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European Heart Journal Advance Access originally published online on February 2, 2006
European Heart Journal 2006 27(9):1032-1037; doi:10.1093/eurheartj/ehi761
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© The European Society of Cardiology 2006. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Interleukin-8 is associated with circulating CD133+ progenitor cells in acute myocardial infarction

Kathrin Schömig, Gabriele Busch, Birgit Steppich, Dominik Sepp, Jan Kaufmann, Andreas Stein, Albert Schömig and Ilka Ott*

Deutsches Herzzentrum und 1. Medizinische Klinik der Technischen Universität München, Lazarettstr. 36, 80636 München, Germany

Received 18 August 2005; revised 13 December 2005; accepted 13 January 2006; online publish-ahead-of-print 2 February 2006.

* Corresponding author. Tel: +49 89 1218 3515; fax: +49 89 1218 4013. E-mail address: ott{at}dhm.mhn.de

See page 1013 for the editorial comment on this article (doi:10.1093/eurheartj/ehi889)


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Supplementary material
 Acknowledgements
 References
 
Aims Release of progenitor cells is observed during inflammatory conditions and contributes to neovascularization. We, therefore, sought to investigate the relationship of circulating progenitor cells and interleukin (IL)-8 in acute myocardial infarction (AMI).

Methods and results From patients with stable angina and AMI, serial venous blood samples were obtained. The number of circulating CD133+CD45– progenitor cells, endothelial progenitor cells (EPCs), and circulating endothelial P1H12+CD45– cells was analyzed by flow cytometry. After stenting in patients with AMI, an increase in plasma IL-8 and vascular endothelial growth factor (VEGF) concentrations was observed, which was only minimal in patients with stable angina. Only in patients with AMI, this was followed by an increase in circulating CD133+CD45– progenitor cells. In contrast, circulating endothelial P1H12+CD45– cells and E-selectin RNA expression in peripheral blood were only elevated early in AMI, indicating shedding of activated endothelial cells. Multivariable analysis revealed an association of IL-8 and circulating CD133+CD45– progenitor cells in AMI, in addition to statin therapy and risk factor profile.

Conclusion In AMI, IL-8 is associated with circulating progenitor cells. In addition to the pro-angiogenic functions of IL-8 and VEGF, this mechanism may contribute to new vessel generation and, thereby, improve myocardial function.

Key Words: Myocardial infarction • Cytokines


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Supplementary material
 Acknowledgements
 References
 
Several lines of evidence suggest that vascular healing may be mediated in part by circulating progenitor cells.1,2 In steady state conditions, progenitor cells are rare and represent <0.01% of all circulating mononuclear cells. During ischaemia, populations of bone marrow-derived endothelial progenitor cells (EPCs) are mobilized and recruited to ischaemic areas, accelerating the neovascularization process.3 Experimental studies have shown that re-introduction of cytokines such as vascular endothelial growth factor (VEGF), angiopoietin-1, SDF-1, G-CSF, or GM-CSF enhance mobilization of the EPC to the ischaemic myocardium, augmenting neovascularization.4,5 Furthermore, ischaemia itself and the release of chemokines and cytokines as a result of vascular injury induce mobilization of EPC,2,6 raising the possibility that the effects of ischaemia on the bone marrow are mediated by these cytokines.7 Therefore, the dynamic changes in EPCs following acute myocardial infarction (AMI) may be the result of myocardial ischaemia per se and systemic inflammatory changes.

Myocardial infarction is associated with an inflammatory response which is a prerequisite for healing and scar formation.8 Previous studies revealed divergent results on the levels of the pro-inflammatory mediators interleukin (IL)-6 and IL-8.9–11 Among those, the CXC chemokine IL-8 is a critical regulator of neutrophil influx and activation of inflammatory processes, but also exerts pro-angiogenic effects and may have a role in wound healing and repair.8,12 Other studies have shown that IL-8 induces stem cell mobilization involving activation of MMP-9 and LFA-1.13 IL-8 upregulation occurs in experimental models of myocardial infarction in inflammatory cells of the infarct border zone and in microvascular endothelial cells.12 Thus, IL-8 may contribute to progenitor cell mobilization in AMI.

Circulating CD34+ cells are mobilized in patients with AMI and correlate with increased VEGF plasma concentrations known to induce stem cell mobilization.14–16,33,34 Moreover, circulating endothelial cells may reflect endothelial injury in acute coronary syndromes.17 As CD34 is expressed on endothelial cells18 as well as on progenitor cells, it is not sufficient for identification of EPCs in AMI19,20 The characterization of circulating progenitors that express the haematopoietic stem cell marker CD133 may allow to identify progenitor cell mobilization from the bone marrow, whereas analysis of cells expressing exclusively endothelial markers may identify endothelial cells released from damaged myocardium. As mobilization and recruitment of circulating endothelial progenitors hold therapeutic potential for new vessel growth in vascular trauma or organ ischaemia, we analyzed the number of circulating progenitor cells and plasma IL-8 concentrations in AMI.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Supplementary material
 Acknowledgements
 References
 
Patient selection
We recruited 22 consecutive patients with AMI and 20 consecutive patients with stable angina undergoing elective PCI for the first part of the study. All patients gave informed consent before inclusion in the study. The study protocol was approved by the institutional Ethics Committee, and all patients gave written informed consent for participation in the study. The diagnosis of AMI was established in the presence of clinical (chest pain for at least 20 min) and electrocardiographic (ST-segment elevation of at least 0.1 mV in two or more limb leads or at least 2 mV in two or more contiguous precordial leads on the surface electrocardiogram) signs for AMI. All patients received 500 mg aspirin, 5000 U heparin intravenously, and 600 mg of clopidogrel orally in the emergency room and then immediately underwent coronary stenting. Twenty patients with stable angina pectoris (SAP) undergoing elective stenting were included in the control group. Venous blood samples were obtained before coronary intervention and 1, 2, and 7 days afterwards. To analyze whether IL-8 is an independent indicator for progenitor cell mobilization, an additional group of 29 consecutive patients with AMI were included. Measurements were completed in all included patients. Blood samples were taken on admission, 1 and 4–7 days after AMI to analyze circulating CD133+ cells, as well as IL-8 and VEGF plasma concentrations. Samples were processed immediately, as indicated subsequently.

Immunoassays
Concentrations of soluble (s) E-selectin, VEGF, and IL-8 were determined by immunoassay (sE-selectin, VEGF, and IL-8 Quantikine, R&D Systems, Minneapolis, MN, USA). Detection limits were 0.1 ng/mL for sE-Selectin, 5 pg/mL for VEGF, and 31.2 pg/mL for IL-8. Intra-assay variability for the lower assay range was <10%.

Flow cytometry
To analyze CD133+CD34+CD45– progenitor cells, 100 µL heparinized blood samples were stained with FITC-conjugated anti-CD34 (Becton Dickinson Biosciences, Heidelberg, Germany), PE-conjugated anti-CD133 (Miltenyi Biotec, Auburn, CA, USA), and APC-conjugated anti-CD45 (Beckman Coulter Krehfeld, Germany), according to a modified protocol of the European Working Group on Clinical Cell Analysis.21 Circulating endothelial cells (P1H12+CD31+CD45–) were determined after labelling with PE-conjugated P1H12 (Abcam, Cambridge, UK), which recognizes endothelial CD146, FITC-conjugated CD31 (Becton Dickinson), and APC-conjugated anti-CD45 (Beckman Coulter Krehfeld, Germany). Staining was performed as described previously.22 Fluorescence isotype-matched antibodies were used as controls. Flow cytometric analysis was performed using a FACS Calibur (Becton Dickinson, Mountain View, CA, USA). Fluorescence intensity of at least 200 000 cells was recorded and analyzed using CellQuest software. Circulating endothelial cells were defined as negative for the haematopoietic marker CD45 and positive for the endothelial markers P1H12 and CD31 (P1H12+CD31+CD45–). Circulating CD133+ progenitor cells were depicted by expression of CD34 and CD133 and lack of expression of CD45 (CD133+CD34+CD45–). The percentage of positive cells in relation to CD45+ cells was calculated. In a subgroup of 10 patients with AMI, additional measurements were performed to determine the absolute numbers using TrueCount beads (Becton Dickinson). Surface expression of P1H12, CD133, and CD34 of EPC and coronary artery endothelial cells (Promocell, Heidelberg, Germany) was analyzed by flow cytometry after detaching the cells with non-enzymatic cell dissociation solution (Gibco) and after staining with PE-conjugated anti-CD133, anti-P1H12, or FITC-conjugated anti-CD34.

RNA preparation and semi-quantitative PCR
Total RNA was extracted from EDTA-anticoagulated blood samples using whole blood RNA extraction assays (RNeasy Blood Mini Kit, Qiagen, Crawley, UK), according to the manufacturer's instructions. RT–PCR was performed as described,22 with denaturing at 95°C for 1 min, annealing at 60°C for 1 min, and extending at 72°C for 1 min repeated in 30 cycles. Primer sequences were: for GAPDH, 5'-GTTGTCATGGATGACCTTGGCC-3' and 5'-CCACCCATGGCAAATTC CATGG-3'; for E-selectin, 5'-CTCTGACAGAAGAAGCCAAF-3' and 5'-ACTTGAGTCCACTGAAGGCA-3'. Serial dilutions of cDNA were examined to ensure that any effects on mRNA induction were not obscured because of a plateau effect in the PCR reactions. The density of the electrophoresed E-selectin PCR product (235 bp) was related to the GAPDH intensity of the same sample (350 bp) and this ratio served as a measure of specific RNA content (NIH Image).

Culture assay of EPCs
Mononuclear cells were isolated from EDTA-anticoagulated blood samples using Ficoll (Pharmacia Biotech, Freiburg, Germany) gradient separation as described.22 Mononuclear cells (4x106 cells) were cultured on fibronectin coated cover slides in EGM-2 medium (Cambrex Clonetics, Baltimore, MD, USA). After 4 days of culture, EPCs were identified by expression of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanineperchlorate-labelled acetylated LDL (DiI-acLDL) incorporation and Ulex europaeus agglutinin-I (Sigma) as described.23 Four randomly selected fields from two cover slides were counted for DiI-ac-LDL and FITC-lectin positive cells as a measure for EPCs.

Other methods
Serum creatine kinase concentrations were determined in the clinical chemistry laboratory. No endotoxin contamination of leukocyte suspensions or buffers was detected (E-toxate, Sigma).

Statistical analysis
Differences between the groups were analyzed by the Mann–Whitney–Wilcoxon rank-sum test. Non-parametric bivariate correlation using Spearman's rank correlation coefficient and multivariable linear regression analysis were applied to correlate the number of circulating CD133+ cells with IL-8, VEGF, risk factors, maximal CK levels, and previous statin therapy (SPSS Inc.).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Supplementary material
 Acknowledgements
 References
 
Clinical and angiographic data
We recruited 22 consecutive patients with AMI and 20 consecutive patients with stable angina undergoing elective PCI. The study group with AMI (n=22) did not differ significantly from the patients with stable angina (n=20) with respect to age, gender distribution, risk factor profile, and extent of coronary artery disease. However, a higher percentage of patients with stable angina were on a regular medication of statins, ß-blocker, aspirin, clopidogrel, or ACE-inhibitors (Supplementary material). The additional group with AMI (n=29) was comparable to the first group with AMI (n=22) with respect to age, gender distribution, risk factor profile, extent of coronary artery disease, or infarct size (Supplementary material). None of the patients suffered re-infarction or death during the hospital stay. Stenting was successful in all patients restoring TIMI grade 3 flow.

Circulating CD133+ progenitor cells and EPC in AMI
When compared with patients with stable angina, a lower percentage of circulating progenitor cells before stenting was observed in AMI (P=0.02, n=22; Figure 1). Yet, the number of circulating CD133+ progenitor cells increased after stenting in AMI, whereas, no changes in circulating of CD133+ cells were found in patients with stable angina. As patients with stable angina were discharged within 3 days, we were not able to extend blood sampling up to 7 days. To ensure that the increase in CD133+ progenitor cells does not merely reflect initial leukocytosis, we analyzed the absolute numbers of circulating CD133+ progenitor cells using TrueCount beads in 10 patients with AMI and found an elevation in CD133+ progenitor cells 6 days after stenting (data not shown). Thus, a similar increase in the absolute and the relative numbers of circulating CD133+ progenitor cells occurred.


Figure 7611
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  		Figure 1 Increase in circulating CD133+ cells in AMI. Serial changes in circulating CD133+ cells in patients with AMI (n=22, closed circles) or in patients with stable angina (open circles) are shown. In whole blood samples, the percentages of CD133+ cells were calculated by immunofluorescence analysis (A). Values are expressed as mean±SEM. In (B), representative immunofluorescence analysis of a patient with AMI is shown. Each dot plot depicts CD133+ cells and isotype control values before coronary intervention and 7 days thereafter.

 
The effect of myocardial infarction on circulating EPCs was analyzed using the differentiation capacity of circulating mononuclear cells into endothelial cells by cell culture assay. Patients with AMI had higher DiI-acLDL+/FITC-lectin+ cells as a measure for EPCs when compared with patients with stable angina.

Two days after stenting in AMI (n=22), we found an increase in EPCs that declined thereafter (Figure 2). In contrast, no changes in EPCs were observed in stable angina. Because the mobilization of CD133+ progenitor cells and EPCs followed systemic inflammatory changes, the underlying mechanisms may be similar. However, the fact that EPCs derive from different sources may explain the differences to CD133+ progenitor cell mobilization.


Figure 7612
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  		Figure 2 Increase in circulating EPCs in AMI. Serial changes in EPCs in patients with AMI (n=22, closed circles) or in patients with stable angina (open circles) are shown. Mononuclear cells were cultured in endothelial medium and DiI-acLDL, and FITC-lectin positive cells were counted as a measure for EPCs (A). Values are expressed as mean±SEM. In (B), representative immunofluorescence analysis of a patient with AMI before coronary intervention and after 7 days is shown.

 
Both circulating EPC and endothelial cells express similar surface antigens. To distinguish these different cell types, we compared surface expression of P1H12, CD34, and CD133 in cultured EPC and coronary endothelial cells. Both cell types were positive for CD34 but only EPC expressed CD133, whereas coronary endothelial cells were highly positive for P1H12 (Supplementary material). Therefore, we used P1H12 as a marker for mature endothelial cells.

Circulating endothelial cells in AMI
Circulating P1H12+ endothelial cells were elevated on admission in AMI (P=0.01, n=22) and decreased within the first day, whereas only minimal amounts of circulating endothelial cells were found in stable angina. Thus, shedding of endothelial cells was not due to the interventional procedure itself but may be due to prolonged myocardial ischaemia and necrosis (Figure 3). As hypoxia is known to induce the expression of the endothelial activation marker E-selectin, we analyzed E-selectin RNA expression within the circulating blood as a measure for circulating, activated endothelial cells. Similar to the elevated numbers of circulating endothelial cells, we found increased E-selectin mRNA expression on admission in AMI (n=22) when compared with patients with stable angina (P=0.01), which decreased thereafter (Figure 4A). Upon activation, E-selectin is shed from endothelial cells. Thus, the amount of circulating soluble E-selectin may reflect endothelial cell activation. In concordance with the flow cytometry analysis and the RT–PCR data, soluble E-selectin was elevated on admission in AMI and decreased thereafter, whereas lower levels were detected in stable angina (P=0.03), which remained unchanged over time (Figure 4B). Elevated levels of P1H12+ cells, E-Selectin mRNA, and soluble E-selectin early in AMI may indicate the release of activated endothelial cells from infarcted myocardium.


Figure 7613
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  		Figure 3 Increase in circulating endothelial cells in AMI. Serial changes in circulating P1H12+ cells in patients with AMI (n=22, closed circles) or in patients with stable angina (open circles) are shown. In whole blood samples, the percentages of P1H12+ cells were calculated by immunofluorescence analysis (A). Values are expressed as mean±SEM. In (B), representative immunofluorescence analysis of a patient with AMI is shown. Each dot plot depicts P1H12+ cells and isotype control values from one patient with AMI before coronary intervention and 7 days thereafter.

 

Figure 7614
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  		Figure 4 Increased E-selectin expression in AMI. Serial changes in E-selectin RNA expression (A) and soluble E-selectin (B) in the circulating blood of patients with AMI (n=22, closed circles and bars) or of patients with SAP (open circles and bars) are shown. Values are expressed as mean±SEM (A).

 
Plasma levels of IL-8 and VEGF in AMI
Baseline IL-8 plasma concentrations were elevated in AMI (n=22) when compared with stable angina (P=0.01). After stenting, a more pronounced increase in IL-8 was found in AMI than in stable angina (Figure 5A). Thus, in addition to the interventional procedure, myocardial infarction may stimulate IL-8 release. A similar increase was observed in plasma levels of VEGF 1 and 2 days after stenting in AMI (n=22), which was less pronounced in patients with stable angina (Figure 5B). As no changes in circulating CD133+ cells were found in patients with stable angina over time, the lower extent of chemokine and cytokine release in patients with stable angina may not be sufficient to induce CD133+ cell mobilization.


Figure 7615
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  		Figure 5 IL-8 and VEGF plasma concentrations in AMI. Serial changes in circulating IL-8 (A) and VEGF (B) levels in patients with AMI (n=22, closed circles) or in patients with stable angina (open circles) are shown (A). Values are expressed as mean±SEM.

 
IL-8 and circulating CD133+ progenitor cells in AMI
As IL-8 is known to contribute to stem cell mobilization, we performed regression analysis of IL-8 with circulating CD133+ progenitor cells in an extended patient group with AMI (n=51). In these patients, circulating CD133+ progenitor cells increased already 1 day after stenting compared with the values before stenting (0.012±0.003–0.025±0.007%, mean±SEM) and remained elevated during 4–7 days hours after stenting (0.048±0.01% mean±SEM). A similar time course was observed for IL-8 and VEGF plasma concentrations (data not shown). Univariate regression analysis revealed a correlation between plasma concentrations of IL-8 and circulating progenitor cells at all time points (Table 1). To analyze whether IL-8 is an independent predictor for circulating CD133+ progenitor cells, multivariable analysis was performed including the following parameters that are known to affect progenitor cell mobilization: infarct size estimated by maximal CK values, number of risk factors, previous statin therapy before admission, and VEGF levels. IL-8, previous statin therapy and the inverse number of risk factors were associated with progenitor cell mobilization (Table 2). Thus, our study provides evidence for an association of IL-8 and progenitor cell mobilization in AMI. Moreover, in line with previous studies, we found that the influence of risk factors and statin therapy on progenitor cell mobilization was maintained in patients with AMI.


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  		Table 1 Univariate correlation between IL-8 and CD133+ cells (n=51)

 

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  		Table 2 Multivariable analysis between maximal CK values, number of risk factors, statin therapy, IL-8, VEGF, and CD133+ cells (day 1 after stenting, n=51)

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Supplementary material
 Acknowledgements
 References
 
Major findings of our study are as follows. (i) Circulating EPCs and CD133+ progenitor cells are increased after stenting in AMI. (ii) In contrast, circulating endothelial cells and E-selectin mRNA expression are elevated before stenting in AMI and decreased thereafter. (iii) Plasma levels of the chemokine IL-8 and the cytokine VEGF increased after stenting in AMI. (iv) IL-8 and circulating CD133+ progenitor cells are correlated in AMI.

Under physiological conditions, only low levels of progenitor cells are released into the peripheral blood. However, inflammation, ischaemia, and vascular injury result in enhanced levels of circulating stem cells.1,24 This mobilization process occurs as a result of proteolytic matrix degradation, enhancement of migration by chemokines and cytokines, and induction of adhesion molecules.25 A number of mediators have been shown to enhance these processes and subsequent mobilization, such as G-CSF, GM-CSF, IL-7, IL-3, IL-2, SCF, IL-8, MIP-alpha, Gro-beta, and SDF. These molecules differ in their time frame, the type of mobilized cell, and their efficiency and may act in a synergistic manner.25 As myocardial infarction is associated with an inflammatory response,22,26,27 we analyzed the role of the pro-inflammatory chemokine IL-8 in progenitor cell mobilization in AMI. IL-8 plasma concentrations were increased in AMI and were associated with CD133+ progenitor cell mobilization. As the underlying mechanism, IL-8 is known to activate MMP-9 and beta-2 integrin LFA-1 and, thereby, mobilize stem and progenitor cells from the bone marrow.13 In parallel with the increase in IL-8, we observed elevated levels of VEGF. Even though VEGF is known to enhance stem cell mobilization4,5 in the multivariable analysis, VEGF levels were not associated with progenitor cell mobilization in AMI. This suggests that a specific cytokine profile including IL-8 may contribute to stem cell mobilization in AMI. Although the interventional procedure itself induced a slight and transient increase in circulating IL-8 and VEGF in patients with stable angina, this did not alter the number of circulating progenitor cells.

The correlation of IL-8 with the mobilized progenitor cells in AMI identifies potential beneficial effects of the inflammatory response. This is in line with previous studies that have shown deleterious effects of anti-inflammatory approaches in AMI.28 The fact that we did not find a correlation of VEGF and progenitor cell release may be due to the variability in VEGF responses. Yet, the similar time course of progenitor cell mobilization and VEGF levels may suggest a role of VEGF in the mobilization process.14 In addition to progenitor cell mobilization, IL-8 and VEGF are known to enhance the adhesive potential of progenitor cells29 and, thus, may not only determine the amount of mobilized cells but also their homing capacity within the myocardium and their potential regenerative ability. There is evidence that bone marrow-derived progenitor cells participate in neovascularization in patients with fatal AMI preceding allogenic bone marrow transplantation.30 As we were not able to determine the fate of the mobilized cells, the role of the mobilization process in these patients remains to be investigated.

The identification and the origin of EPC which are isolated from peripheral blood mononuclear cells by culture in medium favouring endothelial differentiation remain a matter of debate. In peripheral blood mononuclear cells, different sources for endothelial cells may exist: EPCs may derive from haematopoietic stem cells, circulating endothelial, or other progenitors such as side population cells.29 The observation that expansion of CD14+ monocytes yields cells with endothelial characteristics31 and that blood-derived endothelial cells are detected in the culture assay32 may explain our finding in the culture assay that these EPCs are not in accordance with the number of circulating CD133+ cells but may contain a mixed population derived from shed endothelial, monocytic, and progenitor cells.

Because of the antigen promiscuity between circulating endothelial cells and other haematopoietic cells in respect to CD34,18 we aimed to differentiate circulating endothelial cells that may be released from the infarct area and circulating stem cells. In contrast to the mobilization of CD133+ stem cells after AMI, we identified an increase in circulating P1H12+ cells and E-selectin expression only on admission. Thus, analysis of surface CD34+ cells may not merely reflect circulating progenitor cells but also include activated endothelial cells released from ischaemic myocardium.17 These findings may partially explain the divergent results in previous studies regarding the kinetics of circulating CD34+ cells in AMI.14 In addition, differences in blood sampling and processing protocols, peri-procedural medications, risk factor profiles,35 and definition of the control groups may have contributed to the opposing results. Thus, further studies are needed to determine more rigorously the kinetics and modifying factors of progenitor and stem cell mobilization in AMI.

Our study extends previous studies that statin therapy increases36,37 and risk factors decrease circulating progenitor cells35 not only in coronary artery disease but also during the inflammatory response in AMI. Thus, the beneficial effects of statins may expand to the progression of AMI.

To our knowledge, this is the first study that identifies an association of IL-8 with circulating progenitor cells in AMI. Distinct inflammatory stimuli may not only indicate adverse events in acute coronary syndromes but may also induce beneficial effects such as progenitor cell mobilization.


   Supplementary material
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary material
Acknowledgements
 References
 
Supplementary material is available at European Heart Journal online.


   Acknowledgements
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary material
 Acknowledgements
References
 
The study was supported in part by grants from the Wilhelm Sander Stiftung and the Else Kröner Fresenius Stiftung. We thank Mrs B. Campbell, A. Stobbe, and C. Bauer for invaluable technical assistance.

Conflict of interest: none declared.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary material
 Acknowledgements
 References
 

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