Summary
Background
In recent years, the importance of inflammation in restenosis has been recognized gradually. When vascular injury occurs, NF-κB, which controls transcription of many inflammatory genes in restenosis (such as monocyte chemotactic protein-3 [MCP-3]), is activated by IκB degradation, leaving the NF-κB dimer-free to translocate to the nucleus to activate specific target genes.
Aims
To investigate the effect of tissue factor pathway inhibitor (TFPI) on MCP-3 expression and IκB-α degradation stimulated by tumour necrosis factor (TNF)-α in vascular smooth muscle cells (VSMCs), thus further elucidating the mechanism of the inhibitory effect of TFPI on restenosis.
Methods
Dulbecco’s modified Eagle’s medium or human recombinant adenoviruses expressing TFPI or bacterial β-galactosidase (LacZ) were used to infect rat aortic VSMCs in vitro. Enzyme-linked immunosorbent assays were used to detect exogenous TFPI expression and reverse transcription-polymerase chain reactions were used to detect MCP-3 expression after TNF-α stimulation in transfected cells. Western blotting and immunofluorescence microscopy were used to examine IκB-α expression.
Results
TFPI protein was detected in the TFPI group after gene transfer. The cells were stimulated with TNF-α for 6 hours on the third day after gene transfer. MCP-3 messenger ribonucleic acid expression was lower in the TFPI group than in the LacZ group ( P < 0.05) and IκB-α degradation was lower in the TFPI group than in the LacZ group in the cytoplasm ( P < 0.05).
Conclusion
TFPI inhibited MCP-3 expression induced by TNF-α; this effect may be propagated through the NF-κB pathway. TFPI gene transfer may be a new therapeutic strategy for inhibiting restenosis in clinical situations.
Résumé
Justification
Récemment, l’importance de l’inflammation dans la genèse de la resténose a été établie. Lorsqu’une atteinte vasculaire survient, NF-KB, qui contrôle la transcription de nombreux gènes de l’inflammation dans la resténose telle que la protéine 3 (MCP-3), chimiotactique monocytaire est activée avec la dégradation d’IKβ et la libération du dimère NF-KB, et une translocation nucléaire qui aboutit à une activation spécifique des gènes cibles.
Objectif
Investiguer l’effet de l’inhibition de la voie du facteur tissulaire (TFPI) sur l’expression de MCP-3 et la dégradation de IKB-α stimulée par le TNF-α au sein des cellules musculaires lisses vasculaires et ce afin de déterminer les conséquences de l’inhibition du TFPI sur la resténose.
Méthode
Le TFPI humain ou l’adénovirus recombinant LacZ ou le DNEM ont été utilisés pour infecter les cellules musculaires lisses vasculaires (aorte de rat) in vitro. Le test d’Elisa était utilisé pour détecter l’expression du TFPI exogène et la technique RT-PCR a été utilisée pour détecter l’expression de MCP-3 après stimulation du TNF-α dans des cellules transfectées. Les techniques de Western Blot et la microscopie par immunofluorescence ont été utilisées pour examiner l’expression de IKB-α.
Résultats
La protéine TFPI a été détectée dans le groupe TFPI après transfert génique. Les cellules ont été stimulées par le TNF-α pendant six heures, et à j3 après transfert génique. L’expression du MCP-3 ARNm dans le groupe TFPI était plus bas que dans le groupe LacZ ( p < 0,05) et la dégradation du IKB-α dans le groupe TFPI était plus bas que dans le groupe LacZ dans le cytoplasme ( p < 0,05).
Conclusion
Le TFPI peut donc inhiber l’expression de MCP-3 induite par le TNF-α et cet effet peut être propagé au travers de la voie NF-KB. Le transfert de gène TFPI pourrait être une nouvelle stratégie thérapeutique pour favoriser l’inhibition de la resténose en pratique clinique.
Introduction
Restenosis after percutaneous transluminal coronary angioplasty (PTCA) and stent placement is closely related to thrombosis, neointimal hyperplasia, inflammation and vascular remodelling . Coagulation and thrombosis are the initializing phases of restenosis; some active substances generated in this process can lead to the formation of neointima by stimulating vascular smooth muscle cell (VSMC) migration and proliferation, and also by activating an inflammatory response. In recent years, recognition of the importance of inflammation in restenosis has increased gradually. From animal models and clinical experiments, researchers have found that endothelial injury, platelet and leukocyte interactions, cell chemotactic effects and inflammatory mediators are all key factors in the inflammatory response after PTCA and stenting. A method that prevents this inflammatory response may provide a new therapeutic strategy for inhibiting restenosis in clinical situations.
It is well known that the expressions of several genes involved in the inflammatory response and restenosis is regulated at the transcriptional level by NF-κB. In balloon-injured arteries, NF-κB was found to be activated. The classical activation of the NF-κB pathway can be initiated by a wide range of extracellular stimuli. These agents can activate the cells and mediate phosphorylation of IκB, resulting in its degradation and leaving the NF-kB dimer-free to translocate to the nucleus to regulate many target genes, such as chemokines (e.g. monocyte chemotactic protein-3 [MCP-3]). Recently, MCP-3, which is a member of the CC chemokine group that includes MCP-1 and macrophage inflammatory proteins, was found to be involved in the pathological process of atherosclerosis and restenosis after vascular injury .
Tissue factor (TF) is the only initiator of coagulation in the classic extrinsic pathway . Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein found in serum. TFPI inhibits factor Xa and the TF/VIIa complex through the formation of TFPI/FXa/FVIIa/TF, regulates TF activity and exerts anticoagulant and antithrombotic effects. Work by our group and others has demonstrated that TFPI gene transfer or recombinant TFPI (rTFPI) irrigation can significantly reduce restenosis by inhibiting thrombosis and neointimal hyperplasia in balloon-injured arteries . Many in vitro experiments have indicated that TFPI can induce VSMC apoptosis, inhibit VSMC migration and thereby reduce the formation of neointima . At the present time, little is known about the effect of TFPI on inflammation. In this study, we transferred the TFPI gene, mediated by an adenovirus, into VSMCs cultured in vitro. The VSMCs were then stimulated with tumour necrosis factor (TNF)-α and we examined the effect of TFPI on the expression of MCP-3 to further investigate the anti-inflammatory effect of TFPI. To define the anti-inflammatory mechanism of TFPI, we also investigated the effect of TFPI on the degradation of IκB-α because it is known that expression of MCP-3 is regulated by the NF-κB pathway.
Methods
Reagents
The recombinant rat TNF-α was purchased from Peprotech Technologies, Rocky Hill, NJ, USA. The enzyme-linked immunosorbent assay (ELISA) kit for human TFPI protein was purchased from American Diagnostica, Inc., Stamford, CT, USA. The anti-β-actin and anti-IκB-α antibodies were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA. The reverse transcription-polymerase chain reaction kit was purchased from Promega, Madison, WI, USA. The adenoviruses containing the human TFPI gene (Ad-TFPI) and the LacZ gene (Ad-LacZ) (both 5 × 10 8 pfu/mL) were obtained from Dr. Yin Xinhua.
Cell culture
Rat VSMCs were isolated from the media of the thoracic aorta of male Wistar rats (170–200 g) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Cramlington, UK) supplemented with 10% foetal bovine serum (FBS). VSMCs were allowed to grow out from the tissue, which was consequently removed. After confluence was reached, cells were harvested by trypsinase. Cells between passages 3 and 6 were used in the experiments.
Adenovirus infection
The VSMCs were grown in six-well plates using DMEM containing 10% FBS. When 70 to 80% confluence was reached, the cells were serum-deprived for 24 hours. Ad-TFPI or Ad-LacZ was added to the medium at a multiplicity of infection of 100. Two hours later, the cells were washed three times with phosphate buffered saline (PBS), and the medium was changed to DMEM containing 10% FBS. As a control in all experiments, an identical group of cells was left uninfected but was incubated for 2 hours in serum-free DMEM.
Enzyme-linked immunosorbent assay
Cell culture media from each group ( n = 5) were collected, respectively, at the first, third, fifth and seventh days after gene transfer. Quantitative determination of TFPI expression was performed using a specific ELISA kit for human TFPI protein (American Diagnostica, Inc.), following the manufacturer’s instructions.
Stimulation immediately after gene transfer
Immediately after gene transfer, some VSMCs were stimulated with TNF-α (10 ng/mL) for 6 hours and the medium was then changed to DMEM containing 10% FBS. The VSMCs that were not stimulated with TNF-α were considered to be the control group. On the third day, approximately 5 × 10 6 cells were washed with PBS for detection.
Stimulation 3 days after gene transfer
On the third day after gene transfer, some VSMCs were stimulated with TNF-α (10 ng/mL) for 6 hours and were then washed with PBS for detection. The VSMCs that were not stimulated with TNF-α were considered as the control group.
Expression of MCP-3 messenger ribonucleic acid (mRNA)
On the third day after gene transfer, approximately 5 × 10 6 cells in each group (both those stimulated immediately and 3 days after gene transfer) were washed with PBS in preparation for extraction of the total cellular ribonucleic acid (RNA) (each group, n = 6). The cells were then resuspended in 1 mL Trizol (Invitrogen, Carlsbad, CA, USA), and the total RNA in each group was extracted according to the manufacturer’s guidelines . Reverse transcription was performed with 1 μg of isolated RNA using a reverse transcription kit (Promega). The reverse transcription reaction was carried out with random primers according to the manufacturer’s protocol. The complementary DNA (1 μg) was amplified using gene-specific primers. The following primers were designed to detect rat MCP-3 mRNA levels in each group (forward: 5′-CATGGAAGTCTGTGCTGAAG-3′; reverse: 5′-TGAAACTTCAGTAGTCATACA-3′; 494 base pairs) and rat β-actin (forward: 5′-GGCTACAGCTTCACCACCAC-3′; reverse: 5′-GCTTGC TGATCCACATCTGC-3′; 499 base pairs). The PCR amplification cycles for the MCP-3 mRNA were performed as follows: 94 °C for 3 minutes; 94 °C for 30 seconds, 54 °C for 30 seconds, 72 °C for 1 minute for 30 cycles; 72 °C for 5 minutes. The PCR product was electrophoresed using a 1.5% agarose gel. The relative band intensities were measured using the Global Imaging Systems, Inc., Tampa, FL, USA (GIS) detection system. The expression levels of MCP-3 mRNA were expressed as the relative ratio to the amount of β-actin mRNA in each group.
Western blotting
On the third day after gene transfer, VSMCs were stimulated using TNF-α (10 ng/mL) for 6 hours (each group, n = 6). The cells were then washed with PBS and resuspended in cold lysis buffer with phenylmethanesulfonyl fluoride. The cell lysate was incubated on ice for 30 minutes and centrifuged at 12000 g for 15 minutes at 4 °C. The protein content of the supernatant was determined by using a bicinchoninic acid (BCA)-200 protein assay kit (Beyotime, Haimen, Jiangsu, China) . Equal amounts of the proteins (50 μg) were loaded into the gel and separated by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis. The resolved proteins were transferred to polyvinylidene fluoride membranes. After blocking with 5% non-fat milk in tris buffered saline with Tween (TBST) for 1 hour at 37 °C, the blots were incubated overnight at 4 °C with a primary antibody against either actin (1:500 mouse monoclonal) or IκB-α (1:200 rabbit polyclonal) diluted in blocking buffer. The membrane was washed with TBST and probed with a horseradish peroxidase-conjugated secondary antibody for 1 hour at 37 °C. The membrane was washed three times in TBST and treated with 3,3’-diaminobenzidine according to the manufacturer’s protocol. The protein-antibody complexes conjugated with the secondary antibody were visualized using the Global Imaging Systems, Inc. (GIS imaging system).
Immunofluorescence microscopy
VSMCs were grown in 24-well plates in DMEM containing 10% FBS with a slide placed in each well. Either Ad-TFPI or Ad-LacZ was transfected into the VSMCs as described above. At the third day after gene transfer, the VSMCs were stimulated with TNF-α (10 ng/mL) for 6 hours (each group, n = 7). The cells were then washed three times with PBS, fixed with paraformaldehyde for 30 minutes and washed three more times with PBS. After incubating with 0.1% Triton X-100 for 30 minutes and washing, the cells on the slides were incubated overnight at 4 °C with a primary antibody against IκB-α (1:50 rabbit polyclonal) in a humidified chamber and were then rinsed three times in PBS, incubated with goat anti-rabbit IgG-fluorescein isothiocyanate-conjugated antibody diluted 1:50 in PBS for 30 minutes and rinsed three more times with PBS. Cell immunofluorescence was observed under microscopy.
Statistical analysis
All of the experiments were repeated at least five times, with similar patterns of results. All results are expressed as mean ± standard deviation. Analysis of variance was used for the statistical analysis and a value of P < 0.05 was considered statistically significant.
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