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Cardiovascular Research Advance Access originally published online on October 7, 2007
Cardiovascular Research 2008 77(3):471-480; doi:10.1093/cvr/cvm034
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2007.For permissions please email: journals.permissions@oxfordjournals.org

Role of human smooth muscle cell progenitors in atherosclerotic plaque development and composition

Joffrey Zoll1, Vincent Fontaine2, Pierre Gourdy2, Veronique Barateau1, Jose Vilar1, Aurelie Leroyer1, Izolina Lopes-Kam1, Ziad Mallat1, Jean-Francois Arnal2, Patrick Henry3, Gerard Tobelem4 and Alain Tedgui1,*

1 INSERM U689, Hôpital Lariboisière, 41, Bd de la Chapelle, 75010 Paris, France
2 INSERM U589, Institut L. Bugnard,CHU Rangueil, 31403 Toulouse, France
3 Service de Cardiologie, Hôpital Lariboisière, Paris, France
4 Institut des Vaisseaux et du Sang, Hopital Lariboisiere, Paris, France

* Corresponding author. Tel: +33 1 53216695; fax: +33 1 42813128. E-mail address: tedgui{at}larib.inserm.fr

Received 28 March 2007; revised 14 September 2007; accepted 4 October 2007

Time for primary review: 18 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 Funding
 References
 
Aims: We analysed the possible protective role of human endothelial (EPCs) and smooth muscle (SPCs) progenitor cells on atherosclerosis development in apoE–/–RAG2–/– mice. We determined plasma levels of SPCs in coronary patients.

Methods and results: ApoE–/–RAG2–/– mice received four intravenous injections of saline, 5 x 105 SPCs, or 5 x 105 EPCs every other week, one (preventive approach) or 12(curative approach) weeks after starting a high fat diet. Derived-SPC levels were quantified from blood mononuclear cells of patients with stable angina (n = 10) and acute coronary syndromes (ACS, n = 9). SPCs reduced atherosclerosis development by 42% (P < 0.001), but had no effect on lesion progression. In the SPC group, collagen and smooth muscle cell content were increased (+80%, P < 0.001, +46%, P < 0.05, respectively), and macrophage content was decreased (–41%, P < 0.05). In the curative approach, macrophage content decreased by 40.5% (P < 0.05) after SPC injection. EPC injection had no effect on atherosclerosis development or progression. Peripheral blood-derived SPC levels were reduced in patients with ACS compared with stable angina patients (P < 0.05).

Conclusion: We demonstrate that SPCs limit plaque development and promote changes in plaque composition towards a stable phenotype in mice. Our finding in patients suggests that reduced peripheral blood-derived SPC levels might represent a mechanism contributing to plaque destabilization.

KEYWORDS Vascular progenitor cells; Cell therapy; Atherosclerosis; Coronary syndrome


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 Funding
 References
 
Both animal and human experiments have implicated vascular progenitor cells in the pathogenesis of vasculopathy and tissue regeneration.1–5 In several animal models, it has been demonstrated that haematopoietic stem cells are a potential source of smooth muscle (SPCs) or endothelial (EPCs) progenitor cells that can differentiate into smooth muscle cells (SMCs) or endothelial cells, respectively, contributing to pathological arterial remodelling in models of postangioplasty restenosis, transplant-associated atherosclerosis, and hyperlipidaemia-induced atherosclerosis.6 Other experiments suggested that EPCs could play a beneficial role in the development of atherosclerosis. In apolipoprotein E knockout mice (apoE–/–), it has been reported that bone marrow-derived cells may have no effect on atherosclerosis,7 or an atheroprotective effect, which is exhausted with aging and prolonged exposure to risk factors.8 However, the specific effects of vascular progenitor cell population on atherosclerosis development is still unknown, even though George et al.9 recently reported that spleen-derived EPCs may have pro-atherogenic effects.

It has been shown that patients with coronary artery disease (CAD) have reduced levels and functional impairment of EPCs, and that circulating EPC levels are inversely correlated with risk factors for atherosclerosis.10,11 The most accepted view is that EPCs participate in post-ischaemic neovascularization and contribute to tissue repair following ischaemia.12,13 Much less is known regarding the role of SPCs in atherosclerosis. In animal models of transplanted-related atherosclerosis, SMCs originating from circulating bone marrow-derived precursors4 have been shown to attach to the graft endothelia, differentiate into SMCs, proliferate and contribute to neointima formation in coronary arteries of the cardiac allograft.5 Moreover, it has been proposed that progenitor cells in the adventitia could contribute to the accumulation of SMCs in atherosclerotic lesions via direct migration across the media and/or possibly via circulating blood as well.14

However, in natural atherosclerosis, SMCs play a major role in the maintenance of plaque stability. Although rich SMC fibrous caps are able to withstand traumatic mechanical forces, poor SMC fibrous caps present areas of ‘vulnerability’ and plaque destabilization.15

SMC in the fibrous plaque are believed to originate from the underlying media. However, recent studies using gender-mismatched recipients and donors indicate that neointimal SMCs of infiltrating host cell origin can be found in human cardiac allografts.16 Also in bone marrow transplant subjects, SMCs of donor origin are markedly enriched in coronary atherosclerotic plaques.17 Interestingly, circulating SPCs have been identified in human peripheral blood.18 However, the implication of circulating SPCs in atherosclerosis development and plaque stabilization remains to be established. We hypothesized that circulating SPCs could contribute to atherosclerotic plaque stabilization and that a deficiency of these progenitor cells could represent a novel risk factor in patients with CAD.

We therefore studied the effects of injection of human SPCs in the development and progression of atherosclerosis in immune tolerant apoE–/–RAG2–/– mice, and compared with the effects of human EPCs. We also determined peripheral blood-derived SPC levels in peripheral blood of patients with acute coronary syndromes (ACS) as well as in patients with stable CAD.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusion
 Funding
 References
 
2.1 Experimental protocol
This investigation was carried out in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publications no. 85-23, revised 1996).

We used male 14-week-old apoE–/–RAG2–/– mice. Deficiency in the gene RAG2 results in a total deficiency of B and T lymphocytes, making these mice immuno-tolerant to human cells.

Mice were divided in three sets of experiments. In the first set of experiments, we evaluated the effect of vascular progenitor cells on the development of atherosclerosis. One week after starting a high fat diet, 32 apoE–/–RAG2–/– mice received intravenous injection of either saline (n = 10; PBS group), 5 x 105 SPCs (n = 10; SPC group) or 5 x 105 EPCs (n = 12; EPC group). The injections were repeated four times every other week. We used a similar protocol of progenitor cell injection as previously described7,8 with a slight modification of the number of injected cells. 5 x 105 cells instead of 1 x 106 cells were injected every 2 weeks, because in preliminary studies we observed that mice did not well tolerate this high number of cells. Mice were sacrificed 2 weeks after the last injection.

In a second set of experiments, we studied the effect of vascular progenitor cells on the progression of atherosclerosis. Twenty apoE–/–RAG2–/– mice were fed a high fat diet for 12 weeks and then received intravenous injection of either saline (n = 7; PBS group), 5 x 105 SPCs (n = 5; SPC group) or 5 x 105 EPCs (n = 8; EPC group). The injections were repeated four times every other week. The mice were sacrificed 2 weeks after the last injection.

In a third set of experiments, 12 apoE–/–RAG2–/– mice were fed a high fat diet for 6 weeks and then injected with 5 x 105 SPCs (n = 8) or 5 x 105 EPCs (n = 4). The mice were sacrificed 24 h or 4 days after injection in order to evaluate whether early incorporation of progenitor cells occurred in atherosclerotic lesions.

2.2 Smooth muscle and endothelial progenitor cell culture
SPCs and EPCs were obtained and cultured from human umbilical cord blood samples (30–50 mL each) as previously described.19 All experiments conformed with the declaration of Helsinki. Briefly, human umbilical cord blood samples were collected from healthy donors in compliance with French legislation. Mononuclear cells (MNCs) were isolated by density gradient centrifugation with Pancoll (1.077 g/mL) (Dominique Dutscher, Brumath, France). MNCs were cultured in M199 medium (Life Technologies, Cergy Pontoise, France) supplemented with 20% FCS, 15 mM HEPES, antibiotic and antimycotic solution (Life Technologies) and hVEGF (R&D Systems). Phenotypic characterization was assessed by immunocytochemistry and flow cytometry analysis as already described.19 Anti-human CD34 (8G12, Miltenyi Biotech), anti-human CD133 (AC133/2, Miltenyi Biotech), monoclonal anti-human VEGF receptor 2 biotin conjugated (KDR-2, Sigma), anti-human CD64 (22, Beckman Coulter), anti-human CD45 (KC-56, Beckman Coulter), anti-human CD14 (RMO52, Beckman Coulter), polyclonal rabbit anti-human vWF, (DAKO), anti-human CD31 (5.6E, Beckman Coulter), and anti-human calponin (M3556, DakoCytomation) antibodies were used as primary antibodies, and appropriate negative controls were done. Cytospin technique was used for KDR and calponin staining. Cellular uptake of acetylated LDL was measured as previously described19. Culture-expanded SPCs and culture-expanded EPCs used for injection were all from the Passage 6 or 7.

2.3 Quantitative assessment of atherosclerotic lesion size and composition
Mice were anesthetized with isoflurane before being euthanized by cervical fracture. Plasma cholesterol was measured using a commercial cholesterol kit (Sigma). The heart and aorta were removed, fixed in 4% paraformaldehyde, and placed in a PBS sucrose 30% solution overnight at 4°C, before being embedded in a cutting medium and frozen at –70°C. Successive 10-µm transversal sections of aortic sinus were obtained. Lipid deposition and collagen were detected using Oil red O and Sirius red staining, respectively. The presence of macrophages and SMCs was evaluated using a monoclonal rat anti-mouse macrophage antibody (clone MOMA-2, MAB1852; Chemicon), and a monoclonal anti-{alpha}-smooth muscle actin antibody (clone 1A4; Sigma), respectively. Computerized quantifications were performed by two blinded persons, using Histolab software.20 Presence of SPCs or EPCs in atherosclerotic plaques was estimated with a specific monoclonal anti-human nuclei antibody (MAB1281, Chemicon) as previously described.21 Slices of human atherosclerotic plaques were used as positive controls and slices from PBS group as negative controls (Figure 4C and D). At least four sections per mouse were examined for each immunostaining, and appropriate negative controls were done.

2.4 Real-time PCR analysis
Total RNA from lung, spleen, aorta, and liver were isolated using Trizol reagent (Invitrogen, Paris, France). Primer sequences for human and mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as followed: human GAPDH, forward, 5'-GAAGGTGAAGGTCGGAGTC-3', and GAPDH reverse, 5'-GAAGATGGTGATGGGATTTC-3'; mouse GAPDH, forward, 5'-CGTCCCGTAGACAAAATGGTGAA-3', and GAPDH reverse, 5'-GCCGTGAGTGGAGTCATACTGGAACA-3'. The real-time PCR was performed on an ABI prizm 7700 using Taqman Universal PCR master mix (Applied Biosystems) in triplicates. CT for mouse GAPDH was used to normalize the samples.

2.5 Isolation, cultivation and determination of circulating smooth muscle progenitor cells in blood samples of coronary patients
2.5.1 Characteristics of study patients
The characteristics of patients with CAD are summarized in Table 1. Ten patients had stable CAD, defined as angiographically documented CAD, and the absence of ACS for 3 months before blood samples were drawn. Nine patients were studied with ACS defined as troponin T positivity and/or ST-segment myocardial infarction. None of the patients had been treated with a statin before hospitalization.


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  		Table 1 Clinical characteristics of coronary patients

 
Inclusion criteria were age from 50 to 80 years and signed written informed consent. Exclusion criteria were clinical or biochemical evidence for the presence of concomitant inflammatory disease, chronic renal insufficiency, impaired left ventricular ejection fraction (<40%), autoimmune or malignant disease, anaemia (haemoglobin <8.5 g/dL), inability to understand the consent form.

2.5.2 Isolation and cultivation of human mononuclear cells
MNCs were initially isolated from peripheral blood samples (50–60 mL) in Histopaque-1077 followed by washing in PBS. MNCs were depleted of adherent cells by culturing on plastic dishes at 37°C for 24 h and were directly plated into one well of six-well plates coated with type I collagen (Becton Dickinson). MNCs from each patient were cultured in EGM-2 medium (PromoCell). After 7-day culture of MNCs, colonies of derived progenitor cells were counted. Colony consisted of multiple thin, flat cells emanating from a central cluster of rounded cells (Figure 5A). A central cluster alone without associated emerging cells was not counted as a colony. Colonies were counted manually in a minimum of 12 fields per well by observers who were unaware of the subjects’ clinical profiles. At 3 weeks, PDGF BB (50 ng/mL, R&D Systems) was added to the EGM-2 medium in order to facilitate SMC differentiation for 2 additional weeks18.

2.5.3 Immunocytochemistry
After 5 weeks of culture, cells from stable angina patients or patients with ACS were passaged and subsequently cultured on chamber slides (Lab-Tech, Poly Labo, Strasbourg, France) and fixed with cold 90% acetone solution. Nonspecific antibody binding was blocked by incubation with 5% FCS. A monoclonal mouse anti-human smooth muscle {alpha}-actin (Lab vision) and anti-human smooth muscle myosin heavy chain (SMHC, SMMS-1, DakoCytomation) antibodies were used as primary antibodies in order to stain culture-derived SPCs.

2.5.4 FACS analysis
Cells cultured in 6-well plates for 5 weeks were used for FACS analysis in order to quantify the number of SMHC positive cells. SMHC positive cells represent the number of culture-derived smooth muscle progenitor cells. After cell-permeabilization with 50% acetone/methanol, primary SMHC antibodies were used with secondary detection using a FITC-conjugated antibody. Isotype-matched IgG antibodies were used as control. FACS was also performed to verify that MNCs obtained one week after seeding did not expressed SMHC.

2.6 Statistical analysis
Results are expressed as mean ± SEM. One way ANOVA and post hoc Bonferonni's t-test comparisons were used to identify group differences.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusion
 Funding
 References
 
Animals weight and plasma total cholesterol levels did not differ between the three groups of mice receiving progenitor cells 1 week or 12 weeks after high fat diet (Table 2).


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  		Table 2 Serum cholesterol levels, atherosclerotic lesion size, and composition (mean ± SEM) in the aortic sinus of apoE–/–RAG–/– mice after smooth muscle and endothelial progenitor cells injections

 
3.1 Isolation and cultivation of smooth muscle and endothelial progenitor cells from human umbilical cord blood samples
In order to investigate the effect of different types of progenitor cells on atherosclerotic lesion development, we cultured CD34+-enriched cells from human umbilical cord blood samples on type I collagen in a medium containing 10 ng/mL VEGF, 1 ng/mL bFGF and 2 ng/mL IGF-1. We observed isolated starting clones after 7–15 days of culture. These clones, which apparently grew randomly, were of two cell types easily differentiated according to their morphology, one type being cobblestone and the other having a spindle-shaped morphology (Figure 1B). Colonies were picked and expanded for further analysis. SPCs and EPCs were characterized after the 6th or 7th passages. Both culture-expanded SPCs and culture-expanded EPCs expressed CD133, CD34, and KDR progenitor cell markers (Figure 1A). On the other hand, both vascular progenitor cell populations were negative for monocyte markers CD14, leukocyte marker CD45, and granulocyte marker CD64 (Figure 1A). Immunocytochemistry and flow cytometry analysis demonstrated that SPCs expressed calponin, a specific marker for SMC and were negative for specific endothelial cell markers (Figure 1B). Moreover, EPCs were negative for calponin but uniformly positive for endothelial markers including vWF, CD31, and only these cells incorporated acetylated LDL (Figure 1B).


Figure 1
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  		Figure 1 Characterization of progenitor cells obtained from human umbilical cord blood samples. Flow cytometry analysis and fluorescent immunostaining demonstrated that these outgrowth of EPCs and SPCs expressed CD133 and CD34 as well as KDR, but not CD64, CD45, and CD14 (A). Outgrowth progenitor cells morphology analysis showed cobblestone shaped endothelial progenitor cells and spindle-shaped smooth progenitor cells (B). Fluorescent immunostaining for vWF, acetylated-LDL uptake (Dil-ac-LDL) and calponin was studied for each cell type (B). Flow cytometry analysis demonstrated that outgrowth of endothelial progenitor cells expressed selectively CD31 (B). In each flow cytometry graph, the dotted line outlines the region of fluorescent intensity for cells labelled with negative control antibody. Black line outlines the region identifying cells labelled with antibody for the expression marker indicated in each graph.

 
3.2 Effect of cells injection on atherosclerotic lesion size and composition in apoE–/–RAG2–/– mice
3.2.1 Effect of smooth muscle and endothelial progenitor cells on atherosclerosis development
We analysed atherosclerotic lesion size in the aortic sinus of young apoE–/–RAG2–/– mice receiving four successive injections of 5 x 105 SPCs or EPCs (Table 2; Figure 2A) 1 week after starting a high fat diet. Quantitative computer assisted-image analysis showed a significant 42% decrease in plaque area in the aortic sinus of the SPC group of mice compared with controls (PBS group) (P < 0.001), whereas EPC injection had no effect on lesion size (Table 2; Figures 2 and 3).


Figure 2
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  		Figure 2 Extent of atherosclerotic lesions as well as relative macrophage infiltration and relative collagen and actin content in atherosclerotic lesions of young apoE–/–RAG–/– mice (first set of experiments, A) and old apoE–/–RAG–/– mice (second set of experiments, B). Immunohistochemical staining for macrophages and actin was performed using specific antibodies (see Methods). Significantly different from PBS group: *P < 0.05; **P < 0.01; ***P < 0.001.

 


Figure 3
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  		Figure 3 Representative photomicrographs show oil red O staining of atherosclerotic plaques (A), MOMA-2 staining (B, macrophages), Sirius red staining (C, collagen), and {alpha}-actin (D, black) in transversal sections from the aortic sinus of saline-treated (a), smooth progenitor cell-treated (b), or endothelial progenitor cell-treated (c) young apoE–/–RAG–/– mice (first set of experiments). Original magnification x40 (A) and x200 (B–D).

 
Analysis of plaque composition revealed a marked increase in the relative surface area occupied by collagen and SMC (+80%, P < 0.001 and +81%, P < 0.05, respectively, Figure 2A) after injection of SPC, whereas relative macrophage content was significantly decreased (–41%, P < 0.05). Only the absolute content of macrophages was decreased (P < 0.001), whereas the absolute collagen and SMC contents were not modified in SPC group (Table 2). EPC injections did not significantly affect the lesion composition (Table 2; Figures 2 and 3).

The presence of human SPCs and EPCs was analysed in atherosclerotic lesions using anti-human nuclei antibodies. No human SPCs or EPCs were detected in lesions two weeks after the last progenitor cell injection. However, a few injected SPCs were detected in atherosclerotic lesions one and 4 days after injection (Figure 4). No EPCs were detected in lesions even one or four days after injection. Using RT–PCR, we determine the presence of human GAPDH in different organs to assess where most of injected human cells were trapped. One day after injection, human GADPH was found highly expressed in lungs of animals injected with SPC (+430% compared with control). In the other tissues examined, human GAPDH was not significantly different from control.

3.2.2 Effect of smooth muscle and endothelial progenitor cells on atherosclerosis progression
In the second series of experiments, atherosclerotic plaques were already well developed before the first injection of progenitor cells, allowing us to study the effect of SPCs and EPCs on atherosclerotic plaque progression (Table 2; Figure 2B). Late SPC and EPC injections did not significantly affect the progression of atherosclerotic lesions. The absolute content of collagen was increased (+82%, P < 0.05), whereas macrophages and SMCs were not modified after SPCs treatment (Table 2). In the SPC group, concerning the relative surface area, a trend towards a higher relative collagen content (+52%, P = 0.09, Figure 2B) was observed, associated with a significant reduction in the relative macrophage content (–40.5%, P < 0.05, Figure 2B). SMCs were present at a very low level in these advanced plaques and were unchanged after SPC treatment. In the EPC group, there was no significant alteration in the lesion composition.

3.3 Characterization of peripheral blood-derived SPCs from coronary patients
The characteristics of patients with CAD (n = 19) are summarized in Table 1, and show absence of any difference between patients with CAD and patients with ACS in the atherosclerosis profile and medication used.

Peripheral blood-derived progenitor cells have been shown to mostly derive from bone marrow.22 To determine circulating levels of derived progenitor cells able to differentiate in SMC, SPCs were isolated from MNCs and selected ex vivo by culturing in a SMC-specific medium for 5 weeks. After 7-day culture of MNCs in collagen I coated plates with EGM-2 medium, an average of two colonies per field grew out (Figure 5A). We observed no difference in the number of colonies per field between stable patients with CAD and those with ACS (1.5 ± 0.5 vs. 1.9 ± 0.7, respectively). After 3 weeks in EGM-2 culture medium and an additional 2 weeks in PDGF BB-enriched medium, MNCs from stable CAD patients predominantly exhibited SMC characteristics with a ‘hill and valley’ morphology (Figure 5B). MNCs from CAD exhibited similar morphology (data not shown).

To more precisely identify the phenotype of outgrowth cells, cells were cultured on chamber slides and stained using antibodies against SMC-specific markers. Subconfluent cells stained positive for {alpha}-smooth muscle actin (Figure 5C) and SMHC (Figure 5D) in stable CAD patients. Cells stained using an isotype-matched control IgG antibody as well as the freshly isolated MNCs stained negatively for both SMC markers (data not shown).

The percentage and the total number of cells expressing the SMC specific markers were quantified by FACS (Figure 6). An average of 55% of cells were positive for smooth muscle-specific SMHC in the population of stable CAD and ACS patients after 5 weeks of culture, with no difference between the relative content of positive cells from patients with stable CAD and patients with ACS, respectively. More importantly, the absolute number of SMHC-positive cells was markedly reduced in patients with ACS compared with that in patients with stable CAD (5212 ± 2200 vs. 19588 ± 7600 cells, respectively, P < 0.05) (Figure 6D).


Figure 4
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  		Figure 4 Representative photomicrographs showing immunopositive cells stained with anti-human nuclei antibodies 24 h (A) and 4 days (B) after smooth progenitor cell injections (third set of experiments). Negative control was represented in (C), and positive control in (D). Arrows show human nuclei staining. Original magnifications x200.

 


Figure 5
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  		Figure 5 Characterization of cultivated Mononuclear cells from coronary patients. Outgrowth cell morphology at week 1 (A) and at week 5 in stable coronary artery disease patients (B). Spindle-shaped cells were positive for {alpha}-smooth muscle actin (C) and smooth muscle myosin heavy chain (D) in stable coronary patients as demonstrated by immunostaining analysis.

 


Figure 6
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  		Figure 6 FACS analysis of intracellular smooth muscle myosin heavy chain in peripheral blood–derived smooth muscle progenitor cells of stable coronary artery disease patients (B), and in peripheral blood–derived smooth muscle progenitor cells of acute coronary syndrome patients (C). The isotype-matched control IgG antibodies were represented in (A). These graphs are representative of smooth muscle myosin heavy chain positive cells in both groups of patients. The absolute number of smooth muscle myosin heavy chain-positive smooth muscle progenitor cells in patients with stable coronary artery disease and acute coronary syndromes is represented in (D). *P < 0.05 compared with stable coronary artery disease.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusion
 Funding
 References
 
In the present study, we found that only injection of well-characterized SPCs significantly limits the development of atherosclerosis in mice, whereas EPCs injection had no effect. Importantly, SPCs injections promote substantial changes in plaque composition towards a more stable phenotype, both during the development and the progression of atherosclerosis. In coronary patients, we found reduced peripheral blood-derived SPC levels in patients with ACS compared with stable angina patients.

4.1 Beneficial effects of circulating smooth muscle progenitor cells on atherosclerosis development in mice
Studies with gender-mismatched recipients and donors suggest a potential role of putative circulating SPCs in transplant associated atherosclerosis,16,17 and a study has already shown that abundant progenitor cells in the adventitia can differentiate into SMCs, which might contribute to the accumulation of SMCs in atherosclerotic lesions.14

However, the actual contribution of purified population of progenitor cells, especially SPCs, on the development of non-transplant atherosclerosis is currently unknown. So far, only the effects of non-differentiated bone marrow-derived cells have been experimentally investigated in atherosclerosis. One study reported that bone marrow-derived cells have an atheroprotective effect, which is exhausted with aging and prolonged exposure to risk factors.8 In contrast, we observed in apoE–/– mice with hindlimb ischaemia that bone marrow-derived cell therapy promotes further atherosclerotic plaque progression, without affecting atherosclerotic plaque stability,7 whereas stem cells had no effects in control mice without ischaemia. Therefore, the effects of vascular progenitor cells in atherosclerosis still remain controversial and could differ, depending of the vascular progenitor cell type.

In human, it has been shown that some circulating vascular progenitors are capable of differentiating into both endothelial and smooth muscle-like cells1,18,23. However, no studies evaluated the direct effects of vascular progenitor cells purified and differentiated into endothelial and smooth muscle-like cells on atherosclerosis development.

In the present study, we treated apoE–/–RAG2–/– mice with outgrowth cells from human vascular progenitor cells19. We purified CD34+ cells from human cord blood and cultivated them until the 6th passages. We isolated starting clones of two cell types easily differentiated according to their morphology. These cells expressed progenitor cell markers, including CD133, CD34, and KDR. Cells injected to the SPC group had a spindle-shaped morphology, were negative for endothelial and inflammatory cells markers but positive for calponin, suggesting a smooth muscle progenitor cells origin. Cells injected to the EPC group had a cobblestone morphology and displayed features of endothelial cells including CD31, vWF markers, as well as the functional capacity of incorporating acetylated LDL, suggestive of a EPCs origin.

In order to use human progenitor cells, we had to use RAG2-deficient mice that have a persistent combined immunodeficiency with immature, dysfunctional B and T lymphocytes. It is noteworthy that there is no difference in lesion size and progression to fibrous lesions or in the surface area covered by lesions in fat diet-fed apoE–/–RAG2–/– mice compared with apoE–/– mice fed a chow diet24,25.

We showed that chronic injection of SPCs, but not EPCs, limited the development of atherosclerotic lesions. This treatment promoted substantial changes in plaque composition towards a more stable phenotype both during the development and progression of atherosclerosis. In mice receiving SPCs, the marked relative increase in SMC accumulation in the atherosclerotic lesions resulting in higher collagen content compared with control, might account for decreased infiltration of inflammatory cells. On the other hand, in young mice, the absolute content of collagen and SMCs were not significantly altered, whereas the absolute content in macrophages was significantly decreased, suggesting that the decrease of atherosclerotic lesions was essentially due to the decrease of macrophages infiltration. In old mice, lesions were already well developed at the time of progenitor cell injections, and it is known that lesion size progresses very slowly thereafter in apoE–/– mice.26 This can account for the lack of effect of SPCs on lesion size in this series of experiments. An important finding of our work is that SPC injection favored collagen accumulation and decreased macrophage infiltration in late atherosclerosis. Moreover, the macrophage content was reduced without any significant reduction in the lesion area. This was likely due to the parallel increase in collagen content (Table 2). Twenty-four hours later, and to a lesser extend 4 days after SPC injection, we detected a few SPCs present in atherosclerotic lesions. The absence of detection of incorporated SPCs 2 weeks after the last cell injection could be due to their elimination by NK cells. Indeed, these NK cells remain active in RAG animals and could account for the absence of detectable cells. On the other hand we cannot rule out the presence of scattered and undetectable SPCs in the lesions 2 weeks after the last injection for reasons of sensitivity of the method of detection.

The mechanisms allowing beneficial effects of SPCs treatment are largely unknown. In our study, we showed that SPCs could physically contribute to prevent lesion progression in the days following injection. On the other hand, we cannot rule out the possibility that the antiatherogenic activity of these progenitors was due to systemic rather than local effects, especially by releasing cytokines and growth factors that limit inflammation and favour SMC proliferation and production of extracellular matrix proteins.

According to the fact that stem cells have some more subtle roles than cell replacement,27,28 we can hypothesize that after invasion at sites of injury, SPCs release factors that protect other cells, especially synthetic SMCs, and stimulate collagen synthesis, and decrease inflammatory responses22,29.

As opposed to SPC treatment, culture-expanded EPCs had no effect on atherosclerosis development. Moreover, in a recent report, George et al.9 found that transfer of spleen-derived EPCs can even increase atherosclerotic lesions in apoE–/–. Simper et al.18 previously showed that integrin {alpha}5β1 expression was strongly increased in SPCs compared with EPCs, with a eight-fold greater adhesion of SPC to fibronectin than EPCs. This might account for the presence of a few SPCs in plaque while no EPCs were observed. Because EPCs were not found in the atherosclerotic vessels, their effect on local mechanisms of atherogenesis could not be determined in this model. The beneficial effects of SPCs might result from the potential secretion of a number of growth factors that could stimulate collagen synthesis by SMCs in the plaque. Further studies are required to evaluate this hypothesis.

Even though reduced levels of circulating EPCs have been shown to independently predict atherosclerotic disease progression in human,10,11,30 EPC-based strategy seems to be more relevant in order to rescue from ischaemia by promoting repair of blood vessels and/or facilitating neovascularization of ischaemic tissue than directly acting on the development of the atherosclerotic plaque. However, our results have to be interpreted in the light of differences in structure between mouse and human lesions.

4.2 Circulating smooth muscle progenitor cells level in coronary patients
Patients with stable angina and ACS included in our study had similar atherosclerosis profile and medication used. We found outgrowth of peripheral blood-derived SPCs in coronary patients, in agreement with previous observations in healthy human subjects.18 The total number of peripheral blood-derived progenitor cells expressing SMC specific markers was decreased in patients with ACS compared with stable angina patients. This measurement of peripheral blood-derived SPCs suggested an important reduction of these progenitor cells in patients with ACS compared with stable angina patients. These results are in line with studies demonstrating that the number of circulating EPCs, measured in blood or after cultivation, was significantly correlated with the number of risk factors.10,11 It is noteworthy that 70% of patients with ACS have complex plaque morphology or thrombi, whereas no complex lesions are seen in patients with stable angina31. Moreover, it has been suggested that ACS that are refractory to medical treatment can be caused by unstable pathologic processes in the plaque.32 In addition, ACS usually result from rupture of a vulnerable atherosclerotic plaque characterized by a thin fibrous cap and the presence of inflammatory cells.33 Our study suggests that deficiency in peripheral blood-derived SPCs in patients with ACS might contribute to plaque vulnerability. As we found that SPCs specifically decreased the development of atherosclerosis and stabilize atherosclerotic lesions in mice, it is tempting to speculate that circulating SPCs participate in plaque stabilization in human.15 Further experiments are required to establish whether endogenous circulating SPCs are recruited in the atherosclerotic plaques, whether they are able to release growth factors that could protect SMCs from death, and whether they are able to stimulate the production of extracellular matrix proteins leading to increased plaque stability and decreased macrophage infiltration, as in our mice model.

4.3 Limitations
Even though our measurement of peripheral blood-derived SPCs suggested an important reduction of these progenitor cells, we have not identified the actual cause(s) for the decrease of the absolute number of SPCs after 5 weeks of culture. This may result from different proliferation rates, survival rates and/or differences in differentiation propensity. However, we believe that the difference in the absolute number of SMHC-positive cells observed between the two groups of patients is indicative of the marked reduction of the number of SPCs and/or the important changes in their characteristics in patients with ACS. Further studies in large cohorts of patients are needed to confirm and define the precise mechanisms of decreased SPCs in these patients.


   5. Conclusion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
Funding
 References
 
Therapeutic strategies to treat vulnerable plaques are the focus of a large body of experiments and clinical research. We showed that only chronic SPC injection, but not EPC injection, limits atherosclerotic lesion formation and stabilizes the atherosclerotic plaque in mice, suggesting that anti-atherosclerotic effects of progenitors cells is highly depend on the type of vascular progenitor cells injected. All together, our animal and human findings suggest that reduced SPC levels might represent a mechanism contributing to plaque destabilization. Our findings participate to a better understanding of the pathophysiology of plaque vulnerability and rupture, and might eventually lead to a new therapeutic strategy for plaque stabilization.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 Funding
References
 
This work emanates from the European Vascular Genomics Network (http://www.evgn.org), a Network of Excellence supported by the European Community's sixth Framework Programme for Research (contract no LSHM-CT-2003-503254).


   Acknowledgements
 
We wish to thank Sophie Lericousse who gave to us the SPC and EPC.

Conflict of interest: none declared.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 Funding
 References
 

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