2.1

Study population and study design

This prospective cohort study was performed in STEMI patients of the RESPONSE trial , who underwent treatment by PPCI for acute STEMI (≤12 h after symptom onset) and non-invasive assessment of endothelial function with the RH-PAT method after 4 to 6 weeks at Thoraxcentrum Twente in Enschede. PPCIs were performed between October 2007 and December 2008. Of a total of 75 RESPONSE trial participants at Thoraxcentrum Twente, who were treated for STEMI and underwent RH-PAT measurements, 71 had analyzable RH-PAT registrations and 68 patients had laboratory measurements of hs-CRP and LpPLA2. This resulted in a study population of 68 patients.

Details of the randomized RESPONSE trial have previously been reported . In brief, patients had to be 18 to 80 years, without surgery or additional PCI being planned within 8 weeks from PPCI, without congestive heart failure New York Heart Association (NYHA) class III or IV, and with a life expectancy of at least 2 years.

As inflammation and repair processes of the infarcted myocardium might have disturbed endothelial function measurements during the first weeks after the STEMI and endothelial dysfunction had not been fully recovered under medication, endothelial function was assessed 4 to 6 weeks after the PPCI . All patients were seen in the outpatient clinic and the research department of Thoraxcentrum Twente, where non-invasive assessment of the endothelial function was performed according to strict rules in a dedicated laboratory .

All patients provided written informed consent for both participation in the RESPONSE multicenter trial and the present single-center substudy. Trial and substudy complied with the Declaration of Helsinki for investigation in human beings and were approved by accredited Medical Ethical Committees in Amsterdam and Twente, the Netherlands.

2.2

Patient characteristics and follow-up

The following information was collected through interviews during visits in our outpatient clinic 4 to 6 weeks after STEMI: age; sex; body mass index (kg/m ² ); arterial hypertension (blood pressure >140/90 mmHg or treatment with anti-hypertensive medication); history of smoking (previous or current smoker); history of previous myocardial infarction or percutaneous coronary intervention (PCI); presence of diabetes mellitus (patient history and/or treatment with insulin or oral anti-glycemic agents); and history of hyperlipidemia or treatment with lipid-lowering drugs. The Framingham Risk Score (10-year risk prediction for fatal and non-fatal coronary heart disease) was determined to sketch a patient risk profile.

2.3

Laboratory assessment of the biochemical markers

Venous blood samples were drawn after a minimum of 8 h of fasting. The laboratory markers total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, glucose, hemoglobin A1c, creatinine, fibrinogen, apolipoprotein A1 & B100, troponin I and N-terminal pro-B-type natriuretic peptide (NT-proBNP) were assessed according to manufacturer instructions. After centrifugation, plasma and serum aliquots were stored at −80 °C. A commercially available Lp-PLA ₂ enzyme-linked immunosorbent assay kit (third generation PLAC™ Test; diaDexus Inc., South San Francisco, CA) was used to determine the lipoprotein-associated phospholipase A ₂ mass. The inter-assay precision was determined by measuring two controls of known concentration (low and high) in 10 separate assays. The coefficient of variation on all 10 plates was 7% and 6%, respectively. The hs-CRP concentrations were determined by nephelometry using a BN ProSpec® system (Siemens). The detection limit was 0.23 mg/L and the mean inter-assay coefficient of variation was 2.8%.

The thresholds of the hs-CRP tertiles were determined at 1.03 mg/L and 3.84 mg/L; the thresholds of the Lp-PLA ₂ tertiles were 190 μg/L and 239 μg/L comparable to AHA/CDC guidelines and ARIC study .

2.4

Assessment of endothelial function

Endothelial function was evaluated with the RH-PAT method. The finger pulse wave amplitude was assessed with the EndoPAT-2000 sensing device and finger plethysmographic probes (Itamar Medical, Caesarea, Israel), both at baseline and during ischemia-induced hyperemia. All measurements were performed in the early morning in a dedicated laboratory after fasting for at least 8 h. Patients also had to refrain from caffeine consumption, smoking, and vasoactive medications. At least 15 min prior to testing, blood pressure was measured and a blood sample was drawn in the control arm. Before any measurement, patients had an acclimatization period of 20 min in a quiet room, lying in a hospital bed at an ambient temperature of 21 to 23 °C.

The RH-PAT method has previously been reported in detail . In brief, measurements were performed by the use of probes on index fingers of both the study and control arm. Baseline measurements were recorded for 5 min prior to inducing ischemia by inflating a blood pressure cuff on the upper arm of the study arm for 5 min to supra-systolic pressures. This led to NO release from functional endothelium and thus vasodilatation, which was recorded by the sensors in the finger cuff through beat-to-beat finger pulse wave analysis . Following the release of the blood pressure cuff, the ratio of the pulse amplitude of the hyperemic finger and the baseline amplitude was calculated. Subsequently, that ratio was divided by the corresponding ratio, obtained in the control arm, to calculate the RH-PAT index (high values indicate good endothelial function) . Hamburg et al. demonstrated that the maximum hyperemic response can be expected 90 to 120 s after cuff deflation . Therefore, in the present study, the reactive RH-PAT index was calculated as the ratio of the mean hyperemic pulse wave analysis over a period of 30 s, beginning at 90 s after cuff deflation, divided by the baseline pulse wave analysis (mean baseline measurements for 3.5 min), and normalized to the concurrent measurements of the control arm. Based on previously reported data, endothelial function was divided into two groups: endothelial dysfunction (RH-PAT <1.67) and normal endothelial function (RH-PAT ≥1.67) .

2.5

Statistical analysis

Data are presented as frequencies (%) or mean ± standard deviation (SD). We used the Fisher’s exact chi-square test for categorical variables and the T-test for continuous variables to compare patients with high risk versus patients with low-to-intermediate risk. A p -value <0.05 was considered statistically significant. Analyses were performed with SPSS (version 21.0).

2.1

Study population and study design

This prospective cohort study was performed in STEMI patients of the RESPONSE trial , who underwent treatment by PPCI for acute STEMI (≤12 h after symptom onset) and non-invasive assessment of endothelial function with the RH-PAT method after 4 to 6 weeks at Thoraxcentrum Twente in Enschede. PPCIs were performed between October 2007 and December 2008. Of a total of 75 RESPONSE trial participants at Thoraxcentrum Twente, who were treated for STEMI and underwent RH-PAT measurements, 71 had analyzable RH-PAT registrations and 68 patients had laboratory measurements of hs-CRP and LpPLA2. This resulted in a study population of 68 patients.

Details of the randomized RESPONSE trial have previously been reported . In brief, patients had to be 18 to 80 years, without surgery or additional PCI being planned within 8 weeks from PPCI, without congestive heart failure New York Heart Association (NYHA) class III or IV, and with a life expectancy of at least 2 years.

As inflammation and repair processes of the infarcted myocardium might have disturbed endothelial function measurements during the first weeks after the STEMI and endothelial dysfunction had not been fully recovered under medication, endothelial function was assessed 4 to 6 weeks after the PPCI . All patients were seen in the outpatient clinic and the research department of Thoraxcentrum Twente, where non-invasive assessment of the endothelial function was performed according to strict rules in a dedicated laboratory .

All patients provided written informed consent for both participation in the RESPONSE multicenter trial and the present single-center substudy. Trial and substudy complied with the Declaration of Helsinki for investigation in human beings and were approved by accredited Medical Ethical Committees in Amsterdam and Twente, the Netherlands.

2.2

Patient characteristics and follow-up

The following information was collected through interviews during visits in our outpatient clinic 4 to 6 weeks after STEMI: age; sex; body mass index (kg/m ² ); arterial hypertension (blood pressure >140/90 mmHg or treatment with anti-hypertensive medication); history of smoking (previous or current smoker); history of previous myocardial infarction or percutaneous coronary intervention (PCI); presence of diabetes mellitus (patient history and/or treatment with insulin or oral anti-glycemic agents); and history of hyperlipidemia or treatment with lipid-lowering drugs. The Framingham Risk Score (10-year risk prediction for fatal and non-fatal coronary heart disease) was determined to sketch a patient risk profile.

2.3

Laboratory assessment of the biochemical markers

Venous blood samples were drawn after a minimum of 8 h of fasting. The laboratory markers total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, glucose, hemoglobin A1c, creatinine, fibrinogen, apolipoprotein A1 & B100, troponin I and N-terminal pro-B-type natriuretic peptide (NT-proBNP) were assessed according to manufacturer instructions. After centrifugation, plasma and serum aliquots were stored at −80 °C. A commercially available Lp-PLA ₂ enzyme-linked immunosorbent assay kit (third generation PLAC™ Test; diaDexus Inc., South San Francisco, CA) was used to determine the lipoprotein-associated phospholipase A ₂ mass. The inter-assay precision was determined by measuring two controls of known concentration (low and high) in 10 separate assays. The coefficient of variation on all 10 plates was 7% and 6%, respectively. The hs-CRP concentrations were determined by nephelometry using a BN ProSpec® system (Siemens). The detection limit was 0.23 mg/L and the mean inter-assay coefficient of variation was 2.8%.

The thresholds of the hs-CRP tertiles were determined at 1.03 mg/L and 3.84 mg/L; the thresholds of the Lp-PLA ₂ tertiles were 190 μg/L and 239 μg/L comparable to AHA/CDC guidelines and ARIC study .

2.4

Assessment of endothelial function

Endothelial function was evaluated with the RH-PAT method. The finger pulse wave amplitude was assessed with the EndoPAT-2000 sensing device and finger plethysmographic probes (Itamar Medical, Caesarea, Israel), both at baseline and during ischemia-induced hyperemia. All measurements were performed in the early morning in a dedicated laboratory after fasting for at least 8 h. Patients also had to refrain from caffeine consumption, smoking, and vasoactive medications. At least 15 min prior to testing, blood pressure was measured and a blood sample was drawn in the control arm. Before any measurement, patients had an acclimatization period of 20 min in a quiet room, lying in a hospital bed at an ambient temperature of 21 to 23 °C.

The RH-PAT method has previously been reported in detail . In brief, measurements were performed by the use of probes on index fingers of both the study and control arm. Baseline measurements were recorded for 5 min prior to inducing ischemia by inflating a blood pressure cuff on the upper arm of the study arm for 5 min to supra-systolic pressures. This led to NO release from functional endothelium and thus vasodilatation, which was recorded by the sensors in the finger cuff through beat-to-beat finger pulse wave analysis . Following the release of the blood pressure cuff, the ratio of the pulse amplitude of the hyperemic finger and the baseline amplitude was calculated. Subsequently, that ratio was divided by the corresponding ratio, obtained in the control arm, to calculate the RH-PAT index (high values indicate good endothelial function) . Hamburg et al. demonstrated that the maximum hyperemic response can be expected 90 to 120 s after cuff deflation . Therefore, in the present study, the reactive RH-PAT index was calculated as the ratio of the mean hyperemic pulse wave analysis over a period of 30 s, beginning at 90 s after cuff deflation, divided by the baseline pulse wave analysis (mean baseline measurements for 3.5 min), and normalized to the concurrent measurements of the control arm. Based on previously reported data, endothelial function was divided into two groups: endothelial dysfunction (RH-PAT <1.67) and normal endothelial function (RH-PAT ≥1.67) .

2.5

Statistical analysis

Data are presented as frequencies (%) or mean ± standard deviation (SD). We used the Fisher’s exact chi-square test for categorical variables and the T-test for continuous variables to compare patients with high risk versus patients with low-to-intermediate risk. A p -value <0.05 was considered statistically significant. Analyses were performed with SPSS (version 21.0).