A
model for gene therapy of human hereditary lymphedema
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Angiogenesis blood vessel growth), lymphangiogenesis (lymph system growth) are all intrinsically connected with lymphedema and share many of the same genes. We have several pages on both processes.
May 23, 2008
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A model for gene therapy of human hereditary lymphedema
Published
online before print October 9, 2001, 10.1073/pnas.221449198
PNAS | October 23, 2001
| vol. 98 | no. 22 | 12677-12682
* Molecular/Cancer Biology
Laboratory and Ludwig Institute
for Cancer Research, Haartman Institute and Helsinki University
Hospital,
Biomedicum Helsinki, University of Helsinki, P.O.B.
63 (Haartmaninkatu 8),
00014 Helsinki, Finland; Department
of Human Genetics, University of Pittsburgh, 130 DeSoto
Street, Pittsburgh,
PA 15261;
Institute
of Molecular Biology, University of Zürich, Winterthurerstrasse
190, 8057 Zürich,
Switzerland; § Institut d'Embryologie
Cellulaire et Moléculaire
du Centre National de la Recherche Scientifique et du Collège de France
49bis,
Avenue de la Belle Gabrielle, 94736 Nogent-sur-Marne Cedex,
France; ¶ National
Bio-Nuclear Magnetic Resonance Facility and
Department
of Medicine, A.I. Virtanen Institute, University of Kuopio, P.O.B.
1627, 70211 Kuopio,
Finland; and ** Department of
Pediatrics, University of
Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213
Communicated by Erkki Ruoslahti, The Burnham Institute, La Jolla, CA, August 24, 2001 (received for review July 11, 2001)
Abstract
Primary human lymphedema (Milroy's disease), characterized by a chronic and disfiguring swelling of the extremities, is associated with heterozygous inactivating missense mutations of the gene encoding vascular endothelial growth factor C/D receptor (VEGFR-3). Here, we describe a mouse model and a possible treatment for primary lymphedema. Like the human patients, the lymphedema (Chy) mice have an inactivating Vegfr3 mutation in their germ line, and swelling of the limbs because of hypoplastic cutaneous, but not visceral, lymphatic vessels. Neuropilin (NRP)-2 bound VEGF-C and was expressed in the visceral, but not in the cutaneous, lymphatic endothelia, suggesting that it may participate in the pathogenesis of lymphedema. By using virus-mediated VEGF-C gene therapy, we were able to generate functional lymphatic vessels in the lymphedema mice. Our results suggest that growth factor gene therapy is applicable to human lymphedema and provide a paradigm for other diseases associated with mutant receptors
Introduction
Hereditary or primary lymphedema (Milroy's disease) is a
developmental
disorder, in which defective cutaneous lymphatic vessels fail
to
transport lymphatic fluid, resulting in swelling of the extremities.
Primary lymphedema is inherited as an autosomal dominant trait
with
reduced penetrance, variable expression, and variable age
at onset (1).
Several groups have reported linkage of lymphedema to
chromosome 5q (2-4),
and we have shown that mutant, inactive vascular
endothelial growth
factor receptor-3 (VEGFR-3) tyrosine kinase is
responsible for
lymphedema in several such families (5, 6).
Recently, it has also been suggested that lymphedema in
patients
having ectodermal dysplasia with immunodeficiency may
be caused by
defective VEGFR-3 signaling via the nuclear factor (NF)-B
transcription factor (7).
VEGFR3 is one of the rare genes expressed almost exclusively in the lymphatic endothelial cells in adults (8, 9), although it is also needed for proper generation of the embryonic blood vasculature (10). Overexpression of the VEGFR-3 ligands VEGF-C and VEGF-D in the skin of transgenic mice induced the formation of a hyperplastic lymphatic vessel network (11, 12). Similar results were obtained by using the C156S mutant form of VEGF-C (12, 13), which is specific for VEGFR-3, indicating that lymphatic growth is regulated via this receptor. In addition, expression of ligand-blocking concentrations of soluble VEGFR-3 in transgenic mice inhibited the development of the lymphatic vasculature in several organs (14).
Here, we have analyzed the Chy mutant mice that develop chylous ascites after birth (15, 16). We show that, like the human lymphedema patients, these mice have a heterozygous inactivating Vegfr3 mutation and swelling of the limbs because of a lack of s.c. lymphatic vessels. By using viral gene delivery and transgenic approaches, we have explored the possible therapeutic effect of VEGF-C in the Chy mice. We show that VEGFR-3 ligand overexpression induces the growth of functional cutaneous lymphatic vessels in the Chy mice, suggesting that VEGF-C/D therapy is applicable also to human lymphedema.
Materials and Methods
Mouse Lines. The Chy
phenotype was found among the
offspring of a male C3H mouse treated with 250 mg/kg
ethylnitrosourea, in
the Medical Research Council Mammalian Genetics
Unit Embryo Bank
(Harwell, U.K.). To identify the Vegfr3
intron/exon
boundaries, we aligned the Vegfr3
cDNA sequence (Accession no.
L07296)
with the VEGFR3 genomic
sequence (17), and
amplified exons 16 through 26 of Vegfr3,
which were
then sequenced from the C3H, B6, BALB/c, BbxD2, and
PES strains. We
used the K14-VEGF-C156S and Vegfr3+/
mice in the crosses (10, 12).
In Vitro Studies of the Mutant Receptor. We generated the human VEGFR-3(I1053F) expression vector (Accession nos. X68203 and S66407) by using the GeneEditor in vitro Site-Directed Mutagenesis kit (Promega), and the oligonucleotide 5'-CATAGTGAAGTTCTGCGACTT-3', followed by construct transfection into 293T cells, immunoprecipitation, and Western blotting, as previously described (5).
Analyses of Lymphatic and Blood Vessels. To visualize the lymphatic network in the ear, we followed the staining of the lymphatic vessels by fluorescence microscopy after intradermal injection of FITC-dextran (Sigma). For analysis of the deeper lymphatic vessel function, we injected Evans blue (Sigma, 3 mg/ml in PBS) intradermally into the hind footpads. The skin of the limb was then removed to expose the region of the ischiatic vein. To visualize the blood vessels in whole-mount tissue preparations, we used biotinylated Lycopersicon esculentum lectin, as previously described (12, 18).
Immunohistochemistry (IHC). We fixed tissue biopsies in 4% paraformaldehyde, dehydrated them, and embedded them in paraffin. We stained 5-µm sections with antibodies against VEGFR-3 (19), lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1; ref. 20), podoplanin (21), platelet endothelial cell adhesion molecule-1 (PECAM-1) (PharMingen), or hVEGF-C (22), by using a Tyramide Signal Amplification kit (TSA, NEN). We developed the peroxidase activity with 3-amino-9-ethyl carbazole (Sigma), and counterstained the sections with hematoxylin. We used the biotinylated anti-mouse VEGFR-3 Ab (R&D Systems, Oxon, U.K.) for whole-mount stainings.
Magnetic Resonance Imaging (MRI). For high resolution MRI of the feet, mice (Chy, n = 2; and wild-type (WT), n = 2) were anesthetized and externally fixed to a custom-built animal holder. Animals were kept normothermic by blowing warm air to the magnet bore. MRI data were acquired by using a s.m.i.s. console (Surrey Medical Imaging Systems, Guildford, U.K.) interfaced to a 9.4 T vertical magnet (Oxford Instruments, Oxford, U.K.). A single loop surface coil (diameter 35 mm) was used for signal transmission and detection. A T2-weighted [repetition time (TR) 2000 ms, echotime (TE) 40 ms, eight scans perline] multislice spin-echo sequence was used with a field of view of 25.6 × 12.8 mm2 (matrix size 256 × 64) and slice thickness of 1.3 mm in transverse orientation. Diffusion weighted MRI was acquired by using monopolar diffusion gradients (b values 330 and 700 s/mm2) along slice axis in the spin-echo sequence (TR 2000 ms, TE 35 ms), and water apparent diffusion coefficient was computed by fitting the MRI data as function of b-values into a single exponential.
VEGF-C Binding Assays. We assembled the human neuropilin (hNRP)-2(a22) cDNA encoding the extracellular domain (23) from Integrated Molecular Analysis of Genomes and their Expression (IMAGE) Consortium cDNA clones (Incyte Genomics, St. Louis, MO) and cloned it into the pIgplus vector (Ingenius, R&D Systems) in frame with the human IgG1 Fc tail. For receptor-IgG production, we transfected the expression vectors encoding NRP-2-IgG or VEGFR-3-IgG (14) into 293T cells. After 30 h, the cells were starved for 24 h in a medium containing 0.2% BSA, and the medium was used for the binding assays. For growth factor production, we transfected 293EBNA cells with expression vectors encoding human full-length VEGF-C or VEGF, or an empty vector, labeled the cells with 100 µCi/ml [35S]Met/[35S]Cys (Promix, Amersham) for 15 h and immunoprecipitated the media from the VEGF-C and vector-transfected cells with VEGF antibodies (R&D Systems) for depletion of endogenous VEGF. The binding assay was performed as described previously (12). For adeno-associated virus (AAV)-VEGF-C binding assay, we infected ca. 3.5 × 106 HeLa cells with 8 × 1010 particles of AAV-VEGF-C or AAV-EGFP viruses for 8 h in medium containing 2% FCS, glutamine, and antibiotics. After 3 days, the cells were labeled, and the medium was VEGF immunoprecipitated and subjected to binding assays or immunoprecipitation with VEGF-C antibodies.
Production of AAVs Encoding VEGF-C. We cloned the complete coding region of VEGF-C (22) as a blunt-end fragment into the MluI site of psub-CMV-WPRE (24). We cotransfected 293T cells with recombinant rAAV vector plasmid, AAV packaging plasmid pAAV/Ad-rep(ACG), and adenovirus helper plasmid pBS-E2A-VA-E4 (24). Sixteen hours later, we replaced the medium by fresh complete growth medium. We collected the cells 48 h after transfection, and released rAAVs by four freeze-thaw cycles in liquid nitrogen. We purified the rAAV by an Iodixanol-gradient ultracentrifugation and heparin-Sepharose HPLC (25).
Viral VEGF-C Overexpression.
An amount equal to 2-5 × 108
plaque-forming units of the adenoviruses encoding VEGF-C or LacZ, or
5 × 109-1 × 1011
rAAV particles encoding VEGF-C or EGFP were injected intradermally
into
the right ear, the left ear serving as a negative control. We
killed
the mice 2 weeks after adenoviral or 3 to
7 weeks after AAV
gene transfer and confirmed the adenoviral protein expression
in the
ear by 5-bromo-4-chloro-3-indolyl -D-galactoside
(X-Gal) staining for
-galactosidase
activity, and the AAV-EGFP expression by
fluorescent microscopy.
Northern Blotting. We extracted total RNA by using the RNeasy kit (Qiagen, Chatsworth, CA) and electrophoresed 10 µg of RNAs in 1% agarose, followed by transfer to nylon filters (Nytran, Schleicher & Schuell), hybridization with 32P-labeled cDNA probes, and exposure in autoradiography.
Results
A Mouse Model for Primary
Lymphedema. A Chy mouse mutant,
characterized by the accumulation of chylous ascites into the abdomen,
and
swelling of the limbs, was originally obtained
by ethylnitrosourea-induced
mutagenesis (15, 16). This
phenotype was linked to mouse chromosome 11. We sequenced
the Vegfr3
candidate gene of this chromosome in the Chy mice and found
a
heterozygous A3157T mutation resulting in I1053F substitution
in the
tyrosine kinase domain (Fig. 1 A
and B). This
mutation is located in a highly conserved
catalytic domain of the
receptor, in close proximity to the VEGFR-3
mutations in human
primary lymphedema (5,
6). We did
not detect this mutation in the parental C3H
mouse strain, or in the
other strains analyzed.
|
VEGFR-3(I1053F) Mutant Receptor
Is Tyrosine Kinase Inactive.
To analyze how the I1053F substitution affects VEGFR-3 phosphorylation,
we
expressed the corresponding mutant human VEGFR-3 transiently
in
conditions where its overexpression results in ligand-independent
phosphorylation. Unlike for the WT receptor, we
detected no
phosphorylation of VEGFR-3(I1053F) (Fig. 1C).
This
finding is consistent with the results obtained with the
mutant tyrosine
kinase-inactive VEGFR-3s in human primary lymphedema. When
we mated
the Chy mice with the Vegfr3+/
mice, in which one Vegfr3 allele is disrupted by
the LacZ sequence
(10), no offspring
carrying both mutations were born.
At embryonic day (E) 10.5, such
embryos were growth retarded,
suggesting that they die approximately at the
same developmental
stage as the Vegfr3
/
mice. These results support the idea that there is no signaling
via
the VEGFR-3(I1053F).
The Chy Mice Have Defective Lymphatic Vessels. The Chy phenotype was characterized by the appearance of chylous fluid in the abdomen (Fig. 2 A and B). Approximately 10% of the affected pups developed a severe fluid accumulation during the three first postnatal weeks. This condition was associated with histopathological changes in the liver, fibrinous adhesions of the intestinal tract, and lethality. The chylous fluid was spontaneously resorbed from the rest of the mice, which then appeared healthy, developed normally, and were fertile. IHC for the lymphatic endothelial markers showed that their lymphatic vessels were enlarged in the intestinal subserosal tissue (Fig. 2 C-F). These results are consistent with similar findings in human lymphedema (26, 27).
|
IHC for the lymphatic endothelial markers VEGFR-3, lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1; ref. 20), and podoplanin (21) revealed lack of lymphatic vessels in the Chy mouse skin, when compared with the WT littermate (Fig. 2 J and K; and data not shown). However, we observed few enlarged cutaneous lymphatic vessels in the Chy mice, consistent with similar findings in human hereditary lymphedema. We did not detect changes in the blood vessels stained for the platelet endothelial cell adhesion molecule-1 (PECAM-1) (Fig. 2 L and M), or when analyzed by biotin-labeled L. esculentum lectin perfusion (ref. 18 and data not shown). Also, the larger collecting lymphatic vessels and VEGFR-3-positive fenestrated blood vessels appeared normal in the Chy mice (ref. 9; Fig. 2 N and O; and data not shown). In histological examination, the dermis and s.c. adipose tissue were thickened in the Chy mice, when compared with the WT littermates (Fig. 2 P and Q). We also analyzed lymphatic fluid transport by intradermal injection of the Evans blue dye into the hind footpads, and by observing the appearance of the dye in the deeper collecting lymphatic vessels. We detected no transport of the dye in the Chy mice (Fig. 2R), whereas the lymphatic vessels alongside of the ischiatic vein were rapidly stained in the WT mice (Fig. 2S).
Dermal Lymphatic Vessels Lack the VEGF-C Binding Protein NRP-2. One possibility to explain the lack of lymphatic hypoplasia in the visceral organs of the Chy mice is that VEGF-C interacts with a second receptor in these organs. NRP-1 and NRP-2 are transmembrane receptors that are required for axon guidance, and they bind semaphorins as well as certain VEGF family members (29-31). To analyze whether VEGF-C binds NRP-2, we tested the ability of a soluble human NRP-2/IgG1 Fc fusion protein to interact with VEGF-C (Fig. 3A). We detected binding of VEGF-C to NRP-2, and, unlike for VEGF (30), this binding was not affected by heparin (data not shown). Surprisingly, in IHC we obtained a strong NRP-2 signal from the intestinal lymphatic endothelium, but not from the blood vessels (Fig. 3 B and C). In contrast, we did not detect NRP-2 staining in the lymphatic vessels of the skin (Fig. 3 D and E). These results suggest that there is a difference in VEGF-C receptor expression between the affected and unaffected lymphatic vessels.
|
Gene Therapy for Lymphedema via
Adenoviral VEGF-C Expression.
Because the molecular mechanisms leading to the hereditary lymphedema
phenotypes
are only beginning to be resolved, there is no
biologically based
treatment for this disease. We wanted to know
whether we could
stimulate lymphatic growth in the Chy mice by
VEGFR-3 ligand
administration via an adenovirus encoding VEGF-C
(AdVEGF-C; refs. 32
and 33). We infected the ears of
the Chy mice by
intradermal injection of AdVEGF-C or control virus encoding
-galactosidase
(AdLacZ; ref. 34). After
2 weeks, we detected
functional lymphatic vessels in the AdVEGF-C-infected ears,
as shown
by the uptake of FITC-dextran injected intradermally into
the ears
(see Fig. 5A).
We also confirmed the presence of the
lymphatic vessels by IHC (see Fig. 5B).
We also
confirmed the presence of the lymphatic vessels
on by IHC (see Fig. 5B).
AdLacZ-infected Chy ears had strong
-galactosidase
expression but no transport of FITC-dextran
(data not shown). These
results show that a lymphangiogenic response is
obtained by
adenoviral VEGF-C gene transfer in the Chy
mice.
VEGF-C Gene Therapy via AAV. Although we were able to stimulate growth of functional lymphatic vessels by adenoviral VEGF-C expression, no long-term expression could be obtained by using this virus because of a strong immune response against it. Like the adenoviruses, AAV infects both dividing and quiescent cells of several organs, but it gives long-term transgene expression without cell-mediated immune response or toxicity (35). We constructed a rAAV expression vector for VEGF-C, and used AAV encoding enhanced green fluorescent protein (EGFP) as a control (24). We confirmed the AAV-mediated VEGF-C production from rAAV-infected, metabolically labeled HeLa cell culture media by immunoprecipitation and gel electrophoresis. We detected production of the major 30-kDa form of VEGF-C, but very little or no 20-kDa form (Fig. 4A). We also confirmed the binding of AAV-produced VEGF-C to its receptors by using the soluble receptor-IgG fusion proteins. In this binding assay, we detected binding of the major 30-kDa form of VEGF-C to VEGFR-3 and to NRP-2 (Fig. 4A
|
To analyze the in vivo effects of
AAV-mediated VEGF-C expression, we
infected mouse ears by intradermal injection, and analyzed the
effects after 3 to 7 weeks. We confirmed VEGF-C RNA
expression in
the infected ears (Fig. 4B),
and in parallel, detected
AAV-EGFP expression in fluorescence microscopy
(data not shown). In
the fluorescent microlymphography, we detected
functional lymphatic
vessels in the AAV-VEGF-C-infected Chy ears, but not in the
control ears
(Fig. 5 C and
D). The formation of lymphatic
vessels in the AAV-VEGF-C-infected Chy mouse
ears was confirmed by
IHC analysis (Fig. 5
E and F).
Furthermore, the dye was transported into the collecting
lymphatic
vessels in the AAV-VEGF-C-treated but not untreated
Chy ears (Fig. 5
G and H). In the WT mice,
AAV-VEGF-C expression did not
affect the lymphatic vessel function (Fig. 5 I-L),
although in some cases we observed a denser lymphatic network,
resembling that in the transgenic mice overexpressing VEGFR-3
ligands
in the skin (11, 12).
|
Lymphatic Vessel Growth
Stimulated by a VEGFR-3-Specific
Ligand. VEGF-C is also capable of binding to VEGFR-2, and it
may thus affect
blood vessels. We therefore analyzed whether a VEGFR-3-specific
growth
factor can induce lymphatic growth in the Chy model. We mated
the Chy
mice with mice expressing VEGF-C156S in the skin keratinocytes
(12).
In these mice, transgene expression begins between
E14.5 and E16.5,
and thus there is no immune response toward the
encoded proteins (36).
IHC revealed the presence of lymphatic vessels
in the skin of the Chy × K14-VEGF-C156S
mice (Fig. 6
A-C), whereas the blood
vessels were not affected (Fig. 6
D-F).
The VEGF-C156S transgene expression was detected in the hair
follicles by IHC (Fig. 6 G-I).
FITC-dextran
microlymphography of the ears showed that a
WT-like lymphatic
function was restored in the
Chy × K14-VEGF-C156S mice
(Fig. 6 J-L).
These results indicate that
VEGFR-3 stimulation with an excess of its specific ligand is
sufficient to overcome the lymphatic hypoplasia caused by the
mutant
receptor allele.
|
Discussion
The molecular mechanisms and environmental factors affecting the pathogenesis and variable age of onset of lymphedema are largely unclear (28). In human patients, fluorescence microlymphography or lymphoscintigraphy reveal lack of a functional lymphatic vessel network at sites of edema. This finding is consistent with the hypoplasia of the cutaneous lymphatic vessels in the Chy mice. Currently, lymphedema is treated by manual lymphatic drainage and by compressive garments. The discovery of specific genes involved in the pathogenesis of lymphedema now allows us to study more targeted therapies for this disease.
Although the lymphedema patients with heterozygous missense mutations of VEGFR3 retain some receptor activity because of the presence of the WT allele (5), the mutant VEGFR-3 can be classified as a dominant negative receptor similar to certain mutant KIT receptors in piebaldism and rearranged during transportation (RET) receptors in Hirschprung's disease (5, 37). It has been unclear whether ligand therapy could be effective for the treatment of such diseases. Here, by treating the Chy mice with viral VEGF-C gene therapy, we were able to induce the growth of functional lymphatic vessels in their skin. Milroy's disease would thus provide one example of a human hereditary disease where gene therapy seems feasible, and it could provide a paradigm for other diseases associated with mutant receptors.
In the skin, VEGF-D is expressed in the close proximity to the
superficial
lymphatic vessel network (38) and
may be regulated by
cell-cell contacts in the dermis (39),
whereas VEGF-C is
only weakly expressed. However, we did not detect major
differences in
the VEGF-C or VEGF-D levels between the Chy and WT mice. Our
present
results, along with those of Mäkinen et al. (14)
support the idea that the cutaneous lymphatic
vessels are regulated
differently from those in other organs, and
that besides VEGF-C and
VEGF-D, there are additional signals for growth
and maturation of the
lymphatic endothelium. We show here that NRP-2 binds
VEGF-C, and is
expressed in the lymphatic vessels of internal organs, but not
in the
skin. Therefore, it is possible that NRP-2 is involved in
the
VEGFR-3-mediated signal transduction at sites where the two
receptors
are coexpressed, similar to what has been reported for
NRP-1
regulation of VEGFR-2-mediated angiogenic signals (29).
Expression
only in a subset of lymphatic vessels has also been described
for the
-chemokine
receptor D6,
reflecting the heterogeneity of the different
types of lymphatic
vessels (40).
Our results with the Chy mouse model suggest that overexpression of VEGFR-3 ligands could be used also in patients via viral or plasmid vector transfer, or via protein administration into the affected tissues. Such therapy could be even more effective in non-hereditary, more regional forms of lymphedema, resulting from traumas, surgery, or lymphatic vessel destruction e.g., after filarial infection. Because VEGF-C also binds VEGFR-2 on blood vascular endothelium, it is capable of stimulating vascular permeability and, in some conditions, angiogenesis (22, 41). Because of possible complications due to tissue edema or accelerated tumor angiogenesis, the VEGFR-3-specific growth factor VEGF-C156S could thus be a more attractive choice for therapeutic applications. An additional concern would be the fact that tumor lymphangiogenesis has been associated with enhanced lymph node metastasis (42). Thus, treatment of lymphedema arising in the arm after axillary lymphadenectomy in association with breast cancer surgery may pose a problem, because it could enhance the growth and spread of dormant metastases. However, the half-life of VEGF-C in the blood circulation is short (12), and local VEGF-C therapy is thus likely to function without systemic effects.
In conclusion, we have analyzed here a mouse model for Milroy's disease with swelling of the limbs because of a hypoplastic s.c. lymphatic network. Like certain lymphedema patients, the Chy mice carry a heterozygous inactivating Vegfr3 mutation. The pathogenesis of lymphedema, and its consequences, such as fat deposition, fibrosis, and compromised immune function can now be analyzed by using the Chy mice. By overexpressing VEGF-C in the skin, we obtained growth of functional cutaneous lymphatic vessels in the Chy mice. This result suggests that the VEGF-C administration alone or in combination with other lymphangiogenic factors could be a powerful tool in the therapy of various forms of human lymphedema.
Acknowledgements
We gratefully acknowledge the help of P. Glenister with the Chy mice; H. Kubo, D. Jackson, H. Kowalski, and D. Kerjaschki for the antibodies; and T. Tainola, S. Karttunen, R. Kivirikko, K. Makkonen, T. Taina, P. Hyvärinen, R. Kähtävä, A. Parsons, K. Pulkkanen, and S. Furler for excellent technical assistance. This study was supported by grants from the Finnish Cancer Organization, Emil Aaltonen Foundation, Ida Montini Foundation, Paulo Foundation, Finnish Cultural Foundation, Research and Science Foundation of Farmos, Academy of Finland, Novo Nordisk Foundation, the European Union (Biomed Grant PL963380), Swiss Cancer League, Swiss National Foundation, National Institutes of Health Grant HD37243, and a grant from the D. T. Watson Rehabilitation Hospital, Sewickley, PA.
Abbreviations
Ad, adeno; AAV, adeno-associated virus; NRP, neuropilin; IHC, immunohistochemistry; VEGF-C, vascular endothelial growth factor C; VEGFR, VEGF receptor; WT, wild type; PECAM, platelet endothelial cell adhesion molecule; E, embryonic day.
Footnotes
To whom reprint requests should be addressed at: Molecular/Cancer Biology Laboratory, Biomedicum Helsinki, University of Helsinki, Haartmaninkatu 3, SF-00290 Helsinki, Finland. E-mail: kari.alitalo@helsinki.fI .
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SEE LINK BELOW FOR REMAINING REFERENCE
http://www.pnas.org/cgi/content/full/98/22/12677?ijkey=4840e0d53646f0244220c7c32cea039d820ac3a6
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Published 16 September 2002 as 10.1084/jem.20020587
© Rockefeller
University Press, 0022-1007/2002/9/719/ $5.00
The Journal of Experimental Medicine, Volume 196, Number 6, September
16, 2002
719-730
1
Molecular/Cancer Biology Laboratory and Ludwig
Institute for Cancer Research, Biomedicum Helsinki, the Haartman
Institute and
Helsinki University Central Hospital, University of Helsinki, 00014
Helsinki,
Finland
2 Institute of Molecular Biology, University of
Zurich, 8057 Zurich,
Switzerland
3 A.I. Virtanen Institute and Department of
Medicine, University of
Kuopio, 70211 Kuopio, Finland
Address correspondence to Dr. Kari Alitalo, Molecular/Cancer Biology Laboratory, Biomedicum Helsinki, P.O.B. 63 (Haartmaninkatu 8), University of Helsinki, 00014 Helsinki, Finland. Phone: 358-9-1912 5511; Fax: 358-9-1912 5510; E-mail: Kari.Alitalo@helsinki.fi
Abstract
Recent work from many laboratories has demonstrated that the vascular endothelial growth factor-C/VEGF-D/VEGFR-3 signaling pathway is crucial for lymphangiogenesis, and that mutations of the Vegfr3 gene are associated with hereditary lymphedema. Furthermore, VEGF-C gene transfer to the skin of mice with lymphedema induced a regeneration of the cutaneous lymphatic vessel network. However, as is the case with VEGF, high levels of VEGF-C cause blood vessel growth and leakiness, resulting in tissue edema. To avoid these blood vascular side effects of VEGF-C, we constructed a viral vector for a VEGFR-3–specific mutant form of VEGF-C (VEGF-C156S) for lymphedema gene therapy. We demonstrate that VEGF-C156S potently induces lymphangiogenesis in transgenic mouse embryos, and when applied via viral gene transfer, in normal and lymphedema mice. Importantly, adenoviral VEGF-C156S lacked the blood vascular side effects of VEGF and VEGF-C adenoviruses. In particular, in the lymphedema mice functional cutaneous lymphatic vessels of normal caliber and morphology were detected after long-term expression of VEGF-C156S via an adeno associated virus. These results have important implications for the development of gene therapy for human lymphedema.
Key Words: lymphedema • lymphatic endothelium • VEGF-C • VEGFR-2 • VEGFR-3
Introduction
Proangiogenic gene therapy, developed first in the pioneering
work
of Dr. Jeffrey Isner, has shown great promise in the treatment
of
cardiovascular ischemic diseases (1–3).
In such studies, angiogenesis has been
stimulated for example by
overexpression of vascular endothelial growth
factor (VEGF)*
or various fibroblast growth factors (FGFs).
More recent developments
also include the use of modified forms of the
hypoxia-induced
transcription factor (HIF)-1,
which may orchestrate the induction of several angiogenic
mechanisms
(4, 5).
However, although VEGF is a
potent inducer of angiogenesis, the vessels it
helps to create are
immature, tortuous, and leaky, often lacking perivascular
support structures
(6–8).
Only a fraction of the blood
vessels induced in response to VEGF in the
dermis and in subcutaneous
fat tissue were stabilized and functional after adenoviral
treatment of
the skin of nude mice (9, 10), while
intramuscular vessels developed into an
angioma-like proliferation or
regressed with a resulting scar tissue (9, 11).
Furthermore, edema induced by VEGF
overexpression complicates VEGF-mediated
neovascularization, although recent evidence
suggests that it can be
avoided by providing angiopoietin-1 for vessel
stabilization (12,
13).
Lymphatic vessels play an important physiological role in
homeostasis, regulation
of tissue fluid balance, and in the immune responses to
pathogens,
yet the molecular mechanisms that control their development
and
function are only beginning to be elucidated. So
far, only two
peptide growth factors have been found capable of
inducing the growth
of new lymphatic vessels in vivo. These factors,
VEGF-C and VEGF-D (14–16),
belong to the larger VEGF family of growth
factors which also
includes VEGF, placenta growth factor (PlGF),
and VEGF-B. VEGF-C and
VEGF-D are ligands for the endothelial
cell–specific tyrosine
kinase receptors VEGFR-2 and VEGFR-3 (17, 18).
In adult human as well as mouse tissues VEGFR-3
is expressed
predominantly in the lymphatic endothelial
cells which line the inner
surface of lymphatic vessels (19, 20).
Whereas VEGFR-2 is thought to be the main mediator
of angiogenesis,
VEGFR-3 signaling is crucial for the development
and maintenance of
the lymphatic vessels (21).
Inhibition of
VEGFR-3 signaling using soluble VEGFR-3 which competes for ligand
binding with the endogenous receptors led to lymphatic vessel
regression in a transgenic mouse model (22).
Other
molecules that have been reported to be
necessary for normal
lymphatic development include the transcription
factor Prox-1 (23),
the integrin 9 (24),
and angiopoietin-2 (25).
Impairment of lymphatic function, which results in inadequate transport of fluid, macromolecules, or cells from the interstitium, is associated with a variety of diseases and leads to tissue edema, impaired immunity and fibrosis (26). Development of strategies for local and controlled induction of lymphangiogenesis would thus be of major importance for the treatment of such diseases. Adenoviral gene transfer of VEGF-C in the skin has been shown to result in a strong lymphangiogenic response (27, 28), but high levels of VEGF-C also lead to blood vascular effects such as increased vessel leakiness, presumably through the interaction of VEGF-C with the VEGFR-2 expressed on blood vascular endothelium (28). To develop a lymphatic-specific gene therapy approach without the unwanted blood vascular side effects, we have studied the potential of a VEGFR-3–specific mutant form of VEGF-C (VEGF-C156S) as a therapeutic agent in lymphedema. We demonstrate that stimulation of VEGFR-3 alone by VEGF-C156S potently induces lymphangiogenesis both in transgenic embryos and after virus-mediated gene transfer. In a lymphedema mouse model functional cutaneous lymphatic vessels formed after intradermal infection with adeno-associated virus (AAV) encoding VEGF-C156S. Most importantly, VEGF-C156S essentially lacked the blood vascular effects of native VEGF-C.
Materials and Methods
Generation and In Vitro Analysis of Recombinant
Adenoviruses and AAVs.
For the adenovirus construct, the full-length human VEGF-C156S
cDNA (29)
was cloned as a BamHI/NotI fragment into the corresponding sites
of
the pAD BglII vector. Replication-deficient E1-E3 deleted adenoviruses
were produced in 293 cells and concentrated by ultracentrifugation
(30).
Adenoviral preparations were analyzed to be
free of helper viruses,
lipopolysaccharide, and bacteriological contaminants
(31).
The adenoviruses encoding human VEGF-C and nuclear
targeted LacZ
were constructed as described (27,
30).
For the AAV construct, the full-length human VEGF-C156S was
cloned
as a blunt-end fragment into the MluI site of psub-CMV-WPRE plasmid
and the rAAV type 2 was produced as described previously (32).
AAVs encoding human VEGF-C and EGFP were used as controls (32,
33).
For the analysis of protein expression, 293EBNA cells were infected with recombinant adenoviruses for 2 h in serum-free medium or by AAVs for 8 h in 2% FCS medium. After 24–72 h, the cells were metabolically labeled for 8 h and subjected to immunoprecipitation with VEGF-C–specific antibodies or to a binding assay using soluble VEGFR-2-Ig (R&D Systems) and VEGFR-3-Ig (18) fusion proteins. AdLacZ and AAV-EGFP infected cells were used as negative controls. The bound proteins were precipitated with protein G Sepharose, separated in 15% SDS-PAGE, and analyzed by autoradiography. To compare the protein production levels of AdVEGF-C156S and AdVEGF-C viruses, 20-µl aliquots of the media from AdVEGF-C156S, AdVEGF-C, and AdLacZ infected cell cultures were separated in 15% SDS-PAGE gel and subjected to Western blotting using polyclonal anti–VEGF-C antibodies (R&D Systems).
In Vivo Use and Analysis of the Viral Vectors.
All the studies were approved by the Committee for Animal Experiments
of
the University of Helsinki. 5 x
108
pfu of the recombinant adenoviruses or 5 x
109-1 x
1011 rAAV
particles were injected intradermally into the
ears of NMRI nu/nu
mice (Harlan) or Chy lymphedema mice (32). The
infected nude mice were killed 3, 5, 7, 10, 14,
21, 42, or 56 d after
adenoviral infection and 3, 6, or 8 wk after
AAV infection. The AAV-infected
Chy mice were killed 1, 2, 4, 6, or 8 mo after
infection. Total RNA
was extracted from the ears (RNAeasy Kit; QIAGEN)
1 to 8 wk
after adenoviral infection and 10 wk after
AAV-infection. 10 µg
of RNA was subjected to Northern blotting and hybridization
with
a mixture of [32P]dCTP
(Amersham
Biotech) labeled cDNAs specific for VEGF-C. The
glyceraldehyde-3-phosphate dehydrogenase cDNA
probe was used as an
internal control for equal loading. The
adenoviral protein expression
was confirmed by whole mount ß-galactosidase
staining (34)
of the AdLacZ-infected ears 1 to 7 wk after
gene transfer. The
AAV-EGFP-infected ears were studied under the
fluorescence microscope
at 3 wk to 8 mo after infection.
ß-Galactosidase and Lectin Staining of Vessels.
For visualization of the superficial lymphatic vessels in the
K14-VEGF-C156S
and K14-VEGF-C embryos (15, 16),
staged VEGFR-3+/LacZ embryos were dissected,
fixed in 0.2%
glutaraldehyde, and stained with X-gal (
Sigma-Aldrich)
for ß-galactosidase activity at +37°C. For the
analysis of the
adult cutaneous lymphatic phenotype,
ß-galactosidase staining was
performed for dissected adult mouse ear skin.
In some of the adenovirus-infected mice, Lycopersicon esculentum lectin staining was used to visualize the blood vessels in whole mount (12). Biotinylated lectin (1 mg/ml; Vector Laboratories) was injected into the femoral veins of the mice under anesthesia and after 2 min the mice were killed and perfusion fixed with 1% paraformaldehyde (PFA)/0.5% glutaraldehyde in PBS. The tissues were dissected and the biotinylated lectin was visualized by the ABC-DAB peroxidase method (Vectastain and Sigma-Aldrich). Finally the tissues were dehydrated and mounted on slides.
For the gene expression studies of different types of lymphatic vessels, a combination of biotinylated lectin and whole mount ß-galactosidase staining was performed for VEGFR-2+/LacZ (35) and VEGFR-3+/LacZ (36) adult mouse tissues.
Analyses of the Lymphatic and Blood Vessels.
For immunohistochemical analysis the mouse ears were dissected
and
fixed in 4% PFA. Those ears that were analyzed in whole mount
were
incubated in 5% H2O2 in
methanol for 1 h to block endogenous
peroxidase activity. The tissues were then blocked in
3% milk 0.3%
Triton-X in PBS overnight, and antibodies against the
vascular
endothelial marker PECAM-1 (
BD
Biosciences) or VEGFR-3 (R&D
Systems) were applied overnight
at +4°C. The visualization was achieved with
either the ABC-DAB
peroxidase method or with ABC-alkaline
phosphatase using the alkaline
phosphatase substrate kit II (
Vector
Laboratories). Finally the tissues were
flattened and mounted on
slides.
5-µm deparaffinized tissue sections were subjected to heat induced epitope retrieval treatment or to an alternative enzyme treatment. The endogenous peroxidase activity was blocked with 3% H2O2 in methanol for 20 min. Antibodies against VEGFR-3 (19), PECAM-1, podoplanin (a gift from Dr. Miguel Quintanilla, Alberto Sols Biomedical Research Institute, Madrid, Spain), or LYVE-1 (a gift from Dr. Erkki Ruoslahti, Burnham Institute, La Jolla, CA) were applied overnight at +4°C and staining was performed using the tyramide signal amplification kit ( NEN Life Science Products) and 3-amino-9-ethyl carbazole ( Sigma-Aldrich). Hematoxylin was used for counterstaining.
To study the function of the cutaneous lymphatic vessels in the Chy lymphedema mice, a small volume of FITC-labeled dextran (MW 464 000; Sigma-Aldrich) was injected intradermally to the periphery of mouse ear. Drainage of the dye via the lymphatic vessels was followed under a fluorescence microscope.
Quantification of the Lymphangiogenic Response.
To quantify the number of lymphatic vessels and branch points
at 1 wk
after adenoviral infection, six histological sections from
ear
midline with the highest vessel density were chosen from
each study
group (AdVEGF-C156S, AdVEGF-C, AdLacZ). The number
of
LYVE-1–positive vessels and the number of branches in
these vessels
were counted under a high power microscope. The
area analyzed in each
sample was 4 mm2.
Permeability Assay.
The right ear of each mouse was infected with AdVEGF-C156S, AdVEGF-C,
or AdLacZ virus (5 x
108 pfu).
The left ear received either AdLacZ (5 x
108 pfu) or PBS (in the AdLacZ group). 2
wk after the
infection a modified Miles permeability assay was performed
as
described previously (12). 1 µl
per gram of
mouse weight of 3% Evans Blue was injected into the femoral vein
and
after 2 min the mice were perfusion fixed with 0.05 M
citrate buffer
(pH 3.5) in 1% PFA. The ears were dissected, washed,
weighed and
extracted in formamide at +55°C overnight. The
Evans Blue absorbance
of the formamide was then measured with a
spectrophotometer set at
610 nm, and the leakage (ng/mg) was compared
between the right and
left ear of the same mouse
Results
Expression of the Virally Transduced Genes In
Vitro and In Vivo.
The production of active VEGF-C156S and VEGF-C proteins into
the cell
culture media of adenovirus (Ad)- or AAV-infected, metabolically
labeled 293EBNA cells was confirmed by immunoprecipitation and
by
binding to soluble VEGFR-2-Ig and VEGFR-3-Ig fusion proteins.
Both
the partially processed 30 kD and the fully processed 21-kD forms
of
VEGF-C156S and VEGF-C bound to VEGFR-3-Ig, but only the
21-kD form of
VEGF-C was capable of binding to VEGFR-2-Ig (Fig. 1
A, and unpublished data). Furthermore, Western blotting analysis
of
media from the infected cultures confirmed that the
same viral titers
of AdVEGF-C156S and AdVEGF-C gave rise to
comparable levels of the
corresponding proteins in vitro (Fig. 1 A). To
analyze the expression of adenovirus and AAV transduced
genes in
vivo, RNA samples from infected mouse ear skin
were analyzed by
Northern blotting. High levels of human VEGF-C156S
and VEGF-C mRNAs
were detected in the AdVEGF-C156S and AdVEGF-C
infected tissues 1 wk
after infection (Fig. 1 B).
3 wk after infection
transgene expression in the control AdLacZ infected
ears was still
strong (Fig. 1 C). Thereafter
the transgene expression
was gradually down-regulated, and by 8 wk expression was
no longer
detected in the adenovirus-infected ear. Somewhat weaker,
but more
sustained mRNA and protein expression was obtained with
the AAV
vectors (Fig. 1, B and D).
Furthermore, at 8 mo after
infection, the latest time point studied, EGFP fluorescence was
still
detected in the ear skin of the Chy mice infected with
the AAV-EGFP
control virus (Fig. 1 E).
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Comparison of the Lymphangiogenic Potential of
Recombinant VEGF-C156S
and VEGF-C Viruses.
We then wanted to compare the lymphangiogenesis induced by viral
delivery
of VEGF-C156S and VEGF-C. For this purpose, adenoviruses encoding
VEGF-C156S, VEGF-C, or LacZ were injected intradermally into
the ears
of nu/nu mice. The first sprouting lymphatic vessels were
detected in
the infected ear skin by VEGFR-3 whole mount staining
3 to 4 d after
the adenoviral infection. The lymphatic sprouting
was more abundant
in the AdVEGF-C–infected skin than in the
AdVEGF-C156S–infected
skin at all time points studied. In the
AdVEGF-C156S–infected skin
the lymphatic vessels enlarged progressively,
and 1 wk after the
infection several lymphatic vessels appeared to
be splitting to form
new daughter vessels (Fig.
3 A). At this time
point nearly all lymphatic vessels in the
AdVEGF-C–infected skin
exhibited sprouting and bridging; some enlarged
vessels were also
detected, whereas no signs of lymphangiogenesis
were seen in the
AdLacZ-infected control skin (Fig.
3, B and C). 2
wk after AdVEGF-C156S infection the lymphatic
vessels network was
vastly enlarged and most of the large vessels
had formed new sprouts
or bridges (Fig. 3 D).
At the same time point the
AdVEGF-C–infected skin was filled with very
actively sprouting,
relatively small lymphatic vessels, while only
a few enlarged
lymphatic vessels were detected (Fig. 3 E). A few
new lymphatic sprouts were detected in only one
AdLacZ-infected
control mouse around the injection wound (n
= 15; unpublished
data), otherwise the lymphatic vasculature in
the control skin
remained unaltered (Fig. 3 F).
The staining of
histological sections for VEGFR-3, LYVE-1, and podoplanin confirmed
that the sprouting of the lymphatic vessels was more efficient
in the
AdVEGF-C than in the AdVEGF-C156S infected samples
(Fig.
3, G–I, and unpublished data). The newly formed
lymphatic
vessels regressed gradually with time as expression of
the viral
genes was downregulated, and by 8 t to 10 wk after the
infection the
lymphatic vessel network had returned to its normal
architecture
(unpublished data).
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The systemic effects of AdVEGF-C156S, AdVEGF-C, and AdLacZ were studied by injecting 5 x 108 or 1 x 109 pfu of the viruses into the tail veins of C57/Bl6 mice. All mice that had received intravenous AdVEGF-C156S or AdLacZ appeared healthy during the 2-wk follow-up period, whereas all mice that had received different doses of AdVEGF-C became ill on day 3 after infection. Three of the five mice that received the higher dose of AdVEGF-C died during the first week. All mice infected with AdVEGF-C had dose-dependent accumulation of fluid in the thoracic cavity, but intrathoracic fluid was not observed in any of the AdVEGF-C156S or AdLacZ infected mice (Fig. 7, B and C). Two of the five mice that received the higher dose of AdVEGF-C also had fluid in the peritoneal cavity.
AAV-mediated VEGF-C156S Gene Transfer Results in
Formation of
Functional Lymphatic Vessels in the Chy Lymphedema Mice.
Similar to certain patients with Milroy's disease, the Chy lymphedema
mice
have a germline inactivating mutation of VEGFR-3, which results
in
hypoplasia of cutaneous lymphatic vessels (32).
We have
previously reported that adenoviral or AAV-encoded VEGF-C is
capable
of restoring a functional cutaneous lymphatic vessel network
in the
Chy mice (32). To assess the
lymphangiogenic activity
of viral VEGF-C156S in the Chy mice, their ears were injected
with
AAVs encoding VEGF-C156S, VEGF-C, or EGFP. Fluorescent microlymphangiography
using FITC-dextran revealed that functional, normal-looking,
but
slightly dilated lymphatic vessels had formed in
the AAV-VEGF-C156S
infected mice during the 2-mo follow-up period (Fig.
8, A–D); we also observed that in the AAV-VEGF-C–infected
Chy
mice such vessels persisted at least for 8 mo. As a rule, in
Chy mice
infected with AAV-EGFP, FITC-dextran remained at the
injection site,
except that a single draining lymphatic vessel
was seen in 2/12 of
the mice (Fig. 8 C, and
unpublished data). This
finding may reflect the occasional finding of isolated lymphatic
vessel segments in untreated Chy mice.
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Our study shows that overexpression of VEGF-C156S, a VEGFR-3–specific mutant form of VEGF-C, potently induces lymphangiogenesis both during embryonic development and in adult skin. Recombinant VEGF-C156S viruses were capable of inducing growth of lymphatic vessels and the formation of new lymphatic sprouts, although the latter effect was seen only in adults and was less pronounced than with VEGF-C. In addition, we show that VEGF-C156S does not have obvious effects on blood vessels in contrast to VEGF-C, which also binds VEGFR-2 expressed on the blood vascular endothelium. In the Chy lymphedema mice AAV-mediated gene transfer of VEGF-C156S induced the formation of a functional lymphatic vessel network into the skin.
The whole mount images of ongoing embryonic and adult lymphangiogenesis that we present here suggest that the mechanisms of lymphangiogenesis may be very similar to those of angiogenesis (38) in that lymphangiogenesis also appears to proceed via vessel enlargement, sprouting and splitting. Also, like the angiogenic blood vessels, the newly formed lymphatic vessels seem to require a continuous growth factor stimulation in order to be maintained. Although VEGF-C156S and VEGF-C both induced an increase of lymphatic vascularity, their lymphangiogenic mechanisms seemed to differ. The difference between the capacities of VEGF-C156S and VEGF-C to induce new lymphatic sprouts was most obvious in the transgenic skin model. This suggests that activation of VEGFR-2 is required for the induction of lymphatic sprouting during embryonic development. Indeed, we have evidence that VEGFR-2 is expressed in the embryonic lymph sac endothelium (unpublished data). Although VEGFR-3 activation via viral VEGF-C156S expression seems sufficient for the induction of lymphatic sprouting in adult tissues, a more effective sprouting response may require VEGFR-2 signaling also in adult tissues. An interesting possibility is that signaling via VEGFR-2 and VEGFR-3 receptor heterodimers is needed for efficient formation of vessel sprouts. Gene expression studies in cultured lymphatic endothelial cells after VEGF-C156S and VEGF-C stimulation should reveal at least some of the mechanisms behind the differences in such lymphangiogenic phenotypes observed in vivo. On the other hand, it is also possible that VEGF-C can recruit other effector cells and factors needed for sprouting in adult tissues better than VEGF-C156S.
The biological significance of VEGF-C–induced blood vessel dilation and increased permeability is unknown. These effects may at least in part result from receptor-mediated vasodilation as veins and venules in adult skin have been shown to express VEGFR-2 (28). Several angiogenic growth factors including VEGF and bFGF are known to be potent stimulators of nitric oxide production, and in vivo studies have documented endothelium-dependent hypotension in response to treatment with these growth factors (39, 40). VEGF-C has also been reported to stimulate the release of endothelial nitric oxide, which could contribute to enhanced vascular permeability (41). The results we obtained with systemic administration of AdVEGF-C resemble those reported after systemic administration of AdVEGF by Thurston and coworkers (13). Also, patients with tissue ischemia developed edema after VEGF gene transfer (42). In contrast, we have thus far observed minimal blood vascular effects with VEGF-C156S.
The lymphangiogenesis in response to AAV-VEGF-C156S and AAV-VEGF-C was always weaker and slower than lymphangiogenesis obtained with the corresponding adenoviral vectors. This finding probably relates to differences in the biological properties of these two viral vectors. It has been demonstrated that after an intramuscular AAV infection, the level of transgene expression increased gradually over 5 to 10 wk after an initial lag phase of 1 to 2 wk (43). With adenovirus vectors, transgene expression has been detected already 24 h after infection, but after reaching its peak during the first week after infection, the expression is rapidly downregulated (10), probably due to direct cytopathic effects of the virus resulting in the elimination of the virally transduced cells. In the present study adenoviral gene expression was extinguished by eight weeks after infection of nude mice. The lower transgene expression levels and slower kinetics of AAV-VEGF-C156S and AAV-VEGF-C infection seemed to result in more controlled lymphangiogenesis than the acute high-level expression obtained by the use of adenoviral vectors. AAV could therefore be more suited for lymphangiogenic therapy in humans. Importantly, AAV-mediated transgene expression seems very stable as demonstrated by persistent fluorescence in the AAV-EGFP–infected samples. We have followed the AAV-VEGF-C–infected Chy mice for up to 8 mo after infection and found that functional skin lymphatic vessels are still present at this time point. This probably reflects continuous transgene expression from the integrated viral vector rather than actual stabilization of the newly formed lymphatic vessels. At present it is unclear whether a single infection with a recombinant AAV vector will produce life-long transgene expression or whether vector readministration would be required.
The first lymphangiogenic growth factors were discovered some time ago, but the molecular control of the formation of the patterned hierarchy of lymphatic vessels is still largely unknown. Here we report for the first time that the endothelial cells of lymphatic vessels of different calibers express different growth factor receptors on their surface. This probably reflects subtle differences in the function of the lymphatic endothelium of the various vessels and their abilities to respond to different types of stimuli. Interestingly, in the K14-VEGF-C mice only the superficial lymphatic capillaries were hyperplastic, whereas the collecting, VEGFR-2–positive lymphatic vessels appeared normal. Previous microlymphograpy studies have indicated that the function of the superficial lymphatic capillaries in the K14-VEGF-C mice may be partly compromised (15). As both capillaries and collecting vessels are needed for the lymphatic drainage function, more attention should be paid to the development of strategies to specifically target the different types of lymphatic vessels. This issue will be of major importance when designing molecular therapies for human lymphedema.
Angiogenic gene therapy has raised concerns regarding the potential stimulation of dormant tumor growth due to increased tumor angiogenesis in response to elevated systemic VEGF levels. In addition, high local levels of VEGF after viral gene transfer have been shown to result in the formation of hemangioma-like vascular tumors in murine skeletal muscle, heart, and skin (10, 11, 44). VEGF-C has also been shown to increase tumor angiogenesis and the recruitment of inflammatory cells (45, 46), but the increased angiogenesis was reversed by inhibition of VEGFR-2 (45). The safety of angiogenic gene therapy, in particular VEGF gene transfer, would be improved by a brief duration and low level of localized transgene expression. In a lymphatic vascular specific gene therapy without blood vascular side effects the safety margin would be wider. For patients with lymphatic hypoplasia, dysfunction, and edema, VEGF-C156S gene therapy would thus seem like an attractive choice. However, as tumor-induced lymphangiogenesis has been associated with enhanced metastasis to the lymph nodes (47–52) the risk of enhanced growth and spread of dormant metastases needs to be carefully evaluated.
Acknowledgments
We thank Drs. Hajime Kubo, Erkki Ruoslahti, and Miguel Quintanilla for antibodies, Drs. Robert Ferrell and David Finegold for collaboration with the Chy phenotype, and Sanna Karttunen, Kaisa Makkonen, Paula Hyvärinen, Mari Elemo, Paula Turkkelin, and Tapio Tainola for excellent technical assistance.
This study was supported by the Finnish Cultural Foundation, Ida Montini Foundation, the Farmos Research Foundation, the Finnish Academy, the Finnish Medical Foundation, the Helsinki University Central Hospital (TYH 8150), the Novo Nordisk Foundation, the Human Frontier Science Program, the Swiss Cancer League, and the Swiss National Fund.
Submitted: April 12, 2002
Revised: June 25, 2002
Accepted: July 16, 2002
Footnotes
A. Saaristo and T. Veikkola contributed equally to this work.
* Abbreviations used in this paper: AAV, adeno-associated virus; PFA, paraformaldehyde; VEGF, vascular endothelial growth factor.
References
http://www.jem.org/cgi/content/full/196/6/719?ijkey=1e7c28af64421dab92ea0b0ee24c0715321bfe7b
===========================
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master
instead of the sufferer of lymphedema.
http://health.groups.yahoo.com/group/menwithlymphedema/
Subscribe: menwithlymphedema-subscribe@yahoogroups.com
......................
All
About Lymphangiectasia
Support group for parents, patients, children who suffer from all forms
of
lymphangiectasia. This condition is caused by dilation of the
lymphatics. It can
affect the intestinal tract, lungs and other critical body areas.
http://health.groups.yahoo.com/group/allaboutlymphangiectasia/
Subscribe: allaboutlymphangiectasia-subscribe@yahoogroups.com
......................
Lymphatic
Disorders Support Group @ Yahoo Groups
While we have a number
of support groups for lymphedema... there is nothing out there for
other
lymphatic disorders. Because we have one of the most comprehensive
information
sites on all lymphatic disorders, I thought perhaps, it is time that
one be
offered.
DISCRIPTION
Information and support for rare and unusual disorders affecting the
lymph
system. Includes lymphangiomas, lymphatic malformations,
telangiectasia,
hennekam's syndrome, distichiasis, Figueroa
syndrome, ptosis syndrome, plus many more. Extensive database of
information
available through sister site Lymphedema People.
http://health.groups.yahoo.com/group/lymphaticdisorders/
Subscribe: lymphaticdisorders-subscribe@yahoogroups.com
......................
All
About Lymphedema
For
our Google fans, we have just
created this online support group in Google Groups:
Homepage: http://groups-beta.google.com/group/All-About-Lymphedema
Group email: All-About-Lymphedema@googlegroups.com
......................
Lymphedema Friends
http://groups.aol.com/lymphedemafriend
If you an AOL fan and looking for a
support group in AOL
Groups, come and join us there.
===========================
Lymphedema People New Wiki Pages
Have
you seen our new “Wiki”
pages yet? Listed
below are just a
sample of the more than 140 pages now listed in our Wiki section. We
are also
working on hundred more. Come
and
take a stroll!
Lymphedema
Glossary
http://www.lymphedemapeople.com/wiki/doku.php?id=glossary:listing
Lymphedema
http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema
Arm
Lymphedema
http://www.lymphedemapeople.com/wiki/doku.php?id=arm_lymphedema
Leg
Lymphedema
http://www.lymphedemapeople.com/wiki/doku.php?id=leg_lymphedema
Acute
Lymphedema
http://www.lymphedemapeople.com/wiki/doku.php?id=acute_lymphedema
The
Lymphedema Diet
http://www.lymphedemapeople.com/wiki/doku.php?id=the_lymphedema_diet
Exercises
for Lymphedema
http://www.lymphedemapeople.com/wiki/doku.php?id=exercises_for_lymphedema
Diuretics
are not for Lymphedema
http://www.lymphedemapeople.com/wiki/doku.php?id=diuretics_are_not_for_lymphedema
Lymphedema
People Online Support
Groups
http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_people_online_support_groups
Lipedema
http://www.lymphedemapeople.com/wiki/doku.php?id=lipedema
Treatment
http://www.lymphedemapeople.com/wiki/doku.php?id=treatment
Lymphedema
and Pain Management
http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_and_pain_management
Manual
Lymphatic Drainage (MLD) and Complex Decongestive Therapy (CDT)
Infections
Associated with Lymphedema
http://www.lymphedemapeople.com/wiki/doku.php?id=infections_associated_with_lymphedema
How
to Treat a Lymphedema Wound
http://www.lymphedemapeople.com/wiki/doku.php?id=how_to_treat_a_lymphedema_wound
Fungal
Infections Associated with
Lymphedema
http://www.lymphedemapeople.com/wiki/doku.php?id=fungal_infections_associated_with_lymphedema
Lymphedema
in Children
http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_in_children
Lymphoscintigraphy
http://www.lymphedemapeople.com/wiki/doku.php?id=lymphoscintigraphy
Magnetic
Resonance Imaging
http://www.lymphedemapeople.com/wiki/doku.php?id=magnetic_resonance_imaging
Extraperitoneal
para-aortic lymph node dissection (EPLND)
Axillary
node biopsy
http://www.lymphedemapeople.com/wiki/doku.php?id=axillary_node_biopsy
Sentinel
Node Biopsy
http://www.lymphedemapeople.com/wiki/doku.php?id=sentinel_node_biopsy
Small
Needle Biopsy - Fine Needle Aspiration
http://www.lymphedemapeople.com/wiki/doku.php?id=small_needle_biopsy
Magnetic
Resonance Imaging
http://www.lymphedemapeople.com/wiki/doku.php?id=magnetic_resonance_imaging
Lymphedema
Gene FOXC2
http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_foxc2
Lymphedema Gene VEGFC
http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_vegfc
Lymphedema Gene SOX18
http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_sox18
Lymphedema
and Pregnancy
http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_and_pregnancy
Home page: Lymphedema People
http://www.lymphedemapeople.com
Page Updated: May 23, 2008