The
Formation of Lymphatic Vessels and Its Importance in the Setting of
Malignancy
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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 Formation of Lymphatic Vessels and Its Importance in the Setting of Malignancy
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).
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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
.................................
The rediscovery of the lymphatic system: old and new insights into the development and biological function of the lymphatic vasculature
Vol. 16, No. 7, pp. 773-783, April 1, 2002
1 Department of Genetics, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA; 2 Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA
Introduction
The lymphatic system is composed of a vascular network of thin-walled capillaries that drain protein-rich lymph from the extracellular spaces within most organs. A continuous single-cell layer of overlapping endothelial cells lines the lymphatic capillaries, which lack a continuous basement membrane and are, therefore, highly permeable. Lymph returns to venous circulation via the larger lymphatic collecting vessels, which contain a muscular and adventitial layer, and the thoracic duct. The lymphatic system also includes lymphoid organs such as the lymph nodes, tonsils, Peyer's patches, spleen, and thymus, all of which play an important role in the immune response.
The lymphatic
system develops in parallel with the blood vascular system
through a process known as lymphangiogenesis, and lymphatic
vessels
are not normally present in avascular structures such
as epidermis,
hair, nails, cartilage, and cornea, nor in some
vascularized organs
such as brain and retina. Although studies of
normal development and
pathologic growth of the blood vascular system
have thoroughly
elucidated the molecular mechanisms that control
these angiogenic
processes (Gale and Yancopoulos 1999
),
studies of the lymphatic system have been hindered by the
lack of
specific lymphatic markers and growth factors. Consequently,
our
understanding of the development and function of the lymphatic
system
and its role in disease is still emerging.
Recently, the
discovery of molecules that specifically control lymphatic
development and lymphatic vessel growth (lymphangiogenesis) and
the
identification of new lymphatic endothelium-specific markers
(Breiteneder-Geleff
et al. 1999; Wigle and
Oliver 1999; Jackson
et al. 2001;
Sleeman et al. 2001;
Veikkola et al. 2001)
have facilitated key scientific advances and
provided new insights
into the molecular mechanisms that control
lymphatic development and
function. These findings include the
identification of specific
genetic defects in certain hereditary diseases
that are associated
with lymphatic hypoplasia and dysfunction
(i.e., lymphedemas; Milroy
1892;
Meige 1898),
and evidence that malignant tumors can directly activate lymphangiogenesis
and lymphatic metastasis (Karpanen et al. 2001;
Mandriota et al. 2001;
Skobe et al. 2001a;
Stacker et al. 2001).
Historical perspective
Lymphatic
vessels were first described in the seventeenth century by Gasparo
Aselli as "lacteae venae", or milky veins (Asellius 1627),
and the embryonic development of lymphatics was extensively studied
during the beginning of the last century. Since then, however,
this
field has advanced very slowly because of the lack of
specific
lymphatic markers, and the histogenetic origin of lymphatic
vessels
has remained a controversial issue.
Historically,
the most widely accepted view of lymphatic development was
proposed by Sabin in the early twentieth century (Sabin
1902,
1904).
Following results
obtained by ink injection experiments, Sabin
proposed that isolated
primitive lymph sacs originate from endothelial
cells that bud from
the veins during early development. The two
jugular lymph sacs were
proposed to develop in the junction of the
subclavian and anterior
cardinal veins by endothelial budding from the
anterior cardinal
veins (Sabin 1902,
1904).
According to her
view, the remaining lymph sacs originate from
the mesonephric vein
and those in the dorsomedial edge of the
Wolffian bodies in the
junction of the subclavian and anterior
cardinal veins. The
retroperitoneal lymph sac forms near the
primitive inferior vena cava
and mesonephric veins; the cisterna chyli forms
near the Wolffian
bodies; and the posterior lymph sacs appear
near the junctions of the
primitive iliac veins and the posterior
cardinal veins (Fig. 1;
Gray 1985).
|
The model
proposed by Sabin indicated that the peripheral lymphatic system
originates from the primary lymph sacs, then spreads by
endothelial
sprouting into the surrounding tissues and organs, where
local
capillaries are formed (Sabin 1902,
1904;
Gray 1985).
An alternative model suggested that the primary lymph sacs
arise in
the mesenchyme, independent of the veins, and secondarily establish
venous
connections (Huntington and McClure 1910).
Support for this latter model has been recently
obtained in birds,
where it was proposed that the lymphatics of
the early wing buds are
derived not only by sprouting from the lymph
sacs but also from the
embryonic mesenchyme (Schneider et al. 1999).
In the
developing mouse embryo, blood vessels are formed from mesodermally
derived endothelial cell precursors (vasculogenesis). These
vessels
then grow by endothelial sprouting and splitting (angiogenesis).
Because a variety of growth factors and receptors involved
in these
processes have been identified (Gale and Yancopoulos 1999;
Yancopoulos et al. 2000),
the molecular mechanisms that control the
development of the vascular
system are now being deciphered. For example,
vascular endothelial
growth factor (VEGF), the endothelial receptor
tyrosine
kinases Tie1 and Tie2, and
angiopoietin-1 (Ang1) and
angiopoietin-2 (Ang2) participate in the process of
vascular development.
Results obtained from loss- and gain-of-function experiments
performed with some of these factors indicated that VEGF
and
its receptors, VEGFR-1 and VEGFR-2,
are important for the
proliferation, migration, and sprouting of endothelial cells
(Risau
1997;
Gale and
Yancopoulos 1999).
Angiopoietins and their receptor Tie2
appear to play a later
role by controlling the sprouting, remodeling,
and maturation of the
developing vasculature (Gale and Yancopoulos
1999).
In contrast, the mechanisms involved in the
development of the
lymphatic system are still poorly characterized
Lymphatic markers and the current view on lymphatic development: blood versus lymphatic vascular fate determination
Lymphatic and blood vasculature are difficult to differentiate when the histologic morphology of the two systems is the only basis on which the distinction is made. Recently, the identification of several markers that show different profiles of expression in blood and lymphatic vasculature has facilitated detailed analyses of the development and pathologic role of the lymphatic vasculature. The fact that so few lymphatic-specific markers have been identified thus far is a likely indication of the close structural and developmental relationship of the blood and lymphatic vasculature. In fact, the specificity of some of these markers is only acquired as embryonic development progresses, suggesting that the formation of the lymphatic vasculature is a stepwise process.
One of the
first lymphatic markers to be identified was the vascular
endothelial growth factor receptor-3 (VEGFR-3, also
known as Flt4).
The pattern of expression of VEGFR-3 during murine
development has
provided additional support for Sabin's model; VEGFR-3 is initially
expressed in angioblasts of murine head mesenchyme, dorsal
aorta,
cardinal vein, and allantois (Kaipainen et al. 1995;
Dumont et al. 1998).
At embryonic day 12.5 (E12.5), VEGFR-3 is expressed
both in
developing venous and in presumptive lymphatic endothelia,
whereas in
adult tissues, VEGFR-3 is largely restricted to
the lymphatic
endothelium (Kaipainen et al. 1995;
Partanen et al. 2000).
Overexpression of VEGF-C, a ligand of VEGFR-3, in the
skin of
transgenic mice results in hyperplasia of cutaneous lymphatic
vessels
(Jeltsch et al. 1997).
The in vivo application of VEGF-C also
stimulates blood vascular
angiogenesis in the mouse cornea, likely via
interaction with VEGFR-2
expressed on blood vessels (Cao et al. 1998).
The lack of a
viable VEGFR-3-null mouse has hampered the analysis
of
the role of this growth factor receptor in the development of
the
lymphatic system. Inactivation of VEGFR-3 causes
cardiovascular failure
and death of the embryo before the emergence of lymphatic vessels
(Dumont et al. 1998).
However, the identification of nonsense mutations
in VEGFR-3 in
patients with hereditary lymphedema (Karkkainen et
al. 2000)
has provided support for an important role of this gene
in lymphatic
development. These findings suggest that VEGFR-3 may
play a
role in the development of both the blood vascular and
the lymphatic
system (Kukk et al. 1996).
VEGFR-3 is also expressed in some blood
capillaries during tumor
neovascularization and in wound granulation
tissue (Valtola et al.
1999;
Paavonen et al.
2000;
Partanen et al.
2000);
therefore,
depending on the tissue and the developmental
stage, this molecular
marker alone may not precisely discriminate
between blood and
lymphatic vessels.
The lymphatic
endothelial hyaluronan receptor (LYVE-1), a CD44
homolog,
was recently identified as a specific cell surface protein
of
lymphatic endothelial cells and macrophages (Banerji et
al. 1999;
Jackson et al. 2001;
Prevo et al. 2001).
Immunostainings performed using antibodies
against LYVE-1 and the
blood vascular markers PAL-E (Skobe and Detmar
2000)
and CD34 (Prevo et al. 2001)
revealed that they show mutually exclusive vascular
expression patterns
(see also Fig. 4A, below).
Recently, LYVE-1 expression has
also been detected in liver sinusoidal endothelial cells (Carreira
et
al. 2001); however,
LYVE-1 is not expressed by blood vessels in
most organs including the
skin (Skobe and Detmar 2000;
Prevo et al. 2001).
Hyaluronan
might regulate leukocyte migration through the lymphatic
vasculature (Jackson et al. 2001).
In addition, chemokines such as secondary
lymphoid chemokine (SLC,
also termed 6Ckine/exodus-2/CCL21), which is
released by the
lymphatic endothelium and interacts with the CC
chemokine receptor 7 (Gunn
et al. 1998, 1999;
Zlotnik and Yoshie 2000),
attract leukocytes toward the lymphatic vessels. As
the development
of the lymphatic vasculature advances, expression of
SLC is first
detectable at around E11.5 in mice (Wigle et al. 2002),
and SLC becomes uniformly expressed in adult lymphatic endothelium
(Gunn
et al. 1998, 1999).
Podoplanin, a
surface glycoprotein, has been recently described as a novel
marker for the lymphatic vasculature. In humans, podoplanin
is
expressed in osteoblastic cells, kidney podocytes, lung
alveolar type
I cells, and lymphatic endothelial cells (Wetterwald et
al. 1996),
with a strong expression in the lymphatic vasculature of
the skin (Breiteneder-Geleff
et al. 1999). However, a
detailed comparison of the expression of
podoplanin with that of
other lymphatic markers is still lacking, and
expression of LYVE-1
was detected in only a subset of cultured
podoplanin-positive
endothelial cells (Makinen et al. 2001a).
Desmoplakin, a
cytoplasmic protein that attaches intermediate filaments to
the plasma membrane in epithelial cells, has also been
reported to be
a marker of the lymphatic endothelium. By immunoelectron
microscopy,
vessels reacting with an anti-desmoplakin antibody
showed features
characteristic of lymphatic vessels, including
thin endothelial
walls, incomplete basal lamina, open junctions,
and overlapping
endothelial cells. In contrast, blood vessels
did not express
desmoplakin (Ebata et al. 2001).
A detailed comparison of desmoplakin expression
with that of other
lymphatic markers is not yet available.
To achieve a
reliable differentiation between blood and lymphatic vasculature,
combinations of positive and negative molecular markers
for these
tissues have been used. For example, the presence or
absence of any
of the aforementioned gene products, combined with
different
expression levels of vascular basement membrane markers
such as
laminin, collagen IV, and collagen XVIII (Skobe et
al. 2001a),
or that of the blood vascular surface antigens CD34
(see Fig. 4A,
below) or PAL-E (Skobe and Detmar 2000),
can be used to discern between the two
systems.
The homeobox
gene Prox1 was originally cloned by homology to the
Drosophila
gene prospero (Oliver et al. 1993).
Functional inactivation of Prox1
in mice leads to embryonic
lethality and causes phenotypic alterations of
the lens, liver, and
lymphatic vasculature (Wigle and Oliver 1999;
Wigle et al. 1999, 2002;
Sosa-Pineda et al. 2000).
Targeted inactivation of Prox1 resulted in the death
of Prox1+/
mice within 2 to 3 d after birth in all but one of
the tested genetic
backgrounds (Wigle and Oliver 1999).
The intestines of the heterozygous pups, in
contrast to those of
their wild-type littermates, were filled with
chyle, the white fluid
transported by the lymphatic vessels of the
small intestine, a few
hours before death (Wigle and Oliver 1999).
This phenotype suggested a haploinsufficiency effect
of Prox1 during
the normal development of the enteric lymphatic system;
however,
survival of a small percentage of heterozygous Prox1
animals
was obtained by crossing them into mouse strains of
different genetic
backgrounds (Wigle et al. 1999).
The detailed
analysis of the Prox1-null mice revealed that the
expression of Prox1 in a restricted subpopulation
of endothelial cells
in the embryonic veins is required to promote lymphangiogenesis
(Wigle
and Oliver 1999). This
analysis also determined that the initial
localization of the
Prox1-positive lymphatic endothelial cells in
the cardinal vein and
their subsequent migration from there occur in
a polarized manner (Wigle
and Oliver 1999). In
Prox1-null mice, budding and sprouting
of lymphatic endothelial
cells from the veins appears unaffected at E10.5; however,
this process
is arrested prematurely at around E11.5-E12.0, and as a
result, Prox1-null
mice are devoid of lymphatic vasculature (Wigle
and Oliver 1999).
Prox1-null
embryos are the first mutants in which specific alterations
of the development of the lymphatic vasculature were identified.
The
detailed analysis of Prox1 expression in the
lymphatic endothelium
provided strong support for the original model proposed by
Sabin
(1902,
1904).
Although Prox1 is expressed in a variety of
cell types, among
endothelial cells it is exclusively detected in
embryonic lymphatic
endothelial cells (Wigle and Oliver 1999)
and in lymphatic vessels of adult tissues and tumors (Wigle
et al.
2002).
A working model for embryonic lymphatic development in mammals
As mentioned
above, several blood vascular markers are available, but only
recently have markers of the lymphatic vasculature been
identified.
Remarkably, most of the blood vascular markers are
also detected in
the lymphatic vasculature (Sleeman et al. 2001).
The level of expression of most of these markers in the lymphatic
vasculature depends on the developmental stage of the embryo,
the
type of tissue being analyzed, or both. Similar considerations
apply
to other characteristics of the lymphatic vasculature such as
the
lack of a continuous basement membrane, which is reflected by
the low
expression of molecules such as laminin and collagen IV,
or the low
level of expression of surface antigens such as CD34.
During
embryogenesis, the different levels of expression of
these markers
probably reflect the state of differentiation or
commitment of
endothelial cells toward a lymphatic phenotype. For
example, during
early mouse development (E10.5-E11.5), the differences
between the
expression levels of laminin or CD34 in blood
vasculature and in
lymphatic vasculature are not as obvious as
they are later in
development (Sauter et al. 1998;
Wigle et al. 2002).
Similarly,
VEGFR-3 is expressed at comparable levels in blood and lymphatic
vasculature during early embryonic development, but
its expression
later becomes down-regulated in the blood vasculature (Kaipainen
et
al. 1995; Wigle et al.
2002).
Therefore, with
the exception of the few lymphatic markers
listed above, most
available molecular markers can be detected in
both blood and
lymphatic vasculature during early embryonic
development. In late
embryonic and adult tissues, these markers
become cell-type specific,
a finding that suggests that blood and
lymphatic vasculature have
a common origin, or that one is derived from the
other.
Because the
lymphatic vasculature forms after the blood vasculature, and
because only a few lymphatic-specific markers have been
identified,
one could argue that the expression of a limited number
of additional
genes in blood vascular endothelial cells is
sufficient for the
subsequent determination of the lymphatic vasculature.
Support for
this proposal has been provided by further characterization
of the Prox1-null
phenotype. Unlike the lymphatic endothelial
cells that bud from the
veins in E11.5 wild-type embryos, those of Prox1-null
littermates do not coexpress any lymphatic markers.
Instead, the
mutant cells appear to have a blood vascular phenotype,
as determined
by the levels of expression of laminin and CD34
(Wigle et al. 2002).
Therefore, Prox1 activity may be required
not only for
maintenance of the budding of the venous endothelial
cells but also
for their differentiation to the lymphatic phenotype.
These results
suggest that during embryonic development, the
default fate of the
budding venous endothelial cells is to adopt
the blood vascular
phenotype; upon expression of Prox1 and other
factors, these budding
cells then switch to a lymphatic vasculature
phenotype.
Based on the
results presented above, a working model of the early embryonic
steps leading to the development of the lymphatic vasculature
has
been proposed (Wigle et al. 2002).
After the initial formation of the vascular
system, venous
endothelial cells become competent to respond
to a lymphatic-inducing
signal. The first indication that
lymphangiogenesis has begun
(lymphatic bias; Fig. 2)
is the specific expression
of Prox1 in a restricted subpopulation of
endothelial cells located
on one side of the anterior cardinal vein; in
the mouse this
expression occurs at ~E9.5 (Wigle and Oliver
1999).
LYVE-1 is also expressed in endothelial cells in the
cardinal veins
at this stage, but not in a polarized manner. At
~E10.5, the
restricted localization of Prox1 in the veins is still
evident, and
the first lymphatic endothelial cells have started
to bud in a
polarized manner.
|
All venous endothelial cells are probably initially bipotent, and the expression of at least Prox1 causes those cells to initiate the program of lymphatic differentiation. As development proceeds, the subpopulation of LYVE-1- and Prox1-positive endothelial cells starts to bud from the veins in an initially Prox1-independent manner. However, maintenance of the budding requires Prox1 activity. As the cells bud they start to express higher levels of additional lymphatic endothelial markers such as SLC and VEGFR-3, whereas the expression of VEGFR-3 decreases in blood vascular endothelial cells. The expression of Prox1, LYVE-1, SLC, and VEGFR-3 may indicate that the cells are irreversibly committed (specified) to the lymphatic pathway (Fig. 2).
Remaining questions about developmental lymphangiogenesis
The identification of lymphatic-specific markers will allow us to address many unanswered questions: Are lymphoangioblasts present in the mammalian embryo? Are venous endothelial cells initially pluripotent? If so, do pluripotent cells become committed to a lymphatic phenotype once Prox1 is expressed? Or does this commitment occur at an earlier stage in development? Is Prox1 activity sufficient to cause venous endothelial cells to bud from the veins and adopt a lymphatic phenotype? Is the polarized expression of Prox1 in the cardinal vein the consequence of a short-range signaling mechanism that operates only in that side of the vein, or does the polarized expression of Prox1 indicate that the cardinal vein already contains subpopulations of cells with previously determined lymphatic and vascular phenotypes?
Clinical implications of lymphatic system biology
In addition to
its prominent role during embryonic development,
lymphangiogenesis is also an essential feature of tissue repair
and
inflammatory reactions in most organs, and congenital or acquired
dysfunctions
of the lymphatic system, resulting in the formation of
lymphedema,
are frequent and often associated with impaired immune
function
(Witte et al. 2001). In
addition, lymphangiogenesis is a common feature
of vascular
malformations (Witte et al. 2001).
Finally, lymphatics are the primary conduit for malignant
tumor dissemination
to the regional lymph nodes, and recent evidence suggests
an active
role of malignant tumors in the induction of intratumoral
and
peritumoral lymphangiogenesis
Lymphatic regeneration
Successful
tissue repair requires the regrowth and reconnection of a
functional lymphatic vascular system. Early studies showed the
formation of lymphatic vessels in circumferential wounds in the
rabbit, bridging the newly formed scar (Bellman and Oden 1958).
In full-thickness skin wounds, ingrowth of new blood
vessels (angiogenesis)
into the newly formed granulation tissue largely dominates
the delayed
and comparatively less pronounced formation of new lymphatic
vessels
(Paavonen et al. 2000),
which are predominantly located surrounding the
blood vessel-rich
granulation tissue. Lymphangiogenesis in the
adult occurs by
outgrowth from preexisting lymphatics (Clark and
Clark 1932;
Paavonen et al. 2000); it
remains to be established whether
lymphangiogenesis during tissue
repair also involves the incorporation of
progenitor cell
populations, as in blood vessel angiogenesis
(Rafii 2000),
or the budding of lymphatic precursors from
preexisting veins,
similar to lymphatic development during embryogenesis.
The recent
discovery of specific lymphatic markers will
greatly facilitate
studies to address this issue in more detail.
During tissue
repair, lymphatic vessels connect with lymphatic vessels, but
not with blood vessels, and cultured lymphatic endothelial
cells
remain separated from blood vascular endothelial cells
during tube
formation of cocultured cells in vitro (Kriehuber et
al. 2001).
Whereas specific ephrins and their Eph receptors have
been detected
on arteries (Ephrin-B2) and veins (EphB4), specific
molecules
involved in lymphatic identity and homeotypic interactions
remain to
be identified. The establishment of well-characterized populations
of
cultured lymphatic- and blood-vessel-derived endothelial cells
(Kriehuber
et al. 2001; Makinen et
al. 2001b) will now
enable studies to identify such molecules.
VEGF-C and VEGF-D,
activating ligands of VEGFR-3, are prime
candidates for molecules
that control wound-associated
lymphangiogenesis; however, despite the
availability of a number of genetic mouse
models for skin-specific
overexpression or inhibition of bioactivity of
VEGF-C and VEGF-D (Makinen
et al. 2001a;
Veikkola et al. 2001),
the biological importance of these factors for
tissue repair has not
yet been established.
Very recent
evidence suggests that VEGF, a major angiogenic molecule that is
up-regulated during tissue repair (Brown et al. 1992),
might also stimulate lymphangiogenesis under certain conditions,
possibly
via interaction with VEGFR-2 that is also expressed by lymphatic
endothelial cells (Kriehuber et al. 2001;
Makinen et al. 2001b).
Moreover, subcutaneous injection of adenoviral VEGF constructs
into
mouse ear skin resulted both in enhanced formation of
new blood
vessels and in increased numbers of enlarged, proliferating lymphatic
vessels (H.F. Dvorak, pers. comm.). However, because VEGF
also
potently induces vascular leakage and tissue edema, it
remains to be
established whether the lymphangiogenesis observed in
conditions with
enhanced VEGF tissue levels, including tissue repair
and
inflammation, is caused by direct activation of VEGFR-2 on
lymphatic
endothelium or by indirect stimulation of lymphangiogenesis by
enhanced interstitial fluid accumulation.
Lymphedema
Our insights
into the molecular and genetic mechanisms of lymphedema
formation have been greatly enhanced over the last few years.
This is
mainly attributable to: (1) the discovery of gene mutations
in two
different types of lymphedema, (2) the identification of
specific
lymphangiogenesis factors and their receptors on lymphatic endothelium,
and (3) the recent development of genetic mouse models for
cutaneous
lymphedema. Lymphedema is caused by an insufficient transport
function of lymphatic vessels owing to lymphatic hypoplasia,
impaired
lymphatic function, or obstruction of lymph flow (Witte et
al. 2001).
Two recently identified lymphangiogenic factors, VEGF-C
and VEGF-D,
and their lymphatic receptor VEGFR-3 most likely
play an
important role in the pathogenesis of at least some cases of
lymphedema. Primary lymphedema has been classified as Milroy
disease
when present at birth (Milroy 1892),
or as Meige disease, which develops
predominantly after puberty (Meige
1898).
Both diseases
are characterized by a combination of dilated lymphatic capillaries
and interstitial accumulation of lymph fluid leading to
lymphedema.
It has been shown that Milroy disease is linked, at
least in some
families, to the VEGFR-3 locus on distal chromosome
5q
(Ferrell et al. 1998;
Witte et al. 1998; Evans
et al. 1999). Subsequently,
missense mutations in the VEGFR-3 gene that
interfere with the
VEGFR-3 tyrosine kinase signaling function were
identified in
several cases of hereditary, early-onset lymphedema (Karkkainen
et
al. 2000).
Whereas VEGFR-3
inactivating mutations have been found in a relatively
small number of cases of hereditary lymphedema thus far,
additional
supportive evidence for a role of VEGFR-3 in the
pathogenesis
of lymphedema stems from experimental studies in transgenic
mice with
skin-specific overexpression of soluble VEGFR-3 using
a keratin 14 transgene
promoter. In this genetic model, soluble
VEGFR-3 is secreted at high
levels by basal epidermal keratinocytes and
binds both
lymphangiogenesis factors, VEGF-C and VEGF-D
(Fig. 3),
thereby preventing them from activating VEGFR-3 on
lymphatic
endothelium (Makinen et al. 2001b).
K14/soluble VEGFR-3 transgenic mice lack a
functional cutaneous
lymphatic system and are characterized by
lymphedema formation in the
skin.
|
Further
experimental evidence for a role of the VEGF-C/VEGF-D/VEGFR-3 system
in lymphedema formation has been provided by the
identification of a
heterozygous inactivating VEGFR-3 mutation
in the germ line of
Chy mutant mice, which develop chylous ascites
and
lymphedematous limb swelling after birth (Karkkainen et al. 2001).
Remarkably, therapeutic increase of tissue VEGF-C levels by
virus-mediated gene therapy stimulated the growth of functional
lymphatics
in Chy mutant mice, suggesting that growth factor
gene therapy
might be applicable to at least some cases of human lymphedema
(Karkkainen
et al. 2001).
Despite the
important role of VEGFR-3 mutations in a subset of
hereditary lymphedemas, primary lymphedemas are comprised of
a
heterogeneous group of diseases that can be associated with additional
malformations of other organ systems. In one such disease entity,
lymphedema-distichiasis, an autosomal-dominant disorder with
congenital lymphedema, double rows of eyelashes (distichiasis),
and
other complications, inactivating mutations in the FOXC2
gene were
identified in several families (Fang et al. 2000).
FOXC2 is a member of the
forkhead/winged-helix family of
transcription factors involved in diverse
developmental pathways.
Additional lymphatic-specific growth factor
receptors, matrix
molecule receptors, and transcription factors
are likely involved in
other cases of hereditary lymphedema and
lymphatic malformations.
Tumor lymphangiogenesis
In most human
cancers, the lymphatic system serves as the primary conduit for
the metastatic spread of tumor cells to regional lymph
nodes and,
possibly, via the thoracic duct and the blood circulation
to distant
organs. Moreover, tumor cell metastasis to
lymph nodes represents a
major criterion for evaluating the prognosis of
cancer patients and
for the choice of additional chemotherapy
and/or radiation therapy
after excision of primary tumors. However,
despite the importance of
tumor-associated lymphatic vessels for cancer
progression, little
information has been available regarding the
molecular mechanisms by
which tumor cells gain access to the lymphatic
system and
consequently are able to spread. In fact, the
presence of functional
lymphatic vessels within tumors has been
questioned because of the
high interstitial pressure within most cancers
(Jain 1989),
and a widely held view has assigned the
lymphatic system a passive
role during the metastasis process (Folkman 1995;
Carmeliet and Jain 2000).
The major reasons for this lack of insight into the early metastatic process have been: (1) the absence of specific markers for tumor-associated lymphatic vessels, (2) the lack of knowledge about specific lymphangiogenesis factors and the ligand-receptor systems that mediate tumor cell migration toward the lymphatics, and (3) the absence of experimental cancer metastasis models for the quantitative evaluation of lymph node metastasis even at the single-cell level. Based on the discovery of several lymphatic-specific markers, in particular of the hyaluronan receptor LYVE-1, and of several growth factors and chemokines involved in lymphatic growth and function, major scientific advances during the last year have provided new insights into the molecular mechanisms that control lymphatic metastasis. These experimental studies have also provided convincing evidence for an active role of malignant tumor cells in inducing peritumoral and intratumoral lymphangiogenesis, taking advantage of molecular mechanisms operative in the immune response, and for a potential role of tumor lymphangiogenesis as a novel prognostic marker for at least some types of human cancers.
Using an
orthotopic human MDA-435 breast cancer model in immunosuppressed
mice, it was shown that lymphatic vessels are, indeed, present
both
surrounding and within malignant tumors, and that overexpression
of
the lymphangiogenesis factor VEGF-C resulted in
enhanced infiltration
of breast cancers by proliferating lymphatic vessels
that frequently
contained cancer cells (Skobe et al. 2001a).
Moreover, VEGF-C-induced tumor lymphangiogenesis resulted
in enhanced
tumor metastasis to regional lymph nodes, and the extent of
lung metastasis
was highly correlated with the extent of lymphangiogenesis of
the
primary tumor. Whereas VEGF-C selectively induced lymphangiogenesis,
but
not angiogenesis, in breast cancer models (Karpanen et al. 2001;
Skobe et al. 2001a),
subsequent studies of human malignant melanoma
xenotransplants (Skobe
et al. 2001b) revealed
induction of both lymphangiogenesis and
angiogenesis by tumor-derived
VEGF-C. These apparently conflicting biological
effects could be
explained by the detection of the fully
processed, mature 21-kD form
of VEGF-C in melanomas (Skobe et al. 2001b).
The 21-kD form is a cleavage product of the
secreted 31-kD VEGF-C and
activates both VEGFR-2, present on blood
vascular endothelium, and
VEGFR-3 (Joukov et al. 1997),
predominantly expressed in lymphatic vessels, resulting in
both
lymphangiogenesis and vascular angiogenesis (Fig. 3).
In
contrast, only the secreted 31-kD form of VEGF-C, that selectively
activates
VEGFR-3, was found in the breast cancer model. These results
indicate
an important role of the in vivo processing of VEGF-C
in
lymphangiogenesis versus angiogenesis (Fig. 3).
The
pro-metastatic role of growth factor-induced tumor lymphangiogenesis
has
also been reported in xenotransplant models of VEGF-D-transfected
transformed human kidney cells (Stacker et al.
2001),
of VEGF-C-transfected MCF-7 breast cancer cells (Karpanen et
al. 2001),
and in a genetic model of pancreas tumorigenesis in
which VEGF-C
expression, driven by the rat insulin promoter, was
targeted to 
-cells
of the endocrine pancreas, resulting in enhanced
rates of lymph node
metastasis (Mandriota et al. 2001).
Whereas these results indicate that tumor cells can
actively induce tumor-associated
lymphangiogenesis and lymphatic metastasis, a recent
report indicates
that lymphatic vessels might, in turn, actively
promote tumor cell
attraction and lymphatic metastasis (Wiley et
al. 2001).
Secondary lymphoid chemokine (SLC; 6Ckine/exodus-2/CCL21) is
produced
constitutively by lymphatic endothelial cells in the skin
and other
organs (Gunn et al. 1998)
and attracts dendritic cells to the lymphatic
vessels by interaction
with the CCR7 receptor. CCR7 is also expressed
by some human breast
cancer and melanoma cell lines (Muller et al.
2001).
When CCR7-transduced B16 malignant melanoma
cells were injected into
the footpads of mice, a >10-fold increase
in the incidence of
regional lymph node metastases was observed
after 3 wk (Wiley et
al. 2001), and metastasis
was completely blocked by adding neutralizing
anti-SLC antibodies.
SLC, via the CCR7 receptor, selectively
enhanced lymphatic
metastasis, because CCR7-transduced and control
B16 cells
metastasized to the lung at the same frequency
after intravenous
injection (Wiley et al. 2001).
Taken
together, these pioneering studies provide important new insights into
the molecular control of lymphatic cancer metastasis. They
also raise
several new questions: (1) Is the lymphatic marker LYVE-1,
that was
used in most of these studies, specific for tumor-associated
lymphatic
vessels, or might LYVE-1 also be re-expressed by tumor-associated
blood
vessels, similar to the reported re-expression of VEGFR-3 on
some
tumor blood vessels (Valtola et al. 1999)?
(2) Is the presence of intratumoral lymphatic
vessels restricted to
tumor xenotransplant models because of trapping
of lymphatic vessels
in between growing tumor foci in xenografts that start from
an injected
cell population (Karpanen and Alitalo 2001),
or can they also be detected during orthotopic,
multistep
carcinogenesis and in autochthonous human
cancers? (3) Does tumor
lymphangiogenesis also occur in human tumors,
and will the
quantification of tumor-associated lymphatic
vessels serve as a new
prognostic tool for determining the likelihood
of primary human
cancers for metastatic spread? (4) Are the
molecular mechanisms that
control lymphatic tumor metastasis also
important for the survival of
metastatic cancer cells within the lymphatic
system or in distant
organs, and might they, therefore, serve as new
therapeutic targets
for the treatment of advanced cancer?
The answers to
some of these questions are beginning to emerge. LYVE-1 has
indeed been shown to be specifically expressed by
lymphatics in
normal murine and human tissues (with the exception of
liver
sinusoidal endothelial cells that are strongly involved in
hyaluronan
uptake; Carreira et al. 2001)
and in tumors of both murine and human origin
(Wigle et al. 2002).
In normal human skin, LYVE-1 is specifically
expressed by lymphatic
endothelium, but not by blood vascular
endothelium that expresses the
marker CD34 (Fig. 4A).
In contrast to the proposed
lymphatic marker podoplanin that is also
expressed on some
CD34-positive cutaneous endothelial cells
(Kriehuber et al. 2001),
we and others (Jackson et al. 2001)
have consistently found that CD34-positive blood vessels
are negative
for LYVE-1 (Fig. 4A).
|
LYVE-1 is also
specifically expressed by tumor-associated lymphatic vessels,
and there is no evidence, thus far, that tumor-associated blood
vessels reexpress LYVE-1. Double-staining for
LYVE-1 and the
lymphatic-specific transcription factor Prox1 revealed that all
LYVE-1-positive lymphatic vessels within and surrounding human
squamous
cell carcinoma transplants also expressed Prox1 (Wigle et
al. 2002).
Similar results have been recently obtained during chemically
induced
multistep skin carcinogenesis in mice. This experimental
model allows
a detailed analysis of the successive stages of
skin cancer
development and has provided valuable insights into
the importance of
the blood vascular system for tumor progression and
metastasis (Hawighorst
et al. 2001). The
squamous cell carcinomas that develop by
malignant conversion from
benign papillomas in this model are
characterized by slow expansive
growth. Similar to previously reported results
in xenotransplant
models (Skobe et al. 2001a),
double stains for LYVE-1 and Prox1 revealed that both
peritumoral as
well as intratumoral lymphatic vessels were present
in these tumors
and that all LYVE-1-positive vessels expressed Prox1
(Fig. 4B).
Proliferating lymphatic endothelial cells were found
surrounding and
within the tumors (Fig. 4C,D),
showing that active
lymphangiogenesis also occurs in orthotopic tumors.
Although
mounting evidence shows that lymphangiogenesis occurs in
experimental tumor models in mice, with important implications
for
tumor metastasis, it has been questioned whether lymphangiogenesis
also
occurs in human cancer and whether the presence of tumor-associated
lymphatic
vessels might have any functional consequences (Jain 1989;
Carmeliet and Jain 2000;
Carreira et al. 2001).
Recent results suggest that, at least in
certain types of human
cancer, tumor lymphangiogenesis does occur and
that the presence of
tumor-associated lymphatic vessels is
correlated with increased
lymphatic tumor metastasis. Whereas no
lymphatic capillaries were
detected associated with invasive breast cancer
(Jackson et al. 2001),
proliferating intratumoral lymphatics have been
detected in head and
neck squamous cell carcinomas (Jackson et al.
2001).
In addition, in oropharyngeal tumors, a high
density of
LYVE-1-positive lymphatic vessels correlated with
the presence of
regional lymph node metastases (Beasley et al.
2002).
Recent studies using podoplanin as a lymphatic marker determined
a
significant correlation between the lymphatic microvascular density
and the lymph node status in human breast cancer (Schoppmann
et al.
2001);
on the contrary,
increased lymphatic microvessel density was
associated with a
favorable prognosis in early-stage cervical
cancer (Birner et al.
2001).
Although this is
encouraging, further studies will be needed to
compare the
specificity of various lymphatic markers for
tumor-associated
lymphatic vessels and to evaluate the
functionality of
tumor-associated lymphatic vessels in more
detail. Peritumoral and
intratumoral lymphatic vessels are also found
in human malignant
melanomas (Fig. 4E), tumors
that metastasize
frequently and early to the regional lymph nodes via
the lymphatic
system (de Waal et al. 1997;
Clarijs et al. 2001).
Conclusions
Finally, after more than 300 years since the initial description of the lymphatic vessels by Gasparo Aselli, some of the mechanisms controlling the normal and pathological development of the previously neglected lymphatic vasculature are being unraveled. The identification of specific markers for the lymphatic vessels has been instrumental in this advance. In addition, the recent concept of tumor lymphangiogenesis is starting to be considered as an important aspect of cancer metastasis. The future of this field of research is very promising and could eventually lead to better diagnosis and treatment of a variety of lymphatic disorders and certain types of cancer
Acknowledgments
We thank N. Harvey, S. Hirakawa, and T. Hawighorst for helpful discussions and critical reading of the manuscript; and D. Jackson for providing the LYVE-1 antibody. G.O. is supported by grants GM58462 and EY12162 from the National Institutes of Health, by Cancer Center Support (CORE) grant CA21765 from the National Cancer Institute, and by the American Lebanese Syrian Associated Charities (ALSAC). M.D. is supported by NIH/NCI (grants CA69184, CA86410 and CA91861), by the American Cancer Society (Research Project Grant 99-23901), and by the Cutaneous Biology Research Center through the Massachusetts General Hospital/Shiseido Co. Ltd. Agreement.
Footnotes
3 Corresponding authors.
E-MAIL guillermo.oliver@stjude.org; FAX (901) 526-2907.
E-MAIL michael.detmar@cbrc2.mgh.harvard.edu; FAX (617) 726-4453.
Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.975002.
References
====Artricles and Abstracts========
Lymphatic vessels and lymphangiogenesis in female cancer:
Mechanisms, clinical impact and possible implications
for anti-lymphangiogenic therapies (Review)
http://www.spandidos-publications.com/or/9/3/455
========
Lymphatic vessels in cancer lmetastasis: bridging the gaps
http://carcin.oxfordjournals.org/content/27/9/1729.long
=======================
Lymphedema People Angiogenesis Related Pages:
Angiogenesis
http://www.lymphedemapeople.com/thesite/angiogenesis.htm
Angiogenesis and Cancer
http://www.lymphedemapeople.com/thesite/angiogenesis_and_cancer.htm
Angiogenesis and Cancer
Control
http://www.lymphedemapeople.com/thesite/angiogenesis_and_cancer_control.htm
Angiogenesis Inhibitors
and Cancer
http://www.lymphedemapeople.com/thesite/angiogenesis_inhibitors_and_cancer.htm
===========================
Lymphedema People Lymphangiogenesis Related Pages:
The Formation of
Lymphatic Vessels and Its Importance in the Setting of Malignancy
http://www.lymphedemapeople.com/thesite/lymphangiogenesis_formation_of_lymphatic_vessels_and_malignancy.htm
Lymphangiogenesis
Lymphedema and Cancer
http://www.lymphedemapeople.com/thesite/lymphangiogenesis_lymphedema_and_cancer.htm
Lymphangiogenesis and
Gastric Cancer
http://www.lymphedemapeople.com/thesite/lymphangiogenesis_and_gastric_cancer.htm
Lymphangiogenesis in Head
and Neck Cancer
http://www.lymphedemapeople.com/thesite/lymphangiogenesis_in_head_and_neck_cancer.htm
Lymphangiogenesis and
Kaposi's Sarcoma VEGF-C
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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http://www.lymphedemapeople.com/phpBB2/viewforum.php?f=16
===========================
Join us as we work for lymphedema patients everywehere:
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Lymphedema People / Advocates for Lymphedema
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http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema\
For Information about Lymphedema Complications
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===========================
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No matter how you spell it, this is another very little understood and
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Support group for parents, patients, children who suffer from all forms
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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
