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Tissue factor regulation in vascular smooth muscle: A s...
The American Journal of Cardiology

Tissue factor regulation in vascular smooth muscle: A summary of studies performed using in vivo and in vitro models Original Research Article
The American Journal of Cardiology, Volume 72, Issue 8, 9 September 1993, Pages C55-C60
Mark B. Taubman

Abstract
In response to vascular injury, such as occurs in acute or chronic atherosclerosis, vascular smooth muscle cells (VSMCs) proliferate and migrate, ultimately contributing to vessel narrowing. Several growth agonists, including platelet-derived growth factor and fibroblast growth factor, are known to cause VSMCs to profilerate and are believed to be present at sites of vessel injury. Targets for inhibiting VSMC growth or migration in the presence of multiple growth agonists include genes that are induced in common by VSMC growth factors. This article summarizes the in vivo and in vitro findings of experiments designed to investigate the regulation of tissue factor in VSMCs in 2 animal models. The findings demonstrate that tissue factor is rapidly induced by growth factors in VSMC culture and by balloon injury in aortic media, and they suggest that VSMCs may play a major role in mediating the early thrombotic response. Strategies designed to inhibit the response of VSMCs to growth agonists may therefore have important implications for the treatment of intravascular thrombosis.

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p19ARF Deficiency Reduces Macrophage and Vascular Smoot...
Journal of the American College of Cardiology

p19^ARF Deficiency Reduces Macrophage and Vascular Smooth Muscle Cell Apoptosis and Aggravates Atherosclerosis Original Research Article
Journal of the American College of Cardiology, Volume 55, Issue 20, 18 May 2010, Pages 2258-2268
Herminia González-Navarro, Yafa Naim Abu Nabah, Ángela Vinué, María J. Andrés-Manzano, Manuel Collado, Manuel Serrano, Vicente Andrés

Abstract

Objectives

The goal of this study was to investigate the role in atherosclerosis of the tumor suppressor protein ARF (human p14^ARF, mouse p19^ARF) encoded by the CDKN2A gene.

Background

Atherosclerosis is characterized by excessive proliferation and apoptosis, 2 cellular processes regulated by CDKN2A. Although recent genome-wide association studies have linked atherosclerotic diseases to a genomic region in human chromosome 9p21 near the CDKN2A locus, the mechanisms underlying this gene–disease association remain undefined, and no causal link has been established between CDKN2A and atherosclerosis.

Methods

Atherosclerosis-prone apolipoprotein E (apoE)-null and doubly deficient apoE-p19^ARF mice were fed an atherogenic diet and sacrificed to quantify atherosclerosis burden in whole-mounted aortas and in aortic cross-sections. Proliferation and apoptosis were investigated in atherosclerotic lesions and in primary cultures of macrophages and vascular smooth muscle cells obtained from both groups of mice.

Results

Genetic disruption of p19^ARF in apoE-null mice augments aortic atherosclerosis without affecting body weight, plasma lipoproteins, or plaque's proliferative activity. Notably, p19^ARF deficiency significantly attenuates apoptosis both in atherosclerotic lesions and in cultured macrophages and vascular smooth muscle cells, 2 major cellular constituents of atheromatous plaques.

Conclusions

Our findings establish a direct link between p19^ARF, plaque apoptosis, and atherosclerosis, and suggest that human genetic variants associated to diminished CDKN2A expression may accelerate atherosclerosis by limiting plaque apoptosis.

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Cyclosporine a stimulates proliferation of vascular smo...
Transplantation Proceedings

Cyclosporine a stimulates proliferation of vascular smooth muscle cells and enhances monocyte adhesion to vascular smooth muscle cells Original Research Article
Transplantation Proceedings, Volume 31, Issues 1-2, 3 February 1999, Pages 663-665
S. -J. Hu, R. Fernandez, J. W. JonesJr

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Apoptosis of vascular smooth muscle cells in atheroscle...
Atherosclerosis

Apoptosis of vascular smooth muscle cells in atherosclerosis Original Research Article
Atherosclerosis, Volume 138, Issue 1, May 1998, Pages 3-9
Martin R. Bennett, Joseph J. Boyle

Abstract
Apoptosis (programmed cell death) of vascular smooth muscle cells and macrophages has recently been demonstrated in the following: (a) human atherosclerotic plaques; (b) physiological remodelling of the vessel; and (c) a variety of disease states. Apoptosis is a highly regulated mechanism of death, controlled by the interactions between the following: (i) cellular receptors; (ii) cytoplasmic and nuclear gene products; and (iii) the local cytokine and cellular environment within the plaque. The knowledge of the key regulators of apoptosis does, however, offer novel therapeutic targets in both the prevention and treatment of atherosclerosis.

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Vascular smooth muscle cell proliferation and its thera...
American Heart Journal

Vascular smooth muscle cell proliferation and its therapeutic modulation in hypertension Original Research Article
American Heart Journal, Volume 122, Issue 4, Part 2, October 1991, Pages 1198-1203
Vratislav Hadrava, Ursula Kruppa, Roberto C. Russo, Yves Lacourcière, Johanne Tremblay, Pavel Hamet

Abstract
The increased growth potential of vascular smooth muscle cells (VSMCs) represents one of the crucial anomalies responsible for the development of essential hypertension, diabetic macroangiopathy, and atherosclerosis. The exaggerated response to growth factors of VSMC from spontaneously hypertensive rats (SHRs) persists in culture when compared with normotensive Wistar-Kyoto control rats, indicating an intrinsic defect in the hypertension-producing mechanism. This greater proliferation is characterized by two intermediate phenotypes: (1) accelerated entry into the S phase of the cell cycle, which results from hyperresponsiveness to epidermal growth factor and platelet-derived growth factor, and (2) abnormal contact inhibition. The enhanced expression of transforming growth factor β₁ (TGF-β₁) messenger ribonucleic acid in SHRs precedes this altered contact inhibition, and only VSMCs from SHRs respond to exogenously added TGF-β₁ at a high cell density, which suggests that abnormal TGF-β₁ autoregulation may be implicated in the second phenotype. Platelets contain major growth factors for VSMC. Platelet extracts from hypertensive and diabetic patients present augmented growth-promoting activity on VSMCs, which is most evident when both diseases occur simultaneously. Growth-promoting activity may be further influenced by antihypertensive therapy. This growth-promoting activity is increased by hydrochlorothiazide but not by indapamide, atenolol, or captopril in diabetic hypertensive and nondiabetic hypertensive patients. In conclusion, VSMCs in hypertension manifest an intrinsic growth defect that is modulated by extrinsic platelet growth factors and antihypertensive drugs.

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xPharm: The Comprehensive Pharmacology Reference

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 and Duan Ma^b^,^,

^a Department of Cardiology, Huashan Hospital of Fudan University, Shanghai, China, 200040

^b The Key laboratory of molecular medicine, Ministry of Education, Shanghai Medical School of Fudan University, Shanghai, China, 200032

Received 30 September 2010; revised 13 February 2011; accepted 20 February 2011. Available online 1 April 2011.

Abstract

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 (ox-LDL) 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.

Keywords: tissue factor pathway inhibitor-2 (TFPI-2); vascular smooth muscle cells (VSMCs); proliferation; migration; atherosclerosis

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

Article Outline

Materials and methods: Materials; Cell culture; Effects of Ox-LDL on TFPI-2 expression; Construction of recombinant retrovirus; Real-time quantitative PCR analysis; Cell proliferation assay; Apoptosis assay; Cell migration; Scratch-wound assay; Gelatin zymography; SDS-PAGE and western blot analysis; Statistical analysis

Results: TFPI-2 expression is downregulated by ox-LDL in VSMCs; TFPI-2 over expression after retrovirus-mediated gene transduction; Effect of TFPI-2 on VSMCs proliferation and cyclin D1 expression with ox-LDL or FBS treatment; Effects of TFPI-2 on VSMCs apoptosis; TFPI-2 inhibits VSMCs migration; TFPI-2 inhibits MMP-2, 9 activity; TFPI-2 inhibits phosphorylation of FAK

Discussion

Sources of funding

Acknowledgements

References

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 muscle cells (SMCs), and syncytiotrophoblasts [3] and [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] and [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.

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.

Cell culture

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.

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 over-expression 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 Retro-GFP-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). Real-time 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 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.

Apoptosis assay

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).

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. Non-migrating 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).

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 ox-LDL, 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 < 0.05 was considered statistically significant.

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] and [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 ox-LDL. 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].

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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 < 0.05 compared with control; **: P < 0.01 compared with control.

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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.

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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 < 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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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. 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.

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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 < 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 < 0.01 comparison between Retro-TFPI-2 cells and control cells treated with ox-LDL.

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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 < 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 FITC-Annexin 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 > 0.05), 3.05 ± 0.07% and 2.44 ± 0.09% (with ox-LDL treatment, P > 0.05). This indicated no significant effect on apoptosis of TFPI-2 upregulation and ox-LDL treatment. Representative experiments are shown in Fig. 4.

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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.

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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-LDL-stimulated 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 < 0.01).

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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 < 0.01 compared with control.

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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 < 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 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 < 0.01).

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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 ox-LDL 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 < 0.01 compared with control.

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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 < 0.01).

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Fig. 7.

Effect of TFPI-2 on phosphorylation of FAK.

Western blots of extracts from Retro-TFPI-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, TFPI-2 over-expression markedly inhibited phosphorylation of FAK, especially when treated with ox-LDL. The results shown are representative of three separate experiments. #: P < 0.05 comparison between Retro-TFPI-2 cells and control cells treated without ox-LDL. **: P < 0.01 comparison between Retro-TFPI-2 cells and control cells treated with ox-LDL.

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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] and [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 [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-LDL-induced 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 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.

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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.
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