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Inhibition of adhesion molecule expression on human venous endothelial cells by non-viral siRNA transfection.

Walker T, Wendel HP, Tetzloff L, Raabe C, Heidenreich O, Simon P, Scheule AM, Ziemer G - J. Cell. Mol. Med. (2007 Jan-Feb)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Thoracic, Cardiac and Vascular Surgery, Tuebingen University Hospital, Tuebingen, Germany. tobias.walker@med.uni-tuebingen.de

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.

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siRNA-dependent effects on adhesion protein levels. Adhesion protein levels were analysed by immunoblotting as described in ‘Material and methods’ section. Cell treatment is shown on top of each blot, the analysed adhesion proteins are indicated on the left. Uninduced, untreated cells; TNF, TNF-stimulated cells; Mock, TNF-stimulated untransfected cells; siICAM1, ICAM1 siRNA; siVCAM1, VCAM1 siRNA; siSELE, E-selectin siRNA.
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fig03: siRNA-dependent effects on adhesion protein levels. Adhesion protein levels were analysed by immunoblotting as described in ‘Material and methods’ section. Cell treatment is shown on top of each blot, the analysed adhesion proteins are indicated on the left. Uninduced, untreated cells; TNF, TNF-stimulated cells; Mock, TNF-stimulated untransfected cells; siICAM1, ICAM1 siRNA; siVCAM1, VCAM1 siRNA; siSELE, E-selectin siRNA.

Mentions: The Western blot confirmed the FACS results for total adhesion molecule expression (Fig. 3). In all three groups (ICAM1, VCAM1 and SELE) only a weak staining could be observed in the untransfected and non-stimulated cells. After TNF treatment the protein detection was strong. The group of specific siRNA treated cells showed only slight bands after TNF treatment comparable to the group of untransfected and unstimulated cells. In contrast, application of the scramble control siRNA siSCR did not substantially affect adhesion protein levels, when compared to TNF-stimulated untransfected cells.


Inhibition of adhesion molecule expression on human venous endothelial cells by non-viral siRNA transfection.

Walker T, Wendel HP, Tetzloff L, Raabe C, Heidenreich O, Simon P, Scheule AM, Ziemer G - J. Cell. Mol. Med. (2007 Jan-Feb)

siRNA-dependent effects on adhesion protein levels. Adhesion protein levels were analysed by immunoblotting as described in ‘Material and methods’ section. Cell treatment is shown on top of each blot, the analysed adhesion proteins are indicated on the left. Uninduced, untreated cells; TNF, TNF-stimulated cells; Mock, TNF-stimulated untransfected cells; siICAM1, ICAM1 siRNA; siVCAM1, VCAM1 siRNA; siSELE, E-selectin siRNA.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4401227&req=5

fig03: siRNA-dependent effects on adhesion protein levels. Adhesion protein levels were analysed by immunoblotting as described in ‘Material and methods’ section. Cell treatment is shown on top of each blot, the analysed adhesion proteins are indicated on the left. Uninduced, untreated cells; TNF, TNF-stimulated cells; Mock, TNF-stimulated untransfected cells; siICAM1, ICAM1 siRNA; siVCAM1, VCAM1 siRNA; siSELE, E-selectin siRNA.
Mentions: The Western blot confirmed the FACS results for total adhesion molecule expression (Fig. 3). In all three groups (ICAM1, VCAM1 and SELE) only a weak staining could be observed in the untransfected and non-stimulated cells. After TNF treatment the protein detection was strong. The group of specific siRNA treated cells showed only slight bands after TNF treatment comparable to the group of untransfected and unstimulated cells. In contrast, application of the scramble control siRNA siSCR did not substantially affect adhesion protein levels, when compared to TNF-stimulated untransfected cells.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Thoracic, Cardiac and Vascular Surgery, Tuebingen University Hospital, Tuebingen, Germany. tobias.walker@med.uni-tuebingen.de

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.

Show MeSH
Related in: MedlinePlus