Diabetes mellitus (DM) remains the main predictor of restenosis rates and cardiovascular events following successful percutaneous coronary intervention (PCI) despite the use of drug-eluting stents (DES). HbA1c <6.0% is considered an index of optimized metabolic control in patients with DM, but several studies are downsizing its role in the clinical management of these patients. Increasing evidence points at the role of advanced glycation end products (AGEs) in restenosis pathogenesis independently on Hb1AC levels. Thus, we investigated the predictive value of preprocedural AGE levels for in-stent restenosis in a population of euglycaemic diabetic patients undergoing PCI with DES implantation. One hundred twenty-five consecutive patients with DM in optimized glycemic control admitted for stable angina pectoris and treated with elective DES implantation at a tertiary hospital were prospectively included. The primary end point of the ARMYDA-AGEs study was to compare rates of angiographic ISR at 6 months after the intervention according to pre-PCI levels of AGEs. Secondary end points were the correlations of AGE levels with occurrence of periprocedural myocardial damage, major adverse cardiac events, and in-stent late loss at 6-month control coronary angiography. AGE levels >17 μM was found to be an independent predictor of ISR at 6 months and stent lumen loss. AGEs failed to predict occurrence of secondary endpoints. In conclusion, elevated AGE levels predict occurrence of in-stent restenosis after DES implantation in patients with DM on optimized glycemic control and might represent a dosable marker of adverse outcome after PCI.
Diabetes (DM) is widely known to be associated with higher restenosis rates and increased risk of cardiovascular events following successful percutaneous coronary intervention (PCI) or surgical coronary revascularization. High preprocedural glycemic levels and glycated hemoglobin (HbA1c) value >7%, a parameter clinically recognized as indicative of uncontrolled DM, has been correlated with poorer outcome and high rates of restenosis in diabetic patients undergoing PCI. However, the latter observation has not been confirmed in 2 recent studies in which there was no increased risk of target lesion revascularization or other cardiac events after PCI in patients with elevated HbA1c values. Advanced glycation end products (AGEs) are ubiquitous signaling proteins that are associated with vascular and neurologic complications of diabetes influencing inflammatory response, smooth muscle cell proliferation, and matrix production; we previously demonstrated in an in vitro study that even in diabetic patients under optimized glycemic control (HbA1c <6.0%), the presence of a large AGE burden in the vessel wall increases the likelihood of an exaggerated and prolonged inflammatory reaction and determines a prothrombotic state, defining a common mechanism potentially explaining the increased rate of vein graft failure after coronary bypass surgery. Inflammation, smooth muscle cell proliferation, extracellular matrix production, and activation of platelets are involved in the mechanisms of restenosis after stent implantation. Thus, considering the nonconfirmatory reports in the literature on the role of Hb1Ac as an effective marker of metabolic control or restenosis risk prediction, in this study, we prospectively investigated the predictive value of preprocedural AGE levels for in-stent restenosis (ISR) in a population of DM patients considered euglycemic (Hb1AC<6%) undergoing PCI with drug-eluting stent (DES) implantation.
Methods
Consecutive type 2 diabetic patients with optimized glycemic control admitted for stable angina and candidates to elective PCI with DES implantation at Campus Bio-Medico University of Rome were prospectively included. Exclusion criteria are reported in Table 1 . Patients reporting at coronary angiography lesions with characteristics matching the American Heart Association criteria for DES implantation were included ; however, patients with type 2B/C lesions, left main disease, bifurcating lesions, or stenoses requiring at least 3 stents on the same coronary vessel were also excluded to avoid further biases of overestimation of the influence of AGEs serum levels on the primary end point. Because it has been reported a direct biochemical interaction among AGEs and rapamycin in determining endothelial cell proliferation and also because tacrolimus has been reported to affect serum levels of receptors for AGEs, only paclitaxel-eluting stents were included in this study.
| Acute coronary syndrome |
| Previous coronary artery bypass surgery or PCI |
| Nonoptimized glycemic control (HbA1c level >6.0%) |
| Proliferating retinopathy |
| Peripheral neuropathy |
| Diabetic nephropathy (increased UAE in the absence of other renal diseases) or significant microalbuminuria (UAE >20 μg/min and ≤199 μg/min) |
| Severe renal failure (kidney damage or GFR<30 mL/min/1.73 m 2 for ≥3 mo) |
| Leg vein insufficiency or trophic lesions |
| Autoimmune diseases |
| Known presence of malignancy |
| Any therapy with PPAR-γ agonist (i.e., thiazolidinediones) within the past 6 mo |
| Any therapy with steroid within the past 6 mo |
| Any therapy with antitumoral agents within the past 6 mo |
One hundred twenty-five patients fulfilling the enrollment criteria were included. Hb1Ac and AGE serum levels were measured before intervention, and Hb1Ac measurement was repeated every 2 months for a 6-month postprocedural period. All patients underwent ambulatory visit at 30 days and 3 and 6 months after PCI. Routine control coronary angiography was planned at 6 months in the entire study population unless there was earlier clinical indication for angiography because of symptoms recurrence or inducible myocardial ischemia. AGE serum level were measured again at the 6-month follow-up coronary angiogram. All interventions were performed via the femoral approach with standard technique. Glycoprotein IIb/IIIa inhibitors were not used in any patient.
Blood samples were obtained by venipuncture; the serum samples were separated by centrifugation within 1 hour and subsequently stored at −80°C. Serum AGE levels were measured by spectrofluorometry as described in our previous report and according to literature. The concentrations of the AGE products were directly proportional to the fluorescence intensity, and the single fluorescence values were compared with a standard curve obtained with scalar dilutions of AGEs according to the method of Horiuchi et al. This previously validated spectrofluorimetric method is cost-effective and, because it is based on the standard curve with known AGEs concentration, allows for highly replicable and reliable measurements with sensibility ranges adequate for the levels found in human blood.
Hb1AC, creatine kinase-MB (normal limits ≤4 ng/ml), and troponin-I (normal ≤0.08 ng/ml) measurements were performed according to current clinical laboratory practice.
The primary end point of the atorvastatin for reduction of myocardial damage during angioplasty (ARMYDA-AGEs) study was to compare rates of angiographic ISR at 6 months after the intervention according to pre-PCI levels of AGEs. Secondary end points were the correlations of AGEs levels with (1) occurrence of periprocedural myocardial damage, (2) incidence of major adverse cardiac events (MACE) at 6-month follow-up, and (3) in-stent late loss at 6-month control coronary angiography.
DM was defined as either a previous diagnosis of DM treated with diet, oral agents, or insulin or a new diagnosis of DM if the fasting glucose level was >126 mg/dl on 2 occasions during hospitalization. Hb1Ac cut-off level to be considered optimal glycemic control was 6.0%. ISR was defined as restenosis at the site of the stented lesion within 5 mm proximal or distal to the stent edges (in segment), with >50% narrowing of the lumen diameter; intrastent proliferation was considered as a <50% narrowing. Periprocedural myocardial damage was defined as any creatine kinase-MB or troponin-I post-PCI elevation above the upper limit of normal. MACE included all-cause death, myocardial damage, or target lesion revascularization (TLR); TLR included by-pass grafting or percutaneous intervention on the original treated site. Acute myocardial infarction during follow-up was defined as chest pain with >1 mm of ST segment elevation on >2 contiguous electrocardiographic leads, chest pain refractory to medical therapy with associated ST-segment depression in leads V2 to V5 (consistent with posterior injury), or new left bundle branch block. In-stent late loss was calculated as the difference between the minimal lumen diameter at final poststenting and at reangiography, measured by quantitative coronary angiography. The analyses of angiograms were performed by experienced observers who were blind to the AGE status of the patients. Procedural success was defined as the achievement of <30% final diameter without the occurrence of death, infarction, or urgent TLR during the hospital stay.
The local ethics committee approved the protocol, and all individuals provided informed consent to enter the study. The study conformed with the Declaration of Helsinki. The study was not supported by any external source of funding. No stent manufacturer had any role in the study. To estimate the total number of patients required to demonstrate a statistical significant influence of serum AGEs concentration in determining ISR, an inverse power analysis was performed. For this work, data generated by Chello et al analyzing AGEs serum levels in patients undergoing cardiopulmonary bypass surgery in optimized glycemic control have been used. To detect a 70% difference in the 6-month primary end point between patients with elevated and normal AGE levels assuming 2-sided 5% significance level at 80% power, 98 patients were needed. This was increased to a recruitment target of 125 patients assuming up to 20% noncompliance with the study protocol.
Continuous data are presented as mean ± SD; categorical data are presented as the count (percentage). Continuous variables were compared using unpaired Student t test and categorical data with the Fisher exact test. Analysis of binary end points (such as binary restenosis to >50% diameter stenosis and patency) was accomplished using contingency table analysis. Significance of results was assessed with the chi-squared test uncorrected for continuity. To control for the errors that resulted from possible deviations of the continuous variables from a normal distribution, this analysis was verified by the Mann-Whitney U test, which produced similar results.
A multivariate Cox regression model with stepwise removal of nonsignificant variables was used to identify predictors for ISR; the entry and removal probability of stepwise selection were 0.1 and 0.2, respectively. p values <0.05 (2-tailed) were taken to indicate statistical significance. Analysis was performed with the SPSS version 20.0 software for Mac.
Because AGE concentration appeared to be an independent predictor of ISR, a receiver operator characteristic (ROC) curve was plotted, and the area under the curve was calculated to individuate an AGEs serum level cutoff value and determine its predictive ability for the primary end point. The cut-off value identified matched with reports in the literature and in particular with a previous study by Chello et al analyzing AGEs serum levels in patients undergoing cardiopulmonary bypass surgery in optimized glycemic control. Therefore, the cut-off value was used to create a new variable defined as “elevated prePCI AGEs serum levels,” dividing the overall population into 2 subgroups and further inserted in the model for primary and secondary outcomes prediction.
Results
In the study population, mean age was 68 ± 11 years; clinical and procedural data of the population are indicated in Table 2 and Table 3 , respectively.
| Characteristic | (n = 119) |
|---|---|
| Mean age (yrs) | 68 ± 11 |
| Women | 29 (24%) |
| Systemic hypertension | 91 (76%) |
| Previous myocardial infarction | 23 (19%) |
| Familial history of cardiovascular disease | 53 (44%) |
| Total cholesterol (>200 mg/dl) | 31 (26%) |
| Smoker | 39 (33%) |
| Body mass index (kg/m 2 ) | 26.9 ± 4.3 |
| Serum creatinine (mg/dl) | 1.04 ± 1.6 |
| Creatinine clearance (ml/min) | 79.8 ± 5.8 |
| Hemoglobin (g/dl) | 13.7 ± 1.4 |
| Glycated hemoglobin (Hb1Ac) | 5.6 ± 0.8 |
| Left ventricular ejection fraction | 54 ± 9 |
| Therapy | |
| Aspirin | 119 (100%) |
| Clopidogrel | 119 (100%) |
| Beta blocker | 43 (36%) |
| Statin | 119 (100%) |
| ACE inhibitors or ARA | 89 (75%) |
| Calcium antagonist | 30 (25%) |
| Diuretics | 15 (13%) |
| Biguanides | 85 (71%) |
| Sulfanilamides | 23 (19%) |
| Multivessel coronary disease | 47 (39%) |
| Multivessel intervention | 28 (23%) |
| Number of significantly diseased vessels/patient | 1.8 ± 0.7 |
| Coronary vessel treated | |
| Left anterior descending | 69 (58%) |
| Left circumflex | 44 (37%) |
| Right | 31 (26%) |
| Type of procedure | |
| DES (paclitaxel) | 119 (100%) |
| Stent length (mm) | 18.79 ± 5.68 |
| Stent diameter | 2.88 ± 1.02 |
| Direct stenting | 72 (60%) |
| No of predilation | 1.6 ± 2 |
| Stent deployment pressure (atm) | 12.4 ± 4.2 |
| Duration of stent deployment (sec) | 19.2 ± 9.0 |
| Restenosis | |
| Intrastent proliferation (ISP) | 12 (10%) |
| Intrastent restenosis (ISR) | 23 (19%) |
| Combined outcome (ISP + ISR) | 31 (26%) |
| MACE at 6-month follow-up | |
| Death | 6 (5%) |
| Periprocedural myocardial damage | 8 (7%) |
| Target lesion revascularization | 4 (3%) |
| Combined outcome MACE | 12 (10%) |
Procedural success was obtained in all patients; no patient had no-reflow phenomenon or significant (≥2 mm) side branch closure during the intervention. There were no in-hospital major complications (death or need for urgent revascularization).
Patients underwent coronary angiography at 6 months after the procedure; 4 patients received earlier angiography (range 4 weeks–4 months) because of effort angina and evidence of myocardial ischemia at stress test. Among these patients, 3 showed ISR and 1 onset of new lesion on the treated vessel, but no statistically significant difference was observed among ISR and new-onset lesion was found. However, these patients all belonged to the elevated prePCI AGE serum group. Six patients refused the angiographic control. ISR occurred in 23 patients (19%). During the 6-month follow-up, 6 patients died (5%); among these 4 were cardiac-related deaths. Eight patients (7%) had periprocedural myocardial damage, and 4 (3%) had TLR by re-PCI, with 3 patients belonging to those receiving earlier angiography.
Intrastent proliferation occurred in 10% (12 patients) and ISR in 19% (23 patients) of patients. With respect to the primary end point, AGE serum levels at the time of PCI in the no restenosis group and in the restenosis group were 12.70 ± 3.10 μM and 27.85 ± 8.29 μM, respectively (p <0.001). AGEs serum levels at the time of PCI and at 6-month follow-up did not differ significantly in both the restenosis (27.85 ± 8.29 μM and 26.88 ± 9.12 μM, respectively; p = 0.83) and no restenosis groups (12.70 ± 3.10 μM and 13.29 ± 3.71 μM; p = 0.74). HbAC1 levels did not significantly differ between the 2 groups (ISR and no ISR) and remained similar to preprocedural values and throughout the 6-month follow-up.
In the multivariate model, the independent predictors of restenosis included AGE serum levels (p = 0.004) and stent length (p = 0.032; Table 4 ). However, when normalized for number of vessel diseased and number of stents implanted, the variable “stent length” lost its significance. The area under the curve of the ROC derived from the multivariate model was 0.97 (p <0.001).
| Variable | Sig | Odd Ratio | 95% CI Lower | 95% CI Upper |
|---|---|---|---|---|
| Smoker | 0.145 | 0.015 | 0.0 | 4.284 |
| Number of narrowed coronary arteries | 0.5 | 1.638 | 0.390 | 6.876 |
| Stent length | 0.032 | 1.319 | 1.024 | 1.699 |
| AGE serum concentration | 0.004 | 1.653 | 1.178 | 2.318 |
To clarify the relative weight and contribution of AGEs serum level in determining restenosis, the same model was run excluding this variable, and the ROC curve was plotted. Area under the curve was estimated at 0.79 (p >0.001), allowing to infer a contribution of AGEs level of >20% in predicting restenosis.
From the ROC analysis, we identified AGE levels >17 μM as the optimal cut-off value to predict 6-month ISR, with a sensitivity of 87%, a specificity of 90%, and a positive predictive value of 87% (area under the curve was 0.94; 95% confidence interval [CI]: 0.873–1.0; p >0.001). According to this cut-off point, 2 risk groups were identified: patients with elevated pre-PCI AGE levels (n = 52) and those with low pre-PCI AGEs levels (n = 67); main demographic and procedural features were similar between these 2 groups ( Table 5 ). HbAC1 levels did not significantly differ between the 2 groups and remained similar to preprocedural values and throughout the 6-month follow-up.
