ABC | Volume 111, Nº2, August 2018

Original Article Farsky et al Persistent inflammation after stent Arq Bras Cardiol. 2018; 111(2):134-141 Methods Case series The patients admitted to elective CABG with previous stent implantation were consecutively included in this study after signing informed consent. The protocol was approved by the Dante Pazzanese Institute of Cardiology ethics committee (Protocol 4059-2011). This study included 67 patients as follows: 41 patients with previous intracoronary BMS implantation and 26 patients without stent implantation submitted to elective CABG. All patients had stable angina and more than 6 months of the stent implantation, aiming to exclude ongoing restenosis. The exclusion criteria consisted of emergency surgeries, acute coronary syndromes and chronic renal failure in dialysis because of chronic inflammatory reaction. Peripheral blood sample was collected preoperatively from the antecubital vein, using PAXgene tubes (PreAnalytiX®, BD Company, UK) for systemic gene expression analysis. During CABG surgery, a tiny fragment of the bypassed coronary artery was obtained at the arteriotomy site, usually 10 mm after the stent implantation site, to evaluate local inflammation markers. All tissue samples were immediately immersed in buffered formalin for further paraffin-embedded block preparation. Some arterial fragments obtained were not adequate for histological analysis, thus, 176 artery samples were included and grouped as follows: A1- arteries with stent (n = 38); A2- native arteries from a patient with a stent in another artery (n = 68); and A3- arteries from patients without previous stent placement (n = 70). RNA isolation, reverse transcription and real-time polymerase chain reaction (PCR) Total RNA was extracted from peripheral blood collected in PAXgene tubes (PreAnalytiX®, BD Company, UK) using PAXgene blood RNA kit (QIAGEN GmbH, Hilden, Germany), being then quantified using Qubit® 2.0 fluorometer (Life Technologies, Inc., Grand Island, NY). The RNA integrity was performed using Tapestation® 2200 and R6K Screen Tape (Agilent Technologies, Inc. UK). The cDNA was transcribed from 200 ng of total RNA using High Capacity RNA-to-cDNA Master Mix (Applied Biosystems, Foster City, USA). Real-time qPCR was carried out in Rotor-Gene® detection system using the TaqMan® Fast Multiplex PCR kit (QIAGEN GmbH, Hilden, Germany) and primers from Applied Biosystem commercially designed for TaqMan® qPCR (Applied Biosystems, Foster, CA, USA). The expression of the following genes was evaluated: LIGHT (Hs00542477_m1); IL-6 ( Hs00985639_m1); ICAM ( Hs00164932_m1); VCAM ( Hs00174239_m1); CD40 ( Hs01002913_g1); NFKB ( Hs00231653_m1); TNF (Hs01113624_g1); IFNG ( Hs00989291_m1) and GAPDH (Hs00266705_g1). For all genes, we constructed standard curves and determined the slope to calculate the PCR efficiency. Almost equal efficiency for all primer/probe systems was observed. All samples were tested in duplicate using GAPDH as a reference gene, which was previously chosen between the six most common endogenous myocardium genes using geNorm algorithm. 10 The samples amplified after 40 cycles of PCR were considered negative and excluded from further statistical analysis. The expression of the reference GAPDH gene was applied for data normalization, and the relative expression of each mRNA was calculated using the 2 -∆∆CT method. 11 Immunohistochemistry staining Firstly, 4-µm-thick formalin-fixed-paraffin-embedded artery samples were sectioned and fixed in silanized slides, followed by dewaxing at 70°C, in the oven, for 1 hour, and immersion in three xylene baths for 10minutes. They were then rehydrated in decreasing concentrations (100%, 90%, 75%) of ethyl alcohol. Antigen retrieval was performed using the Trilogy® buffer (Cell Marque, California, USA) at a Decloaker equipment at 90°C for 40 minutes (Biocare Medical, CA, USA). The specific blocking reagents (Erviegas EasyPath, DuraEdge, USA) were applied for endogenous peroxidase and protein blocking. In the next step, the slides were incubated with respective primary antibodies previously titrated and diluted in universal thinner (Erviegas EasyPath, DuraEdge, USA). Primary antibodies against interleukin-6 (IL-6) (ab6672), ICAM (ab2213), VCAM (ab106777), TNF-alpha (ab1793), IFN-gamma (ab9657), CD40 (ab58612), and NFKB (ab16502) from Abcam (Cambridge, MA, UK) were used. The immunoperoxidase reaction was detected using Mach4 Kit Universal HRP Polymer + DAB (Biocare Medical, California, USA), and, finally, the slides were stained with Harris hematoxylin (Erviegas EasyPath, DuraEdge, USA) and assembled by synthetic resin Erv-Mount (Erviegas EasyPath, DuraEdge, USA). The positive control of immunohistochemistry reaction was performed using tissues that have the same constructive antigens as the antigen of interest. After immunohistochemistry processing, the slides were scanned through a Scanscope CS System unit (Aperio Technologies, Inc., CA, USA), with an objective 20x Olympus UPlanSApo with specifications 20x/0.75 attached to the scanner, generating image files in svs format. Scanned images were analyzed using the Aperio ImageScope viewing software (Aperio Technologies, Inc., CA, USA) that reports the percentage of positively stained area in relation to the total tissue area. Statistical analysis Continuous variables are reported as mean and standard deviation or median and interquartile interval, depending on the assumption of normality. Categorical variables are reported as absolute and relative frequency. Values between groups were compared by unpaired Student t test after testing for normal distribution by KS test; otherwise, nonparametric Mann-Whitney U tests were used. Fisher exact or chi-square test was used for categorical variables with nominal scales. For comparison of artery tissue markers, Kruskal-Wallis test (or ANOVA, assumption of normality) was used, and, for non-parametric multiple comparisons, Tukey's test. A p-value lower than 0.05 was considered statistically significant. The SPSS version 19 was used. To detect 3 units with standard deviation of 4, 80% test power, and 5% alpha, the sample size calculation is 105 cases. 135