title: Lentiviral-mediated overexpression of Bcl-xL protects primary endothelial cells from ischemia/reperfusion injury-induced apoptosis.
abstract: BACKGROUND
  Endothelial cells (EC) respond to mild injurious stimuli by upregulating anti-apoptotic gene expression to maintain endothelial integrity. EC dysfunction and apoptosis resulting from ischemia/reperfusion injury may contribute to chronic allograft rejection. We optimized conditions for lentiviral vector (LVV) transduction of rat aortic endothelial cells (RAEC) and investigated whether LVV delivery of the anti-apoptotic gene, Bcl-xL, protects RAEC from apoptotic death using in vitro models of hypoxia and ischemia/reperfusion injury.
  
  
  METHODS
  LVV containing Bcl-xL were generated from a human immunodeficiency virus (HIV)-1 construct. EC were prepared from rat aorta. Hypoxia/reperfusion (H/R) or ischemia/reperfusion (I/R) injury was induced in vitro and apoptosis was assessed using caspase-3 activity, Annexin V/PI and TUNEL staining.
  
  
  RESULTS
  After in vitro induction of H/R or I/R injury, RAEC showed duration-dependent apoptosis. We confirmed the damaging effect of the reperfusion phase. Endogenous Bax expression increased with I/R injury, whereas endogenous Bcl-xL remained constant. RAEC transduced with LVV expressing Bcl-xL were protected from early apoptosis caused by I/R injury, correlating with reduced cytochrome c release into the cytosol.
  
  
  CONCLUSIONS
  Overexpressing Bcl-xL protects RAEC from I/R injury. This protective effect may be attributed to altering the balance of pro- and anti-apoptotic proteins, resulting in sequestration of the harmful Bax protein, and may open up new strategies for controlling chronic allograft rejection.
authors.affiliation: Department of Medicine, Addenbrooke's Hospital, University of Cambridge, Cambridge, UK.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhao
authors.forename: Eleanor Mary
authors.initials: EM
authors.lastname: Bolton
authors.forename: John Andrew
authors.initials: JA
authors.lastname: Bradley
authors.forename: Andrew Michael Lindsay
authors.initials: AM
authors.lastname: Lever
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology / Q000502:physiology / Q000821:virology; D017209:Apoptosis; D004195:Disease Models, Animal; D004730:Endothelium, Vascular / Q000166:cytology / Q000293:injuries / Q000502:physiology / Q000821:virology; D005434:Flow Cytometry; D017353:Gene Deletion; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D000860:Hypoxia / Q000503:physiopathology; D016086:Lentivirus / Q000235:genetics*; D010957:Plasmids; D051381:Rats; D015427:Reperfusion Injury / Q000235:genetics / Q000517:prevention & control*; D014162:Transfection / Q000379:methods; D014764:Viral Proteins / Q000235:genetics; D051020:bcl-X Protein / Q000235:genetics*
chemical_list: D014764:Viral Proteins; D051020:bcl-X Protein