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

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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.

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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

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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.

Author information

Authors and Affiliations

  1. Cell and Gene Therapy, Department of Biomedicine and Department of Surgery, Basel University Hospital, ICFS 407, Hebelstrasse 20, 4031, Basel, Switzerland

    Roberto Gianni-Barrera, Marianna Trani, Michael Heberer & Andrea Banfi

  2. Institute of Anatomy, University of Bern, Baltzerstrasse 2, 3000, Bern, Switzerland

    Christian Fontanellaz, Valentin Djonov & Ruslan Hlushchuk

Authors
  1. Roberto Gianni-Barrera
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  2. Marianna Trani
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  3. Christian Fontanellaz
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  4. Michael Heberer
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  5. Valentin Djonov
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  6. Ruslan Hlushchuk
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  7. Andrea Banfi
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Corresponding authors

Correspondence to Ruslan Hlushchuk or Andrea Banfi.

Electronic supplementary material

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10456_2012_9304_MOESM1_ESM.tif

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

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

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)

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Gianni-Barrera, R., Trani, M., Fontanellaz, C. et al. VEGF over-expression in skeletal muscle induces angiogenesis by intussusception rather than sprouting. Angiogenesis 16, 123–136 (2013). https://doi.org/10.1007/s10456-012-9304-y

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