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Short Communications |
From the Molecular/Cancer Biology Laboratory and Ludwig Institute
for Cancer Research,*
Department of Pathology, Haartman
Institute, and Department of
Otorhinolaryngology,
University of Helsinki
and Helsinki University Central Hospital, Helsinki, Finland; and
Children’s and Women’s Hospital and Harvard Medical
School,
Boston, Massachusetts
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The development of a complex vascular system and its ability to respond to changing demands depend on complex molecular regulatory networks. Members of the vascular endothelial growth factor family (VEGF, placenta growth factor, VEGF-B, VEGF-C, and VEGF-D) are currently known as the major inducers of angiogenesis and lymphangiogenesis. In addition, VEGF is more potent than histamine in increasing capillary permeability to plasma proteins.3 VEGF has also been shown to induce fenestrations in, eg, adrenal cortex capillary endothelial cells in culture,4,5 and the inhibition of VEGF activity by specific monoclonal antibodies reduces vascular permeability3,6 and the growth of experimental tumors and their blood vessel density.7 VEGF binds selectively and with high affinity to two transmembrane receptors called VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1).8 VEGFR-2 is expressed in endothelial cells and apparently in human hematopoietic stem cells,9 whereas VEGFR-1 can also be found in monocytes (for reviews, see refs 8 and 10). Results on knockout mice suggest that in embryos, VEGFR-2 is essential for endothelial cell differentiation, whereas VEGFR-1 has a role in regulating the amount of endothelial cells formed.11-13
Recent experiments in transgenic mice have shown that the
VEGF-C/VEGFR-3 signaling pathway is critical for the growth of the
lymphatic vessels.14
Another ligand for VEGFR-3 is
VEGF-D.15
A proteolytically processed form of VEGF-C can
also bind to VEGFR-2, which is expressed in both blood and lymphatic
vessel endothelia.16
Via this receptor, VEGF-C can induce
capillary endothelial cell migration and proliferation in
culture16,17
and stimulate angiogenesis in the cornea and
ischemic muscle.18-20
The proinflammatory cytokines
interleukin-1ß (IL-1ß) and tumor necrosis factor-
(TNF-)
up-regulate VEGF-C mRNA, whereas dexametasone and IL-1 receptor
antagonist inhibited this effect.21
VEGF-C is highly
expressed in the lymph nodes, and it could have an important role in
the regulation of inflammatory conditions through the control of
capillary permeability and lymphatic endothelial functions. The VEGF-C
receptor VEGFR-3 is expressed predominantly in lymphatic endothelium of
adult human tissues22
; in mice its expression starts
during embryonic day (E) 8 in developing blood vessels, but it becomes
largely restricted to the lymphatic vessels after their formation from
E12.5 onward.22,23
However, VEGFR-3 expression was shown
to reappear in the endothelia of angiogenic blood vessels in several
types of cancer,24
and more recently we have also found
VEGFR-3 in the endothelium of certain fenestrated and discontinuous
capillaries in normal human tissues.25
In this study we have analyzed the expression of VEGFR-3 and its VEGF-C ligand in the human upper respiratory tract and its tumors by Northern blot analysis, immunohistochemistry, in situ mRNA hybridization of mouse embryos, and ß-galactosidase staining of embryos heterozygous for a LacZ "knock-in" marker gene in the VEGFR-3 gene locus.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The study was approved by the Ethical Committee of the Helsinki University Central Hospital. The fetal tissues (n = 4, including one frozen sample), obtained from elective abortions, were fixed in 4% paraformaldehyde or frozen in liquid nitrogen. Adult tissues included surgical specimens from normal tissues (n = 10, including six frozen samples); nasal and nasopharyngeal tumors (n = 34), including squamous cell carcinomas (n = 22, five frozen samples); adenocarcinomas (n = 6, two frozen samples); transitional cell carcinoma (n = 2, frozen samples); and angiofibromas (n = 4). The samples from adults were fixed with 4% phosphate-buffered formaldehyde (pH 7.0) or frozen in liquid nitrogen.
mRNA Isolation and Northern Analysis
Total RNAs were isolated from three normal nasal mucosas,
tonsilla, and five nasal and nasopharyngeal tumors, including three
squamous cell carcinomas (of which one was from the nasopharyngeal
area), one adenocarcinoma, and one transitional cell carcinoma. Frozen
tissues were pulverized with a Mikro-Dismembranator U (B. Braun Biotech
International, Melsungen, Germany), and the RNA was isolated using the
guanidium thiocyanate method.26
A total of 15 µg of RNA
samples was size fractionated on 1.0% formaldehyde agarose gels,
transferred to Nytran nylon membrane (Schleicher and Schuell, Dassel,
Germany), UV cross-linked, and then hybridized with the VEGF-C probe.
Poly(A)+ RNA extracted from HT1080 and HMVEC cells was used as positive
and negative control, respectively. The glyceraldehyde-3-phosphate
dehydrogenase cDNA probe was used as an internal control for equal
loading. The probes were labeled with
[-32P]dCTP (Amersham, Buckinghamshire, UK)
by random priming. Prehybridization and hybridizations were performed
at 42°C in a solution containing 50% formamide, 5x Denhardt’s
solution, 5x sodium chloride-sodium phosphate-EDTA buffer, 0.5%
sodium dodecyl sulfate, and 200 µg/ml salmon sperm DNA. The filters
were washed once for 30 minutes at 65°C with 1x standard saline
citrate (SSC) and 0.1% sodium dodecyl sulfate and then exposed to Fuji
Medical X-ray film.
In Situ Hybridization
In situ hybridization of sections from E16.5 mouse embryos was performed as described.22 The mouse VEGF-C antisense RNA probe was generated from linearized pBluescript II SK+ plasmid (Stratagene, La Jolla, CA) containing an EcoRI/HindIII fragment corresponding to nt 558–979 of mouse VEGF-C cDNA (GenBank Acc. X94216). Radiolabeled RNA was synthesized using T7 polymerase and [35S]UTP (Amersham). About 2 million and 1 million cpm of VEGF-C and VEGFR-3 probes were applied per slide, respectively. After an overnight hybridization, the slides were washed first in 2x SSC, 20 mmol/L dithiothreitol for 1 hour at 50°C, followed by a high-stringency wash in 1x SSC/4x SSC, 30/20 mmol/L dithiothreitol for VEGF-C/VEGFR-3 and 50% deionized formamide for 30 minutes at 65°C, followed by treatment with 20 µg/ml RNase A for 30 minutes at 37°C. The high-stringency wash was repeated for 105/45 minutes for VEGFR-3/VEGF-C. Finally the slides were dehydrated and dried for 30 minutes at room temperature, dipped in photography emulsion, dried and exposed in the dark for 4 weeks, developed using Kodak D-16 developer, counterstained with hematoxylin, and mounted with Permount (Fisher Chemical).
ß-Galactosidase Staining of Embryos
Mouse embryos with one VEGFR-3 allele replaced by the LacZ marker gene were generated by a knock-in strategy as described previously.27 Pregnant mice were sacrificed at E16.5. The embryos were dissected, stained for ß-galactosidase, photographed, subsequently dehydrated, and embedded in paraffin. Seven-micrometer sections were counterstained and photographed.
Antibodies
The monoclonal and polyclonal antibodies against human VEGFR-3 and
VEGF-C, respectively, were produced as described
earlier.16,28,29
Antibodies against VEGFR-1 and
VEGFR-230
were kind gifts from Dr. Herbert Weich
(Braunschweig, Germany). Other antibodies used were against CD31
(platelet/endothelial cell adhesion molecule; Dako Immunoglobulins,
Glostrup, Denmark), an as-yet molecularly undefined blood
vascular endothelial antigen PAL-E (0.15 µg/ml; Monosan, Uden, the
Netherlands), Ki-67 (0.5 µg/ml; Dako Immunoglobulins), and -smooth
muscle actin (SMA) (0.5 µg/ml, clone 1A4; Sigma Chemical
Co.). Control experiments used nonimmune mouse IgG1 and staining
without the primary antibody. Specificity of VEGF-C antibodies was
checked by blocking the binding by incubation overnight with a 10-fold
molar excess of recombinant VEGF-C.16
Immunohistochemistry
Five-micrometer sections of deparaffinized tissues were subjected to heat-induced epitope retrieval (Target Retrieval Solution; Dako, Carpinteria, CA) for 20 minutes at 95°C. The endogenous peroxidase was blocked for 20 minutes in methanol containing 3% H2O2. The primary antibody (1.0 µg/ml for VEGFR-3, 5 µg/ml for VEGF-C) was applied for 2 hours at room temperature and detected using the Vectastain Elite ABC kit according to the manufacturer’s instructions. Peroxidase activity was developed with 3-amino-9-ethyl carbazole (Sigma Chemical Co.) for 15 minutes, and the sections were stained with hematoxylin. Adjacent 5-µm cryosections were air-dried and fixed in cold acetone for 10 minutes. The sections were incubated with blocking serum (5% normal horse serum) and then with primary antibody for 2 hours in a humid atmosphere at room temperature and detected as described above. The PAL-E, VEGFR-1, and VEGFR-2 antibodies gave staining of the frozen sections only. The samples were examined by a trained pathologist (J.A.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the expression of VEGFR-3 and its ligand VEGF-C in the
development of the vasculature of the upper respiratory pathway,
in situ mRNA hybridization was carried out on sections from
E16.5 mouse embryos, in which VEGFR-3 mRNA had previously been detected
specifically in the lymphatic endothelium.22
The results,
shown in Figure 1
, illustrate the key
findings. The VEGFR-3 signal was localized to endothelia of vessels
lining the developing nasopharyngeal and nasal cavity (asterisk and
circle in Figure 1A
, respectively) as well as to the vessels of the
snout around the fibrissal follicles (arrowhead). However, the
photographic emulsion covering the in situ hybridized slides
did not allow a clear-cut resolution of the types of vessels containing
the VEGFR-3 mRNA.
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). At higher magnification,
this signal originated from the conchal mucosa and loose connective
tissue (Figure 1, C and D)
. A homogeneously hybridizing connective
tissue area was also present in the snout (S in Figure 1B
). VEGFR-3 Expression in Heterozygous LacZ/VEGFR-3 Mouse Embryos
To better identify the VEGFR-3-expressing structures in the nasal
mucosa we analyzed embryos having one functional VEGFR-3 allele
replaced by the LacZ marker gene, which allows cells
expressing the VEGFR-3 gene to be stained blue (described in
ref. 27
). In whole-mount ß-galactosidase staining of E16.5 embryos,
strong staining of the vascular plexuses of the nasal cavity was
observed (arrows, Figure 1, E and F
). At this point in mouse
development, VEGFR-3 expression elsewhere occurs predominantly in the
lymphatic vessels.22,23,27
In sections of the skin, which
was used as a control tissue, ß-galactosidase staining was indeed
observed only in the lymphatic vessels (data not shown). In contrast,
in sections of the nasal conchae, the endothelial cells of both
lymphatic and blood vessels were positive. That some of the
VEGFR-3-expressing capillaries contain erythrocytes can be seen in
Figure 1G
. However, a subpopulation of blood vessels that was negative
for ß-galactosidase activity was also recognized (data not shown).
VEGFRs in the Vessels of the Human Respiratory Tract
A comparison of the staining of the three different
VEGFRs in the nasal mucosa is shown in Figure 2, A–C
, and in Table 1
. In the lamina propria of the nasal
mucosa the large lymphatic network gave the strongest signals for
VEFGR-3. However, the vascular endothelia of arterioles and venules as
well as endothelial cells of capillaries were also stained (Figure 2A)
.
The blood vessels in the tracheal and bronchial walls were negative for
VEGFR-3, whereas the surrounding lymphatic vessels were positive
(Figure 2A
, inset, and data not shown). VEGFR-2, which functions as an
additional receptor for the proteolytically cleaved, mature form of
VEGF-C, was stained in lymphatic vessels and in small arterioles,
venules, and capillaries (Figure 2B)
. However, several of the CD31
positive vessels were negative for VEGFR-2. Less than half of the
PAL-E-positive blood vessels of this tissue were positive for VEGFR-1
(compare Figure 2, C and D
). The staining was strongest in
the arterioles, whereas the CD31-positive (not shown),
PAL-E-negative lymphatic vessels were all negative for VEGFR-1
(asterisk in Figure 2C
).
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The detection of VEGF-C by immunohistochemistry has been
summarized in Table 1
. Immunoperoxidase staining for VEGF-C occurred in
the cytoplasm of the nasal respiratory epithelial cells as well as in
the mucus-secreting glands (Figure 2E
, arrows, and data not shown).
Moreover, the endothelial cells of the VEGFR-2- and VEGFR-3-positive
vessels stained weakly for VEGF-C, suggesting that the secreted growth
factor becomes concentrated on endothelial cell surfaces displaying its
receptor (Figure 2E
, arrowhead). Antigen-blocking experiments confirmed
that the staining for VEGF-C was specific (Figure 2F)
.
VEGF-C and VEGFR Expression in Nasal and Nasopharyngeal Tumors
Expression of VEGF-C mRNA was detected both in normal nasal
respiratory mucosa and in tonsilla, as well as in all tumors studied
(data not shown). The hybridization signals were strongest in the nasal
squamous cell carcinomas and adenocarcinomas (data not shown). The
detection of VEGF-C and VEGFRs by immunohistochemistry has been
summarized in Table 1
. There was considerable heterogeneity in the
expression pattern of VEGF-C in the tumor types studied (Table 1)
.
Although at least some staining for VEGF-C was seen in all samples
studied, there was heterogeneity in the intensity of staining. In some
cases, most of the carcinoma cells had strong staining for VEGF-C
(Figure 3A)
.
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. VEGF-C and the cell proliferation marker Ki-67 were also
stained in some of such vessels (Figure 3, A and C)
. Staining for all
three VEGFRs and PAL-E in the frozen sections showed that most of the
VEGFR-3-positive vessels surrounding the tumor islets were blood
vessels (Figure 3, E–H)
. Antibodies against smooth muscle actin gave a
very weak staining of these vessels, suggesting that they were
incompletely covered by pericytes/smooth muscle cells (data not shown).
Angiofibroma, a rare benign tumor, showed strong expression of VEGFR-3
in all endothelial cells (data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently we have found VEGFR-3 in the endothelium of certain fenestrated and discontinuous capillaries besides the lymphatic vessels.25 These results suggested that VEGFR-3 might have a role in discontinuous endothelia, which are known to have increased permeability to macromolecules. The present results show that VEGFR-3 is expressed in the capillaries of nasal mucosa, which in electron microscopic studies have been shown to contain such fenestrated or discontinuous endothelia.1 On the other hand, VEGFR-3 was also expressed in rather large arterioles and venules in the nasal mucosa, where its activity could regulate the capacity of these vessels to sustain other physiological responses, such as the release of nitric oxide for the relaxation of smooth muscle cells and an enhanced vessel diameter.
At least some staining for VEGF-C was seen in all tumor samples studied, and the expression could be confirmed by Northern analysis of VEGF-C mRNA. Coexpression of all VEGF receptors occurred in the vessels surrounding the tumor islets. Most of the VEGFR-3-positive vessels also expressed the blood vascular endothelial cell marker PAL-E. Several endothelial cell nuclei in these vessels displayed the nuclear proliferation marker Ki-67, suggesting that the tumor-associated vessels were proliferating. These results are consistent with the previous study, which showed VEGFR-3 up-regulation in the endothelia of angiogenic blood vessels in breast cancer.28
The nasal conchae are surrounded with a rich vascular plexus, which is an important part of nasal physiology. It can be speculated that VEGF-C via VEGFR-3 could regulate the permeability of the vessels needed for the secretion of nasal mucus and regulation of the lumen of the nasal passages. Proinflammatory cytokines have been shown to up-regulate VEGF-C and VEGF mRNAs,8,21 suggesting that they could have important roles in regulating inflammatory conditions. The permeability and angiogenesis-inducing properties of VEGF-C and VEGFR-3 could be of clinical significance in different pathological conditions of the respiratory tract, such as infections and allergic reactions. The expression of VEGF-C and its receptors in nasal and nasopharyngeal tumors and the surrounding angiogenic blood vessels, respectively, suggests that VEGF-C secreted by the tumor cells could act as an angiogenic growth factor in addition to VEGF. However, VEGF-C might also regulate functions, such as the permeability of the lymphatic vessels surrounding the tumor islets, and thereby contribute to the early metastasis of nasopharyngeal squamous cell carcinomas.
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Acknowledgements |
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Footnotes |
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Supported by the Finnish Cancer Organizations, the Finnish Academy, the University of Helsinki Hospital (TYH 8105), and the State Technology Development Center.
Accepted for publication April 3, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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