Skip Navigation

Cardiovascular Research 2003 59(2):460-469; doi:10.1016/S0008-6363(03)00428-0
© 2003 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Wang, B.-W.
Right arrow Articles by Shyu, K.-G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, B.-W.
Right arrow Articles by Shyu, K.-G.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2003, European Society of Cardiology

Induction of matrix metalloproteinases-14 and -2 by cyclical mechanical stretch is mediated by tumor necrosis factor-{alpha} in cultured human umbilical vein endothelial cells

Bao-Wei Wanga, Hang Changb, Shankung Lina, Peiliang Kuana and Kou-Gi Shyua,c,*

aDivision of Cardiology, Shin Kong Wu Ho-Su Memorial Hospital, 95 Wen-Chang Rd., Taipei 111, Taiwan
bDepartment of Emergency Medicine, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan
cGraduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan

* Corresponding author. Tel.: +886-2-2833-2211; fax: +886-2-2836-5775. shyukg{at}ms12.hinet.net

Received 25 January 2003; revised 4 April 2003; accepted 29 April 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Mechanical forces have profound effects on endothelial cells. This study was undertaken to examine the hypothesis that tumor necrosis factor-{alpha} (TNF-{alpha}) is a potential mediator of stretch-induced effects on matrix metalloproteinase (MMP). Methods: Human umbilical vein endothelial cells (HUVECs) grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation, at 60 cycles/min. We used the TNF-{alpha} monoclonal antibody and c-Jun N-terminal kinase (JNK) inhibitor, SP600125, to investigate the cyclical stretch-induced expression of MMP-14 and -2 in cultured HUVECs. Results: Cyclical mechanical stretch significantly increased protein synthesis and mRNA expression for MMP-14 and -2 from 2 to 24 h. The increased MMP-14 and-2 proteins after stretch were completely blocked after the addition of TNF-{alpha} monoclonal antibody (5 µg/ml) or SP600125 (20 µM) 30 min before stretch. By zymography, MMP-2 expression was induced by cyclical stretch and was attenuated by TNF-{alpha} monoclonal antibody and SP600125. Cyclical stretch increased the immunohistochemical labeling of MMP-14 and -2 and significantly increased release of TNF-{alpha} into the culture media from 120±2 to 331±2 pg/ml (P<0.001) after stretch for 12 h. Cyclical stretch increased and SP600125 decreased the phosphorylated JNK. Gel-shifting assay showed that DNA–protein binding activity of AP-1 increased after cyclical stretch and TNF-{alpha} monoclonal antibody and SP600125 abolished the binding activity induced by cyclical stretch. Conclusion: These findings indicate that cyclical stretch augments TNF-{alpha} production and MMP genes expression in HUVECs. TNF-{alpha} mediates the stretch-induced MMP genes expression, at least in part, through the JNK pathway.

KEYWORDS Cell culture; Cytokines; Extracellular matrix; Gene expression; Stretch


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes that are responsible for the degradation of extracellular matrix [1,2]. MMPs are involved in normal physiological processes such as embryogenesis and tissue remodeling and may play an important role in tumor metastasis and angiogenesis [3–6]. Endothelial cells are constantly exposed to mechanical forces and actively remodel the extracellular matrix of basement membrane and mature interstitial tissues. MMP-2 promotes matrix protein degradation in vascular disease remodeling [1] and facilitates vascular smooth muscle cells migration [7]. MMP-2 production by endothelial cells or surrounding cells may be vital to the formation of new functional blood vessels. Like most MMPs, MMP-2, secreted as an inactive proenzyme, requires proteolytic removal of a terminal propeptide domain for its activation. Cleavage and activation of MMP-2 is achieved by a membrane type 1 matrix metalloproteinase (also known as MMP-14). MMP-14 and -2 expression and activation have been associated with neointimal growth after mechanical injury to the vascular wall [8]. Inflammatory cytokines such as interleukin-1 and tumor necrosis factor-{alpha} (TNF-{alpha}) can modulate the expression of MMPs in endothelial cells [9,10]. Endothelial cells, the sensors of increased flow and wall shear stress, can release MMPs [11]. Mechanical stretch has been shown to induce expression and activation of MM-14 in endothelial cells [12,13]. Little is known about the signal pathway mediating the stretch-induced MMP expression [13]. To date, there is no information in endothelial cells on the role of proinflammatory cytokine as a potential mediator of stretch-induced effects on MMPs. Mechanical stretch induced production of TNF-{alpha} in cardiac fibroblasts, but not in cardiac myocytes [14]. It is not reported yet whether mechanical stretch can induce production of TNF-{alpha} in endothelial cells. The present study was designed to test whether cyclical mechanical stretch could induce TNF-{alpha} production in endothelial cells and to investigate whether there is a link between the stretch-induced TNF-{alpha} and MMPs.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Human umbilical vein endothelial cell (HUVECs) culture
HUVECs were isolated and cultured from umbilical cord vein as described previously [15]. HUVECs were used between passages 3 and 5. Each passage took a time of 3 days. Then the third- to fifth-passage HUVECs at a density of 2x105 cells/well were cultured in a Flexcell I flexible membrane dish in medium 199 containing 0.5% fetal calf serum, and cells were incubated for a further 2 days until the cells became confluent before the initiation of each experiment. Purity of endothelial cells was confirmed by positive staining for von Willebrand factor and negative staining for smooth muscle actin. The investigation conformed with the principles outlined in the Declaration of Helsinki (Cardiovascular Research 1997;35:2–3).

2.2 In vitro cyclical stretch on cultured HUVECs
The Flexcell FX-2000 strain unit, which has been characterized and described in detail elsewhere [15,16], consists of a vacuum unit linked to a valve controlled by a computer program. HUVECs cultured on the flexible membrane base were subjected to cyclical stretch produced by this computer-controlled application of sinusoidal negative pressure with a peak level of {cong}15 kPa at a frequency of 1 Hz (60 cycles per min) for various periods of time. Application of the vacuum results in maximum elongation of 20% to cells at the periphery of the wells with strain declining towards the center [16]. To determine the roles of c-Jun N-terminal kinase (JNK), p38 MAP kinase or p42/p44 MAP kinase in the expression of stretch-induced MMP systems expression, HUVECs were pretreated with SP600125 (20 µM, Calbiochem®), SB203580 (3 µM, Calbiochem®), or PD98059 (50 µM) for 30 min, respectively, followed by cyclical stretch. SP600125 is a potent, cell-permeable, selective, and reversible inhibitor of JNK. SB203580 is a highly specific, cell permeable inhibitor of p38 kinase. PD98059 is a specific and potent inhibitor of p42/p44 MAP kinase.

2.3 Western blot analysis
Cells were homogenized in modified RIPA buffer as described previously [16]. Equal amounts of protein (15 µg) were loaded into a 12.5% SDS–polyacrylamide minigel, followed by electrophoresis. Protein samples were mixed with sample buffer, boiled for 10 min, separated by SDS–PAGE under denaturing conditions, and electroblotted to nitrocellulose membranes. The blots were incubated overnight in Tris-buffered saline (TBS) containing 5% milk to block nonspecific binding of the antibody. Proteins of interest were revealed with specific antibodies as indicated (1:1000 dilution) for 1 h at room temperature followed by incubation with a 1:5000 dilution of horseradish peroxidase-conjugated polyclonal anti-rabbit antibody for 1 h at room temperature. Signals were visualized by chemiluminescent detection. Equal protein loading of the samples was further verified by staining monoclonal antibody GAPDH. All Western blots were quantified using densitometry.

2.4 Northern blot analysis
Total RNA was prepared by solubilizing HUVECs in UltraspecTM RNA kit (Biotecx Laboratory, Houston, Texas, USA). Aliquots of 20 µg of total RNA were fractionated in formaldehyde–agarose gels, transferred to Hybond-N+ nylon membrane, and hybridized with [{alpha}32-P]dCTP-labeled cDNA probes for human MMP-2, MMP-14, and TNF-{alpha}. The membrane was prehybridized at 65°C for 1 h, and hybridized with radioactively labeled probes at 65°C for 3 h in Rapid-hyb buffer (Amersham, Buckinghamshire, UK). Post-hybridization wash was performed with a final stringency of 0.2x standard saline citrate containing 0.1% SDS at 65°C. Quantitative analysis was performed with a PhosphorImager.

2.5 Immunohistochemistry
Cells were washed three times with PBS and fixed in 4% paraformaldehyde for 20 min, treated in 3% hydrogen peroxide/PBS for 25 min, blocked in 5% normal rabbit serum for 20 min, blocked with biotin/avidin for 15 min each, and incubated with primary antibodies for 2 h at room temperature as described [15]. Staining was performed with a peroxidase detection kit (Vector Laboratories).

2.6 Zymography
ECM-degrading activity was detected in conditioned media as previously described [17]. Briefly, equal amounts of sample protein were subjected to SDS–PAGE on gelatin-containing acrylamide gels (7.5% polyacrylamide and 2 mg/ml gelatin) under nonreducing conditions. After electrophoresis, the gels were cleared of SDS by incubating for 1 h with two changes of 2.5% Triton X-100. Gels were then incubated overnight in substrate buffer (50 mmol/l Tris, pH 8.0, 50 mmol/l NaCl, 10 mmol/l CaCl2, and 0.05% Brij 35) at 37°C. The gels were then stained with 0.1% Coomassie brilliant blue, and gelatinolytic bands were size-calibrated with a high-molecular-mass standard mixture of protein (Sigma). Zymograms were quantified using densitometry.

2.7 Electrophoretic mobility shift assay (EMSA)
Nuclear protein concentrations from HUVECs were determined by BioRad protein assay. Consensus and control oligonucleotides (Santa Cruz Biotechology) were labeled by polynucleotides kinase incorporation of [{gamma}32P]dATP. Oligonucleotides sequences included the activating protein 1 (AP-1) consensus 5'-CGCTTGATGACTCAGCCGGAA-3'. The AP-1 mutant oligonucleotides sequences were 5'-CGCTTGATGACTTGGCCGGAA-3'. After the oligonucleotide was radiolabeled, the nuclear extracts (4 µg of protein in 2 µl of nuclear extract) were mixed with 20 pmol of the appropriate [{gamma}32P]dATP-labeled consensus or mutant oligonucleotide in a total volume of 20 µl for 30 min at room temperature. The samples were then resolved on a 4% polyacrylamide gel. Gels were dried and imaged by autoradiography. Controls were performed in each case with mutant oligonucleotides or cold oligonucleotides to compete with labeled sequences.

2.8 Cytotoxicity studies
HUVECs were adjusted to 3x104 cells/ml in EBM medium. Aliquots of 20 ml of cell suspension were plated in 40-mm Petri dishes. After incubation for 24 h, the medium was replaced with fresh medium containing SP600125 and TNF-{alpha} monoclonal antibody at a concentration of 20 µM and 5 µg/ml, respectively. After incubation for 24 h, the medium was aspirated off and 0.5 mg/ml MTT solution was added and the incubation continued for another 4 h. At the end of the incubation period, the medium was removed from the attached cells and the converted dye crystals were dissolved with DMSO. Absorbency of converted dye was measured at a wavelength of 570 nm. For detection of cell injury possibly induced by stretch, cell viability after application of cyclical stretch was constantly monitored by trypan blue staining and measurement of release of lactate dehydrogenase (LDH) into culture medium and total HUVECs LDH as described previously [18].

2.9 Measurement of tumor necrosis factor-{alpha} concentration
Conditioned media from HUVECs subjected to cyclical stretch and those from control (unstretched) cells were collected for TNF-{alpha} measurement. The level of TNF-{alpha} was measured by a quantitative sandwich enzyme immunoassay technique. The lower limit of detection of TNF-{alpha} was 5.8 pg/ml.

2.10 Statistical analysis
The data were expressed as mean±S.E.M. Statistical significance was performed with analysis of variance followed by Dunnett's test for experiments consisting of more than two groups and with Student's t-test for stretch at 10 and 20%. A value of P<0.05 was considered to denote statistical significance.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Cyclical stretch enhances MMP protein and mRNA expression in HUVECs
The levels of MMP-14 protein began to increase as early as 2 h after stretch at 20% elongation was applied, reached a maximum of 3-fold over the control by 18 h and remained elevated up to 24 h (Fig. 1). The levels of MMP-2 protein began to increase later than that of MMP-14 and reached a maximum of 3.7-fold over the control by 18 h and remained elevated up to 24 h (Fig. 1). TIMP was not affected by cyclical stretch at 20% elongation. Stretch-induced MMP-14 and MMP-2 proteins expression was load-dependent. When HUVECs were stretched at 10% elongation, the levels of MMP-14 and MMP-2 proteins were similar to that of controls without stretch (Fig. 1). Both MMP protein levels increased earlier than their mRNA expression after cyclical stretch. These findings might indicate that cyclical stretch increases translation of MMP in HUVECs. MMP-2 gelatinolytic activity in the HUVECs increased after cyclical stretch for 2–18 h (Fig. 1C). Cyclical stretch increased both pro- and activated-MMP-2.


Figure 1
View larger version (35K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 (A) Representative Western blots for MMP-14 and MMP-2 in HUVECs subjected to cyclical stretch by 20 or 10% for various periods of time. (B) Quantitative analysis of MMP-14 and MMP-2 protein levels. The values from stretched HUVECs have been normalized to values in control cells (n = 3 per group). *P<0.001 vs. control (non-stretched cells). +P<0.005 vs. control. (C) Representative zymography for pro-MMP-2 and activated MMP-2 in HUVECs subjected to cyclical stretch for various periods of time and in the presence of SP600125 or TNF-{alpha} antibody. Standard (Std) indicates human fibroblast cell-conditioned medium concentrate.

 
The Northern blots showed that MMP-14 and -2 messages increased significantly after 12 h of stretch at 20% elongation (Fig. 2). The GAPDH mRNA levels were relatively constant when HUVECs were subjected to cyclical stretch. To determine if cyclical stretch enhanced the degradation of MMP-14 and -2 mRNA in HUVECs, we used actinomycin D to block RNA synthesis and then measured the half-life of remaining MMP-14 and -2 in HUVECs with or without stretch. As shown in Fig. 2C, cyclical stretch did not increase the turnover rate of MMP-14 and -2 mRNA. These findings indicate that transcriptional control of MMP is likely a major contributor to the increase in mRNA levels induced by stretch. The increases in MMP-14 and MMP-2 protein and mRNA levels after cyclical stretch were also observed in another type of endothelial cells, bovine pulmonary arterial endothelial cells (data not shown). No increase in release of LDH was observed following cyclical stretch at 20% elongation for 24 h and trypan blue staining also did not show any significant cell damage under these conditions. These data demonstrated that cyclical stretch at 20% elongation did not induce cell injury.


Figure 2
View larger version (36K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 (A) Representative Northern blots for MMP-14 and MMP-2 mRNA levels in HUVECs subjected to cyclical stretch by 20% for various periods of time. (B) Quantitative analysis of MMP-14 and MMP-2 Northern blots. The values from stretched HUVECs have been normalized to matched GAPDH measurement and then expressed as a ratio of normalized values to mRNA in control cells (n = 3–5 per group). *P<0.001 vs. control. +P<0.005 vs. control. (C) MMP-14 and MMP-2 mRNA half-life was estimated by analysis of mRNA decay time course following actinomycin D (ActD) in HUVECs with or without stretch. The signals were quantified and normalized to those of GAPDH (n = 3).

 
3.2 Stretch-induced MMP protein expression in HUVECs is mediated by TNF-{alpha}
To investigate the possible signal pathway mediates the stretch-induced MMP in HUVECs, the HUVECs were stretched 20% for 18 h in the presence or absence of inhibitors or antibody. As shown in Fig. 3, the stretch-induced increases of MMP proteins were completely blocked after the addition of TNF-{alpha} antibody (5 µg/ml) or SP600125 (20 µM), 30 min before stretch. However, the MMP proteins induced by stretch were not affected by the addition of PD98059 (50 µM) or SB203580 (3 µM). MAP kinase-activated protein kinase 2, a direct target of p38 MAP kinase was blocked after addition of SB203580 (3 µM) and the phospho-p42/p44 MAP kinase was diminished after addition of PD98059 (50 µM). These findings confirmed the biological activity and correct dose of SB203580 and PD98059. The DMSO alone as a vehicle control did not affect the MMP proteins induced by stretch. Both pro- and activated MMP-2 induced by stretch were largely attenuated by the addition of SP600125 (20 µM) or TNF-{alpha} antibody (5 µg/ml) 30 min before stretch (Fig. 1C). Another inhibitor of JNK, JNK inhibitor 1 (Calbiochem®) also reduced expression of MMP-14 and -2 induced by cyclical stretch. These findings implicated that JNK pathways, but not p38 and p42/p44 MAP kinases mediated the induction of MMP proteins by cyclical stretch in HUVECs. Cyclical stretch increased immunoreactive signals of MMP-2 in HUVECs. The signals began to increase as early as 2 h after stretch at 20% elongation was applied and maximal staining was observed at 18 h after stretch (Fig. 3). The pattern of immunoreactive signals for MMP-14 after cyclical stretch was similar to that of MMP-2. These immunoreactive signals decreased after addition of TNF-{alpha} monoclonal antibody and SP600125 30 min before stretch.


Figure 3
View larger version (43K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 (A) Representative Western blots for MMP-14 and -2 protein levels in HUVECs subjected to cyclical stretch by 20% for 18 h in the absence or presence of TNF-{alpha} antibody, inhibitors, and vehicle (DMSO 0.1%). and quantitative analysis of MMP-14 and -2 Western blots. The values from stretched HUVECs have been normalized to values in control cells (n = 3 per group). *P<0.001 vs. control. (B) Representative microscopic images of the MMP-14 and MMP-2 immunoreactive signal (arrow) in control cells, cyclically stretched HUVECs at 18 h, and after addition of TNF-{alpha} monoclonal antibody, or SP600125 before cyclical stretch. Similar results were observed in another two independent experiments.

 
As shown in Fig. 4, phosphorylated JNK and c-Jun proteins were induced by cyclical stretch for 20% elongation and the increase in phosphorylated JNK and c-Jun proteins were completely inhibited after the addition of SP600125 30 min before stretch. The pattern of increases in phosphorylated JNK and c-Jun proteins after stretch was similar to that of MMP proteins after stretch. To exclude the cytotoxicity of SP600125 and TNF-{alpha} monoclonal antibody, MTT assay was performed. The absorbency at 570 nm demonstrated no difference among control cells and cells treated with SP600125 and TNF-{alpha} at different concentrations for up to 24 h. The data demonstrated that there was no cytotoxicity of SP600125 and TNF-{alpha} on HUVECs.


Figure 4
View larger version (32K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 (A) Representative Western blots for phosphorylated JNK and c-Jun protein levels in HUVECs subjected to cyclical stretch by 20% for various periods of time and in the presence of SP600125. (B) Quantitative analysis of phosphorylated JNK and c-Jun Western blots. (n = 4 per group) *P<0.001 vs. control. +P<0.005 vs. control.

 
3.3 Cyclical stretch increases AP1-binding activity
Cyclical stretch of HUVECs for 2 to 24 h significantly increased the DNA–protein binding activity of AP-1 (Fig. 5). An excess of unlabeled AP-1 oligonucleotide competed with the probe for binding AP-1 protein, whereas an oligonucleotide containing a 3-bp substitution in the AP-1 binding site did not compete for binding. Addition of SP600125 or TNF-{alpha} monoclonal antibody 30 min before stretch abolished the DNA–protein binding activity induced by cyclical stretch.


Figure 5
View larger version (38K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Representative EMSA showing protein binding to the AP-1 oligonucleotide in nuclear extracts of HUVECs after cyclical stretch in the presence or absence of inhibitors. Arrow indicates the mobility of the complex. Similar results were found in another independent experiment. Cold oligo means unlabeled AP-1 oligonucleotides.

 
3.4 Cyclical stretch causes secretion of TNF-{alpha} for HUVECs
As shown in Fig. 6, cyclical stretch significantly began to increase the TNF-{alpha} secretion from HUVECs at 2 h after stretch and reached a maximum at 12 h and remained elevated for 24 h. The mean concentration of TNF-{alpha} rose from 120±2 pg/ml before stretch to 331±2 pg/ml after stretch for 12 h (P<0.001). Cyclical stretch also significantly increased the expression of TNF-{alpha} mRNA (Fig. 6). These data indicate that the increase in TNF-{alpha} produced is due to increased transcriptional activation at the TNF-{alpha} promoter but not increased shedding from the membrane.


Figure 6
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 (A) Cyclical stretch increased release of TNF-{alpha} from HUVECs subjected to cyclical stretch by 20% for various periods of time (n = 3). *P<0.001 vs. control. +P<0.005 vs. control. (B) Cyclical stretch increased TNF-{alpha} mRNA expression (n = 3). *P<0.05 vs. control. +P<0.01 vs. control.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In this study, we demonstrated that cyclical stretch enhanced both MMP-14 and -2 mRNA expressions and protein synthesis in HUVECs. MMP-14 and -2 were upregulated in both a time- and load-dependent manner by cyclical stretch. Yamaguchi et al. [12] recently reported that cyclical strain regulated MMP-14 in rat microvascular endothelial cells. Yun et al. [19], however, demonstrated the inhibitory effect of shear stress on MMP-14 expression in endothelial cells. Previous studies also reported that endothelial cells responded differently to different stress pattern [20] and biomechanical forces induce endothelial structural changes and modulate gene expression [21]. Since rhythmic distension of the vessel wall is a component of pulsatile flow, our and other studies indicate that cyclical stretch is an important factor regulating MMP-14 and -2 in vascular wall cells. We also demonstrated that cyclical stretch also induced TNF-{alpha} protein secretion and mRNA expression in HUVECs. TNF-{alpha} monoclonal antibody blocked the stretch-induced TNF-{alpha} and MMP-14 and -2. These results provide the first evidence for TNF-{alpha} mediating cyclical stretch-induced expression of MMP-14 and -2 in HUVECs. These results confirmed the autocrine or paracrine production of endothelial cells in response to cyclical stretch. Actually, it is not just TNF-{alpha}, but other pro-inflammatory cytokines such as interleukin-8 and monocyte chemotactic protein-1 are also induced during cyclical stretch [22,23].

TNF-{alpha}, a potent pro-inflammatory cytokine, has been shown to modulate a wide spectrum of responses, including the activation of many genes [24]. Vascular endothelial cells have been identified as a principal target of the pro-inflammatory actions of TNF-{alpha}. AP-1 is a principal transcriptional factor that is activated by TNF-{alpha}. In this study, we demonstrated that cyclical stretch increased the AP1 binding activity.

Mitogen-activated protein kinases have been shown to regulate the AP-1 activity [25]. SP600125, a potent inhibitor of JNK [26], inhibited the binding activity of AP-1 induced by stretch, while both inhibitors of p38 and p42/p44 MAP kinases did not have the inhibitory effect. These data implicated that the JNK pathway, but not the p38 and p42/p44 MAP kinases mediated the increased transcriptional activity of AP-1.

The intracellular signaling pathways that regulate MMPs are not well understood. MMP activity is tightly coordinated at several levels including transcriptional regulation, activation of latent zymogen, and interaction with endogenous inhibitors [27]. Han et al. [28] have shown that TNF-{alpha} activated MMP-14 and -2 through nuclear factor {kappa}B in human skin. Wang et al. [29] have demonstrated that ERK and p38 MAP kinases are involved in MMP-9 secretion in brain injury. Recent studies have shown that activation of MMP-14 is mediated by early growth response gene product 1 in endothelial cells [12,30]. The complete inhibition of MMP-14 and -2 expressions by addition of JNK inhibitors indicates that the JNK pathways are possibly involved in the induction of MMP by cyclical stretch.

Matrix degradation mediated by locally produced MMPs may contribute to the genesis and progression of atherosclerotic lesions [31,32] and to the development of intimal lesions in experimental arterial injury [33,34]. Proteolysis of capillary basement membrane proteins by MMPs is a critical component of angiogenesis [5]. The vascular endothelial cell occupies a strategic position at the interface between blood and tissue, and actively participates in the control of leukocyte traffic and the hemostatic and thrombotic systems. Matrix-dependent changes in vascular hemodynamics ultimately affect cardiovascular morbidity and mortality [35]. The increased vascular wall stress that occurs in hypertensive individuals would be sufficient to promote a strain ‘dose-dependent’ increase in TNF-{alpha} production by endothelial cells and an associated increase in matrix accumulation. Increased MMP-14 and -2 expression induced by cyclical stretch through the paracrine and/or autocrine action on endothelial cells may be relevant to pathological states of the cardiovascular system, including atherosclerosis and hypertension.

In summary, our study report for the first time that cyclical mechanical stretch enhances TNF-{alpha} production in cultured HUVECs. The stretch-induced TNF-{alpha} mediates the expression and activation of MMP-14 and -2, at least in part, through JNK and AP-1 pathway.

Time for primary review 22 days.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Woessner J.F. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J (1991) 5:2145–2154.[Abstract]
  2. Matrisian L.M. The matrix-degrading metalloproteinases. Bioassays (1992) 14:455–463.[CrossRef][Web of Science][Medline]
  3. Kugler A. Matrix metalloproteinases and their inhibitors. Anticancer Res (1999) 19:1589–1592.[Web of Science][Medline]
  4. Sounni N.E., Devy L., Hajitou A., et al. MT1-MMP expression promotes tumor growth and angiogenesis through an up-regulation of vascular endothelial growth factor expression. FASEB J (2002) 16:555–564.[Abstract/Free Full Text]
  5. Haas T.L., Milkiewicz M., Davis S.J., et al. Matrix metalloproteinase activity is required for activity-induced angiogenesis in rat skeletal muscle. Am J Physiol (2000) 279:H1540–H1547.[Web of Science]
  6. Hiraoka N., Allen E., Apel I.J., Gyetko M.R., Weiss S.J. Matrix metalloproteinases regulate neovascularization by acting as pericellular fibrinolysis. Cell (2000) 95:365–377.
  7. Pauly R.R., Passaniti A., Bilato C., et al. The 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 (1994) 75:41–54.[Abstract/Free Full Text]
  8. Jenkins G.M., Crow M.T., Bilato C., et al. Increased expression of membrane-type matrix metalloproteinase and preferential localization of matrix metalloproteinase-2 to the neointima of balloon-injured rat carotid arteries. Circulation (1998) 97:82–90.[Abstract/Free Full Text]
  9. Hanemaaijer R., Koolwijk P., Le Clercq L., De Vree W.J.A., Van Hinsbergh V.W.M. Regulation of matrix metalloproteinase expression in human vein and microvascular endothelial cells. Biochem J (1993) 296:803–809.[Web of Science][Medline]
  10. Rajavashisth T.B., Liao J.K., Galis Z.S., et al. Inflammatory cytokines and oxidized low density lipoprotein increase endothelial cell expression of membrane type 1-matrix metalloproteinase. J Biol Chem (1999) 274:11924–11929.[Abstract/Free Full Text]
  11. Haas T.L., Davis S.J., Madri J.A. Three-dimensional type 1 collagen lattices induce coordinate expression of matrix metalloproteinases MT1-MMP and MMP-2 in microvascular endothelial cells. J Biol Chem (1998) 273:3604–3610.[Abstract/Free Full Text]
  12. Meng X., Mavromatis K., Galis Z.S. Mechanical stretching of human saphenous vein grafts induces expression and activation of matrix-degrading enzymes associated with vascular tissue injury and repair. Exp Mol Pathol (1999) 66:227–237.[CrossRef][Web of Science][Medline]
  13. Yamaguchi S., Yamaguchi M., Yattsuuyanagi E., et al. Cyclical strain stimulates early growth response gene product 1-mediated expression of membrane type 1 matrix metalloproteinase in endothelium. Lab Invest (2002) 82:949–956.[Web of Science][Medline]
  14. Yokoyama T., Sekiguchi K., Tanaka T., et al. Angiotensin II and mechanical stretch induce production of tumor necrosis factor in cardiac fibroblasts. Am J Physiol (1999) 276:H1968–H1976.[Web of Science][Medline]
  15. Chang H., Wang B.W., Kuan P., Shyu K.G. Cyclical mechanical stretch enhances angiopoietin-2 and Tie2 receptor expression in cultured human umbilical vein endothelial cells. Clin Sci (2003) 104:421–428.[CrossRef][Web of Science][Medline]
  16. Cheng J.J., Wung B.S., Chao Y.J., Wang D.L. Cyclical strain enhances adhesion of monocytes to endothelial cells by increasing intercellular adhesion molecule-1 expression. Hypertension (1996) 28:386–391.[Abstract/Free Full Text]
  17. O'Callaghan C.J., Williams B. Mechanical strain-induced extracellular matrix production by human vascular smooth muscle cells: role of TGF-β1. Hypertension (2000) 36:319–324.[Abstract/Free Full Text]
  18. Shyu K.G., Chen J.J., Shih N.L., et al. Regulation of human cardiac myosin heavy chain genes by cyclical mechanical stretch in cultured cardiocytes. Biochem Biophys Res Commun (1995) 210:567–573.[CrossRef][Web of Science][Medline]
  19. Yun S., Dardik A., Haga M., et al. Transcription factor Sp1 phosphorylation induced by shear stress inhibits MMT1-MMP expression in endothelium. J Biol Chem (2002) 277:34808–34818.[Abstract/Free Full Text]
  20. Tedgui A., Mallat Z. Anti-inflammatory mechanisms in the vascular wall. Circ Res (2001) 88:877–887.[Abstract/Free Full Text]
  21. Davies P. Flow-mediated endothelial mechanotransduction. Physiol Rev (1995) 75:519–560.[Abstract/Free Full Text]
  22. Okada M., Matsumori A., Ono K., et al. Cyclical stretch upregulates production of interleukin-8 and monocyte chemotactic and activating factor/monocyte chemoattractant protein-1 in human endothelial cells. Arterioscler Thromb Vasc Biol (1998) 18:894–901.[Abstract/Free Full Text]
  23. Wung B.S., Cheng J.J., Chao Y.J., et al. Cyclical strain increases monocyte chemotactic protein-1 secretion in human endothelial cells. Am J Physiol (1996) 39:H1462–H1468.[Web of Science]
  24. Ledgerwood E.C., Pober J.S., Bradley J.R. Recent advances in the molecular basis of TNF signal transduction. Lab Invest (1999) 79:1041–1050.[Web of Science][Medline]
  25. Karin M. The regulation of AP-1 activity by mitogen-activated protein kinases. J Biol Chem (1995) 270:16483–16486.[Free Full Text]
  26. Han Z., Boyle D.L., Chang L., et al. c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis. J Clin Invest (2001) 108:73–81.[CrossRef][Web of Science][Medline]
  27. Fan W.H., Karnovsky M.J. Increased MMP-2 expression in connective tissue growth factor over-expression vascular smooth muscle cells. J Biol Chem (2002) 277:9800–9805.[Abstract/Free Full Text]
  28. Han Y.P., Tuan T.L., Wu H., Hughes M., Garner W.L. TNF-{alpha} stimulates activation of pro-MMP2 in human skin through NF-{kappa}B mediated induction of MT1-MMP. J Cell Sci (2001) 114:131–139.[Abstract]
  29. Wang X., Mori T., Jung J.C., Fini M.E., Lo E.H. Secretion of matrix metalloprotinase-2 and -9 after mechanical trauma injury in rat cortical cultures and involvement of MAP kinase. J Neurotrauma (2002) 19:615–625.[CrossRef][Web of Science][Medline]
  30. Haas T.L., Stielman D., Davis S.J., Apte S.S., Madri J.A. Egr-1 mediates extracellular matrix-driven transcription of membrane type 1 matrix metalloproteinase in endothelium. J Biol Chem (1999) 274:22679–22685.[Abstract/Free Full Text]
  31. Henney A.M., Wakeley P.R., Davies M.J., et al. Localization of stromelysin gene expression in atherosclerotic plaques by in situ hybridization. Proc Natl Acad Sci USA (1991) 88:8154–8158.[Abstract/Free Full Text]
  32. Galis Z.S., Sukhova G.K., Lark M.W., Libby P. Increased expression of matrix metalloproteinases and matrix degrading in vulnerable regions of human atherosclerotic plaques. J Clin Invest (1994) 94:2493–2503.[Web of Science][Medline]
  33. Bendeck M.P., Zempo N., Clowes A.W., Galardy R.E., Reidy M.A. Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat. Circ Res (1994) 75:539–545.[Abstract/Free Full Text]
  34. Zempo N., Kenagy R.D., Au Y.P.T., et al. Matrix metalloproteinases of vascular wall cells are increased in balloon-injured rat carotid artery. J Vasc Surg (1994) 20:209–217.[Web of Science][Medline]
  35. Franklin S.S., Khan S.A., Wong N.D., Larson M.G., Levy D. Is pulse pressure useful in predicting coronary heart disease? The Framingham Heart Study. Circulation (1999) 100:354–360.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
K.-G. Shyu, B.-W. Wang, P. Kuan, and H. Chang
RNA Interference for Discoidin Domain Receptor 2 Attenuates Neointimal Formation in Balloon Injured Rat Carotid Artery
Arterioscler Thromb Vasc Biol, August 1, 2008; 28(8): 1447 - 1453.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
W.-P. Cheng, H.-F. Hung, B.-W. Wang, and K.-G. Shyu
The molecular regulation of GADD153 in apoptosis of cultured vascular smooth muscle cells by cyclic mechanical stretch
Cardiovasc Res, February 1, 2008; 77(3): 551 - 559.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
M. Flamant, S. Placier, C. Dubroca, B. Esposito, I. Lopes, C. Chatziantoniou, A. Tedgui, J.-C. Dussaule, and S. Lehoux
Role of Matrix Metalloproteinases in Early Hypertensive Vascular Remodeling
Hypertension, July 1, 2007; 50(1): 212 - 218.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
P. M. Cummins, N. von Offenberg Sweeney, M. T. Killeen, Y. A. Birney, E. M. Redmond, and P. A. Cahill
Cyclic strain-mediated matrix metalloproteinase regulation within the vascular endothelium: a force to be reckoned with
Am J Physiol Heart Circ Physiol, January 1, 2007; 292(1): H28 - H42.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
A. M. Manso, L. Elsherif, S.-M. Kang, and R. S. Ross
Integrins, membrane-type matrix metalloproteinases and ADAMs: Potential implications for cardiac remodeling
Cardiovasc Res, February 15, 2006; 69(3): 574 - 584.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
J. P.G. Sluijter, D. P.V. de Kleijn, and G. Pasterkamp
Vascular remodeling and protease inhibition-bench to bedside
Cardiovasc Res, February 15, 2006; 69(3): 595 - 603.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
C. M. Dollery and P. Libby
Atherosclerosis and proteinase activation
Cardiovasc Res, February 15, 2006; 69(3): 625 - 635.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
P. Spallarossa, P. Altieri, S. Garibaldi, G. Ghigliotti, C. Barisione, V. Manca, P. Fabbi, A. Ballestrero, C. Brunelli, and A. Barsotti
Matrix metalloproteinase-2 and -9 are induced differently by doxorubicin in H9c2 cells: The role of MAP kinases and NAD(P)H oxidase
Cardiovasc Res, February 15, 2006; 69(3): 736 - 745.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
K.-G. Shyu, W.-H. Ko, W.-S. Yang, B.-W. Wang, and P. Kuan
Insulin-like growth factor-1 mediates stretch-induced upregulation of myostatin expression in neonatal rat cardiomyocytes
Cardiovasc Res, December 1, 2005; 68(3): 405 - 414.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
B. Menon, M. Singh, and K. Singh
Matrix metalloproteinases mediate {beta}-adrenergic receptor-stimulated apoptosis in adult rat ventricular myocytes
Am J Physiol Cell Physiol, July 1, 2005; 289(1): C168 - C176.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
P. Lacolley
Mechanical influence of cyclic stretch on vascular endothelial cells
Cardiovasc Res, September 1, 2004; 63(4): 577 - 579.
[Full Text] [PDF]

Home page
 Cardiovasc ResHome page
N. von Offenberg Sweeney, P. M Cummins, Y. A Birney, J. P Cullen, E. M Redmond, and P. A Cahill
Cyclic strain-mediated regulation of endothelial matrix metalloproteinase-2 expression and activity
Cardiovasc Res, September 1, 2004; 63(4): 625 - 634.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Wang, B.-W.
Right arrow Articles by Shyu, K.-G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, B.-W.
Right arrow Articles by Shyu, K.-G.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?