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Cardiovascular Research 2007 75(1):186-194; doi:10.1016/j.cardiores.2007.02.033
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Copyright © 2007, European Society of Cardiology

c-Myc is essential for urokinase plasminogen activator expression on hypoxia-induced vascular smooth muscle cells

YongZhong Hou, Chikako Okamoto, Kiyotaka Okada, Naoyaki Kawao, Shuhei Kawata, Shigeru Ueshima and Osamu Matsuo*

Department of Physiology, Kinki University School of Medicine, Ohnohigashi 377-2, Osakasayama, Osaka 589-8511, Japan

* Corresponding author. Tel.: +81 72 366 0221(3163); fax: +81 72 366 2853. matsuo-o{at}med.kindai.ac.jp

Received 19 December 2006; revised 26 February 2007; accepted 27 February 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives The purpose of this study was to investigate whether c-Myc regulates expression of urokinase plasminogen activator (uPA) on hypoxia-induced vascular smooth muscle cells (VSMCs).

Methods VSMCs were isolated from thoracic aorta of wild-type (WT), tissue plasminogen activator (tPA) and uPA-deficient mice. Gene and protein expression levels were examined by reverse-transcription PCR and Western blotting, respectively. c-Myc and uPA transcriptional activity were determined by luciferase analysis. Zymography analysis was used to test the activity of matrix metalloproteinases (MMPs), tPA, and uPA.

Results Hypoxia significantly promoted WT and tPA–/– VSMC migration and invasion. However, uPA–/– severely decreased hypoxia-induced VSMC migration and invasion. Hypoxia increased uPA and MMP-2 activity, while uPA–/– decreased hypoxia-induced MMP-2 activity. c-Myc expression and transcriptional activity were increased in response to hypoxia, and silenced c-Myc abolished hypoxia-induced uPA and MMP-2 activity. In addition, hypoxia-induced Bcl2 expression and Bcl2 binding to c-Myc led to enhanced c-Myc-mediated uPA and MMP-2 activity in response to hypoxia.

Conclusions The results show that c-Myc was essential for hypoxia-induced uPA expression and activity, resulting in VSMC migration and invasion. In addition, Bcl2 enhanced the c-Myc-mediated uPA/MMP-2 pathway.

KEYWORDS Hypoxia; c-Myc; uPA; Vascular smooth muscle cell migration and invasion


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Atherosclerosis involves multiple processes including endothelial dysfunction, inflammation, vascular proliferation and matrix alteration. VSMC proliferation and migration into the intima after vascular injury, as well as their formation of neointima, contribute to vessel narrowing and are pivotal to the atherosclerotic process [1,2]. One precursor of lesion development may be focal accumulation of VSMCs within the intima [3,4]. The key stimuli for intimal hyperplasia are injury, inflammation, and increased mean wall stress [5]. Recent report shows that blood-flow cessation induces VSMC migration and invasion, resulting in intimal hyperplasia [6]. Hypoxia is involved in VSMC proliferation [7], endothelial cell migration [8], neovascularization [9], and vascular remodeling [10]. Urokinase plasminogen activator (uPA) plays a significant role in vascular wound healing and arterial neointima formation after injury, most likely by affecting VSMC migration [6,11]. Active uPA converts plasminogen into plasmin, a broadly acting protease that degrades several matrix proteins and can activate latent matrix metalloproteinases (MMPs) [12]. MMPs are associated with embryogenesis, wound healing, inflammation, arthritis, and cardiovascular diseases [13].

c-Myc is involved in the regulation of cellular proliferation, differentiation, and apoptosis [14]. Hypoxia induces c-Myc expression in vivo and in vitro [15,16]. However, it is not known whether c-Myc mediates the activity of uPA and MMP-2 in response to hypoxia. In this paper, our data show that c-Myc expression was increased on hypoxia-induced VSMCs. However, silenced c-Myc led to decreased hypoxia-induced uPA transcriptional activity, suggesting that c-Myc was required for uPA expression.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Smooth muscle cell culture
This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1966). Deficient mice (t-PA–/–, u-PA–/–) were generated by homologous recombination in embryonic stem cells, as described previously [17]. Experimental mice were 8–12 weeks of age. The Institutional Animal Care and Use Committee at the Kinki University School of Medicine approved the animal experiment protocols. Primary culture of wild-type (WT) and deficient mice (t-PA–/–, u-PA–/–) aortic smooth muscle cells (SMCs) was performed as described previously [18]. Cells were placed in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. Hypoxic conditions were achieved by culturing VSMCs in a sealed hypoxia chamber after flushing with a mixture of 3% O2, 5% CO2, and 92% N2.

2.2 Cell migration and invasion assay
Cell migration and invasion were assessed using a QCMTM 24-well colorimetric cell migration assay kit (Chemicon, Temecula, CA) following the manufacturer's instructions. Cells that migrated through the polycarbonate membrane were incubated with "Cell Stain Solution" and then subsequently extracted and detected on a standard microplate reader at 560 nm. Cell invasion was assessed using the Chemicon cell invasion assay kit. This assay was performed in an invasion chamber. The inserts contain an 8-µm pore size polycarbonate membrane over which a thin layer of ECMatrixTM was dried. The extracellular matrix (ECM) layer occludes the membrane pores, blocking non-invasive cells from migrating through. Invasion cells migrate through the ECM layer and cling to the bottom of the polycarbonate membrane. The insert membrane with invaded cells on the bottom was extracted and detected on a standard microplate reader at 560 nm.

2.3 Cell proliferation
Cell proliferation was assessed using a WST-1 kit according to the manufacturer's instructions (Roche Applied Science). This is a colorimetric assay for the quantification of cell proliferation, based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells. The absorbance of the sample was analyzed using a microplate reader.

2.4 Luciferase assay
VSMCs were seeded in the 6-well plate and contransfected with a c-Myc [19] or uPA [20] reporter plasmid and the pRL-CMV-Rluc or pGL2-luc control luciferase reporter vector using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), and subjected to hypoxia or normoxia for 8 h. Cells were lysed in passive lysis buffer and analyzed for firefly luciferase activity with reagents from the Luciferase Reporter Assay System (Promega, Madison, WI) using the luminometer LB96P (Berthold Japan, Tokyo, Japan).

2.5 Reverse-transcription PCR analysis
The mRNA of MMP-2 and uPA was analyzed by RT-PCR. The vascular smooth muscle cells were lysed, and total RNA was isolated by using the RNeasy Mini kit (Qiagen Sciences, Maryland) according to the manufacturer's specifications. Total RNA (0.5 µg) from each sample was reverse transcribed. RT-PCR was performed using the ExcriptTM TR reagent kit (TaKaRa Shuzo Co. Ltd., Kyoto, Japan). Amplified PCR products were resolved in 2% agarose gels, stained with ethidium bromide, and photographed under UV light. For PCR amplification, the following primers were used: MMP-2 forward, 5'-TGTTTACCATGGGTGGCAATGCAG-3'. MMP-2 reverse, 5'-TGTTTGCAGA TCTCCGGAGTGACA-3'; GAPDH forward, 5'-TGCATCCTGCACCACCAACT; GAPDH reverse, 5'-AACACGGAAGG CCATGCCAG-3'; uPA primer was described previously [21].

2.6 RNA interference
VSMCs were seeded in 6-well plates and small interfering RNA for c-Myc or Bcl2 was preformed using silencer c-Myc or Bcl2 siRNA and Silencer Transfection Kit according to the transfection protocol from the manufacturer (Santa Cruz, CA). A control siRNA (non-homologous to any known gene sequence) was used as a negative control.

2.7 Western blot and immunoprecipitation assay
VSMCs were lysed as described previously [18]. VSMCs were washed with ice-cold phosphate-buffered saline and lysed in lysis buffer as above. The indicated antibodies were immunoprecipitated as described previously [22]. The samples were subjected to 10–20% gradient SDS-PAGE, transferred to a nitrocellulose membrane, then probed by Western blot analysis with the indicated antibodies (Santa Cruz, CA) and developed by using an ECL Kit (Amersham Biosciences, Buckinghamshire, UK).

2.8 Zymography assay
The activities of uPA and tPA were performed as described previously [23]. 10% polyacrylamide gel contained 0.55 mg/ml of bovine fibrinogen and 0.056 NIH U/ml of thrombin (Sigma, St. Louis, MO). Protein samples were electrophoresed, subsequently soaked in 2.5% Triton X-100 solution for 60 min, and then incubated in reaction buffer (0.5 M Glycine-HCl, pH 8.4) at 37 °C for 24–36 h. Following development, the gel was stained with Coomassie Blue R-250 for 1 h and destained with multiple changes of destain solution (30% methanol, 10% acetic acid) until light bands appeared on a blue background.

2.9 Metalloproteinase activity assay
The metalloproteinase activity was determined utilizing gelatin substrate zymography. Samples were mixed with SDS loading buffer and incubated for 30 min at 37 °C. Samples and molecular weight markers were electrophoresed in a 10% polyacrylamide gel containing 0.1% gelatin. The gel was then washed in 2.5% Triton X-100 to remove SDS, and then was incubated at 37 °C for 24–48 h in 200 mM NaCl containing 40 mM Tris–HCl and 10mM CaCl2 (pH 7.5). After staining with Coomassie Blue R-250, gelatinases were identified as clear bands.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Hypoxia induces VSMC migration and invasion
Our results show that hypoxia promoted wild-type (WT) and tPA–/– VSMC migration and invasion. However, uPA–/– significantly decreased hypoxia-mediated VSMC migration and invasion (Fig. 1A and B). In the scrape wound-induced migration assay, our data also show that hypoxia promoted WT and tPA–/– VSMC migration. uPA–/– significantly inhibited hypoxia-induced VSMC migration (Fig. 1C). A previous report shows that hypoxia induces VSMC proliferation [7]. Our data show that hypoxia-induced WT and gene-deficient VSMC proliferation (Fig. 1D). The results show that uPA was involved in hypoxia-induced VSMC migration and invasion.


Figure 1
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  		Fig. 1 Hypoxia induces migration and invasion of VSMCs. (A and B) VSMCs were treated with hypoxia for 24 h, and cell migration or invasion was measured using a QCMTM 24-well colorimetric cell migration assay kit or cell invasion kit, respectively. (C) Scrape wound-induced migration for 12 h, and cells were monitored under a microscope equipped with a camera. (D) Cell proliferation was assayed by WST-1 on VSMCs treated with hypoxia for 24 h. *P<0.05 vs normoxic (N) VSMCs, #P<0.05 vs hypoxic (H) WT VSMCs. The data represents at least three determinations.

 
3.2 Hypoxia increases uPA expression and activity
Western blot analysis shows that hypoxia time dependently increased VSMC uPA protein level (Fig. 2A), which was consistent with the increased uPA gene expression (Fig. 2B). To further determine if hypoxia regulates uPA activity, zymography analysis was performed. The results suggest that hypoxia significantly enhanced the uPA activity (Fig. 2C). However, hypoxia had no effect on tPA protein expression (Fig. 2A) and activity (Fig. 2C).


Figure 2
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  		Fig. 2 Hypoxia induces activation of uPA. (A) VSMCs were treated with hypoxia for the indicated time. The protein expression level was analyzed by Western blot using indicated antibodies. (B) uPA gene expression was analyzed by RT-PCR. (C) The uPA and tPA zymography activity were analyzed in conditioned medium, and relative activities were quantified by scanning densitometry. *P<0.05 vs normoxic VSMCs. The data represents at least three determinations.

 
3.3 Hypoxia increases MMP-2 activity in association with uPA
The activation of uPA degrades several matrix proteins and can activate latent MMPs [12]. Reverse-transcription PCR analysis shows that hypoxia markedly increased MMP-2 gene expression (Fig. 3A). Although metalloproteinase activity analysis shows that hypoxia increased the activity of MMP-2, it had no effect on MMP-9 activity (Fig. 3B). To further analyze the interaction of uPA with MMP-2, zymography analysis was carried out. The results show that uPA–/– led to suppress MMP-2 activity on hypoxia-induced VSMCs (Fig. 3C), indicating that hypoxia increased the uPA activity, leading to the activation of MMP-2.


Figure 3
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  		Fig. 3 Hypoxia induces activation of MMP-2 in association with uPA. (A) VSMCs were treated with hypoxia for the indicated time. MMP-2 gene expression was analyzed by RT-PCR. (B) The zymography MMPs activity were analyzed in conditioned medium. (C) WT, uPA–/–, tPA–/– VSMCs were treated with normoxia or hypoxia for 8 h. Zymography shows the MMP-2 activity of conditioned medium. Relative activities were quantified by scanning densitometry. *P<0.05 vs normoxic VSMCs. The data represents at least three determinations.

 
3.4 c-Myc is required for uPA activity
Hypoxia significantly increased c-Myc protein expression (Fig. 4A). To further analyze whether c-Myc regulates the activity and expression of uPA, we knocked down the c-Myc protein expression. Western blot shows that c-Myc siRNA significantly inhibited c-Myc protein expression levels (Fig. 4B). Zymography analysis suggests that silence of c-Myc resulted in decreased uPA and MMP-2 activity (Fig. 4C and D). The inhibition of c-Myc decreased hypoxia-induced VSMC migration and invasion (Fig. 4E and F), which further demonstrated that c-Myc-mediated hypoxia-induced VSMC migration and invasion. The results suggest that c-Myc was required for activation of uPA and MMP-2.


Figure 4
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  		Fig. 4 c-Myc is required for uPA activity. (A) VSMCs were treated with hypoxia for indicated time, and the cells were lysed. Protein expression level was analyzed by Western blot using indicated antibodies. (B) VSMCs were transfected with control siRNA or c-Myc siRNA. Protein expression level was analyzed by Western blot using indicated antibodies. (C and D) VSMCs were transfected with control siRNA or c-Myc siRNA, and the VSMC migration and invasion were analyzed as above. (E) VSMCs were treated as above, tPA and uPA activity of conditioned medium were analyzed. (F) VSMCs were treated as above, and MMP-2 activity of conditioned medium was analyzed. *P<0.05 vs normoxic control siRNA VSMCs, #P<0.05 vs hypoxic control siRNA VSMCs. Relative activities were quantified by scanning densitometry. The data represents at least three determinations.

 
3.5 c-Myc enhances uPA transcriptional activity
The uPA luciferase analysis shows that hypoxia significantly increased uPA transcriptional activity. However, silenced c-Myc markedly decreased hypoxia-mediated uPA transcriptional activity, suggesting that c-Myc was essential for uPA transcriptional activity (Fig. 5).


Figure 5
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  		Fig. 5 c-Myc enhances uPA transcriptional activity. uPA transcriptional activity was analyzed. uPA reporter activity was presented as relative luciferase activity. *P<0.05 vs normoxic VSMCs. #P<0.05 vs hypoxic control VSMCs. The data represents at least three determinations.

 
3.6 Bcl2 enhances c-Myc transcriptional activity
To better understand the mechanism of hypoxia regulating VSMC migration and invasion, Western blot analysis shows that hypoxia-induced VSMC Bcl2 expression (Fig. 6A). The immunoprecipitation analysis suggests that Bcl2 bound to c-Myc protein on hypoxia-induced VSMCs (Fig. 6B). The c-Myc luciferase analysis suggests that hypoxia significantly increased c-Myc transcriptional activity, but silence of Bcl2 (Fig. 6C) decreased hypoxia-induced c-Myc transcriptional activity (Fig. 6D). Our data show that Bcl2 binding to c-Myc enhanced hypoxia-mediated c-Myc transcriptional activity.


Figure 6
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  		Fig. 6 Bcl2 enhances hypoxia-induced c-Myc transcription. (A) VSMCs were treated with hypoxia for the indicated time, and the cells were lysed. Protein expression level was analyzed by Western blot using indicated antibodies. (B) Cell lysates were subjected to immunoprecipitation with antibodies to Bcl2 or with control IgG, and the resulting precipitates were subjected to Western blot analysis with antibodies to Bcl2 or c-Myc. (C) VSMCs were transfected with control siRNA or Bcl2 siRNA. Protein expression level was analyzed by Western blot using indicated antibodies. (D) c-Myc luciferase reporter was analyzed. c-Myc reporter activity was presented as relative luciferase activity. *P<0.05 vs hypoxic control VSMCs. The data represents at least three determinations.

 
3.7 Bcl2 enhances c-Myc-mediated uPA activity
To further analyze the mechanism of Bcl2 association with c-Myc, zymography was performed. The results show that silence of Bcl2 decreased uPA and MMP-2 activity on hypoxia-induced VSMC (Fig. 7A and B), resulting in decreased VSMC migration and invasion (Fig. 7C and D). The data show that Bcl2 binding to c-Myc enhanced c-Myc-mediated VSMC migration and invasion.


Figure 7
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  		Fig. 7 Bcl2 enhances c-Myc function. (A) VSMCs were treated with hypoxia on VSMCs transfected with control siRNA or Bcl2 siRNA. tPA and uPA activity were analyzed as above. (B) VSMCs were treated as above, and MMPs were analyzed as above. (C and D) VSMCs were treated as above, and VSMC migration and invasion were analyzed. *P<0.05 vs hypoxic VSMCs. Relative activities were quantified by scanning densitometry. The data represents at least three determinations.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Intimal hyperplasia is associated with the invasion and proliferation of VSMCs, and is triggered and controlled by numerous stimuli [24]. Blood-flow cessation induces the migration and invasion of VSMC, resulting in intimal hyperplasia [6]. In addition, hypoxia stimulates VSMC proliferation and atherosclerotic lesions [7,8]. In the present investigation, our data show that hypoxia increased VSMC migration and invasion, but uPA–/– significantly decreased VSMC migration and invasion. Further analysis shows that hypoxia increased uPA expression and activity. However, no such change was detected in tPA deficient VSMCs. Previous report shows that loss of uPA decreases hypoxia-induced pulmonary neointimal formation [25], and migration and invasion of VSMC cause intimal hyperplasia [6]. Therefore, our results suggest that uPA-induced VSMC migration and invasion in response to hypoxia was responsible for intimal formation.

MMPs have been implicated in cell migration and invasion [13], and hypoxia induces the cancer cell migration associated with the expression of MMP-2 [26]. In this paper, our results show that hypoxia significantly increased VSMC MMP-2 activity, but hypoxia had no effect on the activity of MMP-9, indicating that MMP-2 may be associated with VSMC migration and invasion in response to hypoxia. The activation of tPA and uPA converts the inactive zymogen plasminogen into the active proteinase plasmin, the latter can activate metalloproteinase activity [12,13]. Our results show that hypoxia increased uPA and MMP-2 activity, while it is unknown whether uPA up-regulates MMP-2 activity in response to hypoxia. The gelatin zymography of uPA–/– VSMCs was performed in response to hypoxia. The results show that uPA–/– decreased hypoxia-mediated MMP-2 activity. Taken together, our results show that hypoxia activated uPA/MMP-2 pathway.

The c-Myc protein plays an important role in regulating multiple cellular processes including cell growth, proliferation, and apoptosis [27]. Hypoxia induces c-Myc expression in vivo [15], and activates PI3K/Rho/ROCK and the c-Myc pathway [16]. MMP-2 activity is increased on hypoxia-induced endothelial cell association with cytosolic phospholipase A2 [28]. However, it is unknown whether c-Myc regulates uPA activity. Our results suggest that hypoxia significantly induced c-Myc expression. Interestingly, silenced c-Myc led to a decrease in hypoxia-induced uPA and MMP-2 activity. Further analysis shows that silenced c-Myc resulted in decreased VSMC migration and invasion, suggesting that c-Myc-mediated hypoxia-induced VSMC migration and invasion by activation of uPA and MMP-2. To better understand the mechanism by c-Myc regulating uPA activity, the luciferase analysis was carried out. The results show that silence of c-Myc decreased hypoxia-induced uPA transcriptional activity, suggesting that c-Myc increased uPA expression and activity by enhancing uPA transcriptional activity in response to hypoxia. Taken together, our results suggest that c-Myc activated the uPA/MMP-2 pathway in hypoxia-induced VSMCs, resulting in VSMC migration and invasion. Other reports show that hypoxia and HIF-1{alpha} promote growth factor-induced proliferation of human VSMCs [7], and overexpression of c-Myc transiently increases the HIF-1{alpha} protein level [15]. Therefore, the c-Myc interaction with HIF-1{alpha} needs to be further investigated.

Bcl2 cooperation with c-Myc increases the expression, secretion, and activation of MMP-2, resulting in cell invasion [29]. For further analysis whether hypoxia induces Bcl2 expression, Western blot analysis showed that hypoxia significantly increased Bcl2 expression. The immunoprecipitation analysis suggests that Bcl2 bound to the c-Myc protein. Silenced Bcl2 decreased c-Myc transcriptional activity in response to hypoxia, suggesting Bcl2 enhanced hypoxia-induced c-Myc transcriptional activity. Further analysis shows that silenced Bcl2 decreased hypoxia-induced uPA and MMP-2 activity, resulting in decreased VSMC migration and invasion, suggesting that Bcl2 enhanced the c-Myc-mediated uPA/MMP-2 signaling pathway.

In conclusion, the results suggest that hypoxia-induced the expression of c-Myc. c-Myc subsequently increased uPA expression by enhancing uPA transcriptional activity, and the activation of uPA resulted in increased MMP-2 activity, leading to VSMC migration and invasion. Therefore, hypoxia activated the c-Myc/uPA/MMP-2 pathway, resulting in VSMC migration and invasion. In addition, hypoxia promoted Bcl2 binding to the c-Myc protein, that enhanced the c-Myc-mediated uPA/MMP-2 pathway.

Time for primary review 27 days


   Acknowledgements
 
We thank Dr. Matsumura for the c-Myc promoter plasmid (Osaka University) and Dr. Nagamine for the uPA promoter plasmid (Friedrich Miescher Institute for Biomedical Research). This work was supported in part by Grants from the Ministry of Education, Culture, Sports, Science and Technology (15590197) and the "High-Tech Research Center" Project for Private Universities: a matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology).


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

  1. Ross R. The pathogenesis of arteriosclerosis, a perspective for the 1990s. Nature (1993) 362:801–809.[CrossRef][Medline]
  2. Schwartz S.M., Virmani R., Rosenfeld M.E. The good smooth muscle cells in atherosclerosis. Curr Atheroscler Rep (2000) 2:422–429.[Medline]
  3. Schwartz S.M., deBlois DO'Brien E.R.M. The intima. Soil for atherosclerosis and restenosis. Circ Res (1995) 77:445–465.[Free Full Text]
  4. Dzau V.J., Braun-Dullaeus R.C., Sedding D.G. Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies. Nat Med (2002) 8:1249–1256.[CrossRef][Web of Science][Medline]
  5. Zwolak R.M., Adams M.C., Clowes A.W. Kinetics of vein graft hyperplasia: association with tangential stress. J Vasc Surg (1987) 5:126–136.[CrossRef][Web of Science][Medline]
  6. Li Y., Minamino T., Tsukamoto O., Yujiri T., Shintani Y., Okada K., et al. Ablation of MEK kinase 1 suppresses intimal hyperplasia by impairing smooth muscle cell migration and urokinase plasminogen activator expression in a mouse blood-flow cessation model. Circulation (2005) 111:1672–1678.[Abstract/Free Full Text]
  7. Schultz K., Fanburg B.L., Beasley D. Hypoxia and hypoxia-inducible factor-1{alpha} promote growth factor-induced proliferation of human vascular smooth muscle cells. Am J Physiol Heart Circ Physiol (2006) 290:2528–2534.[CrossRef]
  8. Tong Q., Zheng L., Li B., Wang D., Huang C., Matuschak G.M., et al. Hypoxia-induced mitogenic factor enhances angiogenesis by promoting proliferation and migration of endothelial cells. Exp Cell Res (2006) 1:3559–3569.
  9. Shweiki D., Neeman M., Itin A., Keshet E. Induction of vascular endothelial growth factor expression by hypoxia and by glucose deficiency in multicell spheroids: implications for tumor angiogenesis. Proc Natl Acad Sci U S A (1995) 92:768–772.[Abstract/Free Full Text]
  10. Lopez-Barneo J., Pardal R., Ortega-Saenz P. Cellular mechanism of oxygen sensing. Annu Rev Physiol (2001) 63:259–287.[CrossRef][Web of Science][Medline]
  11. Carmeliet P., Moons L., Herbert J.M., Crawley J., Lupu F., Lijnen R., et al. Urokinase but not tissue plasminogen activator mediates arterial neointima formation in mice. Circ Res (1997) 81:829–839.[Abstract/Free Full Text]
  12. Van Hinsbergh V.W.M., Koolwijk P., Hanemaaijer R. Role of fibrin and plasminogen activators in repair-associated angiogenesis: in vitro studies with human endothelial cells. EXS (1997) 79:391–411.[Medline]
  13. Chakraborti S., Mandal M., Das S., Mandal A., Chakraborti T. Regulation of matrix metalloproteinases: an overview. Mol Cell Biochem (2003) 253:269–285.[CrossRef][Web of Science][Medline]
  14. Baudino T.A., McKay C., Pendeville-Samain H., Nilsson J.A., Maclean K.H., White E.L., et al. c-Myc is essential for vasculogenesis and angiogenesis during development and tumor progression. Genes Dev (2002) 16:2530–2543.[Abstract/Free Full Text]
  15. Knies-Bamforth U.E., Fox S.B., Poulsom R., Evan G.I., Harris A.L. c-Myc interacts with hypoxia to induce angiogenesis in vivo by a vascular endothelial growth factor-dependent mechanism. Cancer Res (2004) 64:6563–6570.[Abstract/Free Full Text]
  16. Mizukami Y., Fujiki K., Duerr E.M., Gala M., Jo W.S., Zhang X., et al. Hypoxic regulation of vascular endothelial growth factor through the induction of phosphatidylinositol 3-kinase/Rho/ROCK and c-Myc. J Biol Chem (2006) 281:13957–13963.[Abstract/Free Full Text]
  17. Okada K., Ueshima S., Imano M., Kataoka K., Matsuo O. The regulation of liver regeneration by the plasmin/alpha 2-antiplasmin system. J Hepatol (2004) 40:110–116.[Web of Science][Medline]
  18. Hou Y.Z., Yang J., Zhao G.R., Yuan Y.J. Ferulic acid inhibits vascular smooth muscle cell proliferation induced by angiotensin II. Eur J Pharmacol (2004) 19:85–90.
  19. Satoh Y., Matsumura I., Tanaka H., Ezoe S., Sugahara H., Mizuki M., et al. Roles for c-Myc in self-renewal of hematopoietic stem cells. J Biol Chem (2004) 279:24986–24993.[Abstract/Free Full Text]
  20. Miralles F., Parra M., Caelles C., Nagamine Y., Felez J., Munoz-Canoves P. UV irradiation induces the murine urokinase-type plasminogen activator gene via the c-Jun N-terminal kinase signaling pathway: requirement of an AP1 enhancer element. Mol Cell Biol (1998) 18:4537–4547.[Abstract/Free Full Text]
  21. Yang J.N., Allan E.H., Anderson G.I., Martin T.J., Minkin C. Plasminogen activator system in osteoclasts. J Bone Miner Res (1997) 12:761–768.[CrossRef][Web of Science][Medline]
  22. Hurlin P.J., Queva C., Eisenman R.N. A novel max-interacting protein is coexpressed with Myc in proliferating cells and mediates repression at Myc binding Sites. Genes Dev (1997) 11:44–58.[Abstract/Free Full Text]
  23. Matsuo O., Sakai T., Bando H., Okada K., Nakajima S., Takagi O., et al. Plasminogen activator in bronchoalveolar fluid. Haemostasis (1986) 16:43–50.[Web of Science][Medline]
  24. Newby A.C., Zaltsman A.B. Molecular mechanisms in intimal hyperplasia. J Pathol (2000) 190:300–309.[CrossRef][Web of Science][Medline]
  25. Levi M., Moons L., Bouche A., Shapiro S.D., Collen D., Carmeliet P. Deficiency of urokinase-type plasminogen activator-mediated plasmin generation impairs vascular remodeling during hypoxia-induced pulmonary hypertension in mice. Circulation (2001) 103:2014–2020.[Abstract/Free Full Text]
  26. Duong H.S., Le A.D., Zhang Q., Messadi D.V. A novel 3-dimensionalculture system as an in vitro model for studying oral cancer cell invasion. Int J Exp Pathol (2005) 86:365–374.[CrossRef][Web of Science][Medline]
  27. Marcu K.B., Bossone S.A., Patel A.J. Myc function and regulation. Annu Rev Biochem (1992) 61:809–860.[CrossRef][Web of Science][Medline]
  28. Ottino P., Finley J., Rojo E., Ottlecz A., Lambrou G.N., Bazan H.E.P., et al. Hypoxia activates matrix metalloproteinase expression and the VEGF system in monkey choroid-retinal endothelial cells: involvement of cytosolic phospholipase A2 activity. Mol Vis (2004) 10:341–350.[Web of Science][Medline]
  29. Noujaim D., van Golen C.M., van Golen K.L., Grauman A., Feldman E.L. N-Myc and Bcl-2 coexpression induces MMP-2 secretion and activation in human neuroblastoma cells. Oncogene (2002) 21:4549–4557.[CrossRef][Web of Science][Medline]

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Arterioscler Thromb Vasc Biol, July 1, 2008; 28(7): 1257 - 1262.
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