Evaluation of the relationship between anti-inflammatory cytokines and adverse cardiac remodeling after myocardial infarction

Aim To clarify the role of interleukin (IL) – 10 and members of its subfamily (IL-19 and IL-26) in cardiac remodeling during the post-myocardial infarction (MI) period. Material and methods A total of 45 patients with ST-segment elevation MI were enrolled. Serum cytokine concentrations were measured at the first day and 14 days post-MI. Left ventricular (LV) reverse remodeling (RR) was defined as the reduction of LV end-diastolic volume or LV end-systolic volume by ≥ 12 % in cardiac magnetic resonance images at 6-mo follow-up. A 12 % increase was defined as adverse remodeling (AR). Results the RR group compared to the AR group. There was a significant decrease in the concentration of anti-inflammatory cytokines in the AR group from the first to the 14 days post-MI. However, no significant change was observed in the RR group. Regression analysis revealed that a low IL-10 concentration on the post-MI first day was related to RR (OR=0.76, p=0.035). A 1 % increase in change of IL-10 concentration increased the probability of RR by 1.07 times. Conclusion The concentrations of cytokines were higher in the AR group, but this elevation was not sustained and significantly decreased for the 14 days post-MI. In the RR group, the concentrations of cytokines did not change and stable for the 14 days post-MI. As a reflection of this findings, stable IL-10 concentration may play a role the improvement of cardiac functions.


Introduction
Cardiac remodeling occurs as a result of inflam matory / antiinflammatory homeostasis that develops after acu te myocardial infarction (MI). While an excessive inflammatory response induces adverse remodeling (AR), moderate inflammatory and strong antiinflammatory responses can cause reverse remodeling (RR) [1]. Pro inflammatory cytokines, such as tumor necrosis factor (TNF) -α, interleukin (IL) -1β, and IL6, which increa se in the early postMI period, are known to cause apoptosis, collagen production, matrix metalloproteinase (MMP) activation, fibrosis, and ventricular dilation that, in turn, cause ventricular failure [1][2][3]. It is thought that an increased antiinflammatory response during this process would suppress inflammation and improve cardiac function [4].
One of the cytokines thought to play a major role in the antiinflammatory balance in the postMI period is IL10 [5]. It is a powerful antiinflammatory cytokine, as well as a cytokine synthesis inhibitor in humans [6]. In the postMI period, IL10 inhibits lipopolysaccharide induced production of proinflammatory cytokines, such as TNFα, IL1, and IL6 [7,8]. IL10 also acts as an antioxidant. It inhibits the production of reactive oxygen species by TNFα in cardiac myocytes [9]. Experimental studies in mice with IL10 deficiency have confirmed that an increase in neutrophil infiltration increases myocardial infarct size [10].
ОРИГИНАЛЬНЫЕ СТАТЬИ § IL19, one of the IL10 superfamily cytokines, was first discovered in the 2000s [11]. It is mainly secreted by lymphocytes, macrophages, and monocytes. It is a multifunctional cytokine that is also secreted in small amounts by nonimmune cells, such as smooth muscle cells and endothelial cells [12]. IL19 binds to IL20R1 / IL 20R2 on target cells [13,14]. By activating the JAK / STAT pathway, it participates in the progression of many systemic diseases including ischemia / reperfusion injury of the brain and kidneys, psoriasis, and angiogenesis in diabetes. Some recent studies have also focused on the cardioprotective effect of IL19 [15,16].
IL26 is another cytokine of the IL10 superfamily. IL 26 is produced by macrophages, natural killer cells, and T helper (Th) -1 and Th17 cells [17]. Recent studies have demonstrated that IL26 plays a role in the etiopathogenesis of autoimmune diseases, such as colitis, rheumatoid arthritis, and psoriasis [18]. We have not found any study that examined the role of IL26 in acute coronary syndrome and cardiac remodeling.
In this study, we aimed to examine the role of IL10 and members of its superfamily, IL19 and IL26, in cardiac remodeling during the postMI period.

Study population and design
This study was planned as a multicenter, prospective study from June 2015 thorugh June 2016. The study was designed in accordance with the Helsinki Declaration and with good clinical practices guidelines. All participants signed informed, voluntary consent forms. Approval was obtained from the local ethics committee. Assuming an alpha of 0.05, a power of 0.80, and with an 20 % estimated adverse remodeling (AR) rate, in agreement with previous reports, the estimated sample size was at least 40 patients.
Two hundred eighteen STsegment elevation MI (STEMI) patients above the age of 18 who presented within the first 12 hrs after the onset of chest pain ongoing for more than 30 min with >0.1 mV STsegment elevation in two or more related leads, and who underwent primary percutaneous coronary intervention (PCI) were evaluated in the study. The definition of STEMI was according to the third universal definition of myocardial infarction (MI) [19] and was managed according to the latest European Society of Cardiology guidelines [20]. 167 patients were excluded from the study (see exclusion criteria below). In 6 of the remaining 51 patients, no change in volumes or less than 12 % change was observed at the 6mo followup examination, and these patients were also excluded. The remaining 45 patients were included in the study Exclusion criteria: 1) in cardiogenic shock or in need of an intraaortic balloon pump; 2) an estimated glomerular filtration rate (eGFR) ≤30 ml / min / 1.73 m 2 ; 3) Killip class ≥3; 4) a history of silent ischemia or infarct, 5) right coronary artery occlusion; 6) any kind of systemic inflammatory disease; 7) autoimmune disease; 8) a history of chronic corticosteroid or antiinflammatory drugs; 9) pregnancy or delivery within the last 90 days or breastfeeding; 10) emergency or elective coronary artery bypass grafting planned after the angiography; 11) patients who had myocardial reinfarction during follow up. Left ventricular (LV) RR was defined as the reduction of LV enddiastolic volume (LVEDV) or LV endsystolic volume (LVESV) by ≥ 12 % in cardiac magnetic resonance (CMR) images at 6mo followup, and a 12 % increase was defined as AR [21,22]. Clinical, demographic, laboratory, and radiological findings were recorded in a timely manner in the patients' files during followup.

Biochemical analyses
Venous blood samples were taken for hemoglobin, cardiac troponin I, Creactive protein (CRP), and a lipid panel at the time of the patient's admission. Cytokine markers were assessed at the first day and 14 days postMI. Blood samples were centrifuged at 1500 rpm for 10 min and were stored at -80 °C. Analyses were performed after all samples were collected and done in the same laboratory by the same technician in a single session and with the same device. Total cholesterol was determined by the homogeneous enzymatic colorimetric method in a Hitachi Modular P800 autoanalyzer (Roche Diagnostics Corp., Indianapolis, IN, USA). Low density lipoprotein (LDL) was calculated according to Friedewald method [23].
Cytokine quantification was run in duplicate. Serum samples were thawed on ice and concentrations of IL were analyzed according to the manufacturer's instructions for the beadbased multiplex immunoassay system (Bio Plex Pro™ Human Inflammation Panel 1, 37Plex). The formation of different sandwich immunocomplexes on distinct bead sets was measured and quantified using the BioPlex® MAGPIX™ System (BioRad Laboratories, Hercules, CA, USA). The final concentration of analytes was calculated using the BioPlex Manager v5.0 software package (BioRad). For all statistical analyses, values below the detection limit of the assay were replaced with the ОРИГИНАЛЬНЫЕ СТАТЬИ § minimal detectable value for the analyte. The coefficient of va riation (CV%) was <10 %.

CMR imaging
All CMR studies were performed with a 3 Tesla scanner (Magnetom Skyra, Siemens Medical Systems, Erlangen, Germany) at 14 days and six mos postMI. The applied imaging protocol was published previously in detail [24]. The CMR imaging protocol included acquisition of one 4chamber view, cine shortaxis sections (6mm slice thickness at 10mm intervals), and one 2chamber view. The indices of LV systolic function were assessed by using the retrospective electrocardiogramgated, turbofast low angleshot (turboFLASH) sequence. Imaging parameters were as follows: echo time (TE) 1.42 ms, repetition time (TR) 39 ms, flip angle 57°, voxel size 1.67 × 1.67 × 6 mm. Car diac function and volumes were measured using Syngo. via imaging software (Siemens). LVEDV and LVESV were calculated with shortaxis based planimetry from the basal to the apical level. LV stroke volume was calculated as LVEDV minus LVESV, and LV ejection fraction (LVEF) was calculated as: EF= [(LVEDV-LVESV) / LVEDV] x 100.

Statistical analysis
Statistical analyses were performed using SPSS 20 for Windows (IBM Corp., Armonk, NY, USA). The normality data distributions was evaluated by the ShapiroWilk test. Numeric variables with or without normal distribution were plotted as mean±standard deviation or as median (interquartile range (IQR): 25 th 75 th percentile), respec tively. Categorical variables are indicated as numeric and percentage values. StudentT test or Mann-Whitney U test were used for comparison of numeric variables between the two groups according to the distribution normality. Chisquare with Yates correction and Fischer's exact tests were used for comparison of categorical data. A mixed model for repeated measures analysis was performed for comparison of cytokine concentrations in the postMI period and during followup according to the remodeling groups. Logistic regression analysis was used to identify independent predictors of reverse remodeling (RR). Values of p<0.05 (*) were considered statistically significant. Changes in CMR variables and cytokine concentrations in the postMI period are shown by Δ. The relationship between cytokine concentrations and CMR parameters was examined by Spearman correlation analysis.

Results
Detailed demographic, laboratory, and clinical findings at the time of admission of the study population are shown in Table 1. The study population consisted of 5 women and 40 men (mean age: 55.6±7.4 yrs). 20 patients (44.4 %) had hypertension. RR was detected in 26 patients at the end of 6 mos. There was no significant differences in demographic, laboratory, and clinical findings among AR and RR groups.
Cardiac MRI findings of the RR and AR groups did not show significant differences at the 14 days postMI. Mean LVEF was higher (50.5±8.0 % vs. 44.3±10.4 %, p=0.030) and mean LVEDV (144.7±31.0 ml vs. 186.1±33.1 ml, p<0.001) and median LVESV (74.5 ml vs. 107 ml, p=0.012) were lower in the RR group compared to the AR group after 6 mos. At the 6 mos postMI, there was a significant worsening of LV volume and functions in AR group compared to RR group. (Table 2).

ОРИГИНАЛЬНЫЕ СТАТЬИ §
Multivariable regression analysis including IL 10, IL19, and IL26 cytokines on the first day postMI (Model I) revealed IL10 as an independent predictor of RR. Accor ding to this analysis, an increase of 1 pg / ml in the concentration of IL10 would decrease the odds of RR by 1.32fold (OR=0.76, p=0.035). In addition, the regression model in which changes in postMI cytokine concentrations (Model II) were included showed that a 1 % increase in ΔIL10 increased the odds of RR by 1.07fold (OR=1.07, p=0.007). Besides, the model II exhibited a higher goodness of fit compared to Model I (Model I Nagelkerke R2: 0.43 vs Model II Nagelkerke R2: 0.54) ( Table 4).
There was a positive correlation between IL10 concentration and LVEF levels, and a negative correlation between LVEDV in both RR and AR groups (Table 5).

Discussion
In this study, at the first day postMI, the concentration of antiinflammatory cytokines was higher in the AR group than in the RR group. There was a significant decrease in the concentration of antiinflammatory cytokines at the 14 days compared to first day postMI in the AR group. However, no significant decrease was observed in the RR group. Multivariable regression analysis revealed that an increase of IL10 on the first day postMI was related to reduced the odds of RR. On the other hand, multivariable regression analysis showed that a 1 % increase in the change of IL10 concentration increased the odds of RR by 1.07fold. The regression model in which changes in postMI cytokine concentrations exhibited a higher goodness of fit.
In acute STEMI, the process of cardiac repair is molded by the degree of inflammation, oxidative stress, and hemodynamic changes after ischemia. The lower are these risk factors and the higher are the concentrations of antioxidant molecules and antiinflammatory markers, the higher will be the probability of developing RR. In comparison, the likelihood of developing AR will be greater if there is an excessive inflammatory response and increased reactive oxidant radicals in the presence of a weak antioxidant balance and a weak anti inflammatory response [25]. A study by Meng et al. [26] assessed whether amniotic mesenchymal stem cells (AMM / I) regulated by the IL 10 gene contributed to LV function and remodeling after acute MI. They found that AMM / I produced by gene regulation suppresses severe proinflammatory responses by its effects on post  ОРИГИНАЛЬНЫЕ СТАТЬИ § MI angiogenesis and prevents AR, and that AMM / I has a cardioprotective effect. During the postMI period of 30 patients with acute coronary syndrome, a positive correlation between IL8 concentration and LV systolic volume was found. IL10 concentration correlated negatively with LV end diastolic diameter and with left atrial volume [25]. Falcao et al. [27] determined that IL 10 concentrations produced using whole blood cultures in the 72 nd hr postMI were negatively correlated with LVEDV and LVESV. A study in mice demonstrated that endogenous IL10 released from leukocytes during acute MI exerted a cardioprotective action by associated with of endothelial progenitor cells in bone marrow [28].
In a study in rats, the animals were given recombinant IL10 treatment during acute MI [29]. This treatment significantly contributed to cardiac RR in the postMI period in rats. In a Korean study [30], IL10 knockout mice and wildtype mice were given angiotensin II infusions for a week. After this procedure, cardiac function was significantly decreased in the IL10 knockout mice compared to the wildtype mice. The researchers suggested that IL10 plays a protective role against angiotensin II induced cardiac remodeling.
Based on the above findings, it can be postulated that IL 10 decreased the progression to AR by reducing excessive inflammatory responses in the postMI period. Correlation analysis showed that increased IL10 concentration was associated with increased LVEF levels and decreased LVEDV levels in both the RR and AR groups. This suggests that increases in IL10 concentrations in the postMI period are cardioprotective. Meng et al. [26] detected of IL10 concentration in heart tissues after 3 days in AMM / I mice. They found that the not significant improvement in LV function at 2 weeks, but significant improvement in EF at 4 weeks. Falcao et al. [27] measured IL10 concentrations 72 hours after the onset of symptoms, and LV volumes 24-96 hours and 10-14 weeks after admission. They found that the a negative correlation between IL10 concentration and changes in LV volume indices. The results of the correlation analysis in our study showed that there was no correlation between the cytokine concentrations at the first day post MI and LVEF and LV volumes at the 14 days posMI, which were in line with the literature. These findings suggest that the cardioprotective effect of IL10 may be in the long run.
On the other hand, in our study and similar to the studies cited above, CRP levels were higher in the AR group than Table 5. Relationship between IL-10 superfamily concentrations and CMR measurements ОРИГИНАЛЬНЫЕ СТАТЬИ § in the RR group on the first day postMI. At the same time, the IL10 concentration measured was higher in the AR group compared to the RR group. This suggests that the excessive of IL10 concentration measured in the AR group may be associated with an excessive inflammatory response. The presence of excessively increased IL10 concentration on the first day postMI in the AR group explain the association of high IL10 concentration with a reduced the odds of RR in the model I regression analysis. Besides, there was a significant decrease in the concentration of IL 10 at the 14 days compared to first day postMI in the AR group. IL10 concentration did not change in the RR group. As a reflection of this findings, model II regression analysis revealed that the increased concentration of ΔIL10 in the postMI periods was increased the odds of RR.
A main finding of recent studies has been that IL19 has a protective effect on cardiovascular structures due to its antiinflammatory actions [29,30]. In a study conducted in mice by Ellison et al. [15], IL19 treatment was shown to inhibit atherosclerosis by alleviating inflammation in bone marrowderived macrophages, endothelial cells, and vascular smooth muscle cells. In a Chinese study [31], exogenous IL19 was given to mice that had undergone MI. IL19 decreased MI and apoptosis by increasing heme oxygenase1 activity and by decreasing malondialdehyde formation. As a result, it appeared that IL19 would decrease acute ischemic damage and increase survival in mice. In our study, the IL19 concentration was higher in the AR group compared to the RR group on first day postMI. While there was a significant decrease in the IL19 concentration in the AR group from the first day to the 14 days postMI, in the RR group, although not significant, there was an partial increase in the IL19 concentration from the first day to the 14 days postMI. These findings suggest that IL19 has a cardioprotective effect. Although this effect is related to the antiinflammatory actions of IL19, it may also be associated with IL19induced increase in the concentration of IL10, a cardioprotective cytokine [32].
We have not found any studies on the role of IL26, a recently discovered member of the IL10 subfamily, in cardiac remodeling. However, its roles in the pathogenesis of autoimmune diseases, rheumatoid arthritis, psoriasis, and colitis have been studied. In our study, the IL26 concentration was significantly higher in the AR group on the first day postMI, and a significant decrease in IL26 concentration was observed in this group at the 14 days postMI. In the RR group, although not significant, an partial increase was observed in the concentration of IL26 from the first day to the 14 days postMI. In light of these findings, we can postulate that the persistently high concentration of IL26, an antiinflammatory cytokine, for 14 days postMI in the RR group was responsible for cardioprotective effect.
The main limitations of this study are its crosssectional design and the small size of the study population. In this study, we evaluated only the relationships between systemic concentrations of antiinflammatory cytokines and cardiac remodeling. Evaluation of antiinflammatory cytokines in cardiac tissue and their relationship with pathological findings in the postMI period would have made this study stronger.

Conclusion
Cardiac remodeling occurs as a result of inflam ma tory / antiinflammatory homeostasis that develops after STEMI. We found that antiinflammatory cytokine concentrations were higher in the AR group in the postMI period and this elevation was not sustained and significantly decreased for the 14 days postMI. In the RR group, we determined that the rise of antiinflammatory cytokine concentrations in the postMI period was permanent for the 14 days postMI. As a reflection of this findings, stable IL10 concentration may play a role the improvement of cardiac functions.