Advertisement

Angiogenesis

, Volume 16, Issue 1, pp 123–136 | Cite as

VEGF over-expression in skeletal muscle induces angiogenesis by intussusception rather than sprouting

  • Roberto Gianni-Barrera
  • Marianna Trani
  • Christian Fontanellaz
  • Michael Heberer
  • Valentin Djonov
  • Ruslan HlushchukEmail author
  • Andrea BanfiEmail author
Original Paper

Abstract

Therapeutic over-expression of vascular endothelial growth factor (VEGF) can be used to treat ischemic conditions. However, VEGF can induce either normal or aberrant angiogenesis depending on its dose in the microenvironment around each producing cell in vivo, which limits its clinical usefulness. The goal herein was to determine the cellular mechanisms by which physiologic and aberrant vessels are induced by over-expression of different VEGF doses in adult skeletal muscle. We took advantage of a well-characterized cell-based platform for controlled gene expression in skeletal muscle. Clonal populations of retrovirally transduced myoblasts were implanted in limb muscles of immunodeficient mice to homogeneously over-express two specific VEGF164 levels, previously shown to induce physiologic and therapeutic or aberrant angiogenesis, respectively. Three independent and complementary methods (confocal microscopy, vascular casting and 3D-reconstruction of serial semi-thin sections) showed that, at both VEGF doses, angiogenesis took place without sprouting, but rather by intussusception, or vascular splitting. VEGF-induced endothelial proliferation without tip-cell formation caused an initial homogeneous enlargement of pre-existing microvessels, followed by the formation of intravascular transluminal pillars, hallmarks of intussusception. This was associated with increased flow and shear stress, which are potent triggers of intussusception. A similar process of enlargement without sprouting, followed by intussusception, was also induced by VEGF over-expression through a clinically relevant adenoviral gene therapy vector, without the use of transduced cells. Our findings indicate that VEGF over-expression, at doses that have been shown to induce functional benefit, induces vascular growth in skeletal muscle by intussusception rather than sprouting.

Keywords

VEGF Angiogenesis Intussusception Skeletal muscle Gene therapy 

Abbreviations

VEGF

Vascular endothelial growth factor

NG2

Nerve/glial antigen-2

SMA

Smooth muscle actin

KLF-2

Krüppel-like factor-2

eNOS

Endothelial nitric oxide synthase

IRES

Internal rybosomal entry site

FACS

Fluorescence activated cell sorter

SCID

Severe combined immunodeficiency

Notes

Acknowledgments

We are grateful to Werner Graber and Regula Beurgy for valuable technical support. This work was supported by the Swiss National Science Foundation grant 310030_127426 to A.B. and 31003A_135740 to V.D.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standards

The experiments described in this work comply with all applicable laws of Switzerland.

Supplementary material

10456_2012_9304_MOESM1_ESM.tif (3.6 mb)
Supplemental Fig. 1. Enlarged vessels displayed no endothelial structures extending beyond the basal lamina. a-f Immunostaining with antibodies against endomucin (endothelial cells, green), laminin (basal lamina, red) and with DAPI (nuclei, blue) was performed on cryosections of TA and GC muscles 4 days after implantation with VLow and VHigh myoblast clones. Enlarged vessels showed no evidence of endothelial cells protruding in an abluminal direction (sprouting) outside the basal lamina at both VEGF doses. n = 2 muscles per group; size bars = 20 μm. (TIFF 3708 kb)
10456_2012_9304_MOESM2_ESM.tif (2 mb)
Supplemental Fig. 2. The angiogenic effect does not extend beyond the boundary of the VEGF source. Vessels induced by implantation of VLow and VHigh myoblast clones were immunostained with antibodies against CD31 (endothelial cells, red), NG2 (pericytes, green), α-SMA (smooth muscle cells, cyan) and with DAPI (nuclei, blue) in cryosections of implanted TA and GC muscles. a-b By 4 days, vascular enlargements (outlined by white dots) were formed only in close vicinity to VEGF-expressing myoblasts, forming a sharply demarcated boundary with the neighboring muscle fibers at the edge of the implantation sites (outlined by dashed lines). Vascular enlargements were associated with normal NG2+ pericytes with VLow and mainly devoid of mural cell with VHigh. a Enlarged vessel displays emerging pillars (arrowheads) built by intraluminal endothelial protrusions. n = 3 muscles per group, per time-point; size bars = 25 μm. (TIFF 2049 kb)
10456_2012_9304_MOESM3_ESM.tif (1.8 mb)
Supplemental Fig. 3. VEGF induced no angiogenic effect within 2 days after myoblast implantation. TA and GC muscles were implanted with control cells (a), VLow (b) and VHigh (c) myoblast clones. Vessels were immunostained with antibodies against CD31 (endothelial cells, red), α-SMA (smooth muscle cells, cyan) and with DAPI (nuclei, blue). By 2 days after myoblast implantation the pre-existing vessels (arrowheads), which ran parallel to muscle fibers with occasional bridging segments, was not yet affected by either the VLow or VHigh populations. n = 2 muscles per group; size bars = 20 μm. (TIFF 1859 kb)

References

  1. 1.
    Yla-Herttuala S, Rissanen TT, Vajanto I, Hartikainen J (2007) Vascular endothelial growth factors: biology and current status of clinical applications in cardiovascular medicine. J Am Coll Cardiol 49(10):1015–1026PubMedCrossRefGoogle Scholar
  2. 2.
    Ozawa CR, Banfi A, Glazer NL, Thurston G, Springer ML, Kraft PE, McDonald DM, Blau HM (2004) Microenvironmental VEGF concentration, not total dose, determines a threshold between normal and aberrant angiogenesis. J Clin Invest 113(4):516–527PubMedGoogle Scholar
  3. 3.
    von Degenfeld G, Banfi A, Springer ML, Wagner RA, Jacobi J, Ozawa CR, Merchant MJ, Cooke JP, Blau HM (2006) Microenvironmental VEGF distribution is critical for stable and functional vessel growth in ischemia. FASEB J 20(14):2657–2659CrossRefGoogle Scholar
  4. 4.
    Banfi A, von Degenfeld G, Blau HM (2005) Critical role of microenvironmental factors in angiogenesis. Curr Atheroscler Rep 7(3):227–234PubMedCrossRefGoogle Scholar
  5. 5.
    Karvinen H, Ylä-Herttuala S (2010) New aspects in vascular gene therapy. Curr Opin Pharmacol 10(2):208–211PubMedCrossRefGoogle Scholar
  6. 6.
    Yla-Herttuala S, Markkanen JE, Rissanen TT (2004) Gene therapy for ischemic cardiovascular diseases: some lessons learned from the first clinical trials. Trends Cardiovasc Med 14(8):295–300PubMedCrossRefGoogle Scholar
  7. 7.
    Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A, Jeltsch M, Mitchell C, Alitalo K, Shima D, Betsholtz C (2003) VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 161(6):1163–1177PubMedCrossRefGoogle Scholar
  8. 8.
    Brown MD, Hudlicka O (2003) Modulation of physiological angiogenesis in skeletal muscle by mechanical forces: involvement of VEGF and metalloproteinases. Angiogenesis 6(1):1–14PubMedCrossRefGoogle Scholar
  9. 9.
    Egginton S (2011) Physiological factors influencing capillary growth. Acta Physiol (Oxf) 202(3):225–239CrossRefGoogle Scholar
  10. 10.
    Al Haj Zen A, Oikawa A, Bazan-Peregrino M, Meloni M, Emanueli C, Madeddu P (2010) Inhibition of delta-like-4-mediated signaling impairs reparative angiogenesis after ischemia. Circ Res 107(2):283–293PubMedCrossRefGoogle Scholar
  11. 11.
    Makanya AN, Hlushchuk R, Djonov VG (2009) Intussusceptive angiogenesis and its role in vascular morphogenesis, patterning, and remodeling. Angiogenesis 12(2):113–123PubMedCrossRefGoogle Scholar
  12. 12.
    Egginton S, Zhou AL, Brown MD, Hudlicka O (2001) Unorthodox angiogenesis in skeletal muscle. Cardiovasc Res 49(3):634–646PubMedCrossRefGoogle Scholar
  13. 13.
    Hudlicka O, Brown MD (2009) Adaptation of skeletal muscle microvasculature to increased or decreased blood flow: role of shear stress, nitric oxide and vascular endothelial growth factor. J Vasc Res 46(5):504–512PubMedCrossRefGoogle Scholar
  14. 14.
    Milkiewicz M, Kelland C, Colgan S, Haas TL (2006) Nitric oxide and p38 MAP kinase mediate shear stress-dependent inhibition of MMP-2 production in microvascular endothelial cells. J Cell Physiol 208(1):229–237PubMedCrossRefGoogle Scholar
  15. 15.
    Djonov V, Baum O, Burri PH (2003) Vascular remodeling by intussusceptive angiogenesis. Cell Tissue Res 314(1):107–117PubMedCrossRefGoogle Scholar
  16. 16.
    Styp-Rekowska B, Hlushchuk R, Pries AR, Djonov V (2011) Intussusceptive angiogenesis: pillars against the blood flow. Acta Physiol (Oxf) 202(3):213–223CrossRefGoogle Scholar
  17. 17.
    Zhou A, Egginton S, Hudlicka O, Brown MD (1998) Internal division of capillaries in rat skeletal muscle in response to chronic vasodilator treatment with alpha1-antagonist prazosin. Cell Tissue Res 293(2):293–303PubMedCrossRefGoogle Scholar
  18. 18.
    Misteli H, Wolff T, Fuglistaler P, Gianni-Barrera R, Gurke L, Heberer M, Banfi A (2010) High-throughput flow cytometry purification of transduced progenitors expressing defined levels of vascular endothelial growth factor induces controlled angiogenesis in vivo. Stem Cells 28(3):611–619PubMedCrossRefGoogle Scholar
  19. 19.
    Springer ML, Blau HM (1997) High efficiency retroviral infection of primary myoblasts. Somat Cell Mol Genet 23:203–209PubMedCrossRefGoogle Scholar
  20. 20.
    Banfi A, Springer ML, Blau HM (2002) Myoblast-mediated gene transfer for therapeutic angiogenesis. Methods Enzymol 346:145–157PubMedCrossRefGoogle Scholar
  21. 21.
    Gueret V, Negrete-Virgen JA, Lyddiatt A, Al-Rubeai M (2002) Rapid titration of adenoviral infectivity by flow cytometry in batch culture of infected HEK293 cells. Cytotechnology 38(1–3):87–97PubMedCrossRefGoogle Scholar
  22. 22.
    Rando TA, Blau HM (1994) Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. J Cell Biol 125(6):1275–1287PubMedCrossRefGoogle Scholar
  23. 23.
    Springer ML, Blau HM (1997) High-efficiency retroviral infection of primary myoblasts. Somat Cell Mol Genet 23(3):203–209PubMedCrossRefGoogle Scholar
  24. 24.
    Hlushchuk R, Ehrbar M, Reichmuth P, Heinimann N, Styp-Rekowska B, Escher R, Baum O, Lienemann P, Makanya A, Keshet E, Djonov V (2011) Decrease in VEGF expression induces intussusceptive vascular pruning. Arterioscler Thromb Vasc Biol 31(12):2836–2844PubMedCrossRefGoogle Scholar
  25. 25.
    Hlushchuk R, Riesterer O, Baum O, Wood J, Gruber G, Pruschy M, Djonov V (2008) Tumor recovery by angiogenic switch from sprouting to intussusceptive angiogenesis after treatment with PTK787/ZK222584 or ionizing radiation. Am J Pathol 173(4):1173–1185PubMedCrossRefGoogle Scholar
  26. 26.
    Makanya AN, Hlushchuk R, Baum O, Velinov N, Ochs M, Djonov V (2007) Microvascular endowment in the developing chicken embryo lung. Am J Physiol Lung Cell Mol Physiol 292(5):L1136–L1146PubMedCrossRefGoogle Scholar
  27. 27.
    Gussoni E, Blau HM, Kunkel LM (1997) The fate of individual myoblasts after transplantation into muscles of DMD patients. Nat Med 3(9):970–977PubMedCrossRefGoogle Scholar
  28. 28.
    Ellis J (2005) Silencing and variegation of gammaretrovirus and lentivirus vectors. Hum Gene Ther 16(11):1241–1246PubMedCrossRefGoogle Scholar
  29. 29.
    Springer ML, Banfi A, Ye J, von Degenfeld G, Kraft PE, Saini SA, Kapasi NK, Blau HM (2007) Localization of vascular response to VEGF is not dependent on heparin binding. FASEB J 21(9):2074–2085PubMedCrossRefGoogle Scholar
  30. 30.
    Djonov V, Burri PH (2004) Corrosion cast analysis of blood vessels. In: Augustin H (ed) Methods in endothelial cell biology. Springer, BerlinGoogle Scholar
  31. 31.
    Pettersson A, Nagy JA, Brown LF, Sundberg C, Morgan E, Jungles S, Carter R, Krieger JE, Manseau EJ, Harvey VS, Eckelhoefer IA, Feng D, Dvorak AM, Mulligan RC, Dvorak HF (2000) Heterogeneity of the angiogenic response induced in different normal adult tissues by vascular permeability factor/vascular endothelial growth factor. Lab Invest 80(1):99–115PubMedCrossRefGoogle Scholar
  32. 32.
    Banfi A, Von Degenfeld G, Gianni-Barrera R, Reginato S, Merchant MJ, McDonald DM, Blau HM (2012) Therapeutic angiogenesis due to balanced single-vector delivery of VEGF and PDGF-BB. FASEB J 26(6):2486–2497Google Scholar
  33. 33.
    Atkins GB, Jain MK (2007) Role of Kruppel-like transcription factors in endothelial biology. Circ Res 100(12):1686–1695PubMedCrossRefGoogle Scholar
  34. 34.
    Dekker RJ, van Thienen JV, Rohlena J, de Jager SC, Elderkamp YW, Seppen J, de Vries CJ, Biessen EA, van Berkel TJ, Pannekoek H, Horrevoets AJ (2005) Endothelial KLF2 links local arterial shear stress levels to the expression of vascular tone-regulating genes. Am J Pathol 167(2):609–618PubMedCrossRefGoogle Scholar
  35. 35.
    Djonov VG, Kurz H, Burri PH (2002) Optimality in the developing vascular system: branching remodeling by means of intussusception as an efficient adaptation mechanism. Dev Dyn 224(4):391–402PubMedCrossRefGoogle Scholar
  36. 36.
    Greenberg JI, Shields DJ, Barillas SG, Acevedo LM, Murphy E, Huang J, Scheppke L, Stockmann C, Johnson RS, Angle N, Cheresh DA (2008) A role for VEGF as a negative regulator of pericyte function and vessel maturation. Nature 456(7223):809–813PubMedCrossRefGoogle Scholar
  37. 37.
    Schiaffino S, Reggiani C (2011) Fiber types in mammalian skeletal muscles. Physiol Rev 91(4):1447–1531PubMedCrossRefGoogle Scholar
  38. 38.
    Ruhrberg C, Gerhardt H, Golding M, Watson R, Ioannidou S, Fujisawa H, Betsholtz C, Shima DT (2002) Spatially restricted patterning cues provided by heparin-binding VEGF-A control blood vessel branching morphogenesis. Genes Dev 16(20):2684–2698PubMedCrossRefGoogle Scholar
  39. 39.
    Milkiewicz M, Brown MD, Egginton S, Hudlicka O (2001) Association between shear stress, angiogenesis, and VEGF in skeletal muscles in vivo. Microcirculation 8(4):229–241PubMedGoogle Scholar
  40. 40.
    Korpisalo P, Hytonen JP, Laitinen JT, Laidinen S, Parviainen H, Karvinen H, Siponen J, Marjomaki V, Vajanto I, Rissanen TT, Yla-Herttuala S (2011) Capillary enlargement, not sprouting angiogenesis, determines beneficial therapeutic effects and side effects of angiogenic gene therapy. Eur Heart J 32(13):1664–1672PubMedCrossRefGoogle Scholar
  41. 41.
    Ji JW, Tsoukias NM, Goldman D, Popel AS (2006) A computational model of oxygen transport in skeletal muscle for sprouting and splitting modes of angiogenesis. J Theor Biol 241(1):94–108PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Roberto Gianni-Barrera
    • 1
  • Marianna Trani
    • 1
  • Christian Fontanellaz
    • 2
  • Michael Heberer
    • 1
  • Valentin Djonov
    • 2
  • Ruslan Hlushchuk
    • 2
    Email author
  • Andrea Banfi
    • 1
    Email author
  1. 1.Cell and Gene Therapy, Department of Biomedicine and Department of SurgeryBasel University HospitalBaselSwitzerland
  2. 2.Institute of AnatomyUniversity of BernBernSwitzerland

Personalised recommendations