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Blood First Edition Paper, prepublished online March 7, 2014; DOI 10.1182/blood-2013-12-297317
Pressing the right buttons: signaling in lymphangiogenesis
Sanja Coso1, Esther Bovay1 and Tatiana V. Petrova1,2
1
Department of Oncology, CHUV and Department of Biochemistry, University of
Lausanne, Ch. des Boveresses 155, CH-1066 Epalinges, Switzerland, and 2Swiss Institute
for Cancer Research, EPFL, Lausanne, Switzerland
Keywords: signal transduction, lymphangiogenesis
Corresponding author: Tatiana V. Petrova, Department of Oncology, CHUV-UNIL, Ch.
des Boveresses 155, CH-1066 Epalinges, Switzerland. Phone: +41-21-692 58 28; Fax:
+41-21-692 58 72; E-mail: [email protected]
1
Copyright © 2014 American Society of Hematology
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Abstract
Lymphatic vasculature is increasingly recognized as an important actor both in the
regulation of normal tissue homeostasis and immune response, and in many diseases,
such as inflammation, cancer, obesity and hypertension. In the past few years, in addition
to the central role of VEGF-C/VEGFR-3 signaling in lymphangiogenesis, significant new
insights were obtained about Notch, TGFβ/BMP, Ras, MAPK, PI3 kinase and
Ca2+/calcineurin signaling pathways in the control of growth and remodeling of
lymphatic vessels. An emerging picture of lymphangiogenic signaling is complex, and in
many ways distinct from the regulation of angiogenesis. This complexity provides new
challenges, but also new opportunities for selective therapeutic targeting of lymphatic
vasculature.
2
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Overview of lymphatic vasculature
Lymphatic vessel structure and function
The vascular system consists of blood and lymphatic vessels, both of which are lined by
endothelial cells. Lymphatic vasculature is composed of a branched network of
capillaries and collecting lymphatic vessels, present in most organs (Fig. 1 and reviewed
in 1). Unlike the blood vasculature, lymphatic capillaries are blind-ended. Small
capillaries drain into pre-collecting and collecting vessels that join thoracic duct. Lymph
flows through collecting vessels and lymph nodes before ultimately being returned to
venous circulation at junctions with subclavian vein.
The specialized discontinuous “button-like” junctions between endothelial cells of
lymphatic capillaries represent sites of fluid and immune cell entry into lymphatic
vasculature 2. Unlike blood capillaries, lymphatic capillaries have sparse basement
membrane and lack pericytes. They are also attached to extracellular matrix by anchoring
filaments. Collecting lymphatic vessels have continuous cell-cell junctions and they are
covered with basement membrane and smooth muscle cells (SMCs). Collecting
lymphatic vessels contain intraluminal valves, which consist of two semilunar leaflets,
covered by a specialized endothelium attached to the core of extracellular matrix
(reviewed in 3). High lymph pressure upstream of a valve opens the valve and enables
lymph flow, whereas flow in the reverse direction pushes leaflets against each other and
closes the valve, depending on changes in fluid pressure within collecting vessels. SMCs
that cover the lymphangions, contract and regulate the lymph flow 4.
3
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Main steps of lymphatic vascular development in mammals
The early studies of lymphatic vasculature suggested the two potential sources of
lymphatic endothelial cells (LECs) during development: blood vessels
5
and
mesenchymal cells 6. Recent studies, using high resolution imaging of developing mouse
embryos and lineage tracing approaches
7,8
suggest that the majority of LECs in
mammals, are generated through trans-differentiation from embryonic veins. LEC
commitment requires Sry-related Hmg-box 18 (Sox18) and the orphan nuclear receptor
chicken ovalbumin upstream promoter transcription factor (COUP-TFII), which upregulate prospero-related homeodomain transcription factor (Prox1), essential for
lymphatic endothelial-specific programming in mammals, but not zebrafish
9-11
. Prox1
down-regulates blood markers and induces lymphatic-specific genes, receptor tyrosine
kinase Vegfr-3, important for LEC survival, migration and proliferation. LECs then start
to bud by following the gradient of Vegfr-3 ligand, Vegf-c (reviewed in 1). In mouse,
LECs begin to sprout from the cardinal veins and intersomitic vessels around E10
7-9
.
LECs emigrate from veins as non-lumenized, loosely connected strings of cells, so that
the integrity of blood vessel is not disturbed
7,8
. LECs further coalesce to form two large
primordial vessels: the dorsal peripheral longitudinal lymphatic vessel and the ventral
primordial thoracic duct, which are also commonly called “lymph sacs” in earlier
publications 7. Primordial thoracic duct establishes a connection with the cardinal vein, to
provide a path for the return of lymph into the blood circulation. The connection is
protected by the lymphovenous valve, formed by a small subpopulation of LEC
progenitors remaining in the veins 12, and which is important for preventing the backflow
of blood into the thoracic duct.
4
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Embryonic lymphatic vasculature is established by further sprouting from the primordial
lymphatic structures. During late gestation and postnatal period, lymphatic capillary
plexus is remodeled to establish functional compartments of lymphatic vasculature,
capillaries and the collecting lymphatic vessels (reviewed in 1). During this process,
collecting lymphatic vessels develop intraluminal valves and acquire SMCs and basement
membrane coverage, while lymphatic capillaries remodel cell junctions from continuous
“zipper-like” to discontinuous “button-like” state
1,13
. Lymphangiogenesis and vessel
remodeling continues in many organs, including skin and intestine during early postnatal
development, postnatal mouse mammary gland morphogenesis
14
and ovarian follicle
growth in adult mice 15.
Lymphatic vessels in disease
In the adult tissues, the majority of lymphatic vessels are quiescent, with the exception of
reproductive organs during ovarian cycle and gestation 15, however reactivation of vessels
occurs in a variety of pathological conditions. Pathological vessels may show loss of
specialized junctions in capillaries, such as in inflammation
13
and ectopic recruitment of
SMCs to lymphatic capillaries, such as in lymphedema 16.
Lymphangiogenesis has been observed in many human inflammatory diseases including
psoriasis, rheumatoid arthritis and during transplant rejection (reviewed in
17,18
).
Inflammation-induced lymphangiogenesis regulates fluid drainage, immune cell
migration or removal of inflammatory mediators, ultimately accelerating the resolution of
inflammation (e.g.
19
). In contrast, following organ transplantation, enhanced
5
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lymphangiogenesis induces reactivation of immune system in the draining lymph node
resulting in organ rejection (reviewed in
18
). Inflammatory lymphangiogenesis has
frequently been shown to occur via secretion of lymphangiogenic growth factors, such as
Vegf-c, by macrophages (reviewed in
20
). In addition, neutrophils are also recruited to
sites of inflammation where they modulate lymphangiogenesis via Vegf-a bioavailability
and, to a lesser extent, secretion of Vegf-d
21
. Hence, stimulation or inhibition of
lymphangiogenesis represents an attractive novel therapeutic strategy for reducing
chronic inflammation or transplant rejection.
In cancer, lymph node status is an important factor in determining the stage of disease
progression. Many cancers exploit lymphatic vasculature to spread to lymph nodes
(reviewed in
22,23
). Adding to the disease burden is development of secondary
lymphedema in patients who have undergone radical axillary lymph node dissection
24
.
Many cancer types induce lymphangiogenesis via release of VEGF-C or VEGF-D, and
blocking VEGFR-3 signaling inhibits tumor lymphangiogenesis and lymph node
metastasis in animal models (reviewed in
25
). Increased lymphatic vessel density is
correlated with a poor prognosis in a number of solid tumors (reviewed in
22
).
Intratumoral vessels are thought to be collapsed and non-functional due to the high
intratumoral fluid pressure
26
. In contrast, peritumoral lymphatics are often dilated
containing clusters of tumor cells
27,28
. 15-lipoxygenase-1 expressing tumor cells, which
secrete arachidonic acid metabolite 12S-HETE, invade lymphatic vessels inducing the
formation of holes in LEC plasma membrane 27. Dilation of collecting vessels through the
action of tumor-produced VEGF-C and prostaglandins further contributes to cancer
6
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dissemination to lymph nodes (e.g.
29,30
). Lymphangiogenesis in sentinel lymph nodes
even before tumors cells metastasize has been shown in mouse models
31
and the extent
of lymph node lymphangiogenesis correlates with the disease prognosis in several human
cancer types
32,33
. Lymph node lymphatic sinuses, but not peripheral lymphatic vessels
express CCL1 chemokine, which regulates the entry of CCR8+ tumor cells into the lymph
node 34.
Additional functional links emerge between the lymphatic dysfunction and pathogenesis
of obesity and atherosclerosis. Impaired lymphatic function, such as in Prox1+/- mice,
which have leaky lymphatic vessels, leads to adult-onset obesity and inflammation
Conversely, lymphatic function is impaired in obese patients
35
.
36
. Lymphatic vasculature
has a direct role in the reverse cholesterol transport, a process critical for the removal of
cholesterol from peripheral tissues, including arterial wall
37,38
. Interestingly, excessive
accumulation of cholesterol in tissues, such as that found in hypercholesterolemic Apoe−/−
mice, leads to structural and functional defects of lymphatic vessels 39. In the same mouse
model, restoration of lymphatic drainage improved cholesterol clearance 38. Thus, normal
lymphatic function is essential for lipid clearance, and could be a target for prevention or
treatment of atherosclerotic vascular disease.
Skin lymphatic vessels have been also implicated in systemic blood pressure control,
through regulation of the electrolyte clearance. In turn, in hypertension, osmotic stress
leads to inflammatory cell recruitment and increased lymphangiogenic growth factor
production in the skin 40,41.
7
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In summary, lymphatic vasculature plays a key role in a number of physiological and
pathological conditions. Studies of molecular mechanisms and signaling pathways that
regulate development of lymphatic vasculature and the pathological lymphangiogenesis
are actively underway. In this review, we will describe the current view of several critical
pathways regulating lymphangiogenesis with a major focus on the mechanisms in
mammals (Fig. 2). Latest view of lymphangiogenesis in zebrafish is presented in e.g.42,
whereas lymphatic vascular remodeling and the role of non-endothelial cells are reviewed
in 20,1,43.
Cell surface receptors and their ligands in lymphangiogenesis
VEGF-C – VEGFR-3 signaling
VEGF-C, a member of VEGF growth factor family, is a major regulator of formation and
growth of lymphatic vessels both in physiological and pathological contexts (reviewed in
1,43
). In Vegfc-/- mice, LECs fail to migrate out of the veins to form primordial lymphatic
vessels and consequently mice do not develop lymphatic vasculature 44. This function of
Vegf-c is evolutionary conserved as it is also required for the development of the
zebrafish lymphatic vascular system 45. The lymphangiogenic activity of Vegf-c has been
demonstrated in a range of animal models involving transgenic and knockout mice, viral
gene delivery, as well as in in vitro assays, where VEGF-C promotes LECs proliferation,
survival and migration (e.g. reviewed by 25 and 46).
8
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Full length VEGF-C binds receptor tyrosine kinase VEGFR-3 with high affinity, and
further proteolytic processing generates a shorter isoform capable of interacting also with
VEGFR-2
47
. Vegfr-3 is initially expressed in blood vasculature, before becoming
restricted to lymphatic vessels around midgestation. Vegfr-3 knock-out mice die because
of early blood vascular defects 48. Heterozygous missense mutations in Vegfr-3 have been
linked to lymphedema in humans and in Chy mice
49,50
. Loss-of-function VEGF-C
mutation was also recently described in a family with autosomal-dominant form of
lymphedema
51
, and truncation mutation in zebrafish vegf-c severely affected lymphatic
development 52. VEGF-C-dependent activation of VEGFR-3 is enhanced by mechanical
stretch in an integrin β1-dependent manner, providing a mechanism of how increased
interstitial pressure guides expansion of lymphatic vasculature 53.
Neuronal
guidance
molecule
neuropilin-2
(Nrp2)
is
highly
expressed
on
lymphangiogenic vessels, where it acts as a Vegfr-3 co-receptor during Vegf-c-induced
lymphatic sprouting. Nrp2-/- deficient mice have severe hypoplasia of lymphatic
capillaries from E13 to birth 54. Furthermore, reduction of lymphatic vessel sprouting was
observed in Nrp2+/-;Vegfr3+/-, but not in Nrp2+/-;Vegfr2+/- mice, providing a genetic
evidence for the role of Nrp2 in Vegf-c/Vegfr-3 signaling cascade
55
. Nrp2 levels are
reduced in mice with endothelial-specific inactivation of COUP-TFII, and COUP-TFII
acts as a direct regulator of Nrp2 expression in LECs 56. In addition, transcription factors
GATA2 and Lmo2 regulate the expression Nrp2 on endothelial cells in vitro 57. Nrp2 is
re-expressed during tumor lymphangiogenesis and treatment with neutralizing anti-Nrp2
9
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antibody decreases tumor lymphangiogenesis and metastasis to sentinel lymph node and
distant organs 58.
In addition to Vegfr-3, lymphatic vessels also express high levels of Vegfr-2, whereas
Vegfr-1 is predominantly expressed by blood vessels 59. Inactivation of Vegfr-2 in LECs
results in decreased lymphatic sprouting and proliferation, without affecting vessel
caliber
60
.
Interestingly,
VEGFR-2-specific
ligand,
VEGF-E,
rather
induces
circumferential growth without affecting lymphatic sprouting 61. Thus, it is possible that
ligand-dependent Vegfr-2 signaling contributes to LEC proliferation, whereas sprouting
is regulated through a ligand-independent Vegfr-2 modulation of Vegfr-3/Nrp2 signaling,
for example via Vegfr-2/3 heterodimers, present in the LEC filipodia 62.
CCBE1
Collagen- and calcium-binding EGF domains 1 (Ccbe1) is a secreted extracellular
matrix-binding protein, mutated in patients with Hennekam syndrome
63
, a rare disease
that presents with lymphedema, lymphangiectasia, and other pathological features
(reviewed in
64
). The function of Ccbe1 is evolutionary conserved, as it is required for
embryonic lymphangiogenesis in zebrafish
65
and mice 66. Ccbe1 is expressed in somitic
mesoderm during thoracic duct formation in zebrafish and in areas adjacent to anterior
cardinal vein in mouse embryos, where it acts non cell-autonomously to regulate Vegf-c
processing and bioavailability
65,67,68
. Lymphangiogenesis is also potently induced upon
co-administration of Ccbe1 and Vegf-c in a corneal lymphangiogenesis mouse model 66.
Ccbe1-/- mice develop severe anemia because of the defective definitive erythropoiesis,
10
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suggesting that Ccbe1 has additional, Vegf-c-independent function(s) 69. Ccbe1 is a direct
target of atypical E2F transcription factors E2F7/8. Importantly, inactivation of e2f7/8 in
zebrafish
reduced
Ccbe1
expression
and
impaired
venous
sprouting
and
lymphangiogenesis, whereas overexpression of e2f7/8 rescued ccbe1- and vegfr-3dependent lymphangiogenesis 70.
Ephrin-Eph
Eph receptor tyrosine kinase receptors and their ligands, ephrins, are key regulators of
axon guidance during nervous system development, and they also contribute to many
aspects of blood vascular morphogenesis (reviewed in
71
). In addition to acting as Eph-
activating ligands (so called “forward signaling”), ephrins are also able to initiate
“reverse” signaling, which in case of ephrinB ligands is achieved through the recruitment
of adaptor proteins in the cytoplasmic portion. Lymphatic vessels express EphB4, an
ephrinB2 receptor
72,73
. EphrinB2 promotes Vegf-c/Vegfr-3 signaling in the developing
LECs through the regulation of Vegfr-3 internalization, important for full activation of
downstream signaling pathways, such as Akt and ERK
74
. EphrinB2 has a similar
function in the regulation of Vegfr-2 on blood vessels and Pdgfrβ signaling in pericytes
75,76
. Blocking ephrinB2 activity using antibodies prevents tumor angiogenesis and
lymphangiogenesis, suggesting a potential therapeutic approach for targeting tumor
neovascularization 77.
Loss of the C-terminal PDZ domain of ephrinB2, implicated in reverse signaling, also
leads to defective remodeling of primary lymphatic plexus and failure to form collecting
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lymphatic vessels and lymphatic valves
78
. Studies of adult corneal lymphatic vessels
showed that EphB4 is highly expressed in the valves where administration of EphB4-Fc
fusion proteins prevented regeneration of valves after corneal injury 73.
Angiopoietins and Tie receptors
Tie1 and Tie2 endothelial receptor tyrosine kinases and its angiopoietin ligands play
important roles in blood vessel maturation, patterning and stability, both in physiological
and pathological conditions (reviewed in 79). Current view of Ang-Tie signaling in blood
vessels suggests that Ang1 acts to stabilize vessels and limit angiogenesis
79
, although
this was challenged by a genetic study, which found that Ang1 is dispensable for
maintenance of quiescent vasculature in adult animals
80
. Ang2 is in many cases
antagonistic to Ang1, but it can be also agonistic under certain conditions, such as at high
concentration or/and in tumor endothelial cells. Ang1, Tie1 and Tie2, but not Ang2 are
essential for embryonic vascular development and their inactivation leads to embryonic
lethality at E12.5 (reviewed in 79). Decreased dosage of Tie1 leads to embryonic edema
and mispatterning of early lymphatic vasculature, without affecting the lymphatic
endothelial commitment
81,82
. It is not known whether these defects are a result of
modified Ang1 or Tie2 signaling in LECs, or whether Tie1 has an angiopoietinindependent role in lymphatic endothelium. In contrast to its limited role during
physiological angiogenesis, Ang2 plays an essential role in lymphatic vasculature.
Angpt2-/- mice have hypoplastic lymphatic capillaries and fail to form collecting vessels,
which leads to lymphatic dysfunction and impaired postnatal survival
83,84
. Interestingly,
Ang1 rescues the lymphatic vascular, but not blood vascular defects in Angpt2-/- mice and
12
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overexpression of Ang1 induces both physiological and pathological lymphangiogenesis
in a Vegfr-3 dependent manner
83,85,86
, highlighting the differences in the usage of
angiopoietin ligands by the two vascular systems. Studies of blood vasculature revealed
important roles of Ang-Tie pathway in a variety of pathological conditions, such as
inflammation, fibrosis and cancer, underlining their importance as therapeutic targets
(reviewed in 87,79,88). However the relative contribution of Ang-Tie signaling in lymphatic
vessels to these processes still needs to be fully examined.
Notch signaling
Notch is a fundamental signaling pathway, which plays a key role in blood vascular
development and function. Notch pathway is mediated by Notch receptors (Notch1Notch4), as well as Delta-like (Dll1, Dll3, Dll4) and Jagged (Jag1 and Jag2) ligands.
Notch receptors are single-pass transmembrane proteins that function both as cell surface
receptors and transcriptional regulators. Canonical Notch signaling is triggered via the
interaction of Notch receptor and ligands on neighboring cells, which induces
conformational change in Notch receptors, cleavage of the Notch intracellular domain
(NICD) and translocation of NICD in the nucleus. NICD interacts with DNA-binding
protein RbpJ, constitutively recruited to the promoters of Notch target genes, and replaces
the associated co-repressor complex with a co-activator complex containing MAML1-3,
EP300 and SNW1. Primary Notch target genes include Hes and Hey transcription factors
(reviewed in
89
). In blood vessels, Notch signaling regulates angiogenic sprouting, by
controlling the selection of stalk and tip cells, the recruitment of SMC/pericytes and
arterio-venous differentiation (reviewed in 90).
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Cultured human LECs express high levels of DLL4, NOTCH1, NOTCH4 and JAG1 91-93.
Blocking Notch signaling in cultured LECs and in adult mice using Dll4-Fc fusion
protein leads to lymphatic endothelial hypersprouting and increases responsiveness of
LECs to Vegf-a, indicating that under these conditions Notch function is analogous to its
role in blood vessels, i.e. restriction of (lymph)angiogenic potential of endothelial cells 93.
However, inactivation of Notch during early postnatal development or in wound-healing
model in mice using Dll4 or Notch1 blocking antibodies or in zebrafish embryos, using
morpholino oligo knock-down approach, results in decreased lymphatic vessel sprouting
92,94
.
In vitro studies also suggested the role of NOTCH signaling in lymphatic endothelial
differentiation through the repression of PROX1 and COUP-TFII 95. Down-regulation of
Notch activity in vivo resulted in increased generation of LEC progenitors, whereas
overexpression of NICD in LECs between E9.75-E13.5 repressed COUP-TFII and Prox1
96
, demonstrating that Notch acts as a negative regulator of LEC specification. Taken
together, these data reveal a complex picture of Notch signaling in lymphatic vasculature,
where it can be either pro- or anti-lymphangiogenic or yet regulate venous versus
lymphatic endothelial cell-fate decisions, probably depending on the developmental stage
or tissues investigated.
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TGF/BMP signaling
Transforming growth factor (TGF) and Bone morphogenic protein (BMP) signaling
pathways induce pleiotropic tissue-specific responses by regulating proliferation,
differentiation, migration and cellular survival. TGF/BMP ligands (BMP2, BMP7, GDF5,
BMP9, BMP10, AMH, TGF-β1/2/3, Activins and Nodals) bind TGF receptors type II
(TGFRII) (BMPR2, ACVR2A/B and TGFβRII) which phosphorylate TGFRI (Alk1-7)
via Ser/Thr tyrosine kinase activity. Downstream signaling pathways are transmitted to
the cell nucleus via phosphorylation of SMAD transcription factors, which trigger target
gene transcription (reviewed in
). In addition, non-canonical TGFβ signaling pathway
97
regulates diverse cellular responses involving MAP kinases, Rho-like GTPases and
PI3k/Akt
. Endothelial cells signal through two type I TGFRs: Alk5 (TGFβRI) and
98
Alk1 (ACVRL1), which activate SMAD2/3 and SMAD1/5 respectively 98. Mutations of
the components of these signaling pathways cause several human hereditary vascular
diseases, such as hereditary hemorrhagic telangiectasia (ENDOGLIN or ALK1) or
pulmonary arterial hypertension (BMPR2) 98. Studies of genetic mouse models revealed
critical roles of TGF-β/BMP signaling in angiogenic sprouting 99-101. TGF/BMP also play
an important role in the regulation of vascular integrity in the brain
the quiescent vasculature
102
, maintenance of
103
, and SMC differentiation and recruitment to blood
endothelial cells 104-106.
Cultured LECs express Alk1, Alk2, Alk4, Alk5, ACVR2B, BMPR2, Endoglin and
TGFβRII receptors
. In vitro treatment of LECs with TGFβ-1 reduces cell
107
proliferation, cord formation and expression of lymphatic markers Prox1 and Lyve-1
108
.
TGFβ-1 inhibits lymphatic endothelial commitment and down-regulates related markers,
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such as COUP-TFII and Sox18 in murine embryonic stem cell-derived Flk1+
cardiovascular progenitors
, although in another study TGFβ-2 treatment increased
108
expression of Vegfr-3 and Nrp2, both involved in lymphatic sprouting events rather that
lymphatic endothelial commitment
109
. Similarly, BMP9 suppresses the expression of
LYVE1 and PROX1 110,111.
In contrast to in vitro results, endothelial-specific loss of TgfbrI (Alk5) or TgfbrII during
embryogenesis did not affect lymphatic endothelial cell commitment. Instead, it impaired
formation of tip cells and reduced complexity of skin lymphatic network, leading to
hyperplasia of cutaneous lymphatic vessels
109
. These in vivo results suggest that TGFβ
signaling is an important regulator of lymphatic network patterning
109
. BMP2 is known
to interact with BMPR2, ACVR2A or ACVR2B as type II TGF receptors and Alk-2, Alk3 or Alk-4 as type I receptors. In zebrafish embryos, bmp2 signaling pathway acts as a
negative regulator of LEC emergence from veins and over-expression of bmp2b
suppresses formation of lymphatic vascular structures, presumably due to the loss of
prox1a
112
. However, it should be noted that the genetic inactivation of prox1a does not
affect zebrafish lymphangiogenesis 11.
Inhibition of TGFβ/BMP signaling during early postnatal development using Alk1,
Acvr2b and Bmbpr2 blocking antibodies prevented growth of lymphatic vessels in
several organs, including skin and intestine without affecting collecting lymphatic vessels
107
. This effect was further enhanced upon blocking Vegfr-3 signaling. Thus, Alk1
signaling appears to play a major role in postnatal lymphangiogenesis, and in its absence
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lymphatic vessels become more sensitive to Vegfr-3-Vegf-c/d depletion
107
. At present it
is not clear what ligand is responsible for this effect. Indeed, targeted inactivation of high
affinity Alk1 ligand Bmp9 has an opposite effect, as it induces lymphatic vascular
hyperplasia and lymphatic valve defects
110,111
, thus additional Alk1 ligands that may be
contributing to lymphangiogenesis remain to be investigated.
Several studies also addressed the question of the role of TGFβ signaling in pathological
lymphangiogenesis. Treatment using small molecule TGFβRI inhibitor increased
lymphangiogenesis in chronic peritonitis and tumor xenografts
lymphedema,
treatment
with
blocking
TGFβ
antibody
108
. In a model of tail
similarly
increased
lymphangiogenesis and alleviated inflammation, lymphedema and fibrosis 113.
To conclude, TGF/BMP signaling is important for several steps of lymphatic vascular
development, including sprouting/tip cell formation, regulation of cell proliferation and
formation of specialized lymphatic structures, such as intraluminal valves
108,109,114
. The
future challenge will be to elucidate the precise roles of different receptors and ligands in
these processes, as well as to understand potential combinatorial interactions with other
signaling pathways, such as Notch, already revealed in blood vasculature 100,101.
Intracellular signaling pathways in lymphangiogenesis
Lymphangiogenesis is regulated by a number of ligand - receptor interactions, which in
turn activate intracellular signaling pathways responsible for transmitting information
within the cell, and ultimately endothelial cell responses, such as proliferation, survival or
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migration. Much effort is dedicated to studies of such ligands and receptors, because they
represent important targets for drug development. However, in the past few years, novel
knowledge accumulated also on the intracellular pathways, which in many cases revealed
surprising differences between blood and lymphatic vasculature (Table 1). In sections
below, we will describe current knowledge into the major intracellular signaling
pathways in lymphangiogenesis.
Ras/ Raf/MEK/ERK
The Ras family of GTPases (Hras, Nras, and Kras) plays essential roles in a variety of
cellular functions, such as proliferation, migration, differentiation, and apoptosis,
frequently downstream of receptor tyrosine kinases. Ras is small molecule GTPase,
which in its active, GTP bound, state, can activate multiple downstream effectors,
including Raf/MEK/ERK pathway and class I PI3 kinases (PI3K). A variety of associated
proteins (guanine-nucleotide exchange factors, or GEFs) increase Ras GTPase activity by
promoting GDP–GTP exchange, whereas GTPase-activating proteins, or Ras-GAPs,
stimulate the hydrolysis of GTP on Ras, and are important for Ras inactivation (reviewed
in 115). In Raf/MEK/ERK pathway, active Ras interacts with RAF1 Ser/Thr kinase, which
in turn activates downstream MEK kinases. MEK kinases then phosphorylate and
activate ERK1/2 MAP kinases (reviewed in 116).
Ras signaling plays an important role in lymphatic vessel development and function,
likely downstream of VEGFR-3 pathway. Nras+/-Kras+/- mice display lymphatic
hypoplasia and chylous ascites, which could be rescued by overexpression of Hras
117
.
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Conversely, mice that over-express Hras in endothelial cells have lymphatic vessel
hyperplasia, driven by increased levels of Vegfr-3 and enhanced MAPK signaling.
However, such hyperplastic vessels are not fully functional, as transgenic mice develop
edema and chylothorax. In humans, chylous ascites, chylothorax and lymphedema are
associated with activating mutations in KRAS in cardiofaciocutaneous syndrome
HRAS in Costello syndrome
119,120
118
and
. The work in mouse models also revealed differential
sensitivity of blood versus lymphatic vessels to altered Ras signaling, as blood vessel
development or function was not affected upon Hras overexpression or in Kras+/-, Nras+/mice 117.
Negative regulator of Ras activity p120-RasGap or RASA1 is mutated in human
hereditary disease Capillary Malformation–arteriovenous malformation (CM-AVM),
characterized by multiple cutaneous vascular lesions and high risk for development of
fast-flow blood vascular lesions (reviewed in
observed in some CM-AVM patients
122
121
). Lymphatic malformations were
and recent advanced imaging analysis revealed
hyperplastic cutaneous lymphatic vessels in a CM-AVM patient, suggesting that
lymphatic vascular dysfunction may be frequent in CM-AVM
123
. Inactivation of Rasa1
in adult mice causes extensive Vegfr-3-dependent lymphatic vessel hyperplasia and
lymphatic vessel leakage, leading to chylothorax, chylous ascites, and animal death
124
.
Importantly, no blood vasculature phenotype was observed upon loss of Rasa1 in adult
mice, unlike in Rasa1-deficient embryos
125
implying that blood vessels need Ras
regulation only during embryonic angiogenesis, whereas lymphatic vessels require a lifelong suppression of Ras signaling. A number of other Ras signaling effectors and
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regulators have been implicated in vascular development and angiogenesis, such as
Rasip1
126
, therefore it will be interesting to investigate and compare their function in
lymphatic vessels as well.
Studies of blood vasculature revealed important interactions of PI3K-Akt and Erk
signaling in the regulation of arterio-venous differentiation, in which high PI3K-Akt
signaling favors venous fate and vein formation, whereas arterial morphogenesis is
induced by Erk1/2 MAP kinases under conditions of attenuated PI3K-Akt activation
127,128
. In addition to the regulation of lymphatic hyperplasia downstream of VEGFR-3
signaling, as discussed above, MAPK/ERK cascade might be also important for the
establishment of lymphatic endothelial cell fate. Endothelial-specific expression of
activated MAPK upstream regulator Raf1 increased commitment of venous endothelial
cells to the lymphatic fate and even induced lymphatic markers in arterial endothelial
cells, through the induction of Sox18 and Prox1
129
. Spatially localized, yet unknown,
signal may therefore regulate Erk activation in embryonic veins and lead to the
establishment of lymphatic endothelial fate in a subpopulation of venous ECs.
Class I PI3 kinases
PI3K is a family of enzymes, which phosphorylate the 3-OH group of inositol membrane
lipids and produce a variety of 3-phosphorylated phosphoinositides. The latter propagate
intracellular signaling by providing docking sites for pleckstrin-homology domains of
diverse signaling proteins. Downstream effectors of PI3K include serine/threonine kinase
Akt, which, among other pathways, activates mTOR signaling, central for cellular
proliferation, survival and metabolism. PI3K/Akt/mTOR pathway is frequently activated
20
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in cancers and a number of PI3K and mTOR inhibitors are either in clinical trials or
already approved for treatment (reviewed in 130,131).
Based on their primary structure, regulation and substrate specificity, three classes of
PI3Ks (Ia, Ib, II and III) were identified. Class Ia PI3K is a group of dimeric proteins
containing a catalytic subunit (p110α, p110β, or p110δ) and a regulatory subunit (p85α,
p55α, p50α, p85β, or p55γ), which are activated by a variety of receptor tyrosine kinases.
Regulatory p85 subunit directly binds phosphotyrosines on receptor tyrosine kinases or
adaptor proteins, which leads to PI3K activation. PI3K can also be activated directly by
Ras, which interacts with the p110 subunit 132. Studies of animal models have uncovered
multiple functions of class I PI3K in blood vascular development, such as regulation of
blood vessel integrity, angiogenic sprouting and remodeling as well as arterio-venous
differentiation (reviewed by
133
). VEGF-A, VEGF-C and VEGF-D activate PI3K-Akt
signaling cascade in cultured LECs, where this pathway is important for migration 134 via
a direct interaction with VEGFR-3
135
. LECs express p110α, -β, and -δ but not -γ
isoforms, and p110α is a major isoform, responsible for VEGF-C-dependent Akt
activation and LEC migration
136
. Germline deletion of Pik3r1, encoding the regulatory
subunits p85α, p55α, and p50α of class I PI3K, led to chylous ascites, suggesting
lymphatic vascular dysfunction 137. Further analysis of these mice revealed organ-specific
defects in postnatal lymphatic sprouting and lymphatic maturation, without major impact
on blood vessel development
. Mutations in the Pi3kca encoding the catalytic p110α
138
isoform, that block its interaction with Ras, led to lymphatic vessel hypoplasia, reduced
sprouting and perinatal chylous ascites
139
. In a tumor setting, p110α signaling
21
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downstream of VEGF-C is important for the activation of integrin α4β1 on lymph node
lymphatic endothelium and subsequent adhesion of metastatic tumor cells
136
. In human
disease, somatic activating mutations in PI3KCA were found in patients with CLOVES
and Klippel–Trenaunay–Weber syndrome (KTWS), which present with malformation or
overgrowth of lymphatic vessels in addition to tissue overgrowth and other vascular
anomalies 140.
Three highly related isoforms of Akt serine/threonine kinase (Akt1, Akt2, and Akt3)
represent the major signaling arm of PI3K. All isoforms are expressed in LECs, however
Akt1 has a dominant role in lymphatic vasculature, as Akt1-/-, but not Akt2-/- or Akt3-/mice, display reduced capillary density and defective valve development
141
.
Interestingly, VEGF-C-induced lymphangiogenesis is normal in Akt1-/- mice, indicating
either compensation by other isoforms or predominant role in cell survival, rather than
migration or proliferation
141
. As observed in mice with mutant Ras or PI3K signaling
components, the physiological angiogenesis was not affected in the absence of Akt1.
Activating AKT1 mutation underlie Proteus syndrome, characterized by general tissue
overgrowth, as well as cutaneous vascular lymphatic or lympho-venous malformation 142.
Ca2+/Calcineurin/NFAT signaling
Calcineurin is a calcium activated-protein phosphatase, which plays a major role in
calcium signaling in different cells types, and it is a target of immunosuppressive drugs
cyclosporine A and tacrolimus. Increase in intracellular calcium, activates calcineurin and
leads to dephosphorylation and nuclear translocation of the transcription factors of
22
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nuclear factor of activated T cells (NFAT) family, which act as main effectors of
Ca2+/calcineurin pathway. In vitro studies suggested that calcineurin/NFAT mediates
most
VEGF/VEGFR-2-induced
responses
proliferation, migration, and tube formation
of
endothelial
143-145
cells,
including cell
. However, mice with constitutive
endothelium-specific inactivation of calcineurin display coronary vessel-specific, but not
generalized, angiogenesis defects
146
. Angiogenesis proceeds normally upon inducible
inactivation of calcineurin in blood vasculature after E13.5, when the requirements for
calcineurin signaling in heart development are bypassed 147. These data suggest a limited,
vascular bed-specific, role for endothelial calcineurin signaling during physiological
angiogenesis. Calcineurin may be more important for pathological angiogenesis, as mice
deficient in DSRC1, an endogenous inhibitor of calcineurin, have decreased tumor
angiogenesis 148.
To date, of the five members of the NFAT family, NFATc1 (NFAT2) is the only known
transcription factor implicated in lymphatic vascular development
149,150
. In LECs,
expression of NFATc1 is controlled by PROX1 while its nuclear translocation can be
induced by VEGF-C through the activation of VEGFR-2 or by flow shear stress.
Ca2+/calcineurin/NFAT pathway cooperates with FOXC2, a major regulator of collecting
vessel phenotype
147,149
. Pharmacological inhibition or lymphatic-endothelial specific
inactivation of calcineurin prevents maturation of lymphatic vessels and arrests the
development of lymphatic valves
147,149
. Thus, similar to blood vasculature, the role of
calcineurin signaling in lymphatic vessels in physiological conditions is restricted to a
specific vascular compartment.
23
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MicroRNAs and lymphangiogenesis
MicroRNAs (miRs) are short non-coding single stranded RNAs, which act as posttranscriptional regulators, by inhibiting mRNA translation and/or promoting mRNA
degradation. The same miR can regulate many mRNAs, therefore miRs act as broad
regulators of gene networks, akin to the transcription factors, although miRs act not as
binary on- and off-switches, but rather to fine-tune the genetic program. The number of
known miRs that regulate angiogenesis either in cell-autonomous manner or through the
regulation of angiogenic factors in other cells is constantly increasing 151-153. However, in
contrast to the wealth of information on miRs in angiogenesis, little is known about
specific miR functions in LECs. miR-181a is expressed at higher levels in BECs in
comparison to LECs, and it targets Prox1 mRNA for degradation. Increasing miR-181a
levels in LECs reduced Prox1 expression and shifted LECs towards a blood vascular
phenotype, suggesting that miR-181a acts to maintain blood vascular phenotype
154
. In
another study, qRT-PCR profiling of 157 miRs in cultured LECs and BECs identified
LEC-specific miR-95 and miR-326 and BEC-specific miR-137, miR-31, miR-125b, and
miR-99a
155
. Further analysis demonstrated that miR-31 suppresses a number of
lymphatic endothelial-specific genes, and this effect is at least partially due to the direct
suppression of PROX1
155
. Thus, higher expression of miR-31 and miR-181a may be
important for preventing the acquisition of LEC identity by BECs. Factors regulating the
expression of miR-31 and miR-181a in endothelial cells include BMP2 112.
24
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Concluding remarks and open questions
Lymphatic vessels perform a vast array of functions both in physiological and
pathological conditions. Understanding molecular mechanisms regulating lymphatic
vascular growth and remodeling should result in the design of new therapies for many
human diseases. In the past few years, in addition to the well-established roles of VEGFC/VEGFR-3 signaling in lymphangiogenesis, important contributions were uncovered for
Notch, TGFβ/BMP, Ras, MAPK/ERK, PI3K/Akt and Ca2+/calcineurin pathways. It is
noteworthy that in many cases lymphatic and blood vasculature demonstrate differential
responses or usage of these signaling pathways (Table 1). One of the future challenges
will be to decipher the complexity of such signaling in different lymphatic vascular beds
and disease conditions and to identify critical determinants, which could be used for
modulation of lymphatic function.
25
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Acknowledgements
We apologize for not being able to cite all original research contributions due to space
limitations. Authors would like to thank Dr. Jeremiah Bernier-Latmani and Dr. Amélie
Sabine for critical reading of the manuscript.
Authorship
Contribution: SC, EB and TP conceived and wrote the manuscript.
All authors gave final approval for submission.
Conflict of interest statement
Authors declare no conflict of interest.
Funding
The research in T. Petrova’s laboratory is supported by Swiss National Science
Foundation (PPP0033-114898 and CRSII3-141811), Oncosuisse (2863-08-2011),
Leenaards Foundation, Gebert Rüf Foundation and the People Programme (Marie Curie
Actions) of the European Union's Seventh Framework Programme FP7/2007-2013/ under
REA grant agreement n° 317250.
26
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36
Table 1. Comparison of blood and lymphatic vascular phenotypes in loss-of-function genetic mouse models. RBD, Ras-binding domain
Genes
Lymphatic vessel phenotype
Vegfc-/-: Normal developmental angiogenesis44
Vegfc-/-: No sprouting of LECs, absence of lymphatic
vessels44
Ligands and receptors
Vegf-c
Vegfc+/-: Chylous ascites, hypoplasia44
Vegfr-3
Nrp2
Vegfr3-/-: Early blood vessel remodeling defects, death at E9.5-1048
Vegfr3+/Chy: Chylous ascites, hypoplasia157
Vegfr3f/f;Pdgfb-CreERT2: Vessel hypersprouting156
Vegfr3ΔLBD/ΔLBD : No sprouting of LECs due to the
removal of ligand-binding (LBD) domain of Vegfr-3158
Nrp2-/-: Normal developmental angiogenesis54
Nrp2-/-: Hypoplasia of capillaries E13 to birth54
Nrp2+/-;Vegfr3+/-: Decreased vessel sprouting55
EphrinB2
Efnb2f/f;Cdh5-CreERT2: Reduced vascular network complexity,
sprouting and endothelial cell proliferation74
Efnb2
Vegfr-2
ΔV/ΔV
: Normal developmental angiogenesis
159
Vegfr2-/-: Lack of development of the blood islands and embryonic
vasculature, death at E8.5-E9.5160
Efnb2f/f;Cdh5-CreERT2: Decreased vessel sprouting74
Efnb2ΔV/ΔV: Defective lymphatic vascular remodeling,
hyperplasia, valve agenesis74,159
Vegfr f/f;Lyve-1Cre: Hypoplasia, decreased vessel
sprouting60
Vegfr f/f;Lyve-1Cre: Decreased blood vessel density in the yolk sac, liver
and lung60
Bmp9
Bmp9-/-: No phenotype in the retinal blood vessels161
Bmp9-/-: Postnatal vessel hyperplasia, decreased
maturation of valves. Adults: enlarged lymphatics,
decreased number of lymphatic valves, impaired lymph
drainage110,111
TGFβ2
Tgfb2-/-: Blood vascular remodeling occurs normally109
Tgfb2-/-: Mild edema, increased cellular proliferation,
37
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Blood vessel phenotype
TGFβRI
TgfbrIfl/fl;Tie1Cre: Defective vascular network, pericardial effusion in
the heart, abnormalities in the vasculature of the yolk sac at E9.5, lethal
at E10.5104
TGFβRII
TgfbrIfl/fl;Prox1+/GFPCre: Deletion at E9.5-10.5. Edema at
E14.5 with blood filled lymphatics. Lymphatic network
and sprouting reduction107
Tgfbr1flfl;Prox1-CreERT2: Deletion at E12.5 impaired
cell sprouting and hyperplasia107
TgfbrIfl/fl;Alk1GFPCre/+: Enlarged pericardial cavity, underdeveloped heart,
lethal at E14.5163
Tgfbr1flfl;VEC-CreERT2: Deletion at E12.5, mild edema,
hyperplasia, lymphatic network and sprouting
reduction107
TgfbrIIfl/fl;Tie1Cre: Pericardial effusion in the heart, abnormalities in the
vasculature of the yolk sac at E9.5, lethal at around E10.5104
TgfbrIIfl/fl;Prox1+/GFPCre: Deletion at E9.5-10.5. Edema at
E14.5 with blood – filled lymphatics. Lymphatic
network and sprouting reduction107
TgfbrIIfl/fl;Tie2Cre: Cardiac defects and lethality at around E12.5164
TgfbrIIfl/fl;Pdgfb-CreERT2: Hemorrhagic blood vessels in retina,
impaired vascular development97
TgfbrI1flfl;Prox1-CreERT2: Deletion at E12.5 presence
of dysmorphogenic lymphatic vessels and reduced
lymphatic branching107
TgfbrI1flfl;VEC-CreERT2: Deletion at E12.5, mild
edema, hyperplasia, lymphatic network and sprouting
reduction107
Notch1
Notch1f/f;Cdh5-CreERT2: Regional retinal vessel hypersprouting165
Notch1f/f;Prox1CreERT2: Overproduction of LECs,
edema, blood-filled lymphatics and incorporation of
BECs into the peripheral lymphatics96
Ccbe1
Ccbe1-/-: Normal physiological angiogenesis66
Ccbe1-/-: No sprouting of LECs, absence of lymphatic
vessels66
Ccbe1+/-: Early irregular formation of intersegmental
veins; no lymphatic phenotype later7
Angipoietins/Tie
Angpt1-/-, Tie1-/-, Tie2-/-: Defective embryonic blood vessel remodeling,
lethality at E12.548,166
Angpt2-/-: Hypoplasia of lymphatic capillaries, failure to
form collecting vessels83,84
Angpt2-/-: Normal embryonic angiogenesis, defective hyaloid vessel
regression83
Tie1-/-: Abnormal patterning, dilated and disorganized
lymphatic vessels81,82
38
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TgfbrIfl/fl;Tie2Cre: Hypoplastic endocardial cushion in the arterioventricular canal; thinner, poorly trabeculated myocardium, lethal soon
after E13162
decreased complexity and sprouting, enlarged vessels109
Intracellular pathways
Nras+/-;Kras+/-: Normal developmental angiogenesis117
Nras+/-;Kras+/-: Lymphatic hypoplasia, chylous ascites117
Rasa1
Rasa1-/-: Early blood vascular remodeling defects, lethality at E9.5125
Rasa1f/f;UB-ERT2Cre: Systemic inactivation in adults
leading to hyperplasia, increased leakage, chylothorax,
chylous ascites124
Rasa1f/f ;UB-ERT2Cre: Systemic inactivation in adults-normal blood
vessels124
PI3K/Akt1
Pi3kca-/-(p85/p55/p50): Minor defects of developmental angiogenesis138
Akt1-/-: Normal physiological angiogenesis141
Pi3kca-/- (p85/p55/p50): Impaired valve development,
organ-specific hypoplasia138
Pi3kp110Rbd/Rbd: Chylous ascites, decreased branching
and network complexity138
Akt1-/-: Reduced capillary density, defective valve
development141
Cnb1
Cnb1;Tie2-Cre: Defective coronary vasculature146
Cnb1;Pdgfb-CreERT2: Normal physiological angiogenesis
147
Cnb1;Prox1-CreERT2: Defective lymphatic vessel
maturation and valve development147
39
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H/N/Kras
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Figure 1. Organization and the role of lymphatic vasculature in physiology and
human disease. Lymphatic capillaries uptake interstitial fluid, lipids and proteins, and
serve as entry points to immune cells. Lymphatic collecting vessels transport the lymph
towards lymph nodes, and to blood circulation. Intraluminal lymphatic valves and SMCs
coordinate lymph propulsion and the direction of flow.
Figure 2. Signaling pathways in lymphatic vessels. Lymphatic sprouting and
proliferation is regulated by a variety of external stimuli and intracellular signaling
pathways. Loss of Vegf-c/Vegfr-3 or Ccbe1 completely prevents formation of lymphatic
vessels, demonstrating a central role in lymphangiogenesis. Nrp2 and EphrinB2 enhance
VEGF-C-dependent lymphangiogenic sprouting and signaling. Vegfr-2 also contributes
to lymphangiogenic response. TGFβ/BMP and Angiopoietin-2 are important both for
lymphatic capillary patterning and maturation of collecting lymphatic vessels. Dll4/Notch
signaling restricts LEC response to Vegf-a in adult tissues, while it has a prolymphangiogenic function during postnatal development. Ras-RAF-MEK-ERK pathway
promotes lymphatic endothelial proliferation, likely downstream of VEGFR-3. Class I
PI3 kinases are required for growth and remodeling of lymphatic vasculature in some
vascular beds. Although strongly activated by (lymph)angiogenic growth factors in LECs
in vitro, in vivo Ca2+/calcineurin pathway is mostly implicated in lymphatic collecting
vessel development, in cooperation with Foxc2. Some pathways are also important for
establishment
of
lymphatic
endothelial
cell
identity,
such
as
Notch
and
Raf/MEK/ERK1/2.
40
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Blue boxes indicate pathways also involved in sprouting; green box; Foxc2 and
calcineurin/Nfatc1 cooperatively regulate collecting vessel maturation.
41
Structures and functions
of lymphatic vessels
Capillary
Cell morphology
Oak-leaf shape
Role in human diseases
Button-like
junctions
LYMPHEDEMA
Anchoring
filaments
Junctions
Discontinuous button-like
Structures
Flap valves
Anchoring filament
Functions
Fluid, immune cells and
macromolecules uptake
Collecting vessel
Cell morphology
Elongated shape
CANCER
OBESITY
Capillary LECs
HYPERTENSION
Zipper-like
junctions
Environment
Genes
ATHEROSCLEROSIS
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Junctions
Continuous zipper-like
Structures
Lymphatic valves
Smooth muscle cells
Basal membrane
Functions
Collecting vessel LECs
Lymph transport from
tissues to lymph nodes and blood circulation
Figure 1
Basal membrane
SMCs
Lymphatic valve
Lymph node
Fluid and
macromolecules
Lymph flow
Immune cells
Lymphatic endothelial cell fate
Lymphangiogenesis
Ligands and receptors
VEGF-C
VEGF-C;VEGFR-3;CCBE-1
VEGFR-2;VEGFR-2/3
NRP2;EphrinB2
Alk1/5;TgfbrII;TGFß;BMP
Ang1;Ang2;Tie1
DLL4;Notch1
Cardinal
vein
Sprouting
LECs
Endothelial cell
transdifferentiation
Sprouting LECs
Quiescent
LECs
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VEGF-C
Lymphangiogenesis
Intracellular pathways
RAS
RASA1
PI3K;AKT1
RAF1;MEK;ERK
Lymphatic maturation
Ca ;Calcineurin;NFAT
FOXC2
BMP9
Ang2
EphrinB2
2+
Figure 2
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Prepublished online March 7, 2014;
doi:10.1182/blood-2013-12-297317
Pressing the right buttons: signaling in lymphangiogenesis
Sanja Coso, Esther Bovay and Tatiana V. Petrova
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