H-7600) (n=2).

Statistics
All data are expressed as mean ± SE. Comparisons among the three groups were evaluated by one-way ANOVA followed by Bonferroni test. A value of P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cross-circulation was established rapidly and stably between the parabiotic mice
To track the circulating cells without irradiation, a parabiotic model was used, a procedure wherein wild-type C57BL/6 mice and GFP mice are surgically joined without any vascular anastomosis. Peripheral blood was collected from each mouse, and chimerism was determined by flow cytometry to detect the GFP signal. In the wild-type mice joined to the GFP mice, the percentage of GFP-positive leukocytes rapidly increased, reaching 35–45% as early as 10 days after surgery (data not shown). Conversely, the proportion of GFP-positive cells decreased in the peripheral blood of the GFP mice. These data suggest rapid establishment of a common circulatory system between the two mice. The chimerism was maintained in the peripheral blood of the wild-type mice (38.5±3.5%) as long as 20 wk after surgery. A significant number of partner-derived GFP-positive cells was detected also in the bone marrow of the wild-type mice conjoined to the GFP mice at 9 wk (14.0±1.8%) and 21 wk (21.3±0.7%) (Table 1 ). In the wild-type mice that received bone marrow from GFP mice after lethal irradiation, GFP-positive cells were readily detected at 9 wk. At 20 wk after BMT, the majority of peripheral blood cells (80.2±2.3%) and bone marrow cells (74.5±8.1%) were exogenous GFP-positive cells.


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  		Table 1. Proportion of GFP+ cells observed in peripheral blood and bone marrow of wild-type mice conjoined to GFP mice

The parabiotic mice displayed neointima hyperplasia similar to that of untreated mice
To determine whether irradiation had a significant effect on the pathogenesis of vascular lesions, a large wire was induced into the femoral artery of untreated wild-type mice (untreated group), the wild-type mice conjoined to GFP mice (parabiosis group), and the wild-type mice that received bone marrow from GFP mice after lethal irradiation (BMT group). The wild-type mice in the parabiosis group were used at 20–24 wk after surgical conjoining to GFP mice. Vascular injury was induced in the BMT group at 4 wk (4W-BMT) or 8 wk (8W-BMT) after transplantation of BM cells. Neointima hyperplasia was formed in the injured femoral artery in all groups at 4 wk (untreated, parabiosis, and 4W-BMT) or 6 wk (8W-BMT) after vascular injury (Fig. 1 A). Morphometric analysis revealed that the lesion induced at 4 wk after BMT (2.7±0.2x104 µm2, n=5) was significantly smaller than that in the untreated group (4.4±0.5x104 µm2, P < 0.05, n=6) (Fig. 1B ), suggesting that BMT with lethal irradiation had suppressive effects on the process of neointima hyperplasia at 4 wk after BMT (4W-BMT). In contrast, there was no significant difference in lesion size between the parabiosis group (4.7±0.6x104 µm2, n=5) and the untreated group (nonsignificant). When the femoral artery was injured at 8 wk after BMT (8W-BMT), the neointima area (4.7±0.5x104 µm2, n=4) was similar to that in the untreated group.


Figure 1
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  		Figure 1. Morphometric analysis of the neointima induced by wire-mediated vascular injury. Wire-mediated vascular injury was induced in the right femoral artery of a wild-type mouse that had been conjoined to a GFP mouse for 20–24 wk (n=5). The right femoral artery was similarly injured in the wild-type mice (n=6). Wire-mediated injury also was induced in the bone marrow chimeric mice at 4 wk (4W-BMT, n=5) or 8 wk (8W-BMT, n=4) after BMT. The injured and contralateral arteries were harvested and embedded in plastic resin at 4 wk (untreated, parabiosis, and 4W-BMT) or 6 wk (8W-BMT) after vascular injury. A) Cross-sections (3–4 µm) were stained with hematoxylin and eosin. Arrows indicate the internal elastic lamina. Scale bar = 100 µm. B) The neointima area was measured on digitized images using image-analysis software. Three to four sections were measured for each artery. All data are presented as mean ± SE.

Circulating progenitor cells contributed to neointimal formation in nonirradiated parabiotic mice and irradiated BMT mice
The potential contribution of circulating cells to vascular remodeling after severe injury was investigated in the 8W-BMT group and in the parabiosis group. The reconstitution rate in the 8W-BMT group was 91.6 ± 2.0%, as determined by flow cytometry at 14 wk after irradiation (n=5). Bone marrow-derived GFP-positive cells could be detected in the neointima (20.5±5.7%, n=5) and in the media (39.3±3.1%) in the 8W-BMT group (Fig. 2 A). Similarly, partner-derived GFP-positive cells could be detected in the neointima (14.8±4.5%) and in the media (31.1±8.8%) in the vascular lesion induced in the wild-type mice of the parabiotic group. No GFP-positive cells were detected in the uninjured artery in the 8W-BMT group and the parabiosis group (Fig. 2A ).


Figure 2
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  		Figure 2. Contribution of circulating cells to neointima formation in parabiotic and BMT mice. A) In the parabiosis group, wire-mediated vascular injury was induced in the femoral artery of a wild-type mouse that had been conjoined to a GFP mouse for 20–24 wk. The artery was harvested at 4 wk after the injury and embedded in plastic resin. The sections were stained for CD31 or {alpha}-SMA. At 8 wk after BMT (8W-BMT), the femoral artery was injured with a wire. Six weeks after the injury, the femoral artery was harvested and snap-frozen in OCT compound. The frozen sections were stained for GFP (green) and vascular cell marker (red; CD31 or {alpha}-SMA). The sections were observed under a confocal microscope (Fluoview FV300, Olympus). Arrowheads indicate the internal elastic lamina. DIC, differential interference contrast; Uninjured, uninjured left femoral artery; Injured, right femoral artery injured with wire. Scale bar = 20 µm. B) The sections were stained for GFP (green) and CD31 (red). Nuclei were stained with Hoechst 33258 (blue). GFP-positive cells in the luminal side of the neointima expressed an endothelial cell marker (CD31). Arrows indicate the GFP+ CD31+ double-positive cells (yellow). Scale bar = 10 µm. C) The sections were stained for GFP (green) and {alpha}-SMA (red). Arrows indicate the GFP+ {alpha}-SMA+ double-positive cells (yellow). Arrowheads indicate GFP+ {alpha}-SMA– cells (green). Scale bar = 10 µm.

On the luminal side of the injured artery in the 8W-BMT group and the parabiosis group, a double immunoflorescence study readily detected GFP-positive cells that were positive for an endothelial marker (CD31) (Fig. 2B ). A part of CD31-positive cells on the luminal side expressed GFP in the parabiotic group (13.0±3.3%) and the 8W-BMT group (6.3±3.1%) (Table 2 ). Among the {alpha}-SMA-positive cells in the parabiotic group, there were the partner-derived GFP-positive cells in the neointima (3.4±1.1%) and the media (14.8±3.7%) (Fig. 2C and Table 3 ). In the 8W-BMT group, GFP-positive cells were detected also among {alpha}-SMA-positive cells in the neointima (9.8±0.9%, n=5) and the media (25.6±9.3%).


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  		Table 2. Number of GFP+ cells among the CD31+ cells after vascular injuries in the parabiotic and the BMT mice


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  		Table 3. Frequency of GFP+ cells in neointima and media per cross section after vascular injuries in the parabiosis and the BMT mice

Expression of {alpha}-SMA by the circulating GFP-positive cells
To determine that the "GFP+ {alpha}-SMA+" double-positive cells do not result from overlap between GFP+ {alpha}-SMA– inflammatory cells and GFP– {alpha}-SMA+ local SMCs, we performed high-resolution analysis of the colocalization of GFP and {alpha}-SMA in the vascular lesion. A triple immunoflorescence analysis of GFP, {alpha}-SMA, and CD45 revealed that the GFP-positive cells observed in the neointima displayed various cell types (Fig. 3 A and Table 4 ; {alpha}-SMA+ CD45–, 31.7±11.9%; {alpha}-SMA+ CD45+, 24.1±4.2%; {alpha}-SMA– CD45+, 19.8±5.7%; {alpha}-SMA– CD45–, 23.5±8.6%). These results support the notion that there are certain cell populations in the injured artery expressing both GFP and {alpha}-SMA that are distinct from the infiltrating inflammatory cells. Moreover, z-axis analysis of the GFP+ {alpha}-SMA+ double-positive cells in the neointima of the parabiotic mice revealed that red and green fluorescence signals were detected in the same area within a cell (Fig. 3B ). The colocalization signal between GFP and {alpha}-SMA in the neointima of parabiotic mice was similar to that in the aortic media of GFP mice. Finally, an ultra-high-resolution immunoelectron microscopic observation confirmed that {alpha}-SMA and GFP could be colocalized within a cell in the injured artery (Fig. 4 ).


Figure 3
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  		Figure 3. Detection of circulating progenitor-derived {alpha}-SMA-positive cells in the injured artery. A) Immunofluorescence staining was performed on the frozen section of the injured artery in the 8W-BMT group. The frozen sections were stained for GFP (green), {alpha}-SMA (red), and CD45 (blue). The sections were observed under a confocal microscope (Fluoview FV300, Olympus). Small arrowheads indicate a GFP+ {alpha}-SMA+ CD45– cell (yellow). A large arrowhead indicates GFP+ {alpha}-SMA– CD45+ cells (light blue). Arrows indicate GFP+ {alpha}-SMA+ CD45+ cells. Scale bar = 10 µm. B) Z-axis analysis of the double-positive cell in the neointima of parabiosis. Left panel: The series of confocal optical xy images were acquired through the thickness of the double-positive cell in the injured artery of the parabiotic mice. The images were reconstructed in the z axis (0.5 µm pit, 11 slices). Vertical sections in the x–z and y–z planes were generated; x–y, x–z, and y–z images of the double-positive cell. Red and green fluorescence signals were detected in the same area. Right panel: Plastic-embedded sections of the aorta of GFP mice were stained with a Cye3-conjugated anti-{alpha}-SMA antibody and analyzed. Para NI, neointima in the wild-type mice conjoined to GFP mice; GFP Ao Media, aortic media of GFP mice.


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  		Table 4. Expression of {alpha}-SMA and CD45 by the GFP-positive cells in the injured artery of the bone marrow chimeric mice


Figure 4
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  		Figure 4. Immunoelectron microscopic study to detect colocalization of GFP and {alpha}-SMA within a cell. The femoral artery was injured at 8 wk after BMT (8W-BMT). After 6 wk, the injured artery was harvested and embedded in glycol methacrylate. The sections were stained with a rabbit anti-GFP antibody and a mouse anti-{alpha}-SMA antibody, followed by staining with anti-rabbit Ig 15-nm gold colloid and anti-mouse 10-nm gold colloid. The sections were counterstained with uranyl acetate and Reynold’s lead citrate and examined under an electron microscope (Hitachi H-7600). a) A local SMC that is positive for {alpha}-SMA (10-nm gold colloid, white arrows) but not for GFP. b) An infiltrating cell that is positive for GFP (15-nm gold colloid, black arrows) but not for {alpha}-SMA. c) A bone marrow-derived SMC-like cell that is positive for both GFP and {alpha}-SMA. Scale bar = 500 nm.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study successfully tracked circulating progenitor cells without irradiation by conjoining wild-type mice to GFP mice. In this parabiosis model, cross-circulation was established between the two mice for both humoral factors and cellular blood components (17 , 18 , 22) . Partner-derived cells were observed rapidly and stably not only in peripheral blood but also in bone marrow. Circulating cells significantly contributed to the neointima formation and re-endothelialization in the injured femoral artery of the parabiotic mice. Partner-derived cells were detected also in the media of the injured artery.

We and others reported that bone marrow-derived cells potentially contribute to lesion formation after mechanical vascular injury (7 , 12 , 13 , 23 24 25 26) . In these studies, lethal irradiation followed by transplantation of exogenous bone marrow cells was employed to establish a significant chimerism in peripheral blood as well as in bone marrow. Since irradiation potentially causes many deleterious effects on recipient animals, there is a concern regarding the physiological significance of the findings observed in the irradiated mice (27) . Particularly, irradiation is known to cause endothelial dysfunction (28 , 29) and apoptosis (29 , 30) , and possibly may participate in the pathogenesis of gastrointestinal tract damage (29) , carotid artery stenosis (31) , and coronary artery disease (32 33 34 35) . Irradiation also affects viability and proliferation of host cells (36) . For instance, it has been demonstrated that local low-dose irradiation delays wound healing in a dose-dependent manner by inhibiting cell proliferation and inducing apoptotic cell death (37) . Thus, it is possible that vascular healing in irradiated-chimeric mice may not always represent a physiological process that occurs naturally in response to injury in nonirradiated animals. Furthermore, it is unclear whether injected bone marrow cells can completely restore the bone marrow function of both hematopoietic and mesenchymal systems after lethal irradiation (38) . Consistent with these concerns is the finding that the neointima induced by wire injury in irradiated mice (4W-BMT mice) was less than that in untreated mice. Therefore, it remains to be determined whether bone marrow-derived progenitors actually contribute to re-endothelialization and/or lesion formation in mice without irradiation (27) .

In this study, partner-derived circulating cells successfully homed to the bone marrow of the parabiotic mice, whose blood-forming system was not compromised. While neointima formation was partially suppressed in the bone marrow chimeric mice with irradiation, parabiotic mice displayed a lesion size similar to that of untreated mice. The parabiosis model has been used to demonstrate the existence of circulating humoral factors, such as thyroid hormone (39) and leptin (40) . Donskoy and Goldschneider determined the distribution of Evans blue dye and Thy-1-alloantigen-disparate T cells in the blood of parabiotic mice. When Evans blue dye was injected, the mean ratio of the dye concentration at 4 h between the partners was 0.8 ± 0.3, suggesting rapid establishment of cross circulation after parabiosis (17) . Moreover, in this study, chimerism in the thymus reached maximum levels of 16–25%. Common circulation could be established and maintained for both cellular and humoral blood components between 4 and 7 days after parabiosis. Similarly, Wright et al. (18) reported that hematopoietic stem cells rapidly and constitutively migrate through the blood and play a physiological role in the functional re-engraftment of unconditioned bone marrow between the two mice. Consistent with these reports, the results of this study suggest that stable chimerism could be established in peripheral blood cells without irradiation by conjoining two mice.

In the parabiotic model, the maximum chimerism would be less than 50%, whereas higher chimerism could be achieved by BMT with irradiation. In this study, 14.8 ± 4.5% of neointimal cells and 31.1 ± 8.8% of medial cells were partner-derived GFP-positive cells in the injured artery of the parabiosis group, whereas 20.5 ± 5.7% of neointimal cells and 39.3 ± 3.1% of medial cells were bone marrow-derived GFP-positive cells in the BMT group. Given the relatively lower rate of chimerism in the parabiosis model, it is likely that circulating cells contribute to the neointimal hyperplasia and vascular healing after severe vascular injury at a similar rate in the parabiosis and BMT groups. Moreover, a significant number of GFP-positive cells expressed {alpha}-SMA in both the parabiosis group (neointima, 22.7±3.6%; media 22.3±12.8) and the BMT group (neointima, 55.8±7.0%; media 19.7±6.3). It is plausible that previous irradiation may not have dramatic effects on differentiation and proliferation of progenitor cells after they home at injured arteries.

Circulating progenitor cells likely are recruited in response to vascular damage. GFP-positive cells were never found in the untreated contralateral artery in both the parabiosis and BMT groups, while circulating cells spontaneously migrated and homed to hematopoietic-lineage organs such as bone marrow (18) and thymus (17) . A previous report concluded that bone marrow-derived cells significantly participate in vascular remodeling only when the artery is severely injured (12) . Circulating progenitor cells were selectively recruited to the injured artery and participated in the vascular healing and neointimal formation (41 , 42) , probably because no local cells remained for repair after massive apoptosis due to mechanical injury (12 , 20 , 41) . Severe injury was associated with enhanced expression of chemokines and cytokines (12) , which would be important for the recruitment of circulating and/or remote progenitors. Consistently, blocking of chemokines and cytokines reportedly has reduced recruitment of bone marrow-derived cells and suppressed neointimal formation (43 , 44) .

Recently, great controversy has arisen regarding the methodology to detect bone marrow-derived cells in other organs (45 46 47 48 49) . In most of the studies, a double immunofluorescence method is used to detect bone marrow-derived cells that express a lineage marker after transplantation of bone marrow cells that had been genetically labeled with LacZ or EGFP (7 , 12 , 23) . However, other investigators expressed caution regarding the specificity of colocalizing staining for the cell types and the markers (49) . Use of conventional microscopy potentially increases false colocalization signals by the overlap of adjacent different cells. For example, when a GFP+ {alpha}-SMA– inflammatory cell locates close to a GFP– {alpha}-SMA+ media-derived SMC, a "GFP+ {alpha}-SMA+" pseudo-bone-marrow-derived SMC could be imaged artificially. Thus, higher 3-dimensional resolution with confocal/deconvolution microscopy is required to identify colocalization of signal in tissue sections (49) . In this study, we employed high-resolution confocal microscopy with z-axis analysis to convincingly demonstrate that bone marrow-derived cells did express {alpha}-SMA in neointima after wire-mediated vascular injury (12 , 23) . Moreover, an ultra-high-resolution immunoelectron microscopic observation revealed that {alpha}-SMA and GFP could be located within a cell.

There is criticism regarding the markers to be used to identify the SMC-like cells. In most of the studies, {alpha}-SMA is used as a marker of SMCs because anti-{alpha}-SMA antibodies with high specificity and sensitivity are commercially available (50) . It is a well-established view that {alpha}-SMA is not a definitive lineage marker to identify differentiated SMCs, because {alpha}-SMA is reported to be expressed in a wide variety of non-SMC cell types under certain circumstances: 1) in skeletal and cardiac muscle during normal development, 2) in adult cardiomyocytes in association with various cardiomyopathies; 3) in fibroblasts (or so-called myofibroblasts) in a wide range of circumstances, including wound repair; 4) in endothelial cells during vascular remodeling and/or in response to transforming growth factor-beta stimulation; and 5) in numerous tumor cells (51) . In addition, it is known that some macrophages could be positive for {alpha}-SMA (51 , 52) . We also found that some GFP cells expressed both {alpha}-SMA and CD45. Thus, it is unlikely that all {alpha}-SMA-positive cells represent highly differentiated SMCs, although bone marrow-derived {alpha}-SMA-double-positive cells definitively exist in the injured artery, contributing to vascular healing and lesion formation (53) .

There might be limitations of the parabiotic model. A large healing incision elaborates cytokines and growth factors that may affect vascular remodeling and kinetics of circulating vascular progenitors. Therefore, we waited until 20–24 wk postjoining before inducing vascular injury. The skin incision had been healed almost completely at 9 wk after parabiosis operation (Supplemental Fig. 1 ). However, the proportion of granulocytes among peripheral leukocytes were apparently higher at 10 days (37.6±3.2%, n=4) and at 20–22 wk (42.4±4.9%, n=3) in the parabiosis group than that in the untreated C57BL/6 mice (10.1±1.6%, n=4) or in the BMT group (7.7±1.3%, n=5) (Supplemental Fig. 2), suggesting that chronic wounds can occur in the parabiosis model. Thus, it would be plausible that chronic inflammatory response resultant from a parabiotic surgical procedure may somewhat influence the contribution of circulating progenitors to vascular remodeling.

In conclusion, these data demonstrate that a common circulation system can be established for peripheral blood cells without ablating bone marrow with lethal irradiation or chemotherapy. Circulating progenitors contribute, at least in part, to vascular repair and remodeling of the injured artery in nonirradiated animals.


   ACKNOWLEDGMENTS
 
This study was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan; the Ministry of Health, Labor and Welfare of Japan; and the Japan Health Sciences Foundation. The authors declare that they have no conflicts of interest.

Received for publication July 22, 2007. Accepted for publication August 16, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ross, R. (1999) Atherosclerosis–an inflammatory disease. N. Eng. J. Med. 340,115-126[Free Full Text]
  2. Nobuyoshi, M., Kimura, T., Nosaka, H., Mioka, S., Ueno, K., Yokoi, H., Hamasaki, N., Horiuchi, H., Ohishi, H. (1988) Restenosis after successful percutaneous transluminal coronary angioplasty: serial angiographic follow-up of 229 patients. J. Am. Coll. Cardiol. 12,616-623[Abstract]
  3. Kearney, M., Pieczek, A., Haley, L., Losordo, D. W., Andres, V., Schainfeld, R., Rosenfield, K., Isner, J. M. (1997) Histopathology of in-stent restenosis in patients with peripheral artery disease. Circulation 95,1998-2002[Abstract/Free Full Text]
  4. Sarjeant, J. M., Rabinovitch, M. (2002) Understanding and treating vein graft atherosclerosis. Cardiovasc. Pathol. 11,263-271[CrossRef][Medline]
  5. Billingham, M. E. (1987) Cardiac transplant atherosclerosis. Transplant. Proc. 19,19-25[Medline]
  6. Sata, M., Perlman, H., Muruve, D. A., Silver, M., Ikebe, M., Libermann, T. A., Oettgen, P., Walsh, K. (1998) Fas ligand gene transfer to the vessel wall inhibits neointima formation and overrides the adenovirus-mediated T cell response. Proc. Natl. Acad. Sci. U. S. A. 95,1213-1217[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. (2002) Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat. Med. 8,403-409[CrossRef][Medline]
  8. Sata, M. (2003) Circulating vascular progenitor cells contribute to vascular repair, remodeling, and lesion formation. Trends Cardiovasc. Med. 13,249-253[CrossRef][Medline]
  9. Sata, M. (2003) Molecular strategies to treat vascular diseases. Circ. J. 67,983-991[CrossRef][Medline]
  10. Saiura, A., Sata, M., Hirata, Y., Nagai, R., Makuuchi, M. (2001) Circulating smooth muscle progenitor cells contribute to atherosclerosis. Nat. Med. 7,382-383[CrossRef][Medline]
  11. Shimizu, K., Sugiyama, S., Aikawa, M., Fukumoto, Y., Rabkin, E., Libby, P., Mitchell, R. N. (2001) Host bone-marrow cells are a source of donor intimal smooth-muscle-like cells in murine aortic transplant arteriopathy. Nat. Med. 7,738-741[CrossRef][Medline]
  12. Tanaka, K., Sata, M., Hirata, Y., Nagai, R. (2003) Diverse contribution of bone marrow cells to neointimal hyperplasia after mechanical vascular injuries. Circ. Res. 93,783-790[Abstract/Free Full Text]
  13. Fukuda, D., Sata, M., Tanaka, K., Nagai, R. (2005) Potent inhibitory effect of sirolimus on circulating vascular progenitor cells. Circulation 111,926-931[Abstract/Free Full Text]
  14. Caplice, N. M., Bunch, T. J., Stalboerger, P. G., Wang, S., Simper, D., Miller, D. V., Russell, S. J., Litzow, M. R., Edwards, W. D. (2003) Smooth muscle cells in human coronary atherosclerosis can originate from cells administered at marrow transplantation. Proc. Natl. Acad. Sci. U. S. A. 100,4754-4759[Abstract/Free Full Text]
  15. Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T., Nishimune, Y. (1997) Green mice as a source of ubiquitous green cells. FEBS Lett. 407,313-319[CrossRef][Medline]
  16. The National Institutes of Health (1985) Guide for the care and use of laboratory animals NIH Publication 86-23, NIH Bethesda, MD.
  17. Donskoy, E., Goldschneider, I. (1992) Thymocytopoiesis is maintained by blood-borne precursors throughout postnatal life. A study in parabiotic mice. J. Immunol. 148,1604-1612[Abstract]
  18. Wright, D. E., Wagers, A. J., Gulati, A. P., Johnson, F. L., Weissman, I. L. (2001) Physiological migration of hematopoietic stem and progenitor cells. Science 294,1933-1936[Abstract/Free Full Text]
  19. Okada, S., Yoshida, T., Hong, Z., Ishii, G., Hatano, M., Kuro-o, M., Nabeshima, Y., Nabeshima, Y., Tokuhisa, T. (2000) Impairment of B lymphopoiesis in precocious aging (klotho) mice. Int. Immunol. 12,861-871[Abstract/Free Full Text]
  20. 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. (2000) A mouse model of vascular injury that induces rapid onset of medial cell apoptosis followed by reproducible neointimal hyperplasia. J. Mol. Cell. Cardiol. 32,2097-2104[CrossRef][Medline]
  21. Tanaka, K., Sata, M., Fukuda, D., Suematsu, Y., Motomura, N., Takamoto, S., Hirata, Y., Nagai, R. (2005) Age-associated aortic stenosis in apolipoprotein E-deficient mice. J. Am. Coll. Cardiol 46,134-141[Abstract/Free Full Text]
  22. McKinney-Freeman, S., Goodell, M. A. (2004) Circulating hematopoietic stem cells do not efficiently home to bone marrow during homeostasis. Exp. Hematol. 32,868-876[CrossRef][Medline]
  23. Sahara, M., Sata, M., Matsuzaki, Y., Tanaka, K., Morita, T., Hirata, Y., Okano, H., Nagai, R. (2005) Comparison of various bone marrow fractions in the ability to participate in vascular remodeling after mechanical injury. Stem Cell 23,874-878[Abstract/Free Full Text]
  24. Yoshioka, T., Takahashi, M., Shiba, Y., Suzuki, C., Morimoto, H., Izawa, A., Ise, H., Ikeda, U. (2006) Granulocyte colony-stimulating factor (G-CSF) accelerates reendothelialization and reduces neointimal formation after vascular injury in mice. Cardiovasc. Res. 70,61-69[Abstract/Free Full Text]
  25. Han, C. I., Campbell, G. R., Campbell, J. H. (2001) Circulating bone marrow cells can contribute to neointimal formation. J. Vasc. Res. 38,113-119[CrossRef][Medline]
  26. Schafer, K., Schroeter, M. R., Dellas, C., Puls, M., Nitsche, M., Weiss, E., Hasenfuss, G., Konstantinides, S. V. (2006) Plasminogen activator inhibitor-1 from bone marrow-derived cells suppresses neointimal formation after vascular injury in mice. Arterioscler. Thromb. Vasc. Biol. 26,1254-1259[CrossRef][Medline]
  27. Sata, M., Tanaka, K., Nagai, R. (2003) Origin of smooth muscle progenitor cells: different conclusions from different models. Circulation 107,e106-e107[Free Full Text]
  28. Togni, M., Windecker, S., Wenaweser, P., Tueller, D., Kaisaier, A., Maier, W., Meier, B., Hess, O. M. (2004) Deleterious effect of coronary brachytherapy on vasomotor response to exercise. Circulation 110,135-140[Abstract/Free Full Text]
  29. Paris, F., Fuks, Z., Kang, A., Capodieci, P., Juan, G., Ehleiter, D., Haimovitz-Friedman, A., Cordon-Cardo, C., Kolesnick, R. (2001) Endothelial apoptosis as the primary lesion initiating intestinal radiation damage in mice. Science 293,293-297[Abstract/Free Full Text]
  30. Garcia-Barros, M., Paris, F., Cordon-Cardo, C., Lyden, D., Rafii, S., Haimovitz-Friedman, A., Fuks, Z., Kolesnick, R. (2003) Tumor response to radiotherapy regulated by endothelial cell apoptosis. Science 300,1155-1159[Abstract/Free Full Text]
  31. Cheng, S. W., Wu, L. L., Ting, A. C., Lau, H., Lam, L. K., Wei, W. I. (1999) Irradiation-induced extracranial carotid stenosis in patients with head and neck malignancies. Am. J. Surg. 178,323-328[CrossRef][Medline]
  32. Orzan, F., Brusca, A., Conte, M. R., Presbitero, P., Figliomeni, M. C. (1993) Severe coronary artery disease after radiation therapy of the chest and mediastinum: clinical presentation and treatment. Br. Heart J. 69,496-500[Abstract/Free Full Text]
  33. Yeh, E. T., Tong, A. T., Lenihan, D. J., Yusuf, S. W., Swafford, J., Champion, C., Durand, J. B., Gibbs, H., Zafarmand, A. A., Ewer, M. S. (2004) Cardiovascular complications of cancer therapy: diagnosis, pathogenesis, and management. Circulation 109,3122-3131[Abstract/Free Full Text]
  34. Mert, M., Arat-Ozkan, A., Ozkara, A., Aydemir, N. A., Babalik, E. (2003) Radiation-induced coronary artery disease. Z. Kardiol. 92,682-685[CrossRef][Medline]
  35. Veinot, J. P., Edwards, W. D. (1996) Pathology of radiation-induced heart disease: a surgical and autopsy study of 27 cases. Hum. Pathol. 27,766-773[CrossRef][Medline]
  36. Kostanecki, W., Radwan, I., Mroczkowski, T. (1976) The effect of X-ray irradiation on the epithelial melanin unit of the hair bulb in hooded rat. Arch. Dermatol. Res. 256,297-303[CrossRef][Medline]
  37. Liu, X., Liu, J. Z., Zhang, E., Li, P., Zhou, P., Cheng, T. M., Zhou, Y. G. (2005) Impaired wound healing after local soft X-ray irradiation in rat skin: time course study of pathology, proliferation, cell cycle, and apoptosis. J. Trauma 59,682-690[Medline]
  38. Frickhofen, N., Korbling, M., Fliedner, T. M. (1996) Is blood a better source of allogeneic hematopoietic stem cells for use after radiation accidents?. Bone Marrow Transplant. 17,131-135[Medline]
  39. Koneff, A. A., Van Dyke, D. C., Evans, H. M. (1952) Increase in the thyrotropic hormone content of blood after thyroidectomy as shown by parabiosis. Endocrinology 51,249-257[Medline]
  40. Harris, R. B. (1997) Loss of body fat in lean parabiotic partners of ob/ob mice. Am. J. Physiol. 272,R1809-R1815[Medline]
  41. Shoji, M., Sata, M., Fukuda, D., Tanaka, K., Sato, T., Iso, Y., Shibata, M., Suzuki, H., Koba, S., Geshi, E., Katagiri, T. (2004) Temporal and spatial characterization of cellular constituents during neointimal hyperplasia after vascular injury: Potential contribution of bone-marrow-derived progenitors to arterial remodeling. Cardiovasc. Pathol. 13,306-312[CrossRef][Medline]
  42. Sata, M. (2006) Role of circulating vascular progenitors in angiogenesis, vascular healing, and pulmonary hypertension: lessons from animal models. Arterioscler. Thromb. Vasc. Biol. 26,1008-1014[Abstract/Free Full Text]
  43. Ohtani, K., Egashira, K., Hiasa, K., Zhao, Q., Kitamoto, S., Ishibashi, M., Usui, M., Inoue, S., Yonemitsu, Y., Sueishi, K., Sata, M., Shibuya, M., Sunagawa, K. (2004) Blockade of vascular endothelial growth factor suppresses experimental restenosis after intraluminal injury by inhibiting recruitment of monocyte lineage cells. Circulation 110,2444-2452[Abstract/Free Full Text]
  44. Schober, A., Knarren, S., Lietz, M., Lin, E. A., Weber, C. (2003) Crucial role of stromal cell-derived factor-1alpha in neointima formation after vascular injury in apolipoprotein E-deficient mice. Circulation 108,2491-2497[Abstract/Free Full Text]
  45. Hoofnagle, M. H., Wamhoff, B. R., Owens, G. K. (2004) Lost in transdifferentiation. J. Clin. Invest. 113,1249-1251[CrossRef][Medline]
  46. Bentzon, J. F., Weile, C., Sondergaard, C. S., Hindkjaer, J., Kassem, M., Falk, E. (2006) Smooth muscle cells in atherosclerosis originate from the local vessel wall and not circulating progenitor cells in ApoE knockout mice. Arterioscler. Thromb. Vasc. Biol. 26,2696-2702[Abstract/Free Full Text]
  47. Balsam, L. B., Wagers, A. J., Christensen, J. L., Kofidis, T., Weissman, I. L., Robbins, R. C. (2004) Hematopoietic stem cells adopt mature hematopoietic fates in ischemic myocardium. Nature 428,668-673[CrossRef][Medline]
  48. Wagers, A. J., Sherwood, R. I., Christensen, J. L., Weissman, I. L. (2002) Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 297,2256-2259[Abstract/Free Full Text]
  49. Hoofnagle, M. H., Thomas, J. A., Wamhoff, B. R., Owens, G. K. (2006) Origin of neointimal smooth muscle: we’ve come full circle. Arterioscler. Thromb. Vasc. Biol. 26,2579-2581[Free Full Text]
  50. Skalli, O., Ropraz, P., Trzeciak, A., Benzonana, G., Gillessen, D., Gabbiani, G. (1986) A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. J. Cell Biol. 103,2787-2796[Abstract/Free Full Text]
  51. Owens, G. K., Kumar, M. S., Wamhoff, B. R. (2004) Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol. Rev. 84,767-801[Abstract/Free Full Text]
  52. Jabs, A., Moncada, G. A., Nichols, C. E., Waller, E. K., Wilcox, J. N. (2005) Peripheral blood mononuclear cells acquire myofibroblast characteristics in granulation tissue. J. Vasc. Res. 42,174180
  53. Iwata, H., Sata, M. (2007) Potential contribution of bone marrow-derived precursors to vascular repair and lesion formation: lessons from animal models of vascular diseases. Front. Biosci. 12,4157-4167[CrossRef][Medline]



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