Download PDF
Thromb Haemost 2001; 86(04): 1094-1098
DOI: 10.1055/s-0037-1616538
Special Article
Schattauer GmbH

Thrombin Induces Increased Expression and Secretion of VEGF from Human FS4 Fibroblasts, DU145 Prostate Cells and CHRF Megakaryocytes

Y-Q. Huang
1   Department of Medicine and Kaplan Cancer Center, New York University Medical School, New York, NY, USA
,
J-J. Li
1   Department of Medicine and Kaplan Cancer Center, New York University Medical School, New York, NY, USA
,
L. Hu
1   Department of Medicine and Kaplan Cancer Center, New York University Medical School, New York, NY, USA
,
M. Lee
1   Department of Medicine and Kaplan Cancer Center, New York University Medical School, New York, NY, USA
,
S. Karpatkin
1   Department of Medicine and Kaplan Cancer Center, New York University Medical School, New York, NY, USA
› Author Affiliations*Supported by NIH grant HL-13336-28, the Hildegarde D. Becher Foundation, and grants from the Helen Polonsky Research Fund and Dorothy and Seymour Weinstein Research Fund.
Further Information

Publication History

Received 24 April 2001

Accepted after revision 23 May 2001

Publication Date:
09 December 2017 (online)

    Permissions and Reprints

Summary

Angiogenesis is required for tumor growth and metastasis. It has recently been suggested that thrombin is a potent promoter of angio-genesis. We therefore examined the possibility that thrombin could be inducing the expression of vascular endothelial growth factor (VEGF), which promotes endothelial growth. Primary human FS4 fibroblasts as well as tumor cell lines: prostate DU145 and megakaryocyte CHRF were incubated with thrombin (0.25-1 unit/ml) for 1-8 hrs and then examined for mRNA by Northern Analysis. Enhanced mRNA (~3-4 fold over base line) was noted at 2-4 hrs, with 0.5 u/ml thrombin. The effect was specific for thrombin activity on its PAR-1 receptor, since equal units of hirudin completely inhibited the response and the thrombin effect could be mimicked with the 14 mer thrombin receptor activation peptide (TRAP). Upregulation of mRNA was associated with enhanced VEGF protein synthesis and secretion as assayed by immunoblot. Enhanced expression of VEGF mRNA was not secondary to enhanced transcription (nuclear run on experiments), but due to an >3 fold stabilization of mRNA (Actinomycin D chase experiment). Enhanced VEGF mRNA stabilization is promoted by the PI3Kinase and serine/threonine kinase pathways, since thrombin-induced mRNA expression is inhibited by Wortmanin and H7. No effect was noted with the MAPKinase inhibitor, PD98059. Thus, thrombin-induced tumori-genesis and metastasis is associated with enhanced VEGF protein synthesis and secretion via the stabilization of VEGF mRNA promoted by the PI3Kinase and serine/threonine kinase pathways. This could help explain how thrombin promotes angiogenesis.

Keywords

Thrombin - VEGF - angiogenesis - PI3Kinase - serine/threonine kinase
 

  • References

  • 1 Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Review Nat Med 1995; 1: 27-31.
    PubMedGoogle Scholar
  • 2 Folkman J, Shing Y. Angiogenesis. J Biol Chem 1992; 267: 10931-4.
    CrossrefPubMedGoogle Scholar
  • 3 Pepper MS, Ferrara N, Orci L, Montesano R. Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro. Biochem Biophys Res Commun 1992; 189: 824-31.
    CrossrefPubMedGoogle Scholar
  • 4 Tolentino MJ, Mill JW, Gragoudas ES, Chatzistefanou K, Ferrara N, Adamis AP. Vascular endothelial growth factor is sufficient to produce iris neovascularization and neovascular glaucoma in a nonhuman primate. Arch Ophthalmol 1996; 114: 964-70.
    CrossrefPubMedGoogle Scholar
  • 5 Ben-Av P, Crofford L, Wilde RL, Hla T. Induction of vascular endothelial growth factor expression in synovial fibroblasts by prostaglandin E and interleukin-1: a potential mechanism for inflammatory angiogenesis. FEBS Letter 1995; 372: 83-7.
    CrossrefPubMedGoogle Scholar
  • 6 Cohen T, Nahari D, Cerem L, Neufeld G, Levi B. Interleukin 6 induces the expression of vascular endothelial growth factor. J Biol Chem 1996; 271: 736-41.
    CrossrefPubMedGoogle Scholar
  • 7 Frank S, Hubner G, Breier G, Longaker MT, Greenhalgh DG, Werner S. Regulation of vascular endothelial growth factor expression in cultured keratinocytes. Implications for normal and impaired wound healing. J Biol Chem 1995; 270: 12607-13.
    CrossrefPubMedGoogle Scholar
  • 8 Goldman CK, Kim J, Wong WL, King V, Brock T, Gillespie GY. Epidermal growth factor stimulates vascular endothelial growth factor production by human malignant glioma cells: a model of glioblastoma multiforme pathophysiology. Mol Biol Cell 1993; 4: 121-33.
    CrossrefPubMedGoogle Scholar
  • 9 Li J, Perrella MA, Tsai JC, Yet SF, Hsieh CM, Yoshizumi M. et al. Induction of vascular endothelial growth factor gene expression by interluekin-1 beta in rat aortic smooth muscle cells. J Biol Chem 1995; 270: 308-12.
    CrossrefPubMedGoogle Scholar
  • 10 Pertovaara L, Kaipainen A, Mustonen T, Orpana A, Ferrara N, Saksela O. et al. Vascular endothelial growth factor is induced in response to transforming growth factor-beta in fibroblastic and epithelial cells. J Biol Chem 1994; 4: 6271-4.
    PubMedGoogle Scholar
  • 11 Warren RS, Yuan H, Matli MR, Ferrara N, Donne RDB. Induction of vascular endothelial growth factor by insulin-like growth factor 1 in colorectal carcinoma. J Biol Chem 1996; 271: 29483-8.
    CrossrefPubMedGoogle Scholar
  • 12 Ijichi A, Sakuma S, Tofilon PJ. Hypoxia-induced vascular endothelial growth factor expression in normal rat astrocyte cultures. Hypoxia-induced vascular endothelial growth factor expression in normal rat astrocyte cultures. Glia 1995; 14: 87-93.
    CrossrefPubMedGoogle Scholar
  • 13 Mazure NM, Chen EY, Laderoute KR, Giaccia AJ. Induction of vascular endothelial growth factor by hypoxia is modulated by a phosphatidylinositol 3-kinase/Akt signaling pathway in Ha-ras-transformed cells through a hypoxia inducible factor-1 transcriptional element. Blood 1997; 90: 3322-31.
    PubMedGoogle Scholar
  • 14 Levy AP, Levy NS, Goldberg MA. Hypoxia-inducible protein binding to vascular endothelial growth factor mRNA and its modulation by the von Hippel-Lindau protein. J Biol Chem 1996; 271: 2746-53.
    CrossrefPubMedGoogle Scholar
  • 15 Ryuto M, Ono M, Izumi H, Yoshida S, Weich HA, Kohno K. et al. Induction of vascular endothelial growth factor by tumor necrosis factor alpha in human glioma cells. Possible roles of SP-1. J Biol Chem 1996; 271: 28220-8.
    CrossrefPubMedGoogle Scholar
  • 16 Davis S, Aldrich TH, Jones PF, Acheson A, Compton DL, Jain V. et al. Isolation of angiopoietin-1, a ligand for the TIE-2 receptor, by secretion-trap expression cloning. Cell 1996; 87: 1161-9.
    CrossrefPubMedGoogle Scholar
  • 17 Suri C, Jones PF, Patan S, Bartunkova S, Maisonpierre PC, Davis S. et al. Requisite role of angiopoietin-1, a ligand for the TIE-2 receptor, during embryonic angiogenesis. Cell 1996; 87: 1171-80.
    CrossrefPubMedGoogle Scholar
  • 18 Thurston G, Suri C, Smith K, McClain J, Sato N, Yancopoulos GD. et al. Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1. Science 1999; 286: 2511-4.
    CrossrefPubMedGoogle Scholar
  • 19 Maisonpierre PC, Suri C, Jones PF, Bartunkova B, Wiegand SJ, Radziejewski C. et al. Angipoietin-2, a natural antagonist for Tie-2 that disrupts in vivo angiogenesis. Science 1997; 277: 55-60.
    CrossrefPubMedGoogle Scholar
  • 20 Koblizek TI, Weiss C, Yancopoulos GD, Deutsch U, Risau W. Angiopoietin-1 induces sprouting angiogenesis in vitro. Current Biology 1988; 8: 529-32.
    PubMedGoogle Scholar
  • 21 Oh H, Takagi H, Suzuma K, Otani A, Matsumura M, Honda Y. Hypoxia and vascular endothelial growth factor selectively up-regulate Angiopoietin-2 in bovine microvascular endothelial cells. J Biol Chem 1999; 274: 15732-39.
    CrossrefPubMedGoogle Scholar
  • 22 Haralabopoulos GC, Grant DS, Kleinman HK, Maragoudakis ME. Thrombin promotes endothelial cell alignment in matrigel in vitro and angiogenesis in vivo. Am J Physiol 1997; 273: 239-45.
    CrossrefPubMedGoogle Scholar
  • 23 Herbert JM, Dupuy E, Laplace MC, Zini JM, Bar Shavit R. Thrombin induces endothelal cell growth via both a proteolytic and a non-proteolytic pathway. Biochemical Journal 1994; 303: 227-31.
    CrossrefPubMedGoogle Scholar
  • 24 Tsopanoglou NE, Maragoudakis ME. On the endothelial growth factor activity on endothelial cells by up-regulation of its receptors. J Biol Chem 1999; 274: 23969-76.
    CrossrefPubMedGoogle Scholar
  • 25 Tsopanoglou NE, Pipili-Synetos E, Maragoudakis ME. Thrombin promotes angiogenesis by a mechanism independent of fibrin formation. Am J Physiol 1993; 264: C1302-C7.
    CrossrefPubMedGoogle Scholar
  • 26 Trousseau A. Phelgmasia alba dolens. Clinicque Medicale de Hotel-Dieu de Paris, London. New Syndenham Society 1865; 3: 94.
    PubMedGoogle Scholar
  • 27 Merskey C, Johnson AJ, Harris JU, Want MT, Swain S. Isolation of fibrinogen-fibrin related antigen from human plasma by immune-affinity chromatography: its characterization in normal subjects and in defibrinating patients with abruptio placentae and disseminated cancer. Brit J Haematol 1980; 44: 655-70.
    CrossrefPubMedGoogle Scholar
  • 28 Peuscher FW, Cleton FJ, Armstrong L, Stoepman van Dalen EA, van Aken WG. Significance of plasma fibrinopeptide A (FPA) in patients with malignancy. J Lab Clin Med 1980; 96: 5-14.
    PubMedGoogle Scholar
  • 29 Rickles FR, Edwards RC, Barb C, Cronlund M. Abnormalities of blood coagulation in patients with cancer: fibrinopeptide A generation and tumor growth. Cancer 1983; 51: 301-7.
    CrossrefPubMedGoogle Scholar
  • 30 Yoda Y, Abe T. Fibrinopeptide A (FPA) level and fibrinogen kinetics in patients with malignant disease. Thromb Haemost 1981; 46: 706-9.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 31 Klepfish A, Greco MA, Karpatkin S. Thrombin stimulates melanoma tumor cell binding to endothelial cells and subendothelial matrix. Int J Cancer 1993; 53: 978-82.
    PubMedGoogle Scholar
  • 32 Nierodzik ML, Bain RM, Liu L-X, Shivji M, Takeshita K, Karpatkin S. Presence of the seven transmembrane thrombin receptor on human tumour cells: effect of activation on tumour adhesion to platelets and tumour tyrosine phosphorylation. Brit J Haematol 1996; 92: 452-7.
    CrossrefPubMedGoogle Scholar
  • 33 Nierodzik ML, Chen K, Takeshita K, Li J-J, Huang Y-Q, Feng X-S. et al. Pro-tease-Activated Receptor 1 (PAR-1) is required and rate-limiting for thrombin-enhanced experimental pulmonary metastasis. Blood 1998; 92: 3694-700.
    PubMedGoogle Scholar
  • 34 Nierodzik ML, Kajumo F, Karpatkin S. Effect of thrombin treatment of tumor cells on adhesion of tumor cells to platelets in vitro and metastasis in vivo. Cancer Res 1992; 52: 3267-72.
    PubMedGoogle Scholar
  • 35 Grand RJA, Turnell AS, Grabham PW. Cellular consequences of thrombin-receptor activation. Biochem J 1996; 313: 353-68.
    CrossrefPubMedGoogle Scholar
  • 36 Li J-J, Huang Y-Q, Basch R, Karpatkin S. Thrombin induces the release of angiopoietin-1 from platelets. Thromb Haemost 2001; 85: 204-6.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 37 Mohle R, Green D, Moore MAS, Nachman RL, Rafii S. Constitutive production and thrombin-induced release of vascular endothelial growth factor by megakaryoyctes and platelets. Proc Natl Acad Sci USA 1997; 94: 663-8.
    CrossrefPubMedGoogle Scholar
  • 38 Moloney JP, Sillimon CC, Ambruso DR, Wang J, Tuder RM, Voelkel NF. In vitro release of VEGF during platelet aggregation. Amer J Phys 1988; 275: H1054.
    PubMedGoogle Scholar
  • 39 Benjamin LE, Keshet E. Conditional switching of vascular endothelial growth factor (VEGF) expression in tumors: Induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by VEGF withdrawal. Proc Natl Acad Sci USA 1997; 94: 8761-6.
    CrossrefPubMedGoogle Scholar
  • 40 Plate KH, Breier G, Weich HA, Risau W. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature 1992; 359: 845-8.
    CrossrefPubMedGoogle Scholar
  • 41 Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 1992; 359: 843-5.
    CrossrefPubMedGoogle Scholar
  • 42 Zacharski LR, Memoli VA, Morain WD, Schlaepi J-M, Rousseau SM. Cellular localization of enzymatically-active thrombin in intact tissue by hirudin binding. Thromb Haemost 1995; 73: 793-7.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 43 Kim KJ, Li B, Winer J, Armanini M, Gillett N, Phillips HS. et al. Inhibition of vascular endothelial growth factor induced angiogenesis suppresses tumour growth in vivo. Nature 1993; 362: 841-4.
    CrossrefPubMedGoogle Scholar
  • 44 Kondo S, Asano M, Suzuki H. Significance of vascular endothelial growth factor/vascular permeability factor for solid tumor growth, and its inhibition by the natibody. Biochem Biophys Res Commun 1993; 194: 1234-41.
    CrossrefPubMedGoogle Scholar
  • 45 Even-Ram S, Uziely B, Cohen P, Grisaru-Granovsky S, Maoz M, Ginzburg Y. et al. Thrombin receptor overexpression in malignant and physiological invasion processes. Nature Medicine 1998; 4: 909-14.
    CrossrefPubMedGoogle Scholar
  • 46 Karpatkin S, Pearlstein E, Ambrogio C, Coller BS. Role of adhesive proteins in platelet tumor interaction in vitro and metastasis formation in vivo. J Clin Invest 1988; 81: 1012-9.
    CrossrefPubMedGoogle Scholar
  • 47 Pearlstein E, Ambrogio C, Karpatkin S. Effect of anti-platelet antibody on the development of pulmonary metastases following injection of CT26 colon adneocarcinoma, Lewis lung carcinoma and B16 amelanotic melanoma tumor cells in mice. Cancer Res 1984; 44: 3884-7.
    PubMedGoogle Scholar
  • 48 Huang Y-Q, Li J-J, Hu L, Karpatkin S. Thrombin induces the synthesis of VEGF and Angiopoietin-2 (Ang-2). Blood 2000; 96: 34A.
    PubMedGoogle Scholar
  • 49 Ollivier V, Chabbat J, Jm H, Hakim J, de Prost D. Vascular endothelial growth factor production by fibroblasts in response to factor VIIa binding to tissue factor involves thrombin and factor Xa. Arterioscler Thromb Vasc Biol 2000; 20: 1374-81.
    CrossrefPubMedGoogle Scholar
  • 50 Shih SC, Mullen A, Abrams K, Mukhopadhyay D, Claffey KP. Role of protein kinase C isoforms in phorbol ester-induced vascular endothelial growth factor expression in human glioblastoma cells. J Biol Chem 1999; 274: 15407-14.
    CrossrefPubMedGoogle Scholar
  • 51 Finkenzeller G, Technau A, Marmer D. Hypoxia-inducible transcripton of the vascular endothelial grwoth factor gene is independent of functional AP-1 transcription factor. Biochem Biophys Res Commun 1995; 208: 432-9.
    CrossrefPubMedGoogle Scholar
  • 52 Carter AN, Huang R, Sorisk A, Downes CP, Rittenhouse SE. Phosphatidylinositol 3,4,5-trisphosphate is formed from phosphatidylinositol 4,5-bis-phosphate in thrombin-stimulated platelets. Biochem J 1994; 301: 415-20.
    CrossrefPubMedGoogle Scholar
  • 53 Gutkind JS, Lacal PM, Robbins KC. Thrombin-dependent association of phosphatidylinositol-3 kinase with p60c-src and p59fyn in human platelets. Mol Cell Biol 1990; 10: 3806-9.
    CrossrefPubMedGoogle Scholar
  • 54 Ha KS, Exton JH. Differential translocation of protein kinase C isozymes by thrombin and platelet-derived growth factor. A possible function for phosphatidylchline-derived diacylglycerol. J Biol Chem 1993; 268: 10534-9.
    PubMedGoogle Scholar
  • 55 Leach KL, Ruff VA, Jarpe MB, Adams LD, Fabbro D, Raben DM. Alpha-thrombin stimulates nuclear diglyceride levels and differential nuclear localization of protein kinase C isozymes in IIC9 cells. J Biol Chem 1992; 267: 21816-22.
    PubMedGoogle Scholar
  • 56 Levy AP, Levy NS, Loscalzo J, Calderone Takahashi N, Yeo KT. et al. Regulation of vascular endothelial growth factor in cardiac myocytes. Circ Res 1995; 76: 758-66.
    CrossrefPubMedGoogle Scholar
  • 57 Chua CC, Hamdy RC, Chau BH. Upregulation of vascular endothelial growth factor by angiotensin II in rat heart endothelial cells. Biochim Biophy Acta 1401: 187-94.
    PubMedGoogle Scholar