Association of Circulating IgE and CML levels With in-Stent Restenosis After Drug-Eluting Stent Implantation in Type 2 Diabetic Patients With Stable Coronary Artery Disease

Background: We investigated whether serum levels of immunoglobin (Ig) E and Nε-carboxymethyl-lysine (CML) are related to in-stent restenosis (ISR) in patients with stable coronary artery disease and type 2 diabetes mellitus (T2DM). Methods: Serum levels of IgE and CML were measured in 416 stable angina patients with T2DM who received angiographic follow-up 12 months after percutaneous coronary intervention (PCI) with third-generation drug-eluting stent (DES) implantation for de novo lesions. Multivariate logistic regression analysis was performed to assess the association between IgE or CML and ISR. In mice models, femoral artery injury was induced in mice receiving albumin or glycated albumin injection, and immunouorescence staining of the injured artery segment was performed 4 weeks later using CML and IgE antibodies. Results: Both IgE and CML levels were higher in patients with ISR (n=196) compared with non-ISR patients (n=220). The rate of ISR increased stepwise with increasing tertiles of IgE and CML levels, and IgE correlated signicantly with CML. After adjusting for potential confounders, IgE and CML levels remained independently associated with ISR. IgE and CML levels improved the predictive capability of traditional risk factors for ISR, and there existed an interaction between IgE and CML in relation to ISR (P for interaction < 0.01). In mice models, glycated albumin induced increased CML and IgE inltration in the injured femoral artery segment which was associated with a higher degree of neointimal hyperplasia and luminal stenosis. Conclusion: Elevated circulating IgE and CML levels confer an increased risk for ISR after DES-based PCI in type 2 diabetic patients with stable coronary artery disease. advanced glycation end-products; AUC: area under the curve; CML: Nε-carboxymethyl-lysine; drug-eluting stent; HbA1c: glycosylated hemoglobin; IDI: integrated discrimination improvement; Ig: immunoglobin; ISR: in-stent restenosis; HDL: high-density lipoprotein; LDL: low-density lipoprotein; NRI: net reclassication improvement; OR: odds ratio; PCI: percutaneous coronary intervention; QCA: quantitative coronary analysis; RAGE: receptor for AGEs; ROC: receiver-operating characteristic; SD: standard deviation; T2DM: type 2 diabetes mellitus.

extracellular matrix accumulation [7]. Elevated AGEs have been suggested as a risk factor for post-PCI restenosis as well as coronary artery disease progression in type 2 diabetic patients [7,8]. Nεcarboxymethyl-lysine (CML), a major isoform of AGEs, contributes to endothelial dysfunction in diabetes and is associated with cardiovascular mortality [9][10][11]. Nevertheless, the relationship between CML and ISR remains unclear.
Immunoglobin (Ig) E, despite its lowest abundance in vivo, exerts a crucial effect in mediating type I hypersensitivity both systematically and locally and defending against pathogens as the rst line [12]. Previous studies have demonstrated that elevated IgE level is most common in allergy, and interestingly, the risk of cardiovascular diseases such as acute myocardial infarction, heart failure, atrial brillation, and peripheral vascular disease is increased in patients with allergic disorders [13][14][15][16]. Recent data revealed that IgE promotes coronary atherosclerosis [17], participates in abdominal aortic aneurysm formation [18] and coronary artery spasm, independent of atheromatous disease [19]. Likewise, elevated IgE level in serum has been shown to correlate with multivessel disease and contribute to discriminating coronary artery disease severity [20].
Although formation of AGEs has been reported to correlate with immunological and allergic disorders such as asthma and arthritis [21], the exact role of allergic in ammation in both pathogenesis of coronary artery disease and occurrence of adverse events following stent implantation just starts to be noticed in recent years [22]. In addition, knowledge regarding the interaction between IgE and CML on ISR is still scanty. In this study, we sought to examine if circulating levels of IgE and CML are associated with ISR in patients with T2DM after PCI with DES implantation. We also assayed serum IgE and CML levels and performed immuno uorescence staining of injured femoral artery in a high-fat diet/streptozotocin (STZ)induced diabetic mouse model.

Methods
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.

Clinical cohort
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 [23]. 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 modi ed Simpson's rule, and in-hospital management were collected retrospectively, whereas outcomes during follow-up were identi ed 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 nal 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] [24]. Hypertension was diagnosed according to seventh report of the Joint National Committee on prevention, detection, evaluation, and treatment of high blood pressure (JNC 7) [25]. Hyperlipidemia was de ned according to a guideline on the management of blood cholesterol [26].

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 [27]. 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]. Brie y, the outer diameter of contrast-lled 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 de ned 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.

Biochemical assessments
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 pro les, 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 ltration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation [30].
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 coe cient 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 [8]. Then, femoral artery injury was induced with a wire as previously described [31]. 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 immuno uorescence staining using CML and IgE antibodies (ab27684, Abcam; 553416, BD Biosciences,).

Statistical analysis
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 log 2 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 reclassi cation improvement (NRI) and integrated discrimination improvement (IDI) were calculated to assess the added value in reclassi cation of the patients. The 2-sided P value < 0.05 was considered statistically signi cant.

Baseline clinical characteristics
In this clinical cohort, ISR and non-ISR were detected in 196 and 220 patients, respectively. Patients with or without ISR did not differ with respect to age, gender distribution, body mass index, risk factors for coronary artery disease and renal function. Blood concentrations of HbA1c and hsCRP were higher whereas left ventricular ejection fraction and statin use were lower in ISR patients. Despite similar degree of coronary stenosis intervened and average number and length of stents implanted, patients with ISR received smaller stents, and had higher percentages of circum ex or right coronary artery lesion, multivessel disease, and complex lesion morphology ( There was a stepwise increase in incidence of ISR from the lowest tertile to the highest tertile of Ig E or CML (all P for trend <0.001) ( Figure 2). Serum IgE correlated positively with CML levels (all patients: r=0.331, P<0.001; ISR group: r=0.433, P<0.001; non-ISR group: r= 0.153, P=0.023), even after adjusting for confounding factors (Table 2). Logistic regression models were constructed to con rm the association between ISR and IgE or CML level in various subgroups (Figure 3). In multivariable analysis, both serum IgE and CML levels remained independent factors for ISR in patients with T2DM after adjusting for age, sex, body mass index, smoking, dyslipidemia, hypertension, HbA1c, left ventricular ejection fraction, stain use, number of diseased vessel, class B2/C lesion, bifurcation, chronic total occlusion, and stent diameter. The result patterns were similar when serum IgE or CML level was used as a standardized continuous variable with log-transformation and as an ordinal or categorical variable (Table 3).  (Table 4). More importantly, there was a signi cant interaction between IgE and CML in relation to ISR (P for interaction < 0.01). At high tertile of IgE (≥210.7 ng/ml), patients with high tertile of CML (≥215.5 ng/ml) had a signi cantly increased risk of ISR compared with those with low tertile of CML (≤161.5 ng/ml) (adjusted OR=6.784, 95% CI 2.304-19.969, P=0.001) ( Figure 5).

Findings in mice models
Mice receiving glycated albumin injection exhibited elevated CML and IgE levels in serum and increased CML and IgE in ltration in the injured femoral artery segment which were associated with a higher degree of neointimal hyperplasia and luminal stenosis ( Figure.6).

Discussion
This study is the rst to show that in type 2 diabetic patients with stable coronary artery disease, IgE correlated positively with CML. Elevated IgE and CML was associated ISR after DES-based PCI, independent of traditional risk factors.

Role of elevated circulating IgE and CML in ISR
In the bare metal stent era, the stainless-steel struts may act as a foreign body to induce effector cells of hypersensitivity, increasing IgE release and participating in ISR [32,33]. Our study cohort is unique as all patients had stable angina and received third-generation DES which re ects well the current clinical practice. The technology of new DES has been improved dramatically, often consisting of a cobaltchromium alloy platform, an antiproliferative drug (such as everolimus or zotarolimus) and a biodegradable polymer-coating with enhanced biocompatibility [34]. Nevertheless, the stent being an exogenous substance may cause several reactions by promoting proliferation of vascular smooth muscle cells, immune responses and neointima hyperplasia after implantation, leading to ISR and late thrombosis.
The major nding of this study is that serum IgE levels were higher in patients with ISR comparted to non-ISR group. Furthermore, the incidence of ISR increased stepwise across the tertiles of serum IgE, and circulating IgE level remained an independent factor for ISR in patients with T2DM after adjusting for potential confounders. These observations jointly support a notion that allergic in ammation to stent contributes, at least partly, to the development of ISR in type 2 diabetic patients after DES-based PCI. Finn et al reported that allergy-mediated in ammation plays a more critical role with DES-related ISR than with bare metal stent-related ISR [35]. Previous studies have shown that IgE is associated with diabetes status and may be an independent risk factor for pre-diabetes and diabetes [36,37]. IgE induces platelet activation and aggregation [38] and arterial smooth muscle hyperplasia [39], which are essential in the pathophysiology of ISR [40]. More importantly, IgE activates mast cells and basophils by binding to higha nity receptor FcεRI [41], and induces release of preformed in ammatory mediators and de novo synthesis and secretion of cytokines, chemokines, and eicosanoids, which may cause adverse events after stent implantation [22,42]. Based on these ndings and our results, we speculate that in diabetic patients who had elevated serum IgE, vascular injury resulting from balloon dilatation and stent implantation could increase further IgE levels and activate allergic in ammation and relevant effector cells, potentially facilitating the occurrence of ISR.
Another nding of this study is that CML, one of the most typical AGEs that have been implicated in diabetes-related complications [7,9], was correlated with ISR in patients with T2DM. Abundant evidence has demonstrated that production of AGEs is not only a sign of high blood sugar, but also re ects cumulative metabolic burden, oxidative stress and in ammation [43]. AGEs elicit secretion of in ammatory cytokines in basophils which are thought to play a pivotal role in allergic reactions and abundant expression of high-a nity receptors for IgE [44,45]. In the study of food allergy, CML acts as an immunogen by inducing activation and proliferation of various immune cells, and participates in the development of chronic in ammation [46,47]. Our results show that IgE correlated positively with CML and there existed a signi cant interaction of IgE and CML in relation to ISR, suggesting that elevated circulating CML might contribute to activation of granular cells and ampli cation of in ammation, mediating more local and systemic IgE release and, at the same time, leading to the pathogenesis of ISR in type 2 diabetic patients.

Potential clinical implications
The ndings of the present study are of clinical relevance. Our results imply that measurement of IgE and CML is useful for evaluating the risk of ISR in patients with T2DM undergoing DES-based PCI. More importantly, aggressive glycemic control and anti-allergic and anti-in ammatory therapy might be mandatory to reduce ISR, especially for individuals with high level of IgE or CML [48]. Further prospective studies with large cohorts are warranted to con rm these issues.

Study limitations
The present study has several limitations inherited from its retrospective, cross-sectional design for the point of ISR investigation, thereby allowing us to detect association, not to formulate causal link. The sample size in ISR and non-ISR groups was small and all patients were specially selected. Although baseline clinical characteristics and angiographic features were quite homogenously distributed in patients with and without ISR, certain selection biases and unknown confounding factors possibly impacting on IgE and CML could not be excluded. ISR was determined by interpretation of angiography but not through intracoronary imaging (such as intravascular ultrasound), thus we could not provide the details about the degree of ISR. Finally, the relation of serum IgE and CML levels with ISR will be more precisely characterized by serial biochemical measurements.

Conclusions
This study demonstrates that in patients with T2DM, elevated serum IgE and CML levels confer an increased risk of ISR after DES-based PCI. Novel information as such provides new insight into the pathophysiology of ISR and the management of type 2 diabetic patients with stable coronary artery disease.

Declarations
Ethics approval and consent to participate The study protocol was approved by the Institutional Review Board of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. Written informed consent was obtained from all patients, and clinical investigation was conducted according to the principle of the Declaration of Helsinki.

Consent for publication
All authors consent this manuscript for publication.

Availability of data and materials
Data generated or analyzed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.     Figure 1 Flow chart of recruitment procedure. T2DM: type 2 diabetes mellitus; CAD: coronary artery disease; PCI: percutaneous coronary intervention; CABG: coronary artery bypass grafting; ISR: in-stent restenosis Figure 2 Association of serum IgE and CML levels with ISR in T2DM patients. Comparison of serum IgE (A) and CML (C) levels between patients with and without ISR in T2DM patients. In-stent restenosis across the tertiles of IgE (B) and CML (D). ISR: in-stent restenosis; T2DM: type 2 diabetes mellitus. **P < 0.01  Receiver operating characteristic curve analysis between models to verify the predictive values of IgE (A) and CML (B).

Figure 6
Glycated albumin induced greater neointimal hyperplasia and elevated CML and IgE in mice. The mouse arteries were harvested 28 days after injury. Representative images of H&E staining (A) and immuno uorescent staining for CML (red) (B) and IgE (red) (C) of wire-injured femoral artery sections in mice receiving albumin and glycated albumin injection. Serum CML (D) and IgE (E) levels in mice. **P < 0.01, n=6 each group. Scale bar: 100μm