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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 several siRNA sequences and TNF treatment on the fraction of cells positive for VCAM1 and SELE. Each bar represents the mean of three independent experiments, error bars show standard deviations. (A) Effects of four different sequences (S1–S4) siRNA targeting SELE: S1: sense: (GGU UGA AUG CAC CAC UCA A)dTdT antisense: (UUG AGU GGU GCA UUC AAC C)dTdG S2: sense: (UGG UAG AAU UGG AGA GUA A) dTdT antisense: (UUA CUC UCC AAU UCU ACC A) dTdG S3: sense: (CAG UGU GGU UUG UGU UUG A)dTdT antisense: (UCA AAC ACA AAC CAC ACU G) dGdT S4: sense: (CGG AAG CUA UGA CUU AUG A)dTdT antisense: (UCA UAA GUC AUA GCU UCC G)dTdG (B) Effects of five different sequences (S1–S5) siRNA targeting VCAM1: S1: sense: (GGA GGA UAC GGA UAU GAA A)dTdT antisense: (UUU CAU AUC CGU AUC CUC C)dAdA S2: sense: (GAG CUA AAU UAC ACA UUG A)dTdT antisense: (UCA AUG UGU AAU UUA GCU C)dGdG S3: sense: (CAU CUA CGC UGA CAA UGA A)dTdT antisense: (UUC AUU GCU AGC GUA GAU G)dTdG S4: sense: (CUC UAU AUU UAG AUU GUU A)dTdT antisense: (UAA CAA UCU AAA UAU AGA G)dTdG S5: sense: (AAU GCA ACU CUC ACC UUA A)dTdT antisense: (UUA AGG UGA GAG UUG CAU U)dTdT
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fig01: Effects of several siRNA sequences and TNF treatment on the fraction of cells positive for VCAM1 and SELE. Each bar represents the mean of three independent experiments, error bars show standard deviations. (A) Effects of four different sequences (S1–S4) siRNA targeting SELE: S1: sense: (GGU UGA AUG CAC CAC UCA A)dTdT antisense: (UUG AGU GGU GCA UUC AAC C)dTdG S2: sense: (UGG UAG AAU UGG AGA GUA A) dTdT antisense: (UUA CUC UCC AAU UCU ACC A) dTdG S3: sense: (CAG UGU GGU UUG UGU UUG A)dTdT antisense: (UCA AAC ACA AAC CAC ACU G) dGdT S4: sense: (CGG AAG CUA UGA CUU AUG A)dTdT antisense: (UCA UAA GUC AUA GCU UCC G)dTdG (B) Effects of five different sequences (S1–S5) siRNA targeting VCAM1: S1: sense: (GGA GGA UAC GGA UAU GAA A)dTdT antisense: (UUU CAU AUC CGU AUC CUC C)dAdA S2: sense: (GAG CUA AAU UAC ACA UUG A)dTdT antisense: (UCA AUG UGU AAU UUA GCU C)dGdG S3: sense: (CAU CUA CGC UGA CAA UGA A)dTdT antisense: (UUC AUU GCU AGC GUA GAU G)dTdG S4: sense: (CUC UAU AUU UAG AUU GUU A)dTdT antisense: (UAA CAA UCU AAA UAU AGA G)dTdG S5: sense: (AAU GCA ACU CUC ACC UUA A)dTdT antisense: (UUA AGG UGA GAG UUG CAU U)dTdT

Mentions: First steps within the silencing experiments were to find out from several siRNA sequences those with the highest knockdown potency. For SELE we used four and for VCAM1 five different sequences, respectively (Fig. 1). Sequence 1 (Fig. 1A) showed the highest knockdown for SELE and sequence 5 (Fig. 1B) the best results for VCAM1.Therefore, these siRNA sequences were used for all further experiments. For ICAM1 we used a previously tested sequence [17].


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 several siRNA sequences and TNF treatment on the fraction of cells positive for VCAM1 and SELE. Each bar represents the mean of three independent experiments, error bars show standard deviations. (A) Effects of four different sequences (S1–S4) siRNA targeting SELE: S1: sense: (GGU UGA AUG CAC CAC UCA A)dTdT antisense: (UUG AGU GGU GCA UUC AAC C)dTdG S2: sense: (UGG UAG AAU UGG AGA GUA A) dTdT antisense: (UUA CUC UCC AAU UCU ACC A) dTdG S3: sense: (CAG UGU GGU UUG UGU UUG A)dTdT antisense: (UCA AAC ACA AAC CAC ACU G) dGdT S4: sense: (CGG AAG CUA UGA CUU AUG A)dTdT antisense: (UCA UAA GUC AUA GCU UCC G)dTdG (B) Effects of five different sequences (S1–S5) siRNA targeting VCAM1: S1: sense: (GGA GGA UAC GGA UAU GAA A)dTdT antisense: (UUU CAU AUC CGU AUC CUC C)dAdA S2: sense: (GAG CUA AAU UAC ACA UUG A)dTdT antisense: (UCA AUG UGU AAU UUA GCU C)dGdG S3: sense: (CAU CUA CGC UGA CAA UGA A)dTdT antisense: (UUC AUU GCU AGC GUA GAU G)dTdG S4: sense: (CUC UAU AUU UAG AUU GUU A)dTdT antisense: (UAA CAA UCU AAA UAU AGA G)dTdG S5: sense: (AAU GCA ACU CUC ACC UUA A)dTdT antisense: (UUA AGG UGA GAG UUG CAU U)dTdT
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getmorefigures.php?uid=PMC4401227&req=5

fig01: Effects of several siRNA sequences and TNF treatment on the fraction of cells positive for VCAM1 and SELE. Each bar represents the mean of three independent experiments, error bars show standard deviations. (A) Effects of four different sequences (S1–S4) siRNA targeting SELE: S1: sense: (GGU UGA AUG CAC CAC UCA A)dTdT antisense: (UUG AGU GGU GCA UUC AAC C)dTdG S2: sense: (UGG UAG AAU UGG AGA GUA A) dTdT antisense: (UUA CUC UCC AAU UCU ACC A) dTdG S3: sense: (CAG UGU GGU UUG UGU UUG A)dTdT antisense: (UCA AAC ACA AAC CAC ACU G) dGdT S4: sense: (CGG AAG CUA UGA CUU AUG A)dTdT antisense: (UCA UAA GUC AUA GCU UCC G)dTdG (B) Effects of five different sequences (S1–S5) siRNA targeting VCAM1: S1: sense: (GGA GGA UAC GGA UAU GAA A)dTdT antisense: (UUU CAU AUC CGU AUC CUC C)dAdA S2: sense: (GAG CUA AAU UAC ACA UUG A)dTdT antisense: (UCA AUG UGU AAU UUA GCU C)dGdG S3: sense: (CAU CUA CGC UGA CAA UGA A)dTdT antisense: (UUC AUU GCU AGC GUA GAU G)dTdG S4: sense: (CUC UAU AUU UAG AUU GUU A)dTdT antisense: (UAA CAA UCU AAA UAU AGA G)dTdG S5: sense: (AAU GCA ACU CUC ACC UUA A)dTdT antisense: (UUA AGG UGA GAG UUG CAU U)dTdT
Mentions: First steps within the silencing experiments were to find out from several siRNA sequences those with the highest knockdown potency. For SELE we used four and for VCAM1 five different sequences, respectively (Fig. 1). Sequence 1 (Fig. 1A) showed the highest knockdown for SELE and sequence 5 (Fig. 1B) the best results for VCAM1.Therefore, these siRNA sequences were used for all further experiments. For ICAM1 we used a previously tested sequence [17].

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