title: Inhibition of adhesion molecule expression on human venous endothelial cells by non-viral siRNA transfection.
abstract: OBJECTIVE
  Expression of adhesion molecule receptors on venous endothelial cells crucially influences the fate of venous grafts by mediating leukocyte-endothelium interactions. These interactions include adhesion of leukocytes to the endothelium, followed by transendothelial migration, leading to neointimal hyperplasia (NIH) and finally graft occlusion. Therefore, inhibition of adhesion molecule expression may be a promising strategy to improve the quality of venous grafts. We tested the efficiency of non-viral transfection of human venous endothelial cells (HVEC) with short interfering RNA (siRNA) to specifically down-regulate adhesion molecule expression.
  
  
  METHODS
  Primary cultures of HVEC were examined for expression of the adhesion molecules ICAM1, VCAM1 and E-selectin (SELE) after non viral siRNA transfection. Adhesion molecule expression was measured by flow cytometry, real-time polymerase chain reaction and immunoblotting after stimulation with TNF-alpha, an inflammatory cytokine.
  
  
  RESULTS
  Non-transfected cells showed a strong increase of adhesion molecule expression following cytokine stimulation (P < 0.01). Upon transfection with specific siRNAs a sixfold decrease in ICAM1 (P < 0.001) and SELE expression and cell positivity (P < 0.05) and a twofold decrease in VCAM1 expression and cell positivity (P < 0.01) P could be observed. SiRNA-mediated gene suppression of adhesion molecules was also reflected by corresponding decreases in adhesion protein and transcript levels.
  
  
  CONCLUSIONS
  The expression of adhesion molecules on HVECs can be effectively inhibited by specific siRNAs using a safe, non-viral transfection approach. This is a promising tool to pre-condition venous bypass grafts in order to interfere with endothelium-leukocyte interactions and to prohibit neointima thickening or atherosclerosis, which are regarded to be the most important causes of venous graft failure.
authors: Department of Thoracic, Cardiac and Vascular Surgery, Tuebingen University Hospital, Tuebingen, Germany. tobias.walker@med.uni-tuebingen.de
authors: Tobias
authors: T
authors: Walker
authors: Hans P
authors: HP
authors: Wendel
authors: Liane
authors: L
authors: Tetzloff
authors: Claudia
authors: C
authors: Raabe
authors: Olaf
authors: O
authors: Heidenreich
authors: Perikles
authors: P
authors: Simon
authors: Albertus M
authors: AM
authors: Scheule
authors: Gerhard
authors: G
authors: Ziemer
mesh_terms: D015815:Cell Adhesion Molecules / Q000037:antagonists & inhibitors*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000235:genetics*; D012501:Saphenous Vein / Q000166:cytology / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014680:Veins / Q000166:cytology* / Q000378:metabolism
chemical_list: D015815:Cell Adhesion Molecules; D005456:Fluorescent Dyes; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; D016650:Fluorescein-5-isothiocyanate