Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Biomechanical regulation of blood vessel growth during tissue vascularization

Nature Medicine volume 15, pages 657–664 (2009)Cite this article

Abstract

Formation of new vessels in granulation tissue during wound healing has been assumed to occur solely through sprouting angiogenesis. In contrast, we show here that neovascularization can be accomplished by nonangiogenic expansion of preexisting vessels. Using neovascularization models based on the chick chorioallantoic membrane and the healing mouse cornea, we found that tissue tension generated by activated fibroblasts or myofibroblasts during wound contraction mediated and directed translocation of the vasculature. These mechanical forces pulled vessels from the preexisting vascular bed as vascular loops with functional circulation that expanded as an integral part of the growing granulation tissue through vessel enlargement and elongation. Blockade of vascular endothelial growth factor receptor-2 confirmed that biomechanical forces were sufficient to mediate the initial vascular growth independently of endothelial sprouting or proliferation. The neovascular network was further remodeled by splitting, sprouting and regression of individual vessels. This model explains the rapid appearance of large functional vessels in granulation tissue during wound healing.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Vascularization of a fibrin and collagen matrix implanted on the CAM through elongation of the preexisting capillary network.
Figure 2: Neovascularization is preceded by ingrowth of proto- and myofibroblasts.
Figure 3: Matrix contraction is a prerequisite for vascular ingrowth on the CAM.
Figure 4: Time-lapse recordings of vessel remodeling during implant vascularization on the CAM.
Figure 5: Neovascularization of the wounded mouse cornea by recruitment of functional microvascular loops from the limbal capillary network.
Figure 6: Characterization of neovascular growth during wound healing of the injured mouse cornea using whole mount staining.

Similar content being viewed by others

References

  1. Dvorak, H.F. Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. N. Engl. J. Med. 315, 1650–1659 (1986).

    Article  CAS  PubMed  Google Scholar 

  2. Kalluri, R. & Zeisberg, M. Fibroblasts in cancer. Nat. Rev. Cancer 6, 392–401 (2006).

    Article  CAS  PubMed  Google Scholar 

  3. Singer, A.J. & Clark, R.A. Cutaneous wound healing. N. Engl. J. Med. 341, 738–746 (1999).

    Article  CAS  PubMed  Google Scholar 

  4. Gabbiani, G., Ryan, G.B. & Majne, G. Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction. Experientia 27, 549–550 (1971).

    Article  CAS  PubMed  Google Scholar 

  5. Tomasek, J.J., Gabbiani, G., Hinz, B., Chaponnier, C. & Brown, R.A. Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat. Rev. Mol. Cell Biol. 3, 349–363 (2002).

    Article  CAS  PubMed  Google Scholar 

  6. McClain, S.A. et al. Mesenchymal cell activation is the rate-limiting step of granulation tissue induction. Am. J. Pathol. 149, 1257–1270 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Serini, G. et al. The fibronectin domain ED-A is crucial for myofibroblastic phenotype induction by transforming growth factor-beta1. J. Cell Biol. 142, 873–881 (1998).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Gabbiani, G., Hirschel, B.J., Ryan, G.B., Statkov, P.R. & Majno, G. Granulation tissue as a contractile organ. A study of structure and function. J. Exp. Med. 135, 719–734 (1972).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Dudar, T.E. & Jain, R.K. Microcirculatory flow changes during tissue growth. Microvasc. Res. 25, 1–21 (1983).

    Article  CAS  PubMed  Google Scholar 

  10. Fukumura, D. et al. Paracrine regulation of angiogenesis and adipocyte differentiation during in vivo adipogenesis. Circ. Res. 93, e88–e97 (2003).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Gschwandtner, M.E. et al. Microcirculation is similar in ischemic and venous ulcers. Microvasc. Res. 62, 226–235 (2001).

    Article  CAS  PubMed  Google Scholar 

  12. Rendell, M.S. et al. The microvascular composition of the healing wound compared at skin sites with nutritive versus arteriovenous perfusion. J. Surg. Res. 80, 373–379 (1998).

    Article  CAS  PubMed  Google Scholar 

  13. Virag, J.I. & Murry, C.E. Myofibroblast and endothelial cell proliferation during murine myocardial infarct repair. Am. J. Pathol. 163, 2433–2440 (2003).

    Article  PubMed Central  PubMed  Google Scholar 

  14. Thurston, G., Baluk, P. & McDonald, D.M. Determinants of endothelial cell phenotype in venules. Microcirculation 7, 67–80 (2000).

    Article  CAS  PubMed  Google Scholar 

  15. Lorena, D., Uchio, K., Costa, A.M. & Desmouliere, A. Normal scarring: importance of myofibroblasts. Wound Repair Regen. 10, 86–92 (2002).

    Article  PubMed  Google Scholar 

  16. Tonnesen, M.G., Feng, X. & Clark, R.A. Angiogenesis in wound healing. J. Investig. Dermatol. Symp. Proc. 5, 40–46 (2000).

    Article  CAS  PubMed  Google Scholar 

  17. Ausprunk, D.H. & Folkman, J. Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc. Res. 14, 53–65 (1977).

    Article  CAS  PubMed  Google Scholar 

  18. Clark, E.R. & Clark, E.L. Microscopic observations of the growth of blood capillaries in the living mammal. Am. J. Anat. 64, 251–299 (1939).

    Article  Google Scholar 

  19. Burri, P.H. & Djonov, V. Intussusceptive angiogenesis—the alternative to capillary sprouting. Mol. Aspects Med. 23, S1–S27 (2002).

    Article  PubMed  Google Scholar 

  20. Dvorak, H.F. Rous-Whipple Award Lecture. How tumors make bad blood vessels and stroma. Am. J. Pathol. 162, 1747–1757 (2003).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Patan, S. et al. Vascular morphogenesis and remodeling in a model of tissue repair: blood vessel formation and growth in the ovarian pedicle after ovariectomy. Circ. Res. 89, 723–731 (2001).

    Article  CAS  PubMed  Google Scholar 

  22. Pettersson, A. et al. Heterogeneity of the angiogenic response induced in different normal adult tissues by vascular permeability factor/vascular endothelial growth factor. Lab. Invest. 80, 99–115 (2000).

    Article  CAS  PubMed  Google Scholar 

  23. Rogers, P.A. & Gargett, C.E. Endometrial angiogenesis. Angiogenesis 2, 287–294 (1998).

    Article  CAS  PubMed  Google Scholar 

  24. Kilarski, W.W., Jura, N. & Gerwins, P. Inactivation of Src family kinases inhibits angiogenesis in vivo: implications for a mechanism involving organization of the actin cytoskeleton. Exp. Cell Res. 291, 70–82 (2003).

    Article  CAS  PubMed  Google Scholar 

  25. Kilarski, W.W., Jura, N. & Gerwins, P. An ex vivo model for functional studies of myofibroblasts. Lab. Invest. 85, 643–654 (2005).

    Article  CAS  PubMed  Google Scholar 

  26. Lusimbo, W.S., Leighton, F.A. & Wobeser, G.A. Histology and ultrastructure of the chorioallantoic membrane of the mallard duck (Anas platyrhynchos). Anat. Rec. 259, 25–34 (2000).

    Article  CAS  PubMed  Google Scholar 

  27. Westerhausen, A., Kishi, J. & Prockop, D.J. Mutations that substitute serine for glycine alpha 1–598 and glycine alpha 1–631 in type I procollagen. The effects on thermal unfolding of the triple helix are position-specific and demonstrate that the protein unfolds through a series of cooperative blocks. J. Biol. Chem. 265, 13995–14000 (1990).

    CAS  PubMed  Google Scholar 

  28. Woodley, D.T., Yamauchi, M., Wynn, K.C., Mechanic, G. & Briggaman, R.A. Collagen telopeptides (cross-linking sites) play a role in collagen gel lattice contraction. J. Invest. Dermatol. 97, 580–585 (1991).

    Article  CAS  PubMed  Google Scholar 

  29. Eastwood, M., Mudera, V.C., McGrouther, D.A. & Brown, R.A. Effect of precise mechanical loading on fibroblast populated collagen lattices: morphological changes. Cell Motil. Cytoskeleton 40, 13–21 (1998).

    Article  CAS  PubMed  Google Scholar 

  30. Lee, C.R., Grodzinsky, A.J. & Spector, M. The effects of cross-linking of collagen-glycosaminoglycan scaffolds on compressive stiffness, chondrocyte-mediated contraction, proliferation and biosynthesis. Biomaterials 22, 3145–3154 (2001).

    Article  CAS  PubMed  Google Scholar 

  31. Korff, T. & Augustin, H.G. Tensional forces in fibrillar extracellular matrices control directional capillary sprouting. J. Cell Sci. 112, 3249–3258 (1999).

    CAS  PubMed  Google Scholar 

  32. Samolov, B. et al. Delayed inflammation-associated corneal neovascularization in MMP-2-deficient mice. Exp. Eye Res. 80, 159–166 (2005).

    Article  CAS  PubMed  Google Scholar 

  33. Morrison, J.C., Fraunfelder, F.W., Milne, S.T. & Moore, C.G. Limbal microvasculature of the rat eye. Invest. Ophthalmol. Vis. Sci. 36, 751–756 (1995).

    CAS  PubMed  Google Scholar 

  34. Barnhill, R. & Ryan, T. Physical versus chemical factors in angiogenesis. Microvasc. Res. 23, 129–130 (1982).

    Article  Google Scholar 

  35. Ingber, D.E. Mechanical signaling and the cellular response to extracellular matrix in angiogenesis and cardiovascular physiology. Circ. Res. 91, 877–887 (2002).

    Article  CAS  PubMed  Google Scholar 

  36. Mammoto, A. et al. A mechanosensitive transcriptional mechanism that controls angiogenesis. Nature 457, 1103–1108 (2009).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  37. Clark, E.R. Studies on the growth of blood-vessels in the tail of the frog larva––by observation and experiment on the living animal. Am. J. Anat. 23, 37–88 (1918).

    Article  Google Scholar 

  38. Risau, W. Mechanisms of angiogenesis. Nature 386, 671–674 (1997).

    Article  CAS  PubMed  Google Scholar 

  39. Dor, Y., Djonov, V. & Keshet, E. Making vascular networks in the adult: branching morphogenesis without a roadmap. Trends Cell Biol. 13, 131–136 (2003).

    Article  CAS  PubMed  Google Scholar 

  40. Djonov, V., Baum, O. & Burri, P.H. Vascular remodeling by intussusceptive angiogenesis. Cell Tissue Res. 314, 107–117 (2003).

    Article  PubMed  Google Scholar 

  41. Muthukkaruppan, V. & Auerbach, R. Angiogenesis in the mouse cornea. Science 205, 1416–1418 (1979).

    Article  CAS  PubMed  Google Scholar 

  42. Lockhart, A.C. et al. A clinical model of dermal wound angiogenesis. Wound Repair Regen. 11, 306–313 (2003).

    Article  PubMed  Google Scholar 

  43. Kumar, V., Abbas, A. & Nelson, F. Robbins & Cotran Pathologic Basis of Disease—Interactive Case Study. (Saunders, Philadelphia, 2004).

    Google Scholar 

  44. Gabbiani, G. The myofibroblast in wound healing and fibrocontractive diseases. J. Pathol. 200, 500–503 (2003).

    Article  CAS  PubMed  Google Scholar 

  45. Gerhardt, H. et al. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J. Cell Biol. 161, 1163–1177 (2003).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  46. Tammela, T. et al. Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation. Nature 454, 656–660 (2008).

    Article  CAS  PubMed  Google Scholar 

  47. Hlushchuk, R. et al. Tumor recovery by angiogenic switch from sprouting to intussusceptive angiogenesis after treatment with PTK787/ZK222584 or ionizing radiation. Am. J. Pathol. 173, 1173–1185 (2008).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  48. Vosseler, S., Mirancea, N., Bohlen, P., Mueller, M.M. & Fusenig, N.E. Angiogenesis inhibition by vascular endothelial growth factor receptor-2 blockade reduces stromal matrix metalloproteinase expression, normalizes stromal tissue, and reverts epithelial tumor phenotype in surface heterotransplants. Cancer Res. 65, 1294–1305 (2005).

    Article  CAS  PubMed  Google Scholar 

  49. Tong, R.T. et al. Vascular normalization by vascular endothelial growth factor receptor 2 blockade induces a pressure gradient across the vasculature and improves drug penetration in tumors. Cancer Res. 64, 3731–3736 (2004).

    Article  CAS  PubMed  Google Scholar 

  50. Augustin, H.G. Antiangiogenic tumour therapy: will it work? Trends Pharmacol. Sci. 19, 216–222 (1998).

    Article  CAS  PubMed  Google Scholar 

  51. Junghans, B.M. & Collin, H.B. The limbal vascular response to corneal injury. An autoradiographic study. Cornea 8, 141–149 (1989).

    Article  CAS  PubMed  Google Scholar 

  52. Sholley, M.M., Ferguson, G.P., Seibel, H.R., Montour, J.L. & Wilson, J.D. Mechanisms of neovascularization. Vascular sprouting can occur without proliferation of endothelial cells. Lab. Invest. 51, 624–634 (1984).

    CAS  PubMed  Google Scholar 

  53. Scherer, S.S. et al. The mechanism of action of the vacuum-assisted closure device. Plast. Reconstr. Surg. 122, 786–797 (2008).

    Article  CAS  PubMed  Google Scholar 

  54. Segers, V.F. & Lee, R.T. Stem-cell therapy for cardiac disease. Nature 451, 937–942 (2008).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank M. Aronsson for help with cornea experiments, J. Kilarska for help with image processing and analysis and K. Kullander and N. Rabe for help with microscopy of whole-mount corneas. We also thank M. Swartz for allowing us to use her laboratory to perform gel contraction and image analysis and A. Bikfalvi for comments. This study was supported by grants to P.G. from the Göran Gustafsson Foundation, the Swedish Cancer Foundation (project 4422-B04-O5XAB), the Swedish Research Council (project K2005-31X-15348-01A), the Children's Cancer Foundation of Sweden (project 04/037), Lions Cancer Research Foundation in Uppsala, the Magnus Bergvall Foundation, King Gustaf V's 80-Year Foundation and Uppsala Foundation for Medical Research, by grants to A.K. from the Crown Princess Margareta Foundation (KMA), the Edwin Jordan Foundation, Karolinska Institute research grants, Stiftelsen Synfrämjandets Forskningsfond, and Swedish Research Council (project 2006-13828-37891-37) and by grants to B.S. from St. Erik Eye Hospital Research Foundation, Sigvard and Marianne Bernadotte Research Foundation for Children's Eye Care and Stiftelsen Synfrämjandets Forskningsfond.

Author information

Author notes

  1. Present address: Laboratory of Mechanobiology and Morphogenesis, Swiss Federal Institute of Technology, Lausanne, Switzerland.

  2. Branka Samolov and Ludvig Petersson: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Medical Biochemistry and Microbiology, Biomedical Center, University of Uppsala, Uppsala, Sweden

    Witold W Kilarski, Ludvig Petersson & Pär Gerwins

  2. Department of Clinical Neuroscience, Section of Ophthalmology and Vision, Karolinska Institutet, Stockholm, Sweden

    Branka Samolov & Anders Kvanta

  3. Department of Oncology, Radiology, and Clinical Immunology, Section for Radiology, Uppsala University Hospital, Uppsala, Sweden

    Pär Gerwins

Authors
  1. Witold W Kilarski
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Branka Samolov
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ludvig Petersson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anders Kvanta
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Pär Gerwins
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

W.W.K. was involved in the overall design of the study; performed most of the experiments involving the CAM, immunohistochemistry, in vitro gel contraction, whole-mount staining of cornea samples and statistical analysis; analyzed data; and wrote most of the manuscript. B.S. designed and performed the cornea experiments and statistical analysis, and she wrote portions of the manuscript and edited the manuscript. L.P. performed CAM experiments with PVA sponges, in vitro cell analysis, peptide inhibitor studies and cornea stainings. A.K. designed parts of cornea experiments and edited the manuscript. P.G. designed the overall study, analyzed data, and wrote and edited the manuscript.

Corresponding authors

Correspondence to Witold W Kilarski or Pär Gerwins.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11 and Supplementary Methods (PDF 3929 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kilarski, W., Samolov, B., Petersson, L. et al. Biomechanical regulation of blood vessel growth during tissue vascularization. Nat Med 15, 657–664 (2009). https://doi.org/10.1038/nm.1985

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.1985

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing