Author + information
- Received February 14, 2006
- Revision received January 11, 2007
- Accepted January 22, 2007
- Published online June 19, 2007.
- Christine K. Kissel, MD1,
- Ralf Lehmann, MD,
- Birgit Assmus, MD,
- Alexandra Aicher, MD,
- Jörg Honold, MD,
- Ulrich Fischer-Rasokat, MD,
- Christopher Heeschen, MD,
- Ioakim Spyridopoulos, MD,
- Stefanie Dimmeler, PhD and
- Andreas M. Zeiher, MD⁎ ()
- ↵⁎Reprint requests and correspondence:
Dr. Andreas M. Zeiher, Department of Cardiology, University of Frankfurt, Theodor Stern-Kai 7, 60590 Frankfurt, Germany.
Objectives This study investigated whether reduced levels of circulating endothelial progenitors cells (EPCs) in chronic heart failure (CHF) are secondary to an exhaustion of hematopoietic stem cells (HSCs) in the bone marrow or to reduced mobilization.
Background Circulating EPCs presumably originate from bone marrow-derived HSC. Persistent mobilization of EPCs was shown to be associated with favorable left ventricular infarct remodeling processes.
Methods We assessed the number and functional capacity of EPCs in 17 healthy controls, 25 patients with ischemic cardiomyopathy (ICM), and 20 patients with dilated cardiomyopathy (DCM). To document an impairment of HSC function in the bone marrow, the colony-forming unit capacity of bone marrow–derived mononuclear cells and the number of CD34+HSCs were examined in 6 healthy volunteers, 94 ICM patients, and 25 DCM patients.
Results The number of EPCs was reduced in CHF, irrespective of its etiology. In contrast, the migratory capacity was selectively impaired in EPCs of ICM patients (4.8 ± 4.0 migrated cells; DCM 9.7 ± 5.8; p = 0.02). On multivariate analysis, ICM, advanced New York Heart Association functional class, and CHF were independent predictors of functional EPC impairment. The number of bone marrow-derived CD34+cells did not differ between the CHF populations. However, colony-forming units (CFUs) were selectively reduced in ICM patients (54.4 ± 24.6; DCM 68.1 ± 26.9; p < 0.02). Ischemic cardiomyopathy was the only independent predictor of impaired CFU capacity. Impaired CFU capacity was associated with reduced matrix metalloproteinase-9 activity in the bone marrow plasma.
Conclusions Ischemic cardiomyopathy is associated with selective impairment of progenitor cell function in the bone marrow and in the peripheral blood, which may contribute to an unfavorable left ventricular (LV) remodeling process.
Previous studies have identified a population of presumably bone marrow-derived cells, which circulate with the blood (1–3), express a variety of endothelial surface markers (4), incorporate into sites of neovascularization (5–7), and home to sites of endothelial denudation (8–10). Importantly, the level of these so-called circulating endothelial progenitor cells (EPCs) not only correlates with cumulative cardiovascular risk (11) and vascular function (12), but also predicts future cardiovascular events and atherosclerotic disease progression in patients with coronary artery disease (CAD) (13,14).
Recently, advanced stages of heart failure were shown to be associated with reduced levels of circulating EPCs (15). More importantly, persistent mobilization of EPCs correlates with favorable left ventricular (LV) remodeling as evidenced by prevention of LV dilation and enhanced contractile recovery in patients with acute myocardial infarction (MI) (16). These clinical observations were corroborated by experimental data convincingly showing that the failure of mobilizing EPCs in endothelial nitric oxide synthase knockout mice, which show a profound impairment in ischemia-induced mobilization of EPCs (17), abrogates the beneficial effect of statin therapy on LV remodeling and contractile recovery after experimentally induced MI (18). Taken together, these data suggest that circulating EPCs not only contribute to vascular repair, but also may be involved in modulating LV remodeling processes leading to postinfarction heart failure. However, there are currently no data to delineate whether heart failure itself, independent of its etiology, impairs EPC number and function. Moreover, a reduced number of circulating EPCs may be secondary to a variety of mechanisms, including exhaustion of the pool of progenitor cells in the bone marrow, impaired functional capacity within in the bone marrow, reduced mobilization of EPCs, or reduced survival and/or differentiation of mobilized EPCs. Thus, the present study was designed to address the aforementioned questions and to begin to dissect some of the underlying mechanisms driving the differences in progenitor cells derived from patients with chronic heart failure (CHF).
Patients between 18 and 85 years of age were eligible for inclusion in the study. Patients from whom bone marrow-derived cells were obtained were recruited from the patient cohort undergoing intracoronary cell infusion at our institution. Patients in whom circulating EPCs were investigated were recruited from our outpatient heart failure clinic. Patients with ischemic cardiomyopathy (ICM) had to have angiographic evidence of CAD and were required to have had a previous MI at least 3 months before inclusion into the study with persistent well-demarcated regional LV dysfunction by echocardiography or LV angiography and a patent infarct-related artery.
Patients with nonischemic dilated cardiomyopathy (DCM) were required to have angiographically normal coronary arteries and globally reduced LV ejection fraction without segmental wall motion abnormalities. The DCM patients had to be in stable condition by echocardiography and clinical symptoms for at least 3 months before inclusion into the study. There were no specific requirements for global LV ejection fraction to meet a predefined threshold for inclusion into the study. However, patients in New York Heart Association (NYHA) functional class I were only included if they were under intensive pharmacological treatment.
Exclusion criteria were the presence of acutely decompensated heart failure with NYHA functional class IV; a history of leucopenia, thrombocytopenia, or severe hepatic and renal dysfunction; evidence for inflammatory or malignant disease; or unwillingness to participate. The ethics review board of the Johann Wolfgang Goethe University of Frankfurt, Germany, approved the protocol, and the study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from each patient.
Cardiovascular risk factors
The overall risk factor load of an individual patient, using a a risk factor score including age above 40 years, male gender, hypertension, diabetes, smoking, family history for CAD, and hypercholesterolemia, was calculated (modified from Vasa et al. ).
Measurement of EPC number (culture assay)
Mononuclear cells were isolated by density gradient centrifugation with Biocoll (Biochrom, Berlin, Germany) from 20 ml peripheral blood as previously described (11). Immediately after isolation, 4 × 106mononuclear cells were plated on 24-well culture dishes coated with human fibronectin (Sigma-Aldrich, Munich, Germany) and maintained in endothelial basal medium (Cambrex, Walkerville, Maryland) supplemented with endothelial growth medium SingleQuots and 20% fetal calf serum. After 4 days in culture, nonadherent cells were removed by thorough washing with phosphate-buffered saline (PBS).
Characterization of EPCs
To detect the uptake of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (DiLDL), cells were incubated with DiLDL (2.4 μg/ml) at 37°C for 1 h. Cells were then fixed with 2% paraformaldehyde for 10 min and incubated with fluorescein-5-isothiocyanate (FITC)-labeled Ulex europaeusagglutinin I (lectin, 10 μg/ml; Sigma-Aldrich, Munich, Germany) for 1 h. Dual-staining cells positive for both lectin and DiLDL were judged as EPCs and counted per well. The endothelial characteristics were additionally documented by flow cytometry analysis of vascular endothelial growth factor receptor 2 (KDR) and von Willebrand factor (19). The number of EPCs per well was evaluated by counting 3 randomly selected high-power fields.
Measurement of functional capacity of EPCs (migrating capacity)
Isolated EPCs were detached using 1 mmol/l ethylenediaminetetraacetic acid in PBS (pH 7.4), harvested by centrifugation, resuspended in 500 μl endothelial basal medium, counted, and placed in the upper chamber of a modified Boyden chamber (2 × 104cells; BD Bioscience, Heidelberg, Germany). The chamber was placed in a 24-well culture dish containing endothelial basal medium, 20% fetal calf serum, and human recombinant vascular endothelial growth factor (VEGF) (50 ng/ml; R&D Systems, Wiesbaden, Germany). After 24 h incubation at 37°C, the lower side of the filter was washed with PBS and fixed with 2% paraformaldehyde. For quantification, cell nuclei were stained with 4′,6′-diamidino-2-phenylindole. Cells migrating into the lower chamber were counted manually in 3 random microscopic fields.
Bone marrow mononuclear cells
Bone marrow–derived mononuclear cells (BM-MNCs) were isolated from bone marrow aspirates by density gradient centrifugation. After 2 washing steps, cells were resuspended in X-vivo 10 medium (Cambrex, Verviers, Belgium). The cell suspension consists of heterogeneous cell populations, including hematopoietic progenitor cells.
Flow cytometry analysis of BM-MNCs
For the identification of hematopoietic stem/progenitor cell populations, we used directly conjugated antibodies against human CD45 (mouse FITC-labeled; BD Pharmingen, Heidelberg, Germany), human CD34 (FITC-labeled and allophycocyanin-labeled, BD Pharmingen) and human CD133 (allophycocyanin-labeled, Miltenyi Biotec, Bergisch-Gladbach, Germany).
Colony-forming unit assay
The BM-MNCs (1 × 105per dish) were seeded in methylcellulose plates (Methocult GF H4535, Stem Cell Technologies, Vancouver, Canada) including stem cell factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interleukin 3, and interleukin 6. Plates were studied under phase-contrast microscopy, and granulocyte-macrophage colony-forming units (CFU-GM, colonies >50 cells) were counted after 14 days of incubation.
Measurement of cytokine serum levels
At the time of cell isolation (BM-MNC, EPC), serum was collected from all patients and healthy controls. Serum levels for tumor necrosis factor (TNF)-α, TNF-α receptor, interleukin-6, erythropoietin (all from R&D Systems) and N-terminal pro-brain natriuretic peptide (Elecsys, Roche, Mannheim, Germany) were measured by high-sensitive enzyme-linked immunosorbent assays according to the manufacturer’s instructions. High-sensitivity C-reactive protein was measured by means of particle-enhanced immunonephelometry (Dade Behring, Marburg, Germany).
Analysis of bone marrow plasma
Bone marrow plasma was obtained by centrifugation of bone marrow aspirates at 800 gand was kept frozen at −80°C until further use. Bone marrow plasma levels of total matrix metalloproteinase-9 (MMP-9, 92 kDa pro and 82 kDa active forms) and placental growth factor (PlGF) were measured by high-sensitive enzyme-linked immunosorbent assays (R&D Systems) according to the manufacturer’s recommendations.
If not stated otherwise, data are expressed as mean ± SD. The nonparametric Mann-Whitney Utest was used to test for differences between 2 groups. Categorical variables were compared by the chi-square test or the Fisher exact test.
Bivariate correlation was calculated by Pearson correlation. A linear regression model was used to evaluate independent predictors. Statistical significance was assumed if a null hypothesis could be rejected at p ≤ 0.05. All statistical analyses were performed using SPSS for Windows version 12.0 (SPSS Inc., Chicago, Illinois).
Circulating EPCs in patients with heart failure
The characteristics of the study population are summarized in Table 1.The clinical characteristics did not differ between patients with ICM and patients with nonischemic DCM except for the prevalence of hypertension and the more frequent use of statins in patients with CAD, whereas diuretics and digitalis were more frequently used in patients with nonischemic DCM. Likewise, as shown in Table 1, N-terminal pro-brain natriuretic peptide, interleukin 6, high-sensitivity C-reactive protein, and erythropoietin serum levels were similar in both groups, whereas TNF-α and soluble TNF-α receptor were slightly but significantly elevated in patients with ICM.
Compared with healthy controls, both patients with ICM as well as patients with nonischemic cardiomyopathy had significantly lower numbers of EPCs (Fig. 1).However, there was no difference between the 2 patient groups.
Determinants of circulating EPC levels
As summarized in Table 2,for the entire study population, the presence of CHF, advanced NYHA functional class, advanced age, and elevated serum levels of interleukin-6 were correlated with reduced circulating EPC number on univariate analysis. However, on multivariate analysis, only the presence of CHF was an independent predictor of reduced numbers of circulating EPCs, whereas all other parameters lost predictive power (Table 2). Moreover, when the analysis was restricted to patients with CHF, thus excluding the healthy control group, none of the individual parameters remained an independent predictor for a reduced number of circulating EPCs (Table 2). Thus, CHF itself is the most important independent determinant of circulating EPC levels.
Functional capacity of circulating EPCs
The functional capacity of circulating EPCs was assessed by measuring their migratory response to VEGF, which represents a physiological chemoattractant for EPCs. Only EPCs derived from patients with ICM showed an impaired migratory capacity toward VEGF compared with EPCs derived from both healthy controls as well as from patients with nonischemic DCM (Fig. 2).Importantly, the migratory capacity of EPCs derived from patients with nonischemic DCM did not differ from those derived from normal healthy volunteers.
Determinants of functional capacity of EPCs
For the entire study population, the migratory capacity of EPCs was correlated with the presence of ICM, positive family history for CAD, advanced NYHA functional class, advanced age, and elevated levels of interleukin-6, TNF-α, and high-sensitivity C-reactive protein serum levels (Table 3).On multivariate analysis, the presence of ICM, CHF, and advanced NYHA functional class remained independent predictors of impaired functional capacity of EPCs (Table 3). When the analysis was restricted to patients with CHF, again the presence of ICM and advanced NYHA functional class remained as the only independent predictors of impaired EPC function (Table 3).
Taken together, although CHF, independent of its etiology, is associated with a reduced number of EPCs, the presence of ICM is an additional independent predictor for functional impairment of circulating EPCs.
Number of bone marrow–derived hematopoietic progenitor cells in patients with heart failure
The characteristics of the study population, in which bone marrow–derived cells were analyzed, are summarized in Table 4.Patients with nonischemic DCM were slightly but significantly younger, had a lower ejection fraction, and less frequently received statins compared with patients with ICM. Moreover, there was no significant difference in cytokine serum levels except for TNF-α, which was significantly lower in patients with DCM.
The numbers of hematopoietic progenitor cells, which presumably give rise to the circulating EPCs, were determined in the CHF population by flow cytometry analysis of the expression of the marker protein CD34. The number of CD34+CD45+BM-MNCs was similar in both heart failure groups (ICM 0.52 ± 0.27% of total cells, DCM 0.55 ± 0.29% of total cells, p = 0.70). Moreover, the more immature subset of angioblast progenitor cells defined as CD34+CD133+cells did not differ between the 2 subgroups (ICM 0.09 ± 0.08% of total cells, DCM 0.14 ± 0.11% of total cells, p = 0.12) (Fig. 3).
Functional capacity of BM-MNCs
The functional capacity of progenitor cells in the bone marrow aspirates was determined by measuring the colony-forming activity. The BM-MNCs derived from patients with ICM showed a significantly reduced number of CFU-GM compared not only with BM-MNCs from healthy controls, but more importantly also with patients with nonischemic DCM (Fig. 4).
Determinants of functional capacity of BM-MNCs
On univariate analysis of the entire study population, advanced age, the sum of risk factors, CHF, the number of CD34+CD45+and CD34+CD133+cells, and the presence of heart failure of ischemic etiology and advanced NYHA functional class were correlated with the number of CFU-GM (Table 5).However, on multivariate analysis, CHF remained the only independent predictor of impaired hematopoietic stem cell (HSC) function.
When the analysis was restricted to patients with CHF, the presence of ICM was again the only independent predictor of an impaired colony-forming capacity of BM-MNCs (Table 5).
Therefore, the presence of ICM seems to be an independent predictor of impaired HSC function in patients with CHF.
Bone marrow plasma levels of MMP-9 and PlGF
Both MMP-9 and PlGF were experimentally shown to exert crucial functions in the bone marrow niche to mediate mobilization and functional activity of bone marrow progenitor cells (17,20,21). Therefore, we measured total MMP-9 levels and PlGF levels in the bone marrow plasma in a subset of our patients. A trend toward lower levels of MMP-9 could be detected in the bone marrow plasma of patients with postinfarction heart failure (219.0 ± 148.6 ng/ml; n = 29) when compared with patients with nonischemic DCM (342.8 ± 169.8 ng/ml, n = 6, p = 0.12). In contrast, PlGF bone marrow plasma levels did not differ between patient with ischemic heart failure (25.8 ± 9.7 pg/ml, n = 30) and patients with DCM (29.9 ± 12.2 pg/ml, n = 6, p = 0.55).
Cigarette smoking was previously shown to acutely influence MMP-9 serum levels (22,23). Excluding active smokers from the analysis showed a direct positive correlation of MMP-9 bone marrow plasma with CFU capacity of hematopoietic progenitor cells (r = 0.381, p = 0.045, n = 28), indicating a possible direct association between progenitor cell functional activity and MMP-9 activity.
The results of the present study confirm and significantly extend our previous observation that patients with CAD do have a functional impairment of hematopoietic progenitor cells, both in the bone marrow as well as when these cells are mobilized into the blood as circulating progenitors (11,24). Most importantly, the results of the present study show that patients with chronic postinfarction heart failure show a functional exhaustion of their hematopoietic progenitor cell pool in the bone marrow niche, whereas the number of hematopoietic progenitors does not seem to be reduced in the bone marrow. The functional impairment of hematopoietic progenitor cells is mirrored by a reduced migratory capacity of progenitor cells mobilized into the blood. Importantly, while the reduction in circulating EPC numbers seems to be independent of the etiology of heart failure (ischemic vs. nonischemic), the functional impairment is specifically aggravated in patients with postinfarction heart failure.
The present study is the first to address potential effects of CHF on hematopoietic progenitor cell function in the bone marrow. Our previous studies already pointed toward a profound functional impairment of BM-MNCs in patients with chronic ischemic heart disease (24). However, these studies could not answer the question of whether CAD or heart failure itself may have contributed to the observed functional impairment. By choosing a large group of patients with both ischemic and nonischemic etiology of heart failure, the present study enabled us to delineate a specific functional impairment of bone marrow-derived and blood-derived progenitor cells associated with an ischemic etiology of heart failure caused by prior MI.
Moreover, the hypothesis that chronic postinfarction heart failure is associated with a functional exhaustion of hematopoietic progenitor cells is further supported when progenitor cell function in patients with ICM of the present study is compared with progenitor cell function in patients with acute MI, as described previously in the TOPCARE-AMI (Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction) population (25). Patients included in the TOPCARE-AMI trial had a higher migratory capacity of circulating EPC as well as higher colony-forming activity of the bone marrow–derived progenitors compared with patients with ICM. In parallel, the number of CD34+CD45+bone marrow cells was significantly higher when compared with the CHF population of the present study (p < 0.05), whereas the more immature subset of CD133+CD34+BM-MNC did not differ between the 2 patient groups. Thus, progenitor cell function seems to be preserved during acute MI, but deteriorates during the development of ICM.
Obviously, the present clinical study cannot disclose the potential mechanisms underlying the functional impairment of progenitor cells, both in the bone marrow niche as well as in the blood. Interestingly, however, we and others have recently reported that nitric oxide is of crucial importance for both the function of bone marrow-derived progenitor cells as measured by CFU formation (17), as well as for the migrating capacity of circulating EPCs (26), whereas the overall number of hematopoietic progenitor cells in the bone marrow is not affected by genetic ablation of the nitric oxide synthase (17). Thus, these experimental data correspond to our observations in patients with post-infarction heart failure. Indeed, both CAD as well as heart failure are well established to be associated with a profound impairment of systemic nitric oxide bioavailability, which might well extend into the bone marrow niche (27,28). Moreover, experimental data suggested a role for nitric oxide in modulating the activity of MMP-9 (29). The MMP-9 activity is required not only to mobilize progenitor cells from the bone marrow, but also to permit the transfer of endothelial and hematopoietic progenitor cells from the quiescent to the proliferative niche in the bone marrow (21). Importantly, preliminary data analyzing the bone marrow plasma levels of MMP-9 in our patients revealed reduced levels of MMP-9 in patients with postinfarction heart failure when compared to patients with nonischemic etiology of heart failure. Furthermore, MMP-9 levels appear to correlate with HSC function. Thus, the specific impairment in progenitor cell function observed in our patients with postinfarction heart failure may indeed be related to a reduced systemic nitric oxide bioavailability associated with a decreased MMP-9 activity in the bone marrow.
The level of circulating EPCs was previously shown to predict postinfarction LV remodeling (16). We previously have shown that the migrating capacity of blood-derived progenitor cells is an independent determinant of the effect of intracoronary infusion of these cells in patients with acute MI (30). Thus, it is tempting to speculate that a functional exhaustion of bone marrow-derived progenitor cells may causally contribute to the development of postinfarction heart failure caused by impaired LV remodeling. Moreover, it has been reported recently that the level and the functional activity of circulating progenitor cells predict atherosclerotic disease progression (13,14). Thus, one might speculate that the impaired progenitor cell function might be associated with an impaired vascular repair capacity, thereby contributing to aggravation of the atherosclerotic disease process. Importantly, patients with postinfarction heart failure are at substantially increased risk of suffering from recurrence of MI (31).
The major limitation of the present study relates to the fact that we did not simultaneously measure bone marrow– and blood-derived progenitor cell number and function in the same patient. However, the 2 CHF populations in which the different progenitor cell types were analyzed did not differ significantly from each other with regard to patient characteristics and medications. In addition, the patient populations studied seem to be large enough to exclude potentially confounding effects caused by patient selection and sample size. Another limitation is that the healthy volunteers were significantly younger than the examined patients with CHF. Although age did not correlate with EPC number and function as well as with CFU-GM on multivariate analyses, we cannot fully exclude age as a confounding variable. Moreover, although each individual cytokine measured in our patients did not individually and independently predict reduced number or function of circulating EPCs, the exposure of circulating EPCs to a variety of cytokines in a combined fashion may well have contributed to the reduced number of circulating EPCs in patients with heart failure irrespective of its etiology. Nevertheless, although patients with ICM showed elevated serum levels of TNF-α and TNF-α receptor 1 compared with patients with nonischemic DCM, this well-known myelosuppressive cytokine was not independently associated with an impaired function of progenitor cells retrieved from the bone marrow niche. Finally, we cannot comment on an increased rate of apoptosis of circulating EPCs being responsible for the reduced number of EPCs in patients with heart failure irrespective of its etiology. However, given the profoundly upregulated antiapoptotic machinery in circulating EPCs previously reported (32,33), it is unlikely that increased apoptosis or reduced differentiation might have contributed to the effect. Likewise, reduced survival or differentiation of mobilized EPCs cannot account for the reduced migratory capacity selectively aggravated in EPCs derived from patients with postinfarction heart failure.
In summary, the results of the present study show that patients with postinfarction heart failure have a selective functional exhaustion of their hematopoietic progenitor cells in the bone marrow niche characterized by a profoundly impaired colony-forming capacity, but a preserved progenitor cell number. Mechanistically, the functional impairment in the bone marrow niche might be related to reduced activity of MMP-9, which is crucial not only for mobilization of hematopoietic progenitor cells, but also for their proliferative capacity. Given that the mobilization capacity of hematopoietic progenitor cells was both experimentally (18) and clinically (16) shown to be associated with favorable LV remodeling after MI, interventions aiming at improvement of progenitor cell function within the bone marrow niche may provide novel therapeutic targets for the recovery of LV function in patients with MI.
The authors thank Tino Röxe, Tina Rasper, Marga Müller-Ardogan, and Ariane Fischer for their excellent technical assistance.
↵1 Dr. Kissel was supported in part by the Dr. August Scheidel Foundation and the Deutsche Forschungsgemeinschaft (DFG Wa 1461/2-2).
The authors of this article belong to the European Vascular Genomics Network, a Network of Excellence supported by the European Community’s Sixth Framework Programme for Research Priority 1: “Life sciences, genomics and biotechnology for health” (Contract No. LSHM-CT-2003-503254). The first two authors contributed equally to this work.
- Abbreviations and Acronyms
- bone marrow–derived mononuclear cell
- coronary artery disease
- colony-forming unit–granulocyte-macrophage
- chronic heart failure
- dilated cardiomyopathy
- endothelial progenitor cell
- hematopoietic stem cell
- high-sensitivity C-reactive protein
- ischemic cardiomyopathy
- left ventricular
- myocardial infarction
- matrix metalloproteinase
- N-terminal pro-brain natriuretic peptide
- New York Heart Association
- placental growth factor
- tumor necrosis factor
- vascular endothelial growth factor
- Received February 14, 2006.
- Revision received January 11, 2007.
- Accepted January 22, 2007.
- American College of Cardiology Foundation
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