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

Atherosclerosis. 2014 Nov;237(1):146-54. doi: 10.1016/j.atherosclerosis.2014.08.052. Epub 2014 Sep 6.

Abstract

Objective: Endothelial 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.

Methods: A 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.

Results: Carotid 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.

Conclusion: Beclin 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.

Keywords: Apoptosis; Autophagy; Beclin 1; Neointima; Re-endothelialization.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis
  • Apoptosis Regulatory Proteins / genetics
  • Apoptosis Regulatory Proteins / metabolism*
  • Autophagy*
  • Beclin-1
  • Carotid Artery Injuries / pathology*
  • Caspase 3 / metabolism
  • Endothelium, Vascular / pathology
  • Gene Expression Regulation*
  • Heat-Shock Proteins / metabolism
  • Male
  • Microtubule-Associated Proteins / metabolism
  • Neointima*
  • RNA, Small Interfering / metabolism
  • Rats
  • Rats, Sprague-Dawley
  • Sequestosome-1 Protein
  • von Willebrand Factor / metabolism

Substances

  • Apoptosis Regulatory Proteins
  • Beclin-1
  • Becn1 protein, rat
  • Heat-Shock Proteins
  • LC3 protein, rat
  • Microtubule-Associated Proteins
  • RNA, Small Interfering
  • Sequestosome-1 Protein
  • Sqstm1 protein, rat
  • von Willebrand Factor
  • Casp3 protein, rat
  • Caspase 3