molecular biology and pathology of lymphangiogenesis

ANRV335-PM03-14 ARI 16 December 2007 15:29

Molecular Biologyand Pathology ofLymphangiogenesisTerhi Karpanen and Kari AlitaloMolecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research,Biomedicum Helsinki and Haartman Institute, University of Helsinki and HelsinkiUniversity Central Hospital, FI-00014 Helsinki, Finland;email: [email protected], [email protected]

Annu. Rev. Pathol. Mech. Dis. 2008. 3:367–97

First published online as a Review in Advance onOctober 3, 2007

The Annual Review of Pathology: Mechanisms ofDisease is online at pathmechdis.annualreviews.org

This article’s doi:10.1146/annurev.pathmechdis.3.121806.151515

Copyright c© 2008 by Annual Reviews.All rights reserved

1553-4006/08/0228-0367$20.00

Key Words

lymphatic vessel, lymphatic vascular development, tumormetastasis, lymphedema, inflammation

AbstractThe lymphatic vasculature is essential for the maintenance of tis-sue fluid balance, immune surveillance, and adsorption fatty acidsin the gut. The lymphatic vessels are also crucially involved in thepathogenesis of diseases such as tumor metastasis, lymphedema, andvarious inflammatory conditions. Attempts to control or treat thesediseases have drawn a lot of interest to lymphatic vascular researchduring the past few years. Recently, several markers specific for lym-phatic endothelium and models for lymphatic vascular research havebeen characterized, enabling great technical progress in lymphaticvascular biology, and many critical regulators of lymphatic vesselgrowth have been identified. Despite these significant achievements,our understanding of the lymphatic vessel development and patho-genesis is still rather limited. Several key questions remain to be re-solved, including the relative contributions of different pathways tar-geting lymphatic vasculature, the molecular and cellular processes oflymphatic maturation, and the detailed mechanisms of tumor metas-tasis via the lymphatic system.

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Lymphaticmetastasis: spreadof tumor cells fromthe primary tumorthrough lymphaticvessels to lymphnodes

Lymphedema:accumulation ofinterstitial fluid inand swelling of theaffected tissues dueto a failure inlymphatic vascularfunction

LEC: lymphaticendothelial cell

INTRODUCTION

The lymphatic vasculature collects ex-travasated fluid and macromolecules from tis-sues and returns them to the blood circulation.Furthermore, the lymphatic vascular networkplays an essential role in the body’s immunedefense by carrying antigens and antigen-presenting cells from the interstitium to bedisplayed for B and T cells in the lymph nodes.In addition, the lymphatic vessels absorb long-chain dietary triglycerides and lipophilic com-pounds released in the intestine in the form ofchylomicrons.

Reflecting its specialized function in in-terstitial fluid drainage and transport of ex-travasated cells, the lymphatic vasculature isalso implicated in the dissemination of tumorcells to regional lymph nodes. Although lym-phatic metastasis is a common early featureand often the most lethal aspect of humancancers, its mechanisms are still poorly under-stood. The traditional thinking of lymphaticvessels as mere passive channels for tumor celltransit is giving room to the evolving pictureof an active role for lymphatic vasculature infacilitating tumor cell dissemination. Failureof lymphatic function causes accumulation ofprotein-rich fluid in the tissues, which leadsto a chronic and progressive condition knownas lymphedema. Furthermore, lymphatic ves-sels are intimately involved in various inflam-matory conditions. There has been increas-ing focus on prevention or treatment of thesediseases by controlled inhibition or stimula-tion of lymphatic vessel growth, and studies inmice have already produced promising resultsin this respect. Owing to the many achieve-ments in lymphatic vascular research over thepast 10 years, the most important of which in-clude the identification of lymphatic endothe-lial markers, discovery of lymphangiogenicgrowth factors, development of animal mod-els for studying lymphatic vessels, and meth-ods for isolating lymphatic endothelial cells(LECs), the basic mechanisms of lymphan-giogenesis are being revealed. However, manyimportant aspects remain to be resolved.

Here we review the current knowledge onthe molecular regulation of normal lymphaticvascular development and discuss the contri-bution of the lymphatic vessels to the patho-genesis of various diseases. Understanding thedetailed molecular and cellular mechanismsof lymphatic vascular involvement or failurein these pathological processes is necessary toreveal new therapeutic targets and to developefficient methods for prevention, diagnosis,treatment, and management of these diseases.

STRUCTURE ANDDEVELOPMENT OF THELYMPHATIC VASCULAR SYSTEM

In contrast to the closed blood circulatory sys-tem, the lymphatic vasculature functions uni-directionally. The initial lymphatic capillarynetwork is specialized for collecting intersti-tial fluid, macromolecules, and extravasatedleukocytes from tissues. This is reflected inthe distinctive structure of the lymphatic cap-illaries (Figure 1). These drain to the pre-collecting lymphatic vessels, which transportthe lymph to successive sets of lymph nodes.Lymph from the intestinal, hepatic, and lum-bar regions is collected into the cisterna chyli.Finally, the collecting lymphatic vessels draininto the thoracic duct, or from the upper rightquadrant of the human body into the rightlymphatic duct. These are then emptied intothe venous circulation at the junctions of leftor right internal jugular veins and subclavianveins, respectively (reviewed in References 1and 2). Apart from avascular tissues such asthe epidermis, cartilage, and cornea, and somevascularized tissues such as the brain, bonemarrow, and retina, most tissues of higher ver-tebrates contain lymphatic vessels.

The lymphatic vessels arise after thecardiovascular system is established and func-tional. The prevailing view of their originpostulates that LECs are derived from thevenous endothelium (3). Lymphatic vessel de-velopment starts around midgestation, whena distinct subpopulation of endothelial cells

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H2O

ProteinsLipids

Leukocytes

Lymphaticcapillary

Collectinglymphatic

vessel

Bloodcapillary

VeinArtery

Lymph node

Basement membrane

Basementmembrane

Extracellular matrixAnchoring filaments

Basementmembrane

Figure 1Structure of the lymphatic vessels. Compared with blood vessels, lymphatic vessels are thin walled andhave a relatively wide lumen. The endothelial cells of lymphatic capillaries (green) lack tight junctions.Instead, the neighboring endothelial cells partly overlap, forming valve-like openings, which allow easyaccess for fluid, macromolecules, and cells into the vessel lumen. Lymphatic capillaries lack vascularmural cells and have no or only an incomplete basement membrane. Elastic fibers known as anchoringfilaments connect lymphatic capillary endothelial cells to the surrounding stroma and maintain vesselpatency during increased interstitial pressure. The lymph drains from the lymphatic capillaries toprecollecting and collecting lymphatic vessels, which are finally emptied into veins in the jugular region.The precollecting and collecting lymphatic vessels have a basement membrane, are surrounded byvascular smooth muscle cells (vSMCs; red ) with intrinsic contractile activity to promote lymph flow, and,like veins, contain valves that prevent backflow of the lymph. The valve regions are devoid of vSMCs. Onits way, the lymph is filtered through a series of lymph nodes. In contrast, the endothelial cells of bloodvessels form tight and adherence junctions, have a distinct basement membrane, and are surrounded bypericytes/vSMCs, which form one or multiple layers increasing in thickness with vessel size.

(ECs) on one side of the anterior cardinal veincommits to the lymphatic lineage, and thensprouts and migrates to form primary lymphsacs in the jugular region (Figure 2) (re-

viewed in Reference 4). Subsequently, severallymph sacs are formed close to major veins indifferent regions of the embryo. The periph-eral lymphatic vasculature is then generated

www.annualreviews.org • Pathological Lymphangiogenesis 369

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LEC commitmentand differentiation

Budding, migration,and proliferation of LECs

Formation oflymph sacs

Remodeling andmaturation of thelymphatic vasculature

Sprouting and formationof primary lymphatic plexus

Separation of blood andlymphatic vasculature

Primary lymph

sac

?

Prox1Vegfc/Vegfr3

Prox1+

Lyve1+

Vegfc

Prox1+

Lyve1+

Vegfr3+

Podoplanin+

Neuropilin-2+

Syk and Slp76

Cardinalvein

Cardinalvein

Foxc2EphrinB2Angiopoietin-2Integrin α9

Prox1–

E9.0–E9.5 E10–E11 E14.5–postnatalE11.5–E14.5

Figure 2Development of the lymphatic vasculature in mice. Upon stimulation by an as yet unidentified signal(s), asubset of venous endothelial cells becomes committed to the lymphatic endothelial fate. Thesedifferentiating lymphatic endothelial cells (LECs) express Lyve1, Prox1, and Vegfr3. Stimulated byVegfc, which is secreted by the adjacent tissue, they migrate and proliferate to form primary lymph sacs,from which the lymphatic vessels start sprouting. The primary lymphatic vascular plexus ( green) becomesseparated from the blood vessels. It undergoes remodeling and maturation to create the lymphaticvasculature consisting of a lymphatic capillary network, which lacks pericytes, and of collecting lymphaticvessels, which contain valves ( green) and are associated with vascular smooth muscle cells ( purple).Molecules involved at these later stages of the lymphatic development include ephrinB2, neuropilin-2,Angiopoietin-2, podoplanin, integrin α9, and the transcription factors Foxc2, Net, Sox18, and Vezf1.

Endothelial cell(EC): a cell that in asingle layer lines theinner surface ofblood or lymphaticvessels

Lymphangiogenesis:the growth oflymphatic vesselsfrom preexistingones by a processbelieved to be similarto angiogenesis

by centrifugal sprouting of lymphatic vesselsfrom the lymph sacs, followed by merging ofthe separate lymphatic capillary networks, andremodeling and maturation of the primitivelymphatic capillary plexus (Figure 2).

Another theory suggests that LECs arederived from mesenchymal progenitor cells(5). LEC precursors, lymphangioblasts, existin birds and amphibians (6, 7). Although theexistence of lymphangioblasts in mammals isunclear, it is possible that in addition to de-differentiation of LECs from venous ECs andsubsequent sprouting lymphangiogenesis,differentiation of LECs from mesenchymalprecursor cells and lymphvasculogenesis may

contribute to the formation of the lymphaticvasculature during embryonic development.

In adults, ECs are normally in a quies-cent state, but competent to respond to a va-riety of stimuli. Lymphatic vessel growth ap-pears to follow that of the blood vessels dur-ing tissue regeneration, wound healing, tu-mor growth, and in inflammation. New lym-phatic vessels are believed to grow primarilyby sprouting from existing ones. Although theexistence of bone marrow–derived or circulat-ing putative progenitors capable of differen-tiating into LECs has been suggested, theircontribution to adult lymphangiogenesis andtheir exact identity are still controversial. ECs


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of host origin in the lymphatic vessels of in-flamed kidney transplants were apparently de-rived from circulating progenitor cells (8), andmacrophages were suggested to transdifferen-tiate to LECs in inflamed mouse cornea (9).A recent study provides evidence for the con-tribution of hematopoietic endothelial pro-genitor cells in the separation of blood andlymphatic vasculatures (10). However, bonemarrow–derived progenitor cells did not in-corporate significantly into the endotheliumof newly formed lymphatic vessels in tumors(11).

MOLECULAR REGULATIONOF LYMPHATIC VASCULARDEVELOPMENT

With the identification of several critical reg-ulators of lymphatic vascular development,the basic molecular mechanisms of this pro-cess are being revealed. The homeobox tran-scription factor PROX-1 is essential for thedetermination of LEC identity. VEGF-C,a growth factor of the vascular endothelialgrowth factor (VEGF) family, primarily sig-naling through vascular endothelial growthfactor receptor-3 (VEGFR-3), is required forthe proliferation, migration, and survival ofLECs until the postnatal maturation of thelymphatic vasculature occurs. Yet, many cru-cial aspects and the fine-tuning of the lym-phatic development remain to be resolved.In the following, the most important regu-lators and specific markers of lymphatic en-dothelium are discussed in further detail (seealso Table 1). In addition to these, severalother growth factors including VEGF, hepa-tocyte growth factor, insulin-like growth fac-tors 1 and 2, platelet-derived growth factorB (PDGF-B), and fibroblast growth factorhave been described to induce lymphangio-genesis either in a VEGFR-3-independent or-dependent manner. These growth factorsalso stimulate other cells and biological pro-cesses and may often induce lymphangio-genesis indirectly. They have recently beenreviewed elsewhere (12, 13).

VEGF-C: vascularendothelial growthfactor-C

VEGFR-3: vascularendothelial growthfactor receptor-3

E: embryonic day

In contrast to the heterogeneity of theblood vascular endothelium, little is knownabout the molecular heterogeneity of the lym-phatic endothelium in different vessel typesand in distinct organs. Given the multiple andspecialized tasks of the different lymphaticvessels, such as the absorption of dietary lipidsby the central lacteals of the intestinal villi, thetransport of immune cells by the afferent lym-phatic vessels, and the exposure of lymphaticcapillaries to high hydrostatic pressure in theextremities, it is reasonable to presume thatsuch differences exist.

LYVE-1, the Earliest Markerfor Lymphatic EndothelialDifferentiation

LYVE-1, lymphatic endothelial hyaluronanreceptor-1, is one of the most specific andwidely used lymphatic endothelial markers(14). Expressed from embryonic day (E) 9onward in a polarized manner in the ve-nous endothelium differentiating into LECs,LYVE-1 currently provides the first indicatorof lymphatic endothelial competence (4). Inadults its expression in collecting lymphaticvessels decreases and remains high only inlymphatic capillaries (15). LYVE-1 is thoughtto function in hyaluronan turnover or inleukocyte trafficking. Gene targeting in mice,however, indicated that LYVE-1 is not nec-essary for normal lymphatic development orfunction, suggesting a more specific role orthe existence of compensatory receptors (16).

PROX-1, the Master Regulatorof Lymphatic EndothelialDifferentiation

PROX-1 is specific for lymphatic vessels inthe vascular system. In mice, the first Prox1-positive ECs are detected as a restricted sub-population on one side of the anterior cardinalvein at E9.5, and soon thereafter these cellsstart budding from the vein and migrating ina polarized manner, eventually forming lymphsacs (17). Prox1 knockout embryos completely


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Table 1 Genes identified, on the basis of studies in mouse mutants, to be involved in lymphatic vascular developmentand maturation

Gene Lymphatic phenotype Lethality Reference(s)Ang2 −/− Defects in the function and patterning Perinatal-P14 (129/J)

>90% survival (c57bl/6j)(51)

(144)EphrinB2 �V/�V Absence of valves, defective

remodeling, ectopic mural cells

�V/�V Perinatal+/�V Normal

(15)

Fiaf/Angptl4 −/− Dilated, blood-filled lymphatic vesselswith aberrant connections to blood vessels inthe small intestine

−/− <3 weeks+/− Normal

(21)

Foxc2 −/− Absence of valves, abnormal patterning,ectopic mural cells

+/− Hyperplasia (also lymph nodes)

−/− E12.5–perinatal+/− Normal

(61)(145)

Integrin α9−/− Lymphedema, chylothorax −/− Perinatal

+/− Normal(49)

Net mut/mut Lymphangiectasia, chylothorax mut/mut Perinatal+/mut Normal

(146)

Np2 mut/mut Transient absence of lymphaticcapillaries

mut/mut Postnatal or survive+/mut Normal

(46)

p85 subunit of PI3K −/− Chylous ascites −/− Postnatal or survive+/− Normal

(147)

Podoplanin −/− Defects in lymphatic function,lymphangiectasia, lymphedema

−/− Perinatal+/− Normal

(58)

Prox1 −/− No differentiation of LECs+/− Defects in the lymphatic function,

adult-onset obesity

−/− E14.5+/− Perinatal (in most backgrounds)

(17, 18, 22)

Slp76 −/− Failure to separate blood and lymphaticvasculatures, chylous ascites


(63)

Sox18 mut/mut Edema, chylous ascites mut/mut Perinatal+/mut Normal

(148)

Syk −/− Failure to separate blood and lymphaticvasculatures, chylous ascites


(63)

Trisomy 16 Nucheal edema, abnormal size and structureof jugular lymph sacs

E16–E20 (149)

Vegfc −/− No lymph sacs or lymphatic vessels+/− Hypoplasia, chylous ascites

−/− E17–E19+/− Perinatal or normal

(32)

Vegfr3 +/mut Hypoplasia, chylous ascites mut/mut E10+/mut Perinatal or normal

(29)

Vezf1 +/− Lymphatic hypervascularization andedema in the jugular region (incompletelypenetrant and transient phenotype)

−/− E9.5—E16.5+/− Normal

(150)

�V: deletion of a valine residue in the conserved carboxyterminus leading to an inability to bind PDZ-domain-containing proteins.mut: inactivating mutation (Net and Np2, gene targeted; Sox18, spontaneous; Vegfr3, chemically induced).

lack lymph sacs and lymphatic vessels. TheProx1-deficient ECs initially bud and sproutfrom the cardinal vein, although in an unpo-larized manner, but their migration is soon

arrested. These cells fail to express lymphaticendothelial markers and instead retain theirblood vascular endothelial phenotype (17, 18).Overexpression of PROX-1 in human blood


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vascular endothelial cells (BECs) suppressesthe expression of several genes specific forthe blood vascular endothelium and upreg-ulates LEC-specific gene expression, whichfurther suggests a function for PROX-1 asa master control gene that determines LECfate (19, 20). The signals leading to polar-ized expression of PROX-1 in differentiat-ing LECs are currently not known. Prox1 ex-pression is severely reduced in the intestinallymphatic vessels of postnatal mice lackingfasting-induced adipose factor/angiopoietin-like protein 4 (Fiaf/Angptl4). This leads to di-lated and blood-filled lymphatic vessels withabnormal connections to blood vasculature(21). Those Prox1+/− mice that manage tosurvive until adulthood, as well as mice withan endothelial-specific deletion of Prox1, de-velop chylous ascites and obesity, indicatinga link between impaired lymph drainage andtissue adiposity (22).

The VEGF-C/VEGFR-3 Pathway,an Essential Mediator of LymphaticEndothelial Cell Migration,Proliferation, and Survival

Before the generation of lymphatic vessels,VEGFR-3 is expressed in the blood vascu-lar endothelium, where it is essential for theremodeling and maturation of the primarycapillary plexus (23). Later in embryogene-sis, the expression of VEGFR-3 decreases inthe blood vasculature, finally becoming re-stricted primarily to lymphatic vessels andto specialized blood capillaries with fenestra-tions (24). In adults, VEGFR-3 remains ex-pressed mainly in lymphatic capillaries de-void of vascular smooth muscle cells (vSMCs)(15). Vegfr3-deficient mice die at E9.5 owingto cardiovascular failure, which precludes theanalysis of Vegfr3 in lymphatic vascular de-velopment (23). Continuous ligand-inducedVegfr3 signaling is required for the survivaland maintenance of lymphatic vessels dur-ing embryonic development and a postna-tal period of approximately two weeks, afterwhich lymphatic vessels become independent

BEC: blood vascularendothelial cell

Chylous ascites:accumulation of amilky fluid with highfat content in theabdominal cavity dueto a defect oflymphatic vessels

Vascular smoothmuscle cell(vSMC): aspecializednonstriated musclecell that possessesintrinsic contractileactivity andconstitutes the wallof large blood andlymphatic vessels

of Vegfr3 (25, 26). Inactivating missense mu-tations in VEGFR-3 lead to lymphatic hy-poplasia and lymphedema in both mice andhumans, which suggests an important func-tion for VEGFR-3 in lymphatic development(27–29).

The two known ligands of VEGFR-3,VEGF-C and VEGF-D, induce growth oflymphatic vessels rather specifically (30, 31).Although not required for lymphatic en-dothelial commitment, VEGF-C is essentialfor the initial sprouting and directed migra-tion as well as for the subsequent survivalof LECs (32). Deletion of both Veg fc allelesresults in embryonic lethality, and heterozy-gous Veg fc+/− mice display defects in lym-phatic vascular development (32). VEGF-D,however, is dispensable for embryonic devel-opment of both blood vascular and lymphaticsystems, the only defect caused by deletion ofVegfd in mice being a slight reduction in thenumber of lymphatic vessels around the lungbronchioles, the primary site of VEGF-D ex-pression (33).

Although dispensable for blood vasculardevelopment in mice, Veg fc is described asbeing required for angiogenesis in both ze-brafish and Xenopus tadpoles (6, 34). Un-der specific conditions, both VEGF-C andVEGF-D also stimulate the growth of bloodvessels in mammals (35, 36). In vitro, VEGF-C promotes proliferation and migration ofboth LECs and BECs (37, 38). The receptor-binding pattern and affinity and thus the bio-logical activity of VEGF-C and VEGF-D areregulated by stepwise proteolytic processingof the precursor proteins (Figure 3). Uponproteolytic cleavage, their affinities towardVEGFR-3 increase and the fully processed,mature forms of VEGF-C and human VEGF-D also bind to and activate VEGFR-2 (38, 39).The VEGF-C-induced effects are probablymediated by VEGFR-2 on the blood vascu-lar endothelium and primarily by VEGFR-3on the lymphatic endothelium, although thereis increasing evidence that the expression ofVEGFR-2 on lymphatic vessels contributesto mitogenic and chemotactic signaling (31,


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40–42). Thus, the degree of proteolytic pro-cessing of the VEGF-C and VEGF-D pre-cursors might dictate their angiogenic versuslymphangiogenic potential.

VEGFR-3 can heterodimerize withVEGFR-2, leading to differential carboxyter-minal phosphorylation and, potentially,

differential signal transduction properties ofVEGFR-3/VEGFR-2 heterodimers whencompared with VEGFR-3 homodimers (43).Recently, it was suggested that cooperativesignaling between VEGFR-2 and VEGFR-3is required for LEC migration and prolifer-ation, whereas VEGFR-3 is redundant with

Lymphangiogenesis

Lymphatic endothelial cell - sprouting - migration - proliferation - survival

VEGFR-3 VEGFR-3 VEGFR-2 VEGFR-2

Neuropilin-2

Plexin

Integrins

?

Sema3

Extra-cellularmatrix

? ?

VEGF-C

VEGFhomologydomainAminoterminal Carboxyterminal

S-SS-S

S-S

S-S

S-SS-S

Protease(s)

Protease(s)


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VEGFR-2 for the organization of LECsinto functional capillaries (41). However,at least when induced by overexpression ofVEGF or the related VEGFR-2-specificligand VEGF-E, the major contribution ofVEGFR-2 signals is to enlarge lymphaticvessels; however these do not seem sufficientfor the induction of new lymphatic vesselsprouts (Figure 4) (42). Nevertheless, amutant form of VEGF-C, VEGF-C156S,unable to stimulate VEGFR-2 is sufficientfor stimulating lymphangiogenesis (31, 44).

Neuropilin-2 and Integrins,Modulators of VEGFR-3 Signaling

VEGF-C and VEGF-D also bind to neu-ropilins (29, 45). These are nontyrosine ki-nase coreceptors, which together with plex-ins mediate semaphorin signals in repulsiveaxon guidance. Neuropilin-2 is also expressedin lymphatic vessels (46). In vitro, neuropilin-2 is internalized along with VEGFR-3 uponVEGF-C or VEGF-D binding, suggestingthat neuropilin-2 modulates VEGFR-3 sig-naling (45). Neuropilin-2 mutant mice are bornwithout small lymphatic vessels or capillaries,which, however, regenerate during the post-natal period, whereas larger lymphatic vesselsdevelop normally (46). This suggests a tran-sient and selective requirement of neuropilin-2 in the embryonic development of small lym-phatic vessels.

Integrin β1, when in contact with colla-gen or fibronectin, can interact directly withand induce the tyrosine phosphorylation ofVEGFR-3, and stimulate migration of ECsto some extent even in the absence of a cog-nate ligand (47). Integrin α5β1 participates inthe activation of VEGFR-3 by VEGF-C156S,which is essential for fibronectin-mediatedLEC survival and proliferation (48). Mice de-ficient in the integrin α9 chain die during earlypostnatal period owing to chylothorax, whichsuggests an underlying function for integrinα9β1 in lymphatic development or function(49). The exact cellular and molecular mech-anism of this is unknown, but it may be signif-icant that integrin α9β1 binds VEGF-C andVEGF-D, and EC adhesion to and migrationon VEGF-C and VEGF-D is dependent uponthis integrin (50).

Angiopoietins and Tie Receptors AreInvolved in the Remodeling andStabilization of Lymphatic Vessels

The Tie1 and Tie2 receptor tyrosine ki-nases, which are essential for blood vascularremodeling, maturation, and stabilization,are also expressed in LECs. Mice deficientin the Tie2 ligand Angiopoietin-2 (Ang2)fail in postnatal vascular remodeling (51).Ang2-deficient mice also display a generalizedlymphatic dysfunction caused by disorganizedcollecting lymphatic vessels with poorly

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 3Proteolytic processing, receptor binding specificity, and biological effects of VEGF-C. The VEGF-Cprecursor is an antiparaller homodimer covalently coupled by disulfide bonds (S-S) between the amino-and carboxyterminal propeptides. The propeptides are proteolytically removed in a stepwise manner,which increases the affinity of VEGF-C toward VEGFR-3 and provides the mature, noncovalent dimerwith the ability to bind VEGFR-2. The fate of the amino- and carboxyterminal propeptides is currentlyunknown. VEGF-C signaling through VEGFR-3 is modulated by several coreceptors. These includeneuropilin-2, which binds VEGF-C and becomes internalized along with VEGFR-3 upon ligandbinding; integrin β1, which associates with VEGFR-3 and induces its phosphorylation upon ligation withthe extracellular matrix proteins fibronectin or collagen even in the absence of VEGF-C or VEGF-D;and integrin α9, which may bind VEGF-C. The role of plexins, which provide a signaling function forthe neuropilins, in lymphatic endothelium is unknown. Activation of downstream signaling moleculesinduces endothelial cell sprouting, migration, proliferation, and survival, leading to formation of newlymphatic vessels. VEGFR-2 is expressed in at least some lymphatic vessels, but its role as well as the roleof VEGFR-3/VEGFR-2 heterodimers in lymphangiogenesis is still unclear.


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Lyve1 Pecam1

Wild

type

K14

-hV

EG

F-A

165

K14

-mV

EG

F-A

164

a d

b e

c f

100 µm

Figure 4VEGF inducesenlargement oflymphatic vesselsbut no sproutinglymphangiogene-sis. Whole-mountstaining withantibodies againstthelymphatic-specificLyve1 (a–c) andlargely bloodvessel–enrichedPecam1 (d–f ) ofthe ear skin fromtransgenic miceexpressing humanVEGF165 (a and d )or mouse VEGF164(b and e) in the skinunder keratin 14(K14) promoterand from awild-typelittermate (c and f ).The image pairsare from the samemicroscopic field.The scale bar inpanel f applies toall panels. Figurecourtesy of MariaWirzenius.

associated vSMCs and an irregularly pat-terned hypoplastic lymphatic capillarynetwork (51). Interestingly, the lymphaticbut not the blood vascular defects are rescuedby the related Tie2-activating ligand Ang1,suggesting that Ang2 acts as a Tie2 agonistin developing lymphatic vessels but as anantagonist in blood vessels (51). Ang1 hasalso been shown to induce lymphatic vesselgrowth in adult tissues (52, 53).

EphrinB2 Is Required for theRemodeling of the PrimaryLymphatic Vascular Plexus

The primordial lymphatic vascular plexusneeds to undergo extensive remodeling andmaturation to establish a functional lymphaticvasculature (reviewed in Reference 54). Thisprocess continues during the postnatal pe-riod when the superficial lymphatic capillary


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network is formed by sprouting and thedeeper collecting lymphatic vessels mature bydeposition of the basement membrane (BM),recruitment of vSMCs, and development oflymphatic valves (15). Like sprouting BECs,the sprouting LECs also form filopodial ex-tensions, but along the entire length of thelymphatic capillaries, with subsequent elon-gation (15, 55).

EphrinB2 is expressed in collecting lym-phatic vessels, whereas its receptor EphB4is detected both in collecting lymphatic ves-sels and in lymphatic capillaries (15). In-tracellular PDZ-interaction-site-dependentsignaling of ephrinB2 is crucial for the post-natal remodeling of the lymphatic vasculatureinto a hierarchically organized lymphatic ves-sel network (15). Mice with inactivating mu-tation of the carboxyterminal PDZ-bindingdomain of ephrinB2 display hyperplasia of thecollecting lymphatic vessels, defective forma-tion of luminal valves, and disturbed sprout-ing of LECs, leading to blunt-ended protru-sions. This indicates that ephrinB2 reversesignaling is required for the elongation andguidance of sprouting LECs (15). In addition,the specification of collecting versus capillarylymphatic vessel identity fails in these mice, asdemonstrated by persistent LYVE-1 expres-sion in all lymphatic vessels, and by abnormalrecruitment of vSMCs into lymphatic capil-laries (15). Vascular mural cells deficient ofephrinB2 have an abnormal rounded morphol-ogy and make only loose contact with the en-dothelium, which interestingly also leads totheir abnormal migration to lymphatic vessels(56).

Podoplanin Is Essential for NormalLymphatic Vessel Development

Podoplanin is a transmembrane glycoprotein,which within the vascular system is expressedpredominantly in the lymphatic endothelium.There its expression starts around E11 and re-mains high both in collecting lymphatic ves-sels and in lymphatic capillaries in the adult(57, 58). Podoplanin-deficient mice die at birth

Basementmembrane (BM): athin membrane ofproteins, primarilycollagen IV, on thebasal (abluminal) sideof endothelial cells

Vascular mural cell:a nonendothelial cellconstituting the wallof blood andlymphatic vessels;refers mainly topericytes and smoothmuscle cells

Pericyte (PC): amesenchymal cellthat covers the outersurface of smallblood vessels makingclose contacts to andsharing the basementmembrane with theendothelium

owing to respiratory failure and have defectsin lymphatic but not blood vessel formationand patterning, leading to diminished lym-phatic transport and congenital lymphedema(58). In cultured ECs, podoplanin promotescell adhesion, migration, and tube formation(58). Podoplanin is also upregulated in theinvasive front of many human carcinomas,and its expression promotes tumor cell inva-sion in the absence of epithelial-mesenchymaltransition (59).

FOXC2 Is Necessary in LymphaticMaturation

The forkhead transcription factor Foxc2 isexpressed in the developing lymphatic ves-sels and in lymphatic valves of adults (60, 61).In Foxc2−/− embryos, the early lymphatic de-velopment seems to occur normally. At laterstages, the lymphatic vessels of Foxc2-deficientembryos appear irregularly patterned, be-come closely associated with an abnormallyhigh number of pericytes (PCs)/vSMCs, andhave an increased deposition of the BMprotein collagen IV (61). The presence ofPCs/vSMCs around the lymphatic capillar-ies of Foxc2−/− embryos might be due to theincreased expression of Pdgfb and endoglin,both implicated in the recruitment of mu-ral cells to blood vessels. Additionally, thecollecting lymphatic vessels of Foxc2−/− em-bryos lack lymphatic valves. Abnormally pat-terned lymphatic vessels with closely as-sociated PCs/vSMCs are also observed inmice double heterozygous for Foxc2 andVegfr3, suggesting cooperation between thesetwo signaling pathways. These data indicatethat although not required for early lym-phatic development, FOXC2 is essential inlater stages by regulating morphogenesis oflymphatic valves and in controlling inter-actions of LECs with mural cells by sup-pressing the expression of PDGF-B and en-doglin and the deposition of collagen IV,thereby maintaining the lymphatic capillaryphenotype.


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Syk and Slp76 Are Involved in theSeparation of the Lymphaticand Vascular Systems

Lymph is returned to the bloodstream mainlythrough the thoracic and the right lymphaticducts, which make a connection to the veinsin the jugular region. Additional lymphati-covenous communications exist at least in thekidney, adrenal gland, and liver, but they arebelieved to be nonfunctional, except whenthe intralymphatic pressure increases. Lym-phaticovenous anastamoses can be observed inlymphedema, chylous ascites, and chylothorax(reviewed in Reference 62).

Mice deficient of the intracellular signal-ing molecules Syk or Slp76, expressed almostexclusively in hematopoietic but not in en-dothelial cells, display arteriovenous shuntsand abnormal lymphaticovenous connections,leading to blood-filled lymphatic vessels (63).This suggests that circulating hematopoieticcells, presumably endothelial progenitors, arenecessary for the separation of the lymphaticand blood vascular systems (10). Syk and Slp76may be required for integrin-mediated guid-ance of the circulating endothelial precursorcells into the vascular endothelium.

LYMPHATIC VESSELS INPATHOLOGICAL CONDITIONS

Tumor Lymphangiogenesisand Lymphatic Metastasis

Cancer mortality is seldom caused by theprimary tumor but rather by the metastaticspread of malignant cells to distant organs.Although tumor cell dissemination can occurby a variety of mechanisms, including localtissue invasion, direct seeding of body cavi-ties, and spread through the blood vascularsystem, several clinical and pathologicalobservations suggest that for many typesof solid human tumors the most commonpathway of initial metastasis is through thelymphatic system to regional lymph nodes(reviewed in Reference 64). The spread oftumor cells to lymph nodes through the

lymphatic vasculature has long been used asan important prognostic marker of tumor ag-gressiveness and as a criterion for therapeuticchoices (reviewed in References 65 and 66).Despite this, the mechanisms of lymphaticmetastasis are still poorly understood.

Lymphatic vessels, often containing clus-ters of tumor cells, are frequently observed inthe periphery of malignant tumors. However,the existence of functional lymphatic vesselswithin human cancers and the ability of tu-mors to actively stimulate lymphangiogene-sis have been controversial. Intratumoral lym-phatic vessels are detected in some humancancers, and in some cases the presence oflymphatic vessels inside the tumor was re-ported to correlate positively with lymph nodemetastasis and poor prognosis (67, 68). How-ever, intratumoral lymphatic vessels might bepoorly functional and not required for lym-phatic metastasis (69, 70).

Many types of solid human tumors expressthe lymphangiogenic growth factor VEGF-C,and many clinicopathological studies have re-ported a positive correlation between its ex-pression and lymphatic invasion, lymph nodeand distant metastasis, and poor patient sur-vival, but not necessarily with the density oftumor-associated lymphatic vessels (reviewedin References 66, 71, and 72). Furthermore,VEGFR-3 is upregulated in tumor blood ves-sels, and it was suggested to contribute to tu-mor angiogenesis or to vessel integrity (73,74). A recent study clarifies the involvement ofVEGFR-3 in tumor angiogenesis by showingthat blocking antibodies against VEGFR-3decrease tumor angiogenesis and growth (75).

During the past few years, the role ofVEGF-C and VEGF-D in cancer progres-sion has been a focus of intensive exper-imental research. Several studies using ei-ther xenotransplantation or transgenic mousemodels have shown that expression of thesegrowth factors by tumor cells induces growthof new lymphatic vessels—mainly at the tu-mor margin, although in some models alsointratumorally—as well as dilation of preex-isting lymphatic vessels draining the tumor


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a b

dc

Figure 5VEGF-C/VEGFR-3 pathway in tumors. (a) Expression of VEGF-C in MCF-7 human breast cancercells implanted into the mammary fat pads of SCID mice induces abundant lymphangiogenesis at thetumor periphery (red Lyve1 staining) and intralymphatic tumor growth. (b) The control MCF-7 tumorsdo not contain Lyve1-positive lymphatic vessels. (c and d ) A human renal cell carcinoma stained withantibodies against VEGFR-3 (c, red-brown) or control antibodies (d ). VEGFR-3 is predominantlylocalized in endothelial cells of tumor blood and lymphatic vessels but not in tumor cells. Figures inpanels c and d courtesy of Leif Anderson.

(Figure 5) (69, 76–82). Furthermore, tumor-associated lymphangiogenesis increases lym-phatic tumor metastasis (Figure 6) (69, 76–78, 80–82). Tumor lymphangiogenesis andlymphatic metastasis could be efficientlyinhibited by interfering with the ligand-induced VEGFR-3 pathway either by sol-uble VEGFR-3 fusion proteins acting as aVEGF-C/-D trap (55, 79, 83–85), by neutral-

izing VEGFR-3 antibodies (75, 86, 87), bysmall interfering RNA-mediated downregu-lation of VEGF-C (88), and by neutralizingVEGF-D antibodies (77). Inhibition of theVEGF-C/-D/VEGFR-3 pathway does notaffect normal lymphatic vessels in the adultand may thus present a potent and safe methodfor preventing lymphatic tumor metastasis(26). Activated lymphatic vessels in tumors


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VEGF-CVEGF-D

Inhibition of receptor/ligand interaction

Inhibition oftyrosine kinase

signaling

Tyrosine kinaseinhibitors

Anti-VEGFR-3Anti-VEGF-C/-D

Soluble VEGFR-3

a

b

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Figure 6Lymphangiogenesis promotes lymphatic tumor metastasis. (a) Many tumor cells and stromal cells,especially tumor-associated macrophages (stellate red cell), secrete lymphangiogenic growth factors,most importantly VEGF-C and VEGF-D. (b) These stimulate the nearby lymphatic endothelium tosprout toward the tumor, leading to the formation of new lymphatic vessels at the tumor margin and, insome tumor types, occasionally also inside the tumor. (c) The lymphatic vessels in the tumor peripheryfrequently contain clusters of tumor cells, which may be actively entrapped by the sprouting lymphaticendothelium. Tumor-secreted lymphangiogenic factors also stimulate dilation of the preexistinglymphatic vessels draining the tumor area, which leads to increased lymph flow. Furthermore, tumorsmay stimulate lymphangiogenesis and sinusoidal hyperplasia also in the draining lymph nodes alreadybefore the entry of tumor cells (insets in a–c). All these events promote tumor cell dissemination throughthe lymphatic vessels to lymph nodes. From there, further spread to distant organs may occur eitherthrough the efferent lymphatic vessels draining to veins or directly via the blood vessels. Lymphatictumor metastasis may be prevented by blocking the VEGF-C/-D/VEGFR-3 pathway, for example, byinhibiting receptor/ligand interaction or downstream signaling.


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are likely to express specific markers differentfrom normal lymphatic endothelium, whichmay reveal additional specific targets for ther-apy (89, 90).

The VEGF-C-stimulated growth of newtumor-associated lymphatic vessels may elim-inate one rate-limiting step of the metastaticprocess by simply increasing the contact areaof malignant cells with the lymphatic vascu-lature (55, 69, 76–78, 80, 82, 83, 91). The ob-servation that nearby lymphatic endotheliumextensively forms filopodia toward VEGF-C-producing tumor cells suggests that tumoremboli may also be actively entrapped insidethe sprouting lymphatic vessels (55, 92).The enlargement of the existing lymphaticchannels draining the tumor further enhancesthe delivery of tumor cells to sentinel lymphnodes, probably by increasing the lymph flowrate (55, 93). The fact that inhibition of tumormetastasis by soluble VEGFR-3 blocks tumorlymphangiogenesis but does not affect thepreexisting lymphatic vessels suggests thatnewly formed tumor-associated lymphaticvessels or activation of the existing lymphaticvasculature is necessary for lymphatic metas-tasis (55, 83–85). However, some studiessuggest that lymphangiogenesis is unneces-sary and the preexisting lymphatic vesselsare sufficient for metastasis to lymph nodes(70).

VEGF-C might activate the lymphaticendothelium to facilitate tumor cell entryinto the lymphatic vasculature by promot-ing molecular interactions between tumorcells and LECs. This could be mediatedby the secretion of paracrine factors suchas proteases and chemotactic agents by thelymphatic endothelium, which induces de-tachment, migration, or invasion of tumorcells or alteration of the functional proper-ties of the lymphatic endothelium to facili-tate adhesion and intravasation of malignantcells into lymphatic vessels. The chemokineCCL21 secreted by LECs has been shownto promote migration of CCR7-expressingmetastatic malignant melanoma cells (94).Activation of lymphatic endothelium via the

Sentinel lymphnode: the foremostlymph node alongthe lymphatic vesseldraining the tumorand the first reachedby the metastasizingtumor cells

VEGFR-3 pathway is supported by the obser-vation that inhibition of VEGFR-3 signalingmore effectively suppresses lymph node anddistant metastasis than inhibition of VEGFR-2, although either treatment potently sup-presses tumor lymphangiogenesis and block-ade of VEGFR-2 more effectively inhibitstumor angiogenesis and growth (87). Fur-thermore, in several clinicopathological stud-ies, high levels of VEGF-C correlate withlymphatic vessel invasion and lymph nodemetastasis but are not necessarily associatedwith increased lymphatic vessel density. Al-ternatively, VEGF-C might activate the lym-phatic endothelium to produce factors thatpromote survival of malignant cells in thelymphatic system.

Recent experimental studies indicatethat tumors induce lymphangiogenesis inthe sentinel lymph nodes before tumor celldissemination (95, 96). Similar changes wereobserved in the axillary lymph nodes ofbreast cancer patients without evidence ofmetastasis (96). Lymphangiogenic growthfactors secreted by the primary tumor en-hance the growth of lymphatic vessels in andtumor metastasis to the sentinel lymph nodes(97, 98). These observations suggest thattumors start preparing the microenvironmentfor secondary tumors prior to tumor celldissemination. Induction of lymph nodelymphangiogenesis prior to tumor cell arrivalwas suggested to be B cell dependent (95).Lymphangiogenesis in the tumor-draininglymph nodes increases lymph flow, whichmight promote tumor cell transport. Lymphnode lymphangiogenesis might also facilitateextravasation and/or growth of malignantcells in the sentinel lymph nodes.

Although metastasis to distant organs oc-curs via the blood circulation, it might re-quire the initial spread of tumor cells throughthe lymphatic vessels to lymph nodes, fromwhere they metastasize further either throughthe lymphatic system draining into venouscirculation, in which case they would endprimarily in the pulmonary capillaries, ordirectly via the blood vasculature. This is


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supported by the observation that in a chem-ically induced skin carcinoma model, VEGF-C increases tumor metastasis not only to thesentinel lymph nodes but also to distant sites(98). Most importantly, no metastasis in dis-tant organs was observed without lymph nodemetastasis (98). Furthermore, in some cases,lung metastasis can also be inhibited by solu-ble VEGFR-3 (84).

Tumor metastasis is not a random eventbut an extremely complex, highly organized,and tissue-specific process of multiple sequen-tial steps (reviewed in Reference 99). The na-ture of the molecular cues guiding certaintumor types at different stages to metasta-size to specific target organs is being elu-cidated. The vascular endothelium has spe-cific molecular characteristics in each tis-sue, which facilitates tumor cell adhesion andhoming to selected organs (reviewed in Ref-erence 100). Interestingly, the chemokine re-ceptors CCR7 and CXCR4 are highly ex-pressed by at least some human breast can-cer cells, whereas their respective ligands, sec-ondary lymphoid chemokine (SLC/CCL21)and CXCL12, are produced by the lymphaticendothelium in lymph nodes, bone marrow,lung, and liver, which represent the first desti-nations of breast cancer metastasis (101, 102).Inhibition of the CXCL12/CXCR4 interac-tion significantly reduces breast cancer metas-tasis to the lymph nodes and the lung (101).Similarly, expression of CCR7 in murinemelanoma cells promoted metastasis to lymphnodes, which was blocked by neutralizingSLC/CCL21 antibodies (103). These kinds ofmolecular cues could represent a more generalmechanism of guiding tumor cell homing tospecific destinations and might provide a po-tential therapeutic target for the inhibition oftumor progression to a lethal metastatic dis-ease (104).

Lymphedema

Impairment of lymphatic drainage caused bydysfunction of the lymphatic vasculature leadsto interstitial accumulation of proteins and as-

sociated fluid, and finally to lymphedema, achronic progressive swelling of the affectedtissues. Lymphedema can be either hereditaryor with unidentified etiology—in which case itis termed primary lymphedema—or a conse-quence of a disease, trauma, surgery, or radio-therapy when it is referred to as secondary oracquired lymphedema (Figure 7). Commoncomplications of lymphedema include pro-gressive dermal fibrosis, accumulation of adi-pose and connective tissue, impaired woundhealing, decreased immune defense and thusincreased susceptibility to infections, and sub-sequent cellulitis (reviewed in Reference 105).In rare cases, long-term lymphedema canalso place the patient at risk of developinglymphangiosarcoma. Although seldom life-threatening, lymphedema is a disabling anddisfiguring condition severely affecting thequality of life. At the moment, no cure exists.Traditional supporting management of thedisease includes manual lymphatic drainageby physiotherapy, massage, and external com-pression. Recently, preclinical mouse studiesusing viral vectors encoding lymphangiogenicgrowth factors have provided promising re-sults for the development of a prolymphangio-genic therapy (106). The growth of new func-tional lymphatic vessels has been successfullyinduced in a mouse model of lymphedema aswell as in healing diabetic and surgical wounds(29, 107, 108). The VEGFR-3-specific mu-tant form of VEGF-C, VEGF-C156S, is ef-fective in stimulating the growth of lymphaticvessels without having the blood vascular sideeffects such as increase of leakiness causedby wild-type VEGF-C, which also activatesVEGFR-2 (40).

Whereas lymphedema normally affects thebody extremities, chylous ascites and chy-lothorax are rare conditions caused by lymphextravasation as a result of trauma or abnor-mal development of lymphatic vessels leadingto the accumulation of fluid with abundantfatty droplets in the abdomen or thorax.

Primary lymphedema. Although the mostcommon pattern of inheritance of primary


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Normal lymphatic vasculature

Primary lymphedemaLymphatic vascular

hypoplasia or aplasiaLymphedema distichiasis:- Abnormal wall structure- Lack of valves

Secondary lymphedema

InfectionInjury or surgery

Figure 7Lymphedema.Failure in lymphaticvascular function canbe caused by variousdevelopmental oracquired structuraldefects. Hereditarylymphedema can becaused either by totalabsence or severereduction oflymphatic vessels orby abnormallymphatic vesselmorphology.Secondary oracquiredlymphedema isusually due todisruption or traumaof lymphatic vesselsby injury, surgery, orinfection.

lymphedema is autosomal dominant, the pen-etrance is in some cases reduced or variable,suggesting an oligogenic condition or a sub-stantial contribution by modifier genes and/orenvironmental factors (109, 110). Further-more, the classification of lymphedema syn-dromes by phenotypic features does not di-rectly reflect the underlying genetic failure

(111). Primary lymphedemas are estimated toaffect approximately 1 in 6000 people, with asex ratio of approximately one male to threefemales.

Hereditary lymphedema type I, alsoknown as Milroy’s disease or primary congen-ital lymphedema (Online Mendelian Inheri-tance in Man, OMIM, number 153100), is


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an early-onset form of lymphedema, whichbecomes apparent at birth and affects pri-marily the legs and feet. As revealed by lym-phangiography the lymphatic vessels of thesepatients are absent or extremely hypoplasticin the affected areas, but not in the unaf-fected ones (109). Inheritance of this disease,at least in some families, has been linked tothe VEGFR-3 locus in chromosome 5q35.3.Most of the disease-associated alleles con-tain missense mutations encoding tyrosine-kinase-negative VEGFR-3 proteins with ex-tended cellular half-lives, which may alsofunction as dominant negative receptors di-minishing downstream signaling (27, 28).

Hereditary lymphedema type II, alsoknown as Meige disease or lymphedema prae-cox (OMIM 153200), is a late-onset formof primary lymphedema, commonly detectedaround puberty. At least in one family, theinheritance of this disease was linked to aninactivating mutation in the FOXC2 gene(111).

Lymphedema-distichiasis syndrome (LD;OMIM 153400) as well as lymphedemaand yellow nail syndrome (OMIM 153300)are multisymptom disorders characterized bylymphedema of the limbs, with pubertal orvariable age of onset, associated with a varietyof congenital abnormalities. In several fami-lies with LD, inheritance has been linked tomutations in the FOXC2 gene (112). Most ofthese mutations are small insertions or dele-tions leading to a frameshift causing prema-ture, inactivating truncation of the transcrip-tion factor, but two missense single nucleotidesubstitutions have also been identified. In twofamilies, despite linkage to the FOXC2 locus,no mutations in the coding region of FOXC2were found, suggesting a promoter mutation(113).

Individuals with LD have an extra row ofeyelashes, causing corneal irritation. Other as-sociated complications may include cardiacdefects, cleft palate, spinal extradural cysts,varicose veins, and photophobia (114). A re-cent study reported renal disease and diabetesmellitus in combination with LD caused by a

FOXC2 mutation, reflecting the developmen-tal role of FOXC2 in multiple tissues (115).Unlike in congenital lymphedema, wherelymphatic vessels are aplastic or hypoplastic,LD is associated with a normal or even anincreased number of lymphatic vessels, whichhowever display a decreased uptake and refluxof a tracer (116). The uptake of the lymph isprobably hampered by the PCs/vSMCs ab-normally covering the skin lymphatic capil-laries in the legs but interestingly not in theunaffected arms of individuals with LD (61).Furthermore, irregular patterning of the lym-phatic capillaries and increased periendothe-lial deposition of the BM, as well as lack oflymphatic valves observed in Foxc2-deficientmice, in addition to the uncoordinated con-tractility of the vSMCs, might also preventthe uptake and flow of the lymph in LDpatients (61).

An unusual association of lymphedemawith hypotrichosis and telangiectasia (OMIM607823) was recently reported and linkedto mutations in the gene encoding thetranscription factor SOX18 (117). The lo-cus for cholestasis-lymphedema syndrome,also known as Aagenaes syndrome (OMIM214900), has been mapped to chromosome15q, but the gene involved is still unknown(118). The locus responsible for the variablesymptoms of Turner syndrome, frequently as-sociated with lymphedema, has been mappedto region Xp11.2–p22.1, which interestinglycontains the VEGF-D gene (119).

Secondary lymphedema. Postsurgicaledema, especially after mastectomy, repre-sents the primary form of lymphedema inindustrialized countries. Its incidence, ap-proximately 6% to 30% of operated patients,is increased by radiotherapy, but its etiol-ogy and pathophysiology are still not fullyunderstood and appear to be multifactorial(reviewed in Reference 105). Some of thesusceptibility may have a genetic basis.

Worldwide, the most common cause oflymphedema is filariasis, currently affectingover 120 million people, mostly in tropical


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areas. It is caused by mosquito-transmitted in-fection with the parasitic nematodes Wuchere-ria bancrofti, Brugia malayi, or Brugia timori.These parasites live and reproduce in the lym-phatic system, causing a massive dilation oflymphatic vessels and finally a complete andpermanent disruption of lymphatic transport,which leads to a predisposition to long-termrecurrent bacterial infections and a conditionknown as elephantiasis (reviewed in Refer-ence 120). Although several microfilaricidalagents are currently in use, these drugs areonly partially effective against the long-livedadult worms and thus require long-term ap-plication for up to 20 years. Recently, tetracy-clines have been used to target the endosymbi-otic bacteria Wolbachia, which results in filarialgrowth retardation and infertility.

Lymphangiectasia and LymphaticNeoplasms

Lymphangiectasia, local saccular dilation oflymphatic vessels, are visible as translucentvesicles. They are mostly associated with post-surgical lymphedema but can also occur as aconsequence of other local lymphatic damage.Lymphangioma, a benign lymphatic malfor-mation, represents similar symptoms as lym-phangiectasia but has a genetic, mostly inher-ited, cause. Lymphangiosarcomas, malignantlymphatic tumors, are rare and occur mostlyas a consequence of long-lasting lymphedema.Little is known of their molecular geneticbackground.

Kaposi sarcoma, characterized by nod-ules of spindle-shaped tumor cells with aprominent vasculature, is associated withinfection by Kaposi sarcoma herpesvirus(KSHV)/human herpesvirus 8. Kaposi sar-coma spindle cells express both blood andlymphatic endothelial markers and are sug-gested to be of endothelial origin (121). OneKSHV envelope glycoprotein interacts withand activates VEGFR-3 and integrin α3β1,resulting in increased EC growth and migra-tion (122). The transcriptional profile of Ka-posi sarcoma cells closely resembles that of

LAM: lymphangi-oleiomyomatosis

LECs, and furthermore, infection of BECs invitro with KSHV results in the expression ofseveral LEC-specific genes (123, 124).

Lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a rare,progressive, and often fatal cystic lung diseasethat affects women in their reproductive age.It is characterized by the proliferation of ab-normal smooth muscle–like cells (LAM cells)colonizing the lungs and axial lymph nodes,and it is frequently associated with renal an-giomyolipomas (Figure 8). LAM occurs as aconsequence of mutations in one of the tuber-ous sclerosis genes (usually TSC2) and is pre-sumed to depend upon female hormonal stim-uli (reviewed in Reference 125). The LAMcell lesions produce VEGF-C and VEGF-Dand grow along and are closely associated withlymphatic vessels (126). High levels of VEGF-D have been observed in the serum of LAMpatients (127). LAM cell clusters envelopedby LECs are observed frequently in the lym-phatic circulation and chylous fluid of LAMpatients. It is postulated that such shedding ofLAM cell clusters might account for the abil-ity of LAM cells to metastasize to distant sites(128).

Lymphatic Vessels in InflammatoryDiseases

In addition to decreasing inflammation-induced edema, lymphatic vessels activelyregulate inflammatory responses by trans-porting leukocytes from the site of inflamma-tion to secondary lymphoid organs. Severalmolecules have recently been implicated inthe process of leukocyte recirculation throughthe lymphatic system. The chemokine recep-tor CCR7 is essential for the migration ofdendritic cells into afferent lymphatic vessels,which express its ligand SLC/CCL21 (seeReference 130 and references therein). Man-nose receptor 1 and common lymphatic en-dothelial and vascular endothelial receptor 1,


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VEGF-CVEGF-D

PDGF-B

?

Figure 8A hypothetical model for pathogenesis of lymphangioleiomyomatosis (LAM). The origin of theabnormal smooth muscle–like cells (LAM cells, red ) proliferating in the lungs of LAM patients iscurrently unknown. These cells form tumors along the great vessels in the abdominal and lumbarregions, from where they are apparently transported via the thoracic duct into the jugular vein andfurther into the lungs, where they often enter the alveolar spaces and induce cyst formation and fluidaccumulation. LAM cells secrete the lymphangiogenic growth factors VEGF-C and VEGF-D, whichattract lymphatic endothelial cells (LECs; green). An elevated level of PDGF-B has been reported in arelated Gorham’s lymphangiomatosis (129). LECs may produce PDGF-B (61), which may stimulate thegrowth of PDGFR-positive LAM cells, creating a paracrine growth stimulatory loop. This would lead tothe growth of LAM cells along lymphatic vessels, enveloping of LAM cell clusters by LECs, andshedding of these clusters into the lymphatic vessels, which might facilitate the metastatic spread of theLAM cells. Considering the likely paracrine loop in LAM, the patients could profit from therapy withtyrosine kinase inhibitors that block both VEGFR-3 and PDGFR signaling.

both expressed by the lymphatic endothelium,control lymphocyte traffic in lymphatic ves-sels (131, 132).

VEGF-C is upregulated in response toproinflammatory cytokines, suggesting a rolefor the stimulation of lymphatic vessel growthduring inflammation (133). This upregulationpresumably occurs through NFκB, which isan important transcription factor for the sig-nal transduction of proinflammatory factorsand has a putative binding site in the VEGF-Cpromoter (134). Interestingly, NFκB is con-

stitutively active in at least some lymphaticvessels (135).

Macrophages express VEGFR-1 andVEGFR-3 and are thus attracted by angio-genic and lymphangiogenic signals. They alsostimulate further lymphangiogenesis by se-creting VEGF-C and VEGF-D (91, 136,137). Furthermore, recent reports suggestthat macrophages might also contributeto lymphangiogenesis by transdifferentiatinginto LECs and incorporating into the lym-phatic endothelium (8, 9).


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Proliferation of lymphatic vessels has beenreported in human kidney transplants under-going rejection, in a mouse model of chronicairway inflammation, and in corneal mod-els of inflammatory neovascularization (136,138–141). Lymphatic hyperplasia is also ob-served in UVB-irradiation-induced skin in-flammation, in a mouse model of chronicskin inflammation resembling psoriasis, andin human psoriatic skin lesions (142, 143).In kidney transplants, lymphatic vessels con-tribute to the export of the lymphocyte-rich inflammatory infiltrate, but are alsoinvolved in the maintenance of the detri-mental alloreactive immune response by at-tracting CCR7-positive immune cells byproducing SLC/CCL21 (138). Infection ofthe mouse airway epithelia by Mycoplasmapulmonis results in massive lymphangio-genesis induced by VEGF-C/-D-producinginflammatory cells. Inhibition of lymphan-giogenesis by the VEGF-C/-D trap leadsto severe mucosal edema, consistent withthe importance of lymphatic vessels as anexit route for the immune cells and fluid(139).

CONCLUDING REMARKS

Despite the tremendous interest and progressin lymphatic vascular biology during the past

decade and the close resemblance to pro-cesses involved in developmental angiogen-esis, several key players and mechanisms inlymphatic vascular development and matura-tion remain unresolved. Detailed understand-ing of the mechanisms regulating normal andaberrant lymphatic vessel development andthe involvement of lymphatic vessels in var-ious pathological conditions is necessary forthe development of diagnosis and therapiesfor these diseases. The recent elucidation ofimportant mechanisms of blood vessel sprout-ing and recognition of the shared guidancecues in blood vascular and neuronal pattern-ing are likely to shed light on the analo-gous, poorly known processes of lymphaticvascular development. Characterization of thefunctionally and molecularly conserved lym-phatic vessels in model organisms such as ze-brafish and frog may greatly facilitate lym-phatic vascular research in the future (6, 151,152). Markers presently used in the basic re-search may eventually provide diagnostic rel-evance in inflammation and cancer as wellas in acquired and developmental lymphaticvessel disorders. Initial preclinical studies inmouse models have already provided promis-ing results in the inhibition of lymphatic tu-mor metastasis as well as in the stimulation oflymphatic growth in lymphedema and woundhealing.

SUMMARY POINTS

1. Identification of lymphatic-specific markers, discovery of key regulators of lymphaticvessel formation, characterization of animal models, and development of techniquesfor isolation and culture of LECs have greatly facilitated lymphatic vascular researchduring recent years and led to a better understanding of the basic mechanisms oflymphangiogenesis and the significance of the lymphatic vessels in health and disease.

2. Lymphatic development starts after the onset of blood circulation by differentiationof LECs from the venous endothelium and, apparently in some organisms, frommesenchymal progenitor cells.

3. Many tumors secrete lymphangiogenic growth factors and stimulate lymphangiogen-esis at the tumor margin, surrounding tissues, and in sentinel lymph nodes, whichactively promotes lymphatic metastasis.


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4. Tumor lymphangiogenesis and lymphatic metastasis can be effectively inhibited byblocking the VEGF-C/-D/VEGFR-3 signaling pathway in mouse models.

5. Failure in lymphatic vascular function due to a developmental defect or acquireddisruption leads to accumulation of protein-rich fluid and swelling of the affectedtissues.

6. Studies in animal models have provided promising indications for the development ofprolymphangiogenic therapy for the treatment of lymphedema and wound healing.

7. Lymphangiogenesis is stimulated by and involved in the regulation of various inflam-matory conditions.

FUTURE ISSUES

1. Further research is required to elucidate whether and to what extent mesenchymalprecursor cells contribute to the lymphatic endothelium during development and therole of bone marrow–derived cells in adult lymphangiogenesis.

2. The genetic control of lymphatic development and the contribution of physical factorssuch as hydrostatic pressure and interstitial fluid flow should be analyzed in detail tofully understand the processes involved in the formation of the lymphatic vasculature.

3. The detailed molecular and cellular natures of lymphatic vascular remodeling, pat-terning, valve generation, and postnatal maturation remain a challenge for lymphaticvascular research in the future.

4. Deeper knowledge on the complex mechanisms of tumor metastasis is likely to revealadditional targets for efficient inhibition of cancer spread.

5. The exact properties of the various lymphangiogenic factors must be determined todevelop powerful and safe prolymphangiogenic therapies for the treatment of differentforms of lymphedema.

6. Further investigation of the role of lymphatic vessels in inflammation might revealnovel ways to manage inflammatory diseases.

DISCLOSURE STATEMENT

K.A. is a minority shareholder of Lymphatix Ltd. and the Chairman of the Scientific AdvisoryBoard of Vegenics Ltd.

ACKNOWLEDGMENTS

We extend our gratitude to the many colleagues who have contributed significantly to thefield but whose work could not be cited here owing to space limitations. We warmly thankPeter Friedl for valuable discussions, Masabumi Shibuya and Michael Detmar for providingmice and tissues for Figure 4, and Michael Jeltsch for help with computer-related matters. Thework in the authors’ laboratory is supported by the European Union (LymphangiogenomicsLSHG-CT-2004–503573), National Institutes of Health (5 R01 HL075183–02), the Academy


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of Finland (202852 and 204312), the Novo Nordisk Foundation, the Sigrid JuseliusFoundation, and the Finnish Cancer Organizations. Present address of T.K.: Hubrecht In-stitute, Uppsalalaan 8, NL-3584 CT Utrecht, The Netherlands.

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AR335-FM ARI 10 December 2007 18:20

Annual Reviewof Pathology:Mechanismsof Disease

Volume 3, 2008Contents

The Relevance of Research on Red Cell Membranes to theUnderstanding of Complex Human Disease: A Personal PerspectiveVincent T. Marchesi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1

Molecular Mechanisms of Prion PathogenesisAdriano Aguzzi, Christina Sigurdson, and Mathias Heikenwalder � � � � � � � � � � � � � � � � � � � � 11

The Aging BrainBruce A. Yankner, Tao Lu, and Patrick Loerch � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 41

Gene Expression Profiling of Breast CancerMaggie C.U. Cheang, Matt van de Rijn, and Torsten O. Nielsen � � � � � � � � � � � � � � � � � � � � � � 67

The Inflammatory Response to Cell DeathKenneth L. Rock and Hajime Kono � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 99

Molecular Biology and Pathogenesis of Viral MyocarditisMitra Esfandiarei and Bruce M. McManus � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �127

Pancreatic CancerAnirban Maitra and Ralph H. Hruban � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �157

Kidney Transplantation: Mechanisms of Rejection and AcceptanceLynn D. Cornell, R. Neal Smith, and Robert B. Colvin � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �189

Metastatic Cancer CellMarina Bacac and Ivan Stamenkovic � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �221

Pathogenesis of Thrombotic MicroangiopathiesX. Long Zheng and J. Evan Sadler � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �249

Anti-Inflammatory and Proresolving Lipid MediatorsCharles N. Serhan, Stephanie Yacoubian, and Rong Yang � � � � � � � � � � � � � � � � � � � � � � � � � � � � �279

Modeling Morphogenesis and Oncogenesis in Three-DimensionalBreast Epithelial CulturesChristy Hebner, Valerie M. Weaver, and Jayanta Debnath � � � � � � � � � � � � � � � � � � � � � � � � � � � �313

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AR335-FM ARI 10 December 2007 18:20

The Origins of Medulloblastoma SubtypesRichard J. Gilbertson and David W. Ellison � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �341

Molecular Biology and Pathology of LymphangiogenesisTerhi Karpanen and Kari Alitalo � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �367

Endoplasmic Reticulum Stress in Disease PathogenesisJonathan H. Lin, Peter Walter, and T.S. Benedict Yen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �399

Autophagy: Basic Principles and Relevance to DiseaseMondira Kundu and Craig B. Thompson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �427

The Osteoclast: Friend or Foe?Deborah V. Novack and Steven L. Teitelbaum � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �457

Applications of Proteomics to Lab DiagnosisRaghothama Chaerkady and Akhilesh Pandey � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �485

The Pathology of Influenza Virus InfectionsJeffrey K. Taubenberger and David M. Morens � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �499

Airway Smooth Muscle in AsthmaMarc B. Hershenson, Melanie Brown, Blanca Camoretti-Mercado,and Julian Solway � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �523

Molecular Pathobiology of Gastrointestinal Stromal SarcomasChristopher L. Corless and Michael C. Heinrich � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �557

Notch Signaling in LeukemiaJon C. Aster, Warren S. Pear, and Stephen C. Blacklow � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �587

The Role of Hypoxia in Vascular Injury and RepairTony E. Walshe and Patricia A. D’Amore � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �615

Indexes

Cumulative Index of Contributing Authors, Volumes 1–3 � � � � � � � � � � � � � � � � � � � � � � � � � � �645

Cumulative Index of Chapter Titles, Volumes 1–3 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �647

Errata

An online log of corrections to Annual Review of Pathology: Mechanisms of Diseasearticles may be found at http://pathol.annualreviews.org

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molecular biology and pathology of lymphangiogenesis

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