This study was approved by the ethics committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, and conducted according to the Declaration of Helsinki. Written informed consent was obtained from all participants.
A total of 606 consecutive patients with T2DM who received follow-up coronary angiography around 12 months after DES-based PCI of de novo lesions in native coronary artery from January 2017 to December 2020 were recruited from the database of Shanghai Ruijin Hospital PCI Outcome Program. This program utilizes clinical and angiographic information for various cardiovascular diseases to estimate risk-adjusted outcomes. Data on demographics, clinical characteristics and angiographic features, left ventricular function determined by two-dimensional echocardiography according to modified Simpson’s rule, and in-hospital management were collected retrospectively, whereas outcomes during follow-up were identified prospectively. For the purpose of this study and to avoid confounding serum data, we excluded patients with acute coronary syndrome (n=133), familial hypercholesterolemia (n=5), malignant tumor (n =6), renal failure requiring hemodialysis (n=4) or prior coronary bypass grafting (n=27). Patients with history of asthma (n=5), autoimmune disease (n=4), and rheumatic heart disease (n=6) were also excluded. Thus, the remaining 416 patients were eligible and categorized in the final analysis (Figure 1).
The diagnosis of T2DM was made according to the criteria of American Diabetes Association [symptoms of diabetes with casual plasma glucose concentration ≥ 200 mg/dL (11.1 mmol/L) or fasting plasma glucose ≥ 126 mg/dL (7.0 mmol/L), 2h postprandial glucose ≥200mg/dL (11.1 mmol/L) during an oral glucose tolerance test, and currently or previously treated with insulin and/or oral hypoglycemic agents]. Hypertension was diagnosed according to seventh report of the Joint National Committee on prevention, detection, evaluation, and treatment of high blood pressure (JNC 7). Hyperlipidemia was defined according to a guideline on the management of blood cholesterol.
Coronary angiography and quantitative analysis
Coronary angiography and PCI were performed through radial or femoral approach using standard methods. All lesions were stented with a normal-to-normal technique, usually including 5-mm- long, angiographically normal segments proximal and distal to the lesion. The third-generation DES was applied to all patients, but the choice of stent type and technique of deployment were left for the discretion of the operators. A plurality of matching angiographic images was obtained after intracoronary nitrate injection for each patient. All patients were encouraged to take guideline-recommended medications after the procedure.
End-diastolic frames from both baseline and follow-up angiograms were selected with identical angulations that best showed the stenosis at its most severe degree with minimal foreshortening and branch overlap. Quantitative coronary analysis (QCA) of baseline and follow-up angiograms was made using the Cardiovascular Measurement System version 3.0 software (Terra, GE, USA) by two experienced cardiologists, who were blinded to patients’ clinical information and biochemical measurements[28, 29]. Briefly, the outer diameter of contrast-filled catheter was used for calibration to determine absolute measurements in millimeters. Lesion length was measured as the distance from the proximal to distal shoulder. A value of 0 mm was assigned for minimal lumen diameter in the case of total occlusion at baseline. ISR was defined as recurrence of luminal diameter stenosis of > 50% within the stent or in the 5-mm proximal or distal segments adjacent to the stent at follow-up angiography [28, 29]. For patients with multiple coronary lesions, the most severe restenotic lesion at follow-up was included in the analysis.
Peripheral venous blood samples were obtained at the day of angiography after an overnight fasting. To avoid a diurnal variation in IgE concentration and dramatic fasting interval effects, all blood samples were obtained at 8:00 am. Serum levels of glucose, blood urea nitrogen, uric acid, creatinine, and lipid profiles, including triglyceride, total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, lipoprotein (a), apolipoprotein A-I and apolipoprotein B were measured using standard laboratory techniques on a HITACHI 912 Analyzer (Roche Diagnostics, Germany). Blood concentration of glycosylated hemoglobin (HbA1c) was measured using ion-exchange high performance liquid chromatography with Bio-rad Variant Hemoglobin Testing System (Bio-Rad Laboratories, USA). Serum levels of high-sensitivity C-reactive protein (hsCRP) were determined by ELISA (Biocheck Laboratories, Toledo, OH, USA). The estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation .
Serum levels of IgE and CML were determined by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's protocols (BMS2097, eBioscience; STA-816, Cell BioLabs). The average inter-assay coefficient of variance (CV) was 6.2% and 5.8% for IgE and CML, respectively, and the average intra-assay CV was 6.6% or 7.2% for IgE or CML, respectively.
Diabetic mouse model
Animal experiments were approved by Hospital Animal Care Committee and complied with Guide for the Care and Use of Laboratory Animals by the National Institutes of Health. 6-8 weeks old C57BL/6J male mice were housed in a pathogen-free environment and received intraperitoneal injections of albumin (A3139, Sigma Aldrich) (100µg each), or glycated albumin (100µg each) every other day for 12 weeks. The glycated albumin was prepared through a glycation process. Then, femoral artery injury was induced with a wire as previously described . Serum was collected for analysis of CML and IgE 4 weeks later using ELISA kit (STA-816, Cell BioLabs; E99-115, Bethyl Laboratories) and injured femoral artery was harvested for hematoxylin and eosin staining and immunofluorescence staining using CML and IgE antibodies (ab27684, Abcam; 553416, BD Biosciences,).
All statistical analyses were performed with SPSS 26.0 (IBM, Armonk, New York) and R Programming Language 4.0.2. Continuous variables are expressed as mean ± standard deviation (SD) if data were normally distributed, or as median (25th–75th percentile) otherwise, and categorical variables are summarized as frequencies (percentages). IgE and CML levels were presented both as an original skewedly-distributed variable and a log2 transformed normally distributed variable. Continuous variables were compared between two groups using student’s t-test or Mann–Whitney U test. For categorical variables, differences between groups were evaluated with chi-square test. Pearson’s and Spearman’s correlation tests were used to assess the relation between IgE and CML. Logistic regression models were applied to detect the relationship between ISR and serum IgE or CML level. IgE or CML was analyzed as a continuous variable with log- transformation, as an ordinal variable, and as a categorical variable divided into tertiles. Odds ratios (OR) were calculated with unadjusted, adjusted for age, sex, body mass index, smoking, dyslipidemia, hypertension (model 1), and further adjusted by adding HbA1c, left ventricular ejection fraction, statin use, number of diseased vessels, B2/C lesion, bifurcation, chronic total occlusion, and stent diameter (model 2). Receiver-operating characteristic (ROC) curves were plotted to determine the power of IgE and CML for detecting ISR, and the C statistics was compared using Delong method. Category-free net reclassification improvement (NRI) and integrated discrimination improvement (IDI) were calculated to assess the added value in reclassification of the patients. The 2-sided P value < 0.05 was considered statistically significant.