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Endothelin-1 levels predict endothelial progenitor cell mobilization after acute myocardial infarction
Microvascular Research 82 (2011) 177–181
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Microvascular Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y m v r e
Regular Article
Endothelin-1 levels predict endothelial progenitor cell mobilization after acute myocardial infarction Xavier Freixa a,⁎, Monica Masotti a, Marta Palomo b, Maribel Diaz-Ricart b, Ginés Escolar b, Eduard Guasch a, Ander Regueiro a, Marcelo Jiménez a, Amadeo Betriu a, Magda Heras a a b
Cardiology Department, Hospital Clinic, University of Barcelona, Spain Hematology Department, Hospital Clinic, University of Barcelona, Spain
a r t i c l e
i n f o
Article history: Accepted 21 June 2011 Available online 2 July 2011
a b s t r a c t Introduction: Endothelin-1 (ET-1), circulating endothelial cells (CEC) and endothelial progenitor cells (EPC) are well-known modulators of endothelial function with important cardiac effects after an acute myocardial infarction. However, the relationship between them has never been assessed. The objective of the present study was to establish the relationship between ET-1, CEC, and EPC concentrations after ST-elevation myocardial infarction (STEMI). Methods: Endothelin-1, CEC, and EPC levels were measured in 61 patients presenting with a first STEMI. Samples were withdrawn acutely 6–24 h and 1 week after admission. Assessments included reperfusion outcomes (angiography), left ventricular ejection fraction (echocardiography), and 30-day mortality. Results: Mean age was 60.6 ± 12.6 years and 45 (74%) were males. Higher ET-1 plasma levels were associated with lower EPC count after 1 week (7.45 ± 2.53 pg/ml if EPCs in the first quartile vs 5.72 ± 1.49 pg/ml if EPCs in the fourth quartile; P = 0.04). In contrast with CEC and EPC count, higher ET-1 concentrations on admission were associated with Killip ≥ 2 (9.92 ± 2.01 pg/ml vs 7.32 ± 2.13 pg/ml; P b 0.001), post-reperfusion thrombolysis in myocardial infarction (TIMI) b 3 (8.65 ± 2.86 pg/ml vs 5.87 ± 1.93 pg/ml; P = 0.002), myocardial blush grade (MBG) b 3 (7.46 ± 2.48 pg/ml vs 5.99 ± 2.01 pg/ml; P = 0.004) and higher 30-day mortality (10.29 ± 2.02 pg/ml vs 6.57 ± 2.20 pg/ml; P = 0.005). Conclusions: In STEMI patients, high ET-1 levels on admission predict a lower EPC mobilization after 1 week. Endothelin-1 provides better clinical, angiographic and echocardiographic information for prognosis than do CEC and EPC concentrations. © 2011 Elsevier Inc. All rights reserved.
Introduction Endothelial impairment is an independent predictor of acute cardiovascular events in patients with and without coronary artery disease (CAD) (Halcox et al., 2002). Endothelial dysfunction is also considered a predictor of left ventricular ejection fraction (LVEF) (Bae et al., 2004) associated with worse left ventricle remodeling after myocardial infarction (Matsuo et al., 2006). Endothelin-1 (ET-1) is a well-known marker of endothelial dysfunction in patients with CAD (Iglarz and Clozel, 2007). In acute myocardial infarction (AMI), high ET-1 values on admission have been linked to poor angiographic outcomes after primary angioplasty (Eitel et al., n.a.; Niccoli et al., 2006) and poor prognosis, including higher 30-day mortality (Khan et al., 2007; Yip et al., 2005). ⁎ Corresponding author at: Interventional Cardiology Section, Cardiology Department, Thorax Institute, Hospital Clinic, University of Barcelona, Villarroel 170, Barcelona 08036, Spain. Fax: + 34 932275519. E-mail address: [email protected] (X. Freixa). 0026-2862/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2011.06.008
The measurement of circulating endothelial cells (CEC) and endothelial progenitor cells (EPC) also represents an important and novel technique for the assessment of endothelial injury and repair, respectively. Circulating endothelial cells are biomarkers of damage that correlate with several well-established markers of endothelial dysfunction and predict poor outcomes when elevated (Blann et al., 2005; Lee et al., 2005). In contrast, EPC are biomarkers of endothelial repair with potential cardioprotective effects. Low EPC levels after an AMI have been linked to poor outcomes, including worse LVEF and higher NT-proBNP concentrations (Wojakowski et al., 2006). The main mechanism of EPC mobilization from the bone marrow seems to depend on the activation of endothelial nitric oxide synthase (eNOS) in the presence of several mobilizing factors, such as vascular endothelial growth factor and placental growth factor (Li et al., 2006). Endothelin-1 decreases eNOS expression and could therefore play an important role in CEC and EPC mobilization (Sud and Black, 2009). However, no studies to date have assessed this potential interaction. The aim of the present study was to establish the relationship between ET-1 levels and CEC/EPC mobilization patterns in the early hours after an AMI.
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Methods Study population Between June 2007 and January 2009, the study enrolled 61 nonconsecutive patients who underwent primary angioplasty for a first AMI with elevation of the segment ST in the electrocardiogram (STEMI). Inclusion criteria were prolonged chest pain (N30 min) and ST-segment elevation N1 mm in 2 or more adjacent leads within the first 12 h after onset of symptoms. Exclusion criteria were acute or chronic inflammatory disease, malignancy, or recent surgery, trauma, or infection. The study protocol was approved by the institutional Ethics Committee and all patients gave written informed consent before being included. Percutaneous coronary intervention and angiographic analysis A transradial or transfemoral artery 6F approach was employed. Unfractionated heparin was administered at a dose of 60 UI/kg and a 300-mg loading dose of clopidogrel was administered during percutaneous coronary intervention (PCI). Our protocol includes administration of abciximab prior to PCI if no contraindications exist. Angiographic measurements were always performed independently by two experienced angiographers who assessed thrombolysis in myocardial infarction (TIMI) flow grade (Anon, 1985) and myocardial blush grade (MBG) (van 't Hof et al., 1998). Both TIMI and MBG are angiographic scales for measuring the flow and reperfusion after PCI. TIMI 0 is defined as the absence of distal coronary flow and TIMI 3 means normal flow. MBG is defined as no myocardial blush or contrast density and MBG 3 means normal myocardial blush or contrast density. Suboptimal reperfusion was defined as post-procedural TIMI flow ≤2 or MBG ≤2 despite TIMI 3 flow. Blood sampling and laboratory assay Endothelin-1 One blood sample was obtained from a peripheral vein on admission, prior to PCI (mean 10.8 ± 7.5 h after symptoms onset). Samples were decanted into ethylenediaminetetraacetic acid (EDTA) tubes, centrifuged at 3000 rpm for 15 min, and stored at − 80 °C until analysis. Plasma ET-1 levels were determined by radioimmunoassay using rabbit anti-ET-1 antibody (Peninsula Laboratories Inc, San Carlos, California). Samples were run in pairs and the results were averaged. Endothelial progenitor cells and circulating endothelial cells Two 150 μL blood samples were obtained from a peripheral vein, the first on admission (at the same time as ET-1) and the second after 7 days. Samples were collected into low molecular weight heparin (LMWH) tubes and processed in duplicate within 4 h of extraction. The samples were stained with 10 μL of FITC anti-CD34 (Becton Dickinson), PerCP anti-CD45 (Becton Dickinson), PE anti-kinaseinsert domain receptor (KDR) (R&D Systems), and APC anti-CD133 (eBiosciences) monoclonal antibodies for EPC measurements, and with PE anti-CD146 (Biocytex), FITC anti-CD31 (Becton Dickinson), and PerCP anti-CD45 (Becton Dickinson) monoclonal antibodies for CEC measurements. Red blood cells in the samples were subsequently lysed for 15 min (BD FACS Lysing Solution, Becton Dickinson). Samples were then centrifuged, washed twice, and resuspended in PBS with LMWH (50 U/mL), and analyzed using a FACSCalibur flow cytometer (Becton Dickinson). The sensitivity of fluorescence detectors was set and monitored using Calibrite Beads (Becton Dickinson) according to manufacturer's recommendations. Instrumental settings in order to set up accurate positivity of each fluorochrome were established using known cell populations. In addition, cells stained with isotopic controls for IgG conjugated with
the different fluorochromes were used as negative controls. Assessment of 2 × 10 6 events per sample was considered sufficient for statistical analysis. EPC and CEC were defined as negative for CD45 and positive for CD34, KDR, and CD133 (CD45-CD34 + KDR + CD133+ for EPC), and negative for CD45 and positive for CD146, and CD31 (CD45-CD146 + CD31+ for CEC), respectively. In the present study, positivity was established when the mean fluorescence intensity was N10 1 for each fluorochrome. Numbers were calculated multiplying the ratio of EPC and CEC obtained in the flow cytometry analysis by the number of leukocytes/ml in the blood sample to obtain the absolute number of both EPC and CEC per 1 mL of whole blood. Cardiac biomarkers Two common cardiac biomarkers, creatine kinase myocardial band (CK-MB) and troponin-I, were measured every 4 h during the first 24 h and then every 24 h for 3 days. Echocardiography Echocardiographic assessment was performed 3–7 days after PCI with Vivid 7 systems (GE-Vingmed, Milwaukee, Wisconsin). Left ventricle diameter was measured in parasternal and apical views and ejection fraction was evaluated using biplane Simpson's method by an expert cardiologist blinded to the other results. Statistical analysis Results are expressed as mean ± SD or median, depending on normal distribution as assessed by the Shapiro–Wilks test, and considered significant at a P-value b0.05. Comparisons between groups were performed using unpaired t test or Mann–Whitney U test for continuous variables and Chi-square or Fisher's exact test for categorical variables. Correlation analyses were done by Spearman's rank correlation. Assuming that CEC and EPC present at least a 2-fold change after AMI, the sample size was calculated to allow detection of 50% variation with 85% power at P b 0.05. Statistical analyses were done using SPSS package v14.0 (Chicago, Illinois). Results Patient characteristics The clinical characteristics of the study population are shown in Table 1. Mean symptoms-to-angioplasty time was 317 ± 255 min. The clinical and angiographic characteristics of the cohort are listed in Table 2. Suboptimal reperfusion was observed in 43.5% of patients. Three patients died within the first 30 days (2 of heart failure and 1 of cardiogenic shock). Mean peak CK-MB and troponin I after AMI were
Table 1 Baseline characteristics of the study population. Basal characteristics
n = 61
Age (years) Males (%) Smokers (%) Hypertension (%) Hypercholesterolemia (%) Diabetes (%) Peripheral vascular disease (%) Previous PCI/CABG (%) Treatment before admission (%) – Beta-blockers – Statins – ACE inhibitors
60.6 ± 12.6 74% 52% 54% 38% 18% 5% 9%/0% 26% 15% 21%
ACE, angiotensin-converting enzyme; PCI, percutaneous coronary intervention; CABG, coronary artery by-pass grafting.
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Table 2 Acute myocardial infarction characteristics. AMI characteristics Admission hemodynamics Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Heart rate (bpm) Killip I at admission TIMI risk score (points) Ischemia time Symptoms-balloon (min) AMI location: anterior (%) Culprit artery (LAD/RCA) (%) Number of diseased vessels (1/2/3) (%) Pre-PCI TIMI 0 (%) TIMI 1 (%) TIMI 2 (%) TIMI 3 (%) Post-PCI TIMI 0 (%) TIMI 1 (%) TIMI 2 (%) TIMI 3 (%) Post-PCI MBG 0 (%) MBG 1 (%) MBG 2 (%) MBG 3 (%)
n = 61 130.6 ± 32.4 78.7 ± 21.6 77.2 ± 19.1 88.5% 2.9 ± 2.1 317 ± 255 47.5% 47.5/35% 71/23/6% 60.6% 4.9% 14.8% 19.7% 0% 1.6% 4.9% 93.4% 0% 5% 37.7% 57.3%
AMI, acute myocardial infarction; LAD, left anterior descending; RCA, right coronary artery; MBG, myocardial blush grade.
182.8 ± 122.2 ng/ml and 178.65 ± 285.7 ng/ml, respectively. Left ventricular ejection fraction after PCI was 45.8 ± 9.7%. ET-1, CEC, and EPC mobilization patterns Endothelin-1, CEC, and EPC on admission presented a median value of 6.3 pg/ml (25th–75th interquartile range: 4.9–8.5 pg/ml), 220 cells/ml (25th–75th interquartile range: 100–573 cells/ml) and 410 cells/ml (25th–75th interquartile range: 93–1508 cells/ml), respectively. After 1 week, CEC and EPC median concentrations were 116 cells/ml (25th–75th interquartile range: 0–250 cells/ml) and 792 cells/ml (25th–75th interquartile range: 113–1944 cells/ml), respectively. The number of CEC was significantly higher on admission than at 7 days after myocardial infarction onset (459 ± 672 cells/ml on admission and 297 ± 594 cells/ml at 1 week; P b 0.001). Endothelial progenitor cells showed the opposite pattern, with a trend towards higher concentrations after 1 week (1238 ± 1994 cells/ml on admission and 1354 ± 1420 cells/ml at 1 week; P = 0.10) (Fig. 1). We found no correlation between CEC and EPC mobilization. As shown in Fig. 2, lower EPC concentrations after 1 week were associated with higher plasma levels of ET-1 (7.45 ± 2.53 pg/ml in patients with EPCs in the first quartile vs 5.72 ± 1.49 pg/ml in patients with EPCs in the fourth quartile; P = 0.04). Conversely, there was no significant relationship between CEC count and ET-1 levels.
Fig. 1. CEC (A) and EPC (B) levels on admission and 1 week after acute myocardial infarction onset.
(r = − 0.30; P = 0.004) while CEC and EPC failed to demonstrate any significant correlation with the common cardiac biomarkers or LVEF (Table 3).
Clinical, angiographic and echocardiographic outcomes Higher ET-1 levels on admission were associated with worse admission heart failure measured by Killip class (9.92 ± 2.01 pg/ml if Killip ≥ II vs 7.32 ± 2.13 pg/ml if Killip I; P b 0.001), worse postPCI TIMI (8.65 ± 2.86 pg/ml if TIMI b 3 vs 5.87 ± 1.93 pg/ml if TIMI = 3; P = 0.002), worse post-PCI MBG (7.46 ± 2.48 pg/ml if MBG b 3 vs 5.99 ± 2.01 pg/ml if MBG = 3; P = 0.004), and higher 30-day mortality (10.29± 2.02 pg/ml in patients who died vs 6.57 ± 2.20 pg/ml in patients who did not; P = 0.005). In contrast, CEC and EPC did not present any association with admission heart failure, post-PCI angiographic outcomes and 30-day mortality. Endothelin-1 also showed a discrete negative correlation with LVEF
Fig. 2. Relationship between ET-1 levels on admission and EPC counts after 1 week. EPC Q1: first EPC quartile (EPC b 113 cells/ml); EPC Q2: second EPC quartile (EPC 113– 792 cells/ml); EPC Q3: third EPC quartile (EPC 792–1944 cells/ml); EPC Q4: Fourth EPC quartile (EPC N 1944 cells/ml). * P b 0.05. Results are expressed as mean ± SD.
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Table 3 Correlations between ET-1, CEC, EPC, common cardiac biomarkers and LVEF.
Endothelin-1 (on admission) CK-MB Troponin-I LVEF CEC (on admission) CK-MB Troponin-I LVEF EPC (on admission) CK-MB Troponin-I LVEF CEC (1 week after AMI) CK-MB Troponin-I LVEF EPC (1 week after AMI) CK-MB Troponin-I LVEF
Spearman rank correlation
P-value
0.14 0.17 − 0.30
0.16 0.09 0.004
0.00 − 0.07 − 0.06
0.99 0.50 0.57
0.15 − 0.09 − 0.04
0.29 0.53 0.77
− 0.13 − 0.07 − 0.02
0.91 0.54 0.86
− 0.02 − 0.16 0.04
0.85 0.28 0.79
CK, creatin kinase; LVEF, left ventricular ejection fraction; CEC, circulating endothelial cells; EPC; endothelial progenitor cells; AMI, acute myocardial infarction.
Statin treatment and cardiovascular risk factors Statin treatment before AMI onset was associated with lower ET-1 levels on admission (5.60 ± 1.90 pg/ml in patients with statins vs 6.97 ± 2.38 pg/ml in patients without statins; P = 0.04) and a trend towards higher EPC concentrations at 1 week (2312 ± 1945 cells/ml in patients with statins vs 1213 ± 1320 cells/ml; P = 0.09). The linkage between lower EPC concentrations after 1 week and higher ET-1 plasma levels on admission remained significant (P = 0.05) after excluding patients under statin treatment. The extension of CAD (1, 2 or 3 vessels) and the presence of cardiovascular risk factors (smoking, hypertension, dyslipidemia, diabetes and peripheral artery disease) were also explored but no significant association with ET-1, CEC, or EPC concentration was found. Discussion The results of the present study suggest an association between high ET-1 levels on admission and low EPC mobilization after AMI. To our knowledge, this is the first study to assess the potential relationship between ET-1 and both CEC and EPC counts. High ET-1 levels have been associated with poor angiographic (Niccoli et al., 2006) and clinical outcomes (Freixa et al., n.a.; Yip et al., 2005). In agreement with previous studies (Niccoli et al., 2006; Yip et al., 2005), we found a significant association between higher ET-1 levels and worse angiographic outcomes postangioplasty, worse admission Killip class, and higher 30-day mortality. Although reperfusion injury is perhaps the most accepted mechanism of ET-1 action, the present study suggests that impaired EPC mobilization after 1 week is another potential mechanism, thereby offering novel insights into its deleterious effects and new directions for research to characterize EPC modulators after AMI. The main contribution of our findings is to identify a relationship between ET-1 levels on admission and impaired EPC mobilization. Higher ET-1 levels were linked to lower EPC concentrations 1 week after AMI onset. Endothelin-1 impairs nitric oxide signaling in endothelial cells by decreasing eNOS expression (Sud and Black, 2009). The linkage between ET-1 and EPC is not surprising, since the main mechanism of EPC
mobilization from the bone marrow seems to depend on the activation of eNOS (Li et al., 2006). In addition, treatment with eNOS enhancers has been linked to higher EPC mobilization and improved left ventricular remodeling after AMI (Fraccarollo et al., 2008). One potential line of investigation suggested by our findings is the role of endothelin receptor antagonists on EPC mobilization. Although clinical experience is still insufficient, endothelin receptor antagonists have demonstrated a significant reduction of ischemic reperfusion injury after myocardial infarction in animal models (Galiuto et al., 2000; Singh et al., 2006). To our knowledge, ET-1 effects on EPC concentrations have never been assessed. However, they share signaling pathways and therefore it seems feasible that inhibition of ET-1 could modulate EPC mobilization after AMI. It is well established that EPC count is significantly increased in patients with AMI, peaking on the seventh day after onset of symptoms (Shintani et al., 2001). Wojakowski et al. (2006, 2004) suggested that EPCs mobilized from the bone marrow after an AMI played an important role as “committed tissue cells” designed to improve cardiac function, as demonstrated by the significant correlation with post-infarction left ventricular function. Conversely, a recent meta-analysis failed to find any beneficial effect on left ventricular function after an AMI from EPC mobilization by granulocyte colony-stimulating factor (Zohlnhofer et al., 2008); our study also failed to show any correlation between EPC levels and LVEF after 1 week. As with ET-1, CEC count is considered a marker of endothelial damage in acute coronary syndromes (Blann et al., 2005; Lee et al., 2005). Quilici et al. (2004) showed that the highest CEC levels were obtained at the time of infarct presentation. In our study, ET-1 and CEC samples were withdrawn at the same time. Nevertheless, our results suggest that ET-1 levels on admission provide more prognostic information than CEC count, as no significant association was found between CEC levels and clinical, angiographic or echocardiographic outcomes. Another important finding of the current study is the absence of significant correlation between CEC and EPC at both admission and 1 week after AMI onset. Although CEC and EPC mobilization present opposite patterns, our results suggest that the reality is likely to be more complex than simple reciprocity (Hill et al., 2003; Mutin et al., 1999). Treatment with 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors has also been associated with modulation of EPC levels and function. Statin treatment can increase EPC count and improve their function (Werner et al., 2005; Vasa et al., 2001) Intensive statin treatment (atorvastatin 80 mg) after AMI was associated with significantly higher levels of EPC at 4 months of follow-up, compared to patients with standard (atorvastatin 20 mg) treatment (Leone et al., 2008). This finding provides a potential mechanistic explanation for the reduction of acute coronary events associated with intensive statin treatment. Although our study failed to find a significant association between statin treatment prior to AMI and EPC mobilization, we found a strong trend towards higher EPC count at 1 week in patients under statin treatment. Diabetes mellitus also plays a pivotal role in the modulation of CEC/EPC mobilization and function. In comparing diabetic patients with healthy controls, CEC and EPC counts are higher and lower, respectively (Fadini et al., 2005; McClung et al., 2005). Our study did not find any association between diabetic patients and CEC or EPC count, probably due to the limited number of patients and the low incidence of diabetes (18%) in our cohort. Similarly, we found no association between CEC or EPC count and the extension of CAD (1, 2 or 3 vessels) or the presence of peripheral artery disease. Further studies will be needed to clarify this association in STEMI patients.
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Limitations There are several limitations to this study. First, although sample size is similar to previous studies, the number of patients is still relatively small and insufficient to establish the prognostic value of CEC/EPC mobilization for risk prediction in STEMI patients. Additionally, the small size of the sample led to a very low number of events that did not allow for a valid and stable multivariable analysis. Second, without serial ET-1, CEC and EPC measurements, we could not determine actual peak values and time to peak level. Nonetheless, sampling on admission and 1 week after AMI onset seems to be the most accepted timing for measurements. In addition, this time interval represents an achievable target in daily practice. Third, statin treatment could be considered a confounding factor in assessing the relationship between ET-1 and EPC. Nevertheless, the association between ET-1 and EPC remained significant when patients under statin treatment were excluded from analysis. Two final limitations are related to the clinical setting for which this study was designed: nonconsecutive patients were recruited due to a lack of weekend analysis of samples in our laboratory and, although cardiac magnetic resonance is recognized as the most accurate method for LVEF assessment (Longmore et al., 1985), our measurements were done with the echocardiography resources more readily available in our setting. Conclusions In STEMI patients, high ET-1 levels on admission predict a lower EPC mobilization after 1 week. Endothelin-1 provides better clinical, angiographic and echocardiographic information for prognosis than do CEC and EPC concentrations. Acknowledgments Project funded by the Spanish Society of Cardiology, the Hospital Clinic of Barcelona and RD06/0009/1003 (Red HERACLES, Instituto de Salud Carlos III). References The Thrombolysis in Myocardial Infarction (TIMI) trial. Phase I findings. TIMI Study Group. N. Engl. J. Med. 312, 932–936. Bae, J.H., Bassenge, E., Kim, M.H., et al., 2004. Impact of left ventricular ejection fraction on endothelial function in patients with coronary artery disease. Clin. Cardiol. 27, 333–337. Blann, A.D., Woywodt, A., Bertolini, F., et al., 2005. Circulating endothelial cells. Biomarker of vascular disease. Thromb. Haemost. 93, 228–235. Eitel, I., Nowak, M., Stehl, C., et al., 2010. Endothelin-1 release in acute myocardial infarction as a predictor of long-term prognosis and no-reflow assessed by contrast-enhanced magnetic resonance imaging. Am. Heart J. 159, 882–890. Fadini, G.P., Miorin, M., Facco, M., et al., 2005. Circulating endothelial progenitor cells are reduced in peripheral vascular complications of type 2 diabetes mellitus. J. Am. Coll. Cardiol. 45, 1449–1457. Fraccarollo, D., Widder, J.D., Galuppo, P., et al., 2008. Improvement in left ventricular remodeling by the endothelial nitric oxide synthase enhancer AVE9488 after experimental myocardial infarction. Circulation 118, 818–827. Freixa, X., Heras, M., Ortiz, J.T., et al., 2011. Usefulness of endothelin-1 assessment in acute myocardial infarction. Rev. Esp. Cardiol. 64, 105–110.
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Contents lists available at ScienceDirect
Microvascular Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y m v r e
Regular Article
Endothelin-1 levels predict endothelial progenitor cell mobilization after acute myocardial infarction Xavier Freixa a,⁎, Monica Masotti a, Marta Palomo b, Maribel Diaz-Ricart b, Ginés Escolar b, Eduard Guasch a, Ander Regueiro a, Marcelo Jiménez a, Amadeo Betriu a, Magda Heras a a b
Cardiology Department, Hospital Clinic, University of Barcelona, Spain Hematology Department, Hospital Clinic, University of Barcelona, Spain
a r t i c l e
i n f o
Article history: Accepted 21 June 2011 Available online 2 July 2011
a b s t r a c t Introduction: Endothelin-1 (ET-1), circulating endothelial cells (CEC) and endothelial progenitor cells (EPC) are well-known modulators of endothelial function with important cardiac effects after an acute myocardial infarction. However, the relationship between them has never been assessed. The objective of the present study was to establish the relationship between ET-1, CEC, and EPC concentrations after ST-elevation myocardial infarction (STEMI). Methods: Endothelin-1, CEC, and EPC levels were measured in 61 patients presenting with a first STEMI. Samples were withdrawn acutely 6–24 h and 1 week after admission. Assessments included reperfusion outcomes (angiography), left ventricular ejection fraction (echocardiography), and 30-day mortality. Results: Mean age was 60.6 ± 12.6 years and 45 (74%) were males. Higher ET-1 plasma levels were associated with lower EPC count after 1 week (7.45 ± 2.53 pg/ml if EPCs in the first quartile vs 5.72 ± 1.49 pg/ml if EPCs in the fourth quartile; P = 0.04). In contrast with CEC and EPC count, higher ET-1 concentrations on admission were associated with Killip ≥ 2 (9.92 ± 2.01 pg/ml vs 7.32 ± 2.13 pg/ml; P b 0.001), post-reperfusion thrombolysis in myocardial infarction (TIMI) b 3 (8.65 ± 2.86 pg/ml vs 5.87 ± 1.93 pg/ml; P = 0.002), myocardial blush grade (MBG) b 3 (7.46 ± 2.48 pg/ml vs 5.99 ± 2.01 pg/ml; P = 0.004) and higher 30-day mortality (10.29 ± 2.02 pg/ml vs 6.57 ± 2.20 pg/ml; P = 0.005). Conclusions: In STEMI patients, high ET-1 levels on admission predict a lower EPC mobilization after 1 week. Endothelin-1 provides better clinical, angiographic and echocardiographic information for prognosis than do CEC and EPC concentrations. © 2011 Elsevier Inc. All rights reserved.
Introduction Endothelial impairment is an independent predictor of acute cardiovascular events in patients with and without coronary artery disease (CAD) (Halcox et al., 2002). Endothelial dysfunction is also considered a predictor of left ventricular ejection fraction (LVEF) (Bae et al., 2004) associated with worse left ventricle remodeling after myocardial infarction (Matsuo et al., 2006). Endothelin-1 (ET-1) is a well-known marker of endothelial dysfunction in patients with CAD (Iglarz and Clozel, 2007). In acute myocardial infarction (AMI), high ET-1 values on admission have been linked to poor angiographic outcomes after primary angioplasty (Eitel et al., n.a.; Niccoli et al., 2006) and poor prognosis, including higher 30-day mortality (Khan et al., 2007; Yip et al., 2005). ⁎ Corresponding author at: Interventional Cardiology Section, Cardiology Department, Thorax Institute, Hospital Clinic, University of Barcelona, Villarroel 170, Barcelona 08036, Spain. Fax: + 34 932275519. E-mail address: [email protected] (X. Freixa). 0026-2862/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2011.06.008
The measurement of circulating endothelial cells (CEC) and endothelial progenitor cells (EPC) also represents an important and novel technique for the assessment of endothelial injury and repair, respectively. Circulating endothelial cells are biomarkers of damage that correlate with several well-established markers of endothelial dysfunction and predict poor outcomes when elevated (Blann et al., 2005; Lee et al., 2005). In contrast, EPC are biomarkers of endothelial repair with potential cardioprotective effects. Low EPC levels after an AMI have been linked to poor outcomes, including worse LVEF and higher NT-proBNP concentrations (Wojakowski et al., 2006). The main mechanism of EPC mobilization from the bone marrow seems to depend on the activation of endothelial nitric oxide synthase (eNOS) in the presence of several mobilizing factors, such as vascular endothelial growth factor and placental growth factor (Li et al., 2006). Endothelin-1 decreases eNOS expression and could therefore play an important role in CEC and EPC mobilization (Sud and Black, 2009). However, no studies to date have assessed this potential interaction. The aim of the present study was to establish the relationship between ET-1 levels and CEC/EPC mobilization patterns in the early hours after an AMI.
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Methods Study population Between June 2007 and January 2009, the study enrolled 61 nonconsecutive patients who underwent primary angioplasty for a first AMI with elevation of the segment ST in the electrocardiogram (STEMI). Inclusion criteria were prolonged chest pain (N30 min) and ST-segment elevation N1 mm in 2 or more adjacent leads within the first 12 h after onset of symptoms. Exclusion criteria were acute or chronic inflammatory disease, malignancy, or recent surgery, trauma, or infection. The study protocol was approved by the institutional Ethics Committee and all patients gave written informed consent before being included. Percutaneous coronary intervention and angiographic analysis A transradial or transfemoral artery 6F approach was employed. Unfractionated heparin was administered at a dose of 60 UI/kg and a 300-mg loading dose of clopidogrel was administered during percutaneous coronary intervention (PCI). Our protocol includes administration of abciximab prior to PCI if no contraindications exist. Angiographic measurements were always performed independently by two experienced angiographers who assessed thrombolysis in myocardial infarction (TIMI) flow grade (Anon, 1985) and myocardial blush grade (MBG) (van 't Hof et al., 1998). Both TIMI and MBG are angiographic scales for measuring the flow and reperfusion after PCI. TIMI 0 is defined as the absence of distal coronary flow and TIMI 3 means normal flow. MBG is defined as no myocardial blush or contrast density and MBG 3 means normal myocardial blush or contrast density. Suboptimal reperfusion was defined as post-procedural TIMI flow ≤2 or MBG ≤2 despite TIMI 3 flow. Blood sampling and laboratory assay Endothelin-1 One blood sample was obtained from a peripheral vein on admission, prior to PCI (mean 10.8 ± 7.5 h after symptoms onset). Samples were decanted into ethylenediaminetetraacetic acid (EDTA) tubes, centrifuged at 3000 rpm for 15 min, and stored at − 80 °C until analysis. Plasma ET-1 levels were determined by radioimmunoassay using rabbit anti-ET-1 antibody (Peninsula Laboratories Inc, San Carlos, California). Samples were run in pairs and the results were averaged. Endothelial progenitor cells and circulating endothelial cells Two 150 μL blood samples were obtained from a peripheral vein, the first on admission (at the same time as ET-1) and the second after 7 days. Samples were collected into low molecular weight heparin (LMWH) tubes and processed in duplicate within 4 h of extraction. The samples were stained with 10 μL of FITC anti-CD34 (Becton Dickinson), PerCP anti-CD45 (Becton Dickinson), PE anti-kinaseinsert domain receptor (KDR) (R&D Systems), and APC anti-CD133 (eBiosciences) monoclonal antibodies for EPC measurements, and with PE anti-CD146 (Biocytex), FITC anti-CD31 (Becton Dickinson), and PerCP anti-CD45 (Becton Dickinson) monoclonal antibodies for CEC measurements. Red blood cells in the samples were subsequently lysed for 15 min (BD FACS Lysing Solution, Becton Dickinson). Samples were then centrifuged, washed twice, and resuspended in PBS with LMWH (50 U/mL), and analyzed using a FACSCalibur flow cytometer (Becton Dickinson). The sensitivity of fluorescence detectors was set and monitored using Calibrite Beads (Becton Dickinson) according to manufacturer's recommendations. Instrumental settings in order to set up accurate positivity of each fluorochrome were established using known cell populations. In addition, cells stained with isotopic controls for IgG conjugated with
the different fluorochromes were used as negative controls. Assessment of 2 × 10 6 events per sample was considered sufficient for statistical analysis. EPC and CEC were defined as negative for CD45 and positive for CD34, KDR, and CD133 (CD45-CD34 + KDR + CD133+ for EPC), and negative for CD45 and positive for CD146, and CD31 (CD45-CD146 + CD31+ for CEC), respectively. In the present study, positivity was established when the mean fluorescence intensity was N10 1 for each fluorochrome. Numbers were calculated multiplying the ratio of EPC and CEC obtained in the flow cytometry analysis by the number of leukocytes/ml in the blood sample to obtain the absolute number of both EPC and CEC per 1 mL of whole blood. Cardiac biomarkers Two common cardiac biomarkers, creatine kinase myocardial band (CK-MB) and troponin-I, were measured every 4 h during the first 24 h and then every 24 h for 3 days. Echocardiography Echocardiographic assessment was performed 3–7 days after PCI with Vivid 7 systems (GE-Vingmed, Milwaukee, Wisconsin). Left ventricle diameter was measured in parasternal and apical views and ejection fraction was evaluated using biplane Simpson's method by an expert cardiologist blinded to the other results. Statistical analysis Results are expressed as mean ± SD or median, depending on normal distribution as assessed by the Shapiro–Wilks test, and considered significant at a P-value b0.05. Comparisons between groups were performed using unpaired t test or Mann–Whitney U test for continuous variables and Chi-square or Fisher's exact test for categorical variables. Correlation analyses were done by Spearman's rank correlation. Assuming that CEC and EPC present at least a 2-fold change after AMI, the sample size was calculated to allow detection of 50% variation with 85% power at P b 0.05. Statistical analyses were done using SPSS package v14.0 (Chicago, Illinois). Results Patient characteristics The clinical characteristics of the study population are shown in Table 1. Mean symptoms-to-angioplasty time was 317 ± 255 min. The clinical and angiographic characteristics of the cohort are listed in Table 2. Suboptimal reperfusion was observed in 43.5% of patients. Three patients died within the first 30 days (2 of heart failure and 1 of cardiogenic shock). Mean peak CK-MB and troponin I after AMI were
Table 1 Baseline characteristics of the study population. Basal characteristics
n = 61
Age (years) Males (%) Smokers (%) Hypertension (%) Hypercholesterolemia (%) Diabetes (%) Peripheral vascular disease (%) Previous PCI/CABG (%) Treatment before admission (%) – Beta-blockers – Statins – ACE inhibitors
60.6 ± 12.6 74% 52% 54% 38% 18% 5% 9%/0% 26% 15% 21%
ACE, angiotensin-converting enzyme; PCI, percutaneous coronary intervention; CABG, coronary artery by-pass grafting.
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Table 2 Acute myocardial infarction characteristics. AMI characteristics Admission hemodynamics Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Heart rate (bpm) Killip I at admission TIMI risk score (points) Ischemia time Symptoms-balloon (min) AMI location: anterior (%) Culprit artery (LAD/RCA) (%) Number of diseased vessels (1/2/3) (%) Pre-PCI TIMI 0 (%) TIMI 1 (%) TIMI 2 (%) TIMI 3 (%) Post-PCI TIMI 0 (%) TIMI 1 (%) TIMI 2 (%) TIMI 3 (%) Post-PCI MBG 0 (%) MBG 1 (%) MBG 2 (%) MBG 3 (%)
n = 61 130.6 ± 32.4 78.7 ± 21.6 77.2 ± 19.1 88.5% 2.9 ± 2.1 317 ± 255 47.5% 47.5/35% 71/23/6% 60.6% 4.9% 14.8% 19.7% 0% 1.6% 4.9% 93.4% 0% 5% 37.7% 57.3%
AMI, acute myocardial infarction; LAD, left anterior descending; RCA, right coronary artery; MBG, myocardial blush grade.
182.8 ± 122.2 ng/ml and 178.65 ± 285.7 ng/ml, respectively. Left ventricular ejection fraction after PCI was 45.8 ± 9.7%. ET-1, CEC, and EPC mobilization patterns Endothelin-1, CEC, and EPC on admission presented a median value of 6.3 pg/ml (25th–75th interquartile range: 4.9–8.5 pg/ml), 220 cells/ml (25th–75th interquartile range: 100–573 cells/ml) and 410 cells/ml (25th–75th interquartile range: 93–1508 cells/ml), respectively. After 1 week, CEC and EPC median concentrations were 116 cells/ml (25th–75th interquartile range: 0–250 cells/ml) and 792 cells/ml (25th–75th interquartile range: 113–1944 cells/ml), respectively. The number of CEC was significantly higher on admission than at 7 days after myocardial infarction onset (459 ± 672 cells/ml on admission and 297 ± 594 cells/ml at 1 week; P b 0.001). Endothelial progenitor cells showed the opposite pattern, with a trend towards higher concentrations after 1 week (1238 ± 1994 cells/ml on admission and 1354 ± 1420 cells/ml at 1 week; P = 0.10) (Fig. 1). We found no correlation between CEC and EPC mobilization. As shown in Fig. 2, lower EPC concentrations after 1 week were associated with higher plasma levels of ET-1 (7.45 ± 2.53 pg/ml in patients with EPCs in the first quartile vs 5.72 ± 1.49 pg/ml in patients with EPCs in the fourth quartile; P = 0.04). Conversely, there was no significant relationship between CEC count and ET-1 levels.
Fig. 1. CEC (A) and EPC (B) levels on admission and 1 week after acute myocardial infarction onset.
(r = − 0.30; P = 0.004) while CEC and EPC failed to demonstrate any significant correlation with the common cardiac biomarkers or LVEF (Table 3).
Clinical, angiographic and echocardiographic outcomes Higher ET-1 levels on admission were associated with worse admission heart failure measured by Killip class (9.92 ± 2.01 pg/ml if Killip ≥ II vs 7.32 ± 2.13 pg/ml if Killip I; P b 0.001), worse postPCI TIMI (8.65 ± 2.86 pg/ml if TIMI b 3 vs 5.87 ± 1.93 pg/ml if TIMI = 3; P = 0.002), worse post-PCI MBG (7.46 ± 2.48 pg/ml if MBG b 3 vs 5.99 ± 2.01 pg/ml if MBG = 3; P = 0.004), and higher 30-day mortality (10.29± 2.02 pg/ml in patients who died vs 6.57 ± 2.20 pg/ml in patients who did not; P = 0.005). In contrast, CEC and EPC did not present any association with admission heart failure, post-PCI angiographic outcomes and 30-day mortality. Endothelin-1 also showed a discrete negative correlation with LVEF
Fig. 2. Relationship between ET-1 levels on admission and EPC counts after 1 week. EPC Q1: first EPC quartile (EPC b 113 cells/ml); EPC Q2: second EPC quartile (EPC 113– 792 cells/ml); EPC Q3: third EPC quartile (EPC 792–1944 cells/ml); EPC Q4: Fourth EPC quartile (EPC N 1944 cells/ml). * P b 0.05. Results are expressed as mean ± SD.
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Table 3 Correlations between ET-1, CEC, EPC, common cardiac biomarkers and LVEF.
Endothelin-1 (on admission) CK-MB Troponin-I LVEF CEC (on admission) CK-MB Troponin-I LVEF EPC (on admission) CK-MB Troponin-I LVEF CEC (1 week after AMI) CK-MB Troponin-I LVEF EPC (1 week after AMI) CK-MB Troponin-I LVEF
Spearman rank correlation
P-value
0.14 0.17 − 0.30
0.16 0.09 0.004
0.00 − 0.07 − 0.06
0.99 0.50 0.57
0.15 − 0.09 − 0.04
0.29 0.53 0.77
− 0.13 − 0.07 − 0.02
0.91 0.54 0.86
− 0.02 − 0.16 0.04
0.85 0.28 0.79
CK, creatin kinase; LVEF, left ventricular ejection fraction; CEC, circulating endothelial cells; EPC; endothelial progenitor cells; AMI, acute myocardial infarction.
Statin treatment and cardiovascular risk factors Statin treatment before AMI onset was associated with lower ET-1 levels on admission (5.60 ± 1.90 pg/ml in patients with statins vs 6.97 ± 2.38 pg/ml in patients without statins; P = 0.04) and a trend towards higher EPC concentrations at 1 week (2312 ± 1945 cells/ml in patients with statins vs 1213 ± 1320 cells/ml; P = 0.09). The linkage between lower EPC concentrations after 1 week and higher ET-1 plasma levels on admission remained significant (P = 0.05) after excluding patients under statin treatment. The extension of CAD (1, 2 or 3 vessels) and the presence of cardiovascular risk factors (smoking, hypertension, dyslipidemia, diabetes and peripheral artery disease) were also explored but no significant association with ET-1, CEC, or EPC concentration was found. Discussion The results of the present study suggest an association between high ET-1 levels on admission and low EPC mobilization after AMI. To our knowledge, this is the first study to assess the potential relationship between ET-1 and both CEC and EPC counts. High ET-1 levels have been associated with poor angiographic (Niccoli et al., 2006) and clinical outcomes (Freixa et al., n.a.; Yip et al., 2005). In agreement with previous studies (Niccoli et al., 2006; Yip et al., 2005), we found a significant association between higher ET-1 levels and worse angiographic outcomes postangioplasty, worse admission Killip class, and higher 30-day mortality. Although reperfusion injury is perhaps the most accepted mechanism of ET-1 action, the present study suggests that impaired EPC mobilization after 1 week is another potential mechanism, thereby offering novel insights into its deleterious effects and new directions for research to characterize EPC modulators after AMI. The main contribution of our findings is to identify a relationship between ET-1 levels on admission and impaired EPC mobilization. Higher ET-1 levels were linked to lower EPC concentrations 1 week after AMI onset. Endothelin-1 impairs nitric oxide signaling in endothelial cells by decreasing eNOS expression (Sud and Black, 2009). The linkage between ET-1 and EPC is not surprising, since the main mechanism of EPC
mobilization from the bone marrow seems to depend on the activation of eNOS (Li et al., 2006). In addition, treatment with eNOS enhancers has been linked to higher EPC mobilization and improved left ventricular remodeling after AMI (Fraccarollo et al., 2008). One potential line of investigation suggested by our findings is the role of endothelin receptor antagonists on EPC mobilization. Although clinical experience is still insufficient, endothelin receptor antagonists have demonstrated a significant reduction of ischemic reperfusion injury after myocardial infarction in animal models (Galiuto et al., 2000; Singh et al., 2006). To our knowledge, ET-1 effects on EPC concentrations have never been assessed. However, they share signaling pathways and therefore it seems feasible that inhibition of ET-1 could modulate EPC mobilization after AMI. It is well established that EPC count is significantly increased in patients with AMI, peaking on the seventh day after onset of symptoms (Shintani et al., 2001). Wojakowski et al. (2006, 2004) suggested that EPCs mobilized from the bone marrow after an AMI played an important role as “committed tissue cells” designed to improve cardiac function, as demonstrated by the significant correlation with post-infarction left ventricular function. Conversely, a recent meta-analysis failed to find any beneficial effect on left ventricular function after an AMI from EPC mobilization by granulocyte colony-stimulating factor (Zohlnhofer et al., 2008); our study also failed to show any correlation between EPC levels and LVEF after 1 week. As with ET-1, CEC count is considered a marker of endothelial damage in acute coronary syndromes (Blann et al., 2005; Lee et al., 2005). Quilici et al. (2004) showed that the highest CEC levels were obtained at the time of infarct presentation. In our study, ET-1 and CEC samples were withdrawn at the same time. Nevertheless, our results suggest that ET-1 levels on admission provide more prognostic information than CEC count, as no significant association was found between CEC levels and clinical, angiographic or echocardiographic outcomes. Another important finding of the current study is the absence of significant correlation between CEC and EPC at both admission and 1 week after AMI onset. Although CEC and EPC mobilization present opposite patterns, our results suggest that the reality is likely to be more complex than simple reciprocity (Hill et al., 2003; Mutin et al., 1999). Treatment with 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors has also been associated with modulation of EPC levels and function. Statin treatment can increase EPC count and improve their function (Werner et al., 2005; Vasa et al., 2001) Intensive statin treatment (atorvastatin 80 mg) after AMI was associated with significantly higher levels of EPC at 4 months of follow-up, compared to patients with standard (atorvastatin 20 mg) treatment (Leone et al., 2008). This finding provides a potential mechanistic explanation for the reduction of acute coronary events associated with intensive statin treatment. Although our study failed to find a significant association between statin treatment prior to AMI and EPC mobilization, we found a strong trend towards higher EPC count at 1 week in patients under statin treatment. Diabetes mellitus also plays a pivotal role in the modulation of CEC/EPC mobilization and function. In comparing diabetic patients with healthy controls, CEC and EPC counts are higher and lower, respectively (Fadini et al., 2005; McClung et al., 2005). Our study did not find any association between diabetic patients and CEC or EPC count, probably due to the limited number of patients and the low incidence of diabetes (18%) in our cohort. Similarly, we found no association between CEC or EPC count and the extension of CAD (1, 2 or 3 vessels) or the presence of peripheral artery disease. Further studies will be needed to clarify this association in STEMI patients.
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Limitations There are several limitations to this study. First, although sample size is similar to previous studies, the number of patients is still relatively small and insufficient to establish the prognostic value of CEC/EPC mobilization for risk prediction in STEMI patients. Additionally, the small size of the sample led to a very low number of events that did not allow for a valid and stable multivariable analysis. Second, without serial ET-1, CEC and EPC measurements, we could not determine actual peak values and time to peak level. Nonetheless, sampling on admission and 1 week after AMI onset seems to be the most accepted timing for measurements. In addition, this time interval represents an achievable target in daily practice. Third, statin treatment could be considered a confounding factor in assessing the relationship between ET-1 and EPC. Nevertheless, the association between ET-1 and EPC remained significant when patients under statin treatment were excluded from analysis. Two final limitations are related to the clinical setting for which this study was designed: nonconsecutive patients were recruited due to a lack of weekend analysis of samples in our laboratory and, although cardiac magnetic resonance is recognized as the most accurate method for LVEF assessment (Longmore et al., 1985), our measurements were done with the echocardiography resources more readily available in our setting. Conclusions In STEMI patients, high ET-1 levels on admission predict a lower EPC mobilization after 1 week. Endothelin-1 provides better clinical, angiographic and echocardiographic information for prognosis than do CEC and EPC concentrations. Acknowledgments Project funded by the Spanish Society of Cardiology, the Hospital Clinic of Barcelona and RD06/0009/1003 (Red HERACLES, Instituto de Salud Carlos III). References The Thrombolysis in Myocardial Infarction (TIMI) trial. Phase I findings. TIMI Study Group. N. Engl. J. Med. 312, 932–936. Bae, J.H., Bassenge, E., Kim, M.H., et al., 2004. Impact of left ventricular ejection fraction on endothelial function in patients with coronary artery disease. Clin. Cardiol. 27, 333–337. Blann, A.D., Woywodt, A., Bertolini, F., et al., 2005. Circulating endothelial cells. Biomarker of vascular disease. Thromb. Haemost. 93, 228–235. Eitel, I., Nowak, M., Stehl, C., et al., 2010. Endothelin-1 release in acute myocardial infarction as a predictor of long-term prognosis and no-reflow assessed by contrast-enhanced magnetic resonance imaging. Am. Heart J. 159, 882–890. Fadini, G.P., Miorin, M., Facco, M., et al., 2005. Circulating endothelial progenitor cells are reduced in peripheral vascular complications of type 2 diabetes mellitus. J. Am. Coll. Cardiol. 45, 1449–1457. Fraccarollo, D., Widder, J.D., Galuppo, P., et al., 2008. Improvement in left ventricular remodeling by the endothelial nitric oxide synthase enhancer AVE9488 after experimental myocardial infarction. Circulation 118, 818–827. Freixa, X., Heras, M., Ortiz, J.T., et al., 2011. Usefulness of endothelin-1 assessment in acute myocardial infarction. Rev. Esp. Cardiol. 64, 105–110.
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