Matrix Metalloproteinases Expression in the Neointimal Hyperplasia Induced by Drug Eluting Stent (DES) Implantation

Materials and methods Subjects (patients) Patients derived from a population of patients referred to 12 months follow-up coronary angiography for ischemic heart disease who are enable to be performed IVUS will be enrolled in the present trial, between Dec 2017 and Dec 2018. The study was an all comers design involving consecutive enrollment of patients with stable angina or acute coronary syndrome who had at least 1 coronary lesion (defined as a stenosis of >50%) suitable for stent implantation 9 months before. The follow-up coronary angiography was performed according to standard techniques by experienced interventionalists only. In-stent restenosis was defined as a diameter stenosis ≥50% of the vessel reference diameter by visual assessment at the site of the lesion treated with the stent observed in ≥1 multiple projections. The single most severe view was used to categorize the pattern of restenosis as proposed by Mehran et al for classification of in-stent restenotic lesions. Detailed demographic details including anthropometric measurements, cardiovascular risk factors and medication use were recorded for the participants.

Angiographic analysis (QCA-analysis) The off-line quantitative coronary angiographic analysis was performed with an automated edge-detection system (QCA-CMS Version 6.0, Medis, Medical Imaging Systems, Leiden, the Netherlands). The contrast-filled, non-tapered catheter tip was used for calibration. The reference diameter was measured by interpolation. Minimal lumen diameter (before and after PCI) and diameter stenosis (before and after PCI) were measured within the stent and within the 5-mm proximal and distal edges of the stent. Furthermore, we measured vessel size, lesion length, and length of stented segment. All analyses were performed by the same investigator that was blinded to all laboratory results.

IVUS imaging and analysis IVUS imaging was performed after intracoronary administration of 0.2 mg nitroglycerin, using a motorized transducer pullback (0.5 mm/s) and a commercial scanner (Boston Scientific/SCIMED, Minneapolis, MN) consisting of a rotating 40-MHz transducer within a 3.2 Fr imaging sheath. The decision to perform IVUS was at the operator's discretion. All IVUS data were assessed off-line by experienced professionals unaware of the allocated stent type or clinical information. Using computerized planimetry (EchoPlaque 2.7, Indec Systems, Mountain View, CA), stent and reference segments were assessed every 1 mm. In-stent measurements were obtained every 1 mm and included EEM, stent, intra-stent lumen, peristent plaque+media (EEM minus stent), and IH (stent minus intra-stent lumen) areas and volumes. Percent IH was defined as IH divided by stent. All volumes were calculated using the Simpson rule and then normalized by stent length (normalized volume).

Blood samples Blood samples were taken under fasting conditions directly before follow-up angiography. Arterial blood was drawn from the coronary arteries into serum separator tubes and samples serums were allowed to clot for 30 min before centrifugation at 1,000 g for 10 minutes and stored at -70°C until use.

Laboratory measurement Endogenous MMP-2 and MMP-9 were measured in serum using a standardized enzyme-linked immunosorbent assay (ELISA; R\&D systems). This system measures the endogenous activity of the specific MMPs.

Sample size estimations:

Sample size calculation was based on the null hypothesis that MMP-2 and/or -9 activity showed no correlation with neointimal hyperplasia volume. Using the Lachin formula, sample size calculations were performed to show the expected correlation coefficient (r) more than 0.40 between both group, two-sided alpha 0.05 and 80% statistical power. Finally, 47 patients were calculated.

Statistical analysis :

Continuous variables are expressed as mean±SD. Categorical variables are summarized as counts and percentages and were compared by the chi-square or by Fisher exact test. Serum levels of MMPs were compared by Student t test or by Mann-Whitney U test. Pearson's correlation was used to correlate MMP levels with neointimal hyperplasia volume. Multiple logistic regression was used to evaluate the interaction between variables and MMPs in correlation with neointimal hyperplasia volume and in-stent restenosis. A stepwise entry procedure was applied to identify significant or suggestive (p < 0.1) confounders of either patient group or MMP level. Odds ratios were expressed with 95% confidence intervals. A p-value of less than 0.05 was considered statistically significant. All statistical analyses were performed with the statistical software package SPSS version 12.0 (SPSS, Inc., Chicago, Illinois). The authors had full access to the data and take responsibility for its integrity. All authors have read and agree to the manuscript as written.