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Molecular/Cancer Biology Laboratory and Department of Pathology, Haartman Institute, University of Helsinki, 00014 Helsinki, Finland;
Department of Soft Tissue Pathology, Armed Forces Institute of Pathology, Washington, D.C. 20306-6000, USA; and
Angiogenesis Laboratory, Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Royal Melbourne Hospital, Parkville, Victoria, Australia
2Correspondence: Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Haartman Institute, Haartmaninkatu 3, University of Helsinki, 00014 Helsinki, Finland. E-mail: Kari.Alitalo{at}Helsinki.fi
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cells of the islets of Langerhans, prolactin secreting
cells of the anterior pituitary, adrenal medullary cells, and dispersed
neuroendocrine cells of the gastrointestinal tract. VEGF-D was observed
in the innermost zone of the adrenal cortex and in certain dispersed
neuroendocrine cells. These results suggest that VEGF-C and VEGF-D have
a paracrine function and perhaps a role in peptide release from
secretory granules of certain neuroendocrine cells to surrounding
capillaries.—Partanen, T. A., Arola, J., Saaristo, A., Jussila,
L., Ora, A., Miettinen, M., Stacker, S. A., Achen, M. G.,
Alitalo, K. VEGF-C and VEGF-D expression in neuroendocrine cells and
their receptor, VEGFR-3, in fenestrated blood vessels in human tissues.
Key Words: growth substance • receptor tyrosine kinase • vascular endothelium
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Two ligands are known to bind
to VEGFR-3: VEGF-C and VEGF-D (3
4
5
6
7)
. They are related to
VEGF, which is currently known as the major inducer of angiogenesis and
blood vessel permeability (8)
. The lymph angiogenic
effects of VEGF-C in transgenic mice and chick chorioallantoic membrane
and its paracrine expression pattern with VEGFR-3 in mouse embryos have
suggested that this ligand receptor pair functions in the developing
and adult lymphatic system (1
, 2
, 9
, 10)
. Both VEGF-C and
VEGF-D are also ligands for the VEGFR-2/KDR receptor tyrosine kinase
and can induce angiogenesis in certain experimental systems, apparently
via this receptor (11
12
13)
.
The 2.4 kb VEGF-C mRNA has been detected in the human heart, placenta,
muscle, ovary, and small intestine by Northern blotting and
hybridization analysis (4)
. Very little VEGF-C mRNA was
seen in the brain, liver, or thymus, and peripheral blood leukocytes
appeared negative. In in situ hybridization analysis of 8.5
day mouse embryos (E8.5), the VEGF-C mRNA was found to be distributed
in the cephalic mesenchyme, along the somites and in the tail region
and at E12.5, in the mesenchyme surrounding the jugular vessels, the
developing metanephros, and the mesenterium (2)
. At later
developmental stages, VEGF-C mRNA was detected in the mesenterial
connective tissue and around the gut. Other data on VEGF-C expression
in adult tissues come mainly from tumor angiogenesis studies and thus
are limited (14
15
16)
. Northern blotting analyses of human
VEGF-D mRNA have revealed high levels of transcripts in the heart,
lung, skeletal muscle, and small intestine (5
, 6)
. In
in situ hybridization, several organs in midgestation
embryos were found to express VEGF-D mRNA, such as the limb buds,
acoustic ganglions, teeth, heart, anterior pituitary as well as lung
and kidney mesenchyme, liver, skin, and periosteum (17)
.
In E14.5, E17.5 and young adult mice, a more restricted expression
pattern was obtained, mainly from the lungs (18)
.
The exact biological functions of VEGF-C and VEGF-D are still
speculative, and possibilities to resolve some of the key questions
about their effects are limited, for example, because of difficulties
in the in vitro culture of differentiated lymphatic
endothelium. VEGF-C, like VEGF, has also been reported to enhance the
permeability of blood vessels, and at least the latter induces
fenestrations in endothelial cells both in vivo and in
vitro (19
20
21)
. In adults, VEGF is expressed at sites
where no endothelial proliferation can be detected, such as the
glomerulus, choroid plexus, and pancreas (22)
. The
majority of VEGF expressing cells in the pancreas have been shown to be
ß cells, but , 
, and PP cells may also express VEGF and a
paracrine mechanism of action has been suggested by which VEGF
regulates vascular permeability in the capillary network of the
islet-exocrine portal system (23
, 24)
.
Here we report the unexpected finding that in addition to the lymphatic vasculature, VEGFR-3 is also expressed in many fenestrated endothelia. The receptor VEGFR-3 was found in the blood capillaries of tissues where extensive molecular exchange occurs across the blood vessel wall; such tissues include the endocrine glands and the kidney. Furthermore, neuroendocrine cells expressed VEGF-C and VEGF-D in several tissues, suggesting that these growth factors may have a special role in peptide release from secretory granules to surrounding capillaries.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fetal and adult tissues
The fetal tissues were obtained from 5–6 wk, 13 wk, 14 wk, 15
wk, 17 wk, 20 wk, and 27–30 wk fetuses from abortions induced with
prostaglandins or spontaneously. The gestational age was estimated from
the foot length or the crown-rump measurement. The adult tissues were
obtained from surgical specimens, which included normal tissues from
the Soft Tissue Registry of the Armed Forces Institute of Pathology
(Washington, D.C.) and from the Department of Pathology, Helsinki
University Central Hospital. The study was approved by the ethical
Committee of the Helsinki University Central Hospital. All tissues were
fixed with formalin or, in the case of the fetal tissues, with 4%
paraformaldehyde for 
20 h before alcohol dehydration and paraffin
embedding.
Antibodies
The monoclonal antibodies (mAbs) used were against the
extracellular domain of the VEGFR-3 expressed in a baculovirus system
(25)
. Mouse monoclonal anti-VEGFR-2 was a kind gift from
Dr. Herbert Weich (26)
. The monoclonal antibodies
against VEGF-C (clones #9H7F10, 2C1D11, 9H7C12, and 9H7H3) were
produced in mice immunized with the recombinant mature form of VEGF-C
(27)
and the hybridoma culture fluid was used in dilution
1:2 for immunohistochemistry. Polyclonal rabbit antibodies against the
synthetic peptide corresponding to the amino acid residues 2–18 of the
NH2 terminus of the mature form of VEGF-C (#882;
residues 104–120 of the prepropeptide; ref 4
) were
affinity purified using the peptide coupled to epoxy-activated
Sepharose-6B (15)
and used at 1.5 µg/ml. The mouse
monoclonal antibodies and goat polyclonal antibody against VEGF-D were
kind gifts from Dr. Monica Tsang of the R&D Systems, Inc. (Minneapolis,
Minn.). The clones were screened by immunofluorescence staining of
VEGF-D transfected 293EBNA cells as explained below. Three monoclonal
antibodies that gave the strongest signals (78939.11, 78923.11, and
78935.11) were used in immunohistochemical staining at the
concentration of 10 µg/ml. The mouse monoclonal anti-VEGF-D antibody
VD1 (28)
was used to control some of the stainings at 2.9
µg/ml. The polyclonal goat anti-VEGF-D antibody (749–1AP, R&D) was
used at 0.2 µg/ml. For comparison, polyclonal goat anti-VEGF-D (#N19)
antibody, polyclonal goat anti-VEGF-C (#C20), and polyclonal rabbit
anti-VEGF (#A20) were purchased from Santa Cruz Biotechnology (Santa
Cruz, Calif.). Monoclonal anti-human VEGF-antibody (clone 26503.11) was
purchased from R&D. Guinea pig anti-insulin (1 µg/ml), rabbit
anti-glucagon (6 µg/ml), rabbit anti-somatostatin (20 µg/ml),
rabbit anti-chromogranin A (40 ng/ml), rabbit anti-gastrin (1:400),
monoclonal mouse anti-serotonin (3.3 µg/ml), monoclonal mouse
anti-glial fibrillary acidic protein (20 ng/ml), monoclonal mouse
anti-adrenocorticotropin, rabbit anti-growth hormone (5.2 µg/ml), and
rabbit anti-S-100 (50 ng/ml) were purchased from DAKO Corporation
(Carpinteria, Calif.). Monoclonal mouse anti-smooth muscle actin (SMA,
0.5 µg/ml, clone 1A4) was a product of Sigma (St. Louis, Mo.).
Polyclonal rabbit anti-neurofilament 200 (1:10) was obtained from
Boehringer Mannheim (Helsinki, Finland).
Analysis and use of anti-VEGF-C and anti-VEGF-D antibodies
The anti-VEGF-C and anti-VEGF-D antibodies were tested for
specific staining using immunofluorescence of 293EBNA cells transfected
with pREP7 expression vectors containing either VEGF-C or VEGF-D cDNA
(4
, 27)
, using the FuGENETM6
Transfection Reagent (Boehringer Mannheim). After transfection the
cells were plated on glass coverslips at 2 x
104 cells/cm2 and cultured
for 48 h. Fixation was performed in 4% paraformaldehyde in PBS
for 20 min, the cells were permeabilized with 0.1% Triton X-100 for 15
min, and then incubated with the various antibodies at room temperature
for 30 min in 5% fetal calf serum and 0.5% saponin. Anti-goat or
anti-mouse or anti-rabbit IgG coupled to fluorescein (Jackson Immuno
Research Laboratories, West Grove, Pa.) was then incubated on the
coverslips for 30 min at room temperature. After each incubation, the
coverslips were washed several times with PBS, mounted on slides,
analyzed and photographed using a Zeiss Axiophot microscope. The
possibility of a cross-reaction between the antibodies against VEGF-C
and VEGF-D was also studied by Western blotting analysis. Equivalent
(300 ng) amounts of recombinant mature VEGF-C (27)
, VEGF-D
(R&D) or bovine serum albumin were loaded in 12.5% polyacrylamide gel
electrophoresis and immunoblotted with the antibodies.
Immunohistochemistry
Sections of deparaffinized tissues (4 µm) were heated in a
microwave oven in 10 mmol/l citrate buffer, pH 6.0, at 780 W for 5 min,
followed by 450 W for 10 min. The sections were then treated in
methanol containing 3%
H2O2 for 20 min. The
primary antibodies were incubated for 1 h or overnight followed by
detection by the TSA kit (NEN Life Science Products, Inc., Boston,
Mass.) according to the manufacturer’s instructions. Alternatively,
the detection was performed using a biotinylated anti-mouse Ig and
avidin combined with biotinylated peroxidase complex (Vectastain Elite
kit, Vector Laboratories, Burlingame, Calif.); similar results were
obtained with both methods in parallel studies. After incubation with
normal serum, sections from the adrenal gland were incubated with
avidin D and biotin solutions (Vector Laboratories). The color was
developed with 3-amino-9-ethyl carbazole or with diaminobenzidine
(DAKO) supplemented with hydrogen peroxide and the sections were mildly
counterstained with hematoxylin. Negative controls were done by
omitting the primary antibody, using irrelevant primary antibody of the
same isotype, or blocking the anti-VEGF-C by overnight incubation with
a 10-fold molar excess of the immunogen (27)
. VEGF-D
blocking was done with a 40-fold molar excess of the immunogen for
1 h at room temperature.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The 2.3 kb VEGF-D mRNA was detected in all other endocrine tissues
except the thyroid, whereas the pancreas, adrenal, and thyroid gave the
strongest signals for the 7.0 kb VEGFR-2 mRNA. VEGFR-3 was expressed in
the adrenal medulla and cortex, thyroid gland, small intestine, and
stomach as more abundant 5.8 kb mRNA and a minor 4.5 kb mRNA. There
were very small amounts of the VEGFR-2 and VEGFR-3 mRNAs in the thymus
and testis. The signals for VEGF-C and VEGF-D mRNAs varied
independently of each other, as can be concluded from the quantitation
shown in the column diagrams.
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Specificity of anti-VEGF-C antibodies
The rabbit antisera against human VEGF-C have been previously
characterized and found to lack cross-reactivity against VEGF or
VEGF-B. To test for possible cross-reactions of the antibodies against
VEGF-C and VEGF-D, Western immunoblotting was carried out using
purified recombinant proteins. VEGF-C was not recognized by anti-VEGF-D
(Fig. 2A
, lane 1), nor did anti-VEGF-C recognize VEGF-D (lane 2). In
immunofluorescence analysis of the transfected cells, no
cross-reactions were detected either (Fig. 2B
).
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Immunolocalization of VEGFR-3
The immunohistochemical data have been summarized in Table 1
.
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First trimester embryo
In the 5- to 6-wk embryo, the yolk sac had an abundant
VEGFR-3-positive capillary network (data not shown). Cells of the
notochord were also stained for VEGFR-3. Moderate numbers of
VEGFR-3-positive capillaries were seen in the primitive mesenchyme and
adjacent to the epithelial structures in the developing skin and
gastrointestinal tract. The hepatic sinusoids were positive; the
myocardium showed numerous positive vascular channels whereas the
endocardium was negative.
Thirteen- to 30-wk fetuses and adult tissues
The lymphatic endothelium of the thoracic duct was strongly
positive for VEGFR-3 (Fig. 3A
). Capillaries of the vasa vasorum of the adult aorta also
gave a staining signal for VEGFR-3, as did the vascular endothelia in
the cartilage channels of the vertebral bodies (Fig. 3B
).
The more centrally located vessels with wider lumens either did not
stain for VEGFR-3 or gave only a weak signal (asterisk in Fig. 3B
). The cells lining the large sinusoids of bone marrow
from the fetal skull and vertebral bodies and from the adult bone
marrow expressed VEGFR-3 (Fig. 3C
). In the thymus, only the
lymphatic vessels of the connective tissue septa were stained. The
endothelia lining the splenic sinusoids were also VEGFR-3 positive.
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The capillaries of the lungs showed only occasional weak staining,
whereas the deep lymphatic plexuses in the peribronchial and
perivascular connective tissue stained for VEGFR-3 as did the
superficial, subpleural lymphatic plexuses. In the tongue the
superficial capillaries gave a weak staining signal for VEGFR-3 while
the lymphatic network was prominently positive. In the small intestine
the mucosal lymphatics (lacteals) in the cores of the villi, but not
the capillaries, contained VEGFR-3 immunoreactivity. The endothelia
lining the fetal hepatic sinusoids were also VEGFR-3 positive (Fig. 3D
). The distribution of VEGFR-3 in adult tissues was
similar to that seen in the fetal tissues.
In the kidney, the glomerular capillaries expressed VEGFR-3 (Fig. 3E
). However, in afferent arterioles, which give rise to the
glomerular tuft of capillaries, no staining was detected. In the other
genitourinary organs (see Table 1
), the distribution of VEGFR-3 was
restricted to the lymphatic endothelia. The central nervous system was
negative for VEGFR-3 except for the large, thin-walled capillaries of
the choroid plexus, which are at least partially fenestrated (Fig. 3F
).
In endocrine organs, including the thyroid, parathyroid and adrenal
gland as well as the adenohypophysis, a delicate capillary network was
strongly positive for VEGFR-3 (see Fig. 7E
, 7L
).
Furthermore, in the center of the fetal adrenal gland, where the
sinusoidal capillaries join to form larger venous channels,
immunoreactivity for VEGFR-3 was seen in venous endothelia (Fig. 7F
).
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Immunolocalization of VEGF-C and VEGF-D
Endothelial cells were occasionally stained by the
monoclonal anti-VEGF-C antibodies. This may be explained by ligand
binding to VEGFRs expressed on these cells, as has been described for
VEGF and PlGF immunostaining (29)
(Fig. 4A
). Both monoclonal and polyclonal antibody staining of
frozen material indicated that the smooth muscle cells of blood vessels
express small amounts of VEGF-C and VEGF-D (Fig. 4B
, C
).
Smooth muscle cells in certain other locations were also weakly stained
(data not shown). Otherwise, VEGF-C and VEGF-D showed very limited and
distinct distributions in both paraformaldehyde- or formalin-fixed
fetal and adult tissues (summarized in Table 2
), consistent with the data on their differential mRNA expression in the
endocrine organs. Cytoplasmic VEGF-C positivity was also seen in
trophoblasts of the second trimester placenta.
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Strong staining for VEGF-C was obtained in the nondispersed
neuroendocrine (NE) cells of the pancreas, in the mammotrophs of
anterior pituitary, in adrenal medullary cells, and in dispersed NE
cells of the digestive and respiratory mucosa (Fig. 5A
, B
). Such cells in the adrenal gland, digestive, and
respiratory mucosa corresponded in serial sections to the chromogranin
A (CgA) -positive NE cells. In contrast, VEGF-D-positive cells were not
found in the respiratory tract.
|
In detailed analysis of the fetal and adult gastrointestinal
tracts, the serotonin- and ChA-expressing NE cells in the adult colon
were strongly and consistently immunoreactive for VEGF-C (Fig. 5C
, D
). Only a few VEGF-D-positive gastrointestinal NE cells
were found. To better identify such cells, adjacent sections were
stained for gastrin, serotonin, and somatostatin. VEGF-D positivity
could be localized in the gastrin-positive NE cells of the fetal colon
(Fig. 5E
, F
). In the colonic mucosa, there was
apparently no association between VEGF-C- or VEGF-D-expressing cells
and VEGFR-3-positive blood vessel endothelia. However, staining for the
vascular endothelial marker CD31 revealed a rich capillary network
(Fig. 5D
, inset). In the duodenum, only the lacteals were
positive for VEGFR-3.
In adjacent sections of the adult pancreas, the expression patterns of
VEGF-C and glucagon appeared identical, with an accentuation of
labeling in cells localized at the periphery of the islets of
Langerhans (Fig. 6A
, B
, C
, D
). In addition to the cells of the Langerhans islets,
NE cells among the acinar portions were positive for VEGF-C (Fig. 6E
). The expression of VEGFR-3 was restricted to the
lymphatic vessels in the stroma; as an exception among the other
endocrine organs, the fenestrated endothelium of the pancreas was
negative for VEGFR-3 (Fig. 6F
). Pancreatic acinar cells and
NE cells lying basally in the pancreatic ducts were often stained for
VEGF-D, whereas the islets of Langerhans were devoid VEGF-D
immunoreactivity.
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Scattered, migrating medullary cells in fetal adrenal gland were
found to stain for VEGF-C and ChA, as did cells in the adult adrenal
medulla (Fig. 7A
, B
). The medullary cells that were adjacent to the cortical
cell layer often gave the strongest signals. In the adult adrenal
cortex, the reticular cells were also VEGF-D positive (Fig. 7C
, D
), whereas in immature fetal adrenal gland (16 wk), where only
the fetal and definitive zones were present, no VEGF-D was detected.
The cortical sinusoids as well as the venous channels of the adrenal
medulla were VEGFR-3 positive (Fig. 7E
, F
). Staining
of frozen sections indicated that the capillaries adjacent to the
VEGF-C-positive cells were either positive for VEGFR-2 or VEGFR-3 (Fig. 7G
, H
, I
) or both. In comparison, the adrenal medulla did not
stain for VEGF. In the anterior pituitary gland, the prolactin
secreting cells were also found to express VEGF-C, and the adjacent
sinusoids were stained for VEGFR-3. VEGF-C and VEGF had overlapping
patterns of expression in the anterior pituitary gland.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The consistent expression of VEGFR-3 in the lymphatics was shown in
different organs in fetal and adult tissues. For example, the lacteals
located in the core of the intestinal villi were strongly positive for
VEGFR-3. However, no small lymphatics that drain into the lacteals
could be demonstrated, probably due to the limitation of detection
sensitivity. Although the lymphatic system seems to develop from large
central veins in the embryonic jugular, retroperitoneal, and
perimesonephric regions (10
, 30)
, surprisingly few adult
tissues retained VEGFR-3 expression in their venous endothelia. Such
endothelia were seen in the vessels of the cartilage channels in
vertebral bodies, venous canals of the adrenal medulla, and the splenic
venous sinuses.
The capillary endothelia vary structurally according to their location
and functions; the major groups include continuous and discontinuous
endothelia. Continuous endothelium is present in tissues such as
connective tissue, lung, and brain whereas discontinuous endothelium is
typical of tissues undergoing high molecular exchange across the vessel
wall, such as secretion of hormones from endocrine glands and
filtration in glomeruli and in the choroid plexus. Electron microscopic
studies have established that, for example, the capillaries of the
anterior pituitary and glomerulus are fenestrated (31)
. In
this study, we found differences in VEGFR-3 expression between
continuous and discontinuous endothelia, the former typically being
negative and the latter positive. This finding suggests that VEGFR-3
plays a role in the transport functions of the discontinuous and more
permeable endothelia in locations such as in the endocrine organs and
kidney glomeruli.
Nonendothelial expression of VEGFR-3 was apparent in notochordal cells
of 5-wk-old embryos in accordance with earlier observations from avian
embryos (32)
and in the cytotrophoblast layer and
intermediary trophoblasts in the first and second trimester placenta,
but not in term placenta. Cytotrophoblasts lining blood-containing
placental spaces are related to endothelial cells. Recently, it has
become clear that invading cytotrophoblasts transform their receptor
phenotype to resemble the endothelial cells they replace
(33)
. We found that the cytotrophoblasts were stained for
both VEGFR-3 and VEGF-C. It is as yet unknown whether VEGF-C is
produced in these cells or just adsorbed to its receptor on their
surface.
Anti-VEGFR-3 antibodies have been shown to be valuable for
distinguishing vascular vs. lymphatic endothelium in the skin
(25
, 34)
. Furthermore, VEGFR-3 has been demonstrated in
endothelial cells of the proliferating neovasculature in breast cancer
(15)
, other tumors, and vascular tumor cells of
endothelial origin (35)
. It has also been shown that
VEGFR-1 and VEGFR-2 are induced in proliferating neovasculature and
that these receptors are present in fenestrated endothelium (36
, 26)
. Our present results on VEGFR-3 expression are consistent
with these findings. However, although several fenestrated endothelia
were stained for VEGFR-3, the fenestrated capillaries of the pancreas
and intestine were negative. According to our present findings, it is
thus possible that VEGF-C and VEGF-D secreted by the neuroendocrine
cells signal via VEGFR-2 in nearby capillary endothelium.
VEGFR-3 was also expressed at major sites of hematopoiesis or blood cell trafficking, such as in the sinusoids of the liver, spleen, and bone marrow. The endothelium at these sites is capable of regulating the translocation of hematopoietic cells, which may be related to the function of VEGFR-3 in these locations.
In a relatively short time, four distinct markers for lymphatic
endothelium have been characterized. These include VEGFR-3, podoplanin,
Prox1, and LYVE-1 (25
, 37
38
39)
. Podoplanin has been
localized also to podocytes, parietal epithelial cells of Bowman’s
capsule, lung, choroid plexus, leptomeninges, osteocytes, and
osteoblasts (39)
. Podoplanin and VEGFR-3 antigens were
found to overlap in lymphatic endothelium, benign vascular tumors, and
angiosarcomas (40)
. At least during development, Prox1 is
also expressed in the lens, heart, liver, pancreas, and the central
nervous system (37)
. Our unpublished results have also
demonstrated differences between the distributions of the VEGFR-3 and
LYVE-1 markers (the authors and D. Jackson, unpublished results).
The ligands for VEGFR-3, VEGF-C and VEGF-D showed a narrow tissue
distribution. Results obtained from the frozen tissue sections where
smooth muscle cells, and occasionally endothelial cells, were stained
with antibodies against these ligands suggest paracrine functions for
these growth factors in the arterial wall. The high level of expression
of VEGF-C in NE cells of respiratory and especially of digestive mucosa
and endocrine organs lined by fenestrated endothelial cells suggests
that it could play a role in the interaction between the NE cells and
capillary endothelia and perhaps in hormone secretion. Alternatively,
VEGF-C and VEGF-D may have roles unrelated to the vasculature in the
neuroendocrine system. Previously, VEGF was located in the NE cells in
the prostatic verumontanum epithelia (41)
and in the
digestive mucosa (42)
. VEGF and VEGF-C, which are both
potent vascular permeability factors, were partly colocalized in the
anterior pituitary gland. Although most of the VEGF-C-positive cells
were prolactin producing, such cells may also include the follicular
stellate cells of the pituitary gland (43
, 44)
.
In conclusion, we have evaluated the distribution of VEGFR-3 and its ligands, VEGF-C and VEGF-D, in human fetal and adult tissues. Although our results confirm the consistent presence of this receptor in lymphatic endothelia, they also reveal its expression in discontinuous capillary endothelia and production of its two ligands by several types of neuroendocrine cells. These data suggest that VEGFR-3 signals participate in the maintenance on filtration or secretion functions across fenestrated capillary endothelia.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication December 15, 1999.
Revision received April 27, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 Y. Nakamura, H. Yasuoka, M. Tsujimoto, K. Yoshidome, M. Nakahara, K. Nakao, M. Nakamura, and K. Kakudo Nitric Oxide in Breast Cancer: Induction of Vascular Endothelial Growth Factor-C and Correlation with Metastasis and Poor Prognosis Clin. Cancer Res., February 15, 2006; 12(4): 1201 - 1207. [Abstract] [Full Text] [PDF]
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 C. Betsholtz and A. Armulik Homeostatic functions of vascular endothelial growth factor in adult microvasculature Am J Physiol Heart Circ Physiol, February 1, 2006; 290(2): H509 - H511. [Full Text] [PDF]
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T. Kamba, B. Y. Y. Tam, H. Hashizume, A. Haskell, B. Sennino, M. R. Mancuso, S. M. Norberg, S. M. O'Brien, R. B. Davis, L. C. Gowen, et al. VEGF-dependent plasticity of fenestrated capillaries in the normal adult microvasculature Am J Physiol Heart Circ Physiol, February 1, 2006; 290(2): H560 - H576. [Abstract] [Full Text] [PDF]
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 A. Salameh, F. Galvagni, M. Bardelli, F. Bussolino, and S. Oliviero Direct recruitment of CRK and GRB2 to VEGFR-3 induces proliferation, migration, and survival of endothelial cells through the activation of ERK, AKT, and JNK pathways Blood, November 15, 2005; 106(10): 3423 - 3431. [Abstract] [Full Text] [PDF]
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T. Tammela, A. Saaristo, M. Lohela, T. Morisada, J. Tornberg, C. Norrmen, Y. Oike, K. Pajusola, G. Thurston, T. Suda, et al. Angiopoietin-1 promotes lymphatic sprouting and hyperplasia Blood, June 15, 2005; 105(12): 4642 - 4648. [Abstract] [Full Text] [PDF]
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 X. Jimenez, D. Lu, L. Brennan, K. Persaud, M. Liu, H. Miao, L. Witte, and Z. Zhu A recombinant, fully human, bispecific antibody neutralizes the biological activities mediated by both vascular endothelial growth factor receptors 2 and 3 Mol. Cancer Ther., March 1, 2005; 4(3): 427 - 434. [Abstract] [Full Text] [PDF]
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 T. Tammela, B. Enholm, K. Alitalo, and K. Paavonen The biology of vascular endothelial growth factors Cardiovasc Res, February 15, 2005; 65(3): 550 - 563. [Abstract] [Full Text] [PDF]
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V. Kaushal, P. Mukunyadzi, R. A. Dennis, E. R. Siegel, D. E. Johnson, and M. Kohli Stage-Specific Characterization of the Vascular Endothelial Growth Factor Axis in Prostate Cancer: Expression of Lymphangiogenic Markers Is Associated with Advanced-Stage Disease Clin. Cancer Res., January 15, 2005; 11(2): 584 - 593. [Abstract] [Full Text] [PDF]
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 M. Groger, R. Loewe, W. Holnthoner, R. Embacher, M. Pillinger, G. S. Herron, K. Wolff, and P. Petzelbauer IL-3 Induces Expression of Lymphatic Markers Prox-1 and Podoplanin in Human Endothelial Cells J. Immunol., December 15, 2004; 173(12): 7161 - 7169. [Abstract] [Full Text] [PDF]
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 A. Hoeben, B. Landuyt, M. S. Highley, H. Wildiers, A. T. Van Oosterom, and E. A. De Bruijn Vascular Endothelial Growth Factor and Angiogenesis Pharmacol. Rev., December 1, 2004; 56(4): 549 - 580. [Abstract] [Full Text] [PDF]
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 I. NILSSON, C. ROLNY, Y. WU, B. PYTOWSKI, D. HICKLIN, K. ALITALO, L. CLAESSON-WELSH, and S. WENNSTROM Vascular endothelial growth factor receptor-3 in hypoxia-induced vascular development FASEB J, October 1, 2004; 18(13): 1507 - 1515. [Abstract] [Full Text] [PDF]
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B. Sipos, W. Klapper, M.-L. Kruse, H. Kalthoff, D. Kerjaschki, and G. Kloppel Expression of Lymphangiogenic Factors and Evidence of Intratumoral Lymphangiogenesis in Pancreatic Endocrine Tumors Am. J. Pathol., October 1, 2004; 165(4): 1187 - 1197. [Abstract] [Full Text] [PDF]
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Y. Zeng, K. Opeskin, M. E. Baldwin, L. G. Horvath, M. G. Achen, S. A. Stacker, R. L. Sutherland, and E. D. Williams Expression of Vascular Endothelial Growth Factor Receptor-3 by Lymphatic Endothelial Cells Is Associated with Lymph Node Metastasis in Prostate Cancer Clin. Cancer Res., August 1, 2004; 10(15): 5137 - 5144. [Abstract] [Full Text] [PDF]
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 K. Persaud, J.-C. Tille, M. Liu, Z. Zhu, X. Jimenez, D. S. Pereira, H.-Q. Miao, L. A. Brennan, L. Witte, M. S. Pepper, et al. Involvement of the VEGF receptor 3 in tubular morphogenesis demonstrated with a human anti-human VEGFR-3 monoclonal antibody that antagonizes receptor activation by VEGF-C J. Cell Sci., June 1, 2004; 117(13): 2745 - 2756. [Abstract] [Full Text] [PDF]
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J.-L. Su, J.-Y. Shih, M.-L. Yen, Y.-M. Jeng, C.-C. Chang, C.-Y. Hsieh, L.-H. Wei, P.-C. Yang, and M.-L. Kuo Cyclooxygenase-2 Induces EP1- and HER-2/Neu-Dependent Vascular Endothelial Growth Factor-C Up-Regulation: A Novel Mechanism of Lymphangiogenesis in Lung Adenocarcinoma Cancer Res., January 15, 2004; 64(2): 554 - 564. [Abstract] [Full Text] [PDF]
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T. VEIKKOLA, M. LOHELA, K. IKENBERG, T. MAKINEN, T. KORFF, A. SAARISTO, T. PETROVA, M. JELTSCH, H. G. AUGUSTIN, and K. ALITALO Intrinsic versus microenvironmental regulation of lymphatic endothelial cell phenotype and function FASEB J, November 1, 2003; 17(14): 2006 - 2013. [Abstract] [Full Text] [PDF]
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Y. Nakamura, H. Yasuoka, M. Tsujimoto, Q. Yang, S. Imabun, M. Nakahara, K. Nakao, M. Nakamura, I. Mori, and K. Kakudo Flt-4-Positive Vessel Density Correlates with Vascular Endothelial Growth Factor-D Expression, Nodal Status, and Prognosis in Breast Cancer Clin. Cancer Res., November 1, 2003; 9(14): 5313 - 5317. [Abstract] [Full Text] [PDF]
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 J. Dixelius, T. Makinen, M. Wirzenius, M. J. Karkkainen, C. Wernstedt, K. Alitalo, and L. Claesson-Welsh Ligand-induced Vascular Endothelial Growth Factor Receptor-3 (VEGFR-3) Heterodimerization with VEGFR-2 in Primary Lymphatic Endothelial Cells Regulates Tyrosine Phosphorylation Sites J. Biol. Chem., October 17, 2003; 278(42): 40973 - 40979. [Abstract] [Full Text] [PDF]
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J. Rutanen, P. Leppanen, T. T Tuomisto, T. T Rissanen, M. O Hiltunen, I. Vajanto, M. Niemi, T. Hakkinen, K. Karkola, S. A Stacker, et al. Vascular endothelial growth factor-D expression in human atherosclerotic lesions Cardiovasc Res, October 1, 2003; 59(4): 971 - 979. [Abstract] [Full Text] [PDF]
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 B. K. McColl, M. E. Baldwin, S. Roufail, C. Freeman, R. L. Moritz, R. J. Simpson, K. Alitalo, S. A. Stacker, and M. G. Achen Plasmin Activates the Lymphangiogenic Growth Factors VEGF-C and VEGF-D J. Exp. Med., September 15, 2003; 198(6): 863 - 868. [Abstract] [Full Text] [PDF]
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 K. C. Boardman and M. A. Swartz Interstitial Flow as a Guide for Lymphangiogenesis Circ. Res., April 18, 2003; 92(7): 801 - 808. [Abstract] [Full Text] [PDF]
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 M. Ishikawa, J. Kitayama, S. Kazama, and H. Nagawa Expression of Vascular Endothelial Growth Factor C and D (VEGF-C and -D) is an Important Risk Factor for Lymphatic Metastasis in Undifferentiated Early Gastric Carcinoma Jpn. J. Clin. Oncol., January 1, 2003; 33(1): 21 - 27. [Abstract] [Full Text] [PDF]
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P. Salven, S. Mustjoki, R. Alitalo, K. Alitalo, and S. Rafii VEGFR-3 and CD133 identify a population of CD34+ lymphatic/vascular endothelial precursor cells Blood, January 1, 2003; 101(1): 168 - 172. [Abstract] [Full Text] [PDF]
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 C. Cursiefen, U. Schlotzer-Schrehardt, M. Kuchle, L. Sorokin, S. Breiteneder-Geleff, K. Alitalo, and D. Jackson Lymphatic Vessels in Vascularized Human Corneas: Immunohistochemical Investigation Using LYVE-1 and Podoplanin Invest. Ophthalmol. Vis. Sci., July 1, 2002; 43(7): 2127 - 2135. [Abstract] [Full Text] [PDF]
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A. SAARISTO, T. VEIKKOLA, B. ENHOLM, M. HYTONEN, J. AROLA, K. PAJUSOLA, P. TURUNEN, M. JELTSCH, M. J. KARKKAINEN, D. KERJASCHKI, et al. Adenoviral VEGF-C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes FASEB J, July 1, 2002; 16(9): 1041 - 1049. [Abstract] [Full Text] [PDF]
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 L. Jussila and K. Alitalo Vascular Growth Factors and Lymphangiogenesis Physiol Rev, July 1, 2002; 82(3): 673 - 700. [Abstract] [Full Text] [PDF]
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 H. Kubo, R. Cao, E. Brakenhielm, T. Makinen, Y. Cao, and K. Alitalo Blockade of vascular endothelial growth factor receptor-3 signaling inhibits fibroblast growth factor-2-induced lymphangiogenesis in mouse cornea PNAS, June 25, 2002; 99(13): 8868 - 8873. [Abstract] [Full Text] [PDF]
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 A. N. Witmer, J. Dai, H. A. Weich, G. F.J.M. Vrensen, and R. O. Schlingemann Expression of Vascular Endothelial Growth Factor Receptors 1, 2, and 3 in Quiescent Endothelia J. Histochem. Cytochem., June 1, 2002; 50(6): 767 - 778. [Abstract] [Full Text] [PDF]
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 G. Oliver and M. Detmar The rediscovery of the lymphatic system: old and new insights into the development and biological function of the lymphatic vasculature Genes & Dev., April 1, 2002; 16(7): 773 - 783. [Full Text] [PDF]
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 R. K. Jain and B. T. Fenton Intratumoral Lymphatic Vessels: A Case of Mistaken Identity or Malfunction? J Natl Cancer Inst, March 20, 2002; 94(6): 417 - 421. [Full Text] [PDF]
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J. D. White, P. W. Hewett, D. Kosuge, T. McCulloch, B. C. Enholm, J. Carmichael, and J. C. Murray Vascular Endothelial Growth Factor-D Expression Is an Independent Prognostic Marker for Survival in Colorectal Carcinoma Cancer Res., March 1, 2002; 62(6): 1669 - 1675. [Abstract] [Full Text] [PDF]
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M. E. Baldwin, S. Roufail, M. M. Halford, K. Alitalo, S. A. Stacker, and M. G. Achen Multiple Forms of Mouse Vascular Endothelial Growth Factor-D Are Generated by RNA Splicing and Proteolysis J. Biol. Chem., November 16, 2001; 276(47): 44307 - 44314. [Abstract] [Full Text] [PDF]
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C. M. Carreira, S. M. Nasser, E. di Tomaso, T. P. Padera, Y. Boucher, S. I. Tomarev, and R. K. Jain LYVE-1 Is Not Restricted to the Lymph Vessels: Expression in Normal Liver Blood Sinusoids and Down-Regulation in Human Liver Cancer and Cirrhosis Cancer Res., November 1, 2001; 61(22): 8079 - 8084. [Abstract] [Full Text] [PDF]
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M. J. Karkkainen, A. Saaristo, L. Jussila, K. A. Karila, E. C. Lawrence, K. Pajusola, H. Bueler, A. Eichmann, R. Kauppinen, M. I. Kettunen, et al. A model for gene therapy of human hereditary lymphedema PNAS, October 5, 2001; (2001) 221449198. [Abstract] [Full Text] [PDF]
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E. Kriehuber, S. Breiteneder-Geleff, M. Groeger, A. Soleiman, S. F. Schoppmann, G. Stingl, D. Kerjaschki, and D. Maurer Isolation and Characterization of Dermal Lymphatic and Blood Endothelial Cells Reveal Stable and Functionally Specialized Cell Lineages J. Exp. Med., September 17, 2001; 194(6): 797 - 808. [Abstract] [Full Text] [PDF]
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T. Karpanen and K. Alitalo Lymphatic Vessels as Targets of Tumor Therapy? J. Exp. Med., September 17, 2001; 194(6): F37 - F42. [Full Text] [PDF]
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K. ILJIN, M. J. KARKKAINEN, E. C. LAWRENCE, M. A. KIMAK, M. UUTELA, J. TAIPALE KATRI PAJUSOLA LEENA ALHONEN, M. HALMEKYTO, D. N. FINEGOLD, R. E. FERRELL, and K. ALITALO VEGFR3 gene structure, regulatory region, and sequence polymorphisms FASEB J, April 1, 2001; 15(6): 1028 - 1036. [Abstract] [Full Text] [PDF]
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B. Enholm, T. Karpanen, M. Jeltsch, H. Kubo, F. Stenback, R. Prevo, D. G. Jackson, S. Yla-Herttuala, and K. Alitalo Adenoviral Expression of Vascular Endothelial Growth Factor-C Induces Lymphangiogenesis in the Skin Circ. Res., March 30, 2001; 88(6): 623 - 629. [Abstract] [Full Text] [PDF]
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M. J. Karkkainen, A. Saaristo, L. Jussila, K. A. Karila, E. C. Lawrence, K. Pajusola, H. Bueler, A. Eichmann, R. Kauppinen, M. I. Kettunen, et al. A model for gene therapy of human hereditary lymphedema PNAS, October 23, 2001; 98(22): 12677 - 12682. [Abstract] [Full Text] [PDF]
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