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Eur J Cardiothorac Surg 2005;28:816-820
© 2005 Elsevier Science NL
a Department of Thoracic, Cardiac and Vascular Surgery, Tuebingen University Hospital, Hoppe-Seyler-Straße 3, 72076 Tuebingen, Germany
b Department of Molecular Biology, Tuebingen University, Auf der Morgenstelle 5, 72076 Tuebingen, Germany
Received 1 April 2005; received in revised form 12 September 2005; accepted 15 September 2005.
* Corresponding author. Tel.: +49 7071 2986611; fax: +49 7071 293298. (Email: tobias.walker{at}med.uni-tuebingen.de).
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Abstract |
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 Top Abstract 1. Introduction2. Materials and techniques3. Results4. CommentReferences |
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stimulation by flow cytometry. Results: Upon TNF- stimulation, cells transfected with ICAM-1 siRNA showed a six- to seven-fold decreased ICAM-1 expression compared to untransfected cells or cells transfected with the scrambled control siRNA. Conclusions: This is the first report that ICAM-1 expression can be effectively silenced by siRNAs on endothelial cells from human saphenous veins. This new technology may render novel therapeutic concepts to reduce early graft failure by protecting venous bypass grafts against early intra- or postoperative leukocyte infiltration.
Key Words: CABG • Venous grafts • Endothelium • Gene therapy
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1. Introduction |
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TopAbstract1. Introduction2. Materials and techniques3. Results4. CommentReferences |
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Several hypotheses exist accounting for the failure of bypass material. First, reduced bypass flow or limited diameter of the native vessel often causes an early occlusion. Second, technical factors as narrowed anastomosis are involved. However, a more significant alteration can be observed in the venous graft after implantation, namely a neointimal thickening comparable to early lesions seen in atherosclerotic altered vessels [3,4]. Particularly, the intima thickening may be the cause of an early failure of the venous graft. Alternatively, it may be the basis for later development of graft atheroma. Recent investigations demonstrated a close relationship between the endothelial dysfunction and various different stimuli caused by surgery employing cardiopulmonary bypass, all leading to an activation of the endothelium. In consequence, the activated endothelium layer expresses different receptors on the cell surface, enabling leukocytes adherence and penetration through the endothelium. Consecutive reactions like the release of oxygen free radicals and cytotoxic mediators are leading to a stimulation of vascular small muscle cells, and finally, to hyperplasia and migration to the intima layer [5].
Presuming the above-mentioned different factors, the expression of endothelial adhesion molecules can be accentuated as one of the central mediators leading to an intima thickening. They represent a group of different glycoproteins and carbohydrates expressed on the surface of a wide variety of cell types. Within the pathomechanism of intima thickening the intercellular adhesion molecule 1 (ICAM-1) seems to play a central role. ICAM-1, also known as CD54, is expressed on different cell types such as leukocytes, endothelial, and epithelial cells. One of the major functions of this surface glycoprotein is the modulation of a firm adhesion of leukocytes to the endothelium. This step is essential for the later transendothelial diapedesis of the cells.
Small interfering ribonucleic acids (siRNAs) are short double-stranded oligoribonucleotides of 21–25 nucleotides in length, which have been originally identified as intermediates of the RNA interference pathway.
For a practical use the sequence of the targeted gene has to be identified in a gene library. Homologous to the gene-sequence double stranded siRNA is manufactured by commercial providers. SiRNAs may be either directly applied by cell transfection techniques or intracellularly transcribed as small hairpin RNAs from genes inserted by, for instance, lentiviral transduction into the cell genome. In the cytoplasm, one strand of the siRNA becomes part of a ribonucleoprotein complex called RNA-induced silencing complex (RISC). RISC can cleave RNA sequences complementary to the siRNA strand, thereby causing the rapid degradation of the target mRNA. Importantly, and in contrast to longer double-stranded RNAs, siRNAs are in most cases inefficient inducers of an interferon response leading to a general cytotoxicity [6]. However, some siRNAs have been shown to not only inhibit the expression of unintended targets, but also to induce a limited interferon response of the transfected cell in spite of their small size.
Recently, siRNAs have been used to diminish E-selectin expression in human umbilical vein endothelial cells (ECs) resulting in a reduced leukocyte adhesion [7].
The aim of the present study was to evaluate the potency of siRNAs to transiently block ICAM-1 receptor expression in cultured primary venous endothelial cells from human vena saphena magna specimens.
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2. Materials and techniques |
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TopAbstract1. Introduction2. Materials and techniques3. Results4. CommentReferences |
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Isolation and cultivation of endothelial cells from saphenous vein specimens: harvesting and culture of cells were in accordance with other publications [8,9].
All culture plates and flasks were coated overnight with collagen (40%) (Collagen G, Biochrom, IN, USA). After incubation of the vein in RPMI 1640 (containing 0.5%/ml gentamycin) it was rinsed with a buffer solution (137 mM NaCl/5.4 mM KCl/4.2 mM NaHCO3/5 mM D-glucose in 500 ml H2O, pH 7.3, sterile). Endothelial cells were harvested by collagenase (0.1% in PBS, PAA Laboratories GmbH, Cölbe, Germany) and further cultured in EGM-2 (+bullet kit, Cambrex Bio Science Verviers, S.p.r.l., Verviers, Belgium). The cells were splitted after reaching confluence. For all experiments cells from the third passage were used. Purity of the human venous endothelial cells (HVECs) was controlled by staining with a FITC-labeled antibody for human VEGFR-2 and CD 31 (Becton Dickinson GmbH, Heidelberg, Germany).
2.2 siRNA transfections of HVECS
The ICAM-1 siRNA sequences were 5'-GCCUCAGCACGUACCUCUAdTdT-3' (sense) and 5'-UAGAGGUACGUGCUGAGGCdTdT-3' (antisense)[10]; the scrambled siRNA sequences were 5'-UUCUCCGAACGUGUCACGUdTdT-3'(sense) and 5'-ACGUGACACGUUCGAGAAdTdT-3' (antisense). SiRNAs were purchased from CureVac (Tuebingen, Germany) or from Qiagen (Hilden, Germany).
For siRNA transfections, HVECs were cultured in collagen-coated 12-well plates without antibiotics. After reaching 70–80% confluence, HVECs were transfected with 100 nM of siRNA using 2.0 µg/ml Cellfectin (Invitrogen GmbH, Karlsruhe, Germany) for 2 h in serum-free medium followed by the replacement of the supernatants with EGM-1. Nine hours later, HVECs were stimulated with 2.5 ng/ml TNF- (Immunotools, Friesoythe, Germany) for 15 h followed by FACS analysis.
2.3 FACS-analysis
After washing HVECs with EGM-2 media, unspecific binding of antibodies was blocked by incubation in 0.5% FCS. A PE-labeled antibody against human ICAM-1 (Becton Dickinson GmbH) was used to stain the HVECs (4 °C, 1 h). After washing and detaching, the cells were fixed with 2.5% paraformaldehyde in PBS. Flowcytometric analyses (5000 cells/measurement) were performed in a FACScanTM (Becton Dickinson GmbH) and evaluated with the CellQuestPro software (Becton Dickinson GmbH). The results shown represent the averages of three independent experiments. All experiments were performed with cells obtained from the same primary cell culture.
2.4 Statistical procedure
The results were expressed as mean (M) ± standard deviation (SD). Statistical analysis was performed by the statistics software package SPSS (SPSS Software Inc., Chicago, USA). Statistical significance of differences between the groups was examined by ANOVA.
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3. Results |
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TopAbstract1. Introduction2. Materials and techniques3. Results4. CommentReferences |
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concentrations showed a dose-dependent induction of ICAM-1 expression (Fig. 1
). Upon treatment with 78 pg/ml TNF- the percentage of ICAM-1 positive cells raised from less than 2% to 50%, and treatment with 2.5 ng/ml caused a raise to 80%. A further increase of the TNF- concentrations resulted only in a minor increase of ICAM-1 expression as judged by flow cytometry analysis. Therefore, we chose a TNF- concentration of 2.5 ng/ml for ICAM-1 induction for further experiments.
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induced ICAM-1 expression in HVECS. For that, prior to TNF- stimulation, we transfected HVECs either with ICAM-1 siRNA or with a scrambled siRNA serving as sequence-unrelated control.
In this set of experiments, TNF- stimulation of nontransfected HVECs induced ICAM-1 expression in 93 ± 1.2% of cells compared to only 1.7 ± 0.60% positive cells without TNF- stimulation (Fig. 2
). Transfection with the scrambled siRNA hardly affected the extent of ICAM-1 expression (79 ± 3.9%). However, transfection with ICAM-1 siRNA decreased the percentage of stimulated ICAM-1 positive cells to 13 ± 2.7% (p
< 0.0001 compared to nontransfected cells and to scrambled siRNA transfected cells). The decrease in ICAM-1 positive cells was paralleled by a reduction of relative expression levels of ICAM-1 upon ICAM-1 siRNA treatment (Fig. 3
). Whereas TNF- stimulation caused a 13-fold increase of ICAM-1 expression in untransfected cells, and an eight-fold increase in cells transfected with control siRNA, ICAM-1 siRNA treated cells showed only a two-fold increase in ICAM-1 expression (p
< 0.01) compared to unstimulated cells.
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4. Comment |
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TopAbstract1. Introduction2. Materials and techniques3. Results4. CommentReferences |
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to induce adhesion molecule expression; definitely, it can be provoked by others as well. One of the central adhesion molecules that is responsible for transendothelial leukocyte migration is ICAM-1. Zou and coworkers [12] observed a 30–50% reduction of neointimal hyperplasia in ICAM-1 deficient mice in comparison to wild types. These results support the hypothesis that silencing of ICAM-1 expression in venous bypass material may represent a promising approach to reduce the intima thickening that may represent a first step in an occlusion of the venous coronary bypass.
The recent discovery of RNA interference by Tuschl and coworkers [13] opens new techniques with high potential for non-viral and transient gene therapeutic therapies.
This study describes the inhibition of ICAM-1 expression by application of specific siRNA. Cultures of endothelial cells from human saphenous veins were stimulated with TNF-.
In the present study, FACS-analyses showed an ICAM-1 expression after TNF- stimulation of 93% compared to 1.7% in unstimulated HVECs. After a non-viral transfection with ICAM-1-specific siRNA, the stimulated expression of ICAM-1 on the cell surface could be diminished to 13%. These results are encouraging for further investigations analyzing the power of siRNA to reduce the danger of bypass failure.
The minor reduction of ICAM-1 expression seen upon transfection with scrambled siRNA indicates a slight toxic effect generally observed for cationic lipid mediated transfections. The results clearly demonstrate the high potency of designed siRNAs to suppress the expression of the ICAM-1 adhesion molecule in HVECs.
Especially, CABG surgery represents an ideal application for siRNA use. In general, there is a time delay between harvesting and implantation of the venous graft so that the material can be easily manipulated with siRNAs during this period. In addition, the graft can be treated ex vivo so that neither local cross clamping techniques nor systemic application of siRNA is necessary. Thus, a systemic delivery of siRNAs may not be necessary in such a therapeutic setting. First results obtained after ex vivo transfection of venous bypass material with nucleic acids are very encouraging [14,15].
This study demonstrates that siRNA can be transfected by a non-viral carrier in human venous endothelial cells and that it can successfully knockdown ICAM-1 expression. Further animal experiments have to confirm whether the effect of a reduced ICAM-1 expression in venous bypass material also results in a diminished rate of intima thickening and therefore in a reduced rate of an early failure of the bypass material in coronary artery bypass grafting, too. Nevertheless, blocking ICAM-1 function does not completely prevent leukocyte migration. Additional adhesion molecules such as vascular cell adhesion molecule (VCAM), platelet-endothelium cell adhesion molecule (PECAM), endothelium-selectin (E-selectin) or platelet-selectin (P-selectin) are likely to be involved in leukocyte adhesion to the endothelium. We are currently developing siRNAs to suppress each of these surface markers on HVECs.
In the long run, this new technology may offer novel therapeutic concepts to reduce early graft failure in coronary and peripheral vascular surgery by protecting recently implanted venous bypass grafts against leukocyte infiltration and intima thickening.
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Acknowledgments |
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The authors wish to thank Norbert Stefan, MD, for his critical comments and helpful suggestions.
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References |
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TopAbstract1. Introduction2. Materials and techniques3. Results4. CommentReferences |
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