title: Beclin 1 knockdown retards re-endothelialization and exacerbates neointimal formation via a crosstalk between autophagy and apoptosis.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Neurology, Guangdong Key Laboratory for Diagnosis and Treatment of Major Neurological Diseases, National Key Clinical Department, National Key Discipline, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.
authors.forename: Lan-Xiang
authors.initials: LX
authors.lastname: Ye
authors.affiliation: Department of Neurology, Guangdong Key Laboratory for Diagnosis and Treatment of Major Neurological Diseases, National Key Clinical Department, National Key Discipline, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Yu
authors.affiliation: Department of Neurology, Guangdong Key Laboratory for Diagnosis and Treatment of Major Neurological Diseases, National Key Clinical Department, National Key Discipline, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.
authors.forename: Yin-Xing
authors.initials: YX
authors.lastname: Liang
authors.affiliation: Department of Neurology, Guangdong Key Laboratory for Diagnosis and Treatment of Major Neurological Diseases, National Key Clinical Department, National Key Discipline, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.
authors.forename: Jin-Sheng
authors.initials: JS
authors.lastname: Zeng
authors.affiliation: Department of Neurology, Guangdong Key Laboratory for Diagnosis and Treatment of Major Neurological Diseases, National Key Clinical Department, National Key Discipline, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.
authors.forename: Ru-Xun
authors.initials: RX
authors.lastname: Huang
authors.affiliation: Department of Neurology, Guangdong Key Laboratory for Diagnosis and Treatment of Major Neurological Diseases, National Key Clinical Department, National Key Discipline, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. Electronic address: sj_lbbb@163.com.
authors.forename: Song-Jie
authors.initials: SJ
authors.lastname: Liao
mesh_terms: D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D001343:Autophagy; D000071186:Beclin-1; D020212:Carotid Artery Injuries / Q000473:pathology*; D053148:Caspase 3 / Q000378:metabolism; D004730:Endothelium, Vascular / Q000473:pathology; D005786:Gene Expression Regulation; D006360:Heat-Shock Proteins / Q000378:metabolism; D008297:Male; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D058426:Neointima; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D000071456:Sequestosome-1 Protein; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; D000071186:Beclin-1; D053148:Caspase 3; D006360:Heat-Shock Proteins; D008869:Microtubule-Associated Proteins; D034741:RNA, Small Interfering; D000071456:Sequestosome-1 Protein; D014841:von Willebrand Factor; C512829:Becn1 protein, rat; C526109:Anxa3 protein, rat; C491079:Sqstm1 protein, rat; C505410:Casp3 protein, rat
keywords: Apoptosis; Autophagy; Beclin 1; Neointima; Re-endothelialization