Two-Dimensional Fluorescence In-Gel Electrophoresis of Coronary Restenosis Tissues in Minipigs

Abstract

Objective— We aimed to uncover the protein changes of coronary artery in-stent restenosis (ISR) tissue in minipigs with and without streptozotocin-induced diabetes mellitus by quantitative 2-dimensional fluorescence in-gel electrophoresis (2D-DIGE), and to investigate the influences of crucial proteins identified, particularly adipocyte fatty acid binding protein (AFABP), in human arterial smooth muscle cells. Methods and Results— Sirolimus-eluting stents were implanted in the coronary arteries of 15 diabetic and 26 nondiabetic minipigs, and angiography was repeated after 6 months. The intima tissue of significant ISR and non-ISR segments in both diabetic and nondiabetic minipigs was analyzed by 2D-DIGE and MALDI-TOF/TOF mass spectrometry. AFABP level was significantly increased in ISR tissue than in non-ISR tissue in both diabetic and nondiabetic minipigs, with level being higher in diabetic ISR than in nondiabetic ISR tissue. In human arterial smooth muscle cells, overexpression of AFABP significantly altered phenotype and promoted growth and migration, with effects more prominent in high-glucose than in low-glucose medium, whereas AFABP knockdown inhibited these effects. AFABP overexpression increased reactive oxygen species production by upregulating the expression of NADPH oxidase subunits Nox1, Nox4, and P22 through multiple pathways, with elevation of downstream gene cyclin D1, matrix metalloproteinase-2, and monocyte chemoattractant protein-1. However, AFABP-induced effects were inhibited by diphenyleneiodonium, pathway inhibitors, and small interfering RNA. In addition, the supernatant from AFABP-expressing human arterial smooth muscle cells and recombinant AFABP also promoted cellular growth and migration. Conclusion— This study has demonstrated that AFABP is significantly increased in coronary artery ISR segments of both diabetic and nondiabetic minipigs. Increased AFABP expression and secretory AFABP of human arterial smooth muscle cells promote growth and migration via reactive oxygen species-mediated activation.