Abstract 128: Ubiquitin Carboxyl-terminal Hydrolase L1, a Novel Deubiquitinating Enzyme in the Vasculature, Attenuates NF-κB Activation and Plays Pivotal Roles in Vascular Remodeling
We identified a ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) gene, which encodes a deubiquitinating enzyme and is expressed in the vasculature, by functional screening of a human endothelial cell (EC) cDNA library for searching for objective genes with anti-inflammatory action. UCHL1 has been known to be specifically expressed in neurons, and abnormalities in UCHL1 are responsible for inherited Parkinson’s disease via its effects on the ubiquitin-proteasome system. Therefore, the goal of present study was to clarify the role of UCHL1 in vascular remodeling by evaluating NF-κB inactivation in in vitro and in vivo. From Northern blot and immunohistochemical analysis, UCHL1 was endogenously expressed in vascular EC, smooth muscle cells (VSMC), and brain tissue. In atherosclerotic tissue, expression of UCHL1 was markedly increased in the neointima of the balloon-injured common carotid arteries (CCA) (25-fold compared with sham operated common carotid arteries, P<0.05), and was also present in atherosclerotic lesions from human carotid arteries. In an in vitro system, tumor necrosis factor (TNF)-α significantly increased UCHL1 gene expression in VSMC (3.6±0.25 fold, P<0.0001). Over-expression of UCHL1 significantly attenuated TNF-α-induced NF-κB activity in vascular cells and increased inhibitor of kappa B-α(IκB-α) and eNOS protein levels in EC. Of importance, as a deubiquitinating enzyme, UCHL1 attnueted ubiquitination of IκB-α, and thus inhibited the translocation of p65 to nucleus induced by TNF-α. In contrast, knockdown of UCHL1 by small interfering RNA resulted in increased NF-κB activity in VSMC. Accordingly, over-expression of UCHL1 significantly attenuated TNF-α-induced NF-κB-regulated downstream gene expressions such as adhesion molecules and inflammatory cytokines in vascular cells. Furthermore, in vivo gene transfer of UCHL1 into balloon-injuread rat CCA showed attenuation of activated p50 and p65, ICAM-1, and MMP-9 expressed in the neointima 7days after procedure, and inhibition of neointimal formation 14 days after balloon injury (intima to media ratio: control, 1.88 ± 0.44; UCHL1, 1.16 ± 0.24, P<0.001). These data suggest that UCHL1 might partially attenuate vascular remodeling through inhibition of NF-κB activity.