Increased expression of inactive rhomboid protein 2 in circulating monocytes after acute myocardial infarction

Increased expression of inactive rhomboid protein 2 in circulating monocytes after acute myocardial infarction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Increased expression of inactive rhomboid protein 2 in circulating monocytes after acute myocardial infarction Phillip Dijck, Carmen Hannemann, Henryk Dreger, Verena Stangl, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2390961/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Tumor necrosis factor-alpha (TNF-α) blood levels increase following acute myocardial infarction (AMI); TNF-α is involved in impaired recovery of myocardial function following AMI. The interaction of inactive rhomboid protein 2 (iRhom2) with TNF-α converting enzyme (TACE) is required for shedding of TNF-α from the cell surface of immune cells. In this pilot study, we hypothesized that iRhom2 expression increases in circulating monocytes following AMI. Methods Circulating monocytes were MACS-sorted from peripheral blood of 50 AMI patients at admission (day 1) and 3 days after admission. mRNA was isolated from sorted monocytes and expression levels of iRhom2, TACE and TNF-α were evaluated by real-time RT-PCR. Serum TNF-α levels were determined. Circulating monocyte subsets were quantified by flow cytometry. Left ventricular (LV) function was measured by echocardiography. Results We observed a significant increase of iRhom2 mRNA expression in monocytes (p = 0.012), of intermediate monocytes levels (p < 0.001), and of serum TNF-α levels (p < 0.001) at day 3 following AMI compared to day 1. In contrast, TNF-α and TACE mRNA expression in monocytes remained unchanged. At day 3, iRhom2 mRNA expression in monocytes positively correlated with levels of intermediate monocytes (r = 0.37, p = 0.009) and serum TNF-α levels (r = 0.33, p = 0.019). iRhom2 mRNA expression in monocytes at day 3 negatively correlated with LV systolic function (r=-0.34, p = 0.025). Conclusions This study suggests that iRhom2 contributes to the regulation of inflammation and is thereby associated with LV dysfunction following AMI. Thus, iRhom2 modulation should be further evaluated as a potential therapeutic strategy to attenuate adverse cardiac remodeling in AMI patients. myocardial infarction TNF-alpha iRhom2 inflammation ventricular remodeling heart failure Figures Figure 1 Introduction Acute myocardial infarction (AMI) is a leading cause of death worldwide. 1 Patients suffering a non-fatal AMI are exposed to elevated risk for short- and long-term complications including development of heart failure, ventricular arrhythmias, or recurrent AMI. Heart failure represents an important determinant for prognosis and survival in AMI patients. 2 The pathogenesis underlying heart failure development after AMI is closely related to inflammatory processes. 3 , 4 Myocardial cell necrosis following AMI activates innate immunity and triggers a strong local as well as systemic inflammatory reaction that plays a central role in myocardial infarct healing but also in the development of adverse cardiac remodeling if dysregulated, excessive or prolonged. 5 The inflammatory cytokine tumor necrosis factor-alpha (TNF-α) is detectable in the infarcted myocardium already during the early phase of AMI 6 and elevated serum TNF-α levels have consistently been described in patients with AMI. 7 Animal studies have shown that TNF-α is involved in impaired recovery of myocardial function following AMI 8 – 10 whereas different animal models of genetic or pharmacological TNF-α inhibition have shown promise in attenuating important parameters of adverse cardiac remodeling. 11 – 13 Controlled clinical trials addressing the potential of anti-TNF-α therapy in AMI are not available to date. TNF-α is mainly expressed by immune cells (in particular by monocytes and macrophages) as a precursor transmembrane protein located at the cell surface. Transmembrane TNF-α is cleaved by transmembrane metalloprotease TNF-α converting enzyme (TACE; ADAM17) to be released from the cell surface as soluble TNF-α (TNF-α shedding). 14 Soluble TNF-α exerts its biological effects by binding to TNF receptor type 1 and 2 (TNFR1 and 2). 15 TACE maturation in immune cells depends on inactive rhomboid protein 2 (iRhom2; RHBDF2). 16 Genetic deficiency or knock-down of iRhom2 prevents the activation of TACE and subsequently diminishes TNF-α shedding from immune cells. TACE maturation in non-hematopoietic cells remains unaltered as co-expression of closely related iRhom1 compensates for the absence of iRhom2 in these cells. 16 Up to date, the impact of the iRhom2/TACE/TNF-α-axis in AMI is unknown. In a previous study, we have shown that iRhom2-deficiency attenuates atherosclerosis development in low density lipoprotein receptor deficient mice. 17 Given the importance of iRhom2 in the regulation of TACE and TNF-α in immune cells, we aimed to investigate the gene-expression of iRhom2 in patients following AMI. We hypothesized that iRhom2 expression increases in circulating monocytes of patients with AMI, providing first evidence that iRhom2 is involved in the regulation of postischemic inflammation following AMI. Methods Study design and population The study protocol was approved by the Ethics Committee of the Charité-Universitätsmedizin Berlin (EA1/246/14). The study complied with the Declaration of Helsinki. Written informed consent was obtained from each study participant. From June 2015 - February 2017, patients with AMI (n = 50), were recruited at the Charité-Universitätsmedizin Berlin, Germany. The AMI group comprised patients with ST-segment Elevation (STEMI) and Non-ST-segment Elevation Myocardial Infarction (NSTEMI) as defined by the guidelines of the European Society of Cardiology. 18 , 19 Twenty-five young and apparently healthy volunteers (mean age 22.8 years, range 19–27 years; 44% female) without known cardiovascular disease were recruited as control group (controls). AMI patients were recruited within 24 hours after initial hospital admission with completed cardiac catheterization. Inclusion criteria comprised: age ≥ 18 years and diagnosis of AMI (as defined above). Exclusion criteria comprised the need for cardiac resuscitation, fever, acute infection/sepsis, rheumatic or non-rheumatic autoimmune diseases, current immunosuppressive therapy, severe acute or chronic renal failure (GFR < 30 mL/min/1.73 m 2 ), malignant diseases, stroke or surgery within 30 days. AMI patients were evaluated by medical history, clinical examination, blood collection, at study inclusion and at day 3 after study inclusion. Medical history was assessed by interviews and information from medical records. Functional class was assessed according to the New York Heart Association (NYHA) classification. Furthermore, standard transthoracic echocardiographic exams were performed within the first days after hospital admission (mean 2.4 ± 1.6 days after hospital admission) and were digitally stored for offline analysis (TOMTEC-ARENA). Left ventricular ejection fraction (LVEF) was measured using the biplane Simpson’s method as previously described. 20 For patients where image quality was not sufficient for assessment of biplane LVEF, visually estimated LVEF was used. LVEF was categorized into I: less than 30%, severely abnormal; II: 30–39%, moderately to severely abnormal; III: 40–49%, moderately abnormal; VI: 50–53%, mildly abnormal; V: >54%, normal. Controls were evaluated by medical history (interview), clinical examination, and blood collection. Inclusion criteria for controls comprised age ≥ 18 years and < 30 years and no known pre-existing health conditions. Blood sampling and standard laboratory parameters Peripheral blood samples were collected from cubital veins. Standard laboratory parameters including differential blood count, C-reactive protein (CRP), creatine kinase (CK), interleukin-6 (IL-6), N-terminal pro-B-type natriuretic peptide (NT-proBNP), TNF-α, and high sensitivity troponin T (hs-Troponin) were obtained by established assays in the hospital’s laboratory. Monocyte isolation Peripheral blood mononuclear cells (PBMC) were obtained by Ficoll density gradient centrifugation (Ficoll-PaqueTM PLUS, GE Healthcare) of 30 ml peripheral blood collected in EDTA tubes. On average, 30*10 7 PBMCs were isolated per sample. Subsequently, monocytes were isolated by negative selection using the human Pan Monocyte Isolation Kit (Miltenyi Biotec; MACS isolation) according to the manufacturer’s instructions. Purity of enriched monocytes was confirmed by flow cytometry as detailed below. Approximately 7*10 7 monocytes were isolated per sample by MACS isolation. Flow cytometry 200 µL eluate of MACS isolated cell fraction was stained with monoclonal antibodies (CD86 PE [B7-2; Biolegend], CD14 PB [M5E2; Biolegend], CD16 APC [3G8; Biolegend], CD11b PECy7 [ICRF44; Biolegend]) for 15 minutes followed by repeated (twice) washing with 2 ml phosphate-buffered saline (PBS) solution. After centrifugation and removal of supernatant, cells were resuspended in PBS and run on a flow cytometer (CyAn™ ADP Analyzer; Beckman Coulter, Inc.). Data was analyzed using Summit™ 4.4 software applying the following gating strategy: first, peripheral blood mononuclear cells (PBMCs) were chosen and plotted on a CD86 histogram; monocytes were identified as CD86 + cells and purity of enriched monocytes was defined as percentage of monocytes compared to all cells detected. On average, purity of enriched monocytes following MACS isolation was 90%. Secondly, monocytes were divided into three subsets: CD14 + + CD16- (classical), CD14 + + CD16+ (intermediate) and CD14-CD16++ (non-classical) according to the surface expression pattern of CD14 and CD16. 21 To differentiate between intermediate and non-classical monocytes a straight vertical line was drawn to the left of the CD14 staining of the classical monocytes in the CD14 and CD16 dot plot as described previously. 22 Analyses of flow cytometry data was performed by two blinded observers; results represent averaged values from both analyses. Quantitative real-time RT-PCR RNA from MACS sorted monocytes was isolated using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. 500 ng of total RNA was reversed-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). TaqMan assays (Applied Biosystems) were used to quantify the expression of iRhom2 (RHBF2; Hs01078106_m1), TACE (Hs01041915_m1), and TNF-α (Hs01113624_g1). RPL19 (Hs02338565_gH) was used as housekeeping gene. Expression of the target gene relative to housekeeping gene expression was calculated as the difference between the threshold values for the two genes. Relative expression levels were normalized to the control group. Statistical analysis Results are generally expressed as arithmetic mean ± SD for normally distributed data and median with interquartile ranges (IQR = 25th-75th percentile) for non-normally distributed data; Shapiro-Wilk-test was used for testing for normal distribution of the data. Categorical data are presented as absolute numbers with respective percentages. For normally distributed data the Student’s t-test was performed for either paired (one-sample t-test) or unpaired (independent two-sample t-test) observations. For non-normally distributed data the Mann-Whitney-U-test was used for comparison of two independent groups, while the Wilcoxon signed-rank test was used for comparison of paired observations. Pearson’s or Spearman’s correlation coefficient, where appropriate, were calculated for correlation analyses between variables. Frequencies of various groups were compared by chi-square test. Statistical analyses were performed using SPSS 25.0 (SPSS Inc.) software; p < 0.05 was considered statistically significant. Results Clinical Characteristics Clinical characteristics of AMI patients at study inclusion are shown in Table 1 . None of the AMI patients underwent coronary artery bypass grafting (CABG) following cardiac catheterization or received a percutaneous left ventricular assist device. Standard laboratory parameters are shown in Table 2 . The average LVEF of AMI patients was 50.4 ± 9.3%. Patients were treated with guideline directed medical therapy during hospitalization. 18 , 23 Five patients had pre-existing statin therapy before hospital admission. Table 1 Clinical characteristics of AMI patients at study inclusion (day 1) Characteristic Age in years, mean (range) 55 (33–85) Sex, n (%) male 36 (72.0) female 14 (28.0) BMI (kg/m 2 ) 28.6 ± 5.8 NSTEMI, n (%) 16 (32.0) STEMI, n (%) 34 (68.0) CV risk factors IDDM, n (%) 3 (6.0) NIDDM, n (%) 5 (10.0) Active smoking, n (%) 26 (52.0) Hypercholesterolemia, n (%) 29 (58.0) Hypertension, n (%) 29 (58.0) Family history of CVD, n (%) 11 (22.0) CVD history Previous ACS, n (%) 6 (12.0) Previous PCI, n (%) 8 (16.0) Previous CABG, n (%) 0 Previous TIA, n (%) 1 (2.0) Previous Stroke, n (%) 0 PAD, n (%) 0 Catheterization data Initial diagnosis CAD, n (%) 41 (82.0) CAD-1 vessel, n (%) 22 (44.0) CAD-2 vessel, n (%) 11 (22.0) CAD-3 vessel, n (%) 17 (34.0) Main vessel disease, n (%) 2 (4.0) PTCA, n (%) 49 (98.0) Stent, n (%) 46 (92.0) Data are generally expressed as mean ± SD, or absolute numbers and respective percentages. BMI, body mass index; NSTEMI, non-ST-elevation myocardial infarction; STEMI, ST-elevation myocardial infarction; CV, cardiovascular; CVD, CV disease; ACS, acute coronary syndrome; PCI, percutaneous coronary intervention; CABG, coronary artery bypass grafting; TIA, transient ischemic attack; PAD, peripheral artery disease, CAD, coronary artery disease; PTCA, percutaneous transluminal coronary angioplasty; stent, only drug eluting stents were used. Table 2 Laboratory parameters Parameter Controls AMI day 1 AMI day 3 p-value AMI day 1 vs. day 3 Troponin-T high sensitive, ng/L 2.9 (2.9-5.0) 1052.0 (419.0-4403.0) 1233.5 (439.0-2068.0) 0.001 Creatine kinase, U/L 142 (91–203) 593 (265–1242) 181.00 (127–311) < 0.001 NT-Pro-BNP, ng/L 26.0 (8.0–53.0) 642.0 (212.0-1359.0) 569.5 (308.0-1411.0) 0.004 Creatinine, mg/dl 0.83 (0.76–0.86) 0.84 (0.77–0.93) 0.86 (0.77–1.01) 0.021 Data are expressed as median with interquartile ranges. Statistical analysis was performed using the Wilcoxon signed-rank test. NT-proBNP, N-terminal pro-B-type natriuretic peptide. Serum TNF-α levels Serum TNF-α levels of AMI patients on day 1 were significantly higher compared to controls (p = 0.007) and increased significantly from day 1 to day 3 after AMI ( Fig. 1 , Table 3 ) . Serum levels of CRP and IL-6 are shown in Table 3 . Table 3 Blood levels of inflammatory markers Parameter Controls AMI day 1 AMI day 3 p-value, AMI day 1 vs. day 3 TNF-α, pg/ml 5.90 (4.9–6.8) 7.05 (5.4-8.0) 7.45 (6.6–10.0) < 0.001 CRP, mg/L 0.7 (0.4–1.5) 5.3 (2.0-12.3) 19.5 (11.6–41.8) < 0.001 IL-6, ng/L 1.4 (1.40-2.0) 16.95 (10.5–31.1) 11.95 (7.4–19.2) 0.007 Data are expressed as median with interquartile ranges. Statistical analysis was performed using the Wilcoxon signed-rank test. TNF, tumor necrosis factor; CRP, C-reactive protein; IL-6, Interleukin 6. Circulating monocytes subsets The absolute count of circulating monocytes was significantly higher in patients with AMI (on day 1 and 3) compared to controls and did not change significantly from day 1 to day 3 after AMI ( Table 4 ) . In AMI patients, relative levels of circulating intermediate monocytes on day 1 did not differ significantly compared to controls ( Table 4 ) but increased significantly from day 1 to day 3 after AMI. In contrast, relative levels of circulating classical monocytes decreased, while levels of circulating non-classical monocytes remained unaltered from day 1 to day 3 after AMI ( Table 4 ) . Table 4 Absolute monocyte count and relative levels of circulating monocyte subsets. Data are expressed as mean ± SD or as median with interquartile ranges. Statistical analysis was performed using a paired Student's t-test for normally distributed data and the Wilcoxon signed-rank test for non-normally distributed data. Controls AMI day 1 AMI day 3 p-value, AMI day 1 vs. day 3 Monocytes, /µl 374.7 ± 0.11 889.1 ± 0.29 908.8 ± 0.28 0.68 Classical Monocytes (%) 79.3 (75.1–83.9) 87.7 (82.5–91.8) 85.3 (80.8–88.8) 0.025 Intermediate Monocytes (%) 4.90 (3.8–5.9) 5.07 (3.4–7.4) 8.50 (6.3–10.2) < 0.001 Non-Classical Monocytes (%) 11.80 (7.7–17.9) 3.72 (2.3–5.6) 3.93 (2.1–5.7) 0.214 iRhom2, TACE, and TNF-α mRNA expression in circulating monocytes iRhom2 mRNA expression levels in circulating monocytes of AMI patients increased significantly (by 14%) from day 1 to day 3 after AMI ( Fig. 1 and Table 5 ) . iRhom2 mRNA expression levels in circulating monocytes of AMI patients on day 3 were significantly higher compared to controls (p < 0.001). No significant changes in mRNA expression levels of TACE and TNF-α in circulating monocytes of AMI patients were observed between day 1 and day 3 after AMI ( Fig. 1 and Table 5 ) ; there were no significant differences in both parameters compared to controls. We observed a strong correlation between iRhom2 and TACE mRNA expression in circulating monocytes on day 3 after AMI (r = 0.82, p < 0.001). Furthermore, iRhom2 mRNA expression in circulating monocytes correlated moderately with serum TNF-α levels (r = 0.33, p = 0.019) and with relative levels of circulating intermediate monocytes (r = 0.37, p = 0.009) on day 3. Serum TNF-α levels correlated with relative levels of circulating intermediate monocytes on day 1 (r = 0.40, p = 0.004) and day 3 (r = 0.29, p = 0.039). Table 5 Relative mRNA expression of iRhom2, TACE und TNF-α in circulating monocytes Gene Controls AMI day 1 AMI day 3 p -value, AMI d1 vs. d3 iRhom2 1.00 ± 0.21 1.095 ± 0.26 1.254 ± 0.44 0.021 TACE 0.96 (84–1.17) 1.07 (0.90–1.35) 1.10 (0.79–1.45) 0.83 TNF-α 0.91 (0.72–1.23) 1.0 (0.68–1.47) 1.11 (0.82–1.51) 0.406 Data are expressed as mean ± SD or as median with interquartile ranges. Statistical analysis was performed using a paired Student's t-test for normally distributed data and the Wilcoxon signed-rank test for non-normally distributed data. Expression levels were normalized to controls. iRhom2, inactive rhomboid protein 2; TACE, TNF-α converting enzyme; TNF, tumor necrosis factor. Association of iRhom2 mRNA expression in circulating monocytes and LV systolic function Blood levels of NTproBNP, Troponin T, and TNF-α are associated with LV systolic dysfunction following AMI. 24 According to our present observation that iRhom2 expression in monocytes increases in the course of AMI correlation analyses with LV systolic function obtained from transthoracic echocardiography were performed. We found that LV ejection fraction (categorized LVEF) negatively correlated with iRhom2 mRNA expression in circulating monocytes on day 3 after AMI, with the difference (Δ) of iRhom2 mRNA expression in circulating monocytes between day 1 versus day 3, and with serum TNF-α levels on day 3 (Table 6 ). Table 6 Correlations with left ventricular ejection fraction (LVEF) Parameter LVEF (categorized) Spearman rho p-value iRhom2 mRNA expression, day 1 -0.26 0.070 iRhom2 mRNA expression, day 3 -0.34 0.025 Δ iRhom2 mRNA expression, day 1 vs. 3 -0.36 0.010 Serum TNF-α, day 1 -0.19 0.186 Serum TNF-α, day 3 -0.36 0.010 NT-proBNP, day 1 -0.59 < 0.001 NT-proBNP, day 3 -0.57 < 0.001 Troponin-T high sensitive, day 1 -0.47 0.001 Troponin-T high sensitive, day 3 -0.45 0.001 iRhom2, inactive rhomboid protein 2; TNF, tumor necrosis factor; NT-proBNP, N-terminal pro-B-type natriuretic peptide; hs, high sensitivity. Statistical analysis was performed using the Spearman's rank correlation coefficient. Discussion In the present pilot study, we investigated the iRhom2/TACE/TNF-α-axis as a potentially important regulator of postischemic inflammation in patients with AMI. Similar to previous studies, we observed a significant increase of serum TNF-α levels within the first 3 days following AMI as part of a systemic inflammatory response (further indicated by the increase of CRP between day 1 and 3). 25 , 26 The interaction of iRhom2 with TACE is essential for proper shedding of TNF-α from the cell surface of immune cells. 16 Accordingly we found that mRNA expression levels of iRhom2 in circulating monocytes increased in parallel to serum TNF-α levels following AMI. In contrast, TACE and TNF-α mRNA expression in circulating monocytes remained unchanged indicating a central regulatory role of iRhom2 in TNF-α response following AMI. This was further supported by a significant correlation between serum TNF-α levels and iRhom2 expression levels in circulation monocytes on day 3 after AMI. Among monocyte subsets intermediate monocytes are considered the major source of serum TNF-α. 27 In fact, we observed a significant correlation between relative levels of circulating intermediate monocytes not only with serum TNF-α levels but also with iRhom2 mRNA expression levels on day 3 following AMI. Taken together these findings strongly suggest a relevant regulatory role of iRhom2 in the augmented shedding of TNF-α from intermediate monocytes in the early phase of AMI. Given the fact that excess levels of TNF-α are known to impair myocardial recovery and promote postischemic myocardial injury, the observed significant correlation between iRhom2 mRNA expression in circulating monocytes on day 3 following AMI with the extent of LV dysfunction in our patient population appears to be a consistent outcome. These results suggest that modulation of iRhom2 represents a promising novel target to reduce excess TNF-α secretion from immune cells after AMI to attenuate adverse cardiac remodeling. Modulation of iRhom2 is considered a more distinguished strategy compared to non-selective TNF-α blockage as it presumably reduces cardiotoxic effects of TNF-α more selectively while preserving cardioprotective functions of the cytokine. In the setting of AMI modulation of iRhom2 may potentially prevent an excess and therefore detrimental secretion of soluble TNF-α by reducing transmembrane TNF-α shedding selectively from immune cells. 17 Soluble TNF-α primarily binds to TNFR1, which promotes inflammation and apoptosis. 28 In contrast, cardioprotective effects of TNFR2 such as tissue healing, angiogenesis, and anti-inflammatory processes which is primarily activated by transmembrane TNF-α would be preserved. 28 , 29 There are some limitations in the present study. First, due to the observational character of this pilot study the results remain descriptive. Secondly, expression analyses of iRhom2 and TACE were limited to mRNA levels. However, in previous in vitro studies with macrophages we observed that iRhom2 mRNA expression levels parallel levels of soluble TNF-α in a concentration-dependent manner after LPS stimulation, thus indicating a strong association between iRhom2 mRNA expression and TNF-α protein levels. 17 Thirdly, a longer time-course for the evaluation of the iRhom2/TACE/TNF-α-axis beyond day 3 following AMI was not performed due to the generally intended early hospital discharge of patients after AMI. In conclusion, the present study suggests that iRhom2 contributes to the regulation of inflammation and is thereby associated with LV systolic dysfunction following AMI. Thus, iRhom2 modulation should be further evaluated as a potential therapeutic strategy in AMI patients to attenuate adverse cardiac remodeling. Declarations ACKNOWLEDGMENTS We thank all the participants of the study. Our appreciation also goes to Ms. Nicole Rösener and Ms. Andrea Weller for their valuable support and technical assistance. FUNDING This work was supported by grant to BH by the Deutsche Forschungsgemeinschaft (DFG; Sachbeihilfe HE6092/2). BH was a participant in the BIH Charité Clinician Scientist Program funded by the Charité-Universitätsmedizin Berlin and the Berlin Institute of Health. BH received funding by the DZHK (German Centre for Cardiovascular Research). PD was supported by the Deutsche Gesellschaft für Kardiologie (DGK; Otto-Hess-Promotionsstipendium). ETHICS APPROVAL The study protocol was approved by the Ethics Committee of the Charité-Universitätsmedizin Berlin (EA1/246/14). The study complied with the Declaration of Helsinki. Written informed consent was obtained from each study participant. CONFLICT OF INTEREST None declared. AUTHORS’ CONTRIBUTIONS BH: study conception and design, data acquisition, data analysis, interpretation of results, drafting of the manuscript; PvD: data acquisition, data analysis, interpretation of results, drafting of the manuscript; AL: study conception and design, data analysis, interpretation of results, drafting of the manuscript; CH, VS, and KS: interpretation of results, critical revision of the manuscript. DATA AVAILABILITY STATEMENT Data are available from the corresponding author BH upon reasonable request. References Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M, Das SR, de Ferranti S, Després J-P, Fullerton HJ. Heart disease and stroke statistics—2016 update: a report from the American Heart Association. Circulation 2015:CIR. 0000000000000350. Weir RA, McMurray JJ, Velazquez EJ. Epidemiology of heart failure and left ventricular systolic dysfunction after acute myocardial infarction: prevalence, clinical characteristics, and prognostic importance. 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European heart journal 1996;17:1852-9. Belge K-U, Dayyani F, Horelt A, Siedlar M, Frankenberger M, Frankenberger B, Espevik T, Ziegler-Heitbrock L. The proinflammatory CD14+ CD16+ DR++ monocytes are a major source of TNF. The Journal of Immunology 2002;168:3536-42. Monden Y, Kubota T, Inoue T, Tsutsumi T, Kawano S, Ide T, Tsutsui H, Sunagawa K. Tumor necrosis factor-α is toxic via receptor 1 and protective via receptor 2 in a murine model of myocardial infarction. American Journal of Physiology-Heart and Circulatory Physiology 2007;293:H743-H53. Schulz R, Heusch G. Tumor necrosis factor-α and its receptors 1 and 2: Yin and Yang in myocardial infarction? : Am Heart Assoc; 2009. Additional Declarations No competing interests reported. Supplementary Files graphicalabstract.pdf iRhom2 mRNA expression increases in monocytes in parallel to serum TNF-α levels at day 3 following acute myocardial infarction (AMI) compared to day 1. iRhom2 thereby contributes to the regulation of inflammation and is associated with LV dysfunction following AMI. iRhom2, inactive rhomboid protein 2; LVEF, left ventricular ejection fraction; TACE, TNF-a converting enzyme; TNF, tumor necrosis factor; tm, transmembrane. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2390961","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":162488318,"identity":"ba2c86b2-7e98-4d36-957f-0714df73de0f","order_by":0,"name":"Phillip Dijck","email":"","orcid":"","institution":"Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt- Universität zu Berlin, Berlin Institute of Health","correspondingAuthor":false,"prefix":"","firstName":"Phillip","middleName":"","lastName":"Dijck","suffix":""},{"id":162488319,"identity":"89f84e19-2ed4-45c4-b160-91a709035f65","order_by":1,"name":"Carmen Hannemann","email":"","orcid":"","institution":"New York University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Carmen","middleName":"","lastName":"Hannemann","suffix":""},{"id":162488320,"identity":"0e597b82-6bb8-453c-a24b-08a7288c6f9c","order_by":2,"name":"Henryk Dreger","email":"","orcid":"","institution":"Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt- Universität zu Berlin, Berlin Institute of Health","correspondingAuthor":false,"prefix":"","firstName":"Henryk","middleName":"","lastName":"Dreger","suffix":""},{"id":162488321,"identity":"bcb59ccd-3da8-438e-931b-12e39f0e9c67","order_by":3,"name":"Verena Stangl","email":"","orcid":"","institution":"Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt- Universität zu Berlin, Berlin Institute of Health","correspondingAuthor":false,"prefix":"","firstName":"Verena","middleName":"","lastName":"Stangl","suffix":""},{"id":162488322,"identity":"50ee8426-6b77-4989-afbd-82f48143e651","order_by":4,"name":"Karl Stangl","email":"","orcid":"","institution":"Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt- Universität zu Berlin, Berlin Institute of Health","correspondingAuthor":false,"prefix":"","firstName":"Karl","middleName":"","lastName":"Stangl","suffix":""},{"id":162488323,"identity":"923d3bd6-b639-45c7-8418-7706e1d52415","order_by":5,"name":"Antje Ludwig","email":"","orcid":"","institution":"Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt- Universität zu Berlin, Berlin Institute of Health","correspondingAuthor":false,"prefix":"","firstName":"Antje","middleName":"","lastName":"Ludwig","suffix":""},{"id":162488324,"identity":"4b6d27f6-0c28-4c46-8465-a480e851809c","order_by":6,"name":"Bernd Hewing","email":"data:image/png;base64,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","orcid":"","institution":"Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt- Universität zu Berlin, Berlin Institute of Health","correspondingAuthor":true,"prefix":"","firstName":"Bernd","middleName":"","lastName":"Hewing","suffix":""}],"badges":[],"createdAt":"2022-12-18 15:44:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2390961/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2390961/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":30898431,"identity":"c3c31f2b-af80-413c-979a-c4a98e32b09b","added_by":"auto","created_at":"2022-12-29 18:22:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":138130,"visible":true,"origin":"","legend":"\u003cp\u003eRelative mRNA expression of iRhom2 (A), TACE (B), TNF-α (C) in monocytes in AMI patients on day 1 compared to day 3 after hospital admission. (D) Serum TNF-α levels of AMI-patients on day 1 compared to day 3. Statistical analysis for comparison between day 1 and day 3 was performed using the Student’s t-test for normally distributed data and the Wilcoxon signed-rank test for non-normally distributed data. mRNA expression levels were normalized to young and healthy controls (Controls). °, mild outliers (1.5*IQR - 3.0*IQR); *, extreme outliers (\u0026gt;3,0*IQR); AMI, acute myocardial infarction; d1 or 3, day 1 or 3.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2390961/v1/631a7f1022d16e10aa5a7366.png"},{"id":45931771,"identity":"a0287452-831e-4420-8b30-254204b0994b","added_by":"auto","created_at":"2023-11-06 12:22:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":635070,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2390961/v1/f2abcb3b-1966-4559-b573-4cea743c4d21.pdf"},{"id":30898432,"identity":"0b7e5c1d-8db1-4538-bfc2-e3ac3d7a7763","added_by":"auto","created_at":"2022-12-29 18:22:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":853361,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eiRhom2 mRNA expression increases in monocytes in parallel to serum TNF-α levels at day 3 following acute myocardial infarction (AMI) compared to day 1. iRhom2 thereby contributes to the regulation of inflammation and is associated with LV dysfunction following AMI. iRhom2, inactive rhomboid protein 2; LVEF, left ventricular ejection fraction; TACE, TNF-a converting enzyme; TNF, tumor necrosis factor; tm, transmembrane.\u003c/p\u003e","description":"","filename":"graphicalabstract.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2390961/v1/c1530e3d6f688597ad74bab6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Increased expression of inactive rhomboid protein 2 in circulating monocytes after acute myocardial infarction","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcute myocardial infarction (AMI) is a leading cause of death worldwide.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Patients suffering a non-fatal AMI are exposed to elevated risk for short- and long-term complications including development of heart failure, ventricular arrhythmias, or recurrent AMI. Heart failure represents an important determinant for prognosis and survival in AMI patients.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e The pathogenesis underlying heart failure development after AMI is closely related to inflammatory processes.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Myocardial cell necrosis following AMI activates innate immunity and triggers a strong local as well as systemic inflammatory reaction that plays a central role in myocardial infarct healing but also in the development of adverse cardiac remodeling if dysregulated, excessive or prolonged.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e The inflammatory cytokine tumor necrosis factor-alpha (TNF-α) is detectable in the infarcted myocardium already during the early phase of AMI\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and elevated serum TNF-α levels have consistently been described in patients with AMI.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Animal studies have shown that TNF-α is involved in impaired recovery of myocardial function following AMI\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e whereas different animal models of genetic or pharmacological TNF-α inhibition have shown promise in attenuating important parameters of adverse cardiac remodeling.\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Controlled clinical trials addressing the potential of anti-TNF-α therapy in AMI are not available to date. TNF-α is mainly expressed by immune cells (in particular by monocytes and macrophages) as a precursor transmembrane protein located at the cell surface. Transmembrane TNF-α is cleaved by transmembrane metalloprotease TNF-α converting enzyme (TACE; ADAM17) to be released from the cell surface as soluble TNF-α (TNF-α shedding).\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Soluble TNF-α exerts its biological effects by binding to TNF receptor type 1 and 2 (TNFR1 and 2).\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e TACE maturation in immune cells depends on inactive rhomboid protein 2 (iRhom2; RHBDF2).\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Genetic deficiency or knock-down of iRhom2 prevents the activation of TACE and subsequently diminishes TNF-α shedding from immune cells. TACE maturation in non-hematopoietic cells remains unaltered as co-expression of closely related iRhom1 compensates for the absence of iRhom2 in these cells.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Up to date, the impact of the iRhom2/TACE/TNF-α-axis in AMI is unknown. In a previous study, we have shown that iRhom2-deficiency attenuates atherosclerosis development in low density lipoprotein receptor deficient mice.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Given the importance of iRhom2 in the regulation of TACE and TNF-α in immune cells, we aimed to investigate the gene-expression of iRhom2 in patients following AMI. We hypothesized that iRhom2 expression increases in circulating monocytes of patients with AMI, providing first evidence that iRhom2 is involved in the regulation of postischemic inflammation following AMI.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design and population\u003c/h2\u003e \u003cp\u003e The study protocol was approved by the Ethics Committee of the Charit\u0026eacute;-Universit\u0026auml;tsmedizin Berlin (EA1/246/14). The study complied with the Declaration of Helsinki. Written informed consent was obtained from each study participant. From June 2015 - February 2017, patients with AMI (n\u0026thinsp;=\u0026thinsp;50), were recruited at the Charit\u0026eacute;-Universit\u0026auml;tsmedizin Berlin, Germany. The AMI group comprised patients with ST-segment Elevation (STEMI) and Non-ST-segment Elevation Myocardial Infarction (NSTEMI) as defined by the guidelines of the European Society of Cardiology.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Twenty-five young and apparently healthy volunteers (mean age 22.8 years, range 19\u0026ndash;27 years; 44% female) without known cardiovascular disease were recruited as control group (controls). AMI patients were recruited within 24 hours after initial hospital admission with completed cardiac catheterization. Inclusion criteria comprised: age\u0026thinsp;\u0026ge;\u0026thinsp;18 years and diagnosis of AMI (as defined above). Exclusion criteria comprised the need for cardiac resuscitation, fever, acute infection/sepsis, rheumatic or non-rheumatic autoimmune diseases, current immunosuppressive therapy, severe acute or chronic renal failure (GFR\u0026thinsp;\u0026lt;\u0026thinsp;30 mL/min/1.73 m\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e), malignant diseases, stroke or surgery within 30 days. AMI patients were evaluated by medical history, clinical examination, blood collection, at study inclusion and at day 3 after study inclusion. Medical history was assessed by interviews and information from medical records. Functional class was assessed according to the New York Heart Association (NYHA) classification. Furthermore, standard transthoracic echocardiographic exams were performed within the first days after hospital admission (mean 2.4 \u0026plusmn; 1.6 days after hospital admission) and were digitally stored for offline analysis (TOMTEC-ARENA). Left ventricular ejection fraction (LVEF) was measured using the biplane Simpson\u0026rsquo;s method as previously described.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e For patients where image quality was not sufficient for assessment of biplane LVEF, visually estimated LVEF was used. LVEF was categorized into I: less than 30%, severely abnormal; II: 30\u0026ndash;39%, moderately to severely abnormal; III: 40\u0026ndash;49%, moderately abnormal; VI: 50\u0026ndash;53%, mildly abnormal; V: \u0026gt;54%, normal. Controls were evaluated by medical history (interview), clinical examination, and blood collection. Inclusion criteria for controls comprised age\u0026thinsp;\u0026ge;\u0026thinsp;18 years and \u0026lt;\u0026thinsp;30 years and no known pre-existing health conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eBlood sampling and standard laboratory parameters\u003c/h2\u003e \u003cp\u003ePeripheral blood samples were collected from cubital veins. Standard laboratory parameters including differential blood count, C-reactive protein (CRP), creatine kinase (CK), interleukin-6 (IL-6), N-terminal pro-B-type natriuretic peptide (NT-proBNP), TNF-α, and high sensitivity troponin T (hs-Troponin) were obtained by established assays in the hospital\u0026rsquo;s laboratory.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMonocyte isolation\u003c/h2\u003e \u003cp\u003ePeripheral blood mononuclear cells (PBMC) were obtained by Ficoll density gradient centrifugation (Ficoll-PaqueTM PLUS, GE Healthcare) of 30 ml peripheral blood collected in EDTA tubes. On average, 30*10\u003csup\u003e7\u003c/sup\u003e PBMCs were isolated per sample. Subsequently, monocytes were isolated by negative selection using the human Pan Monocyte Isolation Kit (Miltenyi Biotec; MACS isolation) according to the manufacturer\u0026rsquo;s instructions. Purity of enriched monocytes was confirmed by flow cytometry as detailed below. Approximately 7*10\u003csup\u003e7\u003c/sup\u003e monocytes were isolated per sample by MACS isolation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003e200 \u0026micro;L eluate of MACS isolated cell fraction was stained with monoclonal antibodies (CD86 PE [B7-2; Biolegend], CD14 PB [M5E2; Biolegend], CD16 APC [3G8; Biolegend], CD11b PECy7 [ICRF44; Biolegend]) for 15 minutes followed by repeated (twice) washing with 2 ml phosphate-buffered saline (PBS) solution. After centrifugation and removal of supernatant, cells were resuspended in PBS and run on a flow cytometer (CyAn\u0026trade; ADP Analyzer; Beckman Coulter, Inc.). Data was analyzed using Summit\u0026trade; 4.4 software applying the following gating strategy: first, peripheral blood mononuclear cells (PBMCs) were chosen and plotted on a CD86 histogram; monocytes were identified as CD86\u0026thinsp;+\u0026thinsp;cells and purity of enriched monocytes was defined as percentage of monocytes compared to all cells detected. On average, purity of enriched monocytes following MACS isolation was 90%. Secondly, monocytes were divided into three subsets: CD14\u0026thinsp;+\u0026thinsp;+\u0026thinsp;CD16- (classical), CD14\u0026thinsp;+\u0026thinsp;+\u0026thinsp;CD16+ (intermediate) and CD14-CD16++ (non-classical) according to the surface expression pattern of CD14 and CD16.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e To differentiate between intermediate and non-classical monocytes a straight vertical line was drawn to the left of the CD14 staining of the classical monocytes in the CD14 and CD16 dot plot as described previously.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Analyses of flow cytometry data was performed by two blinded observers; results represent averaged values from both analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time RT-PCR\u003c/h2\u003e \u003cp\u003eRNA from MACS sorted monocytes was isolated using RNeasy Mini Kit (Qiagen) according to the manufacturer\u0026rsquo;s instructions. 500 ng of total RNA was reversed-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). TaqMan assays (Applied Biosystems) were used to quantify the expression of iRhom2 (RHBF2; Hs01078106_m1), TACE (Hs01041915_m1), and TNF-α (Hs01113624_g1). RPL19 (Hs02338565_gH) was used as housekeeping gene. Expression of the target gene relative to housekeeping gene expression was calculated as the difference between the threshold values for the two genes. Relative expression levels were normalized to the control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eResults are generally expressed as arithmetic mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD for normally distributed data and median with interquartile ranges (IQR\u0026thinsp;=\u0026thinsp;25th-75th percentile) for non-normally distributed data; Shapiro-Wilk-test was used for testing for normal distribution of the data. Categorical data are presented as absolute numbers with respective percentages. For normally distributed data the Student\u0026rsquo;s t-test was performed for either paired (one-sample t-test) or unpaired (independent two-sample t-test) observations. For non-normally distributed data the Mann-Whitney-U-test was used for comparison of two independent groups, while the Wilcoxon signed-rank test was used for comparison of paired observations. Pearson\u0026rsquo;s or Spearman\u0026rsquo;s correlation coefficient, where appropriate, were calculated for correlation analyses between variables. Frequencies of various groups were compared by chi-square test. Statistical analyses were performed using SPSS 25.0 (SPSS Inc.) software; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eClinical Characteristics\u003c/h2\u003e \u003cp\u003eClinical characteristics of AMI patients at study inclusion are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. None of the AMI patients underwent coronary artery bypass grafting (CABG) following cardiac catheterization or received a percutaneous left ventricular assist device. Standard laboratory parameters are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The average LVEF of AMI patients was 50.4 \u0026plusmn; 9.3%. Patients were treated with guideline directed medical therapy during hospitalization.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Five patients had pre-existing statin therapy before hospital admission.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinical characteristics of AMI patients at study inclusion (day 1)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge in years, mean (range)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55 (33\u0026ndash;85)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36 (72.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14 (28.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eBMI (kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.6\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNSTEMI, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16 (32.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSTEMI, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34 (68.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCV risk factors\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIDDM, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (6.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNIDDM, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (10.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eActive smoking, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26 (52.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eHypercholesterolemia, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29 (58.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eHypertension, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29 (58.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eFamily history of CVD, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11 (22.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCVD history\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003ePrevious ACS, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (12.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003ePrevious PCI, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (16.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003ePrevious CABG, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003ePrevious TIA, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003ePrevious Stroke, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003ePAD, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCatheterization data\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eInitial diagnosis CAD, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41 (82.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCAD-1 vessel, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22 (44.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCAD-2 vessel, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11 (22.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCAD-3 vessel, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17 (34.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eMain vessel disease, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (4.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePTCA, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e49 (98.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStent, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46 (92.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003cem\u003eData are generally expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, or absolute numbers and respective percentages. BMI, body mass index; NSTEMI, non-ST-elevation myocardial infarction; STEMI, ST-elevation myocardial infarction; CV, cardiovascular; CVD, CV disease; ACS, acute coronary syndrome; PCI, percutaneous coronary intervention; CABG, coronary artery bypass grafting; TIA, transient ischemic attack; PAD, peripheral artery disease, CAD, coronary artery disease; PTCA, percutaneous transluminal coronary angioplasty; stent, only drug eluting stents were used.\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLaboratory parameters\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControls\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAMI day 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAMI day 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep-value AMI day 1 vs. day 3\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTroponin-T high sensitive, ng/L\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.9\u003c/p\u003e \u003cp\u003e(2.9-5.0)\u003c/p\u003e\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1052.0\u003c/p\u003e \u003cp\u003e(419.0-4403.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1233.5\u003c/p\u003e \u003cp\u003e(439.0-2068.0)\u003c/p\u003e\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCreatine kinase, U/L\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e142\u003c/p\u003e \u003cp\u003e(91\u0026ndash;203)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e593\u003c/p\u003e \u003cp\u003e(265\u0026ndash;1242)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e181.00\u003c/p\u003e \u003cp\u003e(127\u0026ndash;311)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNT-Pro-BNP, ng/L\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.0\u003c/p\u003e \u003cp\u003e(8.0\u0026ndash;53.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e642.0\u003c/p\u003e \u003cp\u003e(212.0-1359.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e569.5\u003c/p\u003e \u003cp\u003e(308.0-1411.0)\u003c/p\u003e\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.004\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCreatinine, mg/dl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003cp\u003e(0.76\u0026ndash;0.86)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.84\u003c/p\u003e \u003cp\u003e(0.77\u0026ndash;0.93)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003cp\u003e(0.77\u0026ndash;1.01)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.021\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cem\u003eData are expressed as median with interquartile ranges. Statistical analysis was performed using the Wilcoxon signed-rank test. NT-proBNP, N-terminal pro-B-type natriuretic peptide.\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSerum TNF-α levels\u003c/h2\u003e \u003cp\u003eSerum TNF-α levels of AMI patients on day 1 were significantly higher compared to controls (p\u0026thinsp;=\u0026thinsp;0.007) and increased significantly from day 1 to day 3 after AMI \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Serum levels of CRP and IL-6 are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBlood levels of inflammatory markers\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControls\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAMI day 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAMI day 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep-value, AMI\u003c/p\u003e \u003cp\u003eday 1 vs. day 3\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTNF-α, pg/ml\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.90\u003c/p\u003e \u003cp\u003e(4.9\u0026ndash;6.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.05\u003c/p\u003e \u003cp\u003e(5.4-8.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.45\u003c/p\u003e \u003cp\u003e(6.6\u0026ndash;10.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCRP, mg/L\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003cp\u003e(0.4\u0026ndash;1.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.3\u003c/p\u003e \u003cp\u003e(2.0-12.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.5\u003c/p\u003e \u003cp\u003e(11.6\u0026ndash;41.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIL-6, ng/L\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.4\u003c/p\u003e \u003cp\u003e(1.40-2.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.95 (10.5\u0026ndash;31.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.95\u003c/p\u003e \u003cp\u003e(7.4\u0026ndash;19.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.007\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cem\u003eData are expressed as median with interquartile ranges. Statistical analysis was performed using the Wilcoxon signed-rank test. TNF, tumor necrosis factor; CRP, C-reactive protein; IL-6, Interleukin 6.\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCirculating monocytes subsets\u003c/h2\u003e \u003cp\u003eThe absolute count of circulating monocytes was significantly higher in patients with AMI (on day 1 and 3) compared to controls and did not change significantly from day 1 to day 3 after AMI \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. In AMI patients, relative levels of circulating intermediate monocytes on day 1 did not differ significantly compared to controls \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e but increased significantly from day 1 to day 3 after AMI. In contrast, relative levels of circulating classical monocytes decreased, while levels of circulating non-classical monocytes remained unaltered from day 1 to day 3 after AMI \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAbsolute monocyte count and relative levels of circulating monocyte subsets. \u003cem\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD or as median with interquartile ranges. Statistical analysis was performed using a paired Student's t-test for normally distributed data and the Wilcoxon signed-rank test for non-normally distributed data.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControls\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAMI day 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAMI day 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep-value, AMI\u003c/p\u003e \u003cp\u003eday 1 vs. day 3\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMonocytes, /\u0026micro;l\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e374.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e889.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e908.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eClassical Monocytes (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e79.3\u003c/p\u003e \u003cp\u003e(75.1\u0026ndash;83.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e87.7\u003c/p\u003e \u003cp\u003e(82.5\u0026ndash;91.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85.3\u003c/p\u003e \u003cp\u003e(80.8\u0026ndash;88.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.025\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIntermediate Monocytes (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.90\u003c/p\u003e \u003cp\u003e(3.8\u0026ndash;5.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.07\u003c/p\u003e \u003cp\u003e(3.4\u0026ndash;7.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.50\u003c/p\u003e \u003cp\u003e(6.3\u0026ndash;10.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNon-Classical Monocytes (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.80\u003c/p\u003e \u003cp\u003e(7.7\u0026ndash;17.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.72\u003c/p\u003e \u003cp\u003e(2.3\u0026ndash;5.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.93\u003c/p\u003e \u003cp\u003e(2.1\u0026ndash;5.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.214\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eiRhom2, TACE, and TNF-α mRNA expression in circulating monocytes\u003c/h2\u003e \u003cp\u003eiRhom2 mRNA expression levels in circulating monocytes of AMI patients increased significantly (by 14%) from day 1 to day 3 after AMI \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. iRhom2 mRNA expression levels in circulating monocytes of AMI patients on day 3 were significantly higher compared to controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). No significant changes in mRNA expression levels of TACE and TNF-α in circulating monocytes of AMI patients were observed between day 1 and day 3 after AMI \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e; there were no significant differences in both parameters compared to controls. We observed a strong correlation between iRhom2 and TACE mRNA expression in circulating monocytes on day 3 after AMI (r\u0026thinsp;=\u0026thinsp;0.82, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Furthermore, iRhom2 mRNA expression in circulating monocytes correlated moderately with serum TNF-α levels (r\u0026thinsp;=\u0026thinsp;0.33, p\u0026thinsp;=\u0026thinsp;0.019) and with relative levels of circulating intermediate monocytes (r\u0026thinsp;=\u0026thinsp;0.37, p\u0026thinsp;=\u0026thinsp;0.009) on day 3. Serum TNF-α levels correlated with relative levels of circulating intermediate monocytes on day 1 (r\u0026thinsp;=\u0026thinsp;0.40, p\u0026thinsp;=\u0026thinsp;0.004) and day 3 (r\u0026thinsp;=\u0026thinsp;0.29, p\u0026thinsp;=\u0026thinsp;0.039).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRelative mRNA expression of iRhom2, TACE und TNF-α in circulating monocytes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControls\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAMI day 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAMI day 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value, AMI\u003c/p\u003e \u003cp\u003ed1 vs. d3\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eiRhom2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.095\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.254\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.021\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTACE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003cp\u003e(84\u0026ndash;1.17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.07\u003c/p\u003e \u003cp\u003e(0.90\u0026ndash;1.35)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.10\u003c/p\u003e \u003cp\u003e(0.79\u0026ndash;1.45)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTNF-α\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003cp\u003e(0.72\u0026ndash;1.23)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003cp\u003e(0.68\u0026ndash;1.47)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.11\u003c/p\u003e \u003cp\u003e(0.82\u0026ndash;1.51)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.406\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cem\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD or as median with interquartile ranges. Statistical analysis was performed using a paired Student's t-test for normally distributed data and the Wilcoxon signed-rank test for non-normally distributed data. Expression levels were normalized to controls. iRhom2, inactive rhomboid protein 2; TACE, TNF-α converting enzyme; TNF, tumor necrosis factor.\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAssociation of iRhom2 mRNA expression in circulating monocytes and LV systolic function\u003c/h2\u003e \u003cp\u003eBlood levels of NTproBNP, Troponin T, and TNF-α are associated with LV systolic dysfunction following AMI.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e According to our present observation that iRhom2 expression in monocytes increases in the course of AMI correlation analyses with LV systolic function obtained from transthoracic echocardiography were performed. We found that LV ejection fraction (categorized LVEF) negatively correlated with iRhom2 mRNA expression in circulating monocytes on day 3 after AMI, with the difference (Δ) of iRhom2 mRNA expression in circulating monocytes between day 1 versus day 3, and with serum TNF-α levels on day 3 (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelations with left ventricular ejection fraction (LVEF)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eLVEF (categorized)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpearman rho\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eiRhom2 mRNA expression, day 1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.070\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eiRhom2 mRNA expression, day 3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.025\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eΔ iRhom2 mRNA expression, day 1 vs. 3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.010\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSerum TNF-α, day 1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.186\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSerum TNF-α, day 3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.010\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNT-proBNP, day 1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNT-proBNP, day 3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTroponin-T high sensitive, day 1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTroponin-T high sensitive, day 3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003cem\u003eiRhom2, inactive rhomboid protein 2; TNF, tumor necrosis factor; NT-proBNP, N-terminal pro-B-type natriuretic peptide; hs, high sensitivity. Statistical analysis was performed using the Spearman's rank correlation coefficient.\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present pilot study, we investigated the iRhom2/TACE/TNF-α-axis as a potentially important regulator of postischemic inflammation in patients with AMI. Similar to previous studies, we observed a significant increase of serum TNF-α levels within the first 3 days following AMI as part of a systemic inflammatory response (further indicated by the increase of CRP between day 1 and 3).\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e The interaction of iRhom2 with TACE is essential for proper shedding of TNF-α from the cell surface of immune cells.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Accordingly we found that mRNA expression levels of iRhom2 in circulating monocytes increased in parallel to serum TNF-α levels following AMI. In contrast, TACE and TNF-α mRNA expression in circulating monocytes remained unchanged indicating a central regulatory role of iRhom2 in TNF-α response following AMI. This was further supported by a significant correlation between serum TNF-α levels and iRhom2 expression levels in circulation monocytes on day 3 after AMI. Among monocyte subsets intermediate monocytes are considered the major source of serum TNF-α.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e In fact, we observed a significant correlation between relative levels of circulating intermediate monocytes not only with serum TNF-α levels but also with iRhom2 mRNA expression levels on day 3 following AMI. Taken together these findings strongly suggest a relevant regulatory role of iRhom2 in the augmented shedding of TNF-α from intermediate monocytes in the early phase of AMI.\u003c/p\u003e \u003cp\u003eGiven the fact that excess levels of TNF-α are known to impair myocardial recovery and promote postischemic myocardial injury, the observed significant correlation between iRhom2 mRNA expression in circulating monocytes on day 3 following AMI with the extent of LV dysfunction in our patient population appears to be a consistent outcome. These results suggest that modulation of iRhom2 represents a promising novel target to reduce excess TNF-α secretion from immune cells after AMI to attenuate adverse cardiac remodeling. Modulation of iRhom2 is considered a more distinguished strategy compared to non-selective TNF-α blockage as it presumably reduces cardiotoxic effects of TNF-α more selectively while preserving cardioprotective functions of the cytokine.\u003c/p\u003e \u003cp\u003eIn the setting of AMI modulation of iRhom2 may potentially prevent an excess and therefore detrimental secretion of soluble TNF-α by reducing transmembrane TNF-α shedding selectively from immune cells.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Soluble TNF-α primarily binds to TNFR1, which promotes inflammation and apoptosis.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e In contrast, cardioprotective effects of TNFR2 such as tissue healing, angiogenesis, and anti-inflammatory processes which is primarily activated by transmembrane TNF-α would be preserved.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThere are some limitations in the present study. First, due to the observational character of this pilot study the results remain descriptive. Secondly, expression analyses of iRhom2 and TACE were limited to mRNA levels. However, in previous in vitro studies with macrophages we observed that iRhom2 mRNA expression levels parallel levels of soluble TNF-α in a concentration-dependent manner after LPS stimulation, thus indicating a strong association between iRhom2 mRNA expression and TNF-α protein levels.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Thirdly, a longer time-course for the evaluation of the iRhom2/TACE/TNF-α-axis beyond day 3 following AMI was not performed due to the generally intended early hospital discharge of patients after AMI.\u003c/p\u003e \u003cp\u003eIn conclusion, the present study suggests that iRhom2 contributes to the regulation of inflammation and is thereby associated with LV systolic dysfunction following AMI. Thus, iRhom2 modulation should be further evaluated as a potential therapeutic strategy in AMI patients to attenuate adverse cardiac remodeling.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all the participants of the study. Our appreciation also goes to Ms. Nicole R\u0026ouml;sener and Ms. Andrea Weller for their valuable support and technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grant to BH by the Deutsche Forschungsgemeinschaft (DFG; Sachbeihilfe HE6092/2). BH was a participant in the BIH Charité Clinician Scientist Program funded by the Charité-Universitätsmedizin Berlin and the Berlin Institute of Health. BH received funding by the DZHK (German Centre for Cardiovascular Research).\u0026nbsp;PD was supported by the Deutsche Gesellschaft f\u0026uuml;r Kardiologie (DGK; Otto-Hess-Promotionsstipendium).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was approved by the Ethics Committee of the Charit\u0026eacute;-Universit\u0026auml;tsmedizin Berlin (EA1/246/14). The study complied with the Declaration of Helsinki. Written informed consent was obtained from each study participant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone declared.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHORS\u0026rsquo; CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBH: study conception and design, data acquisition, data analysis, interpretation of results, drafting of the manuscript; PvD: data acquisition, data analysis, interpretation of results, drafting of the manuscript; AL: study conception and design, data analysis, interpretation of results, drafting of the manuscript; CH, VS, and KS: interpretation of results, critical revision of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are available from the corresponding author BH upon reasonable request.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M, Das SR, de Ferranti S, Despr\u0026eacute;s J-P, Fullerton HJ. 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The Journal of Immunology 2002;168:3536-42.\u003c/li\u003e\n\u003cli\u003eMonden Y, Kubota T, Inoue T, Tsutsumi T, Kawano S, Ide T, Tsutsui H, Sunagawa K. Tumor necrosis factor-\u0026alpha; is toxic via receptor 1 and protective via receptor 2 in a murine model of myocardial infarction. American Journal of Physiology-Heart and Circulatory Physiology 2007;293:H743-H53.\u003c/li\u003e\n\u003cli\u003eSchulz R, Heusch G. 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The interaction of inactive rhomboid protein 2 (iRhom2) with TNF-α converting enzyme (TACE) is required for shedding of TNF-α from the cell surface of immune cells. In this pilot study, we hypothesized that iRhom2 expression increases in circulating monocytes following AMI.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eCirculating monocytes were MACS-sorted from peripheral blood of 50 AMI patients at admission (day 1) and 3 days after admission. mRNA was isolated from sorted monocytes and expression levels of iRhom2, TACE and TNF-α were evaluated by real-time RT-PCR. Serum TNF-α levels were determined. Circulating monocyte subsets were quantified by flow cytometry. Left ventricular (LV) function was measured by echocardiography.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe observed a significant increase of iRhom2 mRNA expression in monocytes (p\u0026thinsp;=\u0026thinsp;0.012), of intermediate monocytes levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and of serum TNF-α levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) at day 3 following AMI compared to day 1. In contrast, TNF-α and TACE mRNA expression in monocytes remained unchanged. At day 3, iRhom2 mRNA expression in monocytes positively correlated with levels of intermediate monocytes (r\u0026thinsp;=\u0026thinsp;0.37, p\u0026thinsp;=\u0026thinsp;0.009) and serum TNF-α levels (r\u0026thinsp;=\u0026thinsp;0.33, p\u0026thinsp;=\u0026thinsp;0.019). iRhom2 mRNA expression in monocytes at day 3 negatively correlated with LV systolic function (r=-0.34, p\u0026thinsp;=\u0026thinsp;0.025).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study suggests that iRhom2 contributes to the regulation of inflammation and is thereby associated with LV dysfunction following AMI. Thus, iRhom2 modulation should be further evaluated as a potential therapeutic strategy to attenuate adverse cardiac remodeling in AMI patients.\u003c/p\u003e","manuscriptTitle":"Increased expression of inactive rhomboid protein 2 in circulating monocytes after acute myocardial infarction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-12-29 18:21:55","doi":"10.21203/rs.3.rs-2390961/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c2dcb763-bbb8-4868-8256-28c909b4f8f8","owner":[],"postedDate":"December 29th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-11-06T12:14:20+00:00","versionOfRecord":[],"versionCreatedAt":"2022-12-29 18:21:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2390961","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2390961","identity":"rs-2390961","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}