Regulation of lymphatic vascular morphogenesis: Implications for pathological (tumor) lymphangiogenesis

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319 (2013) 1618–1625

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Review Article

Regulation of lymphatic vascular morphogenesis: Implications for pathological (tumor) lymphangiogenesis Ines Martinez-Corral, Taija Makinenn Lymphatic Development Laboratory, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK

article information

abstract

Article Chronology:

Lymphatic vasculature forms the second part of our circulatory system that plays a critical role

Received 14 December 2012

in tissue fluid homeostasis. Failure of the lymphatic system can lead to excessive accumulation

Accepted 26 January 2013

of fluid within the tissue, a condition called lymphedema. Lymphatic dysfunction has also been

Available online 6 February 2013

implicated in cancer metastasis as well as pathogenesis of obesity, atherosclerosis and

Keywords:

cardiovascular disease. Since the identification of the first lymphatic marker VEGFR-3 and

Lymphatic vessel

growth factor VEGF-C almost 20 years ago, a great progress has been made in understanding the

Lymphangiogenesis

mechanisms of lymphangiogenesis. This has been achieved largely through characterization of

Lymphedema

animal models with specific lymphatic defects and identification of genes causative of human

Metastasis

hereditary lymphedema syndromes. In this review we will summarize the current understanding of the regulation of lymphatic vascular morphogenesis, focusing on mechanisms that have been implicated in both developmental and pathological (tumor) lymphangiogenesis. & 2013 Elsevier Inc. All rights reserved.

Contents Lymphatic vascular structure and function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1619 Origin and differentiation of lymphatic endothelial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1620 Lymphatic endothelial cell exit from the vein and vessel sprouting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1620 Venous sprouting of lymphatic endothelial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1620 Lymphatic sprouting from lymph sacs and pre-existing vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1620 Remodeling into a functional lymphatic vasculature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1621 Formation of button-like junctions (‘primary valves’) in lymphatic capillaries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1621 Formation of luminal valves (‘secondary valves’) in collecting vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1622 SMC recruitment and lymphatic vessel wall assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1622 Regulators of lymphangiogenesis in pathological conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1622 Features of pathological lymphatic vessels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1622 Regulators of LEC differentiation in tumor lymphangiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1623 Regulators of lymphatic vessel sprouting in tumor lymphangiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1623 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1623

n

Corresponding author. Fax: þ44 207 269 3417. E-mail address: [email protected] (T. Makinen).

0014-4827/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexcr.2013.01.016

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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1623 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1623

Lymphatic vascular structure and function Lymphatic vasculature consists of a highly organized network of vessels that participate in tissue fluid homeostasis by draining extravasated fluid and macromolecules from the interstitium back to the cardiovascular system. Lymphatic vessels participate also in immune surveillance by serving as a route for immune cell trafficking, as well as in the absorption and transportation of dietary lipids from the gut. Consistent with their important functions, abnormal growth or function of lymphatic vessels has been implicated in various pathological conditions such as lymphedema, inflammation and metastatic spread of certain cancers [1]. Lymphatic dysfunction has also been correlated with late-onset obesity, atherosclerosis and cardiovascular diseases [1]. The lymphatic vasculature is composed of three types of vessels that can be distinguished based on their molecular and morphological characteristics: lymphatic capillaries (also called initial lymphatics), pre-collecting and collecting lymphatic vessels. Lymphatic

capillaries are blind-end vessels composed of oak leaf-shaped lymphatic endothelial cells (LECs) forming discontinuous buttonlike cell–cell junctions and ‘‘flap valves’’ that allow the entry of fluid into the capillary lumen unidirectionally [2]. Lymphatic capillaries lack mural cells and have little or no basement membrane. To prevent the vessels from collapsing under conditions of high interstitial pressure, capillary endothelial cells are anchored to the surrounding extracellular matrix by anchoring filaments. These characteristics make lymphatic capillaries highly permeable and enable the uptake of macromolecules and fluid. From the capillaries the lymph is drained first into pre-collectors that are sparsely covered by smooth muscle cells (SMCs) and then to larger collecting vessels that are characterized by a layer of SMCs, continuous basement membrane, zipper like endothelial junctions and the presence of luminal valves that are essential for preventing backflow of the lymph. Lymph is propelled by the contraction of the SMCs and skeletal muscles and returned to the blood circulation at the junction of the subclavian veins [3].

Developmental lymphangiogenesis

Mature

Vein

vasculature H2O Macromolecules Cells Anchoring filaments

Primary lymphatic plexus

VEGF-C

Button-like junctions

Lymph sac

Smooth muscle cells Luminal valve

LEC progenitors? Prox1 Sox18 COUP-TFII

VEGF-C/VEGFR-3 CCBE1

LEC differentiation

Nrp2

FOXC2 Integrin-α9 Ephrin-B2

?

Zipper-like junctions

Collecting vessel

Remodeling

Sprouting

Sox18 COUP-TFII

Lymphatic capillaries

VEGF-C/VEGF-D/VEGFR-3 Nrp2

Tumor lymphangiogenesis

FGF-2 Tumor angiogenesis

VEGF-C/D

Enlarged collecting vessels

Intratumoral Lymphatic vessels

Peritumoral lymphatic vessels

Lymph node

Fig. 1 – Schematic model of developmental and tumor lymphangiogenesis. Main stages of lymphatic development and the characteristics of normal and pathological vessels are shown. Key regulators of different lymphangiogenic processes are indicated in blue. Blood and lymphatic vessels/endothelial cells are shown in red and blue, respectively. Tumor cells are shown in green.

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Origin and differentiation of lymphatic endothelial cells According to a widely accepted model, originally proposed by Florence Sabin in the early 20th century, lymphatic vessels arise at around E9.5–10 of mouse development (weeks 6–7 of gestation in humans) when a subpopulation of blood endothelial cells in the cardinal veins differentiate into LECs and sprout out to form the first primitive lymphatic structures called ‘lymph sacs’ ([3,4], Fig. 1). Genetic lineage tracing in mice and in vivo imaging of zebrafish embryos have confirmed the venous origin of lymphatic vessels [5,6]. However, recent studies show that LEC budding is not restricted to the major veins (cardinal vein in mice and axial vein in zebrafish) but also occurs in more peripherally located vessels [7–9]. High-resolution imaging of developing mouse embryos further showed that instead of forming lumenized sprouts, the venous-derived LECs bud off as loosely connected strings of cells that subsequently aggregate in the jugular region to two distinct vessel structures: the dorsal peripheral longitudinal lymphatic vessel and the ventral primordial thoracic duct [9]. LEC differentiation is regulated by sequential and combinatorial actions of three transcription factors, Sox18, COUP-TFII and Prox1. Prox1 is considered the master regulator of LEC fate that is critically required both for the differentiation of LECs during embryogenesis and maintenance of LEC fate through development and adulthood [4,10]. Prox1 expression is regulated by Sox18 that is expressed in the cardinal vein prior to Prox1 and directly binds to Prox1 promoter [11]. However, the signal that induces Sox18-mediated Prox1 induction specifically in the cardinal vein but not in other Sox18 positive blood vessels is not known [11]. COUP-TFII is required for establishing venous endothelial cell identity by suppressing Notch signaling and for LEC specification [12–14]. It interacts directly with Prox1 and induces the expression of LEC specific genes such as Vegfr3 and Nrp2 [13,15,16]. After LEC differentiation COUP-TFII appears to regulate lymphatic identity independently of Prox1 [16]. Like COUP-TFII, a member of the Ets family of transcription factors, Ets-2 was shown to physically interact with Prox1 and regulate Prox1-induced expression of the key regulator of LEC sprouting, VEGFR-3 [17]. Ets transcription factors may thus also participate in LEC specification during development, but this needs to be further investigated using in vivo models. An alternative model of lymphatic development, proposed by Hungtington and McClure in 1908, suggests that lymphatic vessels derive from mesenchymal progenitor cells. Such ‘lymphangioblasts’ were described in birds [18] and Xenopus [19]. In addition, mesenchymal Prox1þcells with macrophage characteristics were identified in mouse embryos [20]. However, genetic lineage tracing studies have failed to demonstrate the contribution of hematopoietic cells, including macrophages, to mammalian lymphatic vasculature [21].

319 (2013) 1618–1625

the integrity of the vein during their exit [8,9]. As the cells assemble into the first lymphatic structures, a direct connection is made between the primordial thoracic duct and the cardinal vein [9]. Formation of specialized lympho-venous valves at the connection site prevents blood from flowing into the lymphatic system [9,22]. In addition, at the sites of communication between the cardinal veins and the developing lymph vessels platelet aggregation is induced, which is thought to induce ‘sealing’ and physical separation of the two vascular compartments. This process depends on the interaction between Podoplanin in LECs and Clec-2 in platelets [23].

Venous sprouting of lymphatic endothelial cells Key regulators of venous sprouting of LECs are VEGF-C/VEGFR-3 and CCBE1 signaling pathways. In the absence of Vegfc or Ccbe1 LEC differentiation occurs but venous sprouting is inhibited, which results in a complete lack of lymphatic vessels in mouse and zebrafish embryos [9,24–26]. However, while Vegfc deficient LECs remain completely trapped in the veins, Ccbe1 deficiency leads to aberrant sprout formation which is followed by downregulation of Prox1 and rapid loss of lymphatic structures by E11.5 [9]. The fundamental roles of these genes in lymphatic development is further illustrated by the identification of mutations in CCBE1 and VEGFR3 as underlying genetic causes of primary human lymphedemas Hennekam syndrome and Milroy disease, respectively ([27,28], Table 1). VEGF-C, together with the related VEGF-D, binds and activates the tyrosine kinase receptors VEGFR-2 and VEGFR-3 and their co-receptor Nrp2 on LECs. Although both growth factors can promote migration and proliferation of LECs in vitro and lymphatic vessel hyperplasia in vivo, only VEGF-C is essential for embryonic lymphatic development [24,29,30]. VEGFR-3 is considered the main VEGF-C receptor for lymphangiogenesis. Interestingly, however, while VEGFR-3 kinase activity is necessary for the migration of LEC from the cardinal vein, deletion of the VEGF-C/ VEGF-D binding domain of VEGFR-3 did not affect this early process but only inhibited lymphatic sprouting during later stages of development [31]. This suggests the involvement of the other VEGF-C receptor, VEGFR-2, and possibly signaling from a VEGFR-2/VEGFR-3 heterodimer [32] during venous sprouting of LECs. While it has been reported that VEGFR-2 is expressed in the LECs and that it can mediate circumferential growth of lymphatic vessels in adult mice [33], its direct involvement in developmental lymphangiogenesis has not yet been shown. Like VEGF-C, CBBE1 is a secreted protein expressed in tissues that are in close proximity to the budding venous-derived LECs. Based on its domain structure, CCBE1 is predicted to bind components of the extracellular matrix, however, no binding partners have yet been identified. CCBE1 does not appear to have lymphangiogenic activity on its own but it enhances the effect of VEGF-C in vivo [25].

Lymphatic sprouting from lymph sacs and pre-existing vessels

Lymphatic endothelial cell exit from the vein and vessel sprouting The budding LECs leave the veins as individual cells that are connected to each other via adherens junctions, thus maintaining

After the formation of the primitive lymphatic structures (commonly called as ‘lymph sacs’), further expansion of the lymphatic vasculature occurs via vessel sprouting. These later sprouting processes are also controlled by VEGF-C and its receptor VEGFR-3.

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Table 1 – Genes causative of hereditary lymphedema in humans. Gene

Syndrome

Inheritance

Phenotypea

Defectb

FLT4/ VEGFR-3

Milroy disease (Hereditary lymphedema IA)

Lower limb lymphedema, congenital

FOXC2

Lymphedema-Distichiasis syndrome

AD with reduced penetrance AD

SOX18

Hypotrichosis-Lymphedema-Telangiectasia syndrome

AD

CCBE1

Hennekam lymphangiectasia-lymphedema syndrome

AR

Hypoplasia or dysfunction of lymphatic capillaries [69,70] Valve defect in collecting vessels [47,48] Defect in LEC differentiation and lymph sac formation [11] Defect in venous sprouting of LECs [26]

GJC2

Hereditary lymphedema IC

AD

GATA2

Myelodysplastic syndrome/acute myeloid leukemia-Emberger syndrome

KIF11

Microcephaly, lymphedema, chorioretinal dysplasia syndrome Lymphedema-choanal atresia syndrome

AD with reduced penetrance AD

PTPN14 NEMO

Ectodermal dysplasia, anhidrotic, with inmunodeficiency, osteoporosis and lymphedema-Oledaid syndrome

AR X-linked recessive

Lower limb lymphedema, late onset Lower limb lymphedema, variable age of onset (four to teens) Lymphedema of limbs, genitalia and face, intestinal lymphangiectasia, congenital Four-limb lymphedema, onset during the first or second decade Lower limb lymphedema, onset between infancy and puberty Lower limb lymphedema, congenital Lower limb lymphedema, childhood onset Lymphedema of limbs and genitalia, congenital

No animal model available Defect in lymph sac formation and bloodlymphatic separation [71] No animal model available Hyperplasia of lymphatic capillaries [72] No animal model available

Abbreviations: AD ¼ autosomal dominant, AR ¼ autosomal recessive. Only lymphedema phenotypes are indicated. b Description of lymphatic defects is based on studies of animal models and human patients. a

In addition, Nrp2, which is dispensable for venous sprouting of LECs, is required for lymphatic sprouting during late embryonic and early postnatal stages [34,35]. Similarly, Ephrin-B2 mediated signaling regulates postnatal lymphatic sprouting and remodeling [36]. This function is mediated, at least in part, via modulation of lymphatic endothelial response to VEGF-C signaling by Ephrin-B2 regulation of VEGFR-3 internalization [37]. Notch signaling has been established as a major regulator of blood vascular development (reviewed in [38]). This pathway has recently been shown to play an important role also in lymphangiogenesis, however, it appears to have different functions depending on the stage of development. In analogy to its role in angiogenesis, Notch was found to negatively regulate lymphatic sprouting in adult mice, and its ligand Dll4 to determine tip/ stalk cell patterning of spouting lymphatic vessels [39]. In contrast, blocking of Notch1–Dll4 in zebrafish embryos or early postnatal mice inhibited lymphatic vessel sprouting [40,41]. This was associated with modulation of LEC response to VEGF-C by reduced EphrinB2 expression [39]. In agreement with a positive role in lymphatic sprouting, in early mouse embryos Notch was found to induce VEGFR-3 expression and thereby increase endothelial cell responsiveness to VEGF-C [42]. Notch signaling may therefore have a dual role in lymphangiogenesis: during development it is required for lymphatic vessel formation while in adult tissues its role is to maintain quiescence of the established vessels [39]. Additional function for Notch signaling was suggested during LEC specification. Overexpression of Notch in cultured LECs suppressed Prox1 and COUP-TFII expression [43]. However, in other experiments Notch suppression did not alter Prox1 levels in cultured cells [39]. Furthermore, deletion of Rbpj, a transcriptional co-activator of Notch that is required for

Notch target gene expression, did not inhibit Prox1 expression or LEC specification in vivo [14]. Several other factors with lymphangiogenic activity have recently been described, including FGF, HGF and PDGF-B. However, in some cases their lymphangiogenic effects are secondary to the induction of VEGF-C/D (reviewed in [1]).

Remodeling into a functional lymphatic vasculature Maturation of the primary lymphatic plexus into a functional vascular network involves acquisition of vessel-type specific features that serve their critical functions. These remodeling processes lead to the formation of specialised flap valves in lymphatic capillaries and establishment of pre-collecting and collecting vessel phenotype via SMC recruitment and luminal valve development (Fig. 1).

Formation of button-like junctions (‘primary valves’) in lymphatic capillaries Button-like intercellular junctions between LECs of lymphatic capillaries form flap like openings (also called primary valves) that facilitate entry of fluid and leukocytes into the vessels [2]. In the tracheal lymphatic vessels these specialized junctions were shown to develop from continuous ‘zipper’ junctions during late embryonic and early postnatal period [44]. Interestingly, buttonlike junctions reverted to zippers during inflammation. Such change in junctional organization is likely to affect the ability of the lymphatic capillaries to drain fluid and may contribute to mucosal edema during inflammation [44]. Molecular mechanisms

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controlling maturation of button junctions are poorly understood. Like other endothelial cell–cell junctions, button junctions require VE-cadherin for their stability [2]. In addition, glucocorticoid receptor activation, induced by dexamethasone treatment, was shown to promote button formation both during development and inflammation [44].

Formation of luminal valves (‘secondary valves’) in collecting vessels Formation of luminal valves is initiated by the emergence of clusters of cells expressing elevated levels of Prox1 and Foxc2 transcription factors in defined positions along the developing collecting vessels. Mechanical forces caused by lymph flow may play an important role in establishing the locations of developing valves [45]. Valve-forming endothelial cells subsequently rearrange on the vessel wall and form a transverse ridge, which develops further into mature valve leaflets (reviewed in [46]). Foxc2 is a major regulator of lymphatic valve formation, as its deficiency leads to lymphatic valve aplasia in both mice and humans ([47,48], Table 1). Foxc2 co-operates with another transcription factor NFATc1 that is activated by Calcineurin signaling during early stages of lymphatic valve development [45,49]. Genetic studies in mice have recently revealed a number of other important regulators of valve formation, including Ephrin-B2, Ang2, Connexin and Integrin-a9/Fibronectin-EIIIA signaling pathways that operate during different stages of valve formation (reviewed in [46]).

SMC recruitment and lymphatic vessel wall assembly Pre-collecting and collecting lymphatic vessels are covered by SMCs, except for luminal valve areas. The number of perivascular SMCs increases progressively along the lymphatic vascular tree; while precollecting vessels have only a sparse coverage the largest lymphatic ducts have a continuous smooth muscle coating. The key regulator of vascular SMCs, PDGF-B, may also be involved in lymphatic SMC recruitment [47]. Recruitment of SMCs to collecting vessels coincides with assembly of basement membrane (BM) composed of collagen IV, fibronectin and laminins that also form the blood vascular BMs [49,50]. Additional components that are enriched in lymphatic in comparison to vascular BM include Efemp1 and Reelin, of which the latter was found to play a critical role in the formation of functional collecting lymphatic vessels [50]. Reelin deficiency resulted in reduced SMC coverage and abnormal collecting vessel morphology and function, indicating a critical role for SMCs in lymphatic morphogenesis and function. In addition, the importance of maintaining valve areas free of SMCs was recently demonstrated. Lack of repulsive signaling between LEC-derived Semaphorin 3A and its receptor Nrp1 on SMCs caused enhanced SMC coverage of lymphatic valves and, consequently, abnormal valve morphology and vessel function [51,52]. Unlike collecting vessels, lymphatic capillaries have discontinuous basement membrane containing gaps that allow entry of immune cells [53] and are likely important also for maintaining permeability of lymphatic capillaries to fluid and macromolecules. In agreement, ectopic SMC recruitment to lymphatic capillaries, which is often observed concomitantly with increased basement membrane deposition, has been reported in several mouse mutants with abnormal and/or non-functional lymphatic vessels [36,47,54]. For example,

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Foxc2 deficient mice show SMC coverage of lymphatic capillaries, which is associated with increased deposition of Collagen IV [47]. Capillary LECs are in close contact with and attached to the interstitial matrix by anchoring filaments. The importance of these microfibrils for lymphatic function was highlighted by a study showing that defective ECM anchorage of LECs due to loss of Emilin-1, a connective tissue glycoprotein associated with elastic fibers, leads to dilation of lymphatic vessel lumen and reduced responsiveness of vessels to interstitial pressure variations [55].

Regulators of lymphangiogenesis in pathological conditions Once the functional lymphatic vasculature is established the vessels remain quiescent in adult tissues. However, lymphangiogenesis can be activated in pathological conditions such as inflammation or cancer ([1], Fig. 1). Neo-lymphangiogenesis appears to be driven largely by the same molecular mechanisms that operate during the development of the lymphatic vasculature.

Features of pathological lymphatic vessels Lymphatic vessels that form under pathological stimulus display distinct molecular and morphological features compared with normal lymphatics. For example, intratumoral vessels are generally small and their lumen is collapsed due to the high intratumoral pressure, which may make them non-functional [56]. On the contrary, peritumoral lymphatics are often dilated and packed with tumor cell clusters [57]. Pathological vessels also show loss of specific features of differentiated vessel types, such as the presence of SMCs, a hallmark of collecting vessels, on lymphatic capillaries around the tumor [58], and loss of primary valves in lymphatic capillaries during inflammation [44]. Controversial views exist regarding the functionality of intraand peritumoral lymphatic vessels and their involvement in metastasis. Both clinical and experimental data from various mouse cancer models indicate that lymph node metastasis is associated with tumor lymphangiogenesis [1,59]. However, while in some human tumors high intratumoral lymphatic vessel density correlates with aggressiveness, in others peritumoral lymphatic vessel density is the prognostic factor [57]. These observations suggest that lymphangiogenesis and lymphatic metastasis can differ in different tumor types. Furthermore, it has been well established that tumor location is prognostically important for certain human cancers. In particular, it was recently shown that the proximity of the primary tumor to small lymphatic vessels (capillaries and pre-collecting vessels) is critical [58]. These small lymphatic vessels are able to sprout in response to the lymphangiogenic growth factors, such as VEGF-C and VEGF-D, while collecting lymphatic vessels respond by vessel dilation [58,60,61]. Tumors developing in tissues that are rich in lymphatic capillaries, such as skin, are therefore more likely to induce lymphangiogenesis and metastasize via lymphatics than those located in tissues with fewer lymphatic capillaries [58].

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Regulators of LEC differentiation in tumor lymphangiogenesis Sox18 and COUP-TFII, the two transcription factors regulating LEC differentiation during embryogenesis, are also involved in tumor lymphangiogenesis. Sox18 is down-regulated in postnatal vasculature but re-expressed in both lymphatic and blood vessels of the tumor [16,62]. In melanoma xenograph mouse model Sox18 deficiency led to reduced tumor lymphangiogenesis and metastasis to the regional lymph nodes [62]. Inactivation of COUP-TFII in a mouse mammary gland tumor model similarly resulted in an inhibition of tumor lymphangiogenesis [16]. The lymphangiogenic function of COUP-TFII was suggested to be, at least in part, due to enhanced Nrp2 expression leading to increased VEGF-C signaling [16], while the mechanism by which Sox18 mediates tumor neo-lymphangiogenesis is not known [62]. Induction of Sox18 in tumor blood vessels was suggested to indicate the possibility that it drives the generation of new lymphatic vessels via reprogramming of BEC into LEC [62], raising the question of the origin of tumor lymphatic vessels. While postnatal growth of lymphatics is thought to occur by sprouting of vessels from pre-existing ones, during pathological lymphangiogenesis such as in tumors, haematopoietic cell derived endothelial progenitors and transdifferentiating leukocytes and macrophages were shown to incorporate into growing vessels [63]. In contrast, other studies have found no evidence suggesting trans-differentiation of bone-marrow derived cells into LECs in tumors [64]. Furthermore, genetic lineage tracing showed no contribution of macrophages in tumor-induced lymphangiogenesis [21].

Regulators of lymphatic vessel sprouting in tumor lymphangiogenesis As for embryonic lymphangiogenesis, the best-characterized signaling pathway that contributes to pathological lymphangiogenesis is the VEGF-C/VEGFR-3/Nrp2 pathway. In experimental tumor models, expression of VEGF-C and -D has been shown to induce lymphangiogenesis and correlate with lymphatic invasion and nodal metastasis [57,59]. Conversely, inhibition of VEGFR-3 or the co-receptor Nrp2 leads to reduced tumor lymphangiogenesis without affecting mature vessels [65,66]. Moreover, other growth factors such as FGF-2 promote tumor lymphangiogenesis, at least in part, via an indirect mechanism involving VEGF-C/ VEGFR-3 signaling [67,68].

Concluding remarks During the past two decades, significant progress has been made in identifying genes involved in the regulation of developmental lymphangiogenesis. However, much less is known about the mechanisms controlling pathological lymphatic vessel growth. Most studies so far have focused on the importance of VEGF-C/ VEGF-D/VEGFR-3 signaling pathway, which has been established as a key regulator of both physiological and pathological lymphangiogenesis. The challenge for future studies is to identify additional molecular players, which may provide targets for development of curative treatment strategies for lymphatic

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disorders, including tumor associated lymphangiogenesis and metastasis.

Acknowledgments We apologize to those authors whose work could not be cited here due to space restriction. The authors are supported by Cancer Research UK (IM-C, TM), EMBO Young Investigator Programme (TM) and Fundacio´n Alfonso Martin Escudero (IM-C).

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