Gene silencing of adhesion molecules in venous endothelial cells by non-viral siRNA transfection

Abstract

Objectives: CPB-mediated release of proinflammatory cytokines promotes monocyte transendothelial migration via intercellular adhesion molecules (ICAM) within the venous bypass graft, probably contributing to a diminished early patency rate by hyperplasia of the intima. Non-viral application of short interfering RNA (siRNA) is a powerful technique for post-transcriptional selective knockdown of specific endogenous gene expressions, resulting in a reduced protein synthesis. This study deals with the potential of highly specific siRNA to transiently block leukocyte-endothelial interactions. Material and Methods: Primary cultures of human venous endothelial cells (HVEC) were isolated from saphenous veins by collagenase treatment as previously described. Cells of the third passage were either transfected (reagent: Cellfectin, Invitrogen, Karlsruhe, Germany) with siRNA (100 nM) targeting ICAM-1 or with a scrambled (non targeting) control siRNA (100 nM), or cultured without transfection (n=5). After 9 hours the cells were stimulated with TNF-α for 15 hours to induce expression of ICAM-1. A forth group of HVECs were neither treated with TNF-α nor transfected with siRNA. ICAM-1 receptor expression was analyzed by flow cytometry. Results: The HVECs transfected with ICAM-1 siRNA showed significantly lower expressions of ICAM-1 of 13.06±2.7% compared to 93.18±1.17% in untransfected cells and 79.1±3.9% in cells transfected with the scrambled control siRNA. Cells neither transfected nor stimulated with TNF-α resulted in 1.65±0.6% expression. Conclusions: Non-viral transfection of siRNA effectively protects HVECs against adhesion molecule expression caused by cytokine stimulation. siRNAs tageting ICAMs may offer novel therapeutic concepts to protect new venous bypass grafts against early leukocyte infiltration after CPB causing early obstruction of the venous bypass graft.