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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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Effects of siRNA and TNF treatment on ICAM1, VCAM1 and SELE mRNA levels.Transcript levels were examined by real-time RT-PCR. Each bar represents the mean of at least two independent experiments, error bars show the range of variation in the case of two experiments and standard deviations in the case of three or more experiments. Mock, TNF-stimulated untransfected cells; siICAM1, ICAM1 siRNA; siVCAM1, VCAM1 siRNA; siSELE, E-selectin siRNA.
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fig04: Effects of siRNA and TNF treatment on ICAM1, VCAM1 and SELE mRNA levels.Transcript levels were examined by real-time RT-PCR. Each bar represents the mean of at least two independent experiments, error bars show the range of variation in the case of two experiments and standard deviations in the case of three or more experiments. Mock, TNF-stimulated untransfected cells; siICAM1, ICAM1 siRNA; siVCAM1, VCAM1 siRNA; siSELE, E-selectin siRNA.

Mentions: To assess the effects of siRNA transfection on to ICAM1, VCAM1 and SELE mRNAs, transcript levels were analysed by semi-quantitative real-time RT-PCR. In general, the observed effects of siICAM1, siVCAM1 and siSELE on target protein expression were reflected by changes in the corresponding transcript levels (Fig. 4). All three siRNAs siICAM1, siVCAM1 and siSELE reduced the levels of their corresponding target mRNA 10-fold. In contrast, TNF stimulation of the other two adhesion molecule transcripts was affected only twofold to threefold. Furthermore, the control siRNA siSCR caused a twofold drop in SELE transcript levels and did not affect ICAM1 and VCAM1 levels.


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)

Effects of siRNA and TNF treatment on ICAM1, VCAM1 and SELE mRNA levels.Transcript levels were examined by real-time RT-PCR. Each bar represents the mean of at least two independent experiments, error bars show the range of variation in the case of two experiments and standard deviations in the case of three or more experiments. Mock, TNF-stimulated untransfected cells; siICAM1, ICAM1 siRNA; siVCAM1, VCAM1 siRNA; siSELE, E-selectin siRNA.
© Copyright Policy
Related In: Results  -  Collection

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

fig04: Effects of siRNA and TNF treatment on ICAM1, VCAM1 and SELE mRNA levels.Transcript levels were examined by real-time RT-PCR. Each bar represents the mean of at least two independent experiments, error bars show the range of variation in the case of two experiments and standard deviations in the case of three or more experiments. Mock, TNF-stimulated untransfected cells; siICAM1, ICAM1 siRNA; siVCAM1, VCAM1 siRNA; siSELE, E-selectin siRNA.
Mentions: To assess the effects of siRNA transfection on to ICAM1, VCAM1 and SELE mRNAs, transcript levels were analysed by semi-quantitative real-time RT-PCR. In general, the observed effects of siICAM1, siVCAM1 and siSELE on target protein expression were reflected by changes in the corresponding transcript levels (Fig. 4). All three siRNAs siICAM1, siVCAM1 and siSELE reduced the levels of their corresponding target mRNA 10-fold. In contrast, TNF stimulation of the other two adhesion molecule transcripts was affected only twofold to threefold. Furthermore, the control siRNA siSCR caused a twofold drop in SELE transcript levels and did not affect ICAM1 and VCAM1 levels.

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