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Tissue factor pathway inhibitor-2 is downregulated by ox-LDL and inhibits ox-LDL induced vascular smooth muscle cells proliferation and migration
Thrombosis Research 128 (2011) 179–185
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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
Tissue factor pathway inhibitor-2 is downregulated by ox-LDL and inhibits ox-LDL induced vascular smooth muscle cells proliferation and migration Bilian Zhao a, Xinping Luo a,⁎, Haiming Shi a, Duan Ma b,⁎⁎ a b
Department of Cardiology, Huashan Hospital of Fudan University, Shanghai, China, 200040 The Key laboratory of molecular medicine, Ministry of Education, Shanghai Medical School of Fudan University, Shanghai, China, 200032
a r t i c l e
i n f o
Article history: Received 30 September 2010 Received in revised form 13 February 2011 Accepted 20 February 2011 Available online 1 April 2011 Keywords: tissue factor pathway inhibitor-2 (TFPI-2) vascular smooth muscle cells (VSMCs) proliferation migration atherosclerosis
a b s t r a c t Introduction: Tissue factor pathway inhibitor-2 (TFPI-2) is a member of the Kunitz-type family of serine protease inhibitors, which inhibits several matrix metalloproteinases activity involved in extracellular matrix degradation. Studies have shown low TFPI-2 expression in the shoulder regions of atherosclerotic plaques. But studies evaluating its role in the progression of atherosclerotic plaque are scarce. Vascular smooth muscle cells (VSMCs) are important components of atherosclerotic plaques and oxidized low density lipoprotein (oxLDL) is an important detrimental factor of atherosclerosis. The aim of this study is to elucidate the effect of TFPI-2 on smooth muscle cell proliferation and migration induced by ox-LDL. Methods: Retroviruses expressing human TFPI-2 were constructed. Cell proliferation was determined by CCK-8 assay. Cell apoptosis was analyzed by double staining of FITC-Annexin V and propidium iodide. Cell migration was studied through a Transwell chamber and with a scratch-wound assay. The matrix metalloproteinase-2 and − 9 activities were analyzed by gelatin zymography. Phosphorylation of FAK was analyzed by western blot. Results: TFPI-2 over-expression of mRNA and protein was confirmed in infected cells. CCK-8 assay showed that TFPI-2 inhibit VSMCs proliferation induced by ox-LDL while without cytotoxicity to VSMCs. Transwell and scratch wound assay confirmed TFPI-2 over-expression can inhibit VSMC migration. Zymography assay showed that TFPI-2 can inhibit MMP-2, 9 activity induced by ox-LDL. Western blot assay showed TFPI-2 can inhibit cyclinD1 expression and FAK phosphorylation. Conclusion: TFPI-2 over-expression may strongly inhibit the proliferation and migration of VSMCs and suppresses MMP-2, 9 activity induced by ox-LDL, making it a promising candidate for treatment of atherosclerotic process. © 2011 Elsevier Ltd. All rights reserved.
Proliferation and migration of VSMCs from media to intima is pivotal to atherosclerotic progression [1]. Normally, VSMCs are quiescent and are surrounded by and embedded in an extracellular matrix (ECM) scaffold that acts like a barrier to VSMCs migration. During the process of atherosclerosis, stimulated by environmental stimuli such as ox-LDL, VSMCs secreted several proteinases which degrade the ECM and in turn facilitate VSMCs proliferation and migration [2]. Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor synthesized by endothelial cells (ECs), smooth
Abbreviations: TFPI-2, tissue factor pathway inhibitor-2; VSMCs, vascular smooth muscle cells; ox-LDL, oxidized low density lipoprotein; MMP, matrix metalloproteinase; FAK, focal adhesion kinase. ⁎ Correspondence to: X. Luo, 12 Urumuqi Road, Department of Cardiology, Huashan Hospital, Shanghai, China, 200040. Tel.: +86 21 52887165; fax: +86 21 62480242. ⁎⁎ Correspondence to: D. Ma, 130 Dongan Road, The Key laboratory of molecular medicine, Ministry of Education, Shanghai Medical School of Fudan University, Shanghai, China, 200032. Tel.: +86 21 54237414. E-mail addresses: [email protected] (X. Luo), [email protected] (D. Ma). 0049-3848/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2011.02.025
muscle cells (SMCs), and syncytiotrophoblasts [3,4], which associates through ionic interactions [5] with the extracellular matrix (ECM) [6] and inhibits trypsin, plasmin, and plasma kallikrein among others [7]. The major role of TFPI-2 seems to be plasmin inhibition [8] and thus of proMMP-1, proMMP-3, and proMMP-13 activation [9]. As a direct inhibitor of MMP-2 and MMP-9 [10] and potent inhibitor of both matrix-bound and cell-associated plasmin, TFPI-2 could regulate extracellular proteolysis and ECM remodeling, which are highly relevant both for normal development and for atherosclerosis. It has been shown to inhibit endothelial cell proliferation induced by vascular endothelial growth factor (VEGF) [11]. Its expression has been shown to be lower at the shoulder of atherosclerosis plaque [12,13], which suggests that downregulation of TFPI-2 contributes to atherosclerosis progression. Oxidized low density lipoprotein (ox-LDL), which is an important detrimental factor of atherosclerosis, is well established as an important stimulus of VSMCs proliferation and a chemoattractant directing the migration of VSMCs toward the vessel intima during atherosclerosis and neointima hyperplasia [14]. To test the role of TFPI-2 in atherosclerotic progression, we studied TFPI-2 function on VSMCs under the treatment of ox-LDL.
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Materials and methods Materials Primary human aortic smooth muscle cells were purchased from ATCC. Retroviral plasmid vector pBABE-puro was purchased from Clontech (BD Bosciences, CA). Cell culture media and supplements were from Invitrogen (Carlsbad, CA) and HyClone (Logan, UT). Cell culture disposable materials were purchased from BD. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (MD, USA). Oxidized low density lipoprotein (ox-LDL) and monoclonal antibodies to TFPI-2 were purchased from R&D Systems (Minneapolis, MN). Polyclonal rabbit anti-cyclinD1, anti-GAPDH, anti-focal adhesion kinase (FAK) and phosphorylation specific polyclonal antibody to FAK tyr-397 were purchased from Cell Signal Technology (USA). Transwell with 8-μm pore polycarbonate membrane was purchased from Corning (NY, USA). Crystal violet was purchased from Sigma (St. Louis, USA). All other chemicals and reagents were obtained from commercial source and were of analytic grade.
three step program of 30 s at 94 °C, 30 s at 59 °C and 30 s at 72 °C) and were performed in triplicate. The relative target mRNA levels were assessed with use of ABI Prism 7500 software and normalized to that of internal control β-actin.
Cell proliferation assay CCK-8 cell viability assay was used to evaluate cell proliferation following the manufacturer's instruction. Briefly, 48 hours after infection, Retro-TFPI-2 cells and control cells were trypsinized and harvested, and then 100 μl cells were seeded into 96-well plates both at 5 × 104/ml. After cell adhesion, VSMCs were incubated in DMEM containing 0.5% FBS for 24 hours. Then 40 μg/ml ox-LDL or 10% FBS was added to each well as a stimulator. The medium was replaced every 24 hours. At every 24 hours, 10 μl CCK-8 reagent was added to each well, and then incubated at 37 °C for 4 hours. After that, absorbances in each well were read with a spectrophotometric plate reader at 450 nm.
Cell culture Apoptosis assay VSMCs were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum (FBS). Cultures were maintained at 37 C °C in a humidified 95% air and 5% CO2 atmosphere. VSMCs were passaged by trypsinization with 0.25% trypsin and 0.02% EDTA in PBS. In this study, early passage (four to eight) VSMCs were used. Effects of Ox-LDL on TFPI-2 expression To investigate ox-LDL regulation on TFPI-2 expression, VSMCs were treated with different concentrations of ox-LDL (20-40 μg/ml). 24 hours later, total cell lysates were harvested and western blot analysis was performed.
Cells were stained with FITC-AnnexinV and propidium iodide and then evaluated for apoptosis by flow cytometry according to the manufacturer's protocol (BD Biosciences). Briefly, 24 hours after infection, Retro-TFPI-2 cells and control cells were treated with 40 μg/ml ox-LDL or not for 48 hours, then cells were trypsinized and harvested, washed twice with cold PBS, and centrifuged at 1000 g for 5 min. Cells were resuspended in 400 μl binding buffer at a concentration of 1 × 106 cells per ml, 100 μl of the solution was transferred to a 5 ml culture tube, and 5 μl of FITC-AnnexinV and 5 μl of propidium iodide were added. Cells were gently vortexed and incubated for 15 min at room temperature in the dark. Finally, 400 μl of binding buffer was added to each tube and samples were analyzed by flow cytometry (Becton Dickinson, Heidelberg, Germany).
Construction of recombinant retrovirus Full-length human TFPI-2 was generated from primary VSMCs by RT-PCR. The primers flanking the coding region of TFPI-2 were sense, 5’- ATAGAATTCATGGACCCCGCTCGCCCCCTG-3’, and antisense, 5’CGCGTCGACTTAAAATTGCTTCTTCCGAATTTTC-3’. The recombinant retroviruses were constructed and amplified according to the manufacturer's protocol. The recombinant retroviruses contained human TFPI-2 cDNAs (Retro-TFPI-2) were used to induce overexpression of TFPI-2. A retrovirus carrying green fluorescence protein (GFP) was used as a negative control. For retrovirus-mediated TFPI-2 gene transduction, VSMCs were grown to 60% confluence and infected with Retro-TFPI-2 or RetroGFP-Control separately. After 24 hours, the viral suspension was removed and the cells used as required. Real-time quantitative PCR analysis Real-time reverse transcription (RT)-PCR was used to determine the relative amount of TFPI-2 in infected cells. Forty-eight hours after infected, total RNA was extracted by use of TRIzol reagent (Invitrogen, Carlsbad, CA). Two micrograms of total RNA per sample was reverse-transcribed by the AMV Reverse Transcription System (Promega, Madison, WI). Realtime PCR amplification involved use of an ABI Prism 7500 sequence detector (Applied Biosystems) and SYBR Green reagent. The specific primers for human TFPI-2 were sense, 5’ –GTCGATTCTGCTGCTTTT CC-3’ and antisense, 5’-ATGGAATTTTCTTTGGTGCG-3’; and the primers for human β-actin were sense, 5’-GAAACTACCTTCAACTCCATC- -3’ and antisense, 5’-CGAGGCCAGGATGGAGCCGCC-3’. All amplification reactions were carried out over 40 cycles (an initial stage of 4 min at 94 °C then a
Cell migration The effect of TFPI-2 on VSMCs migration was monitored with Transwell using a modified Boyden's chamber assay. Briefly, 48 hours after infection, the Retro-TFPI-2 and control cells were harvested by trypsinization and suspended in the serum free DMEM supplemented with 0.2% BSA. Cell suspensions were then placed into the upper well at a concentration of 1 × 104 cells/100 μl separately, while the same medium was placed into lower well (500 μl). The lower chamber contained ox-LDL (40 μg/ml) as a chemoattractant. The chamber was incubated at 37 °C in 5% CO2 humidified atmosphere for 4 h. Nonmigrating cells on the upper surface were scraped gently and washed out with PBS three times. VSMCs migrated to the lower surface of the membrane were stained with crystal violet dye and counted per three independent HPFs (×100) with light microscope.
Scratch-wound assay Retro-TFPI-2 cells and control cells were seeded in 6-well plates at a concentration of 10,000 cells per well, and then grown until reaching confluence. A linear wound was gently introduced in the center of the cell monolayer using 1 ml tip, followed by washing with DMEM to remove the cellular debris. Then the cells were incubated with 40 μg/ml ox-LDL and monitored for 48 hours. Cell images were taken at 48 hours, and the number of cells migrated into the wound space were manually counted in three fields per well with light microscope (×100).
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Gelatin zymography 48 hours after infection, Retro-TFPI-2 and control cells were harvested and seeded in 24 well plates at a concentration of 100,000 cells per well with DMEM plus 15% FBS. 12 hours later, the media was changed into DMEM with no serum. Then the cells were treated with 40 μg/ml ox-LDL for 24 hours. Proteins in the conditioned media were separated, without prior boiling, by electrophoresis through 10% SDS-PAGE containing 1 mg/ml gelatin. The proteins in the gel were renatured and stained as described [15]. SDS-PAGE and western blot analysis Retro-TFPI-2 and control cell layers treat with or without 40 μg/ml oxLDL, were rinsed three times with PBS and extracted in ice-cold lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, 1% NP-40, 0.1% SDS, 1 mM PMSF, 1 mg/ml each of aprotinin, leupeptin, pepstatin) for 10 min. Lysates were centrifuged at 12,000 g for 5 min at 4 °C, and supernatants were stored at 80 °C. Protein concentrations were determined by the BCA method. Twenty micrograms of protein from each cell extract was separated by SDS-PAGE and then transferred to nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ). Membranes were blocked with blocking solution (50 mM Tri-HCl, 150 mM NaCl, 5% (w/v) non-fat dry milk and 0.1% Tween-20) overnight at 4 °C. After incubating with appropriate primary and secondary antibodies, the blots were detected with the Supersignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL). The results of western blots were analyzed by the Image J program. Statistical analysis Each experiment was repeated at least three times. All the data analyses were conducted with SPSS 15.0 (SPSS, Inc., Chicago, IL). All the variables were under normal distribution and values were reported as mean ± standard deviation (SD). Significance of differences was analyzed using independent samples t test as appropriate. P b 0.05 was considered statistically significant.
Fig. 1. TFPI-2 expression is downregulated by ox-LDL in VSMCs. VSMCs were treated with ox-LDL at 20-40 μg/ml or not for 24 hours. Total cell lysates were harvested and western blot analysis was performed to detect the expression of TFPI-2. The detected three bands were three different glycosylated isoforms of TFPI-2 (27, 31, 33 kDa). *: P b 0.05 compared with control; **: P b 0.01 compared with control.
Results TFPI-2 expression is downregulated by ox-LDL in VSMCs TFPI-2 has been reported to be expressed in VSMCs and endothelial cells [3,4]. And its expression in endothelial cell was upregulated by vascular endothelial growth factor [11]. Here we were interested in determining whether TFPI-2 may be regulated by oxLDL. Western blot analysis showed ox-LDL treatment decrease TFPI-2 expression in VSMCs in a dose-dependent manner (Fig. 1). We detected three different glycosylated isoforms of TFPI-2 (27, 31, 33 kDa), in accordance with previous observations [11]. TFPI-2 over expression after retrovirus-mediated gene transduction Forty eight hours after infected, both the mRNA and protein expression of TFPI-2 were confirmed by real time reverse transcript PCR and western blot analysis, respectively. Fig. 2A showed the result of real time PCR, which suggesting that Retro-TFPI-2 cells strongly expressed TFPI-2 mRNA than control cells. Consisted with real time PCR, in the Western Blot analysis (Fig. 2B), Retro-TFPI-2 cells had darker bands of TFPI-2 than control cells. Effect of TFPI-2 on VSMCs proliferation and cyclin D1 expression with ox-LDL or FBS treatment CCK-8 assay allows evaluation of cell metabolic activity such as cell number, cell metabolism, and mitochondrial activation. As seen in
Fig. 2. Identification and expression of TFPI-2 after viral infection. A. Real time PCR of TFPI-2 mRNA expression in VSMCs after infected with Retro-GFP viruses or TFPI-2 expression viruses. Real time PCR of β-actin mRNA expression was performed as a control. Data are presented as mean value ± SD, the asterisk indicates a P b 0.01. B. Western blot analysis for the expression of TFPI-2 after infected with Retro-GFP viruses or TFPI-2 expression viruses.
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Fig. 3, when incubated with DMEM plus 10% FBS (A), the 450 nm absorbances of the Retro-TFPI-2 cells and control cells did not differ significantly, indicating that neither the vector construct itself nor TFPI-2 over-expression significantly influenced cell proliferation in normal condition. In contrast, while treated by 40 μg/ml ox-LDL (B), the absorbance of the Retro-TFPI-2 cells were obviously lower than control cells, which suggested that TFPI-2 over expression inhibited VSMCs proliferation induced by ox-LDL. CyclinD1 is a major promoter of the cell cycle. In order to verify the effect of TFPI-2 on VSMCs proliferation, we analyzed CyclinD1 expression in Retro-TFPI-2 cells and control cells with FBS or ox-LDL treatment. As shown in Fig. 3C, with FBS treatment, upregulation of TFPI-2 had no significant influence on cyclin D1 expression. In contrast, when treated by ox-LDL, cyclinD1 expression was strongly inhibited in Retro-TFPI-2 cells in comparison with control cells (P b 0.01). Effects of TFPI-2 on VSMCs apoptosis In order to verify if the induction of apoptosis occurred during TFPI-2 upregulation and ox-LDL treatment, the presence of apoptotic cells was determined by the application of double staining with FITCAnnexin V and propidium iodide. Normal live cells were represented as FITC-negative and PI-positive, early apoptotic cells as FITC- positive and PI- negative, and necrotic cells as FITC-positive and PI-positive. The percentage of apoptotic cell populations in Retro-TFPI-2 cells and control cells were, respectively, 3.22 ± 0.06% and 3.62 ± 0.09% (without ox-LDL treatment, P N 0.05), 3.05 ± 0.07% and 2.44 ± 0.09%
(with ox-LDL treatment, P N 0.05). This indicated no significant effect on apoptosis of TFPI-2 upregulation and ox-LDL treatment. Representative experiments are shown in Fig. 4. TFPI-2 inhibits VSMCs migration It is known to all that ox-LDL is a chemoattractant and proliferative agent for VSMCs. To determine the effect of TFPI-2 on ox-LDLstimulated VSMCs migration, we carried out migration assays in the presence of 40 μg/ml ox-LDL. In the Transwell assay(Fig. 5A), VSMCs migration decreased from 53 ± 3 cells per field for control to 26 ± 1 cells/field for Retro-TFPI-2-infected cells (P b 0.01). In the scratch assay (Fig. 5B), control cells treated with ox-LDL migrated into the wound area and organized a dense cellular network, resulting in nearly complete wound recovery after 48 hours, while migration into the wound area of Retro-TFPI-2 cells was significantly inhibited. The migrated cells in Retro-TFPI-2 cells and control cells were, respectively, 35 ± 4 cells and 86 ± 3cells (P b 0.01). TFPI-2 inhibits MMP-2, 9 activity Matrix metalloproteinase-2, 9 (MMP-2, 9) are known to play several important roles during changes in vascular structure associated with atherosclerotic progression. The stimulation with ox-LDL leads to increased activity of MMP-2 and − 9, and this up-regulation was linked to increased cell migration. To test whether over expression of TFPI-2 could inhibit MMP-2 and MMP-9 activity, we stimulated VSMCs with 40 μg/ml ox-LDL and analyzed the
Fig. 3. Effect of TFPI-2 on ox-LDL induced VSMCs proliferation and cyclin D1 expression. Retro-TFPI-2 cells and control cells were incubated in DMEM containing 0.5% FBS for 24 hours before experiments. A. Cells were incubated with DMEM plus 10% FBS. Absorbances in each well were read with a spectrophotometric plate reader at 450 nm. B. Cells were treated by 40 μg/ml ox-LDL. Absorbances in each well were read with a spectrophotometric plate reader at 450 nm. **: P b 0.01 compared with control. C. Western blot of extracts from VSMCs incubated with 10% FBS or 40 μg/ml ox-LDL for 48 hours (revealed with anti-cyclinD1 and anti-GAPDH antibodies). **: P b 0.01 comparison between Retro-TFPI-2 cells and control cells treated with ox-LDL.
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Fig. 4. Effect of TFPI-2 on apoptosis of VSMCs. Cell apoptosis assessed by double staining of FITC-AnnexinV and propidium iodide and subsequent flow cytometry. 24 hours after infection, Retro-TFPI-2 cells and control cells were treated with 40 μg/ml ox-LDL or not for 48 hours. The presence of apoptotic cells was identified by flow cytometric analysis of cells labeled with FITC-AnnexinV and propidium iodide. Cells in the lower right quadrant correspond to early apoptotic cells, whereas those in the upper right quadrant correspond to late apoptotic or necrotic cells. Numbers in each quadrant are percentage of cells they contain. The results shown are representative of three separate experiments.
Fig. 5. Effect of TFPI-2 on migration of VSMCs. A. 10,000 Retro-TFPI-2 cells or control cells were plated into the upper chamber of Transwells containing an 8 μm barrier with reconstituted basement membrane proteins. Ox-LDL (40 μg/ml) was included in the lower chamber as a chemoattractant. Cells migrating across the filters at 4 hours were stained by crystal violet. Magnification is × 100. Bottom, representative results of three independent experiments is shown. Migrated cells were qualified by the average of three randomly chosen high-power fields of three independent experiments. B. Confluent VSMCs monolayers infected with Retro-TFPI-2 or Retro-GFP were scratch wounded 24 hours after infection. Cells were then treated with 40 μg/ml ox-LDL for 48 hours before imaging (lines indicate wounds edge). The number of cells migrated into the wound space were manually counted in three fields per well with light microscope (×100). **: P b 0.01 compared with control.
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metalloproteinase activity by gelatin zymography. As seen in Fig. 6, without ox-LDL treatment, VSMCs secreted little activated MMP-2 and MMP-9. Ox-LDL treatment strongly induced MMP-2 and MMP-9 activity and TFPI-2 over-expression inhibited VSMCs expression of MMP-2 and MMP-9 induced by ox-LDL (both P b 0.01). TFPI-2 inhibits phosphorylation of FAK Phosphorylation of focal adhesion kinase is critical for cell migration. The decline in VSMCs migration suggested that TFPI-2 might be altering the phosphorylation of FAK. To measure FAK phosphorylation, we immunoprecipitated FAK from Retro-TFPI-2 VSMCs and control cells extracts treated with or without 40 μg/ml ox-LDL and performed an immunoblot with an antibody to detect phosphorylated tyrosine. As shown in Fig. 7, Retro-TFPI-2 cells had a significant decrease in tyrosine phosphorylation of FAK in comparison with control cells, especially when treated with ox-LDL (P b 0.01). Discussion The extensive plasticity of VSMCs makes cells susceptible to various stimuli inducing changing in phenotype that contributes to the etiology of cardiovascular diseases [16]. It is well established that accelerated proliferation of VSMCs and migration of VSMCs from the media to the intima are fundamental events in the development of both intimal hyperplasia and atherosclerosis [17,18]. Therefore, inhibition of VSMCs proliferation and migration represents a potentially promising therapeutic strategy for the treatment of diseases such as atherosclerosis and neointimal hyperplasia. It has been shown that TFPI-2 is induced by fluid shear stress in vascular smooth muscle cells and affects cell proliferation and survival
Fig. 6. Effect of TFPI-2 on secreted MMP-2, 9. 24 hours after ox-LDL treatment, the conditioned media of Retro-TFPI-2 or control cells treated with or without 40 μg/ml oxLDL were analyzed by gelatin zymography. Ox-LDL increased MMP-2 and MMP-9 activity significantly. Compared to control cells, TFPI-2 over-expression markedly inhibited MMP-2 and MMP-9 activity. **: P b 0.01 compared with control.
Fig. 7. Effect of TFPI-2 on phosphorylation of FAK. Western blots of extracts from RetroTFPI-2 and control cell layers treat with or without 40 μg/ml ox-LDL for 48 hours (revealed with anti-FAK and anti-phospho-FAK antibodies). Compared to control, TFPI2 over-expression markedly inhibited phosphorylation of FAK, especially when treated with ox-LDL. The results shown are representative of three separate experiments. #: P b 0.05 comparison between Retro-TFPI-2 cells and control cells treated without oxLDL. **: P b 0.01 comparison between Retro-TFPI-2 cells and control cells treated with ox-LDL.
[19], which suggests that TFPI-2 expression by VSMCs in response to fluid shear stress may play a role in the control of VSMCs turnover in the intima during vascular repair. Our data showed that ox-LDL suppressed TFPI-2 in a dose-dependent manner and over expression of TFPI-2 inhibited ox-LDL induced VSMCs proliferation compared with control. While in normal condition (without ox-LDL treatment), TFPI-2 over-expression had no significant influence on the proliferation of VSMCs. Accordingly, when treated by ox-LDL, cyclin D1 expression was inhibited in Retro-TFPI-2 cells compared to the control cells. Under normal conditions, no significant differences were shown in cyclin D1expression between the two groups. We assume the reason may be that TFPI-2 can inhibit MMP-2 activity induced by ox-LDL, which have been shown to play a pivotal role in ox-LDLinduced activation of the sphingomyelin/ceramide signaling pathway and subsequent VSMCs proliferation [20].While under normal conditions, VSMCs secreted little MMP-2. Our result was different from that of Shinoda [21], who reported TFPI-2 could act as a mitogen for VSMCs. In addition, we found no effect of TFPI-2 on VSMCs apoptosis, unlike Johan [19], who found a pro-apoptotic effect of TFPI-2 in VSMCs. The discrepancy may be because we used viral vector to upregulate TFPI-2 and they used different concentration of recombinant TFPI-2 protein. In this study, we also found TFPI-2 overexpression inhibit VSMCs migration and MMP-2, 9 activity induced by ox-LDL, which is accepted to be an important detrimental factor of atherosclerosis [22]. MMP-2, 9 have been reported as the principle MMPs expressed and secreted by the synthetic VSMCs, which accelerates their migration and increases their invasion ability [23]. As a member of
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the Kunitz-type family of serine proteinase inhibitors, TFPI-2 has been shown to inhibit MMP-2 and MMP-9 activity in several tumor cells. Here we demonstrated that TFPI-2 inhibited MMP-2, 9 activity induced by ox-LDL, suggesting that TFPI-2 has the potential to inhibit the process of atherosclerosis. Ox-LDL is well established as a chemoattractant directing the migration of VSMCs toward the vessel intima during atherosclerosis and neointimal hyperplasia [14]. Our data showed that TFPI-2 strongly inhibited VSMCs migration induced by ox-LDL, as well as into a wound area. TFPI-2's strongly inhibition of plasmin, trypsin and MMPs, which preventing the ECM degradation may be a probable mechanism. FAK is a critically important, non-receptor protein tyrosine kinase that coordinates both integrin and growth factor signaling cascades involved in VSMCs matrix invasion and migration. It has been reported that disruption of FAK signaling may provide a pharmaceutical tool that limits pathological VSMC cell behavior [24]. Our data revealed that tyrosine phosphorylation of FAK in Retro-TFPI-2 cells was diminished in comparison with control cells. We assume this was due to the effect of TFPI-2 on ECM remodeling. These findings suggest a mechanism through which TFPI-2 regulates VSMCs migration. In summary, our data demonstrated that TFPI-2 over-expression may strongly inhibit the proliferation and migration of VSMCs induced by ox-LDL and without cytotoxicity, suppressed MMP-2 and MMP-9 activities and inhibited FAK phosphorylation. These findings suggest TFPI-2 may be a promising therapeutic for treatment of atherosclerotic progression. Further studies are needed to investigate the effect of TFPI-2 downregulation on VSMCs and its role in atherosclerotic progression in vivo. Sources of funding This work was supported by the Shanghai Committee of Science and Technology, China (Grant No. 074119604) and Shanghai Health Bureau, China (Grant No. 2007100). Acknowledgements We thank Chundi Xu, Fenge Deng, Shoudong Ye and Pengliang Mao from The Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai Medical School of Fudan University for technical assistance. References [1] Newby AC, Zaltsman AB. Molecular mechanisms in intimal hyperplasia. J Pathol 2000;190:300–9. [2] Hu J, Van den Steen PE, Sang QX, Opdenakker G. Matrix metalloproteinase inhibitors as therapy for inflammatory and vascular diseases. Nat Rev Drug Discov 2007;6:480–98. [3] Iino M, Foster DC, Kisiel W. Quantification and characterization of human endothelial cell-derived tissue factor pathway inhibitor-2. Arterioscler Thromb Vasc Biol 1998;18:40–6.
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[4] Udagawa K, Miyagi Y, Hirahara F, Miyagi E, Nagashima Y, Minaguchi H, et al. Specific expression of PP5/TFPI2 mRNA by syncytiotrophoblasts in human placenta as revealed by in situ hybridization. Placenta 1998;19:217–23. [5] Liu Y, Stack SM, Lakka SS, Khan AJ, Woodley DT, Rao JS, Rao CN. Matrix localization of tissue factor pathway inhibitor-2/matrix-associated serine protease inhibitor (TFPI-2/MSPI) involves arginine-mediated ionic interactions with heparin and dermatan sulfate: heparin accelerates the activity of TFPI-2/MSPI toward plasmin. Arch Biochem Biophys 1999;370:112–8. [6] Rao CN, Reddy P, Liu Y, O'Toole E, Reeder D, Foster DC, et al. Extracellular matrixassociated serine protease inhibitors (Mr 33,000, 31,000, and 27,000) are singlegene products with differential glycosylation: cDNA cloning of the 33-kDa inhibitor reveals its identity to tissue factor pathway inhibitor-2. Arch Biochem Biophys 1996;335:82–92. [7] Petersen LC, Sprecher CA, Foster DC, Blumberg H, Hamamoto T, Kisiel W. Inhibitory properties of a novel human Kunitz-type protease inhibitor homologous to tissue factor pathway inhibitor. Biochemistry 1996;35:266–72. [8] Rao CN, Cook B, Liu Y, Chilukuri K, Stack MS, Foster DC, et al. HT-1080 fibrosarcoma cell matrix degradation and invasion are inhibited by the matrix-associated serine protease inhibitor TFPI-2/33 kDa MSPI. Int J Cancer 1998;76:749–56. [9] Herman MP, Sukhova GK, Kisiel W, Foster D, Kehry MR, Libby P, et al. Tissue factor pathway inhibitor-2 is a novel inhibitor of matrix metalloproteinases with implications for atherosclerosis. J Clin Invest 2001;107:1117–26. [10] Izumi H, Takahashi C, Oh J, Noda M. Tissue factor pathway inhibitor-2 suppresses the production of active matrix metalloproteinase-2 and is down-regulated in cells harboring activated ras oncogenes. FEBS Lett 2000;481:31–6. [11] Xu Z, Maiti D, Kisiel W, Duh EJ. Tissue factor pathway inhibitor-2 is upregulated by vascularendothelial growth factor and suppresses growth factor-induced proliferation of endothelial cells. Arterioscler Thromb Vasc Biol 2006;26:2819–25. [12] Higashikata T, Yamagishi M, Higashi T, Nagata I, Iihara K, Miyamoto S, et al. Altered expression balance of matrix metalloproteinases and their inhibitors in human carotid plaque disruption: results of quantitative tissue analysis using real-time RT-PCR method. Atherosclerosis 2006 Mar;185(1):165–72. [13] Zawadzki C, Chatelain N, Delestre M, Susen S, Quesnel B, Juthier F, et al. Tissue factor pathway inhibitor-2 gene methylation is associated with low expression in carotid atherosclerotic plaques. Atherosclerosis Jun 2009;204(2):e4–e14. [14] Ishigaki Y, Oka Y, Katagiri H. Circulating oxidized LDL: a biomarker and a pathogenic factor. Curr Opin Lipidol Oct 2009;20(5):363–9 Review. [15] Absence of Akt1 reduces vascular smooth muscle cell migration and survival and induces features of plaque vulnerability and cardiac dysfunction during atherosclerosis. Arterioscler Thromb Vasc Biol 2009;29:2033–40. [16] Yoshida T, Owens GK. Molecular determinants of vascular smooth muscle cell diversity. Circ Res 2005;96:280–91. [17] Hanke H, Strohschneider T, Oberhoff M, Betz E, Karsch KR, et al. Time course of smooth muscle cell proliferation in the intima and media of arteries following experimental angioplasty. Circ Res Sep 1990;67(3):651–9. [18] Andres V. Control of vascular smooth muscle cell growth and its implication in atherosclerosis and restenosis (review). Int J Mol Med Jul 1998;2(1):81–9 Review. [19] Ekstrand J, Razuvaev A, Folkersen L, Roy J, Hedin U. Tissue factor pathway inhibitor-2 is induced by fluid shear stress in vascular smooth muscle cells and affects cell proliferation and survival. J Vasc Surg Jul 2010;52(1):167–75. [20] Augé N, Maupas-Schwalm F, Elbaz M, Thiers JC, Waysbort A, Itohara S, et al. Role for matrix metalloproteinase-2 in oxidized low-density lipoprotein-induced activation of the sphingomyelin/ceramide pathway and smooth muscle cell proliferation. Circulation Aug 3 2004;110(5):571–8. [21] Shinoda E, Yui Y, Hattori R, Tanaka M, Inoue R, Aoyama T, et al. Tissue factor pathway inhibitor-2 is a novel mitogen for vascular smooth muscle cells. J Biol Chem Feb 26 1999;274(9):5379–84. [22] Ishigaki Y, Oka Y, Katagiri H, Monticone R, Cheng L, Papadopoulos N. Circulating oxidized LDL: a biomarker and a pathogenic factor. Curr Opin Lipidol Oct 2009;20(5): 363–9 Review. [23] Pauly R, Passaniti A, Bilato C, et al. Migration of cultured vascular smooth muscle cells through a basement membrane barrier requires type IV collagenase activity and is inhibited by cellular differentiation. Circ Res Jul 1994;75(1):41–54. [24] Brewster L, Ucuzian A, Brey E, et al. FRNK Overexpression Limits the Depth and Frequency of Vascular Smooth Muscle Cell Invasion in a Three-Dimensional Fibrin Matrix. J Cell Physiol. 2010 May 20. [Epub ahead of print].
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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
Tissue factor pathway inhibitor-2 is downregulated by ox-LDL and inhibits ox-LDL induced vascular smooth muscle cells proliferation and migration Bilian Zhao a, Xinping Luo a,⁎, Haiming Shi a, Duan Ma b,⁎⁎ a b
Department of Cardiology, Huashan Hospital of Fudan University, Shanghai, China, 200040 The Key laboratory of molecular medicine, Ministry of Education, Shanghai Medical School of Fudan University, Shanghai, China, 200032
a r t i c l e
i n f o
Article history: Received 30 September 2010 Received in revised form 13 February 2011 Accepted 20 February 2011 Available online 1 April 2011 Keywords: tissue factor pathway inhibitor-2 (TFPI-2) vascular smooth muscle cells (VSMCs) proliferation migration atherosclerosis
a b s t r a c t Introduction: Tissue factor pathway inhibitor-2 (TFPI-2) is a member of the Kunitz-type family of serine protease inhibitors, which inhibits several matrix metalloproteinases activity involved in extracellular matrix degradation. Studies have shown low TFPI-2 expression in the shoulder regions of atherosclerotic plaques. But studies evaluating its role in the progression of atherosclerotic plaque are scarce. Vascular smooth muscle cells (VSMCs) are important components of atherosclerotic plaques and oxidized low density lipoprotein (oxLDL) is an important detrimental factor of atherosclerosis. The aim of this study is to elucidate the effect of TFPI-2 on smooth muscle cell proliferation and migration induced by ox-LDL. Methods: Retroviruses expressing human TFPI-2 were constructed. Cell proliferation was determined by CCK-8 assay. Cell apoptosis was analyzed by double staining of FITC-Annexin V and propidium iodide. Cell migration was studied through a Transwell chamber and with a scratch-wound assay. The matrix metalloproteinase-2 and − 9 activities were analyzed by gelatin zymography. Phosphorylation of FAK was analyzed by western blot. Results: TFPI-2 over-expression of mRNA and protein was confirmed in infected cells. CCK-8 assay showed that TFPI-2 inhibit VSMCs proliferation induced by ox-LDL while without cytotoxicity to VSMCs. Transwell and scratch wound assay confirmed TFPI-2 over-expression can inhibit VSMC migration. Zymography assay showed that TFPI-2 can inhibit MMP-2, 9 activity induced by ox-LDL. Western blot assay showed TFPI-2 can inhibit cyclinD1 expression and FAK phosphorylation. Conclusion: TFPI-2 over-expression may strongly inhibit the proliferation and migration of VSMCs and suppresses MMP-2, 9 activity induced by ox-LDL, making it a promising candidate for treatment of atherosclerotic process. © 2011 Elsevier Ltd. All rights reserved.
Proliferation and migration of VSMCs from media to intima is pivotal to atherosclerotic progression [1]. Normally, VSMCs are quiescent and are surrounded by and embedded in an extracellular matrix (ECM) scaffold that acts like a barrier to VSMCs migration. During the process of atherosclerosis, stimulated by environmental stimuli such as ox-LDL, VSMCs secreted several proteinases which degrade the ECM and in turn facilitate VSMCs proliferation and migration [2]. Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor synthesized by endothelial cells (ECs), smooth
Abbreviations: TFPI-2, tissue factor pathway inhibitor-2; VSMCs, vascular smooth muscle cells; ox-LDL, oxidized low density lipoprotein; MMP, matrix metalloproteinase; FAK, focal adhesion kinase. ⁎ Correspondence to: X. Luo, 12 Urumuqi Road, Department of Cardiology, Huashan Hospital, Shanghai, China, 200040. Tel.: +86 21 52887165; fax: +86 21 62480242. ⁎⁎ Correspondence to: D. Ma, 130 Dongan Road, The Key laboratory of molecular medicine, Ministry of Education, Shanghai Medical School of Fudan University, Shanghai, China, 200032. Tel.: +86 21 54237414. E-mail addresses: [email protected] (X. Luo), [email protected] (D. Ma). 0049-3848/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2011.02.025
muscle cells (SMCs), and syncytiotrophoblasts [3,4], which associates through ionic interactions [5] with the extracellular matrix (ECM) [6] and inhibits trypsin, plasmin, and plasma kallikrein among others [7]. The major role of TFPI-2 seems to be plasmin inhibition [8] and thus of proMMP-1, proMMP-3, and proMMP-13 activation [9]. As a direct inhibitor of MMP-2 and MMP-9 [10] and potent inhibitor of both matrix-bound and cell-associated plasmin, TFPI-2 could regulate extracellular proteolysis and ECM remodeling, which are highly relevant both for normal development and for atherosclerosis. It has been shown to inhibit endothelial cell proliferation induced by vascular endothelial growth factor (VEGF) [11]. Its expression has been shown to be lower at the shoulder of atherosclerosis plaque [12,13], which suggests that downregulation of TFPI-2 contributes to atherosclerosis progression. Oxidized low density lipoprotein (ox-LDL), which is an important detrimental factor of atherosclerosis, is well established as an important stimulus of VSMCs proliferation and a chemoattractant directing the migration of VSMCs toward the vessel intima during atherosclerosis and neointima hyperplasia [14]. To test the role of TFPI-2 in atherosclerotic progression, we studied TFPI-2 function on VSMCs under the treatment of ox-LDL.
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Materials and methods Materials Primary human aortic smooth muscle cells were purchased from ATCC. Retroviral plasmid vector pBABE-puro was purchased from Clontech (BD Bosciences, CA). Cell culture media and supplements were from Invitrogen (Carlsbad, CA) and HyClone (Logan, UT). Cell culture disposable materials were purchased from BD. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (MD, USA). Oxidized low density lipoprotein (ox-LDL) and monoclonal antibodies to TFPI-2 were purchased from R&D Systems (Minneapolis, MN). Polyclonal rabbit anti-cyclinD1, anti-GAPDH, anti-focal adhesion kinase (FAK) and phosphorylation specific polyclonal antibody to FAK tyr-397 were purchased from Cell Signal Technology (USA). Transwell with 8-μm pore polycarbonate membrane was purchased from Corning (NY, USA). Crystal violet was purchased from Sigma (St. Louis, USA). All other chemicals and reagents were obtained from commercial source and were of analytic grade.
three step program of 30 s at 94 °C, 30 s at 59 °C and 30 s at 72 °C) and were performed in triplicate. The relative target mRNA levels were assessed with use of ABI Prism 7500 software and normalized to that of internal control β-actin.
Cell proliferation assay CCK-8 cell viability assay was used to evaluate cell proliferation following the manufacturer's instruction. Briefly, 48 hours after infection, Retro-TFPI-2 cells and control cells were trypsinized and harvested, and then 100 μl cells were seeded into 96-well plates both at 5 × 104/ml. After cell adhesion, VSMCs were incubated in DMEM containing 0.5% FBS for 24 hours. Then 40 μg/ml ox-LDL or 10% FBS was added to each well as a stimulator. The medium was replaced every 24 hours. At every 24 hours, 10 μl CCK-8 reagent was added to each well, and then incubated at 37 °C for 4 hours. After that, absorbances in each well were read with a spectrophotometric plate reader at 450 nm.
Cell culture Apoptosis assay VSMCs were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum (FBS). Cultures were maintained at 37 C °C in a humidified 95% air and 5% CO2 atmosphere. VSMCs were passaged by trypsinization with 0.25% trypsin and 0.02% EDTA in PBS. In this study, early passage (four to eight) VSMCs were used. Effects of Ox-LDL on TFPI-2 expression To investigate ox-LDL regulation on TFPI-2 expression, VSMCs were treated with different concentrations of ox-LDL (20-40 μg/ml). 24 hours later, total cell lysates were harvested and western blot analysis was performed.
Cells were stained with FITC-AnnexinV and propidium iodide and then evaluated for apoptosis by flow cytometry according to the manufacturer's protocol (BD Biosciences). Briefly, 24 hours after infection, Retro-TFPI-2 cells and control cells were treated with 40 μg/ml ox-LDL or not for 48 hours, then cells were trypsinized and harvested, washed twice with cold PBS, and centrifuged at 1000 g for 5 min. Cells were resuspended in 400 μl binding buffer at a concentration of 1 × 106 cells per ml, 100 μl of the solution was transferred to a 5 ml culture tube, and 5 μl of FITC-AnnexinV and 5 μl of propidium iodide were added. Cells were gently vortexed and incubated for 15 min at room temperature in the dark. Finally, 400 μl of binding buffer was added to each tube and samples were analyzed by flow cytometry (Becton Dickinson, Heidelberg, Germany).
Construction of recombinant retrovirus Full-length human TFPI-2 was generated from primary VSMCs by RT-PCR. The primers flanking the coding region of TFPI-2 were sense, 5’- ATAGAATTCATGGACCCCGCTCGCCCCCTG-3’, and antisense, 5’CGCGTCGACTTAAAATTGCTTCTTCCGAATTTTC-3’. The recombinant retroviruses were constructed and amplified according to the manufacturer's protocol. The recombinant retroviruses contained human TFPI-2 cDNAs (Retro-TFPI-2) were used to induce overexpression of TFPI-2. A retrovirus carrying green fluorescence protein (GFP) was used as a negative control. For retrovirus-mediated TFPI-2 gene transduction, VSMCs were grown to 60% confluence and infected with Retro-TFPI-2 or RetroGFP-Control separately. After 24 hours, the viral suspension was removed and the cells used as required. Real-time quantitative PCR analysis Real-time reverse transcription (RT)-PCR was used to determine the relative amount of TFPI-2 in infected cells. Forty-eight hours after infected, total RNA was extracted by use of TRIzol reagent (Invitrogen, Carlsbad, CA). Two micrograms of total RNA per sample was reverse-transcribed by the AMV Reverse Transcription System (Promega, Madison, WI). Realtime PCR amplification involved use of an ABI Prism 7500 sequence detector (Applied Biosystems) and SYBR Green reagent. The specific primers for human TFPI-2 were sense, 5’ –GTCGATTCTGCTGCTTTT CC-3’ and antisense, 5’-ATGGAATTTTCTTTGGTGCG-3’; and the primers for human β-actin were sense, 5’-GAAACTACCTTCAACTCCATC- -3’ and antisense, 5’-CGAGGCCAGGATGGAGCCGCC-3’. All amplification reactions were carried out over 40 cycles (an initial stage of 4 min at 94 °C then a
Cell migration The effect of TFPI-2 on VSMCs migration was monitored with Transwell using a modified Boyden's chamber assay. Briefly, 48 hours after infection, the Retro-TFPI-2 and control cells were harvested by trypsinization and suspended in the serum free DMEM supplemented with 0.2% BSA. Cell suspensions were then placed into the upper well at a concentration of 1 × 104 cells/100 μl separately, while the same medium was placed into lower well (500 μl). The lower chamber contained ox-LDL (40 μg/ml) as a chemoattractant. The chamber was incubated at 37 °C in 5% CO2 humidified atmosphere for 4 h. Nonmigrating cells on the upper surface were scraped gently and washed out with PBS three times. VSMCs migrated to the lower surface of the membrane were stained with crystal violet dye and counted per three independent HPFs (×100) with light microscope.
Scratch-wound assay Retro-TFPI-2 cells and control cells were seeded in 6-well plates at a concentration of 10,000 cells per well, and then grown until reaching confluence. A linear wound was gently introduced in the center of the cell monolayer using 1 ml tip, followed by washing with DMEM to remove the cellular debris. Then the cells were incubated with 40 μg/ml ox-LDL and monitored for 48 hours. Cell images were taken at 48 hours, and the number of cells migrated into the wound space were manually counted in three fields per well with light microscope (×100).
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Gelatin zymography 48 hours after infection, Retro-TFPI-2 and control cells were harvested and seeded in 24 well plates at a concentration of 100,000 cells per well with DMEM plus 15% FBS. 12 hours later, the media was changed into DMEM with no serum. Then the cells were treated with 40 μg/ml ox-LDL for 24 hours. Proteins in the conditioned media were separated, without prior boiling, by electrophoresis through 10% SDS-PAGE containing 1 mg/ml gelatin. The proteins in the gel were renatured and stained as described [15]. SDS-PAGE and western blot analysis Retro-TFPI-2 and control cell layers treat with or without 40 μg/ml oxLDL, were rinsed three times with PBS and extracted in ice-cold lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, 1% NP-40, 0.1% SDS, 1 mM PMSF, 1 mg/ml each of aprotinin, leupeptin, pepstatin) for 10 min. Lysates were centrifuged at 12,000 g for 5 min at 4 °C, and supernatants were stored at 80 °C. Protein concentrations were determined by the BCA method. Twenty micrograms of protein from each cell extract was separated by SDS-PAGE and then transferred to nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ). Membranes were blocked with blocking solution (50 mM Tri-HCl, 150 mM NaCl, 5% (w/v) non-fat dry milk and 0.1% Tween-20) overnight at 4 °C. After incubating with appropriate primary and secondary antibodies, the blots were detected with the Supersignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL). The results of western blots were analyzed by the Image J program. Statistical analysis Each experiment was repeated at least three times. All the data analyses were conducted with SPSS 15.0 (SPSS, Inc., Chicago, IL). All the variables were under normal distribution and values were reported as mean ± standard deviation (SD). Significance of differences was analyzed using independent samples t test as appropriate. P b 0.05 was considered statistically significant.
Fig. 1. TFPI-2 expression is downregulated by ox-LDL in VSMCs. VSMCs were treated with ox-LDL at 20-40 μg/ml or not for 24 hours. Total cell lysates were harvested and western blot analysis was performed to detect the expression of TFPI-2. The detected three bands were three different glycosylated isoforms of TFPI-2 (27, 31, 33 kDa). *: P b 0.05 compared with control; **: P b 0.01 compared with control.
Results TFPI-2 expression is downregulated by ox-LDL in VSMCs TFPI-2 has been reported to be expressed in VSMCs and endothelial cells [3,4]. And its expression in endothelial cell was upregulated by vascular endothelial growth factor [11]. Here we were interested in determining whether TFPI-2 may be regulated by oxLDL. Western blot analysis showed ox-LDL treatment decrease TFPI-2 expression in VSMCs in a dose-dependent manner (Fig. 1). We detected three different glycosylated isoforms of TFPI-2 (27, 31, 33 kDa), in accordance with previous observations [11]. TFPI-2 over expression after retrovirus-mediated gene transduction Forty eight hours after infected, both the mRNA and protein expression of TFPI-2 were confirmed by real time reverse transcript PCR and western blot analysis, respectively. Fig. 2A showed the result of real time PCR, which suggesting that Retro-TFPI-2 cells strongly expressed TFPI-2 mRNA than control cells. Consisted with real time PCR, in the Western Blot analysis (Fig. 2B), Retro-TFPI-2 cells had darker bands of TFPI-2 than control cells. Effect of TFPI-2 on VSMCs proliferation and cyclin D1 expression with ox-LDL or FBS treatment CCK-8 assay allows evaluation of cell metabolic activity such as cell number, cell metabolism, and mitochondrial activation. As seen in
Fig. 2. Identification and expression of TFPI-2 after viral infection. A. Real time PCR of TFPI-2 mRNA expression in VSMCs after infected with Retro-GFP viruses or TFPI-2 expression viruses. Real time PCR of β-actin mRNA expression was performed as a control. Data are presented as mean value ± SD, the asterisk indicates a P b 0.01. B. Western blot analysis for the expression of TFPI-2 after infected with Retro-GFP viruses or TFPI-2 expression viruses.
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Fig. 3, when incubated with DMEM plus 10% FBS (A), the 450 nm absorbances of the Retro-TFPI-2 cells and control cells did not differ significantly, indicating that neither the vector construct itself nor TFPI-2 over-expression significantly influenced cell proliferation in normal condition. In contrast, while treated by 40 μg/ml ox-LDL (B), the absorbance of the Retro-TFPI-2 cells were obviously lower than control cells, which suggested that TFPI-2 over expression inhibited VSMCs proliferation induced by ox-LDL. CyclinD1 is a major promoter of the cell cycle. In order to verify the effect of TFPI-2 on VSMCs proliferation, we analyzed CyclinD1 expression in Retro-TFPI-2 cells and control cells with FBS or ox-LDL treatment. As shown in Fig. 3C, with FBS treatment, upregulation of TFPI-2 had no significant influence on cyclin D1 expression. In contrast, when treated by ox-LDL, cyclinD1 expression was strongly inhibited in Retro-TFPI-2 cells in comparison with control cells (P b 0.01). Effects of TFPI-2 on VSMCs apoptosis In order to verify if the induction of apoptosis occurred during TFPI-2 upregulation and ox-LDL treatment, the presence of apoptotic cells was determined by the application of double staining with FITCAnnexin V and propidium iodide. Normal live cells were represented as FITC-negative and PI-positive, early apoptotic cells as FITC- positive and PI- negative, and necrotic cells as FITC-positive and PI-positive. The percentage of apoptotic cell populations in Retro-TFPI-2 cells and control cells were, respectively, 3.22 ± 0.06% and 3.62 ± 0.09% (without ox-LDL treatment, P N 0.05), 3.05 ± 0.07% and 2.44 ± 0.09%
(with ox-LDL treatment, P N 0.05). This indicated no significant effect on apoptosis of TFPI-2 upregulation and ox-LDL treatment. Representative experiments are shown in Fig. 4. TFPI-2 inhibits VSMCs migration It is known to all that ox-LDL is a chemoattractant and proliferative agent for VSMCs. To determine the effect of TFPI-2 on ox-LDLstimulated VSMCs migration, we carried out migration assays in the presence of 40 μg/ml ox-LDL. In the Transwell assay(Fig. 5A), VSMCs migration decreased from 53 ± 3 cells per field for control to 26 ± 1 cells/field for Retro-TFPI-2-infected cells (P b 0.01). In the scratch assay (Fig. 5B), control cells treated with ox-LDL migrated into the wound area and organized a dense cellular network, resulting in nearly complete wound recovery after 48 hours, while migration into the wound area of Retro-TFPI-2 cells was significantly inhibited. The migrated cells in Retro-TFPI-2 cells and control cells were, respectively, 35 ± 4 cells and 86 ± 3cells (P b 0.01). TFPI-2 inhibits MMP-2, 9 activity Matrix metalloproteinase-2, 9 (MMP-2, 9) are known to play several important roles during changes in vascular structure associated with atherosclerotic progression. The stimulation with ox-LDL leads to increased activity of MMP-2 and − 9, and this up-regulation was linked to increased cell migration. To test whether over expression of TFPI-2 could inhibit MMP-2 and MMP-9 activity, we stimulated VSMCs with 40 μg/ml ox-LDL and analyzed the
Fig. 3. Effect of TFPI-2 on ox-LDL induced VSMCs proliferation and cyclin D1 expression. Retro-TFPI-2 cells and control cells were incubated in DMEM containing 0.5% FBS for 24 hours before experiments. A. Cells were incubated with DMEM plus 10% FBS. Absorbances in each well were read with a spectrophotometric plate reader at 450 nm. B. Cells were treated by 40 μg/ml ox-LDL. Absorbances in each well were read with a spectrophotometric plate reader at 450 nm. **: P b 0.01 compared with control. C. Western blot of extracts from VSMCs incubated with 10% FBS or 40 μg/ml ox-LDL for 48 hours (revealed with anti-cyclinD1 and anti-GAPDH antibodies). **: P b 0.01 comparison between Retro-TFPI-2 cells and control cells treated with ox-LDL.
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Fig. 4. Effect of TFPI-2 on apoptosis of VSMCs. Cell apoptosis assessed by double staining of FITC-AnnexinV and propidium iodide and subsequent flow cytometry. 24 hours after infection, Retro-TFPI-2 cells and control cells were treated with 40 μg/ml ox-LDL or not for 48 hours. The presence of apoptotic cells was identified by flow cytometric analysis of cells labeled with FITC-AnnexinV and propidium iodide. Cells in the lower right quadrant correspond to early apoptotic cells, whereas those in the upper right quadrant correspond to late apoptotic or necrotic cells. Numbers in each quadrant are percentage of cells they contain. The results shown are representative of three separate experiments.
Fig. 5. Effect of TFPI-2 on migration of VSMCs. A. 10,000 Retro-TFPI-2 cells or control cells were plated into the upper chamber of Transwells containing an 8 μm barrier with reconstituted basement membrane proteins. Ox-LDL (40 μg/ml) was included in the lower chamber as a chemoattractant. Cells migrating across the filters at 4 hours were stained by crystal violet. Magnification is × 100. Bottom, representative results of three independent experiments is shown. Migrated cells were qualified by the average of three randomly chosen high-power fields of three independent experiments. B. Confluent VSMCs monolayers infected with Retro-TFPI-2 or Retro-GFP were scratch wounded 24 hours after infection. Cells were then treated with 40 μg/ml ox-LDL for 48 hours before imaging (lines indicate wounds edge). The number of cells migrated into the wound space were manually counted in three fields per well with light microscope (×100). **: P b 0.01 compared with control.
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metalloproteinase activity by gelatin zymography. As seen in Fig. 6, without ox-LDL treatment, VSMCs secreted little activated MMP-2 and MMP-9. Ox-LDL treatment strongly induced MMP-2 and MMP-9 activity and TFPI-2 over-expression inhibited VSMCs expression of MMP-2 and MMP-9 induced by ox-LDL (both P b 0.01). TFPI-2 inhibits phosphorylation of FAK Phosphorylation of focal adhesion kinase is critical for cell migration. The decline in VSMCs migration suggested that TFPI-2 might be altering the phosphorylation of FAK. To measure FAK phosphorylation, we immunoprecipitated FAK from Retro-TFPI-2 VSMCs and control cells extracts treated with or without 40 μg/ml ox-LDL and performed an immunoblot with an antibody to detect phosphorylated tyrosine. As shown in Fig. 7, Retro-TFPI-2 cells had a significant decrease in tyrosine phosphorylation of FAK in comparison with control cells, especially when treated with ox-LDL (P b 0.01). Discussion The extensive plasticity of VSMCs makes cells susceptible to various stimuli inducing changing in phenotype that contributes to the etiology of cardiovascular diseases [16]. It is well established that accelerated proliferation of VSMCs and migration of VSMCs from the media to the intima are fundamental events in the development of both intimal hyperplasia and atherosclerosis [17,18]. Therefore, inhibition of VSMCs proliferation and migration represents a potentially promising therapeutic strategy for the treatment of diseases such as atherosclerosis and neointimal hyperplasia. It has been shown that TFPI-2 is induced by fluid shear stress in vascular smooth muscle cells and affects cell proliferation and survival
Fig. 6. Effect of TFPI-2 on secreted MMP-2, 9. 24 hours after ox-LDL treatment, the conditioned media of Retro-TFPI-2 or control cells treated with or without 40 μg/ml oxLDL were analyzed by gelatin zymography. Ox-LDL increased MMP-2 and MMP-9 activity significantly. Compared to control cells, TFPI-2 over-expression markedly inhibited MMP-2 and MMP-9 activity. **: P b 0.01 compared with control.
Fig. 7. Effect of TFPI-2 on phosphorylation of FAK. Western blots of extracts from RetroTFPI-2 and control cell layers treat with or without 40 μg/ml ox-LDL for 48 hours (revealed with anti-FAK and anti-phospho-FAK antibodies). Compared to control, TFPI2 over-expression markedly inhibited phosphorylation of FAK, especially when treated with ox-LDL. The results shown are representative of three separate experiments. #: P b 0.05 comparison between Retro-TFPI-2 cells and control cells treated without oxLDL. **: P b 0.01 comparison between Retro-TFPI-2 cells and control cells treated with ox-LDL.
[19], which suggests that TFPI-2 expression by VSMCs in response to fluid shear stress may play a role in the control of VSMCs turnover in the intima during vascular repair. Our data showed that ox-LDL suppressed TFPI-2 in a dose-dependent manner and over expression of TFPI-2 inhibited ox-LDL induced VSMCs proliferation compared with control. While in normal condition (without ox-LDL treatment), TFPI-2 over-expression had no significant influence on the proliferation of VSMCs. Accordingly, when treated by ox-LDL, cyclin D1 expression was inhibited in Retro-TFPI-2 cells compared to the control cells. Under normal conditions, no significant differences were shown in cyclin D1expression between the two groups. We assume the reason may be that TFPI-2 can inhibit MMP-2 activity induced by ox-LDL, which have been shown to play a pivotal role in ox-LDLinduced activation of the sphingomyelin/ceramide signaling pathway and subsequent VSMCs proliferation [20].While under normal conditions, VSMCs secreted little MMP-2. Our result was different from that of Shinoda [21], who reported TFPI-2 could act as a mitogen for VSMCs. In addition, we found no effect of TFPI-2 on VSMCs apoptosis, unlike Johan [19], who found a pro-apoptotic effect of TFPI-2 in VSMCs. The discrepancy may be because we used viral vector to upregulate TFPI-2 and they used different concentration of recombinant TFPI-2 protein. In this study, we also found TFPI-2 overexpression inhibit VSMCs migration and MMP-2, 9 activity induced by ox-LDL, which is accepted to be an important detrimental factor of atherosclerosis [22]. MMP-2, 9 have been reported as the principle MMPs expressed and secreted by the synthetic VSMCs, which accelerates their migration and increases their invasion ability [23]. As a member of
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the Kunitz-type family of serine proteinase inhibitors, TFPI-2 has been shown to inhibit MMP-2 and MMP-9 activity in several tumor cells. Here we demonstrated that TFPI-2 inhibited MMP-2, 9 activity induced by ox-LDL, suggesting that TFPI-2 has the potential to inhibit the process of atherosclerosis. Ox-LDL is well established as a chemoattractant directing the migration of VSMCs toward the vessel intima during atherosclerosis and neointimal hyperplasia [14]. Our data showed that TFPI-2 strongly inhibited VSMCs migration induced by ox-LDL, as well as into a wound area. TFPI-2's strongly inhibition of plasmin, trypsin and MMPs, which preventing the ECM degradation may be a probable mechanism. FAK is a critically important, non-receptor protein tyrosine kinase that coordinates both integrin and growth factor signaling cascades involved in VSMCs matrix invasion and migration. It has been reported that disruption of FAK signaling may provide a pharmaceutical tool that limits pathological VSMC cell behavior [24]. Our data revealed that tyrosine phosphorylation of FAK in Retro-TFPI-2 cells was diminished in comparison with control cells. We assume this was due to the effect of TFPI-2 on ECM remodeling. These findings suggest a mechanism through which TFPI-2 regulates VSMCs migration. In summary, our data demonstrated that TFPI-2 over-expression may strongly inhibit the proliferation and migration of VSMCs induced by ox-LDL and without cytotoxicity, suppressed MMP-2 and MMP-9 activities and inhibited FAK phosphorylation. These findings suggest TFPI-2 may be a promising therapeutic for treatment of atherosclerotic progression. Further studies are needed to investigate the effect of TFPI-2 downregulation on VSMCs and its role in atherosclerotic progression in vivo. Sources of funding This work was supported by the Shanghai Committee of Science and Technology, China (Grant No. 074119604) and Shanghai Health Bureau, China (Grant No. 2007100). Acknowledgements We thank Chundi Xu, Fenge Deng, Shoudong Ye and Pengliang Mao from The Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai Medical School of Fudan University for technical assistance. References [1] Newby AC, Zaltsman AB. Molecular mechanisms in intimal hyperplasia. J Pathol 2000;190:300–9. [2] Hu J, Van den Steen PE, Sang QX, Opdenakker G. Matrix metalloproteinase inhibitors as therapy for inflammatory and vascular diseases. Nat Rev Drug Discov 2007;6:480–98. [3] Iino M, Foster DC, Kisiel W. Quantification and characterization of human endothelial cell-derived tissue factor pathway inhibitor-2. Arterioscler Thromb Vasc Biol 1998;18:40–6.
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