Journal Mobile Options
Table of Contents
Vol. 10, No. 5, 2003
Issue release date: September–October 2003
J Biomed Sci 2003;10:526–534

Inhibition of Fibroblast-Induced Angiogenic Phenotype of Cultured Endothelial Cells by the Overexpression of Tissue Inhibitor of Metalloproteinase (TIMP)-3

Ma D.H.-K. · Chen J.-I. · Zhang F. · Hwang D.G. · Chen J.-K.
aDepartment of Ophthalmology, Chang-Gung Memorial Hospital, bDepartment of Physiology, College of Medicine, Chang-Gung University, Kweishan, Taoyuan, Taiwan; cOcular Cell and Gene Therapy Laboratory, Department of Ophthalmology, University of California, San Francisco, Calif., USA

Individual Users: Register with Karger Login Information

Please create your User ID & Password

Contact Information

I have read the Karger Terms and Conditions and agree.

To view the fulltext, please log in

To view the pdf, please log in


In this study, we examined the effect of overexpression of tissue inhibitor of metalloproteinase (TIMP)-3 on the angiogenic phenotype expressed by vascular endothelial cells (ECs). ECs were infected with a recombinant adenovirus carrying the TIMP-3 gene at various multiplicities of infection, and TIMP-3 expression by transfected cells was confirmed by Western blotting and reverse zymography. At transfection doses of 6.25, 12.5, 25, 50 and 100 multiplicity of infection, EC migration was reduced to 66, 45, 25, 17 and 5%, respectively, of that of the control. At the multiplicity of infection of 20, capillary tube length was reduced by 80% compared to that of the control. Thus, expression of TIMP-3 by ECs effectively inhibited EC migration and tube formation. Overexpression of TIMP-3 by ECs may be considered a gene therapy strategy for the treatment of pathological angiogenesis such as cancer and diabetic retinopathy.

Copyright / Drug Dosage

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.


  1. Ahonen M, Ala-Aho R, Baker A, George S, Grenman R, Saarialho-Kere U, Kahari VM. Antitumour activity and bystander effect of adenovirally delivered tissue inhibitor of metalloproteinases-3. Mol Ther 5:705–715;2002.
  2. Ahonen M, Baker AH, Kahari VM. Adenovirus-mediated gene delivery of tissue inhibitor of metalloproteinases-3 inhibits invasion and induces apoptosis in melanoma cells. Cancer Res 58:2310–2315;1998.
  3. Anand-Apte B, Bao L, Smith R, Iwata K, Olsen BR, Zetter B, Apte SS. A review of tissue inhibitor of metalloproteinases-3 (TIMP-3) and experimental analysis of its effect on primary tumor growth. Biochem Cell Biol 74:853–862;1996.
  4. Anand-Apte B, Pepper MS, Voest E, Montesano R, Murphy G, Apte SS, Zetter B. Inhibition of angiogenesis by tissue inhibitor of metalloproteinase-3. Invest Ophthalmol Vis Sci 38:817–823;1997.
  5. Apte SS, Mattei MG, Olsen BR. Cloning of the cDNA encoding human tissue inhibitor of metalloproteinases-3 (TIMP-3) and mapping of the TIMP3 gene to chromosome 22. Genomics 19:86–90;1994.
  6. Baker AH, Edwards DR, Murphy G. Metalloproteinase inhibitors: Biological actions and therapeutic opportunities. J Cell Sci 115:3719–3727;2002.
  7. Baker AH, George SJ, Zaltsman AB, Murphy G, Newby AC. Inhibition of invasion and induction of apoptotic cell death of cancer cell lines by overexpression of TIMP-3. Br J Cancer 79:1347–1355;1999.
  8. Baker AH, Zaltsman AB, George SJ, Newby AC. Divergent effects of tissue inhibitor of metalloproteinase-1,-2, or -3 overexpression on rat vascular smooth muscle cell invasion, proliferation, and death in vitro. TIMP-3 promotes apoptosis. J Clin Invest 101:1478–1487;1998.
  9. Bian JH, Wang YL, Smith MR, Kim H, Jacobs C, Jackman J, Kung HF, Colburn NH, Sun Y. Suppression of in vivo tumor growth and induction of suspension cell death by tissue inhibitor of metalloproteinases (TIMP)-3. Carcinogenesis 17:1805–1811;1996.
  10. Bond M, Murphy G, Bennett MR, Newby AC, Baker AH. Tissue inhibitor of metalloproteinase-3 induces a Fas-associated death domain-dependent type II apoptotic pathway. J Biol Chem 277:13787–13795;2002.
  11. Brogi E, Wu T, Namiki A, Isner JM. Indirect angiogenic cytokines upregulate VEGF and bFGF gene expression in vascular smooth muscle cells, whereas hypoxia upregulates VEGF expression only. Circulation 90:649–652;1994.
  12. Bungo M, Witek B, Bereta J, Bereta M, Edwards DR, Kordula T. Reprogramming of TIMP-1 and TIMP-3 expression profiles in brain microvascular endothelial cells and astrocytes in response to proinflammatory cytokines. FEBS Lett 448:9–14;1999.
  13. Chen JK, Hoshi H, McClure DB, McKeehan WL. Role of lipoproteins in growth of human adult arterial endothelial and smooth muscle cells in low lipoprotein-deficient serum. J Cell Physiol 129:207–214;1986.
  14. Fassina G, Ferrari N, Brigati C, Benelli R, Santi L, Noonan D, Albini A. Tissue inhibitors of metalloproteinase: Regulation and biological activities. Clin Exp Metastasis 18:111–120;2000.
  15. Fernandez HA, Kallenbach K, Seghezzi G, Grossi E, Colvin S, Schneider R, Mignatti P, Gallway A. Inhibition of endothelial cell migration by gene transfer of tissue inhibitor of metalloproteinase-1. J Surg Res 82:156–162;1999.
  16. Fisher C, Gilbertson-Beadling S, Powers EA, Petzold G, Poorman R, Mitchell MA. Interstitial collagenase is reguired for angiogenesis in vitro. Dev Biol 162:499–510;1994.

    External Resources

  17. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1:27–31;1995.
  18. George SJ, Lloyd CT, Angelini GD, Newby AC, Baker AH. Inhibition of late vein graft neointima formation in human and porcine models by adenovirus-mediated overexpression of tissue inhibitor of metalloproteinase-3. Circulation 101:296–304;2000.
  19. Gomez DE, Alonso DF, Yoshiji H, Thorgeirsson UP. Tissue inhibitors of metalloproteases: Structure, regulation and biological functions. Eur J Cell Biol 77:111–122;1997.
  20. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86:353–364;1996.
  21. Hannan RL, Kourembanas S, Flanders KC, Rogelj SJ, Roberts AB, Faller DV, Klagsbrun M. Endothelial cells synthesize basic fibroblast growth factor and transforming growth factor beta. Growth Factors 1:7–17;1988.
  22. Hao YX, Ma DHK, Hwang DG, Kim WS, Zhang F. Identification of antiangiogenic and antiinflammatory proteins in human amniotic membrane. Cornea 19:348–352;2000.
  23. He TC, Zhou S, Da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA 95:2509–2514;1998.
  24. Hlatky L, Tsionou C, Hahnfeldt P, Coleman N. Mammary fibroblasts may influence breast tumor angiogenesis via hypoxia-induced vascular endothelial growth factor upregulation and protein expression. Cancer Res 54:6083–6086;1994.
  25. Hudlicka O, Brown MD, Egginton S. Angiogenesis in skeleton and cardiac muscle. Physiol Rev 72:369–417;1992.
  26. Itoh T, Tanioka M, Yoshida H, Yoshioka T, Nishimoto H, Itohara S. Reduced angiogenesis and tumor progression in gelatinase A-deficient mice. Cancer Res 58:1048–1051;1998.
  27. Jackson CJ, Nguyen M. Human microvascular endothelial cells differ from macrovascular endothelial cells in their expression of matrix metalloproteinases. Int J Biochem Cell Biol 29:1167–1177;1997.

    External Resources

  28. Karelina TV, Eisen AZ. Interstitial collagenase and the ED-B oncofetal domain of fibronectin are markers of angiogenesis in human skin tumors. Cancer Detect Prev 22:438–444;1998.
  29. Kenney MC, Chwa M, Alba A, Saghizadeh M, Huang ZS, Brown DT. Localization of TIMP-1, TIMP-2, TIMP-3, gelatinase A and gelatinase B in pathological human corneas. Curr Eye Res 17:238–46;1998.
  30. Kunz-Schughart LA, Knuechel R. Tumor-associated fibroblasts. II. Functional impact on tumor tissue. Histol Histopathol 17:623–637;2002.
  31. Liotta LA, Steeg PS, Stetler-Stevenson G. Cancer metastasis and angiogenesis: An imbalance of positive and negative regulation. Cell 64:327–336;1991.
  32. Ma DHK, Tsai RJF, Chu WK, Kao CH, Chen JK. Inhibition of vascular endothelial cell morphogenesis in culture by limbal epithelial cells. Invest Ophthalmol Vis Sci 40:1822–1828;1999.
  33. Ma DHK, Zhang F, Shi W, Yao JY, Hsiao CH, Wu HC, Kim WS, Hao YX, Hwang DG, Chen JK, Tsai RJF. Expression of tissue inhibitor of metalloproteinase-4 (TIMP-4) in normal human corneal cells and experimental corneal neovascularization. Ophthalmic Res 35:199–207;2003.
  34. Majid MA, Smith VA, Easty DL, Baker AH, Newby AC. Adenovirus mediated gene delivery of tissue inhibitor of metalloproteinases-3 induces death in retinal pigment epithelial cells. Br J Ophthalmol 86:97–101;2002.
  35. Mignatti P, Rifkin DB. Plasminogen activators and matrix metalloproteases in angiogenesis. Enzyme Protein 49:117–137;1998.
  36. Montesano R, Pepper MS, Orci L. Paracrine induction of angiogenesis in vitro by Swiss 3T3 fibroblasts. J Cell Sci 105:1013–1024;1993.
  37. Murugesan G, Chisolm GM, Fox PL. Oxidized LDL inhibits the migration of aortic endothelial cells in vitro. J Cell Biol 120:1011–1019;1993.
  38. Nakayasu K, Hayashi N, Okisaka S, Sato N. Formation of capillary-like tubes by vascular endothelial cells cocultivated with keratocytes. Invest Ophthalmol Vis Sci 33:3050–3057;1992.
  39. Nehis V, Denzer K, Drenckhahn D. Pericyte involvement in capillary sprouting during angiogenesis in situ. Cell Tissue Res 270:469–474;1992.
  40. Nicosia RF, Villaschi S. Autoregulation of angiogenesis by cells of the vessel wall. Int Rev Cytol 185:1–43;1999.
  41. Pepper MS, Montesano R, Mandriota SJ, Orci L, Vassalli JD. Angiogenesis: A paradigm for balanced extracellular proteolysis during cell migration and morphogenesis. Enzyme Protein 49:138–162;1996.
  42. Sato N, Sawasaki Y, Senoo A, Fuse Y, Hirano Y, Goto T. Development of capillary networks from rat microvascular fragments in vitro: The role of myofibroblastic cells. Microvasc Res 33:194–210;1987.
  43. Smith MR, Kung H, Durum SK, Colburn NH, Sun Y. TIMP-3 induces cell death by stabilizing TNF-alpha receptors on the surface of human colon carcinoma cells. Cytokine 9:770–780;1997.
  44. Villaschi S, Nicosia RF. Paracrine interaction between fibroblasts and endothelial cells in a serum free coculture model: Modulation of angiogenesis and collagen gel contraction. Lab Invest 71:291–299;1994.
  45. Yu WH, Yu S, Meng Q, Brew K, Woessner JF Jr. TIMP-3 binds to sulfated glycosaminoglycans of the extracellular medium. J Biol Chem 275:31226–31232;2000.

Pay-per-View Options
Direct payment This item at the regular price: USD 38.00
Payment from account With a Karger Pay-per-View account (down payment USD 150) you profit from a special rate for this and other single items.
This item at the discounted price: USD 26.50