Heart Vessels DOI 10.1007/s00380-016-0913-z

ORIGINAL ARTICLE

Association of serum microRNA‑21 levels with Visfatin, inflammation, and acute coronary syndromes Faramarz Darabi1,2 · Mahmoud Aghaei1 · Ahmad Movahedian1 · Armin Elahifar2 · Ali Pourmoghadas2 · Nizal Sarrafzadegan2 

Received: 13 June 2016 / Accepted: 21 October 2016 © Springer Japan 2016

Abstract MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression. It seems that microRNA-21 (miR-21) and Visfatin, a novel adipocytokine, play roles in inflammation and atherosclerosis. The aim of this study was to investigate the association of miR-21 with Visfatin, inflammation, atherosclerosis and acute coronary syndrome (ACS). Based on coronary angiography and electrocardiogram (ECG), 53 patients with ACS and 52 patients with stable CAD were enrolled in this study. We assayed serum miR-21, Visfatin, and routine chemistries using quantitative reverse transcriptase polymerase chain reaction (QRT-PCR), enzyme-linked immunosorbent assay (ELISA) and automated analyzer, respectively. We used a regression analysis to describe the relationship between the variables. Serum miR-21 level in 2−ΔCt value was significantly higher in ACS patients (10.52 ± 1.01-fold) than the stable CAD patients (4.4 ± 0.79-fold) (F  = 4.59, p < 0.001). In addition, serum Visfatin was significantly higher in ACS patients (17.5 ± 0.61 ng/ml) than the stable CAD patients (12.7  ± 0.49 ng/ml) (F  = 2.62, p < 0.001). Furthermore, the serum miR-21 level correlated positively with serum Visfatin level (r  = 0.26, p  = 0.008), hs-CRP (r  = 0.29, p = 0.003), age (r = 0.21, p = 0.034) and negatively with HDL-cholesterol (r = -0.28, p = 0.004). We concluded that the increased serum miR-21 and Visfatin may be involved in * Mahmoud Aghaei [email protected] 1

Department of Clinical Biochemistry, School of Pharmacy and Pharmaceutical Sciences and Isfahan Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Isfahan, Iran

2

Isfahan Cardiovascular Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan, Iran





the pathogenesis of ACS through promoting inflammation or may result from inflammatory responses to ACS. Furthermore, the potential role of miR-21 and Visfatin in plaque instability and inflammation warrants further investigations. Keywords  Acute coronary syndrome · miR-21 · Adipocytokines · Visfatin

Introduction Acute coronary syndrome (ACS) is one of the leading causes of death in developed and developing countries [1]. ACS represents a complex phenotype involving the interplay between endothelial dysfunction, proinflammatory, and prothrombotic components and it is influenced by genetic and environmental factors [2, 3]. Many studies have focused on the role of inflammatory factors and extracellular matrix components in plaque erosion, plaque rupture, and plaque instability [4–6]. Acute myocardial infarction and coronary death associated with coronary plaque disruption caused by a fibrous cap of ruptured plaque that leads to thrombus and coronary occlusion. Researchers have shown that collagen degradation of matrix metalloproteinases (MMPs) excreted by macrophages leads to thinning of the fibrous cap of coronary plaque [7]. Studies showed that rupture of atherosclerotic plaques in the coronary artery gradually leads to plaque instability and ACS [7, 8]. It seems that microRNAs (miRNAs) and adipocytokines play a pivotal role in inflammation, plaque instability, and ACS. MicroRNAs are endogenous, non-coding, singlestranded RNAs, 19–25 nucleotides long, encoded by short inverted repeats within the genome [9]. MicroRNAs play key roles in regulating gene expression by degradation, translational inhibition, or translational activation of their

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target mRNAs [10]. MicroRNAs take part in the pathogenesis of neointimal lesion formation, atherosclerosis and coronary artery disease (CAD) [11]. Recently, researchers have focused on circulating miRNAs as a biomarker for early diagnosis of disease. One of the miRNAs, miR-21, is involved in many pathophysiological processes, including development, inflammation, cancer and cardiovascular diseases [12, 13]. In pathological conditions, microRNA-21 is highly expressed in cardiovascular cells, including vascular smooth muscle cells (VSMC) and endothelial cell; whereas under basal conditions, miR-21 is weakly expressed [14, 15]. A recent study showed that miR-21 involved in obesity and adipogenesis [16]. Therefore, the circulating microRNA-21 probably associated with adipocytokines. Adipose tissue secretes cytokines and various proinflammatory mediators called adipocytokines. These adipocytokines regulate different stages of atherosclerosis, from endothelial dysfunction to plaque destabilization and rupture [17]. In addition, the epicardial adipose tissue that covers the surface of the heart in humans is known as a source of proinflammatory cytokines [5]. Visfatin or pre-B cell colony-enhancing factor-1 (PBEF) is an adipocytokine produced by visceral adipose tissue and has insulin-mimetic action. Visfatin acts as an insulin analog on the insulin receptor to exert a mimetic insulin action [18]. Visfatin as a proinflammatory adipocytokine is secreted in response to inflammatory stimuli and upregulates the cytokine production in the monocytes such as IL-1, TNF-α, and IL-6, and probably has a potential role in the pathogenesis of inflammatory disorders [19, 20]. According to Dahl’s study [21], Visfatin should be regarded as an inflammatory mediator, localized to foam cell macrophages within unstable atherosclerotic lesions, that potentially plays a role in plaque destabilization. MicroRNA-21 enhances the gene expression of adipocytokines and plays a role in many biological processes [22–24]. Altogether, studies showed that miR-21 and Visfatin are involved in plaque instability, but the association of serum miR-21, Visfatin and inflammation markers in ACS patients has not been evaluated. It seems that the increased serum miR-21 and Visfatin do participate in plaque instability; therefore, the aim of this study was to investigate the association of serum microRNA-21 levels with Visfatin, inflammation markers, traditional risk factors and ACS. Furthermore, we studied the roles of miR-21 and Visfatin as a diagnostic tool for predicting ACS.

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Heart Vessels

Materials and methods Patient population The study population consisted of 105 patients aged 45–75 years. Samples were collected between August 2015 and February 2016, in the catheterization lab where they underwent coronary angiography in the Chamran Hospital of the Isfahan University of Medical Sciences, Iran. The study population was classified into two groups according to their clinical symptoms, electrocardiography (ECG) and angiography findings: the ACS group (53 patients, 49.5 %) and the stable CAD group (52 patients, 50.5 %). Exclusion criteria were: patients with a previous coronary artery bypass graft surgery, previous myocardial infarction (MI), malignant disease, infectious disease, inflammatory disease, neoplasm, hematological disorders, advanced renal disease (creatinine >2 mg/dl), and liver disease (AST and ALT >2 times upper limit of normal) [25, 26]. A written informed consent was obtained from each patient before study participation. The study was conducted in accordance with guidelines approved by the ethics committee of Isfahan University of Medical Sciences and Chamran Hospital, Isfahan, Iran and according to the Helsinki Declaration. Sample collection and coronary angiography Venous blood samples were collected from the patients before angiography. The blood samples were centrifuged at 2000g for 15 min and the supernatant was transferred to RNase/DNase-free tubes and stored immediately at −80 °C until analysis. All patients underwent selective coronary angiography. Angiography evaluations were performed independently by two cardiologists who were blinded to the clinical features of the patients [27]. Biochemical analysis We measured routine chemistries including blood sugar (BS), high-density lipoprotein cholesterol (HDL-C), lowdensity lipoprotein cholesterol (LDL-C), total cholesterol (TC), triglycerides (TG), and creatinine using an automated analyzer (Hitachi 902, Japan) and commercial kits (Pars Azmoon, Tehran, Iran) in the clinical laboratory of the hospital. In addition, we measured C-reactive protein (hs-CRP) using immunoturbidimetry.

Heart Vessels

Quantification of circulating miRNA The total small RNA, including serum miRNAs, was extracted using an miRNA Purification & Isolation Kit according to the manufacturer’s protocol (Takara Clontech, japan). Briefly, in a microtube, we added 90 μL of lysis buffer to 300 μL of serum, vortex mixed for 5 s, added 30 μL of precipitate buffer, centrifuged for 3 min at 11,000g, added 400 μL of isopropanol to the supernatant, vortex mixed for 5 s, loaded onto the column and centrifuged for 30 s at 11,000g. Finally, 30 μL of RNase-free water was added onto the silica membrane of the column and small RNA was collected in a new collection tube. The quantity and quality of the total small RNA was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Worcester, MA) [28]. The total small RNA was stored at a temperature of −80° C. The Mir-X miRNA First-Strand Synthesis kit (Takara, japan) was used for first-strand cDNA synthesis according to the manufacturer’s instructions. Altogether, RNA is polyadenylated and reverse transcribed using the mRQ buffer and Enzyme Mix that contains poly(A) polymerase. Purified small RNA and mRQ Enzyme Mix were mixed in a reaction volume of 10 μl (3.75 μL RNA sample, 5 μL mRQ buffer and 1.25 μL mRQ enzyme) and incubated in a thermocycler (BIO-RAD, USA) for 1 h at 37 °C, then terminated at 85 °C for 5 min to inactivate the enzymes [29]. We performed absolute quantification of microRNA-21 with quantitative reverse transcriptase polymerase chain reaction (QRT-PCR), using an ABI StepOnePlus Realtime PCR system (Applied Biosystems, USA). We used Real Q Plus 2× Master Mix Green with High ROX™ kit (Ampliqon, A325402) according to the manufacturer’s instructions. miRNA-specific primers were designed on the basis of the miRNA sequences available in the miRBase database (http://microrna.sanger.ac.uk/). For U6 snRNA reaction, we used the forward and reverse primers of U6 snRNA, provided with the kit. For qPCRs, samples were incubated in a 96-well plate at 95 °C for 15 min (1 cycle), followed by 15 s (40 cycles) at 95 and 60 °C for 1 min (40 cycles). At the end of PCR cycles, the melting curve analysis was performed to validate the specificity of PCR product. All samples and blank controls were run in duplicate. For each qPCR, 20 μl of PCR mixture composed of Master Mix Green with High ROX™ (10 μl), forward and reverse primers (0.5 μl of each primer), first-strand cDNA (2 μl) and RNase-free water (7 μl) was prepared. The cycle threshold (Ct) is defined as the number of cycles required for the fluorescent signal to cross the threshold in qPCR. 2 −ΔCt formula was used to calculate the relative gene expression of microRNA compared to an internal control gene, where ΔCt = Ct (miR-21) − Ct (U6 snRNA). Group comparisons were based on mean 2−ΔCt ± standard error (SE)

[30, 31]. Therefore, 2−ΔCt value shows the gene expression of miR-21 relative to the U6 as an internal control gene. Determination of serum Visfatin levels by enzyme‑linked immunosorbent assay (ELISA) The serum Visfatin level was measured with an enzyme immunoassay kit (Hangzhou Eastbiopharm Co. Ltd, China). Briefly, 40 μl of sample or standard, 10 μl of Visfatin antibody and 50 μl of streptavidin–HRP were added to the well (in a 96-well plate), gently shook and incubated for 60 min at 37 °C. Then washing (5 times) was done and chromogens A (50 μl) and B (50 μl) were added, incubated for 10 min at 37 °C and finally a stop solution was added. The optical density (OD) was read at 450 nm wavelength. According to the standard concentration and OD values, a standard curve was drawn and the linear regression equation was calculated, and then the concentrations of each sample were extracted. Statistical analysis Continuous normally distributed variables and continuous variables with little-to-mild skewed distributions are presented as mean ± standard error (SE), and non-normally distributed variables as median. Continuous variables were compared between ACS and stable CAD group with the independent T test. The Pearson correlation analysis was used to investigate the correlations between serum miR-21 with Visfatin and other related variables. To assess the influence of testing the variables, a multiple linear regression analysis was used. All of the statistical analyses were performed using SPSS statistical software, version 16 (USA). p values were two tailed and less than 0.05 was considered a statistically significant difference. The receiver operating characteristic curves (ROC curves) were established for discriminating ACS patients from stable CAD patients by the clinical biomarker.

Results Subject characteristics The baseline clinical characteristics and demographic variables of the study groups are presented in Table 1. The demographic data showed that both groups were comparable statistically. There were no statistically significant differences in age, BMI and blood pressures between ACS group and stable CAD group (p > 0.05). In addition, there were no significant differences in several cardiovascular risk factors, including cigarette smoking, LDL-cholesterol, total

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Table 1  Clinical characteristics of patients with ACS and stable CAD

Heart Vessels Characteristics

ACS group (n = 53)

Stable CAD group (n = 52)

Age (years)

63.1 ± 11.5

62.3 ± 8.7

0.707

Male sex, n (%)

33/20

32/20

0.940

Blood pressure (mmHg)  Systolic  Diastolic Body mass index (kg/m2) BUN (mg/dL) Serum creatinine (mg/dL) Hemoglobin A1c (%) Total cholesterol (mmol/L) LDL-cholesterol (mmol/L) HDL-cholesterol (mmol/L) Triglycerides (mmol/L) C-reactive protein (mg/dL) Glucose (mmol/L)

133.9 ± 19.9 81.33 ± 9.1 24.49 ± 2.45 20.83 ± 2.6 1.07 ± 0.21 6.1 ± 0.61 5.1 ± 0.73 3.34 ± 0.66 0.91 ± 0.13 1.81 ± 0.48 2.1 ± 0.35 6.73 ± 0.93

128.1 ± 13.3 79.13 ± 5.21 24.09 ± 2.24 21.4 ± 2.04 1.01 ± 0.19 6.2 ± 0.45 5.05 ± 0.72 3.18 ± 0.58 1.04 ± 0.12 1.80 ± 0.29 1.7 ± 0.36 6.85 ± 0.76

0.082 0.131 0.382 0.209 0.154 0.362 0.737 0.216 <0.01 0.89 <0.001 0.379

Smoking, n (%)

22 (41)

19 (35)

0.606

Statin (%)

19 (36)

17 (33)

0.677

cholesterol, total triglycerides, creatinine, history of diabetes and smoking status. The patients with ACS had higher serum inflammatory markers, such as hs-CRP compared with the stable CAD patients. Rates of statin use and other drugs were similar among groups. Gene expressions of serum miR‑21 in patients with ACS and stable CAD Quantitative real-time PCR was used for analyses of serum miR-21 expressions in all patients with ACS and stable CAD. Analyses by Δct method [28] showed that the miR21 level in 2−ΔCt value was significantly higher in the ACS group (10.52 ± 1.01-fold) compared with the stable CAD group (4.44 ± 0.79-fold) (F = 4.59, p < 0.001). The serum level of Visfatin in patients with ACS and stable CAD The enzyme-linked immunosorbent assay (ELISA) used for analyses of serum Visfatin in all patients with ACS and stable CAD. The result of ELISA analyses showed that the mean serum Visfatin levels in the ACS group (17.5 ± 0.61 ng/ml) were significantly higher than the stable CAD group (12.7 ± 0.49 ng/ml) (F = 2.62, p < 0.001). The predictive value of serum miR‑21 and Visfatin for ACS We used the receiver operating characteristic curves (ROC curves) to predict ACS. According to this analysis, accuracy is measured by the area under the ROC curve (AUC).

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p value

An area of 1 represents a perfect test. ROC curve also shows the sensitivity and specificity. The cutoff level is defined as the maximal sensitivity and specificity. As a discriminating factor for distinguishing ACS from stable CAD, the ROC curve for the miR-21 level in 2−ΔCt value was analyzed (Fig. 1a). The accuracy (AUC) for miR-21 was 0.781 (95 % CI 0.692–0.869, p < 0.001). The cutoff point for miR-21 in 2−ΔCt value was 5.74 (sensitivity, 71.7 %; specificity, 73.1 %). As a discriminating factor for distinguishing ACS from stable CAD, the ROC curve for Visfatin was analyzed (Fig. 1b). The accuracy (AUC) for Visfatin was 0.829 (95 % CI 0.746–0.912, p < 0.001). The cutoff point for Visfatin was 14.5 ng/mL (sensitivity, 74.00 %; specificity, 76.00 %). Correlations between miR‑21 and Visfatin and other related variables The correlation analysis test showed that the serum miR21 level in 2−ΔCt value was positively correlated with the serum Visfatin level (r = 0.26, p = 0.008) (Fig. 2a). In the correlation analysis of the inflammatory parameter, the serum miR-21 level in 2−ΔCt value was positively correlated with the serum hs-CRP (r  = 0.29, p  = 0.003) (Fig. 2b). In the correlation analysis of the demographic parameter, the serum miR-21 level in 2−ΔCt value only positively correlated with age (r = 0.21, p = 0.034) (Fig. 2c). No association was found with BMI, systolic and diastolic blood pressure. In addition, no association was found with BUN, creatinine, albumin, protein and hemoglobin A1C (HbA1c).

Heart Vessels

Fig. 1  The ROC curve for predicting ACS. Expression of serum miR-21 (2−ΔCt value) and the serum Visfatin level was obtained from the patients with acute coronary syndrome (n  = 53) and patients with stable coronary artery disease (n = 52) as determined by QRTPCR and ELISA, respectively. a The ROC curve for serum miR-21

to predict ACS. The area under the ROC curve was 0.781 (95 % CI 0.692–0.869, p < 0.001). b The ROC curve for serum Visfatin to predict ACS of the study population. The area under the ROC curve was 0.829 (95 % CI 0.746–0.912, p < 0.001)

In the correlation analysis of the lipid profile, the serum miR-21 level in 2−ΔCt value negatively correlated with HDL-cholesterol (r  =  −0.28, p  = 0.004) (Fig. 2d). No association was found with the total cholesterol, triglyceride and LDL-cholesterol (Table 2).

Discussion

Correlations between Visfatin and hs‑CRP, lipid profile, and demographic variables The correlation analysis of the inflammatory markers showed that the serum Visfatin was positively correlated with hs-CRP (r = 0.28, p = 0.003) (Fig. 3a). In addition, the correlation analysis of lipid profile showed that the serum Visfatin was negatively correlated with HDL-cholesterol (r = −0.28, p = 0.004) and positively correlated with LDL-cholesterol. No association was found with total cholesterol and triglyceride (Table 3). The correlation analysis of the demographic variables showed that serum Visfatin was positively correlated with age (r = 0.29, p = 0.003) (Fig. 3b). Multiple linear regression analysis for miR‑21 as a dependent variable A multiple linear regression analysis was performed using miR-21 as the dependent variable, and Visfatin, hs-CRP, total cholesterol, triglyceride, LDL-cholesterol, HDLcholesterol, age, systolic blood pressure, diastolic blood pressure and Body mass index as independent variables (Table 4). Only serum Visfatin and hs-CRP were independently associated with miR-21.

Acute coronary syndrome (ACS) severely threatens human health with increasing morbidity; therefore, early treatment of ACS might improve the adverse events of this disease [32]. The mechanism for ACS involves the rupture of atherosclerotic plaques (plaque instability), platelet aggregation and thrombosis. Instability of coronary plaque leads to progression of stable coronary artery disease (stable CAD) to ACS [33, 34]. The multiple biomarkers in the serum or plasma can provide an appropriate diagnostic tool for this disease. Recently, researchers have focused on miRNAs and adipocytokines as biomarkers [35, 36]. Therefore, we designed this study to investigate the role of serum microRNA-21 and Visfatin as biomarkers for predicting ACS. In the present study, we showed that (a) the serum levels of miR-21, Visfatin and hs-CRP (an inflammation marker) were significantly higher in the ACS group than the stable CAD group. (b) The serum miR-21 had significant positive correlations with Visfatin, hs-CRP, and age. (c) The serum miR-21 had a significant negative correlation with HDL-cholesterol (HDL-C), but no correlations with other traditional risk factors for cardiovascular disease such as total cholesterol, triglyceride, and LDL-cholesterol. (d) The serum Visfatin had significant positive correlations with hs-CRP, LDL-cholesterol, and age. (e) The serum Visfatin had a significant negative correlation with HDL-cholesterol (HDL-C), but no correlations with other traditional risk factors for cardiovascular disease such as total cholesterol and triglyceride.

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Heart Vessels

Fig. 2  Correlation between the serum miR-21 and other parameters. Serum miR-21 level in 2−ΔCt value was positively correlated with: a Visfatin (r = 0. 31, p = 0. 002), b hs-CRP (r = 0.26, p = 0.008), c

age (r  = 0. 22, p  = 0. 03), and negatively with d HDL-cholesterol (r  =  −0.32, p  = 0. 001), solid lines indicate the linear regression lines

Table 2  The correlation between serum miR-21(2−ΔCt) and other parameters related to cardiac disease

Recent reports have indicated that miRNAs play an important role in cardiovascular disease [37, 38]. Dysregulation of miRNAs has been found in many cardiovascular diseases [39]. Circulating miRNAs may serve as noninvasive biomarkers for predicting acute myocardial infarction [40]. MicroRNA-21 is a mammalian microRNA that is encoded by a single gene [41]. Vascular smooth muscle cells (VSMCs) enriched miRNAs including miR-21 [7]. MicroRNA-21 (miR-21) is a highly expressed microRNA (miRNA) in cardiovascular diseases and plays important roles in these disorders [42]. Some miRNAs, such as miR21, associated with the synthetic VSMC phenotype and

Variable

Pearson r

P value

Body mass index Systolic blood pressure Diastolic blood pressure Blood glucose Hemoglobin A1c Total cholesterol Triglyceride

0.11 0.049 0.002 −0.028 −0.076 0.077 −0.048

0.26 0.62 0.98 0.77 0.43 0.43 0.64

Low-density lipoprotein cholesterol

13

0.17

0.07

Heart Vessels

Fig. 3  Correlation between the serum Visfatin and other parameters. Serum Visfatin was positively correlated with: a hs-CRP (r  = 0.28, p = 0.003), b age (r = 0. 29, p = 0. 003), solid lines indicate the linear regression lines

Table 3  Correlation between serum Visfatin and other cardiovascular risk factors Variable

Pearson r

p value

Body mass index Systolic blood pressure Diastolic blood pressure Blood glucose Hemoglobin A1c Total cholesterol Triglyceride

0.065 0.053 0.11 0.084 0.09 0.18 0.16

0.51 0.59 0.27 0.39 0.36 0.06 0.11

Low-density lipoprotein cholesterol

0.25

0.01

Table 4  Multiple linear regression analysis for miR-21 as a dependent variable Variable

Standard error Beta

t

p

Visfatin hs-CRP Total cholesterol Triglyceride LDL-cholesterol HDL-cholesterol Age Systolic blood pressure Diastolic blood pressure

0.145 1.418 4.688 2.641 4.698 6.020 0.058 0.043 0.102

0.276 0.356 0.343 −0.160 −0.216 −0.169 0.142 −0.081 0.017

2.963 3.690 0.678 −1.065 −0.499 −1.378 1.618 −0.754 0.155

0.004 0.000 0.499 0.289 0.619 0.171 0.109 0.453 0.877

Body mass index

0.246

0.141

1.633 0.106

upregulated in atherosclerotic plaques [43]. These studies suggest that miR-21 probably implicated in the pathogenesis of atherosclerosis and cardiovascular diseases.

Tsai et al. [44] found that circulating miR-21 level is a novel and independent biomarker to predict preclinical atherosclerosis. In addition, Fan et al. [45] in an in vitro study showed that the overexpressing miR-21 could induce the expression of pro-Matrix metallopeptidase-9 in human macrophages. They suggested that the increasing of MMP-9 degrades the collagen in the cap of coronary plaque leading to plaque instability. Therefore, these studies suggest that miR-21 involved in plaque destabilization and may have a role in the pathogenesis of ACS, which agree with the results of our study. Prior studies have shown that Visfatin is produced in adipose tissue, bone marrow, skeletal muscle, and liver [46]. Moreover, a study by Dahl et al. [21] suggested that Visfatin is strongly expressed in lipid-loaded macrophages in atherosclerotic lesions that play a role in plaque destabilization because of MMP-9 activity. Visfatin could induce angiogenesis in human vein endothelial cells [47], while progressive angiogenesis in atherosclerotic lesions have been considered one of the causes of plaque vulnerability [48]. In the present study, we showed that serum Visfatin levels were higher in patients with ACS than stable CAD patients. These data suggest that Visfatin has a relationship with plaque destabilization and ACS. Plaque inflammation is an obligatory feature of events leading to plaque rupture [49]. Plaque rupture occurs with multiple inflammatory cells and chemokines that are mainly derived from macrophage foam cells [50]. Studies suggest that Visfatin has probably been one of the clinically important cytokines associated with inflammation and atherosclerosis and maybe have a role in plaque destabilization and ACS [51]. The study showed that Visfatin could activate human leukocytes and upregulate inflammatory cytokines, including TNF-α and IL-6 [19], and increase

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matrix metalloproteinase-9 from monocyte [21]. These studies suggest that Visfatin would play an important role in atherogenesis and plaque destabilization. In agreement with these studies, our study showed that Visfatin significantly associated with hs-CRP and inflammation. HDL-C plays an important role in estimating risk for CVD, in fact increasing levels of HDL-C provides a protective effect [52]. Studies have suggested that the expression of miR-21 associated with lipid profiles [53, 54]. In addition, Visfatin associated with lipid metabolism [55]. Our study showed that HDL-C negatively correlated with ACS. Presumably, serum miR-21 and Visfatin influence HDL-C concentrations. MicroRNA-21 enhances gene expression of some adipocytokines such as leptin and adiponectin [22, 23]. However, no earlier studies have investigated about the relationship between miR-21 and Visfatin. In addition, our study does not demonstrate this relationship, but showed a robust association between them and suggests that miR-21 involved in the metabolism of Visfatin. We recommend that a study is conducted to investigate the role of miR-21 on the gene expression of Visfatin. Taken together, these studies suggest that serum miR-21 and Visfatin have a relationship with plaque instability and ACS. For the first time, we reported that serum miR-21 related to Visfatin and inflammation which indicated that miR-21 and Visfatin are probably involved in a common pathway in the pathogenesis of ACS. However, clarity on this matter warrants further investigations.

Conclusions According to the results of the present study, serum miR-21 highly correlated with Visfatin. IN addition, both factors, serum miR-21 and Visfatin, highly correlated with inflammatory factors such as hs-CRP. Therefore, we concluded that raised serum miR-21 and Visfatin may be involved in the pathogenesis of ACS by promoting inflammation or may result from inflammatory responses to plaque instability and ACS. We suggest that further studies are needed to clarify the cause and mechanism of increased serum miR-21 and Visfatin in patients with ACS. However, raised serum miR-21 and Visfatin have a relationship with plaque instability and ACS; therefore, they can be used as a new diagnostic tool for predicting ACS. Acknowledgments The authors express their heartfelt gratitude to the staff and members of the Isfahan Cardiovascular Research Institute and Isfahan School of Pharmacy and Pharmaceutical Sciences for their assistance in various measurements and other organizational aspects of this study. We also thank all the patients who participated in this study. This project supported by a Grant 394289 from the Isfahan University of Medical Sciences.

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Heart Vessels Compliance with ethical standards  Conflict of interest  The authors declare that there is no conflict of interests regarding the publication of this paper.

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