Beclin 1 knockdown retards re-endothelialization and exacerbates neointimal formation via a crosstalk between autophagy and apoptosis

Abstract

ObjectiveEndothelial regeneration is an essential process for the prevention of excessive neointimal formation following endothelial denudation. Beclin 1, a mammalian autophagy gene, is a link between autophagy and apoptosis. We hypothesized that the interference of Beclin 1 can influence re-endothelialization and ultimately affect neointimal formation by regulating autophagy and apoptosis. MethodsA rat carotid injury model of endothelial denudation was used, and small interfering RNA of Beclin 1 was perivascularly administered. Neointima was evaluated by morphological analysis. von Willebrand factor, Beclin 1, LC3, autophagic substrate p62 and caspase-3 levels were detected by immunofluorescence or Western blotting. Terminal deoxynucleotidyl transferase-mediated digoxigenin–dUTP–biotin nick-end labeling assay was performed to evaluate apoptosis. ResultsCarotid injury induced an upregulation of Beclin 1 protein which was down regulated by more than 50% with small RNA interference. Beclin 1 knockdown significantly retarded re-endothelialization 7 days after injury and subsequently augmented neointima by more than 2 folds at 14 and 21 days. Autophagy and apoptosis were detected to reveal the regulatory effect of Beclin 1. The injury-activated autophagy, shown by the increased levels of punctate LC3 and LC3II as well as decreased p62 expression, was significantly inhibited by Beclin 1 knockdown. Meanwhile, the apoptotic endothelial cell number was increased and caspase-3 was up-regulated, though the expression of truncated BID was not significantly influenced. ConclusionBeclin 1 knockdown exacerbated neointimal formation after rat carotid injury, associated with retarded re-endothelialization due to enhanced apoptosis, while simultaneously prohibiting autophagic activation. The data suggested an essential role of Beclin 1 as a regulator between autophagy and apoptosis in the setting of neointimal formation.