Inflammatory responses involving tumor necrosis factor receptor-associated factor 6 contribute to in-stent lesion formation in a stent implantation model of rabbit carotid artery

Abstract

ObjectiveInflammatory responses are considered to represent a unique property after stent implantation, and we previously demonstrated that inflammatory signaling involving tumor necrosis factor receptor-associated factor 6 (TRAF6) contributes to neointimal formation in a balloon injury model of rabbit carotid artery. The purpose of this study was to examine the role of TRAF6 in in-stent lesion formation after stent implantation in the rabbit carotid artery.MethodsRabbit carotid arteries were injured with a 2F Fogarty catheter, and 28 days later, the same arteries were implanted with a 3-mm-diameter Palmaz-Schatz stent. A dominant negative (DN) form of TRAF6 (pME-FLAG-T6ΔRZ5) was then transferred using a plasmid-based electroporation method. Its effects were evaluated compared with the findings in arteries treated with control plasmid (pME-FLAG).ResultsImmunostaining with anti-FLAG tag antibody showed that an expression plasmid vector containing the DN-TRAF6 sequence was successfully transferred to the arterial intima and media. Morphometric analyses revealed that the increase of intimal area in in-stent lesions was significantly inhibited by DN-TRAF6 14 days after stent implantation (DN-TRAF6 group, 3.01 ± 0.25 × 105 μm2 vs control group, 4.25 ± 0.23 × 105 μm2, P < .01), and the cell density was increased compared with that in the control group. In the DN-TRAF6 plasmid-treated vessels, cell replication was prevented in both the intima and media, and fewer leukocytes adhered to the luminal surface. Moreover, DN-TRAF6 suppressed macrophage infiltration, activation of proteases, and proteoglycan accumulation in the in-stent intima.ConclusionsThese findings suggest that TRAF6 plays an important role in cell replication, inflammatory cell infiltration, protease activity, and extracellular matrix accumulation that contributes to in-stent lesion development.Clinical RelevanceThe purpose of the present study was to determine the role of TRAF6 in in-stent lesion formation after stent implantation in a rabbit carotid artery. A stent was implanted in an artery with a pre-existing intimal lesion, and transfer of plasmid containing TRAF6 was performed using an in vivo electroporation method. We believe that this model mimics the process of in-stent lesion formation in the clinical setting, and this study suggests a possible strategy for preventing in-stent restenosis