Lymphangiogenesis Lymphedema and Cancer
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Angiogenesis (blood vessel growth), lymphangiogenesis (lymph system growth) are all intrinsically connected with lymphedema and share many of the same genes. We have several pages on both processes.
May 23, 2008
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The role of tumor lymphangiogenesis in metastatic spread
(The
FASEB Journal. 2002;16:922-934.)
© 2002 FASEB
Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Victoria 3050, Australia
1Correspondence: Ludwig Institute for Cancer Research, Post Office Box 2008, Royal Melbourne Hospital, Victoria 3050, Australia. E-mail: steven.stacker@ludwig.edu.au
ABSTRACT
The high mortality rates associated with cancer can be attributed to the metastatic spread of tumor cells from the site of their origin. Tumor cells invade either the blood or lymphatic vessels to access the general circulation and then establish themselves in other tissues. Clinicopathological data suggest that the lymphatics are an initial route for the spread of solid tumors. Detection of sentinel lymph nodes by biopsy provides significant information for staging and designing therapeutic regimens. The role of angiogenesis in facilitating the growth of solid tumors has been well established, but the presence of lymphatic vessels and the relevance of lymphangiogenesis to tumor spread are less clear. Recently, the molecular pathway that signals for lymphangiogenesis and relatively specific markers for lymphatic endothelium have been described allowing analyses of tumor lymphangiogenesis to be performed in animal models. These studies demonstrate that tumor lymphangiogenesis is a major component of the metastatic process and implicate members of the VEGF family of growth factors as key mediators of lymphangiogenesis in both normal biology and tumors.—Stacker, S. A., Baldwin, M. E., Achen, M. G. The role of tumor lymphangiogenesis in metastatic spread.
Key Words: metastasis • VEGF-C • VEGF-D • VEGF receptors
INTRODUCTION
EACH YEAR IN
the United States more than 500,000
people die principally as a result of the
metastatic spread of cancer
(1
,
2)
. Cells from malignant primary tumors spread from their sites
of
origin to invade local tissue and enter the systemic circulation
(3
, 4) .
This spread can
occur directly into the local tissue or via
blood vessels (hematogenous
spread) and lymphatics (lymphogenous spread) or
by invasion of body
cavities such as the pleura or peritoneum.
Cells must first invade
either blood or lymphatic vessels to enter the
circulation. In the
case of blood vessels, this requires
penetration of the basement
membrane and migration through the cellular
layers of the vessel (Fig.
1 ). In contrast,
it has been proposed that the entry of tumor cells into the
lymphatic
circulation may be easier due to the nature of lymphatic vessels
and
that the thin and discontinuous basement membrane of
lymphatics (5
6 7)
might not provide a significant barrier to
entry of tumor cells.
|
Tumor cells
can escape from the primary site by entering existing vessels
or new vessels actively recruited into the primary tumor (4)
. The relative importance of the established vessels vs. the
active
invasion of a tumor by new blood and lymphatic vessels for
the
initial metastatic spread of tumor cells is still unclear. Previous
studies have established the role of angiogenesis in solid
tumor
growth (8 9
10 11
12 13
14) ,
and there is some
evidence indicating a direct role for angiogenesis in
hematogenous tumor
spread (15) . The onset
of angiogenesis within small clusters of tumors
cells, known as the
‘angiogenic-switch’, is important for blood
vessel formation in
further development of malignant cells that
have already spread from
the primary tumor (16
, 17) .
However, little
is known about the role of tumor
lymphangiogenesis (the growth and
production of new lymphatic vessels) in the
spread of tumors and
whether this process is important in the
overall context of lymphatic
spread (18 , 19)
.
Clinical and
pathological data point to the spread of solid tumors
via the lymphatics as an important early event in metastatic
disease
(1 , 3)
. Detection of tumor cells in lymphatic vessels and
regional lymph
nodes is a key factor in the staging of human tumors
and forms the
basis for treatment of regional lymph nodes by
surgery and radiation
therapy (1 , 3
, 20 ,
21)
. Although a large body of clinical and
pathological evidence points
to a major role for the lymphatics in the
initial spread of malignant
tumors, the exact mechanism whereby tumor cells enter the
lymphatic system
is uncertain (18 , 22
, 23) .
The role of
lymphangiogenesis in promoting the metastatic spread of
tumor cells via the lymphatics has been an area that has achieved
little publicity in the past decade. This has in part been
due to
difficulty in studying lymphatic vessels because of
their morphology
and a lack of lymphatic-specific markers (24)
. In reviewing this topic, we will emphasize the progress made
in the
past 2 years in identifying novel lymphatic markers, as
well as
lymphangiogenic factors and receptors responsible for
the generation
and maintenance of the lymphatic system. This
will include a summary
of the recent clinical and experimental data
that support the notion
that lymphangiogenic factors influence the
spread of tumors and a
discussion of the potential therapeutic options
for anti-lymphangiogenic
treatment of cancer.
STRUCTURE AND FUNCTION OF THE LYMPHATICS
The lymphatic
system consists of thin-walled, low-pressure vessels, nodes
that occur along the course of lymphatic vessels, aggregations
of
lymphoid tissue, such as the spleen and thymus, and circulating
lymphocytes
(5 , 6
, 25) .
By regulating
fluid absorption from the interstitium,
harboring macrophages, and
providing a passage for lymphocyte trafficking,
the lymphatic system
maintains plasma volume, prevents increases in
tissue pressure, and
has an important role in immune system function
(26)
. Lymphatic vessels are distinct functionally
and ultrastructurally
from their blood vessel counterparts (27)
. Compared to blood vessels, the walls of
lymphatic vessels are
thinner. This is in part due to the highly
attenuated cytoplasm of
lymphatic endothelial cells (Fig. 1)
(5 , 6
, 27 ,
28)
. The endothelium of lymphatic vessels contains
fewer tight junctions
than that of blood vessels and it has been
speculated that this may
be the cause of the greater permeability of the
lymphatic vessels
(25) .
Lymphatic
capillaries have a poorly developed or absent
basal lamina and lack
associated pericytes (5
, 6) .
Their lumens are 
threefold wider than the lumens of blood
capillaries and are more
irregularly shaped, appearing collapsed in
tissue section (5
, 28 ,
29)
. Lymphatic vessels are connected to the
extracellular matrix by
reticular fibers and collagen (5
, 6 ,
30)
. Upon increases in interstitial fluid and
pressure, the connecting
tissue fibers become stretched, thereby opening
the lymphatic lumen
(27 , 29
, 31) .
As the lumen
widens, the endothelial cells, which overlap under normal conditions,
move apart, effectively opening intercellular channels to
aid fluid
and macromolecular uptake into the lymphatic vessel (5
, 6 , 29
, 30)
(Fig. 1)
.
LYMPHANGIOGENESIS
The growth of
lymphatic vessels, lymphangiogenesis, has received considerable
attention in the last 2 years, due to the identification of
proteins
specifically expressed on lymphatic vessels and the
discovery of
molecules that can drive lymphatic vessel growth (32
33 34
35 36
37 38)
. Vascular remodeling associated with lymphangiogenesis
and
angiogenesis is likely to involve similar processes,
although formal
evidence of this assertion has yet to be
published. In response to
molecular mediators, both lymphatic and
vascular endothelial cells
proliferate and migrate toward a stimulus as
the extracellular matrix
is degraded, followed by association of the
endothelial cells into
tube-like structures (31
, 39) .
New production
and realignment of the extracellular matrix and
controlled apoptosis
at appropriate sites are required for blood
vascular and lymphatic
system formation. Besides using similar
processes of remodeling,
blood and lymphatic vessels are closely
associated in vivo. Blood
vascular plexuses often accompany lymphatic
vessels (27
, 40) ,
although the
ratio of lymphatic to blood vessels varies
depending on tissue type
and function (29)
. An abundant and neighboring blood supply provides essential
nourishment for lymphatic vessels that is needed for adequate
function, intrinsic contractility of lymphatic endothelial cells,
and
an ability to regenerate rapidly when required, processes essential
for maintaining fluid balance within an organism (31)
. The close association of blood and lymphatic
vessels and their
coordinated development in vivo suggest that some molecules
may
control both angiogenesis and lymphangiogenesis (41)
.
LYMPHATIC MARKERS
Progress in
understanding lymphangiogenesis has been hampered by
the very similar characteristics of blood and lymphatic vessels
in
tissue section and is confounded by the lack of lymphatic-specific
markers
(24) .
Consequently,
visualization of lymphatic vessels in the past
was restricted to
imaging techniques involving the injection of
dyes that are
specifically taken up by the lymphatics (reviewed
in refs 29
, 31 ).
Vital dyes, such
as Evans blue, trypan blue, and Patent blue,
which are readily taken
up by lymphatic but not blood vessels, are less
toxic than the
materials that were previously used (29)
. These dyes and fluorescent conjugates of high
molecular weight
material such as rhodamine-dextran are now used
routinely in animal
experiments (37 , 42)
. Until recently, immunohistochemical
identification of lymphatic
vessels was achieved, somewhat unreliably, by
comparing staining of
pan-endothelial markers with markers of the basal lamina.
The pan-endothelial
marker PECAM-1/CD31, which is expressed on both blood
and lymphatic
vessels (43 , 44)
, has been used in combination with the
basement membrane markers
laminin and collagen type IV (45
, 46) .
Vessels that
reacted with PECAM-1 antibodies but lacked
basement membrane staining
and red blood cells in their lumens were deemed
lymphatic (44
, 47) .
Use of the blood
vessel-specific marker PAL-E in combination with PECAM-1 has
also
been useful for identifying lymphatic vessels in human tissue
sections (44 , 47)
.
More accurate
and simplified lymphatic vessel identification has
recently been made possible by the discovery of molecules that
are
specifically expressed by lymphatic endothelium (Table 1)
. Vascular endothelial growth factor receptor-3 (VEGFR-3) is
predominantly expressed on lymphatic endothelium in normal adult
tissues (48 , 49)
; it is also up-regulated on blood vessel endothelium
in tumors (44
, 50) and
in wound
healing (51) . The
lymphatic receptor for hyaluronan, LYVE-1, has been reported
to be a
specific marker of lymphatic vessels (52
, 53) and
is
thought to function in transporting hyaluronan from the tissue
to the
lymph (53 , 54)
. Antibodies to LYVE-1 have been used to
localize the receptor to
lymphatic endothelium in normal and tumor
tissue (38
, 52) .
Although
appearing relatively specific for lymphatic
endothelium, staining of
blood vessels in normal lung tissue has been
observed (R. A.
Williams, S. A. Stacker, and M. G. Achen,
unpublished observations)
as has staining of blood vessels in normal
hepatic blood sinusoidal
endothelial cells (55)
. The transcription factor Prox1, although required for
lymphatic
vessel development and expressed on lymphatic endothelium
(56)
, is also expressed in other cell types and tissues,
including
hepatocytes of the liver (57)
and lens tissue (58)
, and is therefore of limited use immunohistochemically to
identify
lymphatic vessels. Podoplanin and desmoplakin have been
reported to
be markers for lymphatic endothelium, but also react
with other cell
types (59 60
61) .
In summary, a more
extensive range of markers for lymphatic endothelium is now
available
that should aid in defining the role of lymphatic vessels
in tumor
biology.
|
LYMPHANGIOGENIC GROWTH FACTORS
Recently
VEGF-C and VEGF-D, members of the VEGF family of secreted glycoproteins
that are ligands for VEGFR-3 (Flt4) (34
, 62 63
64) (Fig.
2
, Table 2 ), have been
identified as regulators of lymphangiogenesis in
mammals (32
, 42) .
Structurally,
these protein growth factors differ from the
angiogenic growth factor
VEGF (65) because of
the presence of amino- and carboxyl-terminal propeptides, but
retain
the central VEGF homology domain (VHD) containing the
cystine knot
motif that is conserved in all VEGF family members
(66
, 67) .
Biosynthetic
processing of the VEGF-C and VEGF-D
polypeptides, by as yet
uncharacterized extracellular proteases,
results in mature growth
factor consisting of dimers of the VHD that
bind VEGFR-3 and VEGFR-2
(KDR/Flk-1) with high affinity (68
, 69) .
The unprocessed
and partially processed forms of VEGF-C and
VEGF-D have reduced
affinity for both receptors, indicating that
processing is important
for receptor binding. VEGF-C and VEGF-D are
mitogenic for lymphatic
and vascular endothelial cells in vitro (34
, 68 ,
70)
, and VEGF-C (62 , 71)
, but not VEGF-D (72)
, can induce vascular permeability. Analysis of VEGF-C
and VEGF-D
function in vivo and in vitro using a range of
animal-based assay
systems, including the chick chorioallantoic membrane,
rabbit cornea
assays, and transgenic mouse models, has
demonstrated the ability of
these factors to drive angiogenesis and
lymphangiogenesis (Table 2)
(32 33
34 , 37
, 38 , 73
74 75)
|
| View this table: [in a new window] |
Table 2. Growth factors and receptors involved in lymphangiogenesis |
VEGF-C
gene expression is induced by a range of growth factors, including
platelet-derived growth factor and epidermal growth factor
(76) ,
and by numerous proinflammatory cytokines (77) . Such
mediators may be responsible for the induction of VEGF-C
gene expression observed in a wide range of human tumors. In
contrast to VEGF, expression of VEGF-C is not induced by
hypoxia (76) . Expression of the X-linked
VEGF-D gene (78) is induced by
the transcription factor c-fos (79) and by signaling resulting from
cell–cell contact that is dependent on cadherin 11 (80) .
As c-fos is induced in a range of human tumors and many tumors
are characterized by high cell density, these forms of regulation
could induce expression of VEGF-D in tumor cells.
VEGF-D has been reported to be regulated by the
transcription factor AP-1 in human glioblastoma
multiforme (81) .
The receptor
for VEGF-C/VEGF-D with specificity for lymphatic endothelium
is VEGFR-3 (34 , 62) . This receptor is expressed
on venous endothelium at sites of lymphatic vessel sprouting
during embryogenesis and, in adults, becomes restricted to
lymphatic endothelium (48 , 49) . VEGFR-3 has been shown to play an
important role in remodeling and maturation of
the primary capillary plexus in the early
embryo (82) , and VEGFR-3 mutations have been
associated with hereditary lymphedema (83) .
Recent studies using a VEGF-C mutant that binds
VEGFR-3 but not VEGFR-2 demonstrated that activation
of VEGFR-3 is sufficient to induce growth of lymphatics (37) .
Studies using a soluble form of the VEGFR-3 extracellular domain
expressed as a transgene under the control of the keratin-14
(skin) promotor have demonstrated the requirement for VEGF-C
and VEGF-D and, by implication, VEGFR-3 signaling, in
lymphangiogenesis (42 , 84) , although the blood vasculature was
unaffected (42) . Evidence is
now emerging from a range of such studies which suggests
that VEGFR-2 is the primary receptor for angiogenesis whereas
VEGFR-3 regulates lymphangiogenesis.
LYMPHATIC INVOLVEMENT IN CANCER
The metastatic
spread of tumor cells is the underlying cause of
most cancer-related deaths (2 , 3) . Clinical and pathological evidence
confirms that the metastatic spread of tumors via lymphatic vessels
to local/regional lymph nodes is an early event in metastatic
disease for many solid human tumors (3 , 20 , 21) . The presence of
tumor cells in local lymph nodes is a significant factor in
the staging of human tumors and forms the basis for surgical
and radiation treatment of regional lymph nodes (3 , 20 , 21 ,
84) . More recently, the use of sentinel
lymph nodes has developed as a promising method
for the diagnosis and staging of such diseases
as breast cancer and melanoma (85 86 87) .
Much
conjecture exists in the literature regarding the existence of
lymphatics within tumors (18 , 22 , 31 , 88) . Until recently, evidence
linking the presence of lymphatic vessels in solid tumors
with the spread of cancer was not compelling (18) . This was due
to the lack of suitable markers to distinguish blood vessels
from lymphatic vessels, the difficulty in identifying these
vessels by injection techniques, and the poorly defined structure
of these vessels. Various reports of the high interstitial pressure
in tumors have been used as a theoretical basis for assuming
there is a lack of functional lymphatic vessels within the
tumor mass (88) . Recent studies using a sarcoma model
demonstrated the lack of functional lymphatic
vessels in a tumor expressing the
lymphangiogenic factor VEGF-C and its receptor, VEGFR-3. The
conclusion of that study was that the physical stress exerted
by the growing tumor cells caused the collapse of the
lymphatic vessels (88) . In contrast, there are many
historical reports of lymphatic vessels in
solid tumors and anecdotal evidence that many
tumors are not edematous in nature, suggesting the existence
of functional lymphatic vessels in tumors (31 , 89) . The recent
explosion of interest in the development of blood vessels
within tumors did for a time overshadow the need for further
study of tumor lymphangiogenesis
LYMPHANGIOGENESIS IN CANCER
The discovery
of the lymphangiogenic factors VEGF-C and VEGF-D raises
the question as to whether these factors are expressed in
human cancers and whether this expression is responsible for
the ability of tumors to metastasize. It is already clear from
a number of studies that members of the VEGF family are expressed
in a variety of human tumors (90 91 92 93 94) . VEGF-C and
VEGF-D expression has been detected in a range of human tumors
including malignant melanoma and lung, breast, colorectal, and
gastric carcinomas (see Table 3 ) (91 , 92 , 94) . These studies
have used either immunohistochemistry or reverse transcription
polymerase chain reaction (RT-PCR) to detect expression of
the genes, and these techniques do not take
into account the need for proteolytic cleavage
to activate the polypeptides. The detection of
mRNA or full-length protein may not in all circumstances reflect
fully active/mature growth factor. Expression of VEGFR-3 is
also an important factor in determining the potential for a
lymphangiogenic response. Some studies have shown the coexpression
of VEGF-C/D with VEGFR-3 in malignant melanoma (94) and
lung cancer (95)
| View this table: [in a new window] |
Table 3. Clinical data showing a relationship between expression of the lymphangiogenic growth factors VEGF-C and VEGF-D and the metastatic spread of tumors |
Tumor
expression studies (Table 3) have allowed a direct comparison
of VEGF-C and VEGF-D expression with clinicopathological
factors that relate directly to the ability of
primary tumors to spread (e.g., lymph node
involvement, lymphatic invasion, secondary metastases,
and disease-free survival) (95 96 97 98) . In many instances,
these studies demonstrate a statistical correlation between
the expression of lymphangiogenic factors and the ability of
a primary solid tumor to spread. For example, levels of VEGF-C
mRNA in adenocarcinoma of the lung are associated with lymph
node metastasis (99) and in breast cancer correlate with
lymphatic vessel invasion and shorter
disease-free survival (100) . Non-small cell
lung cancer showed a significantly increased survival of patients
with tumors lacking VEGF-C compared to VEGF-C-positive tumors
(101) .
Studies by Hashimoto et al. examining samples of
cervical cancer demonstrated that the level of VEGF-C mRNA detected
by RT-PCR was the sole independent factor influencing pelvic
lymph node metastases (102) . The majority of these studies
showed significant correlation between VEGF-C levels and the
clinical parameters of tumor spread (see Table 3 ),
indicating the close association between
expression of this lymphangiogenic factor and
tumor metastasis. Some studies have suggested that expression
of VEGF-D in human tumors is reduced relative to VEGF-C
(99 ,
103) ;
further studies are required to examine this
potential difference.
Although
studies showing a correlation between expression levels of
VEGF-C mRNA and clinical parameters are suggestive
of a role for lymphangiogenic factors in
promoting tumor spread, no direct demonstration
of VEGF-C or VEGF-D involvement had been documented until
recently. Studies using expression of VEGF-C and VEGF-D polypeptides
in various tumor backgrounds in animal models have provided
direct evidence that these factors can indeed promote tumor
lymphangiogenesis and spread (Table 4) . Three studies in
which full-length VEGF-C was overexpressed in tumor cells showed
that the presence of VEGF-C polypeptide induced the formation
of lymphatic metastases in regional lymph nodes (see Table 4 ).
A study by Skobe et al. showed that expression of VEGF-C in
the breast cancer cell line MBA-MD-435 induced increased
lymphangiogenesis, but not angiogenesis, in
tumors grown in immunocompromised mice (36) .
These tumors spread to local lymph nodes and the lung whereas
control tumors lacking VEGF-C did not, demonstrating that
VEGF-C could drive metastatic spread. Others have used Rip1Tag2
transgenic mice to analyze the activity of overexpressed VEGF-C
in a pancreatic ß cell tumor model (35) . In these
tumors, VEGF-C promoted the development of peri-tumoral lymphatics
and this correlated with an increased rate of metastatic spread
to the draining pancreatic lymph nodes. Studies in which the
breast tumor cell line MCF-7 expressing recombinant VEGF-C was
implanted orthotopically in SCID mice have shown the role played
by VEGF-C in promoting tumor growth (104) . Unlike lines expressing
VEGF, which showed increased angiogenesis, the VEGF-C expressing
lines promoted growth of only tumor-associated lymphatics. Inhibition
of this growth by soluble VEGFR-3 protein showed the
potential for tumor growth and metastasis to be inhibited with
reagents that block VEGFR-3 signaling (104) . Other studies have
used the melanoma cell line MeWo to show the effects of VEGF-C
overexpression on the metastatic behavior of tumors grown in
vivo (105) . These studies showed that MeWo cells
expressing VEGF-C induced increased levels of
both lymphangiogenesis and angiogenesis, which
is in contrast to previous experiments in which
VEGF-C was expressed in tumor models. This may reflect the
degree of proteolytic processing of the growth factor, and was
also seen in an earlier study analyzing the effect of VEGF-D
(19 , 38) . This study also reported the
recruitment of macrophages into the tumors as a
result of overexpression of VEGF-C. This reveals
a potential function of VEGF-C and VEGF-D as immune modulators,
a role that has already been demonstrated for the related
angiogenic factor VEGF (106) .
|
Our study in
which cells overexpressing VEGF-D were grown as tumors
in SCID/NOD mice showed that VEGF-D was capable of inducing both
lymphangiogenesis and the spread of tumor cells to local lymph
nodes (38) . This was not seen in tumors
expressing the angiogenic factor VEGF, which
only induced the formation of additional blood
vessels and did not induce spread of the tumor to
local lymph nodes. The dependence of these effects on VEGF-D
was conclusively demonstrated by the use of a monoclonal
antibody (mAb) that blocked the binding of
VEGF-D to both VEGFR-2 and VEGFR-3. This
antibody inhibited the growth and lymphogenous spread
of tumors expressing VEGF-D. In contrast to some of the studies
with VEGF-C (36) , overexpression of VEGF-D resulted
in an increased angiogenic response in the tumors,
presumably via stimulation of VEGFR-2. The
difference between the studies could be due to
the degree of processing of the full-length forms
of the polypeptides in the various models, which may in turn
influence the receptor specificity of the growth factors. Identification
of the proteases that cleave VEGF-C and VEGF-D will
be an important step forward in understanding how these growth
factors induce tumor angiogenesis and lymphangiogenesis. It
will be important to elucidate the biological function of the
propeptides of VEGF-C and VEGF-D and their role in influencing
the balance between angiogenesis and lymphangiogenesis. The
experimental studies carried out so far do show that the
expression of growth factors such as VEGF-C and
VEGF-D can directly influence the development
of lymphatic vessels within tumors and the rate of
metastatic spread. In combination with the expression data derived
from various human tumors (Table 3) , these studies allow
us to postulate that expression of these growth factors and
the proteases that activate them may be critical for determining
the metastatic potential of a tumor. However, molecules
other than those of the VEGF family are likely
to play a role in the development and growth of
lymphatic endothelium.
THERAPEUTIC MANIPULATION OF LYMPHANGIOGENESIS
Recent
experimental approaches with animal models and analysis of
genetic lesions causing hereditary lymphedema in humans have
indicated that the VEGF-C/VEGF-D/VEGFR-3 signaling system
drives lymphatic hyperplasia and/or
lymphangiogenesis during embryonic development
(32 , 33 , 37 , 42 , 83 ,
107) in
adult tissues (108) and in or around tumors (35 , 36 , 38 ,
104) .
Therefore, manipulation of this pathway offers
the opportunity for therapeutic strategies
designed to inhibit or stimulate growth of lymphatic vessels
in conditions such as lymphedema, cancer and infectious diseases.
Inhibition
of lymphangiogenesis
In the context of cancer, it may be beneficial to inhibit
lymphangiogenesis so as to reduce the
occurrence of lymphogenous metastatic spread. Potential
inhibitors of the VEGFR-3 lymphangiogenic signaling pathway
include mAbs that block the binding of VEGF-C and VEGF-D to
VEGFR-3. A neutralizing VEGF-D mAb that blocks binding to both
VEGFR-2 and VEGFR-3 (109) inhibited angiogenesis,
lymphangiogenesis, and metastatic spread via
the lymphatics in a mouse tumor model that
secreted recombinant VEGF-D (38) . Similar studies using neutralizing
VEGF-C mAbs have not yet been reported. A mAb against mouse
VEGFR-3 was found to block the binding of VEGF-C and presumably
VEGF-D. This mAb induced microhemorrhage from tumor blood
vessels in a mouse tumor model, although the
effects on tumor lymphatics were not analyzed
(110) .
An alternative
approach to antibodies would be to sequester VEGF-C
and VEGF-D with a soluble version of the extracellular domain
of VEGFR-3. The potential of this approach is illustrated by
a transgenic mouse study in which a soluble form of the ligand
binding region of the VEGFR-3 extracellular domain was
expressed in the epidermis of the skin (42) .
This protein construct consisted of the first
three immunoglobulin homology domains of VEGFR-3 fused
to the Fc-domain of the human immunoglobulin 
chain. This soluble form
of VEGFR-3 inhibited fetal lymphangiogenesis; consequently, these
mice developed a lymphedema-like phenotype involving swelling
of the feet, edema, and dermal fibrosis (42) .
When delivered via an adenovirus, this soluble
form of VEGFR-3 blocked the growth of
peritumoral lymphatic vessels in a mouse breast cancer model
(104) .
An attractive
approach for inhibiting the VEGFR-3 signaling pathway
would involve identification of orally active small molecules
that interfere with the binding of VEGF-C/D to this receptor.
Rational design of such compounds will be aided by defining
the structure of the complex consisting of the VEGFR-3 ligand
binding domain bound to VEGF-C/D. However, peptidomimetic approaches
based on the structures of the ligands or receptor, as
modeled from the known structure of the VEGF/VEGFR-1 complex
(111) , could also be pursued to generate
such inhibitors. Small molecule inhibitors of
the tyrosine kinase catalytic domain of VEGFR-3
could be useful for blocking this signaling pathway. Tyrosine
kinase inhibitors of the closely related VEGFR-2 have shown
promise for inhibition of tumor angiogenesis, at least in
animal models (112 , 113) . As the catalytic domains of VEGFR-2
and VEGFR-3 are closely related in structure, it is
important to test all known VEGFR-2 tyrosine
kinase inhibitors for activity against VEGFR-3,
although it is already known that one such inhibitor,
PTK787/ZK 222584, inhibits both receptors (113) . A series of
indolinones has recently been reported that inhibit the
kinase activity of VEGFR-3 but not VEGFR-2 (114) .
Inhibitors of
the VEGFR-3 signaling pathway may be useful anti-cancer therapeutics
via mechanisms other than blocking lymphangiogenesis. For
example, Kaposi’s sarcoma (KS) is characterized by the
presence of a core of spindle-shaped cells that may be derived
from lymphatic endothelium. VEGF-C potently induces
proliferation of these cells in vitro (115) and
may play a critical role in controlling KS cell
growth, migration, or invasion (116) . If this is
the case, inhibition of VEGFR-3 signaling may be useful
for inhibiting KS progression by acting on tumor cells directly.
This may also be true for lymphangiomas and lymphangiosarcomas.
One potential problem that could arise from targeting the VEGFR-3 signaling pathway in cancer is that lymphatic vessel function in normal tissues could be compromised if VEGFR-3 signaling is required for the integrity/function of mature lymphatics. The role of VEGFR-3 in mature lymphatics, which express this marker, is unknown.
Therapeutic
lymphangiogenesis
Lymphedema is an impairment of lymph flow from an extremity that
may be caused by lymphatic vessel obstruction, ablation, lymphatic
insufficiency or dysfunction, or parasitic (filariasis)
infection (31 , 117) . Affected areas become swollen and
fibrotic as a consequence of accumulation of
fluid and insufficient protein and
macromolecular uptake by lymph node macrophages (118) . Another
common cause of lymphedema is the removal of the entire breast
(mastectomy) and axillary lymph nodes (radical mastectomy) for
breast cancer treatment (119) . Removal of the axillary lymph
nodes impairs fluid clearance from the upper region of the
chest and arm (117) . Significant edema of the arm occurs
in 20% of
patients who undergo mastectomy and axillary dissection, and
is more common in patients who undergo radiotherapy or suffer
postoperative infection (117) . Late-onset or secondary edema
may occur in radical mastectomy patients as a consequence of
infection that affects lymphatic drainage. The use of limbs
can be severely affected by lymphedema (117) .
It has been proposed that mastectomy patients
may benefit from stimulation of lymphangiogenesis in
the region of lymph node removal to aid fluid drainage and prevent
side effects associated with breast cancer. Therapeutic approaches
to achieve this could be based on gene therapy or direct
protein application to administer VEGF-C or VEGF-D to affected
sites. Alternatively, lymphatic tissue could be transplanted
from internal sites to affected skin and VEGF-C/D
administered to facilitate lymphangiogenesis
from the transplanted tissue. One potential
danger associated with such approaches, at least in
patients who have had surgery to remove primary tumors and affected
lymphatics, is that lymphangiogenesis occurring near the
site of tumor removal could facilitate metastatic spread from
small islands of remaining tumor. It will be important to
test these approaches in appropriate animal models of lymphedema
and metastatic spread before proceeding to clinical trials.
CONCLUDING REMARKS
The metastatic spread of tumor cells causes the vast majority of cancer deaths. Clinicopathological analyses long ago indicated that lymphatic vessels play a very important role in the metastatic spread of cancer. Therefore, metastatic spread to lymph nodes is considered a prognostic indicator and in part determines the therapeutic approaches used for treatment. Nevertheless, it has not been clear whether tumors induce lymphangiogenesis, which facilitates metastatic spread, or whether such spread occurs via preexisting lymphatics.
Recent studies
using mouse tumor models expressing VEGF-C and VEGF-D
indicate that these growth factors can induce hyperplasia of
peritumoral lymphatics, as well as formation of intratumoral
lymphatics (35 , 36 , 38 , 104) , and that these lymphatics facilitate
metastatic spread to lymph nodes (35 , 36 , 38) . VEGF-C
expression in some human tumors correlates with lymphangiogenesis
and dissemination of tumor cells to lymph nodes (120 121 122) .
VEGFR-3 can be up-regulated on tumor blood vessels (92) ; a
study using a neutralizing VEGFR-3 mAb indicated that
signaling via this receptor may be critical for
blood vessel integrity in cancer (110) .
Therefore, the VEGF-C/VEGF-D/VEGFR-3 signaling system
for lymphangiogenesis constitutes a potential new target for
development of anti-cancer therapeutics.
The
development of approaches to block tumor lymphangiogenesis and
treat lymphedema would benefit from the availability of markers
specific for lymphatic endothelium. The absence of such markers
has been a major problem in the field until recently. However,
the advent of lymphatic markers such as VEGFR-3 (44 , 49) ,
podoplanin (59) , prox-1 (56) , and LYVE-1 (52 , 123) will give researchers a much better
opportunity to monitor the effects of potential
therapeutics on lymphangiogenesis in tumors and
normal tissues. Additional requirements for progress in this
field are animal models of lymphangiogenesis, lymphogenous metastatic
spread, and lymphedema. Fortunately, progress has been
made. A recombinant adenovirus encoding VEGF-C that induces lymphangiogenesis
in a rodent model was recently reported (108) and mouse models of lymphedema are now
available: the so-called ‘Chy’ mouse, which has
an inactivating Vegfr3 mutation in
its germline (124) , and another mouse model in which
expression of a soluble form of VEGFR-3 in skin
blocks fetal lymphangiogenesis (42) .
Thus, many of the requisite tools for analysis of therapeutics
designed to inhibit or stimulate lymphangiogenesis are now
available. Clearly this is a field that will
experience rapid progress in the near future.
ACKNOWLEDGMENTS
Note added in proof: Very recent findings have further illustrated the relevance of lymphangiogenesis and VEGF-D to human cancer. Beasley et al. (Cancer Res. 62, 1315–1320, 2002) identified lymphangiogenesis occurring in human head and neck cancer and White et al. (Cancer Res. 62, 1669–1675, 2002) reported that VEGF-D is an independent prognostic indicator for survival in colorectal cancer.
This work was supported by the National Health and Medical Research Council of Australia and the Anti-Cancer Council of Victoria. M.B. is a recipient of the Australian Post-Graduate Research Award and the Anti-Cancer Council of Victoria Post-Doctoral Fellowship. We thank Prof. Tony Burgess for critical analysis of this manuscript and Janna Stickland for assistance with figures
REFERENCES
http://www.fasebj.org/cgi/content/full/16/9/922?ijkey=267e94bab3c50679c5ef875ca3faa96d42c74e40#B36
..............................
Published 16 September 2002 as 10.1084/jem.20021346
Rockefeller University Press, 0022-1007/2002/9/713/ $5.00
The Journal of Experimental Medicine, Volume 196, Number 6, September
16, 2002 713-718
Cutaneous Biology Research Center and Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129
Address correspondence to Michael Detmar, CBRC/Department of Dermatology, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, MA 02129. Phone: 617-724-1170; Fax: 617-726-4453; E-mail: michael.detmar@cbrc2.mgh.harvard.edu
The lymphatic vascular system plays important roles in the maintenance of tissue fluid homeostasis, in the mediation of the afferent immune response, and in the metastatic spread of malignant tumors to regional lymph nodes. It consists of a dense network of blind ending, thin-walled lymphatic capillaries and collecting lymphatics that drain extravasated protein-rich fluid from most organs and transport the lymph via the thoracic duct to the venous circulation (1). Originally discovered as "milky veins" by Gasparo Aselli in the 17th century (2), the mechanisms controlling the normal development of lymphatic vessels and the molecular regulation of their biological function have remained poorly understood in contrast to the rapid progress made in elucidating the formation and molecular control of the blood vascular system (3, 4).
100 yr ago,
Florence Sabin proposed that the lymphatic system develops
by the sprouting of endothelial cells from embryonic veins,
leading to the formation of primitive lymph sacs from which
lymphatic endothelial cells then sprout into surrounding organs
to form mature lymphatic networks (5,
6). Since these pioneering
studies, however, the field of lymphatic research has
remained rather neglected, mainly due to the lack of molecular
tools to specifically detect and functionally characterize
the lymphatic endothelium. The recent
identification of several new markers for
lymphatic endothelial cells and of lymphatic growth
factors and receptors, together with the characterization of
genetic mouse models with impaired lymphatic development and/or
function, has now led to a "rediscovery" of the lymphatic vascular
system and has provided important new insights into the
molecular mechanisms that control its development and biological
function (7).
Importantly, these studies have largely confirmed Sabin's
original hypothesis regarding lymphatic development in
the mammalian system (Fig. 1).
|
Recent studies in angiopoietin-2–deficient mice suggest an important role of the angiopoietins and their receptor Tie2 for the final developmental steps of lymphatic network patterning (Fig. 1) and lymphatic vessel maturation (14). However, the molecular mechanisms controlling the sprouting of lymphatic endothelial cells from primitive lymph sacs and their migration into adjacent organs and tissues (lymphangiogenesis) have remained unclear. In this issue, Saaristo et al. (15) identify VEGF-C as a potent inducer of lymphatic sprouting and provide experimental evidence that in addition to VEGFR-3, VEGFR-2 may also be required for this process. Previously, the authors had shown that signaling via VEGFR-3 was sufficient to induce hyperplasia of cutaneous lymphatic vessels because transgenic mice with skin-specific overexpression of a mutated VEGF-C (K14-VEGF-C156S) that selectively activates VEGFR-3 developed lymphatic vessel enlargement in the skin (17). In contrast, wild-type VEGF-C activates both VEGFR-3 and, after proteolytic processing, VEGFR-2.
In the study by Saaristo et al., K14-VEGF-C or K14-VEGF-C156S transgenic mice were crossed with VEGFR-3+/LacZ mice in which one allele of VEGFR-3 had been replaced by the LacZ gene, thereby enabling the visualization of lymphatic vessels by X-gal staining. Importantly, whereas VEGF-C156S overexpression mainly caused the enlargement of preexisting lymphatic capillaries, wild-type VEGF-C induced lymphatic vessel sprouting during embryogenesis (16). Similarly, an increased number of cutaneous lymphatic vessels was detected in adult VEGF-C transgenic mice and in adult mice that were intradermally injected with an adenovirus encoding VEGF-C, whereas chronic transgenic delivery of VEGF-C156S or intradermal injection of a VEGF-C156S–encoding adenovirus predominantly induced lymphatic enlargement. Moreover, only VEGF-C but not VEGF-C156S also induced angiogenesis and vascular hyperpermeability in these studies, most likely via interaction with VEGFR-2 on blood vascular endothelium. These results indicate that VEGF-C, through interaction with both VEGFR-3 and VEGFR-2, plays an important role in lymphangiogenesis, i.e., the sprouting of lymphatics from preexisting vessels. This is similar to the effects of VEGF-A in angiogenesis where it induces sprouting of new blood vessels (18, 19). Future studies in mice deficient for VEGF-C or VEGF-D, a related lymphangiogenesis factor with comparable VEGFR binding properties, should reveal whether the activation of VEGFR-3 and VEGFR-2 is only sufficient, as shown here, or also necessary for the induction of lymphangiogenesis during normal embryonic development. Moreover, additional studies are needed to investigate whether or not mesenchymal lymphatic progenitor cells might contribute to embryonic (or postnatal) lymphangiogenesis, as has been recently proposed for the early wing bud development in birds (20).
In addition to providing new insights into the mechanisms directing lymphatic development, this study by Saaristo et al. raises new questions regarding the molecular control of angiogenesis versus lymphangiogenesis. In this study, VEGFR-2 was implicated in the induction of lymphatic sprouting and strong expression of VEGFR-2 was detected on collecting lymphatic vessels. Therefore, one might expect that VEGF, thus far thought to specifically induce blood vascular angiogenesis (21), might also be able to activate lymphatic vessel sprouting via the activation of VEGFR-2. Indeed, VEGFR-2 is expressed by cultured lymphatic endothelial cells (22, 23) and VEGF equally stimulates lymphatic and blood vascular endothelial growth in vitro (23 and unpublished data). Moreover, intradermal injection of a VEGF165-encoding adenovirus into mouse ears resulting in high levels of VEGF expression, potently induced the formation of new lymphatic vessels that persisted for up to 1 yr (Dvorak, H.F., personal communication). In contrast, cutaneous wound healing is associated with up-regulated expression of VEGF (24) and the formation of a richly vascularized granulation tissue that initially contains no or only a few lymphatic vessels (unpublished data). Is the formation of VEGFR-3 and VEGFR-2 heterodimers needed for the efficient formation of lymphatic vessel sprouts, as suggested by Saaristo et al. (16)? Does VEGF need simultaneous activation/binding of VEGFR-1, most likely not expressed by lymphatic endothelium in vivo (unpublished data) but by cultured lymphatic endothelial cells (22), and of VEGFR-2 to exert its angiogenic effects under pathological conditions, as suggested by recent findings in placenta growth factor–deficient mice (21)? Does VEGF, via its vascular permeability–inducing activity, create a tissue environment that is permissive for blood vascular endothelial proliferation and sprouting, but not for lymphangiogenesis despite the activation of VEGFR-2, possibly due to the differential expression of extracellular matrix receptors by lymphatic endothelium? Do the observed effects of adenoviral VEGF expression on lymphangiogenesis represent a physiological response of lymphatic endothelium to increased tissue fluid accumulation, or are they caused by the induction of VEGF-C expression in vascular endothelium as has been reported (25)? Future in vivo and in vitro studies, including gene expression profiling, are needed to address this unresolved discrepancy.
Impaired formation of lymphatic vessels results in insufficient fluid drainage from tissues, leading to chronic lymphedema that is characterized by edematous swelling of the skin, epithelial hyperplasia, dermal fibrosis, delayed tissue repair, and impaired immune response (1). Recently, missense mutations in the VEGFR-3 gene have been detected in some cases of primary congenital lymphedema (Milroy disease), indicating an important role of VEGF-C and/or VEGF-D in the normal development of the human lymphatic system (26). Consequently, a heterozygous inactivating VEGFR-3 mutation was identified in Chy mutant mice that develop cutaneous lymphedema and chylous ascites after birth and may serve as a convenient mouse model for primary lymphedema (27). Importantly, virus-mediated VEGF-C gene therapy stimulated the growth of functional lymphatics in this model (27), indicating the potential applicability of growth factor gene therapy to at least some cases of human lymphedema. However, adenoviral VEGF-C gene therapy also induced blood vascular enlargement and increased vascular permeability via interaction with VEGFR-2, unwanted side effects in the context of clinical antilymphedema therapy (28). Saaristo et al. (16) now provide evidence that these blood vascular side effects were avoided by viral gene transfer of a VEGFR-3–specific mutant form of VEGF-C (VEGF-C156S) to wild-type and Chy lymphedema mice. Remarkably, the authors detected functional cutaneous lymphatic vessels as confirmed by their ability to transport intradermally injected FITC-dextran even 8 mo after the injection of the VEGF-C156S-adeno–associated virus into the ear skin of Chy mutant mice, whereas no changes of blood vascularity were observed.
These findings
have potential implications for the development of
novel therapies for human lymphedema, and it will be of interest
to see whether the intradermal injection of naked VEGF-C156S
plasmid cDNA, as previously described for VEGF treatment of
peripheral artery disease (29),
or of recombinant VEGF-C156S protein will also
be able to specifically induce the formation of
functional lymphatics, avoiding potential side effects associated
with the in vivo application of adenoviral constructs.
However, one has to keep in mind that thus far
missense mutations of VEGFR-3 have only been
detected in a minority of all patients with
congenital lymphedema and additional gene mutations are likely
responsible for the majority of these cases. The recent identification
of inactivating mutations of the FOXC2 gene in the
autosomal-dominant disorder lymphedema-distichiasis (30),
together with the detection of lymphedema, chylous ascites,
or chylothorax in an increasing number of mutant mouse
models such as 
9 integrin and angiopoietin-2–deficient mice (15, 31), and the identification of novel
lymphatic-specific markers such as Prox1,
LYVE-1, and podoplanin (32),
suggests the presence of additional
disease-specific targets for the future treatment of
primary lymphedemas.
Secondary lymphedema is frequently induced by the surgical removal or radiation of lymph nodes in cancer patients, whereas filariasis, a chronic infection with the parasitic worms Brugia malayi or Wuchereria bancrofti, is the leading cause in the developing world. Secondary lymphedema after surgery is associated with the interruption of the normal lymphatic drainage system. Recent studies in an experimental postsurgery lymphedema model, involving the removal of lymphatic vessels from rabbit ears, showed that the injection of VEGF-C protein into the wounded area induced the growth of functional lymphatics along with normalization of the tissue structure (33). Therefore, postsurgical lymphedemas might constitute additional targets for VEGF-C– or VEGF-C165S–based protein or gene therapies. The recent discovery of a direct correlation between experimental tumor-associated lymphangiogenesis and enhanced lymph node metastasis (34–37), however, suggests that future studies are warranted to evaluate whether therapeutic regeneration of lymphatic vessels after lymph node removal might increase the risk for enhanced spread of tumor metastases.
Tumor
metastasis to regional lymph nodes represents the first step
of tumor dissemination in many common human cancers and serves
as a major prognostic indicator for the progression of the
disease. In contrast to the extensive molecular and functional
characterization of tumor angiogenesis (38),
i.e., the induction of new blood vessel growth,
little is known about the mechanisms through
which tumor cells gain entry into the lymphatic system. A
widely held view has suggested that lymphatic endothelium only
plays a passive role during this process (38)
and lymphatic invasion only occurs once
stroma-infiltrating tumor cells happen upon
preexisting peritumoral lymphatic vessels (Fig.
2 A). However, the recent
identification of lymphatic growth factors and receptors, together
with the discovery of lymphatic-specific markers and the
development of orthotopic cancer metastasis models, have provided
important new insights into the formation of tumor-associated
lymphatic vessels (7)
and their active contribution to lymphatic tumor
spread (Fig. 2 B). An increasing
number of clinicopathological studies have
shown a direct correlation between tumor expression of
the lymphangiogenesis factors VEGF-C or VEGF-D and metastatic
tumor spread in many human cancers, including cancers of the
breast, lung, prostate, cervix, and colon (for review see
reference 39),
providing circumstantial evidence for the involvement of lymphangiogenesis
in tumor progression.
|
Despite the accumulated evidence for an active role of VEGF-C– or VEGF-D–induced tumor lymphangiogenesis in cancer metastasis to regional lymph nodes, the existence and biological function of lymphatics within experimental and human tumors has remained controversial. High interstitial pressure within tumors has been proposed to prevent intratumoral lymphatic vessel growth and function as assessed by the lack of lymphatic uptake of tracers that were injected in the vicinity of experimental tumors (46, 47). However, the mechanisms controlling metastatic tumor cell invasion and transport within lymphatic vessels are most likely distinct from those involved in fluid uptake and transport. Indeed, proliferating intratumoral lymphatic vessels have been detected in rapidly progressing tumor xenotransplants and in slowly growing, chemically induced orthotopic squamous cell carcinomas in mice and is associated with lymphatic metastasis (7, 9, 34, 45). Proliferating intratumoral lymphatics have also been found in human head and neck squamous cell carcinomas that were characterized by the correlation of the density of LYVE-1+ tumor-associated lymphatic vessels with the presence of regional lymph node metastasis (48). In contrast, no evidence for tumor lymphangiogenesis was found in invasive breast cancer by the same group of investigators (49). Taken together, these results indicate that active tumor-associated lymphangiogenesis induced by VEGF-C, VEGF-D, or other not yet identified growth factors leads to the proliferation and enlargement of peritumoral and, in some cancers, intratumoral lymphatic vessels, likely enhancing the metastatic spread of many different types of human tumors (Fig. 2 B).
Although the mere increase of lymphatic vessel surface area might simply increase the chance for tumor cell invasion and metastasis, lymphatic endothelial cells probably also play an active role in the chemotactic recruitment and intralymphatic transport of tumor cells. Lymphatic endothelium secretes chemokines such as CCL21 (secondary lymphoid chemokine) that binds to CCR7 (13, 22, 50), leading to chemoattraction and migration of mature dendritic cells from the skin to regional lymph nodes. CCR7 and other chemokine receptors are also expressed by some human cancer cell lines including malignant melanomas and breast cancer cells (51). Importantly, the overexpression of CCR7 in B16 malignant melanoma cells led to a >10-fold increase in the incidence of regional lymph node metastases after injection into the footpad of mice, and treatment with CCL21-blocking antibodies completely prevented metastatic tumor spread to lymph nodes (52). These findings indicate that some tumors might take advantage of preexisting molecular mechanisms designed for the physiological immune response to further their metastatic spread.
After several decades of slow progress, the study of lymphatic vessel formation and its role in malignant disease has now led to the identification of several molecular mechanisms involved in the formation and biological function of lymphatic vessels. Although much has still to be learned about the detailed steps of normal and pathological lymph vessel formation, new targets for innovative therapeutic approaches and new tools for the prognostic evaluation of human cancers are now emerging.
Submitted:
August 5, 2002
Accepted: August 13, 2002
References
9ß1. Mol.
Cell. Biol. 20:5208–5215.
http://www.jem.org/cgi/content/full/196/6/713
..............................
Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice
Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Haartman Institute, University of Helsinki, PO Box 21 (Haartmaninkatu 3), 00014 Helsinki, Finland, 1Department of Anatomy and Cardiovascular Research Institute, University of California, San Francisco, CA 94143, USA and 2Ludwig Institute for Cancer Research, PO Box 2008, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia 3Corresponding author e-mail: Kari.Alitalo@helsinki.fiT.Veikkola and L.Jussila contributed equally to this work
Received October 13, 2000; revised December 20, 2000; accepted January 29, 2001.
The
EMBO Journal,
Vol. 20, No. 6 pp. 1223-1231, 2001
© European
Molecular Biology Organization
Abstract
Vascular endothelial growth factor receptor-3 (VEGFR-3) has an essential role in the development of embryonic blood vessels; however, after midgestation its expression becomes restricted mainly to the developing lymphatic vessels. The VEGFR-3 ligand VEGF-C stimulates lymphangiogenesis in transgenic mice and in chick chorioallantoic membrane. As VEGF-C also binds VEGFR-2, which is expressed in lymphatic endothelia, it is not clear which receptors are responsible for the lymphangiogenic effects of VEGF-C. VEGF-D, which binds to the same receptors, has been reported to induce angiogenesis, but its lymphangiogenic potential is not known. In order to define the lymphangiogenic signalling pathway we have created transgenic mice overexpressing a VEGFR-3-specific mutant of VEGF-C (VEGF-C156S) or VEGF-D in epidermal keratinocytes under the keratin 14 promoter. Both transgenes induced the growth of lymphatic vessels in the skin, whereas the blood vessel architecture was not affected. Evidence was also obtained that these growth factors act in a paracrine manner in vivo. These results demonstrate that stimulation of the VEGFR-3 signal transduction pathway is sufficient to induce specifically lymphangiogenesis in vivo.
Keywords: angiogenesis/lymphangiogenesis/vascular endothelial growth factors (VEGFs)/VEGF receptors
Introduction
Growth of new blood and lymphatic vessels by the processes of angiogenesis and lymphangiogenesis requires the activation of specific signal transduction pathways in endothelial cells. These signals are at least in part mediated by members of the vascular endothelial growth factor (VEGF) family via their receptors (VEGFRs) on the surface of endothelial cells. The members of the mammalian VEGF family known to date are VEGF, placenta growth factor (PlGF), VEGF-B, VEGF-C and VEGF-D. They show significant identity at the level of amino acid sequence, but are strikingly different in terms of their mechanisms of regulation, expression patterns and receptor binding profiles (reviewed by Eriksson and Alitalo, 1999). The prototype VEGF regulates vasculogenesis, haematopoiesis and vascular permeability, and is implicated in many physiological and pathological processes (Ferrara, 1999). Like the VEGFs, the three known VEGFRs are differentially expressed. In adult tissues, VEGFR-1 and VEGFR-2 localize predominantly to blood vascular endothelial cells, whereas VEGFR-3 is expressed mainly in lymphatic endothelia (for references see Veikkola et al., 2000).
VEGF-C and VEGF-D, which are the only known ligands for VEGFR-3, are produced as precursor proteins with N- and C-terminal propeptides flanking the VEGF homology domain (VHD; Joukov et al., 1996; Lee et al., 1996; Orlandini et al., 1996; Yamada et al., 1997; Achen et al., 1998). The secreted factors undergo proteolytic processing, resulting in the cleavage of the propeptides and increased affinity for VEGFR-2 (Joukov et al., 1997; Stacker et al., 1999a). The fully processed or mature forms of VEGF-C and VEGF-D consist of the VHD, which acts as a ligand for both VEGFR-2 and VEGFR-3 (Joukov et al., 1997; Achen et al., 1998). Mature VEGF-C and VEGF-D are mitogenic and chemotactic for endothelial cells in culture and angiogenic in vivo (Lee et al., 1996; Joukov et al., 1997; Achen et al., 1998; Cao et al., 1998; Witzenbichler et al., 1998; Marconcini et al., 1999). Importantly, VEGF-C has been shown to induce lymphangiogenesis in transgenic mouse skin and in mature chick chorioallantoic membrane (Jeltsch et al., 1997; Oh et al., 1997). Replacing the second of the eight conserved, characteristically spaced cysteine residues in the VHD (Cys156) with a serine residue in recombinant VEGF-C resulted in a mutant factor (VEGF-C156S), which is a selective agonist of VEGFR-3 (Joukov et al., 1998). VEGF-C156S induced autophosphorylation of VEGFR-3 but not VEGFR-2 in transfected cells, but its activity in primary endothelial cells was not tested.
Transgenic overexpression of VEGF and VEGF-C has yielded important data on their vascular effects (Jeltsch et al., 1997; Detmar et al., 1998), but these studies have not clarified the roles of specific VEGFRs in angiogenesis versus lymphangiogenesis due to the fact that both VEGF and VEGF-C bind more than one receptor. On the other hand, the in vivo studies of VEGFR function have been hampered by the embryonic death of the knockout mice (Fong et al., 1995; Shalaby et al., 1995; Dumont et al., 1998). We wanted to assess the potential in vivo function of VEGF-D and to determine the role of VEGFR-3-specific signals in lymphangiogenesis. For these studies we generated transgenic mouse strains expressing VEGF-D or VEGF-C156S in the skin under the human keratin 14 (K14) promoter. Here we show that VEGF-D is lymphangiogenic. Importantly, stimulation of only VEGFR-3 by VEGF-C156S was sufficient for generating the hyperplastic lymphatic phenotype, demonstrating that a single receptor tyrosine kinase mediates signals sufficient for lymphatic vascular growth.
Results
Receptor
specificity of VEGF-C156S and VEGF-D
We have previously shown that human VEGF-C and VEGF-D bind to
soluble human VEGFR-2 and VEGFR-3 (Joukov
et al., 1997; Achen
et al., 1998) and that VEGF-C156S only
binds to human VEGFR-3 (Joukov
et al., 1998). Human VEGF-D and VEGF-C156S were
assessed for their ability to bind to mouse
VEGFRs using soluble fusion proteins where the
extracellular domains of mouse VEGFR-2 or VEGFR-3
are fused to the immunoglobulin (Ig) 
-chain Fc domain. VEGF-D
was found to bind to both mouse VEGFR-2 and VEGFR-3, whereas
VEGF-C156S and mouse VEGF-D bound only to mouse VEGFR-3 (Figure 1A; M.E.Baldwin, B.Catimel,
E.Nice, S.Roufail, N.E.Hall, K.L.Stenvers,
K.Alitalo, S.A.Stacker and M.G.Achen, in
preparation). Therefore, the receptor binding patterns of human
VEGF-D and VEGF-C156S are retained in mice. In order to test
whether the receptor binding pattern is reflected in VEGFR activation
by ligand-induced dimerization in primary dermal microvascular
endothelial cells, we stimulated the cells with the
purified factors followed by immunoprecipitation of VEGFR-2 and
VEGFR-3 and anti-phosphotyrosine immunoblotting analysis. Tyrosyl
autophosphorylation of VEGFR-2 was stimulated by VEGF-D but
not by VEGF-C156S, whereas VEGFR-3 autophosphorylation was stimulated
by both of these ligands (Figure 1B).
The differential receptor binding of these
factors thus leads to specific receptor activation
in primary endothelial cells.
|
|
|
Selective
hyperplasia of lymphatic, but not blood vessels in transgenic skin
In order to analyse the dermal blood vascular phenotype of K14-VEGF-D
and K14-VEGF-C156S mice, whole-mount tissue preparations of
transgenic and wild-type ear skin were studied after
vascular perfusion with biotin-labelled Lycopersicon
esculentum lectin. When injected
intravenously, this lectin binds to the surface of
the endothelial cells allowing visualization of all blood vascular
structures (Thurston et al.,
1999). The blood vessels of
K14-VEGF-D and K14-VEGF-C156S mice appeared normal in this analysis,
as no differences from wild-type vessels could be found
(Figure 4A–C). In
order to visualize the lymphatic vessels in
whole-mount preparations, the transgenic mice were crossed
with heterozygous mutant VEGFR-3+/LacZ mice (Dumont et
al., 1998). In these mice, one allele coding for
VEGFR-3 has been disrupted by insertion of the LacZ
coding sequence, allowing the localization of
VEGFR-3 expression by staining for
ß-galactosidase activity, which gives a blue signal (Dumont et al., 1998).
The dermal lymphatic vessels in K14-VEGF-D x
VEGFR-3+/LacZ and K14-VEGF- C156S x VEGFR-3+/LacZ
compound heterozygous mice were considerably
enlarged in comparison with the lymphatic
vasculature in VEGFR-3+/LacZ mice (Figure 4D–F). These
phenotypes demonstrate that VEGF-D and the VEGFR-3-specific ligand
VEGF-C156S induce selective hyperplasia of the lymphatic but
not blood vasculature when overexpressed under the K14 promoter.
 View larger version (179K): [in a new window] |
Fig. 4. Whole-mount analysis of skin blood and lymphatic vasculature. Blood vessels were visualized by injecting the mice intravenously with biotin-labelled L.esculentum lectin followed by vascular perfusion (A–F). Lymphatic vessels were stained blue (arrows) in the skin of K14-VEGF-D and K14-VEGF-C156S mice crossed with heterozygous mutant VEGFR-3+/LacZ mice (D–F). Scale bar in (C) for (A–C), 75 µm; in (F) for (D–F), 65 µm. |
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| View this table: [in a new window] |
Table I. Structural parameters of lymphatic vessel network in the tail |
The superficial lymphatic capillaries of the skin drain via connecting vessels into the deeper collecting lymphatic channels. The route of lymphatic transport from the skin can be marked by an intradermal injection of Evans Blue dye and by following the appearance of the dye in the deep collecting lymphatic channels. Upon injection of Evans Blue into the hind limb footpads of wild-type, K14-VEGF-D or K14-VEGF-C156S mice the lymphatic vessels on both sides of the ischiatic vein were rapidly stained blue in all of the mice (Figure 5J–L). Therefore, fluid transport from the skin did not appear to be impaired in K14-VEGF-D and K14-VEGF-C156S mice despite the hyperplasia of the superficial lymphatic capillaries.
Paracrine
effects of the K14-VEGF-D and K14-VEGF-C156S transgene products
To find out whether transgenic overexpression of VEGF-D and VEGF-C156S
had effects beyond the skin, internal organs in the compound
heterozygous K14-VEGF-D x VEGFR-3+/LacZ and
K14-VEGF-C156S x VEGFR-3+/LacZ
mice were stained for ß-galactosidase activity.
When the whole-mount lymphatic staining patterns of various
internal organs were compared with VEGFR-3+/LacZ
mice, no obvious differences were found. As an
example, Figure 6A
shows the pericardial lymphatic vessels of a K14-VEGF-C156S
x VEGFR-3+/LacZ and a VEGFR- 3+/LacZ
mouse. Although variable in architecture, there
were no consistent differences in these vessels
between the normal and transgenic mice.
|
To measure the
systemic half-life of recombinant VEGF-C and VEGF-D
proteins, anaesthetized wild-type mice were given an intravenous
injection of 1 µg of VEGF-D or VEGF-C, and
blood was drawn at different time points after the injection.
As determined by ELISA, the half-life of both VEGF-D and
VEGF-C in mouse circulation was
<15 min (Figure 6B),
and by 30 min the factors had been
cleared below the assay detection level. For
comparison, we also injected recombinant VEGF
into the circulation and measured its clearance rate. VEGF disappeared
in a similar manner to VEGF-D and VEGF-C with a half-life
of 8 min and complete
clearance by 30 min (Figure 6B). Co-injections of VEGF-C and
VEGF-D, or VEGF-C together with VEGF, yielded
similar clearance rates to those for the single
factors (data not shown). As the total blood volume
in mice constitutes 5–6% of body weight (1–1.5 ml in
mice of 20–25 g), the blood concentrations of the
factors after an injection of 1 µg of recombinant protein
should range from 16 to 23 nM, i.e. to be well above
the reported binding constants of VEGF-D and VEGF-C to VEGFR-2
(560 and 410 pM, respectively) and to VEGFR-3 (200 and
135 pM; Joukov et
al., 1997; Stacker
et al., 1999a). The
injected ligand can thus be considered to saturate the receptor,
and the clearance of VEGF-D and VEGF-C from the circulation
after factor co-injection was thus probably not due to
binding to their specific receptors on blood
vessel endothelia. In conclusion, the lymphatic
hyperplasia phenotype may be restricted to the skin
due to the rapid clearance of the transgene products from the
circulation via a receptor-independent mechanism.
Soluble
VEGFR-3 blocks the lymphatic hyperplasia in the skin
Recombinant soluble VEGFR-1 or VEGFR-2 can inhibit both physiological
and pathological angiogenesis, such as retinal
neovascularization, corpus luteum angiogenesis
or tumour growth (Aiello et al.,
1995; Ferrara
et al., 1998; Goldman
et al., 1998; Kong
et al., 1998; Lin et al., 1998;
Takayama et al.,
2000). We wanted to test whether the
lymphatic hyperplasia in the skin of K14-VEGF-D and
K14-VEGF-C156S mice can be neutralized by soluble VEGFR-3. We
therefore mated the mice with K14-VEGFR-3-Ig transgenic mice
expressing a soluble chimeric protein consisting of the
ligand binding portion of the extracellular
part of VEGFR-3 joined to the Fc domain of Ig -chain (Figure 7A; Makinen
et al., 2001). When the
skin of the double transgenic mice was examined histologically,
lymphatic vessels were no longer seen although transgene
expression remained high, as confirmed by northern blotting
(Figure 7B and data
not shown). Therefore, the soluble VEGFR-3 is
capable of inhibiting VEGF-D- and VEGF-C156S-induced lymphatic
hyperplasia in vivo.
|
Discussion
Until now, VEGF-C has been the only growth factor known to target the lymphatic vascular compartment in vivo. VEGF-D is closely related to VEGF-C in structure, and together they are the only known natural ligands for VEGFR-3 (Joukov et al., 1996; Achen et al., 1998). Despite the fact that VEGF-D binds to VEGFR-2 and has been reported to be angiogenic (Marconcini et al., 1999), transgenic overexpression of VEGF-D led to lymphatic hyperplasia but no angiogenesis. In addition, the VEGFR-3-selective mutant factor VEGF-C156S also induced lymphatic hyperplasia, showing that the necessary and sufficient signals for the growth of lymphatic vessels are transduced via VEGFR-3.It is unlikely that the lymphatic hyperplasia in K14-VEGF-D and K14-VEGF-C156S mice would be a secondary consequence of an increased production of lymphatic fluid in response to increased microvascular permeability, as both VEGF-D and VEGF-C156S have been reported to be inactive in the Miles vascular permeability assay (Joukov et al., 1998; Stacker et al., 1999b). The hyperplasia appeared to result from both increased endothelial cell proliferation in the existing lymphatic vessels and from the formation of additional lymphatic vessels, as we observed greater numbers of enlarged lymphatic capillaries in the ear skin of transgenic mice when compared with wild-type mice. This is consistent with the result from the K14-VEGF-C mice where the lymphatic hyperplasia was associated with an increased endothelial cell proliferation rate (Jeltsch et al., 1997).
The enlarged lymphatic capillaries were located in the upper dermis in association with the hair follicles. This localization correlates well with the published K14 promoter expression in the basal cell layer of the epidermis and in the outer root sheaths of the hair follicles (Byrne et al., 1994). The lymphatic nature of these vessels was confirmed by their staining for both LYVE-1 and VEGFR-3, two antigenic markers for the lymphatic endothelium (Jussila et al., 1998; Banerji et al., 1999). Importantly, blood vessels in K14-VEGF-D and K14-VEGF-C156S mice were VEGFR-3 negative. Our results thus indicate that VEGF-D and VEGF-C156S retain their lymphatic specificity in vivo. Interestingly, the hyperplastic lymphatic capillaries of K14-VEGF-D mice were weakly positive for VEGFR-2, whereas those of K14-VEGF-C156S mice and the lymphatic capillaries of wild-type mice expressed no VEGFR-2. This result may reflect a positive feedback loop where VEGFR-2, which is normally expressed only in trace amounts in the endothelia of the lymphatic capillaries, is upregulated upon ligand binding and receptor activation.
High-resolution, whole-mount imaging techniques were used to study the blood vessel morphology of K14-VEGF-D and K14-VEGF-C156S mice, but no differences between wild-type and transgenic mice were found. Even though VEGFR-3 is expressed in blood vessel endothelia early in embryonic development, by embryonic day 13.5–14.5 when the K14 promoter expression becomes widespread in the skin keratinocytes (Byrne et al., 1994), VEGFR-3 has already been downregulated in blood vessels (Kaipainen et al., 1995). In concert with the VEGFR-3 developmental expression pattern, the binding sites of VEGF and VEGF-C in mouse embryos overlap early in the development, but diverge to target the blood vascular and lymphatic compartments, respectively, as the lymphatic vessels develop (Lymboussaki et al., 1999). VEGFR-3 has been shown to be upregulated on blood vascular endothelium in tumours and in chronic wounds (Partanen et al., 1999; Valtola et al., 1999; Paavonen et al., 2000). In these pathological conditions and in tissue ischaemia, VEGF-C and VEGF-D could thus promote angiogenesis. Tissue proteolytic activity in such processes is also enhanced, possibly contributing to the proteolytic processing of both VEGF-C and VEGF-D and formation of their VEGFR-2 binding forms (Achen et al., 1998; Stacker et al., 1999a). The lack of blood vascular effects of VEGF-D in the present model could thus result from both its incomplete proteolytic processing in the skin and from the low levels of VEGFR-2 present on quiescent blood vascular endothelium (our unpublished data).
The in vivo morphology of the K14-VEGF-D and K14-VEGF-C156S lymphatic capillaries was abnormal. In the tail, the characteristic honeycomb-like pattern was preserved, but the transgenic vessel diameter was approximately three times that of the wild-type mice. In the ears of K14-VEGF-D and K14-VEGF-C156S mice, the superficial lymphatic vascularity was increased in comparison with wild-type mice. The hyperplastic lymphatic capillaries were, however, functional, in that they were capable of liquid uptake and transport to the deep collecting lymphatic vessels. Also, these hyperplastic vessels did not appear to be abnormally leaky, as shown by the ferritin transport experiments.
In contrast to the superficial lymphatic vessels, the collecting vessels and visceral lymphatic vessels of K14-VEGF-D and K14-VEGF-C156S mice were seemingly unaffected by transgene overexpression, and no transgene-encoded growth factors could be detected circulating in the bloodstream. The absence of the growth factors in the circulation possibly results from their distribution to the pericellular matrix of the dermis and from their short systemic half-lives, probably due to clearance by the liver, the spleen and the kidneys, as determined by analysis of iodinated VEGF and VEGF-C injected into rat circulation (K.Paavonen and K.Alitalo, unpublished results). Lack of systemic vascular effects in K14-VEGF-D and K14-VEGF-C156S mice suggests that these molecules could be useful in the development of local gene therapy. Local induction of lymphatic capillary growth is of clinical interest as failure to regenerate lymphatic vessels (e.g. after surgery) results in secondary lymphoedema. Targeted delivery of VEGF-D or VEGF-C156S by gene transfer could perhaps also be used to induce lymphatic regeneration after injury.
Primary lymphoedema, a rare early-onset autosomal dominant disorder of the lymphatic system, was recently linked to mutations in the VEGFR-3 tyrosine kinase domain (Karkkainen et al., 2000). Disruption of VEGFR-3 signalling by receptor inactivating mutations or by soluble VEGFR-3 results in lymphatic hypoplasia, underlining the importance of VEGFR-3-mediated signals for the maintenance of lymphatic function after embryonic development (Karkkainen et al., 2000; Makinen et al., 2001). Other growth factor/receptor families are also likely to participate in the formation and maintenance of the lymphatic vessels. The Tie-1 receptor tyrosine kinase was detected on the hyperplastic lymphatic capillaries of K14-VEGF-C mice (Jeltsch et al., 1997), and targeted disruption of the angiopoietin-2 gene in mice results in a complete absence of the lymphatic vessels (C.Suri, M.Witte and G.Yancopoulos, personal communication). However, our results demonstrate for the first time that VEGFR-3 signalling alone is sufficient for the generation of all the necessary secondary signals for the induction of growth of functional lymphatic vessels in vivo. One important conclusion from these results is thus that specific inhibitors of VEGFR-2 that are used to block angiogenesis (see Veikkola et al., 2000) may not suffice to inhibit lymphangiogenesis and possibly associated metastasis in human tumours (Mandriota et al., 2001; Skobe et al., 2001; Stacker et al., 2001).
Materials and methods
Receptor
binding and activation analysis
293T cells were transfected with expression constructs encoding
human full-length VEGF-C, VEGF-D or VEGF-C156S using the
calcium phosphate precipitation method.
Twenty-four hours after transfection the cells
were washed twice with phosphate-buffered saline (PBS) and
incubated in methionine- and cysteine-deficient MEM for 60 min.
The cells were then pulse-labelled for 30 min in
medium containing 100 µCi/ml [35S]Met/[35S]Cys
(Promix, Amersham), subsequently washed with PBS and chased
in Dulbecco’s modified Eagle’s medium supplemented
with 0.2% bovine serum albumin (BSA) and 0.2 mM
each of non-radioactive cysteine and
methionine. After a chasing period of
8 h, the conditioned medium was harvested, supplemented
with 1 mM phenylmethylsulfonyl fluoride (PMSF),
4 µg/ml leupeptin and
0.1 U/ml aprotinin, and cleared by centrifugation. Endogenous
VEGF was depleted using monoclonal anti-human VEGF antibodies
(R&D Systems) and protein G–Sepharose. The
binding of VEGF-C, VEGF-D and VEGF-C156S to the human VEGF receptors
was assessed by precipitation using soluble recombinant proteins
consisting of the first three Ig-like loops of VEGFR-2 or
VEGFR-3 fused to the Fc portion of human IgG (Achen
et al., 1998). The fusion
proteins (200 ng) were incubated with 1 ml
of the pulse–chase-labelled conditioned medium at
+4°C for 2 h in the binding buffer (0.5% BSA, 0.02%
Tween-20 and 1 µg/ml heparin). The complexes were
then precipitated with protein A– Sepharose and
washed twice with the binding buffer and once
with 20 mM Tris pH 7.4. The
bound proteins were analysed by SDS–PAGE. The
binding of human VEGF-C156S to mouse receptors was tested using
200 ng of recombinant soluble mouse VEGFR-2 (R&D
Systems) or 1 ml of conditioned medium from cells
transfected with a construct encoding the first
three Ig-like loops of mouse VEGFR-3 fused to
the Fc portion of IgG.
Receptor stimulation was carried out using passage 4–6 human dermal microvascular endothelial cells (Promocell). Subconfluent cells were starved overnight in microvascular endothelial cell growth medium (Promocell) containing hydrocortisone (1 µg/ml), gentamycin sulfate (50 µg/ml), amphotericin B (50 ng/ml) and 0.2% BSA. The cells were stimulated with 1 µg/ml VEGF-D or VEGF-C156S for 10 min at +37°C, washed twice with ice-cold PBS containing 100 µM sodium orthovanadate and lysed with RIPA buffer (10 mM Tris pH 7.4, 50 mM NaCl, 0.5% sodium deoxycholate, 0.5% NP-40, 0.1% SDS) containing 1 mM sodium orthovanadate, 1 mM PMSF and 0.1 U/ml aprotinin. The lysates were sonicated, clarified by centrifugation at +4°C, and immunoprecipitated with antiserum specific for VEGFR-2 (a kind gift from Lena Claesson-Welsh) or monoclonal antibodies against VEGFR-3 (Jussila et al., 1998). Western blotting analysis was carried out using PY20 phosphotyrosine-specific monoclonal antibodies (Transduction Laboratories) and the ECL method, followed by stripping and analysis using receptor-specific antibodies.
Generation
of transgenic mice and northern blot analysis
The cDNAs encoding full-length human VEGF-D and VEGF-C156S were
cloned into a human K14 promoter expression cassette (Vassar et
al., 1989) and injected into fertilized mouse
oocytes of the strain FVB/NIH. Several
independent inbred lines of transgenic mice
were generated and those lines with high levels of transgene
mRNA expression were used for the study. In the transgenic
mouse lines used there was only one site of
transgene insertion in the genome, and the
phenotype had 100% penetrance.
Total RNA from the back skin of wild-type, K14-VEGF-D and K14-VEGF-C156S mice was extracted using the RNeasy kit (Qiagen). RNA (10 µg) was electrophoresed in agarose gels containing formaldehyde, blotted and hybridized with human VEGF-D or VEGF-C156S, mouse VEGFR-2 or mouse VEGFR-3 cDNA probes.
Analysis
of blood and lymphatic vessels
For immunohistochemistry, biopsies from the back, ear and internal
organs of the transgenic mice were fixed in 4%
paraformaldehyde (PFA), dehydrated and embedded
in paraffin. After rehydration and microwave
treatment for antigen retrieval, 5 µm sections
were stained for VEGFR-3 and VEGFR-2 (Kubo
et al., 2000), VEGF-C (Joukov et al., 1998),
VEGF-D (R&D Systems), LYVE-1 (Banerji et al., 1999),
PECAM-1 (Pharmingen), von Willebrand factor
(DAKO), 
-smooth muscle
actin (Sigma) or collagen IV (Chemicon).
Lectin staining was used to visualize blood vessels in whole-mount tissue preparations (Thurston et al., 1999). One hundred microlitres of 1 mg/ml biotinylated L.esculentum lectin (Sigma) were injected intra venously via the femoral vein into anaesthesized mice and allowed to circulate for 2 min. The mouse was then sacrificed and the tissues were fixed by perfusion with 1% PFA/0.5% glutaraldehyde in PBS. The ears were dissected, washed with PBS, and the cartilage was removed. Bound lectin was visualized by the ABC-DAB peroxidase method, the ears were mounted onto slides and examined by light microscopy. In K14-VEGF-D x VEGFR-3+/LacZ and K14-VEGF-C156S x VEGFR-3+/LacZ compound heterozygous mice blood vessels were visualized as above. Lymphatic vessels were then visualized by incubating the fixed tissues in the ß-galactosidase substrate X-Gal (Sigma) followed by dehydration and mounting.
Studies
on lymphatic transport
Microlymphography was performed to visualize the lymphatic network
in the ear and tail. TRITC-dextran (mol. wt
2000 kDa; Sigma) was injected into the
tip of the tail using a 30 gauge needle,
and the progressive staining of the lymphatic network was
followed by fluorescence microscopy and photographed.
Ferritin (type I ferritin from horse spleen, mol. wt 480 kDa; Sigma) was injected into the ear. Forty-five minutes after injection the mouse was sacrificed and the ears were prepared for histology. The iron component of ferritin was visualized in histological sections by potassium ferrocyanide/HCl (Prussian Blue) followed by counterstaining.
A bolus of 5 mg/ml Evans Blue (Sigma) in PBS was injected intradermally into the hind footpads of mice. The skin from the lateral surface of the hind limb was removed to expose the ischiatic vein. Transport of the dye through the lymphatic vessels along the ischiatic vein was followed under direct microscopic observation and photographed.
The morphometric parameters of the transgenic and wild-type lymphatic capillaries were determined after visualization by an injection of fluorescent dextran into the tail. Lymphatic capillary diameter, as well as the horizontal and vertical mesh sizes of the lymphatic network, was measured from appropriate photomicrographs, and statistically significant variation from wild type was determined by the Mann–Whitney test.
ELISA
For determination of VEGF-D from serum samples, MaxiSorp plates (Nunc) were coated overnight at +4°C with 2.5 µg/ml monoclonal anti-human VEGF-D (R&D Systems) in PBS. The wells were blocked with 5% BSA, 0.05% Tween-20 in PBS for 30 min at room temperature (RT). Serum samples were diluted in 5 mg/ml BSA, 0.05% Tween-20 in PBS and incubated in the wells for 1 h at RT, the wells were washed three times with incubation buffer, and 2.5 µg/ml biotinylated rabbit polyclonal anti-VEGF-D antibody (R&D Systems) was added for 1 h at RT. After washes as above, the wells were incubated with ZyMax Streptavidin-Alkaline Phosphatase (Zymed) at 300 ng/ml for 30 min at RT, washed, and the 4-nitrophenyl phosphate (4-NPP) substrate (Roche) at 1 mg/ml in diethanolamine pH 10.3 was added. Optical density was read at 405 nm.
For quantification of VEGF-D in transgenic tissues, age-matched transgenic and wild-type mice were sacrificed and the hair of back skin was removed. Skin, lung and heart were snap-frozen in liquid nitrogen, pulverized using a dismembranator and lysed in 20 mM Tris pH 7.4, 1 mM EDTA, 50 mM NaCl, 50 mM NaF, 1% Triton X-100, 1 mM PMSF, 4 µg/ml leupeptin and 0.1 U/ml aprotinin. Equal amounts of total protein were used for the ELISA, which was performed as above.
The half-lives of the purified factors in blood circulation were estimated by giving anaesthesized mice an intravenous injection of 1 µg of VEGF-D, VEGF-C or VEGF165 in 100 µl of PBS via the femoral vein. Blood was drawn at different time points after the injection and VEGF-D levels were analysed by ELISA as above. For VEGF-C determination, MaxiSorp plates were coated overnight with 15 µg/ml monoclonal anti-human VEGF-D (clone VD4, cross-reacts with VEGF-C; Achen et al., 2000), and 8 µg/ml rabbit anti-human VEGF-C (Joukov et al., 1997) was used for detection. For VEGF determination, Quantikine human VEGF colorimetric sandwich ELISA (R&D Systems) was used.
Acknowledgements
We thank Eija Koivunen, Pipsa Ylikantola, Riikka Kivirikko, Tapio Tainola, Sanna Karttunen and Sirke Haaka-Lindgren for excellent technical assistance. The K14 expression vector was a kind gift from Dr Elaine Fuchs. Dr David G.Jackson provided the LYVE-1 antibody. This study was supported by grants from the Ida Montini Foundation, the Finnish Cultural Foundation, the Foundation of the Finnish Cancer Institute, the Paolo Fondation, the Finnish Academy of Sciences and the Novo Nordisk Foundation.
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..............................
(Circulation
Research. 2001;88:623.)
© 2001 American Heart Association, Inc
From the Molecular/Cancer Biology Laboratory and Ludvig Institute for Cancer Research (B.E., T.K., M.J., H.K., K.A.), Haartman Institute, University of Helsinki, Finland; Department of Pathology (F.S.), University of Oulu, Oulu, Finland; University of Oxford (R.P., D.G.J.), Molecular Immunology Group, Nuffield Department of Medicine, John Radcliffe Hospital, Headington, Oxford, UK; and A.I. Virtanen Institute and Department of Medicine (S.Y.-H.), University of Kuopio, Kuopio, Finland.
Correspondence to Kari Alitalo, MD, PhD, Molecular/Cancer Biology Laboratory, Biomedicum Helsinki, POB 63 (Haartmaninkatu 8), FIN-00014, Helsinki, Finland. E-mail Kari.Alitalo@Helsinki.fi
Abstract
Abstract— The growth of blood and lymphatic vasculature is mediated in part by secreted polypeptides of the vascular endothelial growth factor (VEGF) family. The prototype VEGF binds VEGF receptor (VEGFR)-1 and VEGFR-2 and is angiogenic, whereas VEGF-C, which binds to VEGFR-2 and VEGFR-3, is either angiogenic or lymphangiogenic in different assays. We used an adenoviral gene transfer approach to compare the effects of these growth factors in adult mice. Recombinant adenoviruses encoding human VEGF-C or VEGF were injected subcutaneously into C57Bl6 mice or into the ears of nude mice. Immunohistochemical analysis showed that VEGF-C upregulated VEGFR-2 and VEGFR-3 expression and VEGF upregulated VEGFR-2 expression at 4 days after injection. After 2 weeks, histochemical and immunohistochemical analysis, including staining for the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), the vascular endothelial marker platelet–endothelial cell adhesion molecule-1 (PECAM-1), and the proliferating cell nuclear antigen (PCNA) revealed that VEGF-C induced mainly lymphangiogenesis in contrast to VEGF, which induced only angiogenesis. These results have significant implications in the planning of gene therapy using these growth factors.
Key Words: angiogenesis • immunohistochemistry • viruses • vessels • revascularization
Introduction
Control of the
vascular system by modulation of growth factor signaling
is essential in attempts to treat diseases such as ischemic
cardiovascular disease and cancer.1
2 3 Perhaps the
most important family of growth factors involved in the
regulation of angiogenesis is the vascular
endothelial growth factor (VEGF) family, which
includes VEGF, VEGF-B, VEGF-C, VEGF-D, Orf virus–encoded VEGF-E,
and the placenta growth factor.4
5 These
ligands bind to VEGF receptors (VEGFR)-1,
VEGFR-2, and VEGFR-3 with partially overlapping
receptor specificities. Both VEGF-C and VEGF-D bind VEGFR-2
and VEGFR-3 but are differentially regulated in cells and
in tissues.6
7 8 9 The affinity
of VEGF-C and VEGF-D toward their receptors is
regulated by proteolytic processing; the affinity
of the mature, proteolytically processed forms toward VEGFR-3
is 
40 times
higher than the affinity toward VEGFR-2.6
10
The importance of VEGFR-3 signals in the vascular system is
indicated by targeted mutagenesis of the VEGFR-3 gene, which
results in embryonic lethality despite the presence of an
intact VEGFR-2.11
The VEGFR-3 gene knockout leads to a disruption of the
remodeling of primitive embryonic vasculature
into a hierarchy of large and small vessels and
results in cardiovascular failure of the embryos.
However, in normal adult tissues, VEGFR-3 is largely absent
from blood vessel endothelia and remains predominantly expressed
in the lymphatic endothelium.9
12 13
Although a variety of angiogenic responses have been shown to be induced by adenoviral expression of VEGF in different mouse tissues,14 the biological functions of VEGF-C in normal adult tissues are thus far less clear. Overexpression of VEGF-C or VEGF in the skin under the keratin 14 promoter induced hyperplasia of lymphatic vessels or angiogenesis, respectively.15 16 17 In addition, recombinant VEGF-C was angiogenic in the early chick chorioallantoic membrane, but it induced exclusively lymphangiogenesis in the differentiated chorioallantoic membrane.18 19 Furthermore, both VEGF and VEGF-C were angiogenic when expressed from a transfected plasmid vector in a rabbit model of hindlimb ischemia.20 VEGF-C expression may thus in general lead to lymphangiogenesis, whereas in early embryonic stages11 or when overexpressed in ischemic tissues, it may stimulate angiogenesis.
The results showing that VEGF-C can induce both angiogenic and lymphangiogenic responses in various settings of gene delivery have raised important questions about the specificity of VEGF-C–induced vascular effects in normal adult tissues. Although in at least some conditions, the goal of proangiogenic gene therapy may be to regenerate all components of the vascular system, in other conditions, such as in secondary lymphedema, only lymphangiogenesis may be desired. In fact, the development of specific lymphangiogenic gene therapy would be an important development for example for the tens of thousands of patients who suffer from lymphedema secondary to axillary evacuation of the lymph nodes or for the millions of patients who develop the disease after filariasis. To resolve questions about the angiogenic versus lymphangiogenic specificity of VEGF-C, we have investigated in the present study the effects of VEGF-C gene transfer on the skin vasculature of adult mice compared with gene transfer of VEGF and ß-galactosidase in the same setting.
Materials and Methods
Generation
of Recombinant Adenoviruses Encoding the VEGFs
For the construction of an adenovirus vector encoding VEGF-C,
the full-length human VEGF-C cDNA (GenBank accession No. X94216)
was cloned under the cytomegalovirus promoter in the pcDNA3
vector (Invitrogen). The SV40-derived
polyadenylation signal of the vector was then
exchanged for that of the human growth hormone gene, and the
transcription unit was inserted into the pAd BglII
vector21
as a BamHI fragment.
Replication-deficient recombinant E1-E3–deleted adenoviruses
were produced in human embryonic kidney 293 cells and
concentrated by ultracentrifugation as previously described.22 Recombinant
adenoviruses encoding VEGF165 and ß-galactosidase
were constructed as previously described.22
23 24 25 26 Adenoviral
preparations were confirmed to be free from helper viruses,
lipopolysaccharide, and bacteriological
contaminants.26
Construction,
Expression, and Purification of VEGFR Ig Fusion Proteins
The expression plasmids encoding human VEGFR-1-Ig and VEGFR-3-Ig
were constructed by polymerase chain reaction-amplifying the
first three Ig homology domains of the extracellular
portions of VEGFR-1 and VEGFR-3 with the primer
pairs 5'-TCTCGGATCCTCTAGT- TCAGGTTCAAAATT-3'
(BamHI site underlined)/5'-GATGAGA-TCTTTATCATATATATGCACTGA-3'
(BglII site underlined) and
5'-CCTGGGATCCCTGGTGAGTGGCTACTCCATGAC-3'/5'-GATGAAGAGATCTTCATGCACAATGACCTCGG-3',
respectively. The products were cloned into the
BglII site of the pMT/BiP ·
V5 · HisC vector (Invitrogen), and the cDNA coding
for the Fc-tail of human IgG1 was cloned in frame with the
VEGFR Ig homology domains into the same vector. The expression
plasmids were cotransfected with the pCO · Hygro selection
plasmid (Invitrogen) into Drosophila S2
cells, and stable cell pools were selected in
150 µg/mL hygromycin B (Calbiochem). The
expression of the Ig fusion proteins was induced with 500 µmol/L
CuSO4 in serum-free DES medium (Invitrogen) and
after 4 days, they were purified from the
conditioned medium by protein A affinity
chromatography (Amersham Pharmacia). VEGFR-2-Ig was obtained
from R&D Systems (catalogue No. 357-KD).
Expression
of Recombinant Adenoviral VEGF-C, VEGF, and ß-Galactosidase
Cells (293EBNA) grown in 10% FCS were transfected with pREP7
(Invitrogen) expression vectors encoding VEGF165
or VEGF-C6
, using the calcium phosphate precipitation method or
infected by incubation with 2x107
pfu/106 cells (multiplicity of infection=20)
of the respective adenoviruses in serum-free medium for 1
hour. The medium was then changed to medium
containing 10% FCS, the cells were incubated
overnight, and metabolically labeled with 35S-methionine
and cysteine (Promix, Amersham) for 6 hours. The media were
collected, and labeled VEGF proteins were precipitated using
soluble VEGFR-Ig domain fusion proteins. Before VEGF-C
precipitation using VEGFR-2-Ig, endogenous VEGF
was removed from the supernatants by
preadsorption using anti-VEGF monoclonal antibodies (R&D
catalogue No. MAB293). The bound proteins were precipitated
with protein G Sepharose, washed three times in PBS,
dissolved in Laemmli sample buffer, and
analyzed by 12.5% or 15% SDS-PAGE. Gels were
then dried and analyzed by phosphor-imaging and autoradiography.
Analysis
of the Adenovirus-Encoded Transcripts In Vivo
Adenovirus (2x108 pfu) encoding VEGF, VEGF-C, or
ß-galactosidase was injected into the tail
veins of two C56/Bl6 mice. The mice were
sacrificed 4 days later and RNA was extracted from the livers
(RNAeasy Kit, Qiagen). Total RNA (15 µg) was subjected to
Northern blotting and hybridization with a mixture of 32P-labeled
cDNAs specific for VEGF (nucleotides 57 to 639, GenBank
accession No. NM003376),
VEGF-C (nucleotides 495 to 1661, GenBank accession No.
X94216),
or LacZ (nucleotides 529 to 977 pBluscript SK+, Stratagene).
All experimental procedures involving laboratory animals were approved by the Helsinki University Ethical Committee and by the Provincial State Office of Southern Finland (permit No. HY 312).
Immunohistochemistry
and Morphometry
Recombinant adenovirus or buffer (2x108 pfu) was
injected subcutaneously into the backs of
C56/Bl6 mice or into the ears of NMRI nude (nu/nu) mice (Harlan).
The mice were sacrificed at various time points after injection.
Skin from the site of injection was fixed in 4%
paraformaldehyde and embedded in paraffin, and
6-µm sections were stained using monoclonal
antibodies against VEGFR-2,27
VEGFR-3,28
or polyclonal antibodies against the lymphatic marker LYVE-1,
a receptor for hyaluronan and a homologue to the CD44
glycoprotein,29
or mouse platelet–endothelial cell adhesion molecule-1
(PECAM-1) (BD Pharmingen, catalogue No. 01951D), the mouse
homologue of the human vascular endothelial
antigen CD31. Sections were also stained using
polyclonal antibodies against laminin.30
The tyramide signal amplification (TSA) kit (NEN Life
Sciences) was used to enhance staining.
Negative controls were done by omitting the
primary antibodies. Double staining of sections was
carried out by first staining sections for proliferating cell
nuclear antigen (PCNA) (ZYMED, catalogue No. 93-1143) and subsequently
for LYVE-1 and PECAM-1 as detailed above. The results were
viewed with an Olympus AX80 microscope and photographed. For
quantification, the vessels in the sections were counted using
square grids (area=0.16 mm2, x200
magnification), and the mean and probability
value were calculated using the Student’s t
test. Eight visual fields were quantified in sites of active
angiogenesis or lymphangiogenesis in five different ears
injected with AdVEGF-C or AdVEGF. For controls,
15 to 20 visual fields in five different ears
injected with AdLacZ were quantified. For
morphometric quantification of vessel volume, quantitative densitometry
of 70 to 80 vessels in 8 to 10 visual fields was performed
according to Weibel’s principles using a CAS200 (Becton-Dickinson)
automated image analyzer and the proprietary software. Blood
vessels were visualized and photographed in situ using a
Leica MZ APO microscope.
Results
Expression
of VEGF-C and VEGF by Recombinant Adenoviruses In Vitro
To confirm that adenoviral gene transfer of VEGF-C results in
secretion of polypeptides that bind to their receptors,
293EBNA cells were infected with the respective
adenoviruses. Cells infected with the VEGF-C
adenovirus (AdVEGF-C) produced major polypeptides of
29/31 kDa that bound to the
VEGFR-3-Ig fusion protein (Figure 1A
,
top). Under nonreducing conditions, these polypeptides migrated
as an 60-kDa band
(Figure 1A, lanes NR). In comparison to cell
cultures transfected with a VEGF-C plasmid expression
vector, very small amounts of the mature,
proteolytically processed 21/23 kDa-form of
VEGF-C were observed in the culture media of Ad
VEGF-C–infected cells, suggesting incomplete proteolytic processing.
However, the mature 21/23-kDa species was the predominant VEGF-C
form that bound to VEGFR-2-Ig (Figure 1A, bottom), as previously
reported.6
|
In an
experiment similar to the one outlined above, we also confirmed
that adenoviral gene transfer of VEGF results in the
expression of VEGF polypeptides that form
disulfide-bonded homodimers and bind to VEGFR-1
and VEGFR-2. Figure 1B
shows SDS-PAGE analysis of
the polypeptides precipitated from the conditioned medium of
metabolically labeled AdVEGF-infected cells. A major VEGF polypeptide
of 24 kDa and a
minor one of 26 kDa are
specifically precipitated using VEGFR-1-Ig or
VEGFR-2-Ig. The former band comigrated with the
major band in similar precipitates from the conditioned media
of cells transfected using a plasmid expression vector for
hVEGF165. The minor band of 22 kDa in the media
of transfected cultures and the 26 kDa-form in
cultures infected with the adenovirus probably
represent differentially glycosylated polypeptide species. The
same bands were also precipitated by monoclonal antibodies against
VEGF (data not shown). Under nonreducing conditions, the
adenovirally expressed polypeptides migrated in the range of
43 to 45 kDa, indicating a disulfide-stabilized dimeric structure
(Figure 1B, lanes NR).
Expression
of Adenovirally Encoded VEGF-C and VEGF In Vivo
The expression of VEGF-C and VEGF adenoviruses in vivo was tested
by injecting the viruses into the tail veins of C56/Bl6
mice. Because most of the gene expression after
intravenous injection of recombinant adenovirus
occurs in the liver,31
we extracted RNA from the liver and analyzed it
by Northern blotting and hybridization with a
combination of probes specific for the adenoviral inserts. As
can be seen in Figure 1C, the adenoviruses efficiently express
mRNAs of 4.5 and 2.4 kb, encoding VEGF and VEGF-C,
respectively, whereas somewhat lower amounts of
mRNA of 6.0 kb encoding ß-galactosidase were
produced by the control virus. The liver of an uninfected mouse
showed no signal.
AdVEGF-C
and AdVEGF Stimulate VEGFR Expression
The effects of the adenoviruses in vivo were tested by subcutaneous
injection into mouse skin and by analyzing skin sections 4
days later by immunohistochemistry for the
VEGF-C receptors VEGFR-2 and VEGFR-3 and for
the vascular marker PECAM-1. As can be seen from
Figure 2A and
from the enclosed insets at higher magnification, adenoviral
expression of VEGF-C for 4 days induced the expression of
VEGFR-2 and VEGFR-3 in endothelial cells of blood vessels (containing
erythrocytes), whereas VEGF gene transfer induced the
expression of VEGFR-2 but not VEGFR-3 (Figure 2D). In contrast, the
blood vessels in mice injected with AdLacZ (Figure 2B) or PBS (Figure
2C)
did not stain for VEGFR-2 or VEGFR-3; only the lymphatic vessels
were positive for VEGFR-3 in these mice. Analysis after 2
weeks showed an inflammatory response in all adenovirus-injected
samples from the C57/Bl6 mice, confounding
immunohistochemical analysis (data not shown).
For this reason, we continued our studies in
the immunocompromised athymic mice.
|
Lymphangiogenic
and Angiogenic Responses to the Adenoviruses
Five ears of three nu/nu mice were injected with each of the
adenoviruses. Shown in Figure 3 are AdVEGF-C, AdVEGF, or AdLacZ
injection sites of mouse ears photographed in situ 3 days
after the injection. As can be seen from this
figure, VEGF induced the formation of enlarged,
tortuous vessels (Figure 3B, arrows) in
contrast to VEGF-C (Figure 3A) or ß-galactosidase (Figure 3C),
which did not seem to affect at least the larger blood vessels.
|
The
adenovirus-injected ears were processed for immunohistochemistry
and stained for PECAM-1 and the lymphatic-specific antigen
LYVE-1. As can be seen from the LYVE-1 staining
shown in Figures 4A and 4B,
AdVEGF-C transfer induced the formation of LYVE-1–positive hyperplastic
lymphatic vessels (arrows), which did not stain for
laminin, a component of the basal laminae of blood vessels (data
not shown), whereas AdVEGF (Figure 4D) or AdLacZ (Figure 4C) did not have
any effects on the lymphatic vessels. In contrast, AdVEGF
induced the formation of blood vessels (Figures 4E and 4F,
arrows) whereas the AdLacZ (Figure 4G) did not have any effects
on the blood vasculature. The effects of AdVEGF-C on blood
vessels were more difficult to evaluate because of the strong
lymphangiogenic response. However, there was a small increase
of PECAM-1–positive vessels in the AdVEGF-C–injected ears
(see Figures 4H and 5B). Some of these may represent newly
formed, very weakly PECAM-1–positive lymphatic vessels
(Figure 4H, asterisk).
|
|
Quantitatative
Analysis of the Adenovirus-Induced Lymphatic and Blood Vessels
As can be seen from the results of counting the LYVE-1–positive
and strongly PECAM-1–staining vessels with lumens in Figure
5A, AdVEGF-C induced an 4-fold increase (P<0.01)
of lymphatic vessel density (Figure 5A)
whereas VEGF induced a 2-fold increase (P<0.01)
of blood vessel density (Figure 5B). The combination of AdVEGF and
AdVEGF-C did not significantly (P>0.5)
potentiate either of these responses. VEGF-C
increased the total volume of the LYVE-1–positive
vessels by 7.5-fold (P<0.01) (Figure
5C), whereas VEGF increased the volume of
the blood vessels by 5.7-fold (P<0.01)
(Figure 5D).
Endothelial
Cell Proliferation in Lymphangiogenesis Induced by VEGF-C
As can be seen in Figure 6A and at higher magnification in Figures
6B and 6C, sequential staining for both LYVE-1
and PCNA revealed that the lymphatic vessels in
AdVEGF-C–injected ears contained proliferating
lymphatic endothelial cells. For example, the lymphatic
endothelial cells surrounding a small arteriole in Figure 6C
stain for PCNA (closed arrowhead), whereas the blood vascular
endothelial cells do not (open arrowhead). Figure 6D
shows PCNA-positive nuclei in the wall of a
blood vessel in an ear injected with AdVEGF. In
contrast, the lymphatic vessels in the ears injected with
AdVEGF or AdLacZ did not stain for PCNA. Approximately 30%
(n=50) of the nuclei in the lymphatic vessels formed in response
to VEGF-C stained positive for LYVE-1, whereas the proportion
of PCNA-positive nuclei in blood vessels in ears injected
with AdVEGF was only 6% (n=50). This low figure
may reflect the fact that the peak in
endothelial cell proliferation in the blood vessels occurs
earlier during angiogenesis induced by VEGF.14
|
Discussion
The present study shows that VEGF-C expressed subcutaneously by adenoviral gene transfer induces proliferation and enlargement of lymphatic vessels in a process that we refer to herein as lymphangiogenesis. VEGF-C also strongly upregulates VEGFR-2 and VEGFR-3 expression in blood vessels. In contrast, adenoviral gene transfer of VEGF induced VEGFR-2 upregulation in the endothelial cells of blood vessels and angiogenesis, as described earlier.14 24
The vessel
density in foci of lymphatic vessel formation in the ears
infected with AdVEGF-C increased 4-fold in comparison to ears injected
with AdVEGF, AdLacZ, or buffer control as measured by
quantification of LYVE-1–positive vessels. The lack of
smooth muscle cells around the vessels and erythrocytes within
the vessels generated in 2 weeks was in accordance with the
lymphatic vessel morphology. Furthermore, these vessels did
not stain for laminin, a component of the basal laminae
(data not shown). The density of strongly
PECAM-1–positive vessels in the ears infected
with AdVEGF increased 2-fold compared with ears
infected with AdVEGF-C, AdLacZ, or buffer. It may also be
noted that LYVE-1 expression was not upregulated in blood vessels
in AdVEGF-induced angiogenesis (eg, see Figure 3A). Thus, the
response to AdVEGF-C was primarily lymphangiogenic, whereas
very little angiogenesis was seen, unlike in the experiments
in which plasmid expression vectors were used in ischemic
rabbit muscle.20
In cell culture, the majority of the adenovirally produced VEGF-C consisted of the partially processed 29/31-kDa form, which binds VEGFR-3 but only very weakly to VEGFR-2.6 In our in vivo assay in normal dermis, this could be the predominant form, whereas in ischemic tissue the 21-kDa form of VEGF-C, which has a higher binding affinity toward VEGFR-2, may predominate because of increased expression of VEGF-C processing enzymes in the latter. A major difference between our assay conditions and those used in experiments using ischemic hindlimb as a target for plasmid delivery is the presence of abundant amounts of endogenous VEGF induced by hypoxia in the latter. However, at least in our initial experiments, simulation of such conditions by coinjection of AdVEGF and AdVEGF-C did not result in a substantial potentiation of the angiogenic response.
The mechanisms of lymphangiogenesis in adult tissues have not been elucidated. The generation of lymphatic vessels could in principle require endothelial cell sprouting from or splitting of preexisting lymphatic vessels or blood vessels, in situ differentiation of endothelial cells, or recruitment and lymphatic differentiation of endothelial precursor cells, as has been described in other models.32 33 34 In embryos, lymphatic vessels are mainly formed by the process of sprouting from certain venous structures, although in the avian species, mesenchymal precursor cells called lymphangioblasts also exist.35 36 We do not yet know the mechanisms of lymphangiogenesis in the adult, but the present results are compatible with the process of sprouting lymphatic vessels from preexisting ones and perhaps splitting of such enlarged lymphatic vessels that we observed in the AdVEGF-C–treated ears. The upregulation of VEGFR-2 and VEGFR-3 in blood vessels in response to VEGF-C raises the interesting possibility that endothelial cells in blood vessels could also participate in lymphangiogenesis by the process of migration and transdifferentiation. Such upregulation of both VEGF-C receptors in the blood vascular endothelium should also be considered when using gene therapy in the setting of tissue ischemia.
It has been shown that the angiogenic response induced by AdVEGF is a highly dynamic process involving the initial formation of mother vessels and endothelial glomeruloid bodies.14 Thus, our analysis at the 2-week time point does not reveal the kinetics of possible transient blood vessel responses. The responses to VEGF-C in blood vessel endothelia, which upregulate both receptors for VEGF-C, remain to be characterized. Therapeutic angiogenesis ultimately requires the induction of entire vascular structures consisting of arteries, veins, and lymphatics. Thus, proangiogenic therapy could consist of different growth factors that cover the entire genetic program for the induction of new vessels.37 Our studies in transgenic mouse embryos and newborn mice have revealed that the developing lymphatic vasculature is dependent on VEGF-C for survival signals and when the embryonic tissues are deprived of such signals by blocking both VEGF-C and VEGF-D, the forming lymphatic vessels regress by specific lymphatic endothelial apoptosis (T. Makinen et al, unpublished observations, 2000). Therefore, further studies are needed to determine the long-term effects of the transient viral expression of VEGF-C, whether this results in permanent and functional lymphatic vasculature and whether stable changes of the blood vasculature can also be observed.
Acknowledgments
This study was supported by grants from the Sigrid Juselius Foundation, the Finska Lakaresallskapet, the Finnish Academy, the University of Helsinki Hospital (TYH 8105), the State Technology Development Center, and EU Biomed Program BMH-98-3380. Dr Jackson is supported by the Medical Research Council and by a grant (00-311) from the Association for International Cancer Research
Footnotes
Original received October 23, 2000; revision received February 2, 2001; accepted February 2, 2001.
References
, c-Kit and Flk1 genes
clustering in mouse chromosome 5 define distinct subsets of nascent
mesodermal cells. Dev Growth Diff. 1997;39:729–740.
[Medline]
4 chain. J Biol
Chem. 1997;272:27862–27868.
=======
Role of Lymphangiogenesis in Cancer
Regional lymph node metastasis is a common event in solid tumors and is considered a marker for dissemination, increased stage, and worse prognosis. Despite rapid advances in tumor biology, the molecular processes that underpin lymphatic invasion and lymph node metastasis remain poorly understood. However, exciting discoveries have been made in the field of lymphangiogenesis in recent years. The identification of vascular endothelial growth factor ligands and cognate receptors involved in lymphangiogenesis, an understanding of the embryology of the mammalian lymphatic system, the recent isolation of pure populations of lymphatic endothelial cells, the investigation of lymphatic metastases in animal models, and the identification of markers that discriminate lymphatics from blood vessels at immunohistochemistry are current advances in the field of lymphangiogenesis, and as such are the main focus of this article. This review also evaluates evidence for lymphangiogenesis (ie, new lymphatic vessel formation in cancer) and critically reviews current data on the prognostic significance of lymphatic vascular density in tumors. A targeted approach to block pathways of lymphangiogenesis seems to be an attractive anticancer treatment strategy. Conversely, promotion of lymphangiogenesis may be a promising approach to the management of treatment-induced lymphedema in cancer survivors. Finally, the implications of these developments in cancer therapeutics and directions for future research are discussed.
===
Stanford Cardiovascular Institute, Stanford, CA.
At the level of the capillaries, the systemic circulation loses about 2-4 liters of fluid and about 100g of protein into the interstitium daily. This ultrafiltrate of the systemic capillaries is returned to the circulatory system by the lymphatics. The lymphatic vasculature is highly specialized to perform this service, beginning with the blind-ended lymphatic capillaries. These vessels are highly permeable to protein, fluid and even cells, due to fenestrations in their basement membrane, and discontinuous button-like junctions rather than tight intercellular junctions as observed in the systemic capillaries(1). The lymphatic capillaries merge into collectors and larger lymphatic conduits that are invested with vascular smooth muscle (capable of contracting and propelling lymph forward) and valves for unidirectional flow. These conduits merge at lymph nodes, delivering antigens to the immune cells and serving as an early warning system of pathogen invasion. The lymph nodes drain into conduits that ultimately merge into the thoracic duct which empties into the left subclavian vein.
http://www.ncbi.nlm.nih.gov/pubmed/22275500
===
1 University of Southern California Keck School of Medicine, Los Angeles, CA;
The lymphatic system plays a key role in tissue fluid homeostasis and lymphatic dysfunction due to genetic defects or lymphatic vessel obstruction can cause lymphedema, disfiguring tissue swellings often associated with fibrosis and recurrent infections without available cures to date. In this study, retinoic acids (RAs) were determined to be a potenttherapeutic agent that is immediately applicable to reduce secondary lymphedema.
We report that RAs promote proliferation, migration and tube formation ofcultured lymphatic endothelial cells (LECs) by activating FGF-receptor signaling. Moreover, RAs control the expression of cell-cycle checkpoint regulators such as p27(Kip1), p57(Kip2) and the aurora kinases through both an Akt-mediated non-genomic action and a transcription-dependent genomic action that is mediated by Prox1, a master regulator of lymphatic development. Moreover, 9-cisRAwas found to activate in vivo lymphangiogenesis in animals based on mouse trachea, matrigel plug and cornea pocket assays. Finally, we demonstrate that 9-cisRA can provide a strong therapeutic efficacy in ameliorating the experimental mouse tail lymphedema by enhancing lymphatic vessel regeneration.
These in vitro and animal studies demonstrate that 9-cisRA potently activates lymphangiogenesis and promotes lymphatic regeneration in an experimental lymphedema model, presenting it as a promising novel therapeutic agent to treat human lymphedema patients.
http://www.ncbi.nlm.nih.gov/pubmed/22275501=====================
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http://www.lymphedemapeople.com/thesite/angiogenesis.htm
Angiogenesis and Cancer
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Lymphangiogenesis in Head
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Lymphangiogenesis and
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http://www.lymphedemapeople.com/thesite/lymphangiogenesis_and_kaposis_sarcoma_vegfc.htm
Lymphangiogenesis in
Wound Healing
http://www.lymphedemapeople.com/thesite/lymphangiogenesis_in_wound_healing.htm
A model for gene therapy
of human hereditary lymphedema
http://www.lymphedemapeople.com/thesite/a_model_for_gene_therapy_of_human_lymphedema.htm
VEGFR-3 Ligands and
Lymphangiogenesis (1)
http://www.lymphedemapeople.com/thesite/vegfr3_ligands_lymphangiogenesis_1.htm
VEGFR-3 Ligands and
Lymphangiogenesis (2)
http://www.lymphedemapeople.com/thesite/vegfr3_ligands_lymphangiogenesis_2.htm
VEGFR-3 Ligands and
Lymphangiogenesis (3)
http://www.lymphedemapeople.com/thesite/vegfr3_ligands_lymphangiogenesis_3.htm
Vascular Endothelial
Growth Factor; VEGF
http://www.lymphedemapeople/thesite/vascular_endothelial_growth_factor_VEGF.htm
VEGF-D is the strongest
angiogenic and lymphangiogenic effector
http://www.lymphedemapeople.com/thesite/VEGFD_Angiogenic_Lymphangiogenic_Effector.htm
Inhibition of Lymphatic
Regeneration by VEGFR3
http://www.lymphedemapeople.com/thesite/Inhibition_of_Lymphatic_Regeneration_by_VEGFR3.htm
VEGFR3 and Metastasis in
Prostate Cancer
http://www.lymphedemapeople.com/thesite/VEGFR3_and_Metastasis_in_Prostate_Cancer.htm
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to help and understand come join us and discover what it is to be the
master instead of the sufferer of lymphedema.
http://health.groups.yahoo.com/group/menwithlymphedema/
Subscribe: menwithlymphedema-subscribe@yahoogroups.com
......................
All About Lymphangiectasia
Support group for parents, patients, children who suffer from all forms
of lymphangiectasia. This condition is caused by dilation of the
lymphatics. It can affect the intestinal tract, lungs and other
critical body areas.
http://health.groups.yahoo.com/group/allaboutlymphangiectasia/
Subscribe: allaboutlymphangiectasia-subscribe@yahoogroups.com
......................
Lymphatic
Disorders Support Group @ Yahoo Groups
While we have a number of support groups for
lymphedema... there is nothing out there for other lymphatic disorders.
Because we have one of the most comprehensive information sites on all
lymphatic disorders, I thought perhaps, it is time that one be offered.
DISCRIPTION
Information and support for rare and unusual disorders affecting the
lymph system. Includes lymphangiomas, lymphatic malformations,
telangiectasia, hennekam's syndrome, distichiasis, Figueroa
syndrome, ptosis syndrome, plus many more. Extensive database of
information available through sister site Lymphedema People.
http://health.groups.yahoo.com/group/lymphaticdisorders/
Subscribe: lymphaticdisorders-subscribe@yahoogroups.com
===========================
Lymphedema People New Wiki Pages
Have
you seen our new “Wiki” pages yet?
Listed below are just a sample of the more than 140 pages
now listed in our Wiki section. We are also working on hundred more. Come and take a
stroll!
Lymphedema Glossary
http://www.lymphedemapeople.com/wiki/doku.php?id=glossary:listing
Lymphedema
http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema
Arm
Lymphedema
http://www.lymphedemapeople.com/wiki/doku.php?id=arm_lymphedema
Leg
Lymphedema
http://www.lymphedemapeople.com/wiki/doku.php?id=leg_lymphedema
Acute
Lymphedema
http://www.lymphedemapeople.com/wiki/doku.php?id=acute_lymphedema
The
Lymphedema Diet
http://www.lymphedemapeople.com/wiki/doku.php?id=the_lymphedema_diet
Exercises
for Lymphedema
http://www.lymphedemapeople.com/wiki/doku.php?id=exercises_for_lymphedema
Diuretics
are not for Lymphedema
http://www.lymphedemapeople.com/wiki/doku.php?id=diuretics_are_not_for_lymphedema
Lymphedema
People Online Support Groups
http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_people_online_support_groups
Lipedema
http://www.lymphedemapeople.com/wiki/doku.php?id=lipedema
Treatment
http://www.lymphedemapeople.com/wiki/doku.php?id=treatment
Lymphedema
and Pain Management
http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_and_pain_management
Manual Lymphatic Drainage (MLD) and
Complex Decongestive Therapy (CDT)
Infections
Associated with Lymphedema
http://www.lymphedemapeople.com/wiki/doku.php?id=infections_associated_with_lymphedema
How
to Treat a Lymphedema Wound
http://www.lymphedemapeople.com/wiki/doku.php?id=how_to_treat_a_lymphedema_wound
Fungal
Infections Associated with Lymphedema
http://www.lymphedemapeople.com/wiki/doku.php?id=fungal_infections_associated_with_lymphedema
Lymphedema
in Children
http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_in_children
Lymphoscintigraphy
http://www.lymphedemapeople.com/wiki/doku.php?id=lymphoscintigraphy
Magnetic
Resonance Imaging
http://www.lymphedemapeople.com/wiki/doku.php?id=magnetic_resonance_imaging
Extraperitoneal para-aortic lymph node
dissection (EPLND)
Axillary node biopsy
http://www.lymphedemapeople.com/wiki/doku.php?id=axillary_node_biopsy
Sentinel
Node Biopsy
http://www.lymphedemapeople.com/wiki/doku.php?id=sentinel_node_biopsy
Small Needle Biopsy -
Fine Needle Aspiration
http://www.lymphedemapeople.com/wiki/doku.php?id=small_needle_biopsy
Magnetic Resonance Imaging
http://www.lymphedemapeople.com/wiki/doku.php?id=magnetic_resonance_imaging
Lymphedema Gene FOXC2
http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_foxc2
Lymphedema Gene VEGFC
http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_vegfc
Lymphedema Gene SOX18
http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_sox18
Lymphedema
and Pregnancy
http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_and_pregnancy
Home page: Lymphedema People
http://www.lymphedemapeople.com
Page Updated: Dec. 24, 2011
