VEGF over-expression in skeletal muscle induces angiogenesis by intussusception rather than sprouting
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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.
KeywordsVEGF Angiogenesis Intussusception Skeletal muscle Gene therapy
Vascular endothelial growth factor
Smooth muscle actin
Endothelial nitric oxide synthase
Internal rybosomal entry site
Fluorescence activated cell sorter
Severe combined immunodeficiency
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.
The experiments described in this work comply with all applicable laws of Switzerland.
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