, Volume 4, Issue 1, pp 11–20 | Cite as

TGFβ is required for the formation of capillary-like structures in three-dimensional cocultures of 10T1/2 and endothelial cells

  • D.C. Darland
  • P.A. D'Amore


New vessels form de novo (vasculogenesis) or from pre-existing vessels (angiogenesis) in a process that involves the interaction of endothelial cells (EC) and pericytes/smooth muscle cells (SMC). One basic component of this interaction is the endothelial-induced recruitment, proliferation and subsequent differentiation of pericytes and SMC. We have previously demonstrated that TGFβ induces the differentiation of C3H/10T1/2 (10T1/2) mesenchymal cells toward a SMC/pericyte lineage. The current study tests the hypothesis that TGFβ not only induces SMC differentiation but stabilizes capillary-like structures in a three-dimensional (3D) model of in vitro angiogenesis. 10T1/2 and EC in Matrigel™ were used to establish cocultures that form cord structures that are reminiscent of new capillaries in vivo. Cord formation is initiated within 2–3 h after plating and continues through 18 h after plating. In longer cocultures the cord structures disassemble and form aggregates. 10T1/2 expression of proteins associated with the SMC/pericyte lineage, such as smooth muscle α-actin (SMA) and NG2 proteoglycan, are upregulated in these 3D cocultures. Application of neutralizing reagents specific for TGFβ blocks cord formation and inhibits expression of SMA and NG2 in the 10T1/2 cells. We conclude that TGFβ mediates 10T1/2 differentiation to SMC/pericytes in the 3D cocultures and that association with differentiated mural cells is required for formation of capillary-like structures in Matrigel™.

angiogenesis EC Matrigel™ 10T1/2 TGFβ 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Adamis AP, Aiello LP, D'Amato RA. Angiogenesis and ophthalmic disease. Angiogenesis 1999; 3: 9-14.PubMedCrossRefGoogle Scholar
  2. 2.
    Zetter BR. Angiogenesis and tumor metastasis. Annu Rev Med 1998; 49: 407-24.PubMedCrossRefGoogle Scholar
  3. 3.
    Isner JM (ed) Angiogenesis. Philadelphia: Lippincott-Raven 1998.Google Scholar
  4. 4.
    Risau W. Differentiation of endothelium. FASEB J 1995; 9: 926-33.PubMedGoogle Scholar
  5. 5.
    Lindahl P, Johansson BR, Levéen P, Betsholtz C. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science 1997; 277: 242-5.PubMedCrossRefGoogle Scholar
  6. 6.
    Hirschi K, Rohovsky SA, D'Amore PA. PDGF, TGF-β and heterotypic cell-cell interactions mediate the recruitment and differentiation of 10T1/2 cells to a smooth muscle cell fate. J Cell Biol 1998; 141: 805-14.PubMedCrossRefGoogle Scholar
  7. 7.
    Hellström M, Kalén M, Lindahl P et al. Role of PDGF-B and PDGFR-β in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. Development 1999; 126: 3047-55.PubMedGoogle Scholar
  8. 8.
    Ferrara N, Henzel WJ. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun 1989; 161: 851-5.PubMedCrossRefGoogle Scholar
  9. 9.
    Gospodarowicz D, Abraham JA, Schilling J. Isolation and characterization of a vascular endothelial cell mitogen produced by pituitary-derived folliculostellate cells. Proc Natl Acad Sci USA 1989; 86: 7311-5.PubMedCrossRefGoogle Scholar
  10. 10.
    Senger D, Ledbetter S, Claffey K et al. Stimulation of endothelial cell migration by vascular permeability factor/vascular endothelial growth factor through cooperative mechanisms involving the α v β 3 integrin, osteopontin, and thrombin. Am J Pathol 1996; 149: 293-305.PubMedGoogle Scholar
  11. 11.
    Shalaby F, Rossant J, Yamaguchi TP et al. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 1995; 376: 62-6.PubMedCrossRefGoogle Scholar
  12. 12.
    Fong G-H, Zhang L, Bryce D-M, Peng J. Increased hemangioblast commitment, not vascular disorganization, is the primary defect in flt-1 knock-out mice. Development 1999; 126: 3015-25.PubMedGoogle Scholar
  13. 13.
    Drake CJ, Little CD. Exogenous vascular endothelial growth factor induces malformed and hyperfused vessels during embryonic neovascularization. Proc Natl Acad Sci USA 1995; 92: 7657-61.PubMedCrossRefGoogle Scholar
  14. 14.
    Soriano P. Abnormal kidney development and hematological disorders in PDGF β-receptor mutant mice. Genes Dev 1994; 8: 1888-96.PubMedGoogle Scholar
  15. 15.
    Gale NW, Yancopoulos GD. Growth factors acting via endothelial cell-specific receptor tyrosine kinases: VEGFs, angiopoietins, and ephrins in vascular development. Genes Dev 1999; 13: 1055-66.PubMedGoogle Scholar
  16. 16.
    Suri C, Jones PF, Patan S et al. Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 1996; 87: 1171-80.PubMedCrossRefGoogle Scholar
  17. 17.
    Dumont DJ, Gradwohl G, Fong G-H et al. Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo. Genes Dev 1994; 8: 1897-907.PubMedGoogle Scholar
  18. 18.
    Maisonpierre PC, Suri C, Jones PF et al. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 1997; 277: 55-60.PubMedCrossRefGoogle Scholar
  19. 19.
    Dickson MC, Martin JS, Cousins FM et al. Defective haematopoiesis and vasculogenesis in transforming growth factor-β1 knock-out mice. Development 1995; 121: 1845-54.PubMedGoogle Scholar
  20. 20.
    Oshima M, Oshima H, Taketo MM. TGF-β receptor type II deficiency results in defects of yolk sac hematopoiesis and vasculogenesis. Dev Biol 1996; 179: 297-302.PubMedCrossRefGoogle Scholar
  21. 21.
    Barbara NP, Wrana JL, Letarte M. Endoglin is an accessory protein that interacts with the signaling receptor complex of multiple members of the Transforming Growth Factor-β superfamily. J Biol Chem 1999; 274: 584-94.PubMedCrossRefGoogle Scholar
  22. 22.
    Li DY, Sorensen LK, Brooke BS et al. Defective angiogenesis in mice lacking endoglin. Science 1999; 284: 1534-7.PubMedCrossRefGoogle Scholar
  23. 23.
    McAllister KA, Grogg KM, Johnson DW et al. Endoglin, a TGF-beta binding protein of endothelial cells, is the gene for hereditary haemorrhagic telangiectasia type 1. Nat Gen 1994; 8: 345-51.CrossRefGoogle Scholar
  24. 24.
    Shah NM, Groves AK, Anderson DJ. Alternative neural crest cell fates are instructively promoted by TGFβ superfamily members. Cell 1996; 85: 331-43.PubMedCrossRefGoogle Scholar
  25. 25.
    Jain M, Layne M, Watanabe M et al. In vitro system for differentiating pluripotent neural crest cells into smooth muscle cells. J Biol Chem 1998; 273: 5993-6.PubMedCrossRefGoogle Scholar
  26. 26.
    Grainger DJ, Metcalfe JC, Grace AA, Mosedale DE. Transforming growth factor-β dynamically regulates vascular smooth muscle differentiation in vivo. J Cell Sci 1998; 111: 2977-88.PubMedGoogle Scholar
  27. 27.
    Antonelli-Orlidge A, Saunders KB, Smith SR, D'Amore PA. An activated form of transforming growth factor β is produced by cocultures of endothelial cells and pericytes. Proc Natl Acad Sci USA 1989; 86: 4544-8.PubMedCrossRefGoogle Scholar
  28. 28.
    Wakui S, Furusato M, Muto T et al. Transforming growth factor-β and urokinase plasminogen activator presents at endothelial cell-pericyte interdigitation in human granulation tissue. Microvas Res 1997; 54: 262-9.CrossRefGoogle Scholar
  29. 29.
    Skalli O, Pelte M-F, Feclet M-C et al. Alpha-smooth muscle cell actin, a differentiation market of smooth muscle cells, is present in microfilamentous bundle of pericytes. J Histochem Cytochem 1989; 37: 315-21.PubMedGoogle Scholar
  30. 30.
    Schlingemann RO, Rietveld FJR, Kwaspen F et al. Differential expression of markers for endothelial cells, pericytes and basal lamina in the microvasculature of tumors and granulation tissue. Am J Pathol 1991; 138: 1335-47.PubMedGoogle Scholar
  31. 31.
    Grako KA, Stallcup WB. Participation of the NG2 proteoglycan in rat aortic smooth muscle cell responses to platelet-derived growth factor. Exp Cell Res 1995; 221: 231-40.PubMedCrossRefGoogle Scholar
  32. 32.
    Grako KA, Ochiya T, Barritt D et al. PDGF α-receptor is unresponsive to PDGF-AA in aortic smooth muscle cells from NG2 knockout mouse. J Cell Sci 1999; 112: 905-15.PubMedGoogle Scholar
  33. 33.
    Sims DE. The pericyte — a review. Tissue Cell 1986; 18: 153-74.PubMedCrossRefGoogle Scholar
  34. 34.
    Fujiwara T, Uehara Y. The cytoarchitecture of the wall and the innervation pattern of the microvessels in the rat mammary gland: A scanning electron microscopic observation. Am J Anat 1984; 170: 39-54.PubMedCrossRefGoogle Scholar
  35. 35.
    Vukicevic S, Kleinman HK, Luyten FP et al. Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components. Exp Cell Res 1992; 202: 1-8.PubMedCrossRefGoogle Scholar
  36. 36.
    Papapetropoulos A, Garcia-Cardena G, Dengler TJ et al. Direct actions of angiopoietin-1 on human endothelium: Evidence for network stabilization, cell survival, and interaction with other angiogenic growth factors. Lab Invest 1999; 79: 213-23.PubMedGoogle Scholar
  37. 37.
    Vernon RB, Sage EH. A novel, quantitative model for study of endothelial cell migration and sprout formation within three-dimensional collagen matrices. Microvas Res 1999; 57: 118-33.CrossRefGoogle Scholar
  38. 38.
    Madri JA, Pratt BM, Tucker AM. Phenotypic modulation of endothelial cells by transforming growth factor-beta depends upon the composition and organization of the extracellular matrix. J Cell Biol 1988; 106: 1375-84.PubMedCrossRefGoogle Scholar
  39. 39.
    Davis G, Camarillo CW. Regulation of endothelial cell morphogenesis by integrins, mechanical forces, and matrix guidance pathways. Exp Cell Res 1995; 216: 113-23.PubMedCrossRefGoogle Scholar
  40. 40.
    Nehls V, Schuchardt E, Drenckhahn D. The effect of fibroblasts, vascular smooth muscle cells, and pericytes on sprout formation of endothelial cells in a fibrin gel angiogenesis system. Microvas Res 1994; 48: 349-63.CrossRefGoogle Scholar
  41. 41.
    Nicosia RF, Ottinetti A. Modulation of microvascular growth and morphogenesis by reconstituted basement membrane gel in three-dimension cultures of rat aorta: A comparative study of angiogenesis in matrigel, collagen, fibrin, and plasma clot. In Vitro Cell Dev Biol 1990; 26: 119-28.PubMedGoogle Scholar
  42. 42.
    Iruela-Arispe ML, Sage EH. Endothelial cells exhibiting angiogenesis in vitro proliferate in response to TGF-β1. J Cell Biochem 1993; 52: 414-30.PubMedCrossRefGoogle Scholar
  43. 43.
    Merwin JR, Anderson JM, Kocher O et al. Transforming growth factor beta1 modulates extracellular matrix organization and cell-cell junctional complex formation during in vivo angiogenesis. J Cell Physiol 1990; 142: 117-28.PubMedCrossRefGoogle Scholar
  44. 44.
    Burg MA, Pasqualini R, Arap W et al. NG2 Proteoglycan-binding peptides target tumor neovasculature. Cancer Res 1999; 59: 2869-74.PubMedGoogle Scholar
  45. 45.
    Schlingemann RO, Rietveld FJR, de Waal RMW et al. Expression of the high molecular weight melanoma-associated antigen by pericytes during angiogenesis in tumors and in healing wounds. Am J Pathol 1990; 136: 1393-405.PubMedGoogle Scholar
  46. 46.
    Schlingemann RO, Oosterwijk E, Wesseling P et al. Aminopeptidase A is a constituent of activated pericytes in angiogenesis. J Pathol 1996; 179: 436-42.PubMedCrossRefGoogle Scholar
  47. 47.
    Goretzki L, Lombardo CR, Stallcup WB. Binding of the NG2 proteoglycan to kringle domains modulates the functional properties of angiostatin and plasmin(ogen). J Biol Chem 2000; 275: 28625-33.PubMedCrossRefGoogle Scholar
  48. 48.
    Benjamin LE, Hemo I, Keshet E. A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF. Development 1998; 125: 1591-8.PubMedGoogle Scholar
  49. 49.
    Roberts AB, Sporn MB, Assoian RK et al. Transforming growth factor type-beta: Rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci USA 1986; 83: 4167-71.PubMedCrossRefGoogle Scholar
  50. 50.
    Roberts A. Transforming growth factor-β: Activity and efficacy in animal models of wound healing. Wound Rep Reg 1995; 3: 408-18.CrossRefGoogle Scholar
  51. 51.
    Oh SP, Seki T, Goss KA et al. Activin receptor-like kinase 1 modulates transforming growth factor-β1 signaling in the regulation of angiogenesis. Proc Natl Acad Sci USA 2000; 97: 2626-31.PubMedCrossRefGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2001

Authors and Affiliations

  • D.C. Darland
    • 1
  • P.A. D'Amore
    • 1
  1. 1.The Schepens Eye Research Institute and the Department of OphthalmologyHarvard Medical SchoolBostonUSA

Personalised recommendations