Symptomatic patients with stable angina pectoris and a flow-limiting SVG stenosis (n=40) were recruited. The study was conducted in accordance with the ethical guidelines of the Declaration of Helsinki 1975, and the investigation was approved by the Institutional Review Board (GZ.: 07–3387) and registered at ClinicalTrials.gov (NCT01430884). All patients gave informed consent prior to their inclusion in the study. Using a position statement of the American Diabetes Association on Diagnosis and Classification of DM , patients were classified according to their hemoglobin (Hb)A1c-value and their use of antidiabetic medications (with DM: HbA1c ≥ 6.5% and with use of antidiabetic medications vs. without DM: HbA1c < 5.7% and without use of antidiabetic medications).
Quantitative coronary angiography
Patients were on aspirin (100 mg/day) and received 10.000 I.U. heparin intravenously. 17 patients each with and without DM were on clopidogrel (75 mg/day). Coronary angiography was performed using the femoral approach and 6F or 8F guiding catheters. Stenosis severity was quantified using off-line caliper measurements (QCA-MEDISR, Leiden, NL) , and thrombolysis in myocardial infarction (TIMI) flow was measured before and after stent implantation . Minimal lumen diameter and reference diameter were determined before, immediately after, and at follow-up 6 months after stent implantation, and the percent diameter stenosis was calculated.
IVUS and virtual histology (VH) analysis
IVUS was performed before and after stent implantation with a commercially available electronic IVUS catheter (Eagle-EyeTM 20 MHz catheter and R-100 pullback device, Volcano Corporation, Rancho Cordova, CA, USA). The site and the length of the target lesion before stent implantation were retrospectively identified after stent implantation from landmarks in the vascular profile [34, 35]. Plaque composition was categorized with VH using a customized software (pcVHTM2.1, Volcano Corp.). All detected plaque components (fibrotic, fibro-fatty, necrotic core, dense calcium) were presented as a fraction of total plaque volume (%) .
Implantation of balloon-expandable bare metal stents was performed with direct stenting without prior dilatation/debulking and a stent-to-vessel diameter ratio of 1:1.15, because stenting with predilatation eventually increases plaque mobilisation and debris embolism . To prevent microembolization, a distal balloon occlusion extraction device (GuardWireR Temporary Occlusion & Aspiration System; Medtronic Inc., Minneapolis, MN USA)  was used. Before stent implantation, the balloon of the device was inflated at 2 atm with contrast agent. After stent implantation, the catheter with the stent-balloon was removed, and the aspiration catheter was loaded onto the monorail GuardWireR. During slow withdrawal of the aspiration catheter, the blood column was retrieved. Then, the balloon was deflated. After PCI patients were loaded with 600 mg of clopidogrel and medication was continued at a dose of 75 mg/day for the next 4 weeks.
Coronary arterial blood and coronary aspirate
Coronary arterial blood was obtained through the respective aspiration catheter (10 mL into Heparin S-Monovette, SARSTEDT AG & Co, Nümbrecht, Germany) distal to the lesion before stent implantation and served as control. Coronary aspirate (between 10 and 20 mL) was filtered ex vivo through a 40 μm mesh filter. The aspirate dilution by contrast agent was corrected for by reference to the hematocrit. Visible particulate debris was retained on the filter and weighed.
The filtered coronary arterial and aspirate samples were immediately centrifuged (800 g, 10 min, 4°C). Both, particulate debris and plasma samples were quickly frozen in liquid nitrogen and stored at −80°C until further use.
Total calcium, vasoconstrictors, tissue factor, TNFα, C reactive protein (CRP), and troponin I
Total calcium concentration (sum of ionized and bound/complexed calcium) was measured in coronary arterial and aspirate plasma and in particulate debris after extraction with HCl by atomic absorption spectrophotometry .
The serotonin concentration in particulate debris and plasma was measured using an enzyme immunometric assay kit (Assay Designs, Michigan, USA). The TxB2 concentration in particulate debris and plasma was determined using the ACE™ enzyme immunoassay (Cayman Chemical Company, Ann Arbor, USA). The TNFα concentration in particulate debris and plasma was determined using a sandwich enzyme immunoassay (Cayman Chemical Company, Ann Arbor, USA). The plasma concentration of endothelin was detected using the immunometric endothelin assay kit (ACE™ enzyme immunoassay, Cayman Chemical Company, Ann Arbor, USA). The plasma concentrations of epinephrine and norepinephrine were determined by HPLC with electrochemical detection (EC 41.000 Chromsystems, München, Germany) using a kit and a reverse phase analytical column (Chromsystems, München, Germany). To determine plasma tissue factor concentration the IMUBIND Tissue Factor Elisa Kit was used, as described by the manufacturer (American diagnostica inc, Stamford, USA).
Peripheral venous blood was taken before and between 6 and 48 h after stent implantation. Serum CRP was determined in peripheral venous blood taken before stent implantation using an immunometric assay kit (ADVIA Clinical Chemistry System, Siemens, Tarrytown, USA). Serum troponin I was measured using a specific 2-side immunoassay detected with the DimensionR RxL MaxR Integrated System (Dimension Flex, Dade Behring GmbH, Marburg; and Siemens, Eschborn, Germany) [7, 35].
Human coronary arteries and rat mesenteric arteries are characterized by a comparable receptor arrangement for serotonin and TxA2[5, 7, 8]. Therefore, we used rat mesenteric arteries with intact and denuded endothelium (+E/–E). Segments of 2 mm length were mounted in a Mulvany myograph and equilibrated with Krebs-Henseleit buffer. After verification of functionality vessels were incubated with coronary arterial and aspirate plasma, which was diluted to a final ratio of 1:10 vol/vol (after correction for dilution by the hematocrit). Constrictor responses were recorded over 8 min and normalized to the maximum vasoconstriction induced by KCl (% of KClmax =100%) [7, 35].
Continuous data are presented as mean±standard error of mean (SEM), categorical data as absolute numbers. Patient characteristics were compared using unpaired t test (continuous data) and 2-tailed Fisher’s exact test (categorical data). Mediator concentrations in particulate debris and serum CRP were compared between patients with and without DM using unpaired t test. Serum troponin I and TIMI flow grading before and after stent implantation, mediator concentrations in and vasoconstrictor responses to coronary arterial and aspirate plasma, minimal lumen diameter and the percent diameter stenosis before, immediately after and 6 months after stent implantation were compared between patients with and without DM using 2-way repeated measures ANOVA followed by Bonferroni’s post-hoc tests. Linear regression analysis was calculated between the increase in TNFα immediately after stent implantation and angiographic diameter 6 months later in patients with and without DM. All statistics were performed with SPSS Statistics 19.0; SPSS Inc., Chicago, IL, USA. P<0.05 was considered significant.