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LYMPHATIC RESEARCH AND BIOLOGYVolume 4, Number 2, 2006© Mary Ann Liebert, Inc.

Lymphatic Endothelial Cells, Lymphangiogenesis, andExtracellular Matrix

RUI-CHENG JI, M.D., Ph.D.

ABSTRACT

Exciting studies involving the molecular regulation of lymphangiogenesis in lymphatic-as-sociated disorders (e.g., wound healing, lymphedema and tumor metastasis) have focused re-newed attention on the intrinsic relationship between lymphatic endothelial cells (LECs) andextracellular matrix (ECM) microenvironment. ECM molecules and remodeling events play akey role in regulating lymphangiogenesis, and the “functionality”-relating molecules, espe-cially hyaluronan, integrins, reelin, IL-7, and matrix metalloproteinases, provide the most fun-damental and critical prerequisite for LEC growth, migration, tube formation, and survival,although lymphangiogenesis is directly or/and indirectly controlled by VEGF-C/-D/VEGFR-3- Prox-1-, Syk/SLP76-, podoplanin/Ang-2/Nrp-2-, FOXC2-, and other signaling pathways inembryonic and pathological processes. New knowledge regarding the differentiation of ini-tial lymphatics should enable improvements in understanding of a variety of cytokines,chemokines, and other factors. The lymphatic colocalization with histochemical staining byusing the novel molecular markers (e.g., LYVE-1), along with subsequent injection techniquewith ferritin or some tracer, will reveal functional and structural features of newly formedand preexisting lymphatics. Growing recognition of the multiple functions of ECM and LECmolecules for important physiological and pathological events may be helpful in identifyingthe crucial changes in tissues subjected to lymph circulation and ultimately in the search forrational therapeutic approaches to prevent lymphatic-associated disorders.

83

INTRODUCTION

THE LYMPHATIC SYSTEM IS ESSENTIAL for main-tenance of normal fluid balance, draining

extracellular fluid from the tissues to blood cir-culation, and providing an exclusive environ-ment in which immune cells can encounter andrespond to foreign antigen in peripheral lym-phoid tissues as an important immunologicaldefense. Architectural characteristics of thelymphoid tissues are the rich reticular fibermeshworks composed of various molecules inextracellular matrix (ECM). Previous studieshave shown an abundant presence of the mainECM components, vitronectin, fibronectin,

laminin, type IV collagen, and types I and IIIcollagen in the reticular fibers of spleen, lymphnodes, and tonsil. Recent findings have dem-onstrated that the interstitial matrix, which oc-cupies the space between the cells, is a very or-dered structure of the tissue. Wrapped aroundthe collagen fibers and perhaps attached tothem are long molecules of hyaluronan.1 TheECM plays an important role in fetal and post-natal tissue development, and in the attraction,differentiation,, and retention of monocytesand macrophages that are able to interact withECM proteins such as laminin and fibronectinvia the expression of specific integrin recep-tors.2 During embryonic development, specific

Department of Anatomy, Biology and Medicine, Oita University Faculty of Medicine, Oita, Japan

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changes can be distinguished in the ECM com-position,3 which fulfill diverse physiologicalroles in addition to providing structural sup-port to tissues. The highly developed bioactivesubstance serves as a medium for cell–cell in-teractions. Moreover, a variety of processes inlymphatic-associated diseases, (e.g., in thelymphedema, wound healing, and tumor me-tastasis) in which lymphangiogenesis is a crit-ical factor to initiate and coordinate the se-quence of events, are closely related with themolecule composition and functional microen-vironment of the ECM.4 As a rapid progress inthe lymphatic field, a better understanding ofhow ECM molecules influence endothelial cellbehaviors will necessitate full insights into howlymphangiogenesis occurs and contributes tolocal lymph drainage in disordered tissues.

Recently, a novel connection between inte-grin-mediated adhesion and critical endothelialcell was suggested to function via VEGFR-3, thetransmembrane receptor for vascular endothe-lial growth factor-C (VEGF-C) and VEGF-D.5 Al-though VEGF-C/-D are primary lymphatic en-dothelial cell (LEC) determinants, the behaviorof LECs is also mediated by signals from extra-cellular environment molecules. The particularlocal ECM environment is assumed to triggerdifferent VEGF receptors, resulting in distinctsignaling pathways that promote lymphangio-genesis. The ECM component feature may thusaffect lymphocyte migration, adhesion, and pro-liferation.6 However, the physiological signifi-cance of the regulation of ECM on the differen-tiation of endothelial cells and on the lymphaticdevelopment remains to be determined. In thiscontext, this review will outline the main cyto-kines, chemokines, and other components relat-ing to ECM and lymphatic growth, and high-light some of the recently published findings inECM and lymphangiogenesis. The advances inour understanding of the lymphatic biology willlead to new applications in the foreseeable fu-ture.

DIFFERENTIATION BETWEEN INITIALLYMPHATICS AND BLOOD VESSELS

During embryonic development, lymphaticsprovide a complementary and distinct path-

way for maintaining the integrity of tissues.Hypoplasia or dysfunction of the lymphaticscan lead to lymphedema, whereas hyperplasiaor abnormal growth of these vessels is associ-ated with lymphangiomas and lymphangiosar-comas. Importantly, renewed attention on themechanisms by which tumor cells enter initiallymphatics has focused on the identification ofinitial lymphatics and blood capillaries in mor-phological and molecular biology, which willprovide a fundamental prerequisite for the in-vestigation of lymphangiogenesis and its rolein lymphatic-related disorders.

The lymphatics act as a one-way transportsystem, in which initial lymphatics are optimallysuited for fluid, protein, particle, and cell uptakefrom the interstitium. Although initial lymphat-ics have many properties in common with bloodcapillaries, they also have distinct morphologi-cal features reflecting their specific functions.Initial lymphatics with blind-ended structuresare composed of a single nonfenestrated endo-thelial layer, showing a wide irregular lumen.The endothelial cells have abundant cell mem-brane invaginations and cytoplasmic vesicles, aswell as obvious overlapping intercellular junc-tions, although interendothelial tight junctionsare usually infrequent.7 The basement mem-brane of blood capillaries is developed with en-circling pericytes and commonly existed tightjunctions. In contrast, initial lymphatics lackmural cells and are characterized by an incom-plete or absent basement membrane. The lym-phatic endothelial cytoskeleton is directly con-nected to adjacent ECM by anchoring filamentscomposed mainly of fine strands of elasticfibers.8 Interestingly, an elastin microfibril pro-tein that is normally expressed in the elastin-richtissues,9 has been found to be selectively andabundantly expressed in LECs.

The transendothelial pathways may be usedas a mechanism for the entry of molecules intolymphatics. The study previously demon-strated that lymphatic endothelium is charac-terized by a high density of anionic sites on cellmembranes, particularly along intercellularjunctions,10 which have been suggested to fa-cilitate movement of small solutes and mole-cules into the lymphatic lumen. The initial lym-phatics may thus have the capacity to removeselectively molecules from the interstitium and

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actively control the composition of lymph andinterstitial fluid. In addition, the intercellularopen junction is necessary for fluid and pro-teins into the lymphatics,7 the entry of whichis driven by pressure gradients across the en-dothelial wall.11

Changes in components and strains of lym-phangiogenic ECM may affect the function ofboth preexisting and newly formed lymphat-ics, which may become actively involved in tu-mor cell chemotaxis, promoting tumor cell in-vasion and dissemination.4 If the activatedinterstitial–lymphatic interface is destroyedand initial lymphatics cannot function, nolymph will be drained from that local regiondespite the baseline systemic drainage forces,12

which seems to inhibit tumor cell migrationthrough the lymphatic endothelial walls in thetumor tissues. However, a complex mechanismin the interaction of intratumoral and peritu-moral interstitial pressure, upregulation ordownregulation of cell adhesion molecules,specific ultrastructure of initial lymphatics willdirectly force the lymph that carries tumor cellsinto regional lymph nodes.13 Recently, the lym-phatic colocalization with histochemical stain-ing by using novel molecular markers, alongwith subsequent intratumoral injection tech-nique with ferritin or some tracer, were widelyemployed to reveal functional and structuralfeatures of newly formed and preexisting lym-phatics.

In tumors, once the metastatic cells reach ad-jacent initial lymphatics, they move along the

external surface of the endothelium, led by finecytoplasmic processes. The cells migrate to-ward and invade into the lumen either throughopen junctions or by inducing the opening ofclosed junctions.14 Intratumoral interstitialfluid pressure increases as tumors increase insize,15 and lymph flow is in the direction of theperitumoral lymphatics; as a result, the inter-stitial fluid volume increases. Anchoring fila-ments keep the initial lymphatics open, evenwhen the peritumoral interstitial fluid volumeand pressure rise. As the interstitium swells,the anchoring filaments not only increase lym-phatic lumen diameters but also pull open theintercellular junctions, producing intercellularclefts. Consequently, it is easy for fluid, parti-cles, and cells to pass into the lymphatic lumen.Once the pressure outside the initial lymphat-ics decreases, these junctions begin to close,preventing retrograde flow into the intersti-tium.11 Therefore, a more extensive range offeatures for initial lymphatics is available to aidin distinguishing blood capillaries (Table 1)(Fig. 1).

The unique structural and functional differ-ences of initial lymphatics from blood capillar-ies indicate a significant discrimination at themolecular expression level.16 The identificationof differentially expressed molecules is criticalto a better understanding of the mechanismsthat modulate lymphatic development andfunction (Table 2). These lymphatic endothelialmarkers mainly include VEGFR-3, a highly glycosylated and relatively stable fms-like

LYMPHATIC ENDOTHELIAL CELLS 85

TABLE 1. MORPHOLOGICAL COMPARISON OF INITIAL LYMPHATICS AND BLOOD CAPILLARIES

Initial lymphatics Blood capillaries

Size (diameters) Uneven (20–120 �m) Even (7–9 �m)Lumen (section) Irregular Regular (elliptical)Network organization Loose (100–800 �m) Dense (10–40 �m)Cell outline (boundary) Oak-leaf-like (wavelike) Spindle-shaped (straight-shaped)Intercellular junctions Zonula adherens (loose) Zonula occludens (tight)Overlapping junctions Present AbsentEnd-to-end junctions Present ScantInterdigitating junctions Present ScantMarginal fold Present AbsentCytoplasmic vesicles Abundant ScantInvaginations Abundant ScantFenestration None PresentWeibel–Palade body Infrequent PresentBasal lamina Absent or undeveloped Developed (continued)Pericytes None Present

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tyrosine kinase receptor for VEGF-C/-D;17

podoplanin, a glomerular podocyte membranemucoprotein;18 Prox-1, the homeobox geneproduct;19 and LYVE-1, a lymphatic endothe-lial receptor for ECM/lymph fluid glycosami-noglycan hyaluronan.20 Furthermore, the paceof research into the LEC continues to acceler-ate with the availability of new molecularmarkers (e.g., LyP-1 molecular marker of tu-mor lymphatics,21 Nrp2 coreceptor of VEGF-Cin LECs, integrin�9�1 necessary for embry-ologic development of lymphatics.22).The Tie/angiopoietin system and neuropilin-2 may alsobe involved in lymphangiogenesis. In initiallymphatics, constitutive expression of Ang-2by endothelial cells may account for the char-acteristic lack of pericytes in these vessels. Ini-tial lymphatics are further distinguished by thespecific organization of its intercellular junc-tions, including the presence of adherens junc-tions.23 Several genes encoding proteins thatconstitute adherens junctions were identified,such as plakophillin 2, H-cadherin, and zonaoccludens 2.24–26

Classification of the differentially expressedgenes into functional groups revealed thatLECs express remarkably high levels of genesimplicated in protein metabolism, sorting, andtrafficking, indicating a more active role of lym-phatic endothelium in uptake and transport ofmolecules than previously anticipated.16 Par-ticularly highly represented were genes en-coding proteins that control specificity of vesi-cle targeting and fusion, such as proteins of theSNARE family, rab GTPases, AAA ATPases,and sec-related proteins,27,28 and hematopoi-etic signaling proteins for regulating blood vas-cular and lymphatic separation,29 such as SLP-76 and Syk, indicating pronounced vesicular

transport in LECs and separating ability in lym-phatic development. 5�-nucleotidase (5�-Nase)is recognized as a maturation marker (differ-entiation antigen CD73) for both T and B lym-phocytes. 5�-Nase enzyme expression dependshighly on the physiological state of cells, its ac-tivity is much more strong in the LECs thanBECs.30–33 The ecto-5�-Nase may play a role inthe interaction of activated cells with either the ECM (especially laminin and fibronectin)and/or with other cells.34 The application of 5�-Nase antibody may provide further informa-tion on endothelial cell-ECM interaction.35

The successful use of lymphatic-specific anti-bodies to receptors for the isolation and purifi-cation of primary LECs represents a major ad-vance in lymphatic biology. The LECs fromhuman cultured cells and dermal cell suspen-sions were established by immunoselection with antibodies to podoplanin, LYVE-1, andVEGFR3.16,36,37 Two cell lineages were demon-strated to express distinct sets of endothelialmarkers and respond differently to growth fac-tors and ECM components. These LECs, whichare defined as LYVE-1�/podoplanin�/CD31�/CD34�/low/CD45�, were distinct from BECs,defined as LYVE-1�/podoplanin�/CD31�/CD34�/CD45�. Phenotypic analyses of LECsindicate these cells also express the adherensjunction adhesion molecule VE-cadherin andsynthesize the leukocyte chemoattractants SLC(CCL21) and MIP-3 (CCL20, Exodus), which, respectively, induce the migration of CCR7-positive and CCR6-positive lymphocytes anddendritic cells to the T-cell areas of lymphnodes.36,38,39 The studies in vitro demonstratedthat LECs maintained expression of their char-acteristic markers in cultured condition. How-ever, the slightly different expression in distinct


FIG. 1. Morphological and histochemical features of lymphatics in tissues of the monkey (a–i) and mouse (j). (a, b)The pleural lymphatic networks in the whole mount preparation and the irregular uterine lymphatic vessel in thecryosection are stained with 5�-Nase-lead medium, showing strong highlights under the observation of a backscat-tered electron image-scanning electron microscope. (c–f) The lymphatic endothelial cells represent typical intercellu-lar junctions [i.e., end-to-end (c), overlapping (d), interdigitating (e), and open (f) junctions in the urinary bladder(c–e) and uterine endometrium (f, 5�-Nase-cerium staining). (g, h) 5�-Nase-cerium reaction product is evenly distrib-uted in the intrinsic lymphatics of the urinary bladder (g) and the collecting lymphatics of falciform ligament (h), butnot found in the blood vascular endothelial cells (g). Note the two-flap (arrows) and bunch valves (arrowheads) in thecollecting lymphatic vessel (h). (i, j) The immunogold reactive particles of VEGFR-3 are located in the lymphatic en-dothelial cells, mainly on the luminal and abluminal surfaces, in the developing gastric wall (i, without silver en-hancement) and wound healing skin (j, gold-silver double treatment). L, lymphatic; B, blood vessel. Bars � 100 �m(a); 10 �m (b); 5 �m (h); 1 �m (f, g); 0.5 �m (c, d, e, i, j).

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LEC subpopulations may come from differentselecting strategies and sources of the tissuesused (i.e., neonatal vs. adult skins), as well asdifferent cultured passages. It is becoming clearthat multiple molecules are necessary to ensureproper differentiation and patterning of LECsinto a functional lymphatic system.40

Lymphatics and blood vessels provide com-plimentary but distinct pathways for main-taining the integrity of tissues.22,41 LECs andBECs appear to be distinct populations thatseparately serve in lymphangiogenesis and an-giogenesis. Lymphangiogenic factors and ECMare two major contributors to a series of LECfunctions. The identification of a large numberof genes selectively expressed by LECs shouldfacilitate the discovery of hitherto unknownlymphatic markers and provide a basis for theanalysis of the molecular mechanisms ac-counting for the characteristic functions of ini-tial lymphatics.16 Evidently, improved distin-guishing between the two capillary types iscrucial for addressing questions regarding thepathology of the lymphatic system. In this re-spect, clear localization and structure of initiallymphatics, which can also be demonstrated byother immunohistochemical staining antibod-ies,42–49 is necessary for analyzing local envi-ronment changes in lymphatic-relating dis-eased tissues (Fig, 2; Table 2). It is anticipatedthat biochemical analysis of primary LECs, par-ticularly the application of cDNA microarray,quantitative real-time RT-PCR, OctoChromefluorescence in situ hybridization, and gene-chip technologies for RNA profiling, might fur-ther reveal the molecular features that distin-guish initial lymphatics from blood capillaries.

ENVIRONMENT FORLYMPHANGIOGENESIS

Lymphangiogenesis is a term that can be em-ployed to designate the embryonic and post-natal development, and proliferation of newlymphatics by sprouting from veins or/and denovo from lymphangioblasts by extension fromthe preexisting vessels.4,22,50–55 The primarylymphatic networks continue to develop into afunctional lymphatic system through theirbudding, sprouting, and remodeling. A well-

balanced process in embryogenesis is impor-tant to promote primary lymphatic network aswell as adequate initial lymphatics from de-veloping organs, to serve as a functional re-quirement for lymph drainage in the region.32

In pathological disorders, lymphangiogenesiswhich frequently occurs in wound healing, in-flammation, lymphedema, and in the estab-lishment and spread of malignant tumors, caninduce their own lymph absorption from thepreexisting lymphatics in a route that is closeto normal lymphangiogenesis.4 Lymphangio-genesis is thus a central element in a variety ofphysiological and pathological processes to ini-tiate and coordinate the sequence of events.

Like the process of angiogenesis,56 lym-phangiogenesis might be stimulated by a shiftin the balance between lymphangiogenic andantilymphangiogenic factors and depends onspecific molecular interactions between theLECs and their surrounding ECM. The endo-thelial cells need to receive appropriate signalsfrom the changing microenvironment. Severalimportant molecular regulators have been implicated in lymphangiogenesis, includingVEGF-C/-D and their receptor VEGFR-3, andProx1, FOXC2, Tie2/Ang2, podoplanin, LYVE-1, and basic fibroblast growth factor (bFGF viaVEGF-C).57–60 VEGF-C is perhaps the most im-portant of prolymphangiogenic cytokine be-cause of its ability to regulate most of the stepsin the lymphangiogenic cascade. In embryoge-nesis, VEGF-C works as a paracrine signal toestablish a concentration gradient towardwhich the first LECs migrate to form the lymphsac.61 Study of cell cultures indicated thatVEGF-C selectively promoted survival andtube formation of LECs. Interestingly, in the ab-sence of exogenous growth factors, LECs in-corporated into collagen type I scaffolds ex-hibited a significantly higher survival rate thanBECs. This difference in response to collagentype I may reflect differences in the type of theECM that each vessel type is exposed to in itsnatural environment.16 In a mouse model oflymphedema, the VEGFR-3-specific mutantform of VEGF-C called VEGF-C156S lacksblood vascular side effects but is sufficient for therapeutic lymphangiogenesis. As VEGF-C156S is a specific lymphatic endothelialgrowth factor in the skin, it provides an at-


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tractive molecule for prolymphangiogenictherapy.62 However, VEGF-C does not enhanceLEC migration through a proteolytically sensi-tive ECM, although in vivo it may do so indi-rectly through the recruitment of proteolyti-

cally active macrophages.63 Consistent with theprevious observations, recent findings indi-cated that excess VEGF-C does not augment thephysiological rate of migration or functional-ity, and by itself cannot sustain any lasting ef-

JI90

FIG. 2. Immunohistochemical staining in paraffin sections of diabetic (a–d) and tumor-model mice (e, f). (a, b) LYVE-1-expressing lymphatic compartments are shown in the thymus, which increase and extend from cortex to medullaas the insulitis progresses. (b) is further magnification of the (a). (c, d) The strong expression of LYVE-1 appears inthe medullary sinus of the lymph node (c) and in the inter- or/and intralobular lymphatics of the pancreas (d). (e, f)In the hybridoma metastatic pancreas, the lymphatic vessels containing a cluster of tumor cells show both the ex-pressions of LYVE-1 (e) and podoplanin (f) in the serial sections. Bars � 50 �m

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fects on lymphatic size, density, or organiza-tion in regenerating adult skin, although it mayenhance early LEC proliferation and cause lym-phatic hyperplasia.64 These studies highlightimportant differences in the relative roles ofVEGF-C versus matrix proteolysis and fluidchannel formation in directly inducing LEC mi-gration.

In addition, the first stage of commitment tolymphatic lineage is marked by cells express-ing LYVE-1, VEGFR-3, and Prox-1, which startbuds, again in a polarized fashion.40 The home-odomain transcription factor Prox-1, an impor-tant LEC fate-determining factor, can induceLEC-specific gene transcription even in BECs.This suggests that the distinct phenotypes ofcells in the adult vascular endothelium areplastic and sensitive to transcriptional repro-gramming, which might be useful for futuretherapeutic applications involving endothelialcells.62 In mice with heterozygous and ho-mozygous disruptions of the T1�/podoplaningene, lymph transport was impaired and de-tectable cutaneous lymphedema occurred inthe lower limbs,65 indicating podoplanin mightinvolved in lymphatic patterning and matura-tion. FOXC2, which is mutated in the humanhereditary disease lymphedema–distichiasis,controls the late stages of lymphatic develop-ment.66 FOXC2 is highly expressed in all de-veloping lymphatics, but in adults the highestexpression is observed in the endothelial cellsof lymphatic valves.60,66,67 Mice lacking Ang-2also exhibit major lymphatic defects in the re-modeling and function.68 Taken together, it isquite evident that some key molecular regula-tors or genes tell LECs to sprout or not. Prox-1, VEGF-C, Syk, and SLP-76 are involved in polarization, budding and sprouting, and sep-aration to form lymph sac, respectively. The de-veloping lymphatics continue their remodelingand maturation through the regulation ofpodoplanin, Ang-2 and Nrp-2.40,57,68 Indeed,the apparent lymphangiogenic property ofmany other cytokines and chemokines (e.g.,FGF-2) may be mediated by VEGF-C/-D.36,69

In lymphatic-associated disorders, moreover,far from these factors will participate in envi-ronment-fitting lymphangiogenesis.

It is generally accepted that a preexistingblood vascular bed may be necessary to guide

lymphangiogenesis. In embryonic tissues, lym-phatics develop shortly after blood vessels andmight share a common origin with the latter,22

although new ECM produced and realigned atappropriate sites, provides the structural in-tegrity and participates in several key eventssuch as cell migration and proliferation for bothangiogenesis and lymphangiogenesis.

Histochemical studies demonstrated that theblood vessels often accompany the lymphaticnetworks, but the structures of both vessels andthe ratio of lymphatics to blood vessels variesdepending on tissue type and function.7,32 It isquite evident that an abundant and neighbor-ing blood supply provides essential nourish-ment for slender lymphatic walls to have ade-quate intrinsic contractility and ability toregenerate rapidly when required, processesessential for maintaining fluid balance.51 Lackof accompanying lymphatic growth would re-sult in increasing tissue edema. Spatio-tempo-ral studies showed that new blood vessels werefollowed by lymphatics and that the lymphat-ics grew alongside new veins.70 In transgenicmice, VEGF-A promoted wound-associatedlymphatic formation through VEGFR-2 signal-ing. The growth of newly formed lymphaticsinto the wound granulation tissue occurred 1week later than the sprouting of blood ves-sels.71 Recently, an animal experiment of the tumor transfected by human lung cancer demonstrated that angiogenesis has alwayspreceded lymphangiogenesis.72 The preexist-ing lymphatics have been supposed to have animportant role in guiding the lymphaticgrowth, however, the experiments in vitro sug-gested that LECs have the ability to proliferate,migrate, and invade to form a new vessel in theabsence of a preexisting lymphatic matrix.36,66

These observations and others have led to thebelief that a preexisting blood vascular bedmay be necessary to guide lymphangiogene-sis.69 Nevertheless, the morphological, spatial,and temporal aspects of lymphangiogenesis re-main poorly understood. The lymphatic andblood vascular systems might be coordinatelyregulated by different molecules in theirgrowth and development because they arefunctionally interconnected and act together tomaintain fluid homeostasis in tissues and cellnutrition.


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ECM has a mechanical role in supportingand maintaining tissue architecture but canalso be described as a dynamic meshwork ac-tively regulating critical cellular functions suchas migration, survival, proliferation, and dif-ferentiation.73 However, less attention has beenpaid to the role of insoluble ECM molecules incontrolling lymphangiogenesis. In an in vitroexperiment, T1�/podoplanin-overexpressingmurine hemangioendothelioma-derived cellclones showed a significantly increased abilityto form tube-like structures after plating on toMatrigel.65 In order to analyze the propertiesof human LECs, dermal microvascular endo-thelial cells were transfected with a retroviruscontaining the coding region of telomerase re-verse transcriptase.74 During invasion and tubeformation, however, endothelial cells degradetheir basement membrane, and proliferate andmigrate into the surrounding collagen-rich ma-trix. This complex process requires the coordi-nated activities of many different molecules, including VEGFRs, integrins, and matrix-degrading proteinases. During lymphangio-genesis, the endothelial cells need to interactwith matrices composed of changing molecu-lar components. To accomplish this, endothe-lial cells use different sets of matrix-recogniz-ing receptor molecules, such as integrins,75,76

that mediate signals from specific ECM com-ponents. For example, endothelial cells upreg-ulate expression of �V�1 that binds to severalmatrix molecules within the interstitial matrix.5Moreover, an important role of keratinocytesand fibroblasts, which constitutively secreteVEGF in vitro,77 should not be neglected in theregulation of lymphangiogenesis in the skinhomeostasis and disease.

Temporal and spatial regulation of ECM re-modeling is not only a critical event in the for-mation and stability of newly formed lym-phatic networks but is also involved in lymphnode metastasis of tumors. Far beyond theknowledge of lymphangiogenesis, the signifi-cance of endothelial cell–matrix interactionshas been supported by several ongoing clinicaltrials that analyze the effects of drugs block-ing this interaction on angiogenesis-dependentgrowth of human tumors.56 Recent experimen-tal evidence on the important influence of lym-phangiogenetic growth factors on intralym-

phatic cancer growth and metastasis raiseshopes that these factors could serve as an ad-ditional target for possible lymphatic-associ-ated tumor therapy.78 The pertinent questionactually is how newly formed lymphatics in-terplay with ECM microenvironment. One pos-sible explanation is that organ-specific struc-ture-functional property of the LECs gaugestheir response to survival and proliferative fac-tors. Antilymphangiogenic factors might blockendothelial cell–receptor interactions, inhibitthe activity of lymphangiogenic factors, and in-terfere with ECM remodeling. Obviously, theidentification of the specific mechanisms regu-lating LEC–ECM interactions during embry-onic and pathological lymphatic growth maylead to novel therapeutic applications for dis-eases characterized by excessive or insufficientlymphangiogenesis. The following sectionswill be given in more details to gain a betterunderstanding of the LEC-ECM interaction.

HYALURONAN AND LYMPHATICS

The recent progress in lymphatic biologygreatly promotes the understanding of molec-ular players involved in the uptake and degra-dation of dissolved macromolecules and lym-phangiogenesis. Hyaluronan, a high-molecularweight and cell-surface glycosaminoglycan, isone of the major components of the ECM andplays an important role in maintaining tissueintegrity, as well as in facilitating the adhesion,migration, and differentiation of cells duringinflammation, wound repair, and embryonicdevelopment.55,79,80 Unlike most ECM compo-nents in the tissues, however, hyaluronan un-dergoes rapid metabolic turnover, with a half-life of approximately 24 hours.79 Tissuehyaluronan is released from the ECM, de-graded within distant lymph nodes via drain-ing afferent lymph, and removed through ef-ferent lymphatics for terminal hydrolysiswithin the liver sinusoids.55,81,82 The presenceof hyaluronan in lymph and its circulationthrough the lymphatics is interesting in viewof the role of this glycosaminoglycan as a sub-stratum for leukocyte and tumor cell traffick-ing in perivascular tissues,83 particularly inpathological states of lymphangiodysplasia or

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lymph damage.84 With implant of a biocom-patible device capable of guiding lymphaticproliferation in patients, micropatterned sur-faces of hyaluronan proved to orient the LECgrowth adequately, allowing the regenerationin the desired direction.85 Hyaluronan in bloodand lymph at very low levels (520 ng/ml) riseafter increased flux in infection, injury, or neoplasia, or when blockage of the lymphaticvasculature prevents transit of hyaluronan tothe lymph nodes during lymphedema.55,86

Hyaluronan promotes tumor cell adhesion tolaminin and may thereby facilitate invasion ofthe basement membrane and metastasis incolon carcinoma.87 High tumoral cytosolichyaluronan level is suggested to be associatedwith lesions of unfavorable outcome in gastriccancer patients,88,89 involving in the growthand progression of malignant tumors. Inhibi-tion of hyaluronan synthases by transfectionwith antisense cDNA or removal of hyaluro-nan by digestion with hyaluronidase wouldimpair adhesion and prevent tumor metastasis,as well as avoid inflammatory insults.

LYVE-1, the lymphatic vascular endothelialhyaluronan receptor-1, has been widely ap-plied in the study of embryonic and patholog-ical lymphangiogenesis. The lymphatics, whichalso transport CD44�ve leukocytes, expresstheir own hyaluronan receptor, the distinct, butrelated molecule.20,90 Being a member of theLink protein family whose only other majorhyaluronan receptor is directly involved inleukocyte migration and tumor metastasis,LYVE-1, expressed on both the luminal and ab-luminal surfaces of the LECs, might be in-volved in either blocking or facilitating cell ad-hesion, entry, or migration in lymphatics andtrafficking of cells within lymph nodes.55 It isclearly possible that LYVE-1 functions primar-ily as an hyaluronan transporter, although suchtransport does not appear to be required inlymphatics due to their free permeability na-ture to macromolecules. The hyaluronan-de-pendent transport may be critical in lymphnodes, and in liver or spleen sinusoids, whereactive uptake and hyaluronan degradation oc-cur. Interestingly, in the thymus of nonobesediabetic mice, the most remarkable biologicalfeature is occurrence of LYVE-1-positive lym-phatic compartments (Fig. 2), which are packed

with CD4- and CD8-positive T lymphocytes.These LYVE-1-expressing lymphatics increasein number and size, and extend from the cor-ticomedullary boundary into the medulla withage (unpublished data). In the autoimmunedisorder, nevertheless, the alteration may as-sess a thymic dysfunction, which ultimatelyleads to abnormal autoreactive regulatory oreffector T cells. Recent studies in vitro indicatedthat a chimeric form of cartilage oligomeric ma-trix protein-Ang-1, promotes LYVE-1-positivelymphatic formation in mouse cornea, andstimulates colony formation of CD45�CD31�

CD34�/lowLYVE-1� LECs.91 However, the cellanalysis in LYVE-1-defined microenviron-ments and their interaction with chemokines,cytokines, and other factors, has not been fullydetermined as yet, especially whether it pos-sesses potent and selective attractant activityfor circulating naive lymphocytes.55

INTEGRINS, ENDOSTATIN, REELIN, IL-7, MMPS, AND LYMPHATICS

Endothelial cell–ECM interactions may pro-vide distinct spatial and molecular signals forproliferation, survival, migration, and differ-entiation of endothelial cells during angiogen-esis and lymphangiogenesis. Integrin-medi-ated cell–matrix interactions are critical for adiversity of cellular processes (e.g., lack of con-tact with the matrix may induce apoptosis).92

Integrins are heterodimeric transmembraneproteins composed 18 known � and 8 � sub-units. They are expressed on endothelial cellsand mediate adhesion to a variety of ECM proteins including vitronectin, fibronectin,laminin, collagen, fibrinogen, and von Wille-brand factor,93 among which �9 has been pre-viously shown to be involved in lymphatic formation.94 In lymphangiogenesis, the en-gagement of integrins is essential for new lym-phatic development in a selective manner, butinvolvement of specific integrins and growthfactor receptors seems important for the pro-cess. The modifications in the ECM compart-ment or in the cellular integrin profile duringthe perinatal period, might affect both the normal differentiation of tissue cells as well asthe migration, retention, and differentiation of


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monocytes and macrophages in the developingtissues.95

Recent progress in endothelial cell biologyhas paved the way for studying the importanceof integrins by using transgenic or gene-knock-out animals. In VEGF-A transgenic mice, thesignificantly enlarged lymphatics were in-creased in numbers within the granulation tis-sue. VEGF-A potently upregulated the expres-sion of the �1 and �2 integrins in culturedhuman LECs, promoting their capacity to formcords and haptotactic migration. Interestingly,blockade of both these integrins in vitro inhib-ited VEGF-A-induced cord formation and LECmigration toward collagen but not toward fi-bronectin.71 Incubation of LECs with an integ-rin�9-specific blocking antibody inhibited hep-tocyte growth factor-induced migration.75 Micehomozygous for a null mutation of the integ-rin�9 subunit die 6–12 days after birth from bi-lateral chylothoraces, suggesting an underlyingdefect in lymphatic development. When em-bryonic fibroblasts of mouse and tumor cells ofhuman were transfected to express �9�1, thesecells adhered and/or migrated on VEGF-C/-Din a concentration-dependent fashion, showingthat VEGF-C/-D are ligands for the integ-rin�9�1.76 The study on the interaction be-tween�9�1 and VEGF-C and/or -D may beforceful to explain the abnormal lymphaticphenotype of the�9 knock-out mice.

Both integrin-mediated and receptor tyro-sine kinase-mediated signaling pathways areinvolved in endothelial cell growth and sur-vival.96 The process for angiogenesis indicatedthat endothelial cell–ECM interactions directlyregulate endothelial behavior either throughreceptor-mediated signaling or by modulatingthe cellular response to growth factors.97 Inte-grins may thus transmit intracellular signalsand activate signaling proteins, includinggrowth factor receptor tyrosine kinases. In lym-phangiogenesis, recent study showed a novelconnection between integrin-mediated adhe-sion and critical endothelial cell functions viaVEGFR-3, which with its ligands are believedto be powerful lymphangiogenic factors. Theintegrin �5�1 subunit may activate bothVEGFR-3 and its downstream PI3 kinase/Aktsignaling pathway, which is essential for fi-

bronectin-mediated LEC survival and prolifer-ation. The fibronectin, vice versa, the ligand for integrin �5�1, was found to transactivateVEGFR-3 and significantly enhanced the phos-phorylation of VEGFR-3 induced by VEGF-C156S as compared to vitronectin.5 Further-more, the interaction of �5�1 or �6�1 on tumorcells, which appropriate levels of integrins,with fibronectin in several tissues is involvedin lymph node metastases.98–100

A good example of the interplay between thedifferent classes of molecules participating inECM recognition and remodeling is the regu-lation of the synthesis and function of the en-dogenous protein endostatin. The polypeptideis distinguished by strong antiangiogenic ac-tivity in vivo restricting the growth of solid tu-mors and metastasis, and the migration of endothelial cells. Endostatin is C-terminal frag-ment of the noncollagenous domains of colla-gen XVIII, a component of the vascular base-ment membranes.101 The cellular signalinginduced by endostatin is characterized by alarge number of individual genetic signals,which are highly coordinated and interdepen-dent.102 Endostatin may exert its function bybinding to endothelial cell surface integrins,such as �V�3 and �5�1,103 or by blocking acti-vation or catalytic activity of endothelial ma-trix metalloproteinases (MMPs), such as MMP-2 and MMP-14.104 Recently, the experiment invitro indicated that endostatin is able to inhibitthe migration and proliferation of LECs, andthe effect on these cells is dose-dependent.105

Reelin, a glycoprotein secreted by Cajal–Ret-zius cells, is a serine protease of the extracellu-lar matrix, which rapidly degrades fibronectinand laminin.106 The immunohistochemicalstaining in human and rat fetus demonstratedthe expression of reelin on LECs, but not onBECs,107 indicating it may contribute to the spe-cial phenotype both during lymphangiogene-sis and in mature lymphatics. Kaposi’s sar-coma-associated herpesvirus infection ofhuman dermal microvascular endothelial cellsinduces significant upregulation of the lym-phatic lineage-specific gene, reelin,108 indicat-ing the origin of Kaposi’s sarcoma from LECs.IL-7 specifically increases the expression oflymphatic markers, LYVE-1, podoplanin, and

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Prox-1 in endothelial cells, and induces the lymphangiogenic properties of breast cancercells in vivo, probably by upregulation ofVEGF-D.109

Degradation of the ECM components byMMPs will greatly influence the LEC–ECM in-teraction. MMP-9, a member of the family ofMMP genes, encodes zinc-dependent enzymesthat break down ECM through the degradationof type IV collagen.110 The prognostic value ofMMP-9 expression by tumor metastasis hasbeen reported in relation to a variety of can-cers.111,112 In an in vitro experiment, the activ-ity of both MMP-2 and MMP-9 produced bycancer cells was expressed by murine LECs, butstrongly inhibited by MMI270, the MMP in-hibitory compound,78 suggesting that MMI270might be involved in tumor metastasis on thebasis of its downregulation in the lymphan-giogenesis-related properties of LECs and theinvasive properties of cancer cells. Attentionshould be directed to the fact that MMPs act asmediators of lymphangiogenesis and tumormetastasis, although there is a great possibilitythat different MMPs serve individual and spe-cific roles during the different stages of the cap-illary or/and metastatic formation.

Indeed, a diversity of cell types and variouslocal ECM environments exist in different tis-sues, therefore, a unique intracellular signalingpathway relating to lymphangiogenic pheno-types, and an intrinsic relationship between theECM protein, molecular regulator on LECs andtumor biology, need to be urgently clarified.

MORPHOGENETIC RESPONSES OF LECS TO INTERSTITIAL FLOW—BIOPHYSICAL ENVIRONMENT

The extent of similarity between the pro-cesses of lymphangiogenesis and angiogenesisis poorly understood, although they sharemany molecular regulators and serve comple-mentary functions. In contrast to the blood cir-culation, the primary function of the lymphaticsystem is to maintain interstitial fluid balanceand provide lymph clearance of interstitialfluid and macromolecules, thereby sustainingosmotic and hydrostatic gradients from blood

capillaries through the interstitium and stimu-lating convection for interstitial protein trans-port. The management and control of tissuefluid balance depends on the highly regulatedorchestration of various interstitial factors. Inparticular, lymphatic function, biology and de-velopment (lymphangiogenesis), and the ECMall contribute to interstitial fluid balance. It isplausible to suggest that an important physio-logical regulating factor of lymphangiogenesismay be the maintenance of interstitial fluid bal-ance and protein convection and, vice versa, thatinterstitial fluid flow may play a role in lym-phangiogenesis.113

It is well accepted that mechanical forces suchas fluid shear and matrix strain take part in reg-ulating blood capillary morphogenesis and en-dothelial cell morphology.114,115 Interstitial flow,as a powerful morphoregulatory stimulant, is afunctionally critical component of the circula-tion, in which high shearing flows are known toinduce endothelial cell remodeling, whereasvery low interstitial flow rates trigger endothe-lial cell morphogenesis in three-dimensional col-lagen gel cultures. The latter are usually in-volved in microvascular organization andstabilization in vitro, promoting the formation oflarge vacuoles with geodesic actin networks andlong dendritic extensions in LECs, and greatlyenhance networking and multicellular tubulo-genesis, particularly in BECs.116 In a regenerat-ing region of skin, the interstitial flow was ob-served to drive the formation of fluid channelsalong which LECs migrate, proliferate, and fi-nally reorganize into a functional lymphatic net-work. Moreover, increased MMP activity in themodel of lymphatic development could lead topreferential cell migration in the direction offlow.117 The improved lymphatic function per-sisted in the VEGF-C treated mice, despite thefact that adenoviral VEGF-C gene expressionwas downregulated and part of the generatedlymphatics regressed during the follow-up pe-riod.118 The lymphatics that become functionalremain stable in the tissue even without contin-uous growth factor stimulation. It is due to thatinterstitial flow is present to some degree in alltissues and, importantly, constitutes the bio-physical environment in tissues undergoinglymphatic development.


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CONCLUSIONS

The lymphatic research in recent years hasrevolutionized the field of cell and molecule bi-ology, with the accelerating emerging aware-ness of the complex interplay between LECsand ECM. Critical and important events re-garding lymphangiogenesis are strongly af-fected by changing ECM microenvironmentand are supposed to play a key role in lym-phatic-associated disorders. The new insightsinto lymphangiogenesis and ECM compositionand organization will lead to a better under-standing of the mechanisms of unique lym-phatic-specific growth factors, cytokines, andchemokines.

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Address reprint requests to:R.C. Ji, M.D., Ph.D.

Division of Morphological AnalysisDepartment of Anatomy, Biology and Medicine

Oita University Faculty of MedicineOita 879-5593, Japan

E-mail: [email protected]

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