In the present study, we have investigated the effect of (i) ET-1 (endothelin-1) and its precursor, big ET-1, on MMP (matrix metalloproteinase)-2 and MMP-9 synthesis and activity in osteosarcoma tissue, and (ii) ET-1 receptor antagonists on cell invasion. Using Western blotting, zymography, RT-PCR (reverse transcription–PCR), immunohistochemistry, immunofluorescence and Northern blotting, we have shown that ET-1 and ET-1 receptors (ETA and ETB) were expressed in these cells. Additionally, we have demonstrated that ET-1 markedly induced the synthesis and activity of MMP-2, which was significantly increased when compared with MMP-9. Furthermore, inhibition of NF-κB (nuclear factor κB) activation blocked MMP-2 production and activity, indicating the involvement of NF-κB, a ubiquitous transcription factor playing a central role in the differentiation, proliferation and malignant transformation. Since ET-1 acts as an autocrine mediator through gelatinase induction and because inhibition of ETA receptor is beneficial for reducing both basal and ET-1-induced osteosarcoma cell invasion, targeting this receptor could be an attractive therapeutic alternative for the successful treatment of osteosarcoma.
- endothelin-1 (ET-1)
- nuclear factor κB (NF-κB)
- matrix metalloproteinase (MMP)
Osteosarcoma is the most frequent primary bone malignancy and the third most common type of cancer among youngsters ; it develops mainly during adolescence, especially in boys, and is characterized by the production of osteoid by cancer cells. It shows a predilection for the metaphysis of the long bones, most commonly at the distal femur, proximal tibia, proximal humerus and distal radius in decreasing order of frequency. Classic osteosarcoma demonstrates an aggressive and rapid growth with frequent findings such as skip metastases and early pulmonary metastases. Osteosarcoma treatment consists of a combination of multidrug chemotherapy and surgery. There has been little progress over the last 20 years in survival rates for osteosarcoma. The cure rate is approx. 65% for patients with localized diseases. When presenting with metastases at the time of diagnosis, survival is 25% [1,2].
Osteosarcoma invasion involves the infiltration of neighbouring tissues surrounding the tumour as a consequence of abnormal cellular proliferation and, for the development of metastases, cancerous cells must degrade the components of the ECM (extracellular matrix). This crucial step in tumour progression involves MMP (matrix metalloproteinase) activity. Reich and Liotta, pioneers in this field of research, have described the concept by stating that the proteolytic degradation of the matrix barrier is critical for tumour cell invasion and metastasis to distant sites [3,4]. A significant role in cancerous progression was quickly allotted to the MMPs, because of their capacity to degrade ECM thus supporting tumour invasion.
This provided the rationale for clinical trials using MMP inhibitors, unfortunately with inconclusive results in the outcomes for osteosarcoma and other neoplasia . This is probably due to the fact that the inhibitors were not employed in the early stages of cancer to prevent its progression . Also, the MMP inhibitors used only had effects on MMP activity, but did not inhibit MMP synthesis.
Among all of the MMPs, gelatinases (MMP-2 and MMP-9) are recognized to be particularly involved in the degradation of ECM components, such as collagen type IV and V of cell basal membrane. In osteosarcoma tissue, both MMP-2 and MMP-9 are overexpressed in comparison with their expression in non-affected stromal tissue . In the tumour micro-environment, the regulation of MMPs is crucial, and factors controlling this regulation appear to be even more important. According to previous studies , ET-1 (endothelin-1) is such a regulating factor.
ET-1 is a potent vasoconstrictor peptide, initially discovered in endothelial cells , that also plays a role in cell differentiation and migration. ET-1 promotes both mitogenesis and angiogenesis, and its overexpression by several cancer tissues and cells, including osteosarcoma cells, has been demonstrated previously [7–9]. Increasing evidence points to ET-1 as a relevant mediator in tumour progression in a variety of malignancies. High plasma levels of ET-1 were found among patients with various solid tumours [7–9], where ET-1 levels were higher among patients with metastasis. In addition, in vitro synthesis of ET-1 has been observed in many cancer cell lines [10,11]. This phenomenon was also reflected in vivo where an increase in immunoreactivity for ET-1 was demonstrated in several types of cancer [6,12,13]. These findings suggest that the modulation of ET-1 is involved in the tumour process. It has also been shown that ET-1 acts as an MMP inducer , which may facilitate the invasion of various tumours. Among the MMPs, gelatinases were suggested to be predictive markers for the clinical outcome of affected patients. Indeed, gelatinase B (MMP-9) was suggested as a prognostic factor for the development of metastasis in high-grade osteosarcoma .
MMPs in the tumour micro-environment appear crucial and factors controlling their regulation are even more important. Therefore, in the present study, the role of ET-1 and its receptors and expression of gelatinases were explored in osteosarcoma cells as well as in human osteosarcoma tissues.
MATERIALS AND METHODS
MG-63, Saos-2, MNNG/HOS and SJSA-1 (osteosarcoma cell lines) and SK-ES-1 (Ewing sarcoma cell line), purchased from the American Type Culture Collection, as well as eight primary cell cultures derived from human osteosarcoma tissues obtained intra-operatively (Table 1), were investigated in the present study. Tissues were collected with the consent of patients following approval by the Institutional Ethics Committee Board of Sainte-Justine Hospital, Montreal, Canada. Tissues were incubated in culture medium [DMEM (Dulbecco's modified Eagle's medium; Gibco BRL) supplemented with 10% (v/v) heat-inactivated FCS (fetal calf serum; Hyclone), 100 units/ml penicillin (Gibco BRL) and 100 μg/ml streptomycin (Gibco BRL)] at 37 °C in a humidified atmosphere of 5% CO2/95% air and primary cells were derived. Cells were then plated in tissue-culture flasks (25 cm2) and cultured until confluence was reached. At 24 h before cell stimulation, cells were incubated in serum-free culture medium and then incubated with ET-1 (10 nmol/l), big ET-1 (10 nmol/l), IL-1β (interleukin-1β; 200 pg/ml; all from Sigma–Aldrich) and PDTC (pyrrolidine carbodithioic acid; Calbiochem EDM Biosciences) from 0–10 μmol/l in DMEM containing 2.5% (v/v) FCS for various lengths of time [24 h for RT-PCR (reverse transcription–PCR), Northern blotting and Western blotting, and 5 min, 30 min and 4 h for NF-κB (nuclear factor κB) determination].
Total RNA was extracted from cells using TRIzol reagent (Gibco BRL), as described previously , and processed according to the manufacturer's instructions. The extracted RNA was quantified by the RiboGreen method .
For RT-PCR, 1 μg of total RNA was used and reverse transcription and PCR were carried out as described by Lange et al. . The ET-1 primers used were: forward primer, 5′-GTCAACACTCCCGACCACGTT-3′; and reverse primer, 5′-CTGGTTTGTCTTAGGTGTTCCTC-3′ (yielding amplified products of 304 bp). The GAPDH (glyceraldehyde-3-phosphate dehydrogenase) primers used were: forward primer, 5′-CAGAACATCATCCCTGCCTCT-3′; reverse primer, 5′-GCTTGACAAAGTGGTCGTTGAG-3′ (yielding amplified products of 315 bp). PCR was carried out as for the ET-1 PCR. The ETA primers used were: forward primer, 5′-ACCACAGTCCATGCCATCAC-3′; and reverse primer 5′-TCAACATCTCACAAGTCATGAG-3′ (yielding amplified products of 383 bp). The ETB receptor primers used were forward primer, 5′-TTGGAGCTGAGATGTGTAAGC-3′; and reverse primer, 5′-CAGTGAAGCCATGTTGATACC-3′ (yielding amplified products of 450 bp).
MG-63 cells were lysed in 0.2 ml of boiling 0.5% SDS. The protein concentration of the lysate was determined with the Bradford dye assay (Bio-Rad Laboratories). For Western blotting, the supernatant or 10 μg of lysate protein was electrophoresed on an SDS discontinuous gradient polyacrylamide gel (10%, w/v) and transferred electrophoretically on to a nitrocellulose membrane (Hybond C extra; Amersham Biosciences). The membranes were immersed overnight in the Super Block Blocking buffer (Pierce) and were then rinsed and incubated for 24 h at 4 °C with antibodies specifically recognizing: (i) MMP-2; (ii) TIMP (tissue inhibitor of metalloproteinases)-1; (iii) TIMP-2 (all at a dilution of 0.1 μg/ml; all from Oncogene Research Products) and (iv) p65 subunit of NF-κB (0.2 μg/ml; Santa Cruz Biotechnology). Following incubation with the primary antibody, membranes were carefully washed and re-incubated for 1 h at room temperature with a secondary antibody. Anti-(mouse IgG)–horseradish peroxidase conjugate (1:40000 dilution) was used for the detection of the MMP-2, TIMP-1 and TIMP-2, and anti-(rabbit IgG)–horseradish peroxidase conjugate (1:40000 dilution) was used for the detection of the p65 subunit of NF-κB. After careful washing, detection was performed using the Super Signal Ultra Western blotting chemiluminescence system (Pierce).
After RNA extraction, 5 μg of total RNA was resolved on 1.2% (w/v) formaldehyde/agarose gel and transferred electrophoretically on to a nylon membrane (Hybond-N; Amersham Biosciences) in 10 mmol/l sodium acetate buffer (pH 7.8) containing 20 mmol/l Tris/HCl and 0.5 mM EDTA overnight at 4 °C. RNA was cross-linked to the membrane by exposure to UV light. After prehybridization for 24 h, hybridization was carried out at 68 °C with MMP-2 and MMP-9 RNA probes. The RNA probes were transcribed and labelled with digoxigenin-11-2′-deoxyUTP (DIG-11 UTP; Roche Diagnostic) according to the manufacturer's specifications. Detection was carried out by chemiluminescence with CDP substrate (Roche Diagnostic) and revealed by exposure to Kodak X-AR5 film (Eastman Kodak). After the first hybridization, the membrane was stripped and rehybridized with a digoxigenin-labelled probe specific to GAPDH RNA, which served as a housekeeping gene. The autoradiographs were scanned with the Chemi-Imager 4000 imaging system (Alpha Innotech).
Gelatinase activity was assayed by zymography analysis as described by Xu et al. . Briefly, culture medium was separated by SDS/PAGE on a 10% (w/v) gel containing 1 mg/ml gelatin. The gel was washed twice in 2.5% (w/v) Triton X-100 solution and was incubated overnight at 37 °C in developing buffer [50 mmol/l Tris/HCl (pH 7.4), 10 mmol/l CaCl2, 5 mmol/l ZnCl2 and 0.05% Brij-35], stained with 0.5% Coomassie Blue and then destained in a 40% (v/v) methanol/10% (v/v) acetic acid solution.
Briefly, specimens were fixed in 4% (v/v) paraformaldehyde and embedded in paraffin. Sections (5 μm) of paraffin-embedded specimens were de-paraffinized in toluene and hydrated in a degraded series of ethanol. The tissue sections were incubated with primary antibody [monoclonal anti-(ET-1) antibody (5 μg/ml; Research Diagnostic), anti-ETA and -ETB antibodies (8 μg/ml; Sigma–Aldrich) and anti-(MMP-2) and anti-(MMP-9) antibodies (2 μg/ml; Oncogene Research Products)] and stained using the avidin–biotin complex method (Vectastain ABC kit; Vector Laboratories). The colour was developed by DAB (diaminobenzidine; Dako Diagnostics) containing H2O2, and the slides were counter-stained with eosin (Digene Diagnostics). The specificity of staining was evaluated by omission of the primary antibody following the same experimental protocol.
The osteosarcoma (MG-63) cells were grown in monolayer on glass slides (Nagle Nunc) and were then treated with 10 nmol/l ET-1 (Sigma-Aldrich), 10 nmol/l big ET-1 (Sigma-Aldrich) or 200 pg/ml IL-1β (Sigma–Aldrich) at 37 °C for 24 h in DMEM/2.5% (v/v) FCS. Cells were fixed in a solution of methanol/acetic acid (1:1, v/v) for 10 min. The fixed cells were incubated with a rabbit polyclonal antibody against the p65 subunit of NF-κB (2 μg/ml; Santa Cruz Biotechnology) overnight at 4 °C. The cells were washed and incubated with FITC-conjugated anti-(rabbit IgG) (1:100 dilution) for 1 h at room temperature. Cells were washed, mounted with cover slips using ProLong® Gold antifade reagent with DAPI (4′,6-diamidino-2-phenylindole; Molecular Probes) and were analysed using a confocal microscope. Positive staining was revealed by Alexa fluor probe 488 for green cytoplasm staining and DAPI for blue nuclear staining.
The invasion of osteosarcoma cells expressing both ETA and ETB receptors (MG-63) and those expressing only the ETA receptor (MNNG/HOS) was evaluated. Invasion was quantified by in vitro cell transmigration after 16 h using the Cell Invasion Assay Kit (Chemicon Temecula). Cells were incubated without or with selective antagonists of ETA receptor (1 μmol/l BQ123), ETB receptor (1 μmol/l BQ788), both ETA and ETB antagonists (1 μmol/l BQ123+1 μmol/l BQ788) and with the specific inhibitor of NF-κB activity (1 μmol/l PDTC). In this experiment, cells were not exposed to exogenous ET-1 in order to determine the autocrine effects of this peptide (endogenous ET-1 invasive properties). In another set of experiments, cells were exposed to exogenous ET-1 (10 nmol/l) without or with the ETA antagonist (1 μmol/l BQ123), and invasive cells were quantified. Invasive cells (migrating through the polycarbonate membrane) were incubated with Cell Stain solution, and then subsequently extracted and detected on a standard microplate reader (at 560 nm).
Statistical significance was assessed by Mann–Whitney U test, and P<0.05 was considered significant.
Expression and synthesis of ET-1 and ET-1 receptors (ETA and ETB) in human osteosarcoma
The presence of ET-1 was demonstrated in both osteosarcoma cells and osteosarcoma tissues. In osteosarcoma tissues, ET-1 protein was localized by immunohistochemistry (Figure 1a). All of the tissues investigated (n=4) had specific immunoreactivity. Positive staining was observed preferentially in matrix-localized cells with an osteoblastic phenotype. In osteosarcoma cells, using RT-PCR, specific mRNA coding for ET-1 was found in all cell lines investigated, except MNNG/HOS and SK-ES-1 (Figure 1b, lanes 1–5), as well as in cells derived from biopsy tissue (Figure 1b, lanes 6–13). Immunolocalization of ET-1 receptors (ETA and ETB) is shown in Figure 2(a–c). In osteosarcoma tissues, 49±10.57% of the cells were positive for ETA, whereas only 11.5±7.3% of the cells were positive for ETB (P<0.005). Positive staining for ETB receptors was principally observed in vascular cells. Using RT-PCR, specific mRNA coding for ETA (Figure 2d) was found in all of the cells investigated (except for OS#8). However, as shown in Figure 2(e) (lanes 3–5), specific mRNA coding for ETB was detected only in MG-63, SK-ES-1 and SJSA-1 cells.
MMP-2 and MMP-9 expression is increased in human osteosarcoma tissues by ET-1
To assess the importance of ET-1 and gelatinases in osteosarcoma, we analysed basal expression of MMP-2 and MMP-9 as well as the effect of ET-1 on MMP-2 and MMP-9 synthesis. The effects of ET-1 were explored at the level of mRNA and enzymatic activity. Basal expression of MMP-2 and MMP-9 in osteosarcoma tissue is shown in Figures 3(a) and 3(b). By immunohistochemistry, 81±18.7% of cells were positive for MMP-2, whereas only 3.67±1.53% of cells were positive for MMP-9 (P<0.05). mRNA transcripts for MMP-2 and MMP-9 (Figure 3c), as well as for gelatinase activity (Figure 3d), were detected in both stimulated and non-stimulated osteosarcoma cells. In Figure 3(c), ET-1-induced expression levels of MMP-2 and MMP-9 were similar, as determined by Northern blotting. By zymography, ET-1 acted as an inducer of both MMP-2 and MMP-9 (Figure 3d). Induction of the enzymatic activity of MMP-2 and MMP-9 by ET-1 was observed for both latent and active forms of MMP-2 and MMP-9. In addition, enzymatic activity corresponding to MMP-2 was increased when compared with MMP-9. This could be explained by the fact that MMP-2 is physiologically expressed by the stroma cells of many tissues and, conversely, MMP-9 expression is weak or absent in normal tissue and induced and secreted in the case of tissue turnover, such as tumour invasion .
NF-κB inhibition down-regulates ET-1-induced MMP-2, TIMP-1 and TIMP-2 in osteosarcoma cells
NF-κB is a ubiquitous transcription factor that regulates MMP gene activation . Therefore the effect of NF-κB inhibition by PDTC on the expression of MMP-2 was investigated. Figure 4(a) shows that ET-1-induced MMP2 expression was inhibited in a dose-dependent manner by treatment of cells with PDTC. Furthermore, the expression of TIMP-1 (Figure 4b) and TIMP-2 (Figure 4c), which was also induced by ET-1, was inhibited by PDTC in a similar manner to that observed for MMP-2. PDTC also diminished the enzymatic activity of ET-1-induced MMP-2 and MMP-9, as determined by gelatin zymography (Figure 4d).
ET-1 up-regulates NF-κB (p65 subunit) and causes NF-κB translocation to the nucleus in osteosarcoma cells
To assess the effect of ET-1 on the expression of the p65 subunit of NF-κB, osteosarcoma cells (MG-63) were treated with ET-1. Using a specific antibody against the p65 subunit of NF-κB, protein expression was determined by Western blotting. As shown in Figure 5, exposing MG-63 cells to ET-1 (10 nmol/l) and its precursor big ET-1 (10 nmol/l) induced protein expression of the p65 subunit of NF-κB. In other experiments, osteosarcoma cells (MG-63) were treated with ET-1 (10 nmol/l), big ET-1 (10 nmol/l) or IL-1β (200 pg/ml) for 24 h in basal medium. Figure 6 shows that ET-1, big ET-1 and IL-1β (used as a positive control) induced the translocation of the p65 subunit of NF-κB into the nucleus. In control cells, the majority of p65 staining resided in the cytoplasm (Figure 6a). Nuclear accumulation of p65 was observed in the treated cells (Figures 6b, 6c and 6f), suggesting a role of ET-1 and its precursor in p65 activation.
ETA and NF-κB inhibition reduces osteosarcoma cell invasion
To assess the direct effect of endogenous ET-1 on cellular invasion, selective antagonists of two ET-1 receptors (ETA and ETB) and of NF-κB were used. Selective antagonist of ETA receptor alone significantly reduced (P<0.05) cellular invasion (Figure 7a). In contrast, the ETB antagonist did not significantly modulate cellular invasion. In combination, ETA and ETB antagonists did not demonstrate additional or synergistic effects on osteosarcoma cell invasion (Figure 7a). As shown in Figure 7(b), exposure of osteosarcoma cells to exogenous ET-1 (10 nmol/l) increased the level of invasive cells which was significantly decreased in the presence of 1 μmol/l ETA antagonist BQ123. Similar results were obtained for cells expressing both ETA and ETB receptors (MG63 cells) and those expressing only ETA receptors (MNNG/HOS).
The present study shows that ET-1 has a stimulatory effect on two MMPs, MMP-2 and MMP-9, which are among the most important MMPs involved in ECM degradation, a process which leads to tumour invasion. As ECM remodelling is a major event in osteosarcoma invasion, these results provide evidence of the importance of ET-1 in osteosarcoma malignancy. Our present study also demonstrates that targeting the ETA receptor is beneficial in abolishing osteosarcoma cell invasion.
ET-1 may have various roles in the tumour process such as: (i) facilitating growth of cancerous cells (mitogenesis effect); (ii) protecting cancerous cells from apoptosis; (iii) promoting angiogenesis; and (iv) supporting tumour progression and metastasis . Rosano et al.  have shown that ET-1 induces the secretion and activation of various MMPs, such as MMP-2, -3, -9, -7, -13 and MT1-MMP, in ovarian carcinoma cells. These results correlate with our findings in which we demonstrate that ET-1 induces MMP-2 and MMP-9 in osteosarcoma cells. Bagnato et al.  have also shown that ET-1 inhibits the secretion of TIMP-1 and TIMP-2, thus modifying the MMP/TIMP ratio prevailing under physiological conditions. The regulation of MMPs by TIMP is a significant factor controlling ECM catabolism, and modifications of this regulation are responsible for several pathological states, particularly cancer . However, Rosano et al.  did not observe an induction of TIMP by ET-1, whereas, in the present study, it was found that in osteosarcoma cells ET-1 acted as an inducer of both TIMP-1 and TIMP-2. This can be explained by the fact that TIMP may play a dual role: they not only bind to the active form of MMPs to inhibit their proteolytic activity, but they can also bind to the proform of some MMPs to facilitate enzyme activation . Indeed, TIMP-1 forms a preferential complex with proMMP-9, and TIMP-2 forms a preferential complex with proMMP-2 [21–23]. Thus a vicious circle is created: ET-1 induces MMPs, which in turn induce the formation of ET-1, including the alternative forms, such as ET-11–32. This allows us to propose a dual inhibition in order to diminish the invasiveness of osteosarcomas of ET-1 on one hand and of the MMPs on the other.
Originally, MMPs were considered to be important almost exclusively in tumour invasion and metastasis; however, recent studies have shown  that MMPs play a crucial role in generating growth-promoting molecules and signalling factors. Among these, the transcription factor NF-κB could play a critical role. The action of ET-1, which leads to a significant increase in MMPs, could be connected to the activation of the NF-κB. NF-κB is a ubiquitous transcriptional factor, the basal signalling of which is enhanced in cancer cells. NF-κB plays a central role in the process of differentiation and cellular proliferation and could also have a role in the transformation of cancer cells to a malignant state. NF-κB is activated by many stimuli and could constitute a point of convergence between the various mechanisms leading to tumour progression. The present study demonstrates that ET-1 and big ET-1 are among the stimuli activating NF-κB (Figures 5 and 6). It has been shown previously  that NF-κB up-regulates MMP-9 and down-regulates TIMP-1. NF-κB is also important in the regulation of many genes involved in matrix degradation. Among these, MMPs play a central role in matrix degradation. In addition, the present study shows that the blockade of NF-κB by PDTC inhibits MMP-2 expression. These results correlate with the observations of Meyer et al. , who showed a down-regulation of MMP RNA expression when using PDTC. This leads one to believe that PDTC is an efficient inhibitor of MMP induction by NF-κB, which is also induced by ET-1 and its immediate precursor.
Finally, our present study provides new evidence for the ET-1 receptor as a potential therapeutic target in the treatment of osteosarcoma. In osteosarcoma cells, the ETA receptor is the principal receptor involved in their invasive activity. The results obtained by measuring in vitro cell invasion using selective antagonists for each receptor subtype (ETA and ETB) alone clearly demonstrate that ETB does not influence the invasive behaviour of osteosarcoma cells. Interestingly, we observed that inhibition of both ETA and ETB does not have a synergistic or additive effect on osteosarcoma cell invasion. An explanation for this observation could be due to ETA and ETB receptor dimerization, which is a phenomenon well known in the family of G-protein-coupled receptors to which ETA and ETB belong. This interpretation is supported by an interesting study by Harada et al.  on the functional binding capability of ET-1 to ETA and ETB receptor subtypes. Harada et al.  concluded that the ETB receptor does not independently recognize ET-1 without the aid of the ETA receptor, thus drawing attention to the crucial role of the ETA receptor in the biological activity of ET-1. In light of this, our present results demonstrate that exposure of osteosarcoma cells to exogenous ET-1 results in increased invasive capacity of these cells, which is diminished when the ETA receptor is blocked.
In conclusion, the present study has demonstrated MMP-2 and MMP-9 induction by ET-1 in osteosarcoma cells and human osteosarcoma tissues and has shown a critical role for NF-κB transcription factor in this activity. We also established that ET-1, as well as the two ET-1-specific receptors, is expressed ex vivo in human osteosarcoma tissues and that the ETA receptor is involved in the invasive ability in osteosarcoma cells. ET-1 receptors and MMPs have been detected previously in rat and canine osteosarcoma tissues [28,29], but our present study is the first one to demonstrate their presence in human osteosarcoma. Additionally, MMPs, including, MMP-2 and MMP-9, were also found in these tumours. These molecules (ET-1 and MMP) are also present in normal cells (including normal osteoblasts). In accordance with these findings, it appears reasonable to attribute a predominant role to the presence or absence of the ET-1 receptors, rather than ET-1 itself. As the ETA receptor controls invasion and the ETB receptor has a recycling role, the ETA receptor would be a significant target. Indeed, there is evidence demonstrating the antitumoral activity of ET-1 receptor antagonists in combination with other tumour inhibitors . As interactions between ET-1, MMPs and the TIMPs play a major role in tumour invasion, the ET-1 axis could be an attractive target in the development of new therapies against bone tumours such as osteosarcoma.
This work was supported by grants from the Canadian Orthopaedics Foundation, Mentor, a Canadian Institutes of Health Research programme. F. M. is the recipient of a scholarship from the Fonds de Recherche en Santé du Québec (FRSQ) and the Carroll A. Laurin Award 2004. We thank Fazool Shipkolye and Heather Yampolsky for their technical assistance.
Abbreviations: DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix; ET-1, endothelin-1; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MMP, matrix metalloproteinase; NF-κB, nuclear factor κB; PDTC, pyrrolidine carbodithioic acid; RT-PCR, reverse transcription–PCR; TIMP, tissue inhibitor of metalloproteinases
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