ABC | Volume 111, Nº2, August 2018

Original Article Farsky et al Persistent inflammation after stent Arq Bras Cardiol. 2018; 111(2):134-141 Table 1 – Biodemographic data of the studied groups: with previous stent implantation and controls Variables Stent Group Control Group p value Cases 41 26 Age (years) 60.2 ± 7.1 6.3 ± 8.69 0.004 Female (%) 13 (31.3) 8 (30.8) 0.58 Blood platelet count 237.355 ± 70.831 269.560 ± 74.461 0.12 Hypertension (%) 35 (85.4) 22 (84.6) 0.60 Diabetes (%) 17 (42.5) 8 (36.4) 0.42 Current smoking (%) 4 (10.5) 3 (14.3) 0.48 Stroke (%) 2 (5.3) 0 0.43 CKD (%) 2 (5.3) 0 0.43 Dyslipidemia (%) 25 (62.5) 16 (64) 0.56 MI (%) 14 (35.9) 5 (22.7) 0.22 LVEF < 50% (%) 14 (41.2) 2 (9.1) 0.009 ASA (%) 41 (100) 26 (100) 1.00 Statins (%) 41 (100) 26 (100) 1.00 CKD: chronic kidney disease; MI: myocardial infarction; LVEF: left ventricular ejection fraction; ASA: acetylsalicylic acid. Age and blood platelet count were expressed as mean ± standard deviation. Sex, hypertension, diabetes, smoking, stroke, CKD, dyslipidemia, MI, LVEF < 50%, ASA use, and statin use are expressed in numbers and percentages of the patients studied. The statistical significance level adopted was p < 0.05. Results Clinical characteristics of study group The frequencies of clinical characteristics (Table 1), such as sex, diabetes, dyslipidemia, smoking, previous stroke and heart attack, are similar in both groups. However, the stent group was younger and had a higher prevalence of ventricular dysfunction, characterized by an ejection fraction lower than 50%. The control group had a higher blood platelet count (269,560 ± 74,461) than the stent group (237,355 ± 70,831), but with no statistical significance (p = 0.12). All patients were on statins and acetylsalicylic acid treatment. The time between stent implantation and CABG was over 6 months. Nine patients (22%) had stent implanted within 6 and 12 months, 25 patients (61%) had only one stent, and 16 patients (39%) had two or more stents implanted. Gene expression in peripheral blood cells by real-time PCR Total RNA from peripheral blood cells was obtained and the expression of the following genes was evaluated: LIGHT , IL-6, ICAM, VCAM, CD40, NFKB, TNF , IFNG and GAPDH . Of eight genes, the expression of only two differed between the stent and control groups: the expression of TNF was significantly higher (p = 0.0308) in the stent group (Figure 1-f) and that of CD40 was higher in the control group (p = 0.0106) (Figure 1-a). No difference was detected in the expression of IL-6 , IFNG , LIGHT , NFKB , ICAM and VCAM genes (Figure 1). Quantitative analysis by immunohistochemistry Thequantificationofproteinsstainingbyimmunohistochemistry is presented in Table 2 and illustrated in Figures 2 and 3. The TNF-alpha staining in the arteries of the adipose tissue was higher in group A1 than in group A2 (6.69 ± 3.87 vs 2.27 ± 4.00; p < 0.001) (Figure 2-a). In addition, group A1 had higher TNF‑alpha staining in the intima-media region than group A3 did (5.16 ± 5.05 vs 1.90 ± 2.27; p = 0.023) (Figure 2-b). A large amount of TNF-alpha was detected in the cytoplasm of inflammatory cells and around the lipid core (Figure 3-C and D). Higher IL-6 was detected in arteries of the adipose tissue of group A1 than in those of group A3 (2.29 ± 1.96 vs 0.28 ± 0.33; p = 0.048) (Figure 2-c), also observed under the microscope (Figure 3-E and F). There was no difference between groups in the quantification of the CD40, ICAM, VCAM, NFKB and IFN-gamma staining. On histological examination, we identified major histocompatibility complex class II (MHCII) positive cells surrounding the lipid core, probably macrophages (Figure 3-A and B). These infiltrate cells also stained for TNF-alpha, being detected in adipose tissue and in intima layer (Figure 3-C and D). Fewer IL-6-positive infiltrate cells were also observed in the same layer of artery tissue (Figure 3-E and F). Discussion This study analyzed both the gene expression in peripheral blood cells and the protein localization in coronary artery tissues, to evaluate simultaneous systemic and local inflammation. A persistently higher TNF-alpha systemic expressionwas observed in peripheral blood cells, in addition to a local exacerbated TNF‑alpha and IL-6 production in coronary arteries. Our study evaluated, in stable patients, local coronary and systemic inflammation after stent implantation as compared to controls. All patients included had undergone PCI more than 6 months before and had CABG indication. Regarding stent implantation time, 9 of 41 patients (22%) were within one year 136