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Our previous study suggested that heart-type fatty acid-binding protein (HFABP) levels were greatly elevated in the conditioned medium of explant culture of in-stent restenosis (ISR) tissue from diabetic minipigs compared with those of non-ISR tissue. We here verified this result in animal tissues and investigated the impact of HFABP overexpression in human aortic smooth muscle cells (hASMCs).In Western blot and real-time RT-PCR, HFABP protein and mRNA levels were significantly higher in ISR than in non-ISR tissues from minipigs, and higher in the ISR tissue from diabetic minipigs than that from nondiabetic minipigs. The mRNA microarray and cellular effects of hASMC retroviral overexpression of HFABP and vector was analyzed. Compared with vector, HFABP transduction activates multiple signaling pathways (e.g. adipokine, TGF-β, Toll-like receptor, Wnt, Hedgehog, ErbB and Notch) and promotes inflammation, growth and migration in hASMCs whereas the knockdown of HFABP by small hairpin RNA attenuates these effects.HFABP expression is significantly higher in ISR tissue than in non-ISR tissue from diabetic and nondiabetic minipigs. Overexpression of HFABP induces multiple pathway-related promotion of inflammation, growth and migration in vascular SMCs, suggesting a potential role in coronary artery ISR.