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A multi-site coronary sampling study on CRP in non-STEMI: Novel insights into the inflammatory process in acute coronary syndromes

Open AccessPublished:September 19, 2018DOI:https://doi.org/10.1016/j.atherosclerosis.2018.09.024

      Highlights

      • In NSTEMI patients, there was a trans-cardiac decrease in CRP across the myocardium.
      • This decrease was irrespective of time of presentation, infarct size and culprit lesion location.
      • There was no trans-lesional gradient across the culprit coronary artery lesion.
      • Both injured and non-injured myocardium contributes to the decrease in CRP.

      Abstract

      Background and aims

      Inflammation has become a key element in cardiovascular disease, and recently, new anti-inflammatory interventions have shown promising results. In this context, CRP levels have been thoroughly studied in vitro and in animals, but studies in humans are scarce and insights into its release, site(s) of production and uptake are not uniform.

      Methods

      We performed a biomarker study with multi-site sampling in the coronary circulation, in non-ST elevation MI (NSTEMI) patients with coronary angiography and right-sided catheterisation. Trans-lesional gradients were obtained by sampling distal to the culprit lesion, in patients with a suitable anatomy. To asses trans-cardiac gradients, blood was sampled from the systemic circulation, coronary sinus (CS) and great cardiac vein. Concentrations of CRP were measured with a high-sensitivity assay.

      Results

      In 42 patients, a median systemic venous CRP concentration of 4.97 mg/L was observed. There was no evidence of a trans-lesional gradient (4.59 mg/L versus 4.56 mg/L, p = 0.278; n = 14). A significant decrease in CRP concentration was observed between systemic arterial and CS samples (4.88 mg/L versus 4.44 mg/L; p < 0.001; n = 42). This trans-cardiac gradient was irrespective of time of presentation, infarct size and culprit lesion location. The gradient was not only driven by blood that ran through the injured myocardium, but also by lower CRP concentrations in the coronary veins that drain non-infarcted myocardium.

      Conclusions

      In the context of NSTEMI, we observed a trans-cardiac decrease in CRP, which may indicate the first human in vivo proof of a net CRP uptake by the myocardium, with a role for CRP both in the injured and adjacent myocardium.

      Keywords

      1. Introduction

      The inflammatory process has become a key issue of interest in the development of atherosclerosis and the progression to atherothrombosis [
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      ]. Despite clear associations between C-reactive protein (CRP) and the risk of future events in coronary artery disease [
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      • Monassier J.P.
      Prognostic value of C-reactive protein and cardiac troponin I in primary percutaneous interventions for ST-elevation myocardial infarction.
      ], the role of CRP in the acute phase of myocardial infarction still requires further investigation.
      In general, there is consensus that CRP production mainly occurs in the hepatocytes in response to an extra-hepatic stimulus [
      • Black S.
      • Kushner I.
      • Samols D.
      C-reactive protein.
      ]. Moreover, systemic CRP levels are generally higher in myocardial infarction (MI) patients than in stable coronary artery disease [
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      ]. Appreciating that CRP binds to injured cells and activates inflammation, an evident role for CRP was suggested in the aftermath of MI [
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      ]. Several studies focused on coronary CRP levels, varying from trans-lesional assessments (proximal and distal to the culprit lesion) [
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      Inflammatory markers at the site of ruptured plaque in acute myocardial infarction: locally increased interleukin-6 and serum amyloid A but decreased C-reactive protein.
      ,
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      • Nik Ibrahim N.N.
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      • Yusof Z.
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      • Abdul Rahman A.R.
      • Yvonne-Tee G.B.
      Systemic and coronary levels of CRP, MPO, sCD40L and PlGF in patients with coronary artery disease.
      ,
      • Kirbis S.
      • Breskvar U.D.
      • Sabovic M.
      • Zupan I.
      • Sinkovic A.
      Inflammation markers in patients with coronary artery disease--comparison of intracoronary and systemic levels.
      ,
      • Aggarwal A.
      • Schneider D.J.
      • Terrien E.F.
      • Sobel B.E.
      • Dauerman H.L.
      Increased coronary arterial release of interleukin-1 receptor antagonist and soluble CD40 ligand indicative of inflammation associated with culprit coronary plaques.
      ,
      • Inoue T.
      • Kato T.
      • Uchida T.
      • Sakuma M.
      • Nakajima A.
      • Shibazaki M.
      • Imoto Y.
      • Saito M.
      • Hashimoto S.
      • Hikichi Y.
      • Node K.
      Local release of C-reactive protein from vulnerable plaque or coronary arterial wall injured by stenting.
      ] to studies on trans-cardiac gradients, i.e. from the aorta to the coronary sinus (CS) [
      • Inoue T.
      • Kato T.
      • Uchida T.
      • Sakuma M.
      • Nakajima A.
      • Shibazaki M.
      • Imoto Y.
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      • Hashimoto S.
      • Hikichi Y.
      • Node K.
      Local release of C-reactive protein from vulnerable plaque or coronary arterial wall injured by stenting.
      ,
      • Forte L.
      • Cimmino G.
      • Loffredo F.
      • De Palma R.
      • Abbate G.
      • Calabro P.
      • Ingrosso D.
      • Galletti P.
      • Carangio C.
      • Casillo B.
      • Calabro R.
      • Golino P.
      C-reactive protein is released in the coronary circulation and causes endothelial dysfunction in patients with acute coronary syndromes.
      ,
      • Cimmino G.
      • Ragni M.
      • Cirillo P.
      • Petrillo G.
      • Loffredo F.
      • Chiariello M.
      • Gresele P.
      • Falcinelli E.
      • Golino P.
      C-reactive protein induces expression of matrix metalloproteinase-9: a possible link between inflammation and plaque rupture.
      ,
      • Taylor A.J.
      • Bobik A.
      • Richards M.
      • Kaye D.
      • Raines G.
      • Gould P.
      • Jennings G.
      Myocardial endothelin-1 release and indices of inflammation during angioplasty for acute myocardial infarction and stable coronary artery disease.
      ,
      • Leite W.F.
      • Ramires J.A.
      • Moreira L.F.
      • Strunz C.M.
      • Mangione J.A.
      Correlation between C-reactive protein in peripheral vein and coronary sinus in stable and unstable angina.
      ,
      • Wang Y.
      • Li L.
      • Tan H.W.
      • Yu G.S.
      • Ma Z.Y.
      • Zhao Y.X.
      • Zhang Y.
      Transcoronary concentration gradient of sCD40L and hsCRP in patients with coronary heart disease.
      ].
      Despite evidence of high CRP mRNA content in unstable plaques [
      • Forte L.
      • Cimmino G.
      • Loffredo F.
      • De Palma R.
      • Abbate G.
      • Calabro P.
      • Ingrosso D.
      • Galletti P.
      • Carangio C.
      • Casillo B.
      • Calabro R.
      • Golino P.
      C-reactive protein is released in the coronary circulation and causes endothelial dysfunction in patients with acute coronary syndromes.
      ] and a trans-lesional increase of CRP in patients with unstable angina [
      • Inoue T.
      • Kato T.
      • Uchida T.
      • Sakuma M.
      • Nakajima A.
      • Shibazaki M.
      • Imoto Y.
      • Saito M.
      • Hashimoto S.
      • Hikichi Y.
      • Node K.
      Local release of C-reactive protein from vulnerable plaque or coronary arterial wall injured by stenting.
      ], other studies on trans-lesional concentrations generally reported no evidence of a gradient in acute coronary syndrome (ACS) [
      • Fong S.W.
      • Few L.L.
      • See Too W.C.
      • Khoo B.Y.
      • Nik Ibrahim N.N.
      • Yahaya S.A.
      • Yusof Z.
      • Mohd Ali R.
      • Abdul Rahman A.R.
      • Yvonne-Tee G.B.
      Systemic and coronary levels of CRP, MPO, sCD40L and PlGF in patients with coronary artery disease.
      ,
      • Kirbis S.
      • Breskvar U.D.
      • Sabovic M.
      • Zupan I.
      • Sinkovic A.
      Inflammation markers in patients with coronary artery disease--comparison of intracoronary and systemic levels.
      ,
      • Aggarwal A.
      • Schneider D.J.
      • Terrien E.F.
      • Sobel B.E.
      • Dauerman H.L.
      Increased coronary arterial release of interleukin-1 receptor antagonist and soluble CD40 ligand indicative of inflammation associated with culprit coronary plaques.
      ]. The few studies on trans-cardiac gradients showed conflicting results and varied in study design and population [
      • Inoue T.
      • Kato T.
      • Uchida T.
      • Sakuma M.
      • Nakajima A.
      • Shibazaki M.
      • Imoto Y.
      • Saito M.
      • Hashimoto S.
      • Hikichi Y.
      • Node K.
      Local release of C-reactive protein from vulnerable plaque or coronary arterial wall injured by stenting.
      ,
      • Forte L.
      • Cimmino G.
      • Loffredo F.
      • De Palma R.
      • Abbate G.
      • Calabro P.
      • Ingrosso D.
      • Galletti P.
      • Carangio C.
      • Casillo B.
      • Calabro R.
      • Golino P.
      C-reactive protein is released in the coronary circulation and causes endothelial dysfunction in patients with acute coronary syndromes.
      ,
      • Cimmino G.
      • Ragni M.
      • Cirillo P.
      • Petrillo G.
      • Loffredo F.
      • Chiariello M.
      • Gresele P.
      • Falcinelli E.
      • Golino P.
      C-reactive protein induces expression of matrix metalloproteinase-9: a possible link between inflammation and plaque rupture.
      ,
      • Taylor A.J.
      • Bobik A.
      • Richards M.
      • Kaye D.
      • Raines G.
      • Gould P.
      • Jennings G.
      Myocardial endothelin-1 release and indices of inflammation during angioplasty for acute myocardial infarction and stable coronary artery disease.
      ,
      • Leite W.F.
      • Ramires J.A.
      • Moreira L.F.
      • Strunz C.M.
      • Mangione J.A.
      Correlation between C-reactive protein in peripheral vein and coronary sinus in stable and unstable angina.
      ,
      • Wang Y.
      • Li L.
      • Tan H.W.
      • Yu G.S.
      • Ma Z.Y.
      • Zhao Y.X.
      • Zhang Y.
      Transcoronary concentration gradient of sCD40L and hsCRP in patients with coronary heart disease.
      ]. Although several histopathological studies have shown that CRP is deposited in infarcted regions of the myocardium [
      • Lagrand W.K.
      • Niessen H.W.
      • Wolbink G.J.
      • Jaspars L.H.
      • Visser C.A.
      • Verheugt F.W.
      • Meijer C.J.
      • Hack C.E.
      C-reactive protein colocalizes with complement in human hearts during acute myocardial infarction.
      ,
      • Nijmeijer R.
      • Lagrand W.K.
      • Lubbers Y.T.P.
      • Visser C.A.
      • Meijer C.J.L.M.
      • Niessen H.W.M.
      • Hack C.E.
      C-reactive protein activates complement in infarcted human myocardium.
      ], most studies on trans-cardiac gradients did not report on the association between the extent of myocardial damage and CRP changes. This may be of particular importance, as previous studies have suggested that the trans-cardiac gradient of inflammatory markers depends on the presence or absence of injured myocardium [
      • Cusack M.R.
      • Marber M.S.
      • Lambiase P.D.
      • Bucknall C.A.
      • Redwood S.R.
      Systemic inflammation in unstable angina is the result of myocardial necrosis.
      ,
      • Ohashi Y.
      • Kawashima S.
      • Mori T.
      • Terashima M.
      • Ichikawa S.
      • Ejiri J.
      • Awano K.
      Soluble CD40 ligand and interleukin-6 in the coronary circulation after acute myocardial infarction.
      ]. In this context, a more detailed study could improve insight into the role of CRP in MI patients.
      We therefore conducted a multi-site coronary sampling study in non-ST elevation MI (NSTEMI) patients to assess both trans-lesional and trans-cardiac CRP gradients. In the abovementioned context, we focussed on the impact of single versus multivessel disease, infarct size and had specific interest for injured versus non-injured myocardium on changes in CRP levels across the heart.

      2. Patients and methods

      2.1 Patient population

      The TRans-cardiac Assessment of Myocardial Injury and Coronary Inflammation (TRAMICI) study is a prospective mechanistic study performed in the catheterisation laboratory of the Radboud University Medical Center (Radboudumc, Nijmegen, The Netherlands) in patients presenting with a NSTEMI. Patients from the Radboudumc and three referring centers were eligible if they presented early after symptom onset, were diagnosed with a NSTEMI and had evidence of elevated (i.e. above the 99th percentile reference limit of normal) and rising cardiac troponin levels based on conventional troponin assays used in the respective hospitals. Subsequently, patients were referred for a clinically indicated coronary angiography (CAG) in the Radboudumc. Upon identification of the culprit coronary artery by the interventional cardiologist, patients were included and study procedures were started. Exclusion criteria were: an indication for emergency percutaneous coronary intervention (PCI) at presentation, prior PCI or CABG (<3 months), prior anginal complaints (<3 weeks), killip class III and IV, other suspected life-threatening disease at presentation, peripheral arterial disease (Fontaine III and IV), presence of a pacemaker, main stem stenosis (>50%), anomalous coronary anatomy, serum creatinine >150 μmol/L, systemic infection, hematologic disorder or treatment with an immunosuppressive agent, treatment with NSAID or antibiotics. The presence of collateral coronary artery filling was considered a relative exclusion criterion. After obtaining oral informed consent prior to the procedure, participants provided written informed consent. The protocol was approved by the local ethical committee and study procedures were in accordance with the Declaration of Helsinki.

      2.2 Study procedures

      The coronary venous anatomy was recorded during CAG by filming the complete washout of contrast dye. Access to the CS was gained by means of a right-sided catheterisation procedure. For cannulation and blood sampling of the coronary venous system, a Terumo wire (Terumo Europe NV, Leuven, Belgium) and CHAMP multipurpose catheter (Medtronic, Santa Rosa, CA, USA) were used, respectively.
      Coronary venous system samples: After cannulation of the CS, the CHAMP catheter was advanced into the great cardiac vein (GCV). This vein primarily drains blood from the anteroseptal walls [
      • Ganz W.
      • Tamura K.
      • Marcus H.S.
      • Donoso R.
      • Yoshida S.
      • Swan H.J.
      Measurement of coronary sinus blood flow by continuous thermodilution in man.
      ]. Consequently, in patients with a culprit lesion in the left anterior descending artery (LAD), a selective blood sample from this site represents biomarker concentrations in blood that just ran through injured myocardium. Alternatively, in patients with a non-LAD culprit lesion, a selective blood sample from this site represents biomarker concentrations in a vein that drains blood from myocardium not supplied by the infarct related artery. After sampling in the GCV, the catheter was pulled back towards the CS at the point where the middle cardiac vein merges with the CS. Being the rendezvous point of all blood that passed the myocardium, the CS sample represents the trans-cardiac gradient.
      Blood sampling was performed according to the following protocol driven procedures. First, the position of the catheter was confirmed using contrast dye, after which the catheter was flushed. Second, there was a waiting period to allow for sufficient blood reflux. Then, a first blood sample of 3–4 mL was drawn to perform blood gas analysis, as a double check of the catheter position. Subsequently, the blood sample of interest was obtained. Fig. 1 illustrates the different sampling sites within the coronary venous system.
      Fig. 1
      Fig. 1Schematic view of the coronary venous system.
      LAD: left anterior descending artery. GCV: great cardiac vein. RCX: ramus circumflex artery. PLV: posterolateral vein. RCA: right coronary artery. MCV: middle cardiac vein. SVC: superior caval vein. IVC: inferior caval vein. The asterixes depict the sampling sites in the great cardiac vein and coronary sinus.
      Systemic blood samples: After removal of the CHAMP catheter from the coronary venous system, systemic blood samples were obtained from the femoral venous and arterial sheaths.
      Culprit artery samples: Patients with a suitable anatomy, and in whom the preferred revascularisation strategy was PCI, were eligible for an additional sample from the culprit coronary artery distal to the culprit lesion. Prior to the PCI, a guiding catheter was inserted in the ostium of the culprit coronary artery. A wire was then advanced beyond the culprit lesion followed by an over-the-wire balloon- (MAVERICK) or microcatheter (Boston Scientific, Marlborough, MA, USA). After removal of the wire while keeping the catheter in place, blood was aspirated distal from the culprit lesion.
      Follow-up samples: Finally, at 6 and 12 h post-procedure, additional venous samples were obtained from an antecubital vein.
      All collected blood samples were divided over serum and plasma tubes, centrifuged, aliquoted and stored at −80 °C until thawed for further analysis.

      2.3 Coronary angiogram analysis

      All angiograms were reviewed by a second team of doctors (SD, GC and HG), unaware of biomarker analysis. They performed a standardised evaluation of left ventriculography for wall motion abnormalities, lesion characteristics, and coronary flow to identify the culprit lesion. Lesion severity was based on visual estimation. In case of a discrepancy with regard to the decision on the culprit artery between the interventional cardiologist and the second team, data were analysed by a third cardiologist (MB), unaware of any former decision on culprit location. When the third reviewer agreed with either of the two previous teams, the location of the culprit artery was decided on. If not, the patient was excluded from the present analysis.

      2.4 Measurement of biomarkers

      Biomarker analysis was performed at the clinical chemistry department in the Isala Clinics (Isala Clinics, Zwolle, The Netherlands) and the Maastricht University Medical Center+ (MUMC, Maastricht, The Netherlands). The biomarkers involved in the current analysis are: high-sensitivity CRP, high-sensitivity cardiac troponin T (hs-cTnT), CK-MB and albumin. All assays were provided by Roche Diagnostics, and analyses performed on a Cobas analyser (Roche, Mannheim, Germany). With respect to CRP concentrations the lower limit of detection is 0.15 mg/L, and at 0.3 mg/L the coefficient of variation is <10%. The hs-cTnT assay has a limit of blank of 3 ng/L and a coefficient of variation of <10% at 13 ng/L (limit of quantification). With regard to the CK-MB immunoassay a lower limit of detection of 0.1 ng/mL is reported. Given the well-described correlation between infarct size and peak CK-MB, this biomarker was chosen to define infarct size in our population [
      • Dohi T.
      • Maehara A.
      • Brener S.J.
      • Genereux P.
      • Gershlick A.H.
      • Mehran R.
      • Gibson C.M.
      • Mintz G.S.
      • Stone G.W.
      Utility of peak creatine kinase-MB measurements in predicting myocardial infarct size, left ventricular dysfunction, and outcome after first anterior wall acute myocardial infarction (from the INFUSE-AMI trial).
      ]. Patients were divided in small (lower 50th percentile) and large (upper 50th percentile) MI based on the peak CK-MB concentration measured during admission. As for albumin, the measuring range was 2–100 g/L defined by the limit of detection and the maximum of the master curve.

      2.5 Statistical analysis

      The present analysis on CRP is a prospectively defined secondary objective of the TRAMICI project, and is performed on a subset of the entire study population. The total number of participants in TRAMICI was based on the required number of patients for the primary objective, with troponin measurements as primary outcome. Continuous data were analysed for Gaussian distribution and were expressed as medians with interquartile ranges (IQR). Numerical data were described as a number with a percentage. Paired data were compared using the Wilcoxon signed rank test. The Mann-Whitney U test was performed for comparisons of biomarker concentrations between subgroups of patients. Fisher's exact test was used for comparison of proportions of patients. To assess the correlation between the per-patient trans-cardiac CRP and albumin gradients we calculated the Pearson correlation coefficient. A p-value less than 0.05 was considered statistically significant. All analyses were performed using IBM SPSS Statistics software (version 22.0, IBM Corp., Armonk, NY, USA).

      3. Results

      The study population for the current analysis consists of 42 patients (Supplementary Figure 1). Baseline clinical and angiographic characteristics are shown in Table 1. The median duration of symptoms was 236 (interquartile range: 99–485) minutes, with a median duration of symptom onset to the start of CAG of 25 (19–35) hours. The median baseline peripheral venous concentration of hs-cTnT was 175 (91–314) ng/L, which increased to a median peak concentration of 229 (131–398) ng/L.
      Table 1Baseline clinical and angiographic characteristics.
      All patients n = 42
      Age at admission65 (53–73)
      Gender (male)32 (76%)
      Smoking22 (52%)
      Hypertension21 (50%)
      Diabetes mellitus4 (10%)
      Aspirin therapy
      Every patient received a loading dose of aspirin.
      14 (33%)
      Statin therapy13 (31%)
      History of MI9 (21%)
      History of coronary revascularisation4 (10%)
      hs-cTnT on admission (ng/L)175 (91–314)
      CK-MB on admission (ng/mL)7.13 (3.0–15.9)
      Time between onset complaints and CAG (hours)25 (19–35)
      Duration of anginal symptoms (min)236 (99–485)
      Number of diseased vessels
       115 (36%)
       219 (45%)
       38 (19%)
      Culprit artery
       RCA10 (24%)
       RCX16 (38%)
       LAD16 (38%)
      Severity culprit stenosis
       50–70%0 (0%)
       70–90%20 (48%)
       >90%22 (52%)
      PCI performed27 (64%)
      Values are medians (interquartile range) or numbers (percentage).
      MI: myocardial infarction. hs-cTnT: high-sensitivity cardiac troponin T. CK-MB: creatinin kinase myocardial brain. URL: upper reference limit. CAG: coronary angiography. RCA: right coronary artery. RCX: ramus circumflex artery. LAD: left anterior descending artery.
      a Every patient received a loading dose of aspirin.

      3.1 Peripheral CRP concentrations

      The CRP concentrations at baseline showed no significant difference between the arterial and venous samples (4.88 [2.35–10.42] mg/L vs. 4.97 [2.29–10.16] mg/L; p = 0.164). The median peripheral venous CRP concentration increased to 5.35 [2.62–13.11] mg/L (p < 0.001) at 6 h and to 6.87 [3.32–12.10] mg/L (p < 0.001) at 12 h after the baseline measurements. At baseline, patients with a large MI had non-significantly higher CRP concentrations compared to small MI patients (7.52 [3.44–41.26] mg/L vs. 5.22 [2.06–11.9] mg/L; p = 0.170).

      3.2 Trans-cardiac gradients

      CRP concentrations in the CS and systemic circulation are depicted in Table 2. A decrease in median CRP concentration was observed between systemic arterial and CS blood (4.88 mg/L [2.35–10.42] versus 4.44 mg/L [2.14–9.42]; p < 0.001) (see Supplementary Materials for the individual CRP concentrations). No difference in trans-cardiac gradient was observed between statin users and statin naive patients and between aspirin users and aspirin naive patients. In addition, we assessed trans-cardiac albumin gradients to ascertain whether sample dilution might have affected the results. Although a median trans-cardiac albumin decrease of 4.8% was observed, there was no correlation with the trans-cardiac CRP gradient (correlation coefficient 0.044; p = 0.783). In the patients who showed both a trans-cardiac CRP decrease and a trans-cardiac albumin decrease, no correlation was observed between the per-patient trans-cardiac CRP and albumin gradients (correlation coefficient 0.028; p = 0.895).
      Table 2CRP concentrations in systemic artery, coronary sinus and great cardiac vein.
      Systemic arteryCSp-valueaGCVp-valueb
      All patients (n = 42)4.88 (2.35–10.42)4.44 (2.14–9.42)<0.0014.43 (2.23–9.50)<0.001
      LAD (n = 16)4.87 (1.33–12.25)4.65 (1.36–10.87)0.0034.80 (1.45–9.60)0.134
      Non-LAD (n = 26)4.88 (2.53–10.42)4.44 (2.31–9.30)0.0024.28 (2.39–9.65)0.003
      Concentrations of CRP depicted as medians (IQR) in mg/l. a: comparison between peripheral artery and CS. b: comparison between peripheral artery and GCV.
      CS: coronary sinus. GCV: great cardiac vein.LAD: left anterior descending culprit artery. Non-LAD: right coronary artery or ramus circumflex culprit artery.
      In Fig. 2, the relative changes in CRP concentration are depicted in the dark grey boxes in relation to time-interval between symptom onset and blood sampling (2A), infarct size based on peak CK-MB concentration (2B), the number of diseased vessels involved (2C), and the location of the culprit lesion (2D). The observed relative trans-cardiac decrease in CRP concentration in the overall population was present in all subgroups. Of the 21 patients with a large MI, five (24%) had a trans-cardiac increase in CRP concentration. Two of the 21 patients (10%) with a small MI had a trans-cardiac increase (p = 0.410).
      Fig. 2
      Fig. 2Trans-cardiac gradients of CRP according to subgroups.
      Median percentage relative change in CRP with interquartile range. *Significant gradients. (A) Early vs. late presentation: based on time interval between symptom onset and blood sampling: early (lower 50th percentile) and late (upper 50th percentile). (B) Small MI vs. large MI: patients divided based on peak CK-MB infarct size in small MI (lower 50th percentile) and large MI (upper 50th percentile). (C) Single vs. multi-vessel disease: patients divided based on the coronary atherosclerotic burden in single vessel disease (one coronary artery with stenosis >50%) or multi vessel disease (>1 coronary artery with stenosis >50%). (D) LAD vs. non-LAD: patients divided based on the location of their culprit artery in LAD or non-LAD. Gradient - to coronary sinus: median relative change between systemic arterial and coronary sinus sample. Gradient - to GCV: median relative change between systemic arterial and great cardiac vein sample. hs-CRP: high sensitivity C-reactive protein. GCV: great cardiac vein. MI: myocardial infarction. LAD: left anterior descending artery.

      3.3 Culprit lesion

      In a selected group of 14 patients, blood was obtained from the culprit coronary artery distal to the culprit lesion. No significant difference was observed in CRP concentration between the systemic arterial and post-stenotic compartment sample (4.59 mg/L [2.07–7.73] versus 4.56 mg/L [2.07–7.90], p = 0.278).

      3.4 Selective sampling of the great cardiac vein

      Patients were categorised according to the infarct related artery, LAD (n = 16) versus non-LAD (n = 26; RCA or RCX). As the GCV selectively drains blood from the anteroseptal wall, measurements of CRP derived from the GCV represent a trans-cardiac gradient across ‘injured’ myocardium for patients with a culprit in the LAD. For patients in the non-LAD group, the measurements reflect a gradient across ‘non-injured’ myocardium. Concentrations of CRP in the GCV are depicted in Table 2.
      There was a decrease in CRP concentration towards the GCV, which was only significant in patients with a non-LAD culprit lesion. Relative changes in CRP towards the GCV are depicted in Fig. 2D (light grey boxes).

      4. Discussion

      In this elaborate multi-site coronary sampling study in patients presenting with an NSTEMI, we showed a significant trans-cardiac decrease in CRP from the systemic circulation to the coronary venous system. At the same time, CRP concentrations across the culprit lesion were not different from those measured in the systemic circulation. Our in vivo finding of a trans-cardiac decrease are in line with ex vivo observation of CRP deposition, and suggest a net uptake at the level of the myocardial tissue.
      In general, systemic CRP concentrations are known to increase after MI [
      • Ferraro S.
      • Ardoino I.
      • Boracchi P.
      • Santagostino M.
      • Ciardi L.
      • Antonini G.
      • Braga F.
      • Biganzoli E.
      • Panteghini M.
      • Bongo A.S.
      Inside ST-elevation myocardial infarction by monitoring concentrations of cardiovascular risk biomarkers in blood.
      ]. This corroborates with our CRP concentrations six and 12 h after the CAG. In view of this, the overall response to myocyte injury in the setting of NSTEMI is that of CRP production. Besides hepatic secretion, several studies also suggested a role for the coronary plaque as production site for CRP. This was based on an in vitro study that demonstrated CRP production by coronary artery smooth muscle cells [
      • Calabro P.
      • Willerson J.T.
      • Yeh E.T.
      Inflammatory cytokines stimulated C-reactive protein production by human coronary artery smooth muscle cells.
      ]. Moreover, an ex vivo experiment showed the presence of CRP mRNA in culprit plaques from patients with unstable coronary artery disease [
      • Forte L.
      • Cimmino G.
      • Loffredo F.
      • De Palma R.
      • Abbate G.
      • Calabro P.
      • Ingrosso D.
      • Galletti P.
      • Carangio C.
      • Casillo B.
      • Calabro R.
      • Golino P.
      C-reactive protein is released in the coronary circulation and causes endothelial dysfunction in patients with acute coronary syndromes.
      ]. In accordance with this, it has also been shown that CRP concentrations increased across the culprit lesion in a group of patients with unstable angina [
      • Inoue T.
      • Kato T.
      • Uchida T.
      • Sakuma M.
      • Nakajima A.
      • Shibazaki M.
      • Imoto Y.
      • Saito M.
      • Hashimoto S.
      • Hikichi Y.
      • Node K.
      Local release of C-reactive protein from vulnerable plaque or coronary arterial wall injured by stenting.
      ], and across the coronary circulation (aorta to CS) in another group of patients [
      • Forte L.
      • Cimmino G.
      • Loffredo F.
      • De Palma R.
      • Abbate G.
      • Calabro P.
      • Ingrosso D.
      • Galletti P.
      • Carangio C.
      • Casillo B.
      • Calabro R.
      • Golino P.
      C-reactive protein is released in the coronary circulation and causes endothelial dysfunction in patients with acute coronary syndromes.
      ,
      • Cimmino G.
      • Ragni M.
      • Cirillo P.
      • Petrillo G.
      • Loffredo F.
      • Chiariello M.
      • Gresele P.
      • Falcinelli E.
      • Golino P.
      C-reactive protein induces expression of matrix metalloproteinase-9: a possible link between inflammation and plaque rupture.
      ]. Based on these observations, it has been suggested that there is local vascular production in addition to systemic production. Interestingly, several other studies investigating trans-lesional gradients did not reproduce an increase of CRP across the culprit lesion and disputed the finding of local vascular production [
      • Fong S.W.
      • Few L.L.
      • See Too W.C.
      • Khoo B.Y.
      • Nik Ibrahim N.N.
      • Yahaya S.A.
      • Yusof Z.
      • Mohd Ali R.
      • Abdul Rahman A.R.
      • Yvonne-Tee G.B.
      Systemic and coronary levels of CRP, MPO, sCD40L and PlGF in patients with coronary artery disease.
      ,
      • Kirbis S.
      • Breskvar U.D.
      • Sabovic M.
      • Zupan I.
      • Sinkovic A.
      Inflammation markers in patients with coronary artery disease--comparison of intracoronary and systemic levels.
      ,
      • Aggarwal A.
      • Schneider D.J.
      • Terrien E.F.
      • Sobel B.E.
      • Dauerman H.L.
      Increased coronary arterial release of interleukin-1 receptor antagonist and soluble CD40 ligand indicative of inflammation associated with culprit coronary plaques.
      ]. Our study, in which a trans-lesional CRP gradient could not be shown, corroborates with these findings. Additionally, our analyses on the burden of atherosclerotic disease - as expressed by single versus multivessel disease - had no influence on the observed trans-cardiac CRP decrease (Fig. 2). This suggests that the trans-cardiac gradient we observed is mainly driven at the level of the myocardium. Interestingly, and in contrast to the multi-site sampling procedures in our study, the majority of studies on CRP gradients did not simultaneously assess both trans-lesional and trans-cardiac gradients, which complicates the integration of the available study data into a more comprehensive concept. Moreover, it is important to note that, in contrast to cardiac production, there have also been reports on myocardial CRP uptake in the setting of myocardial injury. In histopathological studies on myocardial tissue of patients who suffered from fatal ST-elevation MI, it has consistently been shown that CRP was deposited in the infarcted parts of the myocardium [
      • Lagrand W.K.
      • Niessen H.W.
      • Wolbink G.J.
      • Jaspars L.H.
      • Visser C.A.
      • Verheugt F.W.
      • Meijer C.J.
      • Hack C.E.
      C-reactive protein colocalizes with complement in human hearts during acute myocardial infarction.
      ,
      • Nijmeijer R.
      • Lagrand W.K.
      • Lubbers Y.T.P.
      • Visser C.A.
      • Meijer C.J.L.M.
      • Niessen H.W.M.
      • Hack C.E.
      C-reactive protein activates complement in infarcted human myocardium.
      ,
      • Kushner I.
      • Rakita L.
      • Kaplan M.H.
      Studies of acute-phase protein. II. Localization of Cx-reactive protein in heart in induced myocardial infarction in rabbits.
      ].
      Intriguingly, we are the first to have observed a clear trans-cardiac decrease in an in vivo setting of NSTEMI patients, which suggests a net myocardial CRP uptake. From previous reports on the function of CRP, it is well known that CRP a-specifically binds to injured tissue after exposure of phosphocholine groups, which appear on the cell membrane of necrotic cells. After binding, CRP activates the complement system promoting opsonisation [
      • Du Clos T.W.
      Function of C-reactive protein.
      ,
      • Agrawal A.
      • Gang T.B.
      • Rusinol A.E.
      Recognition functions of pentameric C-reactive protein in cardiovascular disease.
      ], and therefore acts as an important pro-inflammatory mediator [
      • Nijmeijer R.
      • Lagrand W.K.
      • Lubbers Y.T.P.
      • Visser C.A.
      • Meijer C.J.L.M.
      • Niessen H.W.M.
      • Hack C.E.
      C-reactive protein activates complement in infarcted human myocardium.
      ]. CRP provides an early defence mechanism through rapid activation of the innate immune system. By attracting other inflammatory cytokines, it ultimately plays a role as an initial step in the healing process of injured tissues [
      • Du Clos T.W.
      Function of C-reactive protein.
      ]. Given the extra-cardiac production in the presence of MI, and appreciating the physiologic function of CRP, binding to damaged myocardial cells is a plausible explanation for the observed trans-cardiac decrease in CRP concentrations across the myocardium in the present study.
      In contrast to our findings, the majority of studies on trans-cardiac gradients found no difference, and two reported an increase in CRP across the heart [
      • Inoue T.
      • Kato T.
      • Uchida T.
      • Sakuma M.
      • Nakajima A.
      • Shibazaki M.
      • Imoto Y.
      • Saito M.
      • Hashimoto S.
      • Hikichi Y.
      • Node K.
      Local release of C-reactive protein from vulnerable plaque or coronary arterial wall injured by stenting.
      ,
      • Forte L.
      • Cimmino G.
      • Loffredo F.
      • De Palma R.
      • Abbate G.
      • Calabro P.
      • Ingrosso D.
      • Galletti P.
      • Carangio C.
      • Casillo B.
      • Calabro R.
      • Golino P.
      C-reactive protein is released in the coronary circulation and causes endothelial dysfunction in patients with acute coronary syndromes.
      ,
      • Cimmino G.
      • Ragni M.
      • Cirillo P.
      • Petrillo G.
      • Loffredo F.
      • Chiariello M.
      • Gresele P.
      • Falcinelli E.
      • Golino P.
      C-reactive protein induces expression of matrix metalloproteinase-9: a possible link between inflammation and plaque rupture.
      ,
      • Taylor A.J.
      • Bobik A.
      • Richards M.
      • Kaye D.
      • Raines G.
      • Gould P.
      • Jennings G.
      Myocardial endothelin-1 release and indices of inflammation during angioplasty for acute myocardial infarction and stable coronary artery disease.
      ,
      • Leite W.F.
      • Ramires J.A.
      • Moreira L.F.
      • Strunz C.M.
      • Mangione J.A.
      Correlation between C-reactive protein in peripheral vein and coronary sinus in stable and unstable angina.
      ,
      • Wang Y.
      • Li L.
      • Tan H.W.
      • Yu G.S.
      • Ma Z.Y.
      • Zhao Y.X.
      • Zhang Y.
      Transcoronary concentration gradient of sCD40L and hsCRP in patients with coronary heart disease.
      ]. Appreciating that the reported group effect is a composite of observed individual increases and decreases, we hypothesised that differences in infarct size between studies may have contributed to the discrepant findings. Especially, given the previous evidence that the presence or absence of myocardial injury has an effect on trans-cardiac gradients of inflammatory markers [
      • Cusack M.R.
      • Marber M.S.
      • Lambiase P.D.
      • Bucknall C.A.
      • Redwood S.R.
      Systemic inflammation in unstable angina is the result of myocardial necrosis.
      ,
      • Ohashi Y.
      • Kawashima S.
      • Mori T.
      • Terashima M.
      • Ichikawa S.
      • Ejiri J.
      • Awano K.
      Soluble CD40 ligand and interleukin-6 in the coronary circulation after acute myocardial infarction.
      ]. With regard to the impact of the extent of myocardial injury on our observed trans-cardiac gradient, we compared patients with a larger-sized MI to those with a smaller-sized MI according to CK-MB peak concentrations. On average, we observed a decrease in CRP concentration from the systemic circulation to the CS for both groups, irrespective of whether infarct size was based on peak CK-MB, baseline or peak troponin levels (latter two not reported). Interestingly, in a few patients we saw a trans-cardiac increase in CRP. Most of these patients were among those with a larger-sized MI. From this we contemplate that in a different population, represented by patients with larger-sized MIs, trans-cardiac gradients might show net increases in CRP concentration more often. Although speculative, this could be one of the explainations for the non-uniformity of available evidence in ACS [
      • Forte L.
      • Cimmino G.
      • Loffredo F.
      • De Palma R.
      • Abbate G.
      • Calabro P.
      • Ingrosso D.
      • Galletti P.
      • Carangio C.
      • Casillo B.
      • Calabro R.
      • Golino P.
      C-reactive protein is released in the coronary circulation and causes endothelial dysfunction in patients with acute coronary syndromes.
      ,
      • Cimmino G.
      • Ragni M.
      • Cirillo P.
      • Petrillo G.
      • Loffredo F.
      • Chiariello M.
      • Gresele P.
      • Falcinelli E.
      • Golino P.
      C-reactive protein induces expression of matrix metalloproteinase-9: a possible link between inflammation and plaque rupture.
      ]. Unfortunately, the majority of previous studies did not report on infarct size and the impact on the trans-cardiac CRP gradients. The only study in acute ST elevation MI patients, to our knowledge, that performed CRP measurements in CS blood did not observe a trans-cardiac gradient [
      • Taylor A.J.
      • Bobik A.
      • Richards M.
      • Kaye D.
      • Raines G.
      • Gould P.
      • Jennings G.
      Myocardial endothelin-1 release and indices of inflammation during angioplasty for acute myocardial infarction and stable coronary artery disease.
      ]. Notably, the median time-interval between symptom onset and study procedures was only 3.2 ± 0.5 h, whereas in our study it was 25 [19–35] hours. Importantly, ex vivo examinations of STEMI patients have shown that the process of CRP uptake occurs between 12 h and 5 days after symptom onset. This time-interval is considered the phase of polymorphonuclear leukocyte infiltration [
      • Lagrand W.K.
      • Niessen H.W.
      • Wolbink G.J.
      • Jaspars L.H.
      • Visser C.A.
      • Verheugt F.W.
      • Meijer C.J.
      • Hack C.E.
      C-reactive protein colocalizes with complement in human hearts during acute myocardial infarction.
      ,
      • Nijmeijer R.
      • Lagrand W.K.
      • Lubbers Y.T.P.
      • Visser C.A.
      • Meijer C.J.L.M.
      • Niessen H.W.M.
      • Hack C.E.
      C-reactive protein activates complement in infarcted human myocardium.
      ]. Probably, and in contrast to our findings, blood sampling was too early to result in a measurable amount of CRP uptake in this study of acute MI patients. Given the aforementioned aspects, future research investigating inflammatory gradients in MI patients should adequately address infarct size and timing between symptom onset and blood sampling.
      In contrast to previous studies, our sampling protocol allowed for determination of CRP changes across injured and non-injured myocardium, and we found a decrease in CRP in both subgroups. This demonstrates that non infarcted myocardium is involved in the CRP physiology as well. In support of this observation is an imaging study that showed that high increases in CRP concentration after suffering MI resulted in larger adjacent peri-infarct zones [
      • Quinaglia e Silva J.C.
      • Coelho-Filho O.R.
      • Andrade J.M.
      • Quinaglia T.
      • Modolo R.G.
      • Almeida B.O.
      • van der Geest R.J.
      • Jerosch-Herold M.
      • Coelho O.R.
      • Sposito A.C.
      • Brasilia Heart Study G.
      Peri-infarct zone characterized by cardiac magnetic resonance imaging is directly associated with the inflammatory activity during acute phase myocardial infarction.
      ]. Interestingly, in a murine study of ischemia and reperfusion, the impact of CRP injections was studied in a controlled design. In the group with injections of CRP, higher rates of apoptosis were not confined to the infarcted part of the myocardium but included the entire area at risk [
      • Oh S.J.
      • Na Kim E.
      • Jai Kim C.
      • Choi J.S.
      • Kim K.B.
      The effect of C-reactive protein deposition on myocardium with ischaemia-reperfusion injury in rats.
      ]. In addition, in a transgenic mouse model of MI, there was increased macrophage infiltration and apoptosis in the border zones of the infarcted myocardium in the mice with CRP expression [
      • Takahashi T.
      • Anzai T.
      • Kaneko H.
      • Mano Y.
      • Anzai A.
      • Nagai T.
      • Kohno T.
      • Maekawa Y.
      • Yoshikawa T.
      • Fukuda K.
      • Ogawa S.
      Increased C-reactive protein expression exacerbates left ventricular dysfunction and remodeling after myocardial infarction.
      ]. Importantly, an adverse effect on left ventricular function recovery was observed in the mice with versus without CRP expression, despite equal infarct sizes [
      • Takahashi T.
      • Anzai T.
      • Kaneko H.
      • Mano Y.
      • Anzai A.
      • Nagai T.
      • Kohno T.
      • Maekawa Y.
      • Yoshikawa T.
      • Fukuda K.
      • Ogawa S.
      Increased C-reactive protein expression exacerbates left ventricular dysfunction and remodeling after myocardial infarction.
      ]. Therefore, our observation of a decrease in CRP over non-injured myocardium, might be the first in vivo proof of a pathobiological role for CRP in non-infarcted myocardium.

      4.1 Implications

      Our observations support the hypothesis that CRP binds to myocardial cells in the setting of a NSTEMI. This opens new insights in the role of inflammation in the aftermath of MI. New drugs targeting upstream biomarkers, interacting with the interleukin-CRP axis, are investigated in patients with stable cardiovascular disease (CIRT trial and CANTOS trial) [
      • Everett B.M.
      • Pradhan A.D.
      • Solomon D.H.
      • Paynter N.
      • Macfadyen J.
      • Zaharris E.
      • Gupta M.
      • Clearfield M.
      • Libby P.
      • Hasan A.A.
      • Glynn R.J.
      • Ridker P.M.
      Rationale and design of the Cardiovascular Inflammation Reduction Trial: a test of the inflammatory hypothesis of atherothrombosis.
      ,
      • Ridker P.M.
      • Thuren T.
      • Zalewski A.
      • Libby P.
      Interleukin-1beta inhibition and the prevention of recurrent cardiovascular events: rationale and design of the Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS).
      ,
      • Ridker P.M.
      • Everett B.M.
      • Thuren T.
      • MacFadyen J.G.
      • Chang W.H.
      • Ballantyne C.
      • Fonseca F.
      • Nicolau J.
      • Koenig W.
      • Anker S.D.
      • Kastelein J.J.P.
      • Cornel J.H.
      • Pais P.
      • Pella D.
      • Genest J.
      • Cifkova R.
      • Lorenzatti A.
      • Forster T.
      • Kobalava Z.
      • Vida-Simiti L.
      • Flather M.
      • Shimokawa H.
      • Ogawa H.
      • Dellborg M.
      • Rossi P.R.F.
      • Troquay R.P.T.
      • Libby P.
      • Glynn R.J.
      • Group C.T.
      Antiinflammatory therapy with canakinumab for atherosclerotic disease.
      ]. Their target is to slow down the progression of coronary artery disease. In view of the potential roles of CRP in LV remodelling after MI, it remains to be answered whether and how these drugs might interfere with infarct healing. Interestingly, it was shown that the IL-6 receptor antagonist tocolizumab reduced the levels of CRP, and cardiac troponin release after NSTEMI [
      • Kleveland O.
      • Kunszt G.
      • Bratlie M.
      • Ueland T.
      • Broch K.
      • Holte E.
      • Michelsen A.E.
      • Bendz B.
      • Amundsen B.H.
      • Espevik T.
      • Aakhus S.
      • Damas J.K.
      • Aukrust P.
      • Wiseth R.
      • Gullestad L.
      Effect of a single dose of the interleukin-6 receptor antagonist tocilizumab on inflammation and troponin T release in patients with non-ST-elevation myocardial infarction: a double-blind, randomized, placebo-controlled phase 2 trial.
      ]. Notably, together with our findings of a trans-cardiac CRP decrease across the myocardium in the acute phase of myocardial infarction, the concept that more insight in the role of inflammatory processes after ACS might provide novel therapeutic targets is strongly supported [
      • Ridker P.M.
      From C-reactive protein to Interleukin-6 to Interleukin-1: moving upstream to identify novel targets for atheroprotection.
      ,
      • Sager H.B.
      • Heidt T.
      • Hulsmans M.
      • Dutta P.
      • Courties G.
      • Sebas M.
      • Wojtkiewicz G.R.
      • Tricot B.
      • Iwamoto Y.
      • Sun Y.
      • Weissleder R.
      • Libby P.
      • Swirski F.K.
      • Nahrendorf M.
      Targeting Interleukin-1beta reduces leukocyte production after acute myocardial infarction.
      ].

      4.2 Limitations

      Appreciating the mechanistic design of the study, our sample size is limited and therefore results on trans-lesional findings, subgroup analyses, and correction for confounders may have been hampered by lack of statistical power. The present study question concerns a prospectively defined secondary analysis of the TRAMICI protocol, and the primary outcome measure was the within patient difference in the trans-cardiac gradient of CRP. In retrospect, incorporation of more upstream inflammatory biomarkers (e.g. IL-1 and IL-6) would have been interesting to gain additional information on the pathobiology of inflammatory processes in acute myocardial infarction. The observed absence of a trans-lesional CRP difference might have been caused by local catabolism or uptake of CRP in the vessel wall [
      • Maier W.
      • Altwegg L.A.
      • Corti R.
      • Gay S.
      • Hersberger M.
      • Maly F.E.
      • Sutsch G.
      • Roffi M.
      • Neidhart M.
      • Eberli F.R.
      • Tanner F.C.
      • Gobbi S.
      • von Eckardstein A.
      • Luscher T.F.
      Inflammatory markers at the site of ruptured plaque in acute myocardial infarction: locally increased interleukin-6 and serum amyloid A but decreased C-reactive protein.
      ]. However, the CRP assay used has high-sensitivity characteristics. Thus, small differences were detectable, even in the setting of relatively high systemic production. Despite several protocol driven precautions, it can not be ruled out completely that dilution may have affected the observed trans-cardiac decrease in CRP in some cases, to some extent. However, prior to actual sampling, the protocol not only dictated a temporal halt, to allow for sufficient blood reflux; it also incorporated a first sample of 3–4 mL for blood gas analysis, before the sample of interest was obtained. To further address this issue, we performed analyses on other plasma proteins, i.e. albumin. In the absence of a per-patient correlation between the trans-cardiac gradients of albumin and CRP, it is unlikely that sample diluton markedly affected our findings. With regard to possible confounders, none were identified for the trans-cardiac CRP gradient. In addition, no difference in trans-cardiac gradient was observed for patients on long-term aspirin and statin use. Finally, our study lacks a control group of patients without evidence of MI. Appreciating the different studies reporting no CRP gradient in patients with stable angina [
      • Kirbis S.
      • Breskvar U.D.
      • Sabovic M.
      • Zupan I.
      • Sinkovic A.
      Inflammation markers in patients with coronary artery disease--comparison of intracoronary and systemic levels.
      ,
      • Forte L.
      • Cimmino G.
      • Loffredo F.
      • De Palma R.
      • Abbate G.
      • Calabro P.
      • Ingrosso D.
      • Galletti P.
      • Carangio C.
      • Casillo B.
      • Calabro R.
      • Golino P.
      C-reactive protein is released in the coronary circulation and causes endothelial dysfunction in patients with acute coronary syndromes.
      ,
      • Cimmino G.
      • Ragni M.
      • Cirillo P.
      • Petrillo G.
      • Loffredo F.
      • Chiariello M.
      • Gresele P.
      • Falcinelli E.
      • Golino P.
      C-reactive protein induces expression of matrix metalloproteinase-9: a possible link between inflammation and plaque rupture.
      ,
      • Leite W.F.
      • Ramires J.A.
      • Moreira L.F.
      • Strunz C.M.
      • Mangione J.A.
      Correlation between C-reactive protein in peripheral vein and coronary sinus in stable and unstable angina.
      ], and the expected additional clinical/scientific benefit in this particular group of patients, we decided not to expose these patients to an invasive, rather extended procedure with an elaborate sampling protocol.

      4.3 Conclusion

      In the present population of patients with ongoing NSTEMI, we observed a net trans-cardiac CRP decrease in conjunction with increases of CRP in the systemic circulation over time, without evidence of local production at the site of the culprit coronary lesion suggesting myocardial uptake of CRP. Due to elaborate blood sampling from both the systemic and cardiac circulation, we were able to observe that CRP uptake was not limited to the infarcted myocardium but was also prominent in regions adjacent to injured myocardium.

      Conflicts of interest

      The authors declared they do not have anything to disclose regarding conflict of interest with respect to this manuscript.

      Financial support

      The laboratory assays were kindly provided to use by Roche diagnostics.

      Acknowledgements

      We would like to thank Rein van der Sluis, lab technician of the Isala Clinics, for the analysis of the blood samples. We would like to thank Joris Nas M.D. for critically revising the manuscript.

      Appendix A. Supplementary data

      The following are the supplementary data to this article:
      supplementary1
      vessel pairs individual2

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