Inhibition of adhesion molecule expression on human venous endothelial cells by non-viral siRNA transfection.
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.
Walker
Tobias
T
Department of Thoracic, Cardiac and Vascular Surgery, Tuebingen University Hospital, Tuebingen, Germany. tobias.walker@med.uni-tuebingen.de
Wendel
Hans P
HP
Tetzloff
Liane
L
Raabe
Claudia
C
Heidenreich
Olaf
O
Simon
Perikles
P
Scheule
Albertus M
AM
Ziemer
Gerhard
G
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
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