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To determine the relationship between the number of CD14+ cells, myocardial infarct (MI) size and left ventricular (LV) volumes in ST segment elevation MI (STEMI) and non-ST segment elevation MI (NSTEMI) patients.
A total of 62 patients with STEMI (n=34) or NSTEMI (n=28) were enrolled. The number of CD14+ cells was assessed at admission. Infarct size, left ventricular ejection fraction (LVEF) and LV volumes were measured using magnetic resonance imaging five days after MI and six months after MI.
In STEMI patients, the number of CD14+ cells was positively and significantly correlated with infarct size at day 5 (r=0.40; P=0.016) and after six months (r=0.34; P=0.047), negatively correlated with LVEF at day 5 (r=−0.50; P=0.002) and after six months (r=−0.46; P=0.005) and positively correlated with end-diastolic (r=0.38; P=0.02) and end-systolic (r=0.49; P=0.002) volumes after six months. In NSTEMI patients, no significant correlation was found between the number of CD14+ cells and infarct size, LVEF or LV volumes at day 5 or after six months.
The number of CD14+ cells at admission was associated with infarct size and LV remodelling in STEMI patients with large infarct size, whereas in NSTEMI patients, no relationship was observed between numbers of CD14+ cells and LV remodelling.
Left ventricular (LV) remodelling after acute myocardial infarction (MI) is characterized by increased LV volumes, which result in impairment of myocardial function (1,2). Many factors influence LV remodelling, particularly infarct location and the extent of necrosis (3).
Acute MI is associated with an increased release of monocytes into the bloodstream; these monocytes reportedly contribute to ischemic tissue healing after acute MI, which should help to prevent LV remodelling (4–6). Recently, new research has provided more insight into the sequential mobilization and respective roles of the different monocyte subsets in acute MI (7–9). Particularly, Tsujioka et al (8) demonstrated that the CD14+CD16− subset is associated with the extent of myocardial salvage in ST segment elevation MI (STEMI) patients with large infarctions. However, the role of CD14+ monocytes has not been studied in the context of non-ST segment elevation MI (NSTEMI), as emphasized by Kavsak and Jaffe (10). Thus, given the hematopoietic origin of CD14+ monocytes, which increase in peripheral blood in acute MI, we aimed to assess the relationship among the number of circulating CD14+ cells, infarct size and LV remodelling in both STEMI and NSTEMI patients, as assessed by magnetic resonance imaging (MRI) five days after MI and six months after MI.
The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology (11).
Patients younger than 75 years of age who were referred to the department for a first acute MI and were admitted within 12 h of the onset of symptoms were considered eligible for inclusion in the present study. MI was defined by the guidelines of the joint Task Force of the European Society of Cardiology, the American College of Cardiology, the American Heart Association, and the World Heart Federation (12). This definition includes both STEMI and NSTEMI that were confirmed by detection of elevated levels of creatine kinase (CK) and troponin I (TnI) (at least one value above the 99th percentile of the upper reference limit), together with evidence of myocardial ischemia (ie, new ST-T changes or new left bundle branch block, or development of pathological Q waves in the electrocardiogram). Exclusion criteria were previous cardiovascular or pulmonary disease, previous treatment with statins, a history of renal or hepatic disease, hematological or coagulative disorders, anemia (defined as hemoglobin <120 g/L), thrombocytopenia (platelet count <150 × 109/L), immune suppression, acute or chronic inflammatory disease, pregnancy, recent trauma, underlying infection, cancer and cardiogenic shock. All patients were referred to the catheterization laboratory within the first 24 h for a coronary angiogram, and received medication according to current guidelines (13,14). The study protocol was approved by the local ethics committee and informed consent was obtained from all participants.
Serum levels of TnI, creatine kinase (CK) and creatine kinase isoenzyme myocardial and brain (CK-Mb) were determined at admission, and at 6 h, 12 h, 24 h and 48 h after the onset of MI. TnI levels were expressed as peak level and area under the curve (AUC). Creatinine, glycosylated hemoglobin, glycemia, high sensitivity C-reactive protein (hs-CRP), brain natriuretic peptide, total cholesterol, low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol and triglycerides were also determined on admission. Interleukin-8 (IL-8) levels were determined from samples that had been stored at −80°C on admission using commercially available kits (R&D Systems, France). Concentrations of plasma IL-8 were calculated from a standard curve, following the manufacturer’s instructions.
Peripheral blood samples were drawn on admission after the onset of MI. Twenty millilitres of heparinised blood was diluted with phosphate buffered saline and centrifuged on a Hypaque-Ficoll density gradient at 800 g for 30 min. Isolated mononuclear cells were stained with fluoroscein isothiocyanate-conjugated monoclonal antibodies against CD14 (Becton Dickinson, USA) and analyzed using a FACsort flow cytometer equipped with CellQuest software (Becton Dickinson, USA).
All MRI studies were conducted at 3.0 field strength (General Electric, USA) with a standard 40 mT/m gradient, using an eight-element phased-array surface coil. LV function was assessed by electrocardiography-gated cine steady-state free precession breath-hold sequences in the two-chamber and four-chamber views as well as in the short cardiac axis from base to apex (30 phases per cardiac cycle; repetition time [TR] 3.5 ms, echo time [TE] 1.2 ms, flip angle 45°, typical voxel size 1.92 mm × 1.25 mm × 8.0 mm) (15). T2-weighted images (triple inversion recovery; TE 60 ms, TR 2x R-R interval, inversion time [TI] 170 ms, slice thickness 7 mm, flip angle 180°, pixel size 2.3 mm × 1.3 mm) were acquired in the short-axis plane. First-pass perfusion imaging was performed using a T1-weighted fast gradient echo sequence (fast gradient echo TR/TE = 3.5 ms/1.5 ms) with a notched saturation pulse, after the injection of a bolus of gadolinium (Dota-Gd, Guerbet, France) in a brachial vein at the single dose of 0.2 mL/kg. The field of view was 400 mm and the matrix size was 256 × 224, interpolated to 256 × 256. The heart was imaged in the short-axis plane with four to six slices 8 mm thick with a gap of 1 mm. One image per slice was acquired every two cardiac cycles, leading to a temporal resolution of 2 RR per imaging plane. Forty frames were obtained from each imaged plane within 80 RR. For late gadolinium enhancement imaging at 15 min, a breath-hold ECG-gated T1-weighted sequence was used (TE = MinFull, field of view 440 mm, TI optimised to obtain an optimal myocardial nulling, matrix 256×224 interpolated to 256×256, slice thickness 8 mm, gap 1 mm). The number of slices and position of slices were the same as used for the perfusion first-pass imaging.
Image analysis was performed by two fully blinded operators using a dedicated offline workstation (General Electric, USA). LV ejection fraction (LVEF), end-diastolic volume (LVEDV), end-systolic volume (LVESV) and stroke volume were calculated from cine steady-state free precession short-axis views by drawing endocardial and epicardial contours (16). Infarct size was manually traced and calculated from the late gadolinium enhancement short-axis images (17). Myocardial regions were considered infarcted if the infarct size signal intensity was >2 SD above the remote myocardium. Measurements are expressed as % LV. The portion of the heart exhibiting late enhancement was classified according to the segmentation established by the American Heart Association, which divides the LV into 17 segments (18).
Continuous variables were tested for normal distribution using the Kolmogorov-Smirnov test. Data are expressed as mean ± SD or as median and interquartile range, as appropriate. The number of CD14+ cells was analyzed after log transformation to normalize distribution. Categorical variables were compared using the χ2 test or Fisher’s exact test. Between-group comparisons were performed using an unpaired t test or the Mann-Whitney U test and Kruskal-Wallis test for continuous data, as appropriate. The correlations between the log-transformed number of CD14+ cells and TnI AUC, infarct size, LVESD, LVEDV, stroke volume and LVEF were assessed using the Spearman rank test. Differences were considered to be statistically significant at P<0.05. Statistical analysis was performed using SAS software version 9.1 (SAS Institute, USA).
During the six-month enrollment period, 311 patients were admitted with STEMI or NSTEMI. A total of 62 patients with STEMI (n=34) or NSTEMI (n=28) were enrolled in the study after consideration of inclusion and exclusion criteria and obtaining patient consent. The clinical characteristics of the study population are summarized in Table 1. All patients underwent coronary angiography within the first 24 h. All patients who survived the in-hospital phase were followed up after six months; none had been lost to follow-up. No significant difference was observed between NSTEMI and STEMI groups in terms of age, cardiovascular risk factors and previous medication. The levels of CK, CK-Mb and TnI were significantly lower in NSTEMI patients.
CD14+ cells were counted at admission in both groups and were expressed as percentage of positive cells (number of positive cells per 105 events) (Figures 1A and 1B).
The number of CD14+ cells was lower in the NSTEMI group compared with the STEMI group, but the difference was not statistically significant (43.3±19.0% versus 50.8±22.4%; P=0.18). No difference in the levels of hs-CRP and IL-8 was observed between groups. In addition, no relationships among the number of CD14+cells, hs-CRP levels and IL-8 levels were observed between STEMI and NSTEMI patients at admission.
Baseline and follow-up measurements of infarct size, LVEF, LV and stroke volumes are shown in Table 2. A significant difference in infarct size was observed between STEMI and NSTEMI patients at day 5 and after six months, resulting in a difference in LVEF between these groups at the same timepoints. In addition, no significant LV remodelling was observed between day 5 and after six months in either group, despite a tendency toward increased LVEDV and LVESV in both groups. LVESV was greater at day 5 and after six months in STEMI patients compared with NSTEMI patients.
In STEMI patients, a significant positive correlation between the number of CD14+ cells and infarct size at day 5 (r=0.409; P=0.016) and after six months (r=0.342; P=0.047) was observed (Figures 2A and 2B). In addition, the number of CD14+ cells was significantly correlated with peak TnI (r=0.34; P=0.044) and AUC (r=0.37; P=0.020). In NSTEMI patients, no significant correlation was found between the number of CD14+ cells and infarct size at day 5 (r=0.23; P=0.21) or after six months (r=0.05; P=0.79) (Figures 2C and 2D) or with levels of TnI.
At day 5 and after six months, the number of CD14+ cells was significantly correlated with LVEF (r=−0.50, P=0.002; r=−0.46, P=0.005, respectively) and LVESV (r=0.39, P=0.020; r=0.49, P=0.002, respectively) in STEMI patients. In addition, the number of CD14+ cells was significantly correlated with LVEDV after six months (r=0.38, P=0.02) but not at day 5. Figures 3A, 3B and 3C present the correlations between LVEF and LV volumes and numbers of CD4+ cells after six months in STEMI patients. In NSTEMI patients, there was no relationship between the number of CD14+ cells and either LVEF (r=−0.022, P=0.90; r=0.13, P=0.50, respectively) or LV volumes at day 5 or after six months. Figures 3D, 3E and 3F show the nonsignificant correlations between the number of CD14+ cells and LVEF and LV volumes after six months in NSTEMI patients.
In the present study, we showed that CD14+ cell counts were significantly correlated with larger infarct size in STEMI patients, as assessed by MRI and TnI levels. In addition, the number of CD14+ cells was higher in STEMI than in NSTEMI patients, although this was not statistically significant. These results suggest that the number of CD14+ cells recruited at day 0 increases with the size of infarction, as assessed at day 5, and predicts the extent of healing after six months. Our results also partially confirm the findings of Tsujioka et al (8) regarding the impact of different subsets of CD14+ cells on myocardial salvage and LVEF in STEMI patients. However, our results do not support the validity of these findings in NSTEMI patients, because the number of CD14+ cells was not associated with infarct size or LVEF in our study.
Numerous studies have shown that acute MI is associated with the mobilization of a large variety of hematopoietic stem/progenitor cells (19). Cells mobilized from the bone marrow into peripheral blood are a mix of stem/progenitor cells and cells of committed lineages such as monocytes/macrophages that express hematopoietic and endothelial cell markers, including CD14. CD14 is a coreceptor of toll-like receptors 4 and 2, can detect antigenic molecules such as lipopolysaccharide, and is expressed by mature monocytes and macrophages (5). In STEMI patients, infarct size and infarct healing are associated with peripheral recruitment of monocytes (4). However, no data concerning the role of CD14+ cells in NSTEMI patients have been available until the present study.
As reported by other authors (7,8,20,21), a larger infarct size is associated with a more severe inflammatory process and, consequently, a higher white blood cell count in peripheral blood in acute MI. In the present study, the lack of significant correlations between the number of CD14+ cells and infarct size or LVEF in NSTEMI patients could be explained by a lesser extent of necrosis and, subsequently, lesser inflammatory phenomenon during acute MI, in accordance with previous hypotheses (22). However, we did not observe any differences between STEMI and NSTEMI groups in terms of inflammatory markers such as hs-CRP and IL-8. This contradictory result may be explained by the small number of patients in each group.
In STEMI patients, the large infarct size and subsequent infarct healing is associated with peripheral recruitment of monocytes/macrophages that play a key role in LV remodelling (4). The results of the present study demonstrate a significant difference between STEMI and NSTEMI patients in terms of LVESV. It is well established that LVESV is a prognostic factor of LV remodelling in MI, with greater LV remodelling observed in STEMI patients than in NSTEMI (23). The same tendency was observed with LVEDV, albeit without attaining statistical significance. The number of CD14+ cells was related to LV dilation after six months in STEMI patients.
Our results are in agreement with those of Maekawa et al (24), who showed an association between high levels of monocytes and LV dysfunction after acute MI. Conversely, our findings are in contrast with those of Hojo et al (25), who showed an improvement in LV function related to a higher monocyte count.
Taken together, the results of the present study suggest that mobilization of CD14+ cells is associated with infarct size and LV remodelling in STEMI patients with large infarct size, but not in NSTEMI patients with relatively small infarct size.
There are several limitations to the present study. First, the number of patients included was small for an observational study. Second, our results do not provide any mechanistic explanation for the role of CD14+ cells in infarct size and LV remodelling. Finally, because CD14+ cells are a heterogenous population of hematopoietic cells, the exact characterisation of CD14+ cells would be required in further studies.
The results of the present study show that the impact of the presence of CD14+ cells is different in STEMI and NSTEMI patients. The number of CD14+ cells is associated with larger infarct size and LV remodelling in STEMI patients, whereas no relationship was found between the number of CD14+ cells and either infarct size or LV volumes in NSTEMI patients. These results suggest that the impact of CD14+ recruitment is different in LV remodelling in STEMI and NSTEMI patients.