Activated Forms of VEGF-C and VEGF-D Provide
Improved Vascular Function in Skeletal Muscle
Andrey Anisimov, Annamari Alitalo, Petra Korpisalo, Jarkko Soronen, Seppo Kaijalainen,
Veli-Matti Leppa¨nen, Michael Jeltsch, Seppo Yla¨-Herttuala, Kari Alitalo
Abstract—The therapeutic potential of vascular endothelial growth factor (VEGF)-C and VEGF-D in skeletal muscle has
been of considerable interest as these factors have both angiogenic and lymphangiogenic activities. Previous studies
have mainly used adenoviral gene delivery for short-term expression of VEGF-C and VEGF-D in pig, rabbit, and mouse
skeletal muscles. Here we have used the activated mature forms of VEGF-C and VEGF-D expressed via recombinant
adeno-associated virus (rAAV), which provides stable, long-lasting transgene expression in various tissues including
skeletal muscle. Mouse tibialis anterior muscle was transduced with rAAV encoding human or mouse VEGF-C or
VEGF-D. Two weeks later, immunohistochemical analysis showed increased numbers of both blood and lymph vessels,
and Doppler ultrasound analysis indicated increased blood vessel perfusion. The lymphatic vessels further increased at
the 4-week time point were functional, as shown by FITC-lectin uptake and transport. Furthermore, receptor activation
and arteriogenic activity were increased by an alanine substitution mutant of human VEGF-C (C137A) having an
increased dimer stability and by a chimeric CAC growth factor that contained the VEGF receptor-binding domain
flanked by VEGF-C propeptides, but only the latter promoted significantly more blood vessel perfusion when compared
to the other growth factors studied. We conclude that long-term expression of VEGF-C and VEGF-D in skeletal muscle
results in the generation of new functional blood and lymphatic vessels. The therapeutic value of intramuscular lymph
vessels in draining tissue edema and lymphedema can now be evaluated using this model system. (Circ Res. 2009;104:
1302-1312.)
Key Words: VEGF-C


VEGF-D


adeno-associated virus


angiogenesis


lymphangiogenesis


skeletal muscle
Blood and lymphatic vessels integrate to form the circu-latory system, which provides oxygen and nutrients to
the tissues and removes carbon dioxide and waste metabo-
lites. Endothelial cells (ECs) form a surface barrier between
the intra- and extravascular spaces, and EC number deter-
mines the functional capacity of the vasculature in each
tissue. Vascular endothelial growth factors (VEGFs) are
considered to be the main mitogenic and survival factors for
ECs, obligatory for embryonic angio- and lymphangiogenesis
and capable of activating ECs in the adult.1–3 In mammals, the 5
known VEGFs are VEGF-A, -B, -C, -D, and placenta growth
factor. Of these, VEGF-C and VEGF-D activate primarily
lymphatic ECs, which express VEGF receptor (VEGFR)-3.4,5
After biosynthesis, full-length (fl) VEGF-C and VEGF-D un-
dergo proteolytic cleavage of their C- and N-terminal propeptide
domains.6,7 This releases the mature growth factor (
N
C, here
short form [sf]), with an increased affinity toward VEGFR-3 and
toward VEGFR-2, which is expressed mainly in blood vessels.
VEGFR-2–binding has been assumed to be responsible for the
angiogenic properties of VEGF-C and VEGF-D in a number of
experimental conditions.8–10 However, no systematic studies
have addressed the capacity of the proteolytically activated,
mature VEGF-C/-D forms to stimulate angiogenesis versus
lymphangiogenesis in vivo.
The ability to stimulate both VEGFR-2 and VEGFR-3 in
blood and lymphatic vessels has made VEGF-C and VEGF-D
attractive candidates for therapeutic improvement in many
types of vascular insufficiencies, including skeletal and car-
diac muscle ischemia, lymphedema, and lymphatic vessel
hypoplasia.11 Adenoviral gene transfer was effective in de-
livering vascular growth factors to pig myocardium,12 to
rabbit and mouse skeletal muscle,9,13 and to the periadventi-
tial tissue of rabbit carotid arteries.14 Transgene expression
was found to be robust but transient, lasting only 1 to 3
weeks, yet it resulted in significant improvement of tissue
perfusion and capillary size. However, declining levels of
transgene expression lead to significantly decreased perfusion
and to pruning of newly formed blood vessels.9,12 In contrast
to adenovirus, recombinant adeno-associated virus (rAAV) is
an effective vehicle for delivering transgenes into many types
Original received December 30, 2008; resubmission received March 23, 2009; revised resubmission received April 23, 2009; accepted April 30, 2009.
From the Molecular/Cancer Biology Laboratory (A. Anisimov, J.S., S.K., V.-M.L., M.J., K.A.), Biomedicum Helsinki, Department of Pathology,
Haartman Institute and Helsinki University Central Hospital, University of Helsinki, Finland; and Department of Biotechnology and Molecular Medicine
(A. Alitalo, P.K., S.Y.-H.), A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, Finland.
Correspondence to Kari Alitalo, MD, PhD, Molecular/Cancer Biology Laboratory, Biomedicum Helsinki, PO Box 63, (Haartmaninkatu 8), University
of Helsinki, FI-00014, Helsinki, Finland. E-mail Kari.Alitalo@helsinki.fi
© 2009 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.109.197830
1302
at USACO REF: NX68155 on August 8, 2011http://circres.ahajournals.org/Downloaded from