Distinct Architecture of Lymphatic Vessels Induced by
Chimeric Vascular Endothelial Growth Factor-C/Vascular
Endothelial Growth Factor Heparin-Binding Domain
Fusion Proteins
Tuomas Tammela,* Yulong He,* Johannes Lyytikka¨, Michael Jeltsch, Johanna Markkanen,
Katri Pajusola, Seppo Yla¨-Herttuala, Kari Alitalo
Abstract—Vascular endothelial growth factor (VEGF)-C and VEGF-D are composed of the receptor-binding VEGF
homology domain and a carboxy-terminal silk homology domain that requires proteolytic cleavage for growth factor
activation. Here, we explored whether the C-terminal heparin-binding domain of the VEGF165 or VEGF189 isoform also
containing neuropilin-binding sequences could substitute for the silk homology domain of VEGF-C. Such VEGF-C/
VEGF–heparin-binding domain chimeras were produced and shown to activate VEGF-C receptors, and, when
expressed in tissues via adenovirus or adeno-associated virus vectors, stimulated lymphangiogenesis in vivo. However,
both chimeras induced a distinctly different pattern of lymphatic vessels when compared with VEGF-C. Whereas
VEGF-C–induced vessels were initially a dense network of small diameter vessels, the lymphatic vessels induced by the
chimeric growth factors tended to form directly along tissue borders, along basement membranes that are rich in heparan
sulfate. For example, in skeletal muscle, the chimeras induced formation of lumenized lymphatic vessels more
efficiently than wild-type VEGF-C. We conclude that the matrix-binding domain of VEGF can target VEGF-C activity
to heparin-rich basement membrane structures. These properties may prove useful for tissue engineering and attempts
to regenerate lymphatic vessels in lymphedema patients. (Circ Res. 2007;100:1468-1475.)
Key Words: VEGF-C


VEGF-A


heparin-binding


lymphangiogenesis
The 5 mammalian vascular endothelial growth factor(VEGF) family members identified to date, VEGF,
VEGF-B, VEGF-C, VEGF-D, and placenta growth factor, are
key effectors of physiological and pathological regulation of
vasculogenesis, hematopoiesis, angiogenesis, lymphangio-
genesis, and vascular permeability.1–3 VEGF is a key growth
factor for blood vessel formation and plays an essential role
in this process via VEGF receptor (VEGFR)-1 and VEGFR-
2.1 VEGF-C and VEGF-D activate primarily VEGFR-34–8
and induce lymphangiogenesis in transgenic mice and in
other in vivo models.8–11
VEGF is expressed as multiple forms, including the major
forms VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206,
which result from alternative RNA splicing.1 An important
biological property that distinguishes these VEGF isoforms
from each other is their different binding affinity to heparin
and other heparan sulfates. Except for VEGF121, all of the
other forms described above contain a heparin-binding do-
main (HBD) encoded by exon 6 and/or exon 7. The 24-aa
residues encoded by exon 6 contain the HBD and also
elements that enable its binding to the extracellular matrix.12
VEGF molecules containing the cationic polypeptide se-
quence encoded by exon 7 (44 aa) are also heparin-binding
and remain bound to the cell surface and the extracellular
matrix.13 VEGF exon 7–encoded domain also enables its
binding to neuropilin-1 (NP-1).14 Other members of the
VEGF family that contain a HBD include VEGF-B16715 and
placenta growth factor-2.16,17 There is increasing evidence
pointing to the importance of the HBD for the biological
activity of VEGF.18,19
VEGF-C and VEGF-D have a C-terminal domain homol-
ogous to certain silk proteins, plus an amino terminal propep-
tide. A proteolytic cleavage between the growth factor
domain and the silk domain activates VEGF-C binding to
VEGFR-3, and the N-terminally cleaved mature form
(VEGF-C
N
C) can also activate VEGFR-2 in blood vessel
endothelium, resulting in angiogenic activity.20–24 However,
in transgenic models in which both wild-type and mutant
Original received February 5, 2007; revision received March 30, 2007; accepted April 25, 2007.
From the Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research (T.T., Y.H., J.L., M.J., K.P., K.A.), Biomedicum Helsinki,
the Haartman Institute and Helsinki University Central Hospital, University of Helsinki; and Department of Biotechnology and Molecular Medicine (J.M.,
S.Y.-H.), A.I. Virtanen Institute, University of Kuopio, Finland.
*Both authors contributed equally to this work.
Correspondence to Dr Kari Alitalo, University of Helsinki, Molecular/Cancer Biology Program, Biomedicum Helsinki, POB 23 (Haartmaninkatu 8),
Helsinki 00014, Finland. E-mail Kari.Alitalo@helsinki.fi
© 2007 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/01.RES.0000269043.51272.6d
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