model for gene therapy of human hereditary lymphedema
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
A model for gene therapy of human hereditary lymphedema
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)
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
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.
A Mouse Model for Primary
Lymphedema. A Chy mouse mutant,
characterized by the accumulation of chylous ascites into the abdomen,
swelling of the limbs, was originally obtained
mutagenesis (15, 16). This
phenotype was linked to mouse chromosome 11. We sequenced
candidate gene of this chromosome in the Chy mice and found
heterozygous A3157T mutation resulting in I1053F substitution
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
the infected ears (Fig. 4B),
and in parallel, detected
AAV-EGFP expression in fluorescence microscopy
(data not shown). In
the fluorescent microlymphography, we detected
vessels in the AAV-VEGF-C-infected Chy ears, but not in the
(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
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
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
factor can induce lymphatic growth in the Chy model. We mated
mice with mice expressing VEGF-C156S in the skin keratinocytes
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
The VEGF-C156S transgene expression was detected in the hair
follicles by IHC (Fig. 6 G-I).
microlymphography of the ears showed that a
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
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.
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.
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.
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: email@example.comI .
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SEE LINK BELOW FOR REMAINING REFERENCE
Published 16 September 2002 as 10.1084/jem.20020587
University Press, 0022-1007/2002/9/719/ $5.00
The Journal of Experimental Medicine, Volume 196, Number 6, September 16, 2002 719-730
Molecular/Cancer Biology Laboratory and Ludwig
Institute for Cancer Research, Biomedicum Helsinki, the Haartman
Helsinki University Central Hospital, University of Helsinki, 00014
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
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
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.
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
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).
Comparison of the Lymphangiogenic Potential of
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).
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
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.
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.
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
A. Saaristo and T. Veikkola contributed equally to this work.
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