ABC | Volume 111, Nº2, August 2018

Original Article Farsky et al Persistent inflammation after stent Arq Bras Cardiol. 2018; 111(2):134-141 Table 2 – Quantification of CD40, ICAM, VCAM, MHC-II, TNF-alpha, NFKB, IL-6 and IFN-gamma proteins in arterial intima-media layer, adventitia and adipose tissue by immunohistochemistry Protein Artery layers Group A1 Group A2 Group A3 p value (α = 0.05) n mean Std. dev n mean Std. dev n mean Std. dev 3 groups 1 vs 2 1 vs 3 2 vs 3 CD40 intima-media 16 1.37 2.02 27 1.51 1.73 23 1.11 1.56 0.55 ns ns ns Adventitia 13 0.70 0.77 26 0.82 0.71 19 1.27 1.66 0.58 ns ns ns Adipose tissue 3 0.73 0.12 6 0.62 0.58 9 0.58 0.74 0.45 ns ns ns ICAM intima-media 18 3.27 3.00 27 3.47 3.77 20 2.81 4.23 0.16 ns ns ns Adventitia 14 4.07 3.67 25 4.22 4.82 19 3.99 5.39 0.76 ns ns ns Adipose tissue 1 5.93 - 7 1.99 1.37 5 1.52 1.64 0.22 ns ns ns VCAM intima-media 14 11.88 16.01 24 10.33 9.64 23 7.88 6.12 0.76 ns ns ns Adventitia 11 4.69 8.39 19 4.09 4.74 21 2.56 2.26 0.68 ns ns ns Adipose tissue 2 4.31 5.28 4 1.76 1.22 4 0.32 0.36 0.10 ns ns ns MHC II total area 7 0.74 0.59 8 0.47 0.12 30 0.75 0.52 0.307 ns ns ns TNF-alpha intima-media 15 5.16 5.05 24 3.11 3.01 21 1.90 2.27 0.03 ns 0.023 ns Adventitia 14 4.05 2.82 21 2.28 2.24 20 3.57 5.95 0.10 ns ns ns Adipose tissue 4 6.69 3.88 4 1.27 0.84 9 2.27 4.00 0.05 0.001 ns ns NFKB intima-media 14 1.11 1.14 24 0.93 0.88 20 0.92 1.07 0.96 ns ns ns Adventitia 14 0.64 0.61 21 0.83 0.78 19 0.76 0.55 0.65 ns ns ns Adipose tissue 2 1.18 0.87 6 0.52 0.44 6 1.63 2.58 0.66 ns ns ns Interleukin-6 intima-media 16 1.15 1.12 23 1.27 1.61 21 0.66 0.88 0.17 ns ns ns Adventitia 15 1.65 2.04 20 1.65 2.02 21 0.91 0.74 0.69 ns ns ns Adipose tissue 4 1.12 1.03 3 2.29 1.96 11 0.28 0.33 0.01 ns 0.061 0.048 IFN-gamma intima-media 14 0.67 0.70 25 0.66 0.69 22 0.56 0.84 0.36 ns ns ns Adventitia 12 0.52 0.41 20 0.59 0.54 21 0.40 0.46 0.36 ns ns ns Adipose tissue 3 0.54 0.53 4 0.14 0.10 7 0.10 0.11 0.13 ns ns ns The comparison was performed among groups (Kruskal-Wallis test or ANOVA): A1 (arteries with stent), A2 (native arteries from patients with a stent in another artery), and A3 (control, patients without previous stent placement). The statistical significance level adopted was p < 0.05. Blood Analysis The analysis of mRNA expression from circulating blood cells pointed out a significant higher expression of the TNF gene in the group with previous stent implantation than in controls (Figure 1-f), suggesting a greater activation of this gene in leukocytes from stented patients. This gene encodes a pleiotropic cytokine involved in a broad range of biological activities, including inflammation, cell survival, cell proliferation, and, paradoxically, cell death. 16 We also observed a significantly lower CD40 gene expression in blood cells from the stent group than from controls (Figure 1-a). The CD40 is the receptor for CD40L, being present in platelets. Gerdes et al. 17 have demonstrated in knockout mice for CD40 and ApoE that platelet plays a crucial role in inflammation by stimulating leukocyte and endothelial cells activation, thereby promoting atherosclerosis. Coronary artery tissue samples We analyzed the arterial tissue separated in three layers (adventitia, intima-media and adipose tissue) by h ematoxylin‑eosin and immunohistochemistry staining. It is worth pointing that, although only few samples collected contained adipose tissue, due to the difficulty to obtain all layers in such small fragments, we could discriminate a greater amount of TNF‑alpha and IL-6proteins in the adipose tissue from groups A1 and A2 than in that from controls (Figure 2-a and 2-c). The white adipose tissue is considered to be an endocrine gland, and the main feature is insulin and leptin resistance, as well as the production of inflammatory cytokines (TNF-alpha and IL-6) and monocyte chemoattractant protein, 18,19 which are involved in atherogenesis. 20,21 Interestingly, on the histological study, we observed activated immune cells, with MHCII expressed in membrane (Figure 3), surrounding the lipid core. Furthermore, these cells were colocalized with TNF-alpha and IL-6 staining, suggesting a greater inflammatory response in the adipose tissue around the artery from individuals with previous stent placement. The TNF-alpha protein was also expressed in higher quantity in intima-media layer from group A1 than from groups A2 and A3 (Figure 2-b). Probably immune cells migrated from circulation to that layer, mainly macrophages, which 138