F o c u s o n va s c u l a r d i s e a s e

review

The lymphatic vasculature in disease

Kari Alitalo

Blood vessels form a closed circulatory system, whereas lymphatic vessels form a one-way conduit for tissue fluid and leukocytes.
In most vertebrates, the main function of lymphatic vessels is to collect excess protein-rich fluid that has extravasated from blood
vessels and transport it back into the blood circulation. Lymphatic vessels have an important immune surveillance function, as
they import various antigens and activated antigen-presenting cells into the lymph nodes and export immune effector cells and
humoral response factors into the blood circulation. Defects in lymphatic function can lead to lymph accumulation in tissues,
dampened immune responses, connective tissue and fat accumulation, and tissue swelling known as lymphedema. This review
© 2011 Nature America, Inc. All rights reserved.

highlights the most recent developments in lymphatic biology and how the lymphatic system contributes to the pathogenesis of
various diseases involving immune and inflammatory responses and its role in disseminating tumor cells.

Structure, function and development of lymphatic vessels
Blood plasma is continuously filtered from the arterial side of the
capillary bed into the interstitial space, where excess fluid and mac-
romolecules drain through the permeable lymphatic endothelial cell
junctions into lymphatic vessels (Fig. 1). Lacteal lymphatic vessels inside
the intestinal villi absorb dietary lipids. Tissues such as the skin and
mucous membranes, which often encounter foreign antigens, are also
particularly rich in lymphatic vessels. Lymphatic capillaries are thin-
walled vessels of approximately 30–80 mm in diameter, composed of a
single layer of oak-leaf–shaped lymphatic endothelial cells (LECs) that
differ in many ways from blood vascular endothelial cells (BECs)1. In
contrast to blood capillaries, lymphatic capillaries lack pericytes, which
are connective tissue cells surrounding blood capillaries, and have a
discontinuous basement membrane containing portals for cells, such as
dendritic cells (DCs), that can migrate into the vessels2. Instead of the
zipper-like endothelial junctions that occur between BECs, LECs have
discontinuous button-like junctions, allowing fluid and certain leuko-
cytes to enter into the vessel lumen through flap-like openings that form
the primary lymphatic valves3–6. Anchoring filaments attach LECs to
collagen fibers and regulate the valve-like opening into the lymphatic
vessel lumen (Fig. 1).
Lymph moves from the lymphatic capillary bed into precollector ves-
sels, which are sparsely covered by smooth muscle cells. The precollector
vessels drain into collecting lymphatic vessels, which are characterized
by the presence of a periendothelial smooth muscle cell layer, a basement
membrane, continuous zipper-like interendothelial junctions and bileaf-
let valves (Fig. 1)4,7. The intrinsic contractility of smooth muscle cells,
the contraction of surrounding skeletal muscles, and arterial pulsations
are necessary for lymph propulsion, whereas the valves prevent lymph
backflow. In this respect, the collecting lymphatic vessels resemble small
veins.
Molecular/Cancer Biology Program, Faculty of Medicine, Institute for Molecular
Medicine Finland and Helsinki University Central Hospital, Biomedicum
Helsinki, University of Helsinki, Helsinki, Finland.
Correspondence should be addressed to K.A. (kari.alitalo@helsinki.fi).
Published online 07 November 2011; doi:10.1038/nm.2545
Although the blood and lymphatic vascular systems are structurally
related and function in concert, they develop at least partially by separate
molecular mechanisms. The blood vasculature is formed by vasculogen-
esis and angiogenesis, driven chiefly by vascular endothelial growth fac-
tor (VEGF), whereas lymphangiogenesis is induced by VEGF-C, which
acts on LECs that differentiate from venous endothelial cells at mid-
gestation8,9. LECs express specific molecules such as prospero-related
homeodomain transcription factor (Prox1), vascular endothelial growth
factor receptor-3 (VEGFR-3), the membrane glycoprotein podoplanin
and lymphatic vessel hyaluronan receptor-1 (refs. 1,8–10). In embryonic
day 9.5 (E9.5) mouse embryos, the transcription factor SOX18 induces
expression of Prox1, which interacts with the venous nuclear receptor
COUP-TFII and initiates the LEC differentiation program in a segmen-
tal pattern in the anterior cardinal vein11,12 (Fig. 1). The dorsolateral
sprouting, migration and survival of the first LECs and the formation of
lymph sacs are dependent on paracrine secretion of the VEGFR-3 ligand
VEGF-C8. The VEGF-C co-receptor neuropilin-2 (NRP-2) and the Eph
tyrosine kinase ligand ephrin B2 are required for efficient sprouting of
lymphatic capillaries13,14. The Notch1-Dll4 signaling pathway is essential
for postnatal lymphatic development15. However, in adult tissues VEGF
does not promote efficient lymphatic sprouting unless Notch signaling
is inhibited, whereas sprouting induced by VEGF-C is not restricted by
Notch16. In addition, the angiopoietins and their Tie receptors have been
implicated in lymphangiogenesis17. Angiopoietin-1 (Ang1) induces lym-
phatic capillary sprouting in mice, and deletion of Ang2 in developing
mouse embryos leads to defective maturation of the collecting lymphatic
vessels17.
Recent studies show that the collagen and calcium binding EGF
domains 1 protein (CCBE1) enhances lymphangiogenesis induced by
VEGF-C and is also required for the budding and sprouting of lymphan-
gioblasts from the venous endothelium in mouse embryos and for tho-
racic duct formation in zebrafish18,19. Interstitial matrix signals can also
stimulate some VEGFR-3 activation when complexed with collagen and
b1 integrin in LECs20,21 (Fig. 2). The points where the lymphatic circula-
tion separates from blood circulation in the developing mouse embryo
(for example, the E11.5 cardinal vein) are sealed by platelet aggregates
that form when blood comes in contact with podoplanin expressed in
LECs22. This separation requires the hematopoietic signaling proteins

nature medicine volume 17 | number 11 | november 2011 1371

 review

Figure 1 The lymphatic vasculature and

molecular mechanisms involved in its
development and growth. (a) Lymphatic
capillaries are derived from venous endothelial
cells. After the arteriovenous differentiation
controlled by Notch and COUP-TFII transcription
factors, Sox18 activates Prox1, which interacts
with COUP-TFII and induces lymphatic
endothelial differentiation9,12, involving elevated
expression of VEGFR-3. VEGF-C then induces the
sprouting of the LECs to generate new vessels8.
VEGF-C and VEGF can also increase the size of
lymphatic vessels by stimulating circumferential
growth. EC, endothelial cell. (b) The formation
of lymphatic valves requires a calcium-induced
signal via phospholipase C-g (PLC-g) and
calmodulin to calcineurin that dephosphorylates
the NFATc1 transcription factor, which enters
the nucleus and induces valve-specific genes
in a complex with FoxC2 (T. Petrova (University
of Lausanne), personal communication)28.
VEGFR-2 and VEGFR-3 and ephrin B2 are
upstream regulators of the pathways necessary
© 2011 Nature America, Inc. All rights reserved.

for valve development32. PDGF-B and collagen

IV (ColIV) production is inhibited simultaneously.
ItgA9, integrin a9; FN-EIIIA, embryonic
form of fibronectin; BM, bone marrow. (c) In
developing lymph nodes, lymphangiogenesis is
first induced when IL-7 and TRANCE stimulate
LTi cells that develop from lymphatic precursor cells (LPCs) under the influence of the chemokine CXCL13 (ref. 78). The LTi cells produce LTa1b2,
which activates VEGF-C expression via the LTb receptor in stromal organizer cells48. In adult lymph nodes, B cell proliferation stimulates VEGF-mediated
lymphangiogenesis62, whereas T cell–derived cytokines restrict lymphangiogenesis by producing interferon-g 63. LN, lymph node.

Syk and Slp-76, which are activated in platelets by the transmembrane
podoplanin receptor Clec2 (refs. 22,23). After their formation, lymphatic
vessels mature and remodel into a branched network that covers the skin
and most internal organs of the body. The development of a hierarchy of
vessels resembling blood capillaries and veins enables the specialization
of the absorptive functions of the lymphatic capillaries and the transport
and immune functions of the collecting vessels.
Analyzing lymphatic malfunction in lymphedema
Techniques for direct imaging of the lymphatic system have devel-
oped significantly over the last twenty years, allowing new insights to
be derived into the pathogenesis of lymphatic-related disorders such
as lymphedema. Lymphoscintigraphy is routinely used for diagnostic
imaging of lymphatics and involves injecting radioactive colloid par-
ticles intradermally or intraparenchymally, allowing the visualization
of their accumulation in the lymphatic plexus and sentinel lymph
nodes. However, this technique is not well suited for imaging lymph
flow. Instead, optical imaging with near-infrared fluorescence dyes such
as indocyanine green has a higher sensitivity that permits quantitative
imaging of lymph flow, especially when stabilized liposomal formula-
tions of the dye are used to prevent toxicity24,25. Other promising new
approaches are also under development, for example optical frequency-
domain imaging, which overcomes the limitations of fluorescent trac-
ers such as their tissue penetration and extravasation and is also suited
for high-resolution imaging of tumor microvasculature26. Ultrasound
array-based real-time photoacoustic microscopy enables noninvasive
high-speed three-dimensional imaging of sentinel lymph nodes27.
Recent molecular genetic studies of lymphangiogenic growth factors,
receptors and signaling pathways have also illuminated the pathogen-
esis of lymphedema, allowing the identification of candidate targets for
lymphedema treatment. These studies have led to a better understand-
ing of the molecular mechanisms of lymphatic vessel development and
regulation. The identification of mutant genes in hereditary human
lymphedemas has pinpointed crucial pathways during the development
of the lymphovascular system1. For example, the most common form
of primary lymphedema, lymphedema-distichiasis, is caused by muta-
tions in FOXC2, which encodes a transcription factor that interacts with
the calcineurin-NFATc1 signal transduction pathway during normal
development of the collecting vessels and valves28 (Fig. 1). Analyses
of Foxc2-mutant mice and individuals with lymphedema-distichiasis
have indicated that the underlying cause for the lymphatic failure in
lymphedema-distichiasis is defective lymphatic valve development and
aberrant recruitment of periendothelial smooth muscle cells and basal
lamina components to lymphatic capillaries. Downstream of Foxc2,
specific connexins are expressed in the developing lymphatic vessels,
including the valve areas, and defective connexins are associated with
lymphatic disorders in mice and man29,30. Interestingly, both mice
with Foxc2 mutations as well as humans with lymphedema-distichiasis
also show venous valve insufficiency in addition to lymphedema31,32.
Although the development of venous valves is initiated by the expression
of ephrin B2, an arterial marker, these valves later express several genes
that are key regulators of lymphangiogenesis, indicating that there are
similar morphogenetic mechanisms for valve development in veins and
lymphatic vessels32.
Another example is provided by analysis of Milroy’s disease
(Supplementary Fig. 1), which is characterized by early-onset congeni-
tal lymphedema. This disease commonly involves heterozygous mis-
sense mutations that inactivate the kinase activity of VEGFR3, which
acts upstream of the FoxC2-NFATc1 pathway (Fig. 1)1,7. In a mouse
model of Milroy’s disease, the resulting defective lymphangiogenesis
can be rescued by therapeutic delivery of excess VEGF-C, which gen-
erates functional lymphatic capillaries33. Genes so far implicated in
various lymphedema syndromes have been listed in Table 1(refs. 1,34).
The molecular mechanisms of pathogenesis behind several of these

1372 volume 17 | number 11 | november 2011 nature medicine


conditions are still poorly understood. In addition, lymphedema may
be a component of other rare syndromes, such as Turner’s syndrome,
Noonan’s syndrome and cholestasis-lymphedema syndrome1.
Rational treatment of secondary lymphedema
Lymphangiogenic growth factor therapy may be most appropriate to
treat common forms of secondary lymphedema after lymphatic ves-
sel damage by surgery, infections or radiation therapy. For example,
one of the most common causes of secondary lymphedema is radical
axillary lymph node dissection during breast cancer surgery. Twenty to
thirty percent of these patients develop lymphedema of the arm on the
same side of the body as the lymph node dissection, and patients with
a high peripheral blood vascular filtration rate seem to be predisposed
to this complication35,36. Preclinical studies of lymphedema treatment
have employed transfer of the gene encoding VEGF-C (or VEGF-D) via
adenoviruses, adeno-associated viruses or naked plasmids or adminis-
tered VEGF-C protein, which all stimulate the formation of new lym-
phatic capillaries and, after an initial increase in lymph extravasation,
reduce edema7. In mice, VEGF-C therapy of damaged collecting lymph
vessels has been shown to lead to lymphatic capillary growth, which was
© 2011 Nature America, Inc. All rights reserved.
followed by intrinsic remodeling, differentiation and maturation into
functional vessels that had normal zipper-like endothelial cell-cell junc-
tions and intraluminal valves and were covered by smooth muscle cells37.
Combining VEGF-C therapy with lymph node transplantation in a
mouse model of limb lymphedema showed that the growth factor–trans-
duced lymph nodes formed both afferent and efferent connections with
the preexisting lymphatic vessel network and could even trap metastatic
tumor cells37. In agreement with earlier findings indicating that a con-
tinuous influx of lymph is required for the maintenance of organized
lymph nodes, control-treated lymph nodes regressed, and their follicular
structure was replaced by adipose and fibrotic tissue37,38. Similar strat-
egies have also been used to repair damaged lymphatic networks in a
large animal model. In pigs, VEGF-C or VEGF-D therapy effectively
restored lymphatic vasculature to the site of surgical damage and greatly
increased the structural preservation and functionality of the transferred
lymph nodes (Supplementary Fig. 2)38. Both growth factors increased
lymphatic drainage when analyzed two months postoperatively.
A number of surgical approaches to lymphedema have also been
reported that are based on small numbers of patients, use nonstandard-
ized or inconsistent measurement techniques and lack long-term follow-
up39. Although lymph node transfer without growth factor therapy has
been shown to provide at least some benefit for humans with lymph-
edema40,41, autologous lymph nodes incorporate into existing lymphatic
vasculature at a low frequency. However, the new findings in mouse and
pig models described above suggest that the transfer of chains of lymph
nodes from another location in the patient could be combined with
VEGF-C treatment to form lymphatic microvascular anastomoses37,42.
Table 1 Genes identified behind various lymphedema syndromes1,34
Gene Disease name (OMIM number)
FLT4 (VEGFR-3) Hereditary lymphedema IA (OMIM 153100, AD)
GJC2 Hereditary lymphedema IC (OMIM 613480, AD)
FOXC2 Lymphedema-distichiasis syndrome (OMIM 153400)
CCBE1 Hennekam lymphangiectasia-lymphedema syndrome (OMIM 235510, AR) Lymphedema of the extremities, intestinal lymphangiectasia, mental
SOX18 Hypotrichosis-lymphedema-telangiectasia syndrome (OMIM 607823, AD)
PTPN14 Lymphedema-choanal atresia syndrome (OMIM 608911, AR)
GATA2 Emberger’s syndrome (OMIM 614038, AD)
AD, autosomal dominant; AR, autosomal recessive.
nature medicine volume 17 | number 11 | november 2011
review
These promising findings could provide a basis for clinical trials combin-
ing autologous transplantation and VEGF-C therapy in patients with,
for example, postoperative lymphatic insufficiency.
Proteolytic processing of VEGF-C and VEGF-D generates short forms
of these growth factors that bind and activate VEGFR-2, which potently
promotes angiogenesis (Fig. 2). These forms can be used for increas-
ing blood and lymphatic vessel size and density in mouse skeletal and
cardiac muscle43,44. Recent structural studies have indicated that the
short forms of VEGF-C and VEGF-D have an extended aminoterminal
alpha helix that is not present in VEGF45,46. Furthermore, one of the two
VEGF-D isoforms was found to bind VEGFR-2 but not VEGFR-3 (ref.
47). Accordingly, this form with a truncated N-terminal helix induced
angiogenesis but not lymphangiogenesis in skeletal muscle, making it a
candidate molecule for proangiogenic therapy in tissue ischemia.
Lymphatic vessels and immune function
The collection of interstitial fluid and associated antigens by lymphatic
vessels permits downstream immune monitoring by lymph nodes. In
mouse embryonic development, lymph node induction is initiated at
E12.5 along large veins by lymphoid tissue inducer (LTi) cells of hema-
topoietic origin. The tumor necrosis factor family cytokine TRANCE
and interleukin 7 (IL-7) induce the LTi cells to produce lymphotoxin
(LT)-a1b2, which binds the LTb receptor of stromal organizer cells and
stimulates production of chemokines, adhesion receptors and cytokines
that attract naive lymphocytes into the developing lymph node (Fig. 1).
Activation of the LTb receptor further induces VEGF-C production in
stromal organizer cells, recruiting lymphatic vessels toward the nascent
lymph node48. However, extranodal lymphatic vessel development is
not driven via the LTb receptor pathway, nor does lack of lymph sacs
prevent the early stages of lymph node development49.
The lymphatic vessels are the principal conduit for soluble antigens
and antigen-presenting cells from peripheral tissues to the lymph nodes
for the mounting of immune responses. They also help to clear other
types of leukocytes from sites of resolving inflammation. The LECs
of afferent lymphatic vessels attract activated DCs, T cells and B cells
expressing the chemokine receptors CCR7 and CCR10 by secreting their
ligands CCL21 (also known as secondary lymphoid chemokine) and
CCL27 (precollector vessels), respectively50,51. Leukocyte trafficking in
the lymphatic vessels is also regulated by LEC products such as com-
mon lymphatic endothelial and vascular receptor-1, which mediates
migration of T and B lymphocytes to the draining lymph nodes, and
sphingosine-1-phosphate (S1P), which stimulates egress of B and T cells
from lymph nodes52,53. S1P also regulates the maturation of lymphatic
vessel button and zipper-like junctions either directly or by lymphocyte-
mediated mechanisms52.
Antigen-presenting cells such as DCs and small soluble antigens in
afferent lymphatic vessels can reach the lymph node subcapsular sinus. A
Findings
Congenital lymphedema
Lymphedema of the extremities, onset at <15 years of age
Lymphedema of mainly lower limbs, triple row of eyelashes, varicose
veins
retardation
Lymphedema, alopecia, teleangiectasias
Lymphedema of lower limbs in children, lack of nasal airways
Lymphedema of lower extremities and genitalia, immune dysfunction,
cutaneous warts, deafness
1373
 review

a S S
CT VHD NT b D1
S S
SS SS
?
S S S S VEGF-C/D VEGF-C
CCBE1
Ab
D2
PlGF VEGF Inhibiting
VEGF-B Collagen Ang1–4 ligand
binding

VEGFR-2
D3

D4

NRP-1 CLP24 Ephrin B2 NRP-2 Integrin Dimerization

b1 inhibiting
PDZ
Src allosteric Ab

VEGFR-1 VEGFR-2 Internalization VEGFR-3 Tie1 Tie2 D5

vesicular trafficking
© 2011 Nature America, Inc. All rights reserved.

Lymphangiogenesis Endothelial survival,

Angiogenesis
Arteriogenesis Angiogenesis vessel stabilization

Figure 2 Lymphangiogenic growth factor–endothelial receptor interactions and binding sites of blocking antibodies. (a) Schematic view of the endothelial
cell–specific growth factors (VEGF, VEGF-B, placental growth factor, VEGF-C, VEGF-D, Ang1, Ang2 and Ang3/4) and receptor tyrosine kinases (VEGFR-1,
VEGFR-2, VEGFR-3 and Tie1 and Tie2)120. Lymphangiogenic signals (in color) are transduced by VEGF-C and VEGF-D, which undergo stepwise proteolytic
activation and bind VEGFR-2 and VEGFR-3. The further functions of the VEGF-C propeptides are unknown and indicated by a question mark. VEGF binds
VEGFR-1 and VEGFR-2. NRP-2, ephrin B2 growth factor and claudin-like protein-24 (CLP24) 121 contain C-terminal PDZ binding domains, which in
NRP-2 and ephrin B2 are involved in the internalization and vesicular trafficking of the receptors. The lymphangiogenic factor CCBE1 binds pericellular
matrix components, such as some collagens19. Collagen can also transduce signals via integrins, VEGFR-3 and Src to LECs20. (b) A structural model of the
VEGF-C–VEGFR-2 complex (in color), as determined by X-ray and small-angle X-ray scattering studies 45,46. In the model, the crystal structure of VEGF-C
in complex with VEGFR-2 domains 2 and 3 are shown in orange and in blue, respectively, in combination with the related Kit receptor domains 1, 4 and 5
in gray, suggesting that VEGFR-2 domains 4 or 5 or both may contribute additional interactions for dimerization upon ligand-induced activation. VEGFR-2
dimerization and activation involves homotypic interactions between the membrane proximal immunoglobulin-like domains 7 (ref. 122). Antibodies (Ab)
that block ligand activation of the receptor interact either with the ligand binding site or allosterically interfere with the region involved in the formation of
active receptor homo- or heterodimers95 (see Fig. 4 for details).

system of conduits that extend into the follicles mediates delivery of small
soluble antigens to cognate B cells and follicular DCs54. Larger antigens
are captured by cortical DCs and subcapsular sinus macrophages for
translocation, B cell encounter and transfer of opsonized antigen along
macrophage processes to initiate early T cell priming events within the
first four hours after antigenic challenge55,56. It is possible that cytoplas-
mic microparticles released by various cells are also transported in lymph
and signal to remote lymph nodes57. Importantly, recent studies have
indicated that lymph node–resident stromal cells and LECs can induce
tolerance to host peripheral tissue antigens by direct antigen cross-pre-
sentation to antigen-specific CD8+ T cells, leading to their deletion58. It is
still unclear how important this mechanism is overall in the maintenance
of peripheral tolerance, but its failure could contribute, for example, to
the chronic inflammation that develops in individuals with lymphedema.
Lymphatic vessels in inflammation
Striking changes in lymphatic vessels are associated with acute tissue
inflammation, such as that in bacterial infection. Proinflammatory
cytokines and bacterial lipopolysaccharide induce VEGF-C expression
in several cell types via the downstream nuclear factor-kB pathway3,59.
High levels of lymphangiogenic factors are also produced by macro-
phages and granulocytes in inflamed tissue, and blocking these factors
suppresses lymphangiogenesis and reactive inflammation of the lymph
node, lymphadenitis3. Lymphangiogenesis in inflammatory settings
facilitates the resolution of tissue edema and promotes macrophage and
DC mobilization3,60,61. Lymph node lymphangiogenesis is stimulated by
VEGF produced by activated follicular B cells, whereas T cell–derived
interferon-g signals restrict lymphangiogenesis62,63 (Fig. 1).
Lymphangiogenesis is also associated with chronic inflammation,
for example in psoriasis or rheumatoid arthritis64. Furthermore, lym-
phatic vessel dysfunction and lymphangiogenesis may be involved in
Crohn’s disease, where inflammatory granulomas and ‘fat wrapping’
around the intestinal lymphatic structures may result from a failure of
the lymphatic vessels to transport inflammatory cells and lipids65. In
kidney transplants, CCL21 produced by host-derived lymphatic ves-
sels attracts CCR7-expressing DCs66, which elicit primary alloantigen
recognition events in the lymph nodes that drain the graft (Fig. 3). In
contrast, blocking lymphangiogenic signals mediated by VEGFR-3
reduces CCL21 expression in allograft LECs and suppresses adaptive
immune responses toward heart transplants in a rat model67. Inhibition
of lymphangiogenesis also promotes corneal and pancreatic endocrine
islet allograft survival68,69. Thus, whereas abundant lymphatic vessels
provide resolution of the inflammatory infiltrate and reduce signs of
inflammation in peripheral tissues, the blocking of lymphangiogenesis
and associated DC migration provide an anti-inflammatory mechanism
for the prevention of alloimmunization. The therapeutic concept that
emerges from these studies suggests that a lymphangiogenic growth
factor such as VEGF-C would clearly be beneficial in dampening tis-
sue inflammation associated with lymphedema, whereas inhibition of
lymphangiogenesis in the context of heterologous tissue transplantation
would be beneficial by dampening the development of a full-blown
acquired immune response toward the graft.

1374 volume 17 | number 11 | november 2011 nature medicine


Interestingly, because the lymphatic basement membrane has pre-
formed holes and the lymphatic endothelium has flaps, lymphatic entry
of DCs may not require proteolysis or integrins2,70. Sessile DCs located
along blood vessels are mobilized by inflammation, move direction-
ally toward lymphatics, contact CCL21 deposits and migrate through
connective tissue portals and endothelial flaps into the lumen6. Inside
lymphatic vessels, DCs extend lamellipodia to crawl along lymphatic
endothelium, and, finally, in the collecting lymphatic vessels they start
drifting freely along the fluid flow6. DC migration can be promoted by
VEGF-C, increased transmural fluid flow, delayed type hypersensitiv-
ity reactions and tumor necrosis factor, all of which upregulate CCL21
secretion, as well as the ICAM-1 leukocyte adhesion receptor by the
LECs71–73. CCL21 binds heparan sulfate, which seems essential for
DC intravasation; podoplanin and collagen type IV may be involved
as well6,74. However, although ICAM-1 contributes to lymphatic trans-
migration in at least some inflammatory processes73, inflamed LECs
were reported to suppress DC maturation in an ICAM-1–dependent
manner, perhaps to prevent undesired (auto)immune responses during
inflammatory processes75. Additionally, D6, a decoy receptor, which
is expressed in LECs, scavenges inflammatory CC chemokines and is
© 2011 Nature America, Inc. All rights reserved.
thus capable of reducing the inflammatory response in various organs76.
Although lymph nodes are highly plastic organs, complete lymph
nodes cannot form de novo postnatally. However, ectopic, ‘tertiary’
lymphoid organs consisting of clonally expanding B cell follicles, T
cell compartments and high endothelial venules can be found at sites
of chronic inflammation, even in atherosclerosis, and their formation
seems to involve many of the same chemokines that operate in lymph
node development77,78. LTa can promote inflammation-associated lym-
phangiogenesis, and CD11c-positive DCs are crucial in the induction of
lymphangiogenesis in the tertiary lymphoid structures79,80.
Tissue inflammation links lymphatic vessels to obesity and
cardiovascular disease
A functional link has emerged between lymphatic malfunction and
the pathogenesis of obesity, atherosclerosis and cardiovascular disease,
which are leading causes of disability and mortality in developed coun-
tries. Lymphatic vessels are important in lipid metabolism, as intestinal
lymphatics take up dietary lipids in the form of chylomicrons, large
lipoprotein particles, to transport them to the bloodstream. Lymph
nodes and collecting lymphatic vessels are commonly embedded in
subcutaneous or visceral fat, and the perinodal adipose tissue undergoes
dynamic changes in response to low-level inflammatory processes81,
which are known to contribute to obesity-associated metabolic disease
involving insulin resistance, type 2 diabetes and cardiovascular disease.
A link between lymphatic dysfunction, inflammation and adipogenesis
is evident from the development of ectopic subcutaneous adipose tis-
sue in edematous sites in individuals with chronic lymphedema82, but
the molecular mechanisms of these changes are poorly known. Mice
with a heterozygous Prox1-inactivating mutation were found to have
leaky lymphatic vessels and to develop obesity and inflammation with
features associated with late-onset obesity in humans, such as higher
levels of leptin and insulin and fatty liver83. Furthermore, mice with
a heterozygous mutation in Vegfr3 (so-called Chy mice) had lymph-
edema and chronic tissue inflammation in association with abnormal fat
accumulation33,84. Conversely, therapeutic lymphangiogenesis induced
by VEGF-C alleviated tissue inflammation and edema in the mouse
models33,85.
Lymphatic vessels are also found at sites of atherosclerosis, which
is associated with inflammation and lipid accumulation in arterial
walls86,87. Arterial smooth muscle cells constitutively produce lymphan-
giogenic factors, and lymphatic vessels are present in the adventitia of
nature medicine volume 17 | number 11 | november 2011
review
Organ transplantation
Soluble
antigen
Lymphangiogenesis APC
CCR7+
VEGF-C VEGF-C
VEG VEGFR3+
Macrophage
T cell
CCL21
Blood
Blood vessels Lymphatic
and
nd circulation
an vessels
Spleen
Lymph
ph n
nodes
Figure 3 A schematic model of lymphatic vessel function in organ
transplantation. Inflammatory cells such as macrophages in organ
transplants can produce high levels of VEGF-C66. The resulting lymphatic
vessel activation is associated with enhanced secretion of the CCL21 ligand
of the chemokine receptor CCR7, improved flow of lymph that contains
soluble antigens, and mobilization of activated antigen-presenting cells
(APCs) that express CCR7; some APCs also express VEGFR-3 (ref. 67). APCs
migrate to lymph vessels, then to lymph nodes, and further, via the blood
vessels, to the spleen to activate T cells for immune rejection of the graft,
being subsequently delivered to the graft via the blood vessels. The blocking
of lymphangiogenesis, which has so far not shown adverse side effects in
normal tissue, inhibits acute graft rejection in the context of heart, corneal
and pancreatic islet cell transplantation survival67–69.
arteries adjacent to small blood vessels, called the vasa vasorum, which
are expanded in atherosclerotic plaques77,88. Mice lacking apolipopro-
tein E have not only atherosclerotic plaques and tertiary lymphoid tissue
in the adventitia of arteries but also dysfunctional lymphatic vessels77,89.
Lymphatic vessels could perhaps provide a protective pathway for lipid
and inflammatory cell efflux from the arterial wall, which could oppose
the development of atherosclerotic plaques. Further studies should
reveal whether manipulation of lymphangiogenesis can provide a means
to slow down fat deposition and tissue inflammation associated with the
development of atherosclerotic lesions.
Inhibition of the VEGF-C receptor VEGFR-3 acts as a lymphatic
vessel–targeted immunomodulatory therapy for the arteriosclerosis that
develops in a cardiac allograft model67. The heart itself has lymphatic
vessels, but virtually nothing is known about their role in the healthy
or failing heart. Cardiac contraction generates considerable transmural
pressures that are predicted to result in cardiac or pericardial edema if
the lymphatic vessels do not remove the excess fluid. However, it remains
to be seen whether increased lymphatic function improves myocardial
performance in, for example, infarcted or dilated heart, in which the
wall tension is severely elevated.
Recent results have shown that hypertension induced by salt overload
leads to inflammatory cell recruitment and increased lymphangiogenic
growth factor production in the skin90. These studies have suggested
a VEGF-C–mediated mechanism in the skin that maintains normal
blood pressure in states of interstitial salt accumulation. An excess of
salt accumulated in the skin without associated water retention recruits
macrophages, which are induced to secrete VEGF-C. Selective blockade
of VEGF-C and VEGFR-3–mediated signals resulted in salt-sensitive
1375
 review
VEGF-C/D trap
VEGF trap
Ab
VEGF Ab VEGF-C Ab Pb Ang2
Ab
Ab*
VEGF/Ang2
trap
TKI
VEGFR-1 VEGFR-2 VEGFR-3 Tie1 Tie2
© 2011 Nature America, Inc. All rights reserved.
Angiogenesis Lymphangiogenesis Angiogenesis
Figure 4 Inhibitors of VEGF, VEGF-C, Ang2 and their receptors. The blocking
antibodies (Ab) against VEGF (bevacizumab) and VEGF-C and against the
ligand-binding parts of VEGFR-2 (Ramucirumab) and VEGFR-3 (http://
clinicaltrials.gov/ct2/show/NCT01288989) are shown in red. The antibodies
marked Ab* are allosteric inhibitors of active VEGFR dimer formation95,123.
The VEGF trap (aflibercept) contains the ligand-binding immunoglobulin
homology domain 2 (Ig2) of VEGFR-1 fused to the Ig3 domain of VEGFR-2
and further to the Fc part of human IgG (http://clinicaltrials.gov/ct2/show/
NCT00561470?term=aflibercept&phase=2&rank=4). The VEGF-C/VEGF-D
trap consists of a fusion of the Ig1–3 domains of VEGFR-3 with the IgG Fc
domain42. The VEGF/Ang2 trap fuses the Ig1 and Ig2 domains plus the EGF
homology domains of Tie2 with the Ig2 domain of VEGFR-1 and further with
the IgG Fc domain124. The peptibody (Pb) against Ang2 is a fusion of an
inhibitory Ang2 binding peptide with the IgG Fc domain. Ang2 inhibitors and
VEGF inhibitors together result in increased reduction of tumor vascularity
and growth125,126. VEGF-B, PlGF and VEGFR-1 are outside of the scope of
this review. TKI, tyrosine kinase inhibitor.
hypertension despite increased expression of endothelial nitric oxide
synthetase90. These findings suggest that macrophages are extrarenal
regulators of electrolyte, volume and blood pressure homeostasis via a
unique regulatory feedback control system involving lymphangiogenic
factors.
Lymphovascular tumors
A number of tumor types can form in the lymphatic tissues, and
researchers are just beginning to understand the pathogenesis of these
tumors. Studies investigating lymphovascular tumors have highlighted
that manipulation of lymphangiogenesis and angiogenesis may be of
potential therapeutic benefit for lymphatic tumors. Lymphangiosarcoma
is a malignant tumor that occurs in long-standing cases of primary or
secondary lymphedema, but its molecular pathogenesis is unclear. LECs
may also give rise to Kaposi’s sarcoma, which is an angiogenic tumor
common especially in the HIV-infected populations of sub-Saharan
Africa. Kaposi’s sarcoma is caused by infection with a gammaherpes-
virus (KSHV) that has previously been shown to induce reprogramming
of BECs to LECs (and vice versa)91. Correspondingly, global transcrip-
tomic analysis has demonstrated that the gene expression signature
in the Kaposi’s sarcoma tumors resembles more that of LECs than of
BECs. More recently, KSHV was shown to induce transcriptional repro-
1376
gramming of primary LECs to mesenchymal, invasive ‘spindle’ cells via
activation of the Notch signal transduction pathway92,93. Moreover, a
component of the KSHV capsid has been shown to activate VEGFR-3
and induce endothelial proliferation and migration94. Inhibition of
VEGFR-3 with a combination of the two blocking antibodies schemati-
cally illustrated in Figure 4 was effective in reducing (lymph)angiogenic
sprouting and growth of KSHV-infected LECs in three-dimensional cul-
ture, suggesting that this approach could provide a potential treatment
option for Kaposi’s sarcoma95.
Lymphangioleiomyomatosis (LAM) is characterized by the infiltra-
tion of abnormal, smooth muscle–like LAM cells through the pulmo-
nary interstitium, perivascular spaces and the lymphatics of young
females, leading to cystic structures that obstruct airways and lead to
lymphatic disruption with effusion of fluid containing lymph and lipids,
finally resulting in respiratory failure. In about one-third of the cases,
LAM is associated with mutations in the tuberous sclerosis tumor sup-
pressors TSC1 or TSC2 involved in mTOR signaling and also with the
formation of renal angiomyolipomas (tumors made up of LAM cells,
adipose tissue and poorly developed blood vessels)96. Although some
patients with LAM benefit from the mTOR inhibitor rapamycin97, pul-
monary transplantation is needed for treatment of progressive disease.
High, often diagnostic levels of VEGF-D are secreted by LAM cells that
frequently become covered by LECs and invade the lymphatic system
and lungs98. Although an animal model of LAM is lacking, VEGFR-3–
blocking antibodies, which are currently in phase 1 clinical trials (http://
clinicaltrials.gov/ct2/show/NCT01288989), may provide another agent
for LAM treatment.
Lymphatic metastasis and its inhibition
The lymphatic vessels also provide a route for tumor cells to metastasize.
Solid tumors have high interstitial pressure, which may at least partly
have a role in enabling tumor cells to enter lymphatic vessels in the
tumor periphery and to metastasize99. When injected into tumor tissue,
tracer compounds are readily taken up by the lymphatic vessels that
drain the tumor, providing the means for sentinel lymph node mapping
for tumor staging, for example in breast cancer and melanoma42,100.
However, growth of tumor cells after their arrival in the marginal sinus of
the sentinel lymph node may impede lymph flow and reroute the lymph
flow around the blocked lymph node, thus compromising the mapping
of sentinel nodes24,100,101. Despite these considerations, lymph node
metastasis continues to be used as an important parameter in tumor
staging and therapeutic decision-making.
Growth in the lymph node microenvironment may select tumor cells
for increased metastatic potential. Current cancer treatments comprise
surgical removal of the primary tumor and metastatic lymph nodes fol-
lowed by various types of adjuvant therapy. A tumor-positive sentinel
lymph node biopsy frequently leads to complete surgical resection of
lymph nodes. However, several large clinical studies have failed to con-
firm a survival advantage despite the previously held view that axillary
lymph node dissection and irradiation improve the overall survival of
individuals with breast carcinoma whose sentinel lymph node biopsy
contains tumor cells102,103. Overall, the studies suggest that there is
tumor type–specific variation between the contribution of lymphatic
vessels and the formation of distant metastases.
Recent progress in tumor imaging, high-throughput analysis of
genomic aberrations and clonal single-cell analysis has provided new
insights into early tumor cell dissemination, and tumor cell dormancy
versus metastasis outgrowth104,105. Considerable heterogeneity is evi-
dent when the primary tumor and different lymphatic and distant organ
metastases are compared106,107. This has enabled the drawing of phylo-
genetic trees across metastases, which show the organ-specific branches
volume 17 | number 11 | november 2011 nature medicine
 review

Figure 5 Mechanisms contributing to lymphatic

metastasis. (a) Top left, the normal lymph node
a b Tumor Sentinel LN
(LN) has been stained with antibodies detecting B LN Metastasis in LN VEGF-C, VEGF, Ang2↑ LN lymphangiogenesis
cells (red), T cells (green) and lymphatic sinusoid Tolerance induction?
VEGF, VEGF-C↑
(white)63. Top right, the eosin-stained (blue) LN Lymphangiogenesis
Lipoxygenase↑
Angiogenesis Collecting
shows metastatic foci in gray. Bottom, increased CCL21↑
CCL21↑
lymphatic vessels
VEGF-C and D secretion by tumor cells or tumor- Increase in size
associated inflammatory cells induces tumor Increased flow
lymphangiogenesis, hyperplasia of collecting Mø
LTi
lymphatic vessels and tumor cell translocation Treg Distant
Circumferential
into lymphatic vessels and lymph nodes10,42,64. hyperplasia LN
(b) Aberrant expression of chemokine receptors Mø
such as CCR7 may increase tumor cell migration
toward lymphatic vessels that produce its
Angiogenesis and
ligand CCL21 (refs. 50,108). Tumors may also tumor growth
stimulate LN lymphangiogenesis (sinusoidal Inhibitors:
hyperplasia) 64,112 in the draining (sentinel) Anti–VEGF
Anti–VEGFR-2
lymph node and invade the generated vessels Anti–VEGFR-3
in the lymph nodes by a lipoxygenase-mediated VEGF trap
VEGF-C/VEGF-D trap
mechanism that contributes to further metastasis
TKI
to secondary lymph nodes112. VEGF and Ang2 Tumor Intralymphatic Anti–Ang-2
can also contribute to these lymphangiogenic lymphangiogenesis tumor growth Ang2 peptibody
VEGF/Ang2 trap
changes, whereas macrophages (MØ) are recruited
© 2011 Nature America, Inc. All rights reserved.

Lymphangiogenesis
to the tumor stroma. VEGF-C also upregulates
Metastasis
lymph flow and CCL21 expression, which has
been shown to lead to recruitment of CCR7-
expressing immune cells, including regulatory
T cells (Treg) and LTi cells, resulting in a shift of the host immune response from immunogenic to tolerogenic, which facilitates tumor progression 108. Some of
the tumor-reactive CD8+ T cells may also be eliminated in the sentinel lymph node111.

and genetic heterogeneity among metastasis-initiating cells and indicate
that driver mutations may be required for metastasis development107. In
different tumor types, the density of tumor-associated lymphatic vessels
varies and correlates with the incidence of lymph node metastasis and
poor prognosis10,42,64. However, it is not clear to what extent lymph
node metastases promote spreading of the tumor cells by expanding the
tumor and serving as a launch pad for further systemic metastasis105.
Intratumoral lymphatic vessels have been considered to be poorly
functional as a result of high intratumoral pressure, whereas lymphatic
vessels in the tumor periphery are functional99. Because of the lower
mechanical stress inside peritumoral lymphatic vessels, they may pro-
vide a more favorable environment for metastasis dissemination than
blood vessels. In addition, LECs secrete ‘lymphangiocrine’ chemokines,
such as CCL21, that can promote tumor cells expressing the cognate
receptor CCR7 to migrate toward the lymphatic vessels. Another
important chemokine produced by LECs is stromal-derived factor-1
(also known as CXCL12), which binds CXCR4 on tumor cells, promot-
ing a lymphatic microenvironment that supports tumor growth108,109.
Although tumor cells can invade lymphatic vessels, it has become evi-
dent that tumors overexpressing either VEGF-C or VEGF-D in tumor
cells or stromal cells, such as tumor-associated macrophages, induce
sprouting of lymphatic vessels, enlargement of collecting lymph vessels
and lymph node lymphangiogenesis (also known as sinusoidal hyper-
plasia) in the draining lymph nodes42,64,110. Such tumors are particularly
prone to developing lymph node metastases10,42,64. When overexpressed,
several other growth factors, such as VEGF, fibroblast growth factors,
platelet-derived growth factor-B (PDGF-B), hepatocyte growth factor
and insulin-like growth factor-1, can also induce lymphangiogenesis
and metastasis, presumably via more indirect pathways, such as inflam-
matory cell recruitment and VEGF-C induction10,42. It has been sug-
gested that the tumor-draining lymph nodes are capable of promoting
immune tolerance toward tumor antigens by antigen cross-presentation
to CD8+ T cells that can lead to the destruction of the tumor antigen-
specific T cells110. Furthermore, although metastases form initially in,
for example, axillary sentinel lymph nodes, they progress via connecting
lymphatic vessels into postsentinel lymph nodes of the same chain (Fig.
5). In human mammary carcinomas and their matching axillary lymph
nodes, intrametastatic lymphatic vessels, lipoxygenase expression and
bulk tumor cell invasion into these vessels highly correlates with the
formation of postsentinel metastasis112.
Inhibition of lymphangiogenesis with a soluble form of VEGFR-3 or
monoclonal antibodies that block this receptor inhibits 50–70% of tumor
metastasis to lymph nodes in mouse and rat models42,105. VEGFR-3 sig-
naling can also contribute to angiogenesis in tumors where the receptor
is expressed on both tumor blood and lymphatic vessels113. The involve-
ment of VEGFR-3 in the growth of both blood vessels and lymphatics
makes this receptor a promising candidate for cancer therapy, as it is pos-
sible that, by blocking this receptor, a dual effect on lymphangiogenesis
and angiogenesis may be obtained. Consistent with this, monoclonal
antibodies that block the binding of VEGF-C and VEGF-D to VEGFR-3
can suppress angiogenesis, and antibodies against VEGFR-3 and
VEGFR-2 in combination resulted in additive inhibition of lymphangio-
genesis114, angiogenesis and tumor growth113. Furthermore, the efficacy
of angiogenesis inhibition was increased through the simultaneous use
of an antibody that blocks growth factor binding to VEGFR and another
antibody that allosterically inhibits the formation of kinase-activated
VEGFR dimers95. Also, NRP-2–blocking antibodies have recently been
shown to reduce tumor lymphangiogenesis and lymph node metastasis,
at least partly by suppressing LEC migration13,115. However, the safety
of a treatment targeting VEGFR-2 and VEGFR-3 needs to be carefully
addressed as, for example, hematopoietic reconstitution after irradiation
for bone marrow depletion may require VEGFR-2 and VEGFR-3 expres-
sion in the bone marrow sinusoidal endothelium116. Further validation
of the efficacy and safety of antibody combinations in preclinical models
could lead to inhibitors that block both tumor angiogenesis and lymph
node metastasis in patients with cancer.
Despite complete removal of the primary tumor, approximately one-
fifth of individuals with melanoma develop local metastasis, termed
satellite metastasis or in-transit metastasis, after surgery117, and the
lymphatic system may contribute to this. Indeed, clinical studies have

nature medicine volume 17 | number 11 | november 2011 1377

 review
suggested that tumor-draining collecting lymphatic vessels host tumor
cells. We have recently shown that photodynamic therapy with the ben-
zoporphyrin derivative verteporfin can be used to destroy lymphatic
vessels and small tumor nodules inside them that have the capacity to
develop into secondary tumors118. In addition, the photodynamic ther-
apy in combination with blocking of the VEGFR-3 pathway prevented
metastasis of mouse melanoma cells as well as subsequent relapse118.
These studies show considerable potential for the development of clini-
cal antimetastasis therapy.
Clinical translation of lymphatic biology
The essential role of the lymphatic vessels in a multitude of biological
functions such as in the maintenance of tissue fluid balance, immuno-
surveillance and transport of dietary fats, and as a gateway for tumor
metastasis, make them excellent targets for drug development in vari-
ous diseases. Through the discovery of lymphangiogenic factors and
studies of their signal transduction pathways, it has become possible to
stimulate or inhibit lymphatic vessel formation in adult tissues. This has
provided new possibilities for the treatment of secondary lymphedema
in humans and for the inhibition of lymphatic metastasis with the benefit
© 2011 Nature America, Inc. All rights reserved.
of additional inhibition of tumor angiogenesis.
The first inhibitor of lymphangiogenesis, the VEGFR-3–targeting
monoclonal antibody IMC-3C5, has recently entered phase 1 clinical
trials for patients with advanced solid tumors that are refractory to stan-
dard therapy or for which no standard therapy is available. The animal
data predict that clinical studies using VEGFR-3–blocking antibodies
should provide benefit as antiangiogenic and antimetastatic agents.
Further trials using lymphangiogenesis blockers seem equally straight-
forward for the treatment of Kaposi’s sarcoma or LAM, and they may
aid in prolonging graft survival in, for example, corneal transplantation.
These inhibitors may also be useful in treating dry eye disease118.
A clinical trial using VEGF-C growth factor therapy is imminent
for the regeneration of lymphatic vessels in the treatment of secondary
lymphedema. The ability of VEGF-C to regulate the growth of lym-
phatic vessels should facilitate successful lymph node transfer when
combined transplant and VEGF-C therapy is used to treat lymphedema.
This strategy should result in more durable lymph node transplants,
reducing the need for pressure bandages, which are sometimes painful.
In addition, broader applications of VEGF-C and VEGF-D may become
possible in regenerative medicine and tissue engineering for patients
with tissue defects, including those with lymphatic vessel damage due
to skin burn injuries.
Knowledge of the genetics of inherited syndromes involving the lym-
phatic vessels has provided insight into the pathogenesis of lymphatic
vascular diseases. There is now increased attention on the mechanisms
coupling lymphatic vessels to immune regulation, host defense to infec-
tions, allo- and autoimmune responses and perhaps also obesity, meta-
bolic disease and atherosclerosis. Such research should result in the
design of new therapies for these conditions. Improved knowledge on
lymphatic vascular biology could aid also the development of better vac-
cination strategies. Furthermore, new questions are continuously emerg-
ing from the findings obtained from preclinical studies. For instance, do
the persistently enlarged lymphatic vessels in airway submucosa after
respiratory infections provide more efficient secondary responses upon
re-infection, or do they predispose to inflammatory diseases3? What are
the pathogenenetic mechanisms behind intestinal lymphangiectasias
associated with protein-losing enteropathy? Answering such questions
should provide additional clinical concepts for the therapeutic targeting
of the lymphatic system in various human diseases.
Note: Supplementary information is available on the Nature Medicine website.
1378
ACKNOWLEDGMENTS
I am grateful to M. Bry, L. Eklund, S. Jalkanen, D. Kerjaschki, G.Y. Koh,
V.-M. Leppänen, T. Mäkinen, A. Nykänen, M. Öhman, P. Ojala, P. Saharinen,
M. Swartz and T. Tammela for useful discussions of the topics of this review.
The lymph node images in Figure 5a were kindly provided by D. Kerjaschki
and by G.Y. Koh, and draft figures were produced by H. Schmidt. The studies in
my laboratory are currently supported by the Academy of Finland, the Finnish
Cancer Organisations, the Association for International Cancer Research, the
Sigrid Juselius Foundation, Seventh Framework Program of the European Union
(ERC Advanced Grant), Finnish Foundation for Cardiovascular Research and
Biocentrum Finland. I apologize to the many authors whose important work could
not be cited because of space restrictions and the focus on the recent developments;
older references appear in the many excellent reviews that have been cited.
COMPETING FINANCIAL INTERESTS
The author declares competing financial interests: details accompany the full-text
HTML version of the paper at http://www.nature.com/naturemedicine/.
Published online at http://www.nature.com/naturemedicine/.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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volume 17 | number 11 | november 2011 nature medicine