The number and function of circulating CD34+CD133+ progenitor cells decreased in stable coronary artery disease but not in acute myocardial infarction
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* Takahisa Kondo
* Satoshi Shintani
* Kengo Maeda
* Mutsuharu Hayashi
* Yasuya Inden
* Yasushi Numaguchi
* Kaichiro Sugiura
* Yasuhiro Morita
* Tomoya Kitamura
* Haruo Kamiya
* Takahito Sone
* Miyoshi Ohno
* Toyoaki Murohara

## Abstract

**Objective** Circulating CD34+CD133+ cells are one of the main sources of circulating endothelial progenitor cells (EPCs). Age is inversely related to the number and function of CD34+CD133+ progenitor cells in stable coronary artery disease (CAD), but the relationship remains unclear in acute myocardial infarction (AMI). The authors aimed to clarify how ageing affects the number and function of mobilised CD34+CD133+ progenitor cells in AMI.

**Design and results** Circulating CD34+CD133+ progenitor cells were measured by flow cytometry. Measurements were made at admission for CAD, or on day 7 after the onset of AMI. In stable CAD (n=131), circulating CD34+CD133+ cells decreased with age (r=−0.344, p<0.0001). In AMI, circulating CD34+CD133+ cells did not correlate with age (n=50), and multivariate analysis revealed that the decreased number of circulating CD34+CD133+ cells was associated with male sex and higher peak creatinine kinase. The ability to give rise to functional EPCs, which show good migratory and tube-forming capabilities, deteriorated among stable CAD subjects (n=10) compared with AMI subjects (N=6).

**Conclusions** In stable CAD, the number and function of circulating CD34+CD133+ progenitor cells decreased with age, whereas those mobilised and circulating in AMI did not.

*   CD34
*   CD133
*   EPCs
*   chronic stable angina
*   acute myocardial infarction
*   age
*   angina—stable
*   vascular biology

Bone marrow-derived endothelial progenitor cells (EPCs) are known to maintain cardiovascular homeostasis.1–3 In a non-ischaemic condition, circulating EPCs constitute a pool of progenitor cells that repair the injured endothelium.3 In an ischaemic condition, EPCs are mobilised from bone marrow to peripheral blood, home to the ischaemic sites, and proliferate at the local foci.4 5 Studies have demonstrated that circulating EPC levels are reduced by the accumulation of cardiovascular risk factors such as diabetes,6 smoking,7 hypertension8 and hypercholesterolaemia,8 and that the number and function of circulating EPCs are inversely correlated with the endothelial dysfunction.2 Furthermore, circulating EPC levels are reported to predict future cardiovascular events.9 10 Therefore, EPC is now considered to be a biomarker of cardiovascular diseases.

Ageing is one of the strongest risk factors for cardiovascular disease. Previous investigations demonstrated that age is associated with a reduced number and function of EPCs, circulating CD34+/KDR+ cells, or CD133+ cells in healthy individuals and patients with coronary artery diseases (CAD).8 11 However, there are very few reports concerning the effects of ageing on CD34+CD133+ progenitor cells in acute myocardial infarction (AMI) subjects.12

Therefore, we aimed to clarify the potential effects of ageing on circulating CD34+CD133+ progenitor cells in patients with stable CAD and in patients with AMI. We also sought to examine whether the functions of CD34+CD133+ cell-derived EPCs differ between CAD and AMI. We examined only never-smoking subjects, because smoking exerts detrimental effects on circulating CD34+CD133+ cells and EPCs.7

## Methods

### Study patients

Patients were recruited from Nagoya University Hospital, Japanese Red Cross Nagoya Daiichi Hospital or Ogaki Municipal Hospital from January 2004 to December 2006. We examined circulating CD34+CD133+ cell levels in 131 consecutive non-smoking patients with stable CAD and circulating CD34+CD133+ cell levels in 50 AMI non-smoking patients. We previously demonstrated that circulating EPC and CD34+ cell levels peaked at day 7 after the onset of AMI.13 Therefore, we investigated circulating EPC and CD34+CD133+ cell levels on day 7 after the onset of AMI.14 Definitions of stable CAD or AMI and criteria for coronary risk factors were written in detail in the online data supplement files (please see [http://heart.bmj.com/](http://heart.bmj.com/)).

### Blood sampling and medical treatment

In subjects with stable CAD, peripheral blood (PB) samples were collected on the day before the day of coronary angiography. In subjects with AMI, PB samples were collected 7 days after the onset of AMI. Detailed description regarding blood examination and medical treatment are written in the online supplement files (please see [http://heart.bmj.com/](http://heart.bmj.com/)).

### Circulating CD34+CD133+ progenitor cells and functional assessments of endothelial progenitor cells

Methods regarding quantification of CD34+CD133+ progenitor cells and functional assessments of endothelial progenitor cells (EPCs) are described in detail in the online supplement files (please see [http://heart.bmj.com/](http://heart.bmj.com/)).

### Statistical analysis

Data are presented as the mean±SD or median (25–75th percentile) for continuous variables, and as percentages for categorical variables. Correlations between the variables were assessed using the Pearson correlation analysis (r) or the Spearman rank correlation coefficient (ρ) as appropriate. Multiple stepwise regression analyses were performed to determine the factor to affect the circulating CD34+CD133+ cell levels in AMI patients. Comparisons between the in vitro experimental groups were performed using ANOVA followed by Tukey–Kramer test. In all analyses, p values <0.05 were considered statistically significant. All statistical analysis was performed using Statview 5.0-J (SAS Institute, Cary, North Carolina).

## Results

The characteristics of the study patients are shown in tables 1, 2. The number of circulating CD34+CD133+ cells was significantly lower in subjects with stable IHD (734 counts/ml) compared with subjects with AMI (1655 counts/ml) (p<0.0001).

View this table:
[Table 1](http://heartasia.bmj.com/content/2/1/20/T1)

Table 1 
Characteristics of patients with stable coronary artery disease

View this table:
[Table 2](http://heartasia.bmj.com/content/2/1/20/T2)

Table 2 
Characteristics of patients with acute myocardial infarction

### CD34+CD133+ cells decreased with age in subjects with stable CAD

In subjects with stable CAD, the number of circulating CD34+CD133+ cells decreased linearly with age (ρ=−0.360, p<0.0001) (figure 1A). Multiple regression analysis, using diabetes mellitus, hypertension, sex, ‘statin use’ and age as dependent variables, revealed that age was the only and significant factor determining the circulating CD34+CD133+ cell levels in subjects with CAD.

![Figure 1](http://heartasia.bmj.com/https://heartasia.bmj.com/content/heartasia/2/1/20/F1.medium.gif)

[Figure 1](http://heartasia.bmj.com/content/2/1/20/F1)

Figure 1 
(A) Relationship between circulating CD34+CD133+ cell levels and age in subjects with stable coronary artery disease (CAD). The number of circulating CD34+CD133+ decreased linearly with age (ρ=−0.0360, p<0.001). (B) Relationship between circulating CD34+CD133+ cell levels and age in subjects with acute myocardial infarction (AMI). The number of circulating CD34+CD133+ cells 7 days after AMI was not associated with age.

### Relationship between CD34+CD133+ cell levels and age in patients with AMI

In contrast to subjects with stable CAD, circulating CD34+CD133+ cell levels on day 7 after the onset of AMI did not correlate with age (figure 2B). Multiple stepwise regression analyses using sex, age, hypertension, diabetes mellitus, ‘statin use,’ peak CK values, BNP, CRP and VEGF as dependent variables indicated that only sex (male) and high peak CK value were the significant and negative factors to predict circulating CD34+CD133+ cell levels on day 7 after the onset of AMI. There were no interaction effects between sex and peak CK levels.

![Figure 2](http://heartasia.bmj.com/https://heartasia.bmj.com/content/heartasia/2/1/20/F2.medium.gif)

[Figure 2](http://heartasia.bmj.com/content/2/1/20/F2)

Figure 2 
Migratory activity and network forming activity of endothelial progenitor cells (EPCs) differing between subjects with chronic coronary artery disease (CAD) and subjects with acute myocardial infarction (AMI). (A) Migratory activity of CD34+CD133+ cell-derived EPCs assessed by modified Boyden chamber methods. Both in subjects <65 years old and in subjects ≥65 years old, migratory activity was significantly enhanced in EPCs from AMI compared with EPCs from stable CAD (p<0.05). Interestingly, enhanced migratory activity of EPCs was observed even in AMI subjects ≥65 years old. (B) Similar to migratory activity, the ability of network-forming activity of CD34+CD133+ cell-derived EPCs with human umbilical vein endothelial cells was improved in AMI compared with stable CAD. The statistically significant difference was only observed in subjects ≥65 years old.

### Functional assessment of CD34+CD133+ cell-derived EPCs

EPCs were obtained after cultivation of circulating CD34+CD133+ cells. The migratory activity of EPCs from male subjects with AMI, whether younger or elder, was significantly enhanced compared with that of EPCs from the corresponding age male subjects with stable CAD (p<0.05, respectively) (figure 2A). Similarly, the number of EPCs incorporated into the endothelial network formation was significantly increased in older adult subjects with AMI compared with older adult subjects with stable CAD (p<0.05) (figure 2B).

## Discussion

Major findings in this study were as follows: (1) In subjects with stable CAD, circulating CD34+CD133+ cell levels decreased with age. (2) In subjects with AMI, (a) there was no significant relationship between the number of mobilised CD34+CD133+ cells and age, and (b) peak CK level and sex (male) were negatively associated with the number of mobilised CD34+CD133+ cells. (3) As for the functions of CD34+CD133+ cell-derived EPCs, both the migratory activity and the network-forming ability with HUVECs were superior in subjects with AMI to those with stable CAD.

Circulating EPCs are known to play an important role not only in ischaemia-induced angiogenesis but also in healing the injured endothelium.1 3 4 However, the results from clinical trials involving administration of autologous bone marrow cells to patients with critical limb ischaemia or myocardial infarction are not consistent.15–18 One of the reasons for this discrepancy may be related to the fact that most patients were targeted at older subjects, whereas in animal models, most experiments were performed in young mice.19–21

Circulating CD34+ or CD133+ cells are reported to decrease with age in subjects with stable CAD.3 This explains, in part, the varying efficacy of autologous bone marrow cell transplantation into critical ischaemic limb or ischaemic myocardium. However, in the present study, the inverse relationship between age and circulating CD34+CD133+ cells was not confirmed in subjects with AMI. Our findings are consistent with the description about CD34+/CD117+ and CD34+/CXCR4+ cells in AMI by Wojakowski *et al.*22 These findings imply that, although circulating CD34+CD133+ cells decrease in patients with stable CAD, some patients still have the capacity to release CD34+CD133+ cells into the circulation from bone marrow in the event of AMI. Signals from ischaemic tissue may be responsible for stimulating the release of CD34+CD133+ cells. So far, VEGF is known to be one of the major angiogenic cytokines released from ischaemic tissues.3 However, VEGF did not correlate with circulating CD34+CD133+ levels in the present study. Because VEGF levels increase quickly after the onset of AMI and return to the normal range,23 the relationship between CD34+CD133+ levels and VEGF could not be observed as late as day 7. Alternatively, other angiogenic chemokines such as Angiopoietin-124 and SDF-13 may be a dominant factor affecting the mobilisation of CD34+CD133+ cells around 7 days after the onset of AMI.

In the present study, the migratory activity of EPCs and network-forming ability of EPCs were superior in subjects with AMI compared with those with stable CAD. In AMI, increased inflammatory and angiogenic cytokine/chemokine, such as VEGF13 and SDF-1,3 might improve the functions of the EPCs mobilised from bone marrow. A similar phenomenon was reported by Vöö *et al*, who found an enhanced chemotactic response of CD133+ circulating progenitor cells following AMI.25

Recently, the precise origin of these EPCs has become the subject of debate.26 Nonetheless, the importance of EPCs in maintaining vascular homeostasis remains unchanged, because they have been used clinically to induce angiogenesis,15–18 We can safely state that circulating CD34+CD133+ cells and these cell-derived EPCs are maintained in at least some AMI subjects.

The peak CK level and sex (male) were negatively associated with CD34+CD133+ cell mobilisation in AMI. Our findings are consistent with the study by Wojakowski *et al* indicating that the number of mobilised stem cells is negatively correlated with cardiac necrosis markers (Troponin I, CK-MB), and that the peak number of CD34 cells was shown to be significantly lower than in patients with LVEF≤40%.22 The high value of peak CK in the present study was associated with lower LVEF, which made for the mobilisation of CD34+CD133+ cells after AMI decreased.

The fact that the number of mobilised CD34+CD133+ was lower in men was also consistent with the report by Hoetzer *et al*, which clarified that the EPC colony-forming capacity and migratory activity were higher in women than in men.27 Although oestrogen is known to mobilise EPC from bone marrow, our female subjects were all in postmenopause. Thus, it is unlikely that oestrogen affected the mobilisation of EPCs in the present study. The circulating EPC level might be intrinsically higher in women than in men, which would explain one mechanism of slow atherosclerotic progression in women.

Limitations of our study include the fact that we measured CD34+CD133+ cell levels and EPC levels at a single time after the onset of AMI. However, we previously confirmed that EPC and CD34+ levels peaked during 4–7 days after the onset.13 Therefore, the number measured on day 7 after the onset could be used to indicate the circulating CD34+CD133+ cell level in subjects after AMI. Second, other types of EPCs derived from myeloid and mesenchymal were not taken into consideration. Serial blood samplings would be warranted to elucidate the behaviour of CD34+CD133+ cell and EPC levels in both CAD and AMI. Additionally, different types of EPCs should also be investigated.

In conclusion, the number of circulating CD34+CD133+ cells decreased with age in non-smoking subjects with stable CAD, but not in subjects with AMI. Furthermore, the functions of CD34+CD133+ cell-derived EPCs in subjects with AMI did not deteriorate, even in older people. Taken together, some older adults with CAD may well have the capacity to mobilise and utilise residual EPCs in the event of AMI, even if the circulating CD34+CD133+ cell level is low.

## Acknowledgments

We thank M Aoki and R Miura for their technical assistance.

## Footnotes

*   Funding This work was supported by grants from Nagoya University Graduate School of Medicine, and a grant from the Smoking Research Foundation.

*   Competing interests None.

*   Ethics approval Ethics approval was provided by the ethical committee of Nagoya University Graduate School of Medicine (Institutional No 68).

*   Provenance and peer review Not commissioned; externally peer reviewed.

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    [PubMed](http://heartasia.bmj.com/lookup/external-ref?access_num=17196460&link_type=MED&atom=%2Fheartasia%2F2%2F1%2F20.atom) 
    
    [Web of Science](http://heartasia.bmj.com/lookup/external-ref?access_num=000243283000009&link_type=ISI)