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Cardiovascular Research Advance Access first published online on September 25, 2008
This version [Corrected Proof] published online on October 22, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn265
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

The origin of post-injury neointimal cells in the rat balloon injury model

Luis Rodriguez-Menocal1, Melissa St-Pierre1, Yuntao Wei1, Sheik Khan1, Dania Mateu1, Marian Calfa1, Amir A. Rahnemai-Azar1, Gary Striker1,2, Si M. Pham1 and Roberto I. Vazquez-Padron1,*

1 Division of Cardiothoracic Surgery, Department of Surgery, Vascular Biology Institute, University of Miami, Miller School of Medicine, 1600 NW 10th Avenue, RMSB 1063, Miami, FL 33136, USA
2 Mt. Sinai School of Medicine, 1 Gustave Levy Place, New York, NY 10029, USA

* Corresponding author. Tel: +1 305 243 1154; fax: +1 305 243 5636. E-mail address: rvazquez{at}med.miami.edu

Received 17 June 2008; revised 10 September 2008; accepted 23 September 2008

Time for primary review: 44 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Aims: The origin of post-injury neointimal cells is still a matter of debate. This study aims to determine the anatomic source of neointimal cells in one of the most important animal models for the study of vascular stenosis in response to injury, the rat balloon injury model.

Methods and results: Chimeric rats were generated by rescuing lethally irradiated animals with green fluorescent protein (GFP)+ bone marrow (BM) cells from transgenic rats. Neointimal formation was induced in the right iliac artery of these animals using a balloon angioplasty catheter. Injured and non-injured contra-lateral arteries were harvested at 7, 14, and 30 days post-surgery. BM-derived monocytes/macrophages (CD68+ GFP+) were abundant in the media and adventitia of injured vessels harvested at 7 days as determined by immunofluorescence and confocal microscopy. The number of GFP+ cells declined in the vascular wall with time. Post-injury neointimal cells were mostly GFP–/smooth muscle actin (SMA)+, which indicated that those cells originated in the recipient. Only a few neointimal cells seemed to come from circulating progenitors (GFP+ SMA+, 2.34% ± 1.61). The vascular origin of cells in the neointima was further confirmed by transplanting injured GFP arteries into wild-type recipients. In these grafts, 94.23 ± 0.44% of medial and 92.95 ± 19.34% of neointimal cells were GFP+ SMA+. Finally, we tested the capacity of vascular smooth muscle cells (VSMC) to migrate through the vascular wall using a novel in vivo assay. As expected, VSMC migrated and populated the neointima only in response to injury.

Conclusion: Our results suggest that neointimal cells in the rat balloon injury model mostly derive from pre-existing vascular cells and that only a small population of those cells come from BM-derived progenitors.

KEYWORDS Restenosis; Neointima; Balloon injury; Rat; Angioplasty; Cell origin


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Atherosclerosis and post-angioplasty restenosis are histologically characterized by the development of an enlarged neointima. The main cellular component of this neointima is the synthetic smooth muscle cell. The initial hypothesis explaining the pathophysiology of neointima formation proposed that neointimal cells originated from quiescent medial vascular smooth muscle cells (VSMC) that switch from a contractile to a synthetic phenotype to migrate to the intima and proliferate, in response to injury.1–3 This theory was supported by the apparent increase in proliferation among the tunica media cells after injury4–7 and by the ex vivo development of neointima in arterial culture systems.8–10 It was also supported by the fact that neointima cells expressed VSMC markers, smooth muscle alpha actin (SMA), and vimentin.11–13 This theory has recently been challenged by Sata et al.14,15 who proposed that VSMC of injured and atherosclerotic arteries are of bone marrow (BM) origin. On the other hand, the idea that cells within the vascular microenvironment are more plastic is becoming more prevalent. Current data have revealed the plasticity of periadventitial fibroblasts,16,17 adventitial progenitors,18 and/or blood-borne cells19 to differentiate into neointimal cells.

Using one of the most important animal models in studying vascular stenosis, the rat balloon injury model, we demonstrate that the participation of BM derived-cells in the healing of injured arteries is limited to inflammatory cells and that only a small population of neointimal cells come from circulating progenitors. Our results clearly show that in this animal model neointimal cells have mostly a vessel wall origin. We also demonstrate that synthetic VSMC have the ability to migrate across the vascular wall and populate the neointima in response to injury.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
2.1 Transgenic animals
Transgenic inbred Lewis rats that express green fluorescent protein (GFP) under the control of ubiquitin-C promoter20 were obtained from the Rat Resource and Research Center (Columbia, MO) and bred in our laboratories. Wild-type (WT) inbred Lewis rats were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN). Animal procedures were approved by the Institutional Committee for Use and Care of Laboratory Animals at the University of Miami and conform to the Guide for the Care and Use of Laboratory Animals published by US National Institutes of Health (NIH publication No. 85-23, revised 1996).

2.2 Generation of chimeric rats
GFP BM mononuclear cells were isolated from femurs and tibias of transgenic and WT rats. Recipient animals were lethally irradiated with a single dose of 1025 cGy from a Cs-137 source (Nordion, Ontario) and immediately received one dose of 8 x 107 GFP BM cells via the jugular vein. Control chimeric rats were reconstituted with WT BM cells. The chimerism was assessed by flow cytometry (BD LSR System I) in peripheral blood 1 month after transplant and in the BM after sacrificing. The 7ADD staining was used to gate out dead cells and debris. Flow cytometry data was analysed using Flowjo 7.2 (Ashland, OR).

2.3 Balloon injury model
All surgeries were under isoflurane anaesthesia. Vascular injury was performed according to Gabeler et al.21 Briefly, an aortotomy in the abdominal aorta was made to insert a 2F Fogarty embolectomy catheter to the level of the right iliac artery. The balloon was inflated to 1.5 atmospheres and retracted to the arteriotomy site three times to assure a good vascular injury. The aortic excision was repaired with 8.0 sutures. The abdominal cavity was closed by planes using interrupted suture pattern.

2.4 Aortic transplantation
Descending thoracic aortas were balloon-injured before harvesting. Vascular grafts included adventitia and most of the surrounding connective tissue. GFP and WT thoracic aortas were transplanted to the abdominal aorta of WT and GFP recipients, respectively, by side to-side with interrupted anastomoses. In the control group, non-injured GFP and WT arteries were transplanted into WT recipients. WT injured aortas were also transplanted into chimeric rats. No immunosuppressive or anticoagulant treatment was used in this study.

2.5 Tissue processing
Arterial specimens were collected 7–30 days after injury or transplant and fixed in 4% formalin–PBS. Arteries were cut in three pieces and paraffin-embedded in the same block. Sections of 5 µm thick were mounted on Superfrost/Plus glass slides.

2.6 Morphometric analysis
The area of each vascular layer and lumen was measured on Elastic Van Gieson stained slides. These measurements were used to calculate the neointima to media ratio [N/M = N/(M + N)]. All morphometric measurements were performed on digital images using the Image Pro Plus (Media Cybernetics, Inc., Bethesda, MD) computer software.

2.7 Immunofluorescence and scanning confocal microscopy
Sections were re-hydrated by serially immersing them in xylene, alcohol, and water. Antigens were retrieved by boiling slides in 10 mM citrate buffer, pH 6.0 for 20 min. Unspecific binding was avoided using TBS-FBS 15% (Tris–Borate Saline buffer supplemented with 15% Fetal Bovine Serum) for 20 min. Sections were incubated overnight with goat anti-GFP polyclonal antibodies (1:100, Abcam, Cambridge, MA) and mouse anti-human SMA clone 1A4 (1:50, Dako) in TBS-FBS 10%. Bound antibodies were detected with Alexa Fluor 488 donkey anti-goat and Alexa Fluor 546 goat anti-mouse (Invitrogene, Carlsbad, CA). Alternatively, mouse anti-rat CD68 (1:100, AbD Serotech, Raleigh, NC) monoclonal antibody followed by goat anti-mouse Alexa Fluor 546 (Invitrogene) was used to detect infiltrated monocytes/macrophages. Sections were DAPI counterstained (Sigma, St Louis, MO) and mounted in Cytoseal (Richard-Allan Scientific, Kalamazoo, MI). The immunofluorescent staining protocol was validated for potential cross-reactivity of primary and secondary antibodies in sections of injured arteries from WT rats. The specificity of anti-GFP antibodies were further validated by western blot.

The stained sections were examined with a confocal scanning laser microscope Zeiss LSM 510 META (Carl Zeiss MicroImaging, Inc., Thornwood, NY) in an inverted configuration. The system is equipped with four lasers and three confocal detectors, and data were captured and analysed with Zeiss LSM 510 Meta and Image Browser software (Carl Zeiss). Dual antibody-stained images were acquired with the use of sequential capture mode to avoid potential fluorescence bleed-through between channels. All images were captured with a plain-neofluor 40x/1.3 Oil DIC objective lens. Up to 12 optical slices of 0.5 µm in depth each were recorded for every sample. The three grayscale images were electronically merged to produce a pseudocolored image in which blue depicts cell nuclei, red depicts either SMA or CD68 immunoreactivity, and green depicts GFP.

2.8 Vascular smooth muscle cells isolation and characterization
VSMC were isolated from thoracic aortas of GFP rats and were kept under conventional culture conditions. Primary cell lines were characterized by immunostaining using anti-SMA monoclonal antibody (1:10, Dako) and by RT–PCR. The primers and amplification conditions for rat GAPDH (450 bp), SMA (292 bp), SM22{alpha} (179 bp), SM1/2 (299 bp), SM-MHC (85 bp), CD34 (362 bp), CD31 (225 bp), and eNos (226 bp) have been describe already.22,23

2.9 In vivo migration assays
Thirty million GFP VSMCs between passages 4–10 were embedded in 1 mL of BD Matrigel Matrix (BD Biosciences, San Jose, CA). The cell suspension (250 µL) was applied on the perivascular area of both injured and non-injured iliac arteries. Matrigel was allowed to solidify for 10 min before closing the surgical excision. VSMC migration and engraftment in the neointima was detected by immunofluorescence and confocal microscopy with anti-GFP and SMA antibody as described earlier.

2.10 Statistical analysis
Results were expressed as mean±SEM. Initially, all morphometric and histopathological parameters were tested for normality (Shapiro–Wilk test) and homogeneity of variances (Hartley’s F max test). Most of variables in this study were neither normally distributed nor homoscedastic, even after data transformations. Therefore, overall variability among groups was assessed with a non-parametric Kruskal–Wallis one-way ANOVA model. If the null hypothesis was rejected, the Dunn’s multiple comparison test was used to delineate differences among groups. All statistical procedures were calculated and/or plotted with Statistic 7.0 (StatSoft, Tulsa, OK) and GraphPad Prism 5 (GraphPad Software, La Jolla, CA) computer software.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Supplementary material
 Funding
 References
 
3.1 The origin of neointimal cells in chimeric rats
First, we validated that GFP was constitutively expressed in the vasculature of transgenic rats during arterial remodelling. GFP expression was tracked using immunofluorescence and confocal microscopy. The neointimal development was similar between transgenic and WT rats in response to balloon injury (N/M ratio of 0.19 ± 0.08 vs. 0.21 ± 0.05, n = 5, n.s). GFP was homogenously expressed in endothelial, smooth muscle, and neointimal cells before and after arterial remodelling (Figure 1A–F). No cross-reactivity was observed in WT sections from injured and non-injured arteries, which were used as controls (Figure 1G–I). Anti-GFP antibodies were specific for the transgene and no cross-reactivity to rat arterial endogenous proteins was detected by western blot (see Supplementary material online, Figure S1). Therefore, GFP expression and its detection using antibodies was a reliable, sensitive method for detecting BM-derived cells.


Figure 1
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  		Figure 1 Assessment of GFP transgene expression in rat iliac arteries by immunofluorescence and confocal microscopy. Green fluorescent protein (green) and smooth muscle actin (SMA, red) were detected in injured (A–C) and non-injured (D–F) arteries of green fluorescent protein transgenic rats. Green fluorescent protein was co-localized with smooth muscle actin in medial and neointimal cells of transgenic rats (yellow). Injured arteries of wild-type rats were used as negative controls for staining with anti-green fluorescent protein polyclonal antibody to demonstrate that there was no cross-reactivity with local antigens (H–I). Nuclei were counterstained with DAPI (blue). The internal elastic lamina (IEL) that limits the boundaries between the intima and the media is indicated. Magnification x760.

 
To assess the role of BM-derived cells in the remodelling of the arterial wall after injury, chimeras were created by rescuing lethally irradiated WT rats with GFP+ BM cells from transgenic animals. Flow-cytometric analysis 1 month following cell transplantation revealed that in chimeras 95.40 ± 8.21% of blood peripheral cells were from donor BM (see Supplementary material online, Figure S2). This agreed with the 92.43 ± 0.36% of GFP+ cells found in the BM of those animals at sacrifice. Once chimeras were established, neointimal development was induced by balloon injuring the right iliac artery. Chimeras were sacrificed at 7, 14, and 30 days post-surgery and injured and uninjured arteries were harvested for histopathological analysis.

Seven days after injury, GFP+ cells were found in the media and in the adventitia of the remodelled arteries (Figure 2A). These GFP+ cells were mainly monocytes/macrophages as determined by double immunostaining for CD68 and GFP (Figure 3). Furthermore, those GFP+ cells did not stain positively for SMA, showing that they were not of the smooth muscle cell lineage (Figure 2A). The numbers of infiltrating BM-derived GFP+ cells in the media and adventitia decreased as the arteries remodelled. GFP+ cells decreased three-fold from 7–30 days after injury (Figure 2D and E). At this time, only BM-derived cells remained in the perivascular region of the injured arteries. These cells were negative for macrophage and VSMC markers (data not shown). Only a few GFP+ SMA+ neointimal cells (34.12 ± 25.10 cells per mm2 or 2.3%, n = 6) were found. No GFP+ cells were found in the vascular wall of non-injured arteries, although they were present in the adventitial connective tissue. Interestingly, the arterial neointima of the chimeras was less thickened than that in the WT rats (N/M ratio 0.1784 ± 0.03 vs. 0.29 ± 0.03, P = 0.04, n = 5) (see Supplementary material online, Figure S3). Neointimas of control chimeras (lethal irradiated WT rats reconstituted with WT BM) were also thinner than those developed in the injured arteries of non-irradiated rats (N/M ratio 0.15 ± 0.01 vs. 0.29 ± 0.03, P = 0.02, n = 5). The neointimal development was similar between the GFP and control chimeras (N/M ratio 0.17 ± 0.03 vs. 0.15 ± 0.01, P = 0.43). It excludes the possibility that the transgene in the BM cells was playing a role in the different outcome of the N/M ratio between GFP chimeras and the WT non-irradiated rats.


Figure 2
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  		Figure 2 Post injury neointimal cells are of local origin in the rat balloon-injury model. Injured and non- injured iliac arteries of chimeric rats were harvested at 7, 14, and 30 days post-injury and stained with anti-green fluorescent protein (green) and anti-smooth muscle actin (red) antibodies (A–C). Positive cells were visualized by immunofluorescence and confocal microscopy. Typical GFP+ SMA– cells at 7 days are labelled with white arrowheads (A). One of the few GFP+ SMA+ cells is labelled with a yellow arrowhead. Green fluorescent protein cells significantly diminished in the vascular wall at 14 days after surgery (B). Neointimal cells were GFP– SMA+ (C). The internal elastic lamina (IEL) limits the boundaries between intima and the media is indicated. Nuclei were DAPI counterstained (blue). Magnification x760. Bar graphs at the bottom represent the number of GFP+ cells in the media (D) and adventitia (E) of injured arteries of chimeric rats at different time points. Bars represent the mean±SEM of n = 5–6.

 


Figure 3
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  		Figure 3 Inflammatory cells involved in the arterial response to balloon-injury are of BM origin. The lineage of BM-derived cell in the arterial wall was determined by immunofluorescence and confocal microscopy with specific antibodies. GFP+ cells (green) in the arterial wall of injured arteries at 7 days post-injury (A). Most of those cells were positive for the macrophage marker CD68 (red, B). Typical GFP+ CD68+ cells are labelled with arrowheads (C). Nuclei were counterstained with DAPI (blue). Magnification x760.

 
3.2 The origin of neointimal cells in arterial grafts
We used an arterial transplant model to assess the contribution of vascular wall cells and recipient cells to neointimal development in rats. Arterial grafts were balloon-injured before transplantation to accentuate development of the neointima after placing the graft. Donor injured aortas were transplanted including the adventitia and most of the surrounding connective tissues. Grafted and native arteries were harvested 1 month after transplant. The N/M ratios of the GFP and WT aortic grafts transplanted into WT and GFP recipients, respectively, were similar (0.38 ± 0.03 vs. 0.45 ± 0.03, P = 0.14). The graft neointima was thicker than that found in the balloon injury model noted above (N/M ratio of 0.45 ± 0.02 vs. 0.17 ± 0.03, P = 0.002). Most of the medial (94.23 ± 0.44% or 395 ± 27 cells per mm2) and neointimal (92.95 ± 19.67% or 1495 ± 127 cells per mm2) cells of aortas from GFP rats transplanted into WT recipients were GFP+/SMA+ as determined by immunofluorescence and confocal microscopy (Figure 4A–C). In contrast, when WT injured aortas were transplanted into GFP rats, only 2.68 ± 1.2% (41.32 ± 11.23 cells per mm2) of the medial cells and 4.88 ± 2.13% (83.33 ± 31.22 cells per mm2) of the neointimal cells were doubly positive (Figure 4D–F). Macrophages (CD68+ cells) were rarely found in the vascular wall of the grafts at sacrifice (data not shown). The control groups where non-injury GFP and WT aortic grafts were transplanted into WT recipients developed no neointima, ruling out the existence of GFP-induced alloreactivity that could interfere with our results (see Supplementary material online, Figure S4).


Figure 4
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  		Figure 4 Neointimal cells of aortic isografts are of donor origin. Green fluorescent protein and wild-type injured aortas were transplanted in the abdominal aorta of wild-type (A–C) and green fluorescent protein (D–E) recipients, respectively. GFP+ smooth muscle cells were detected by immunofluorescence and confocal microscopy using anti-green fluorescent protein (green) and anti-smooth muscle actin antibodies (red). Nuclei were DAPI counterstained (blue). The internal elastic lamina (IEL) limits the boundaries between the intima and the media is indicated. Magnification x760.

 
The contribution of BM-derived cells to the origin of neointimal cells was further assessed by transplanting injured WT aortas into GFP chimeric recipients. The neointima of these grafts was significantly smaller than those of non-irradiated recipients (N/M ratios 0.24 ± 0.06 vs. 0.41 ± 0.02, P < 0.01, All n = 5). Only 3.20 ± 1.13% (58.71 ± 24.87 cells per mm2) of the neointimal and 5.81 ± 3.38 (18.47 ± 14.35 cells per mm2) of the medial cells of those grafts were GFP and SMA positive (see Supplementary material online, Figure S5).

3.3 Migration and engraftment of synthetic vascular smooth muscle cells to the neointima
The above results strongly suggest that neointimal cells originate in the local vascular wall. Therefore, the migration of smooth muscle cells into the intima might be a necessary prerequisite for the formation of a neointima.24 We developed a model to determine if VSMC migrate across the vascular wall in response to injury. This consisted of seeding Matrigel-embedded GFP+ VSMC or BM cells outside of injured and control arteries. Control rats received Matrigel which contained no cells. Prior to seeding, we had found that 99.23% of the VSMC used for seeding were GFP positive by FACS and fluorescence microscopy and expressed the VSMC markers, SMA, SM22a, SM1/2, and SM-MHC by IHC or RT–PCR (Figure 5A–C). These cells were free of endothelial contamination as shown by RT–PCR (Figure 5C).


Figure 5
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  		Figure 5 Vascular smooth muscle cells migrated through the media to the neointima in response to vascular injury. Vascular smooth muscle cells constitutively expressed the green fluorescent protein transgene (A) and smooth muscle cell actin (B) as detected by immunohistochemistry. These cells expressed the smooth muscle cell markers {alpha}-SMA, SM22a, SM1/2, and SM-MHC by RT–PCR, but not the endothelial cell markers, CD34, CD31, and eNos (C). Matrigel-embedded GFP+ vascular smooth muscle cells were seeded around injured arteries of wild-type rats. The GFP+ SMA+ cells (arrowheads) were detected in the neointima 3 weeks after balloon injury by immunofluorescence and confocal microscopy (D–F). Injured artery treated with BM-embedded cells (H–I). No GFP+ cells were found in the neointima of those animals. The internal elastic lamina (IEL) limits the boundaries between intima and the media is indicated. Magnification x380.

 
The N/M ratio was larger in injured arteries that received embedded VSMC than in those treated with BM cells (0.28 ± 0.04 vs. 0.17 ± 0.02, P = 0.08) or matrigel alone (0.15 ± 0.02, P = 0.02). The matrigel-embedded cells did not induce visible vascular lesions in the control, non-injured arteries. The number of VSMC (GFP+ and SMA+) which migrated and engrafted into the neointima in response to injury was 10 times greater in arteries that received Matrigel-embedded VSMC cells than in those in which the Matrigel contained BM cells (21.79 ± 5.15 vs. 1.58 ± 0.45%, P < 0.01, Figure 5D–G). No GFP cells were present in the media of rats which received Matrigel containing BM cells (data not shown).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Supplementary material
 Funding
 References
 
The initial studies of vascular restenosis suggested that the tunica media was the main source of neointimal cells based on the observation that VSMC were rapidly labelled with thymidine after injury.4,7 The current study supports this initial hypothesis and utilizes new experimental approaches. Our data indicate that most of post-injury neointimal VSMC originate from local vascular cells and not from circulating progenitors in rats. The participation of the BM in arterial remodelling after injury appears to be limited to the inflammatory stage of the vascular remodelling process. Only a minor population of VSMC in the neointima had a BM origin. Our results also showed that VSMCs migrate across the vascular wall in response to injury and participate in the formation of the neointima.

Herein, we demonstrated by transplanting injured arteries between GFP and transgenic rats and by injuring the arteries of GFP BM reconstituted chimeras that the main anatomic source of cells in the rat post-injury neointima is the vascular wall. The outcomes from these experiments showed that the neointima of balloon-injured GFP arterial grafts transplanted into WT recipients were mainly populated with GFP+/SMA+ cells. Furthermore, only few GFP+/SMA+ cells were found in the neointimas of chimeric rats that received GFP BM after lethal irradiation. These experimental evidences suggest that neointimal cells in this animal model were either from local tunica media cells or from adventitial pericytes/smooth muscle progenitors.

These findings contribute substantially to the very confusing literature existing in the area of the neointimal cell origin. Our results are in agreement with a series of recent studies which show little or no contribution of BM-derived cells to SMC lineages in a variety of models including arteriogenesis,25 atherosclerosis,26 tumour angiogenesis,27,28 and graft vasculopathy.18,29 For instance, the fact that post-injury neointimal cells are of local origin concurs with Bentzon et al.26 studies which showed that VSMC in atherosclerotic plaques came from the local vessel wall and not from circulating progenitors as claimed by Sata et al.14,15 In addition, neointimal cells in the mouse vein-to-artery graft models were found to originate from adventitial progenitors and not from the BM.18,30 However, our results are in disagreement with other studies suggesting that a majority of cells in the neointima comes from BM-derived circulating progenitors that engraft the vascular wall in response to atherogenic stimuli.28,31 Although these results have been partially reproduced, they remain controversial.32,33

There are substantial biological and methodological differences between our experiments and those that support a BM origin of neointimal cells that may account for the contrasting outcomes. Our studies were performed in rats and vascular injury was induced using an embolectomy balloon. Reports finding great contribution of marrow cells to the neointimal cell population were performed with the wire-injury model in the mouse. It is well-accepted that species have different mechanisms of vascular remodelling in response to injury.34 On the other hand, it is also known that the method of inducing arterial damage can affect the recruitment of BM-derived cells to the site of injury. For example, Tanaka et al.35 found that wire injury, but not blood cessation or cuff placement, mobilized BM cells to the neointima. Furthermore, a profound difference exists between our study and those that claimed that most of neointimal cells come from BM with respect to the histological methods utilized to track cells into the vascular wall. Data that support a BM origin of neointimal cells were derived from the direct observation of GFP+ cells in unfixed tissue. This histopathological procedure does not provide enough resolution to discriminate between GFP+ positive and the negative cells in the arterial background.36–38 The use of unfixed tissues may also lead to diffusion of the tracer marker from sectioned cells.39 We detected GFP+ cells in fixed tissues by immunofluorescence microscopy using antibodies which do not cross-react with self antigens of injured and non-injured arteries from WT animals. This allowed the differentiation of GFP+ cells from the neighbouring negative cells. The GFP+ cells had a defined morphology and the target protein was always intracellularly located.

The outcomes of this study also differ from human data. Using sex mismatched specimens, it has been shown that there are smooth muscle cells of donor origin in the plaques of human coronary atherosclerosis.40 The discrepancies between these findings and our results using the balloon-injury models could be explained by the different pathophysiological mechanisms underlying atherosclerosis and post-injury stenosis. Even though these vascular occlusive diseases share certain mechanistic elements including VSMC proliferation, they have different dynamics and pathological characteristics. In addition, the specimens were collected from patients exposed to variables like immunosuppression, chemotherapy, and graft-vs.-host disease that were not included in the current study. The differences between the human data and our studies could also be explained through the known limitation of animal models used for vascular stenosis/restenosis. Those major limitations in our case are the absence of a primary lesion (atheroma) in the target vessel and dyslipidaemia that modifies VSMC biology.

The source of the neointimal cells in the rat balloon injury model appeared to be from a local site. However, it is not yet proved that neointimal cells are derived from pre-existing contractile VSMC. The existence of stem cells have been recently documented in the vascular wall,41 perivascular area,42 and adventitia.18 The fact that neointimal cells would have an origin in local progenitor cells would explain the arterial repopulation after the massive apoptosis that occurs after injury.43 It is possible that neointimal cells have a clonal origin from contractile VSMC precursors that develop a synthetic phenotype after injury.44 Further research is necessary to find out which of the cell types in the arterial wall serve as the source of the neointima.

The fact that neointimal vascular smooth muscle cells do not appear to originate from the BM does not imply that BM-derived cells have no role in the development of the neointima. BM-derived inflammatory cells, mainly macrophages, are abundant in the arterial wall early after injury. The number of those cells decreases with time. Thus, cells derived from the BM may control the remodelling and initiation of healing in the injured blood vessel and these cells may also provide signals for the mobilization and recruitment of the VSMC that repopulate the injured arteries. This hypothesis is supported by the observation that inhibition of macrophage infiltration after injury significantly diminishes neointimal formation.45

Finally, we assessed whether VSMC or BM cells can migrate across the arterial wall in response to injury. We found that VSMC from primary cultures, but not BM mononuclear cells, migrate from the adventitial region into the intima in response to injury. The fact that BM cells do not appear to migrate through the arterial wall has previously been shown in the vein-to-graft transplant model.18 It is also notable that injured arteries treated with embedded VSMC developed a thicker neointima than those treated with BM or matrigel alone. This suggests that actively proliferating VSMC derived from the outer layers of the artery could contribute to the population of neointimal cells.

In conclusion, our results demonstrate that the majority of neointimal cells are of local origin in the balloon-injury model of the rat. We also found that the function of BM-derived cells in the pathological vascular remodelling is to serve as a source of inflammatory cells. Therefore, further experiments that dissect molecular mechanisms by which local progenitors are recruited and differentiate at the site of injury are warranted.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
Funding
 References
 
Supplementary material is available at Cardiovascular Research online.

Conflict of interest: none declared.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
References
 
This work was supported by awards from the American Heart Association (Scientist Development Award 0535167B) and Stanley Glaser Foundation.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 

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  4. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium. Lab Invest (1983) 49:327–333.[Web of Science][Medline]
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