Lymphatic vasculature: development, molecular regulation and role in tumor metastasis and inflammation

Review

TRENDS in Immunology

Vol.25 No.7 July 2004

Lymphatic vasculature: development, molecular regulation and role in tumor metastasis and inflammation Pipsa Saharinen, Tuomas Tammela, Marika J. Karkkainen and Kari Alitalo Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Biomedicum Helsinki and Helsinki University Central Hospital, University of Helsinki, P.O.B. 63 (Haartmaninkatu 8), 00014 Helsinki, Finland

The lymphatic vascular system is important for immune surveillance, tissue fluid homeostasis and fat absorption, and is involved in many pathological processes, including tumor metastasis and lymphedema. The recent success in the isolation of lymphatic endothelial cells has shed light on their molecular characteristics. Lymphatic commitment and growth during embryonic development is dependent on the activities of the homeodomain transcription factor Prox-1 and vascular endothelial growth factor-C (VEGF-C). VEGF-C and VEGF-D are involved in adult inflammation-associated lymphangiogenesis, wound healing and tumor metastasis. Administration of lymphangiogenic growth factors or their antagonists provides the possibility of targeting lymphatic vessels in human disease. The lymphatic vasculature complements the blood vascular network, but rather than forming a circulatory system, the lymphatic network comprises a tree-like hierarchy of vessels that transports extravasated fluid and macromolecules unidirectionally from tissues back to the blood circulation [1]. Lymphocytes and antigen-presenting cells (APCs) enter lymphatic capillaries in the periphery and migrate through the lymphatic vessels to the lymph nodes to elicit acquired immune responses in the body. In the small intestine, the lymphatics serve a specialized function in the absorption of dietary fat. The lymphatic capillaries are blind-ended vessels, with a single thin, non-fenestrated lymphatic endothelial-cell (LEC) layer, which is not invested by pericytes (PCs) or smooth muscle cells (SMCs) (Figure 1a). The basement membrane of the lymphatic vessels is incomplete; instead, the LECs are anchored to the extracellular matrix (ECM) through elastic fibers, which keep the vessels from collapsing during changes in interstitial pressure. Characteristic overlapping intercellular junctions serve as valves in the lymphatic capillaries, and an increasing interstitial fluid pressure exerts a pulling force through the anchoring filaments, causing these junctions to open and permitting the uptake of fluid and particles. As the pressure equalizes, the valves close, blocking fluid flow back to the interstitium. The larger collecting lymphatics, however, contain Corresponding author: Kari Alitalo ([email protected]).

perivascular SMCs with an intrinsic contractile function for propulsion of lymph within the vessels.

Lymphatic commitment, sprouting and growth The development of the lymphatic vasculature during embryogenesis lags behind that of the blood vessels, suggesting that these processes are regulated by different signals [2]. A century ago, Florence Sabin had already proposed a widely accepted theory concerning the venous origin of the lymphatic vasculature [3]. Recent studies of mice deficient in the homeobox transcription factor Prox-1 or vascular endothelial growth factor C (VEGF-C) support this model [4,5] (Figure 2). Prox-1 is first detected at embryonic day (E) 10.5 in a polarized manner in a subset of endothelial cells (ECs) in the cardinal veins. Prox-1þ ECs normally bud from veins, giving rise to primitive lymph sacs, initially in the jugular and mesonephric regions. In Prox1 2/2 mouse embryos, this sprouting is arrested [4]. Prox1 2/2 venous ECs lack LEC markers, such as the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) or secondary lymphoid tissue chemokine (SLC), but do express low levels of the VEGF receptor-3 (VEGFR-3, the receptor for VEGF-C and VEGF-D), and contain blood endothelial cell (BEC) markers, including laminin and CD34 [6]. Vegfc 2/2 embryos lack the primitive lymph sacs and all other lymphatic vessels, dying after E15.5 [5]. A closer analysis shows that the Prox-1þ venous ECs of Vegfc 2/2 embryos do not sprout, but remain associated with the cardinal vein and later disappear, possibly by apoptosis [5]. VEGF-C-containing microbeads inserted into whole mount explants of the knockout embryos induced the migration of the Prox-1þ venous ECs, whereas VEGF did not [5]. These results suggest that Prox-1 activity is required for the commitment of the venous ECs to lymphatic differentiation, whereas VEGF-C provides the essential signals through VEGFR-3 for the sprouting to occur (Figure 2). The secondary lymphoid organs, such as lymph nodes and Peyer’s patches of the small intestine, are superimposed on the lymphatic vessels. The lymph nodes initially develop from the primitive lymph sacs formed by migrating Prox-1þ ECs [7]. At , E12.5 – 13.5, connective tissue starts to protrude into the sprouting lymph sacs

www.sciencedirect.com 1471-4906/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.it.2004.05.003

Review

388

TRENDS in Immunology

(a) Lymphatic vessel

Blood vessel PC

EC BM Valve-like structures DC

Lymphocyte Anchoring filaments (b)

(c)

(d)

(e)

TRENDS in Immunology

Figure 1. Visualization of the lymphatic and blood capillaries. (a) Presentation of blood and lymphatic vessels. The lymphatic capillaries are characterized by a single, non-fenestrated endothelial cell (EC) layer, which is not invested by pericytes (PCs), and compared to blood capillaries, contains a more irregular and wider lumen. The EC –EC junctions are tight in blood vessels, whereas the lymphatic capillaries have intercellular valve-like structures that facilitate the uptake of fluid, particles and immune system cells, including dendritic cells (DCs) and lymphocytes. Lymphatic vessels either lack or have an incomplete basement membrane (BM), and are anchored to the extracellular matrix through anchoring filaments, which help to preserve the patency of the vessels. (b,c) Confocal microscopic images of lymphatic (green) and/or blood vessels (red) in mouse ear stained in whole mount with fluorochrome conjugated antibodies specific to lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1), respectively. Note the blind ends of lymphatic vessels. (b) Staining of blood and lymphatic vessels in normal mouse ear. (c) Adenoviral expression of vascular endothelial growth factor-C (VEGF-C) in mouse ear induces lymphatic hyperplasia. The lymphatic vessels are stained. (d,e) Whole mount staining of mouse skin with anti-VEGF receptor-3 (anti-VEGFR-3) antibody. (d) Normal mouse skin. (e) Adenoviral expression of VEGF in mouse skin results in the enlargement of lymphatic vessels. Parts (d) and (e) have been reproduced with permission from Ref. [20].

forming the first anlage of the lymph nodes [7]. Thereafter, the secondary lymphoid organs develop sequentially, and are regulated by several genes distinct from those regulating lymphangiogenesis. For example, lymphotoxin signaling is required for the development of lymph nodes and Peyer’s patches, however, in mice lacking the lymphotoxin-b receptor, the lymphatic vessels develop normally [7]. www.sciencedirect.com

Vol.25 No.7 July 2004

Molecular regulation of lymphangiogenesis VEGFR-3 was one of the first LEC-specific cell surface molecules to be characterized. However, before midgestation, VEGFR-3 is also expressed in BECs, and Vegfr3 2/2 embryos die at E9.5 due to abnormal remodeling of the primary vascular plexus [8]. In adults, the expression of VEGFR-3 becomes confined to the lymphatic endothelium [9]. In addition, fenestrated capillaries and veins in certain endocrine organs, as well as monocytes, macrophages and some dendritic cells (DCs), continue to express VEGFR-3 in adults [10 –12]. VEGF-C and VEGF-D, which bind to, and activate, VEGFR-3, stimulate lymphangiogenesis (e.g. in the differentiated chick chorioallantoic membrane and when delivered to mouse skin using adenoviruses or by transgene expression) [13 – 15] (Figure 1b and c). Conversely, inhibition of these ligands by the expression of a soluble VEGFR-3 – Ig fusion protein in mice starting at E15 caused regression of the developing lymphatic vessels by EC apoptosis [16]. The proteolytically processed forms of VEGF-C and VEGF-D also stimulate VEGFR-2 [17,18], and induce blood vessel growth in mouse cornea and skin, possibly as a result of VEGFR-2 activation in BECs [19 – 21]. Interestingly, mouse VEGF-D is unable to activate VEGFR-2 [22]. VEGFR-2 is the main signal transducer for VEGFmediated angiogenesis, and is mainly expressed in BECs, but also in the collecting lymphatic vessels and in lymphatic capillaries undergoing active lymphangiogenesis [13,15]. VEGF stimulates angiogenesis in the chorioallantoic membrane assay and in mouse skin [14]. However, depending on the target tissue, VEGF also induces hyperplastic, large lymphatic vessels [20,23] (Figure 1d and e) and supports the growth of isolated LECs [24]. These studies suggest that, under certain circumstances, VEGF might induce lymphangiogenesis. At least some of the effects of VEGF on the lymphatic vessels might be indirect, secondary to edema or to recruitment of inflammatory cells that produce VEGF-C and VEGF-D [12,25]. In addition to Prox-1, VEGF-C, VEGF-D and VEGFR-3, several molecules are known to be important for later stages of lymphatic development. Mice deficient in angiopoietin 2 (Ang2), a primarily antagonistic ligand for the endothelial-specific receptor tyrosine kinase Tie-2, show defects in the patterning and function of the lymphatic vasculature and develop subcutaneous edema and chylous ascites after birth [26]. Ang1 can rescue these effects, although the abnormal angiogenesis also observed in Ang2 2/2 mice is not corrected [26]. Interestingly, VEGF-C induces Ang2 expression in cultured LECs through VEGFR-2, indicating a possible connection between the VEGF and angiopoietin families during lymphangiogenesis [27]. The vascular expression pattern of neuropilin 2 (NRP2) resembles that of VEGFR-3. It is first expressed in the ECs of embryonic veins and later becomes abundant in the lymphatic endothelium. VEGF-C binds to NRP-2, suggesting that NRP-2 could act as a co-receptor for VEGFR-3 [28]. Nrp2 deficient mice have reduced numbers of small lymphatic vessels and capillaries, which, however, grow back during postnatal life [29]. Similarly, Vegfcþ/2 embryos largely lack lymphatic vessels, but regrowth

Review

TRENDS in Immunology

Lymphatic endothelial cell sprouting

Lymphatic commitment VEGF-C

Lymph sac formation/ lymphangiogenesis

VEGF-C

VEGF-C Lymph sac/ vessel

Induction of migration Jugular vein

389

Vol.25 No.7 July 2004

Jugular vein

Lymphatic markers: Prox-1 VEGFR-3 LYVE-1 Podoplanin SLC

Survival and migration signals

Blood vessel

TRENDS in Immunology

Figure 2. Venous origin of the lymphatic vasculature. As shown by Oliver et al., Prox-1 is expressed in a polarized manner in a subpopulation of endothelial cells (ECs) of the jugular vein at around E10.5 during mouse embryogenesis [2]. These ECs upregulate lymphatic endothelial specific genes, including vascular endothelial growth factor receptor-3 (VEGFR-3), whereas they downregulate blood vessel endothelial specific genes [4]. The committed ECs migrate along a VEGF-C gradient emanating from nearby mesenchymal cells to form primitive lymph sacs [5]. Consecutive formation of the lymphatic vascular system occurs in a centrifugal fashion from the lymph sacs. Abbreviations: LYVE-1, lymphatic vessel endothelial hyaluronan receptor-1; SLC, secondary lymphoid tissue chemokine.

occurs after birth, leaving only cutaneous lymphatics hypoplastic [5]. The mechanism for the reactivation of lymphatic growth is not yet understood. Recent studies on gene-modified mice have revealed novel candidate molecules, which seem to have a role in lymphatic development. Podoplanin is a cell surface glycoprotein first expressed at around E11.0 in Prox1þ ECs [30]. Podoplanin knockout mice have defects in lymphatic, but not blood vessel patterning, show symptoms of lymphedema and die at birth due to respiratory failure [31]. Mice deficient in Net, a member of the Ets transcription factor family, have dilated lymphatic vessels, develop chylous fluid in the thoracic cavity and die after birth [32]. By contrast, mice deficient in the integrin a9 subunit show edema, extravascular lymphocytes surrounding the thoracic duct and other lymphatic vessels, and die shortly after birth [33]. Because integrin b1 can stimulate some degree of VEGFR-3 activation, the integrin a9b1 complex might be involved in lymphatic vessel stabilization [34]. Characteristics of LECs Although the blood vascular endothelium and its tissuespecific heterogeneity have been the subject of several studies, similar studies of the lymphatic endothelium have been hampered by difficulties in isolating LECs from different tissues. Recently, LECs have been purified to homogeneity from the skin microvasculature, using immunomagnetic isolation based on LEC surface markers, such as VEGFR-3, podoplanin or LYVE-1 [24,35 – 38], thus enabling more detailed studies of LECs (Figure 3). VEGF-C, secreted by BECs in microvascular EC co-cultures, acts as a growth and survival factor for LECs [24]. In vivo, VEGF-C secreted by BECs or SMCs can, indeed, be involved in lymphangiogenesis in the vicinity of blood vessels. A VEGFR-3-specific form of VEGF-C, VEGF-C156S, also supports the growth of LECs, but not of BECs, presumably owing to the lack of expression of VEGFR-3 on BECs [24,39]. However, the morphological changes mimicking LEC sprouting require VEGF-C – VEGFR-2 signaling [27]. Unlike VEGF-C, VEGF-C156S does not promote lymphatic vessel www.sciencedirect.com

invasion into the corneal collagen matrix and the vessels generated in transgenic mouse skin with this VEGFR-3 specific factor show sprouting problems [15,19]. This suggests a role for VEGFR-2 in the LEC invasion of connective tissue. VEGFR-3 is expressed by cultured human umbical vein ECs (HUVECs) but not human aortic ECs [27]. VEGFR-3 expression is significantly reduced when HUVECs are grown in spheroid co-culture with SMCs [27]. This is consistent with the cessation of VEGFR-3 expression in venous ECs during embryonic development, when the vessels mature and acquire a SMC or PC coating. N-cadherin, which is important for EC – SMC interactions, is expressed exclusively in BECs [36,38]. Bone morphogenetic protein-2 (Bmp-2), which is one of the factors inducing SMC chemotaxis, is upregulated by VEGFR-2 in BECs, but not in LECs, suggesting that these cell types use different transcriptional programs downstream of VEGFR-2 [27]. Signaling through VEGFR-2 is mediated by multiple signal transduction proteins, such as protein kinase C (PKC) and Fak, and by src homology domain-2-containing proteins phospholipase C-g (PLC-g), phosphoinositide 3-kinase (PI3-K), Shc, and Shp2 [40]. Much less is known about signaling mediated by VEGFR-3. VEGFR-3 induces the PI3-K pathway leading to Akt activation, as well as the MAP (mitogen-activated protein) kinase pathway, including activation of p42/p44 kinases, probably by PKC [24]. Five tyrosyl phosphorylation sites have been identified in the VEGFR-3 carboxyl-terminal tail, including tyrosyl residue 1337, which binds the adaptor proteins Shc and Grb2 [41]. VEGFR-2 forms heterodimers with VEGFR-3 in primary LECs. Y1337 is not phosphorylated in the heterodimers, indicating that VEGFR-3 signaling can be modulated by VEGFR-2 [42]. However, the more detailed signal transduction mechanisms of VEGFR-2 and VEGFR-3 have not yet been compared in LECs and BECs. Thus, the specific signals emanating from these receptors that are potentially responsible for LEC and BEC specific functions, such as tube formation, remain unknown. Gene expression profiles of BECs and LECs have been compared [35– 38] (Table 1). The most prominent

Review

390

TRENDS in Immunology

[O2]

Vol.25 No.7 July 2004

VEGF-C/D VEGF

VEGFR-1 VEGFR-3 Mo

MCP-1

VEGF-B PlGF

VEGF

VEGFR-1

VEGFR-2

DC

VEGF-C VEGF-D

IL-8 IL-6 PECAM-1

NRP-1

VEGFR-3 NRP-2

LYVE-1

ICAM-2

BEC

SLC

MRC-1

PECAM-1 Integrin α9 Podoplanin

LEC

Stat6

Prox-1 Angiogenesis

Net

Lymphangiogenesis TRENDS in Immunology

Figure 3. Molecular characteristics of blood vascular endothelial cells (BECs) and lymphatic endothelial cells (LECs). Vascular endothelial growth factor receptor-C (VEGF-C) is secreted by smooth muscle cells and pericytes but also by BECs, which can recruit LECs to the vicinity of newly formed blood vessels. Hypoxic conditions in tumors induce VEGF expression, and many tumors also produce VEGF-C and VEGF-D, resulting in recruitment of VEGFR-1 and VEGFR-3 positive macrophages (Mo). Macrophages and inflammatory dendritic cells (DCs) secrete VEGFs and other factors that can boost the angiogenic and lymphangiogenic responses, especially because the expression of VEGF and VEGF-C is induced by proinflammatory cytokines [86,87]. VEGFR-2, which binds VEGF, VEGF-C and VEGF-D, is expressed mainly in BECs. VEGFR-1 is expressed in BECs but also in macrophages and monocytes and binds VEGF-B, PlGF and VEGF. VEGFR-3 is expressed mainly in LECs, and VEGF-C- and VEGF-D-induced VEGFR-3 signaling is one of the proximal regulators of lymphangiogenesis. Among members of the VEGF family, VEGF is the main inducer of angiogenesis, which is mediated by VEGFR-1 and VEGFR-2. Neuropilin-2 (NRP-2) binds VEGF-C, whereas NRP-1 functions as a co-receptor for VEGF. BECs express the intercellular adhesion molecule-2 (ICAM-2) and platelet endothelial cell adhesion molecule-1 (PECAM-1) that is also weakly expressed by LECs. BECs also express Stat6 (signal transducer and activator of transcription 6), whereas Prox-1, a homeodomain transcription factor (TF) and Net, an Ets family TF, are specific for LECs. BECs secrete interleukin-6 (IL-6), IL-8 and monocyte chemoattractant protein-1 (MCP-1), whereas LECs secrete secondary lymphoid tissue chemokine (SLC). LECs also express the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), integrin a9 and podoplanin, a mucin-type transmembrane protein originally characterized in kidney podocytes. The mannose receptor C-1 (MRC-1) is highly expressed in macrophages, where it mediates the endocytosis of glycoproteins. MRC-1 is also expressed in LECs, and in tumor associated lymphatic vessels [88]. Abbreviation: PIGF, placental growth factor.

differences were detected in genes coding for proinflammatory cytokines and chemokines, as well as molecules involved in cytoskeletal or cell-matrix interactions [36]. Interleukin-8 (IL-8), IL-6, the IL-4 receptor and CXCR4, the receptor for stromal cell-derived factor-1 (SDF-1), are expressed at higher levels in the BECs, whereas the LECs show increased expression of IL-7 and SDF-1b [36]. The BECs express Stat6 (signal transducer and activator of transcription 6), an IL-4 inducible transcription factor, whereas Prox-1 is specifically expressed in the LECs. b-catenin and plakoglobin, which connect cadherins to the actin cytoskeleton, show specificity for BECs and LECs, respectively, and the whole actin cytoskeleton is differently organized in the two cell types. Integrin a5, which is part of the fibronectin receptor, is mainly expressed in BECs, whereas integrins a1 and a9, which are subunits of the receptors for laminin and collagen and for osteopontin and tenascin, respectively, are expressed in the LECs. Prox-1 seems to function as a master regulator of lymphatic endothelial cell phenotype because adenoviral expression of Prox-1 in the BECs induced a shift in their gene expression pattern towards the LEC phenotype [36,43]. , 20% of the LEC-specific genes were upregulated www.sciencedirect.com

and 40% of the BEC-specific genes were downregulated on Prox-1 expression in the BECs [36]. These results support the conclusion from knockout studies that the default EC phenotype in the absence of Prox-1 is the BEC phenotype, and that Prox-1 expression signifies LEC commitment. The LECs display heterogeneity in regard of the thus far identified lymphatic markers. For example, LYVE-1 is found in many, but not all, VEGFR-3þ dermal microvascular LECs [24]. It is as yet unclear whether the different LEC populations, defined by such expression patterns, represent functionally different cell types. ECs are highly dependent on their microenvironment. For example, high endothelial venules lose their phenotype and assume a flat morphology when the lymph nodes are deprived of afferent lymph [44]. Similar plasticity in EC phenotype can be reflected in the results from the gene profiling studies of cultured ECs, and in vivo studies are required for a better evaluation of these results. Conversely, because ECs tend to loose their molecular characteristics when propagated in culture, the actual number of differentially regulated genes thus far discovered for the cultured LECs and BECs could be an underestimate for in vivo conditions.

Review

TRENDS in Immunology

391

Vol.25 No.7 July 2004

Table 1. LEC- and BEC-specific gene expressiona,b,c

Adhesion and transmembrane molecules

Cytoskeletal proteins

ECM proteins

ECM modulation

Cytokines, chemokines and their receptors

Growth factors and their receptors

Transcription factors and signalling proteins

BEC

LEC

Integrin a5 (-) Integrin b5 ICAM-1, ICAM-2 (-) N-cadherin Selectin P (-), selectin E CD44 (-) Vinculin Claudin 7 Actin, a2 Profilin 2 b-catenin Collagens 8A1, 6A1, 1A2 Laminin, g2, a5 Versican (-) Proteoglycan (-) MMP-1, MMP-14 (-) uPA (-), PAI-1 (-) Cathepsin C IL-8, IL-6 (-) MCP-1 (-) CXCR4, CCRL2 IL-4 receptor VEGF-C, PlGF Axl (-) NRP1 (-) Stat6 (-)

Integrin a9 Integrin a1 Macrophage mannose receptor I LYVE-1 Podoplanin Desmoplakin I and II (þ) Plakoglobin a-actinin-2 associated LIM protein (þ)

Matrix Gla protein Reelin

TIMP-3

IL-7 SDF-1 SLC VEGFR-3 (þ)

Prox-1

a

The expression pattern of genes shown in bold has been confirmed by northern blotting, immunoblotting, immunofluorescence or immunohistochemistry. b Where known, downregulation (-) or upregulation (þ ) of gene expression by Prox-1 is indicated. c Abbreviations: BEC, blood vascular endothelial cell; ECM, extracellular matrix; ICAM-1, intercellular adhesion molecule-1; IL-8, interleukin-8; LEC, lymphatic endothelial cell; LYVE-1, lymphatic vessel endothelial hyaluronan receptor-1; MCP-1, monocyte chemoattractant protein-1; MMP-1, matrix metalloproteinase-1; NRP1, neuropilin 1; PAI-1, plasminogen activator inhibitor-1; PIGF, placental growth factor; SDF-1, stromal cell-derived factor-1; SLC, secondary lymphoid tissue chemokine; Stat6, signal transducer and activator of transcription 6; TIMP-3, tissue inhibitor of metalloproteinases-3; uPA, urokinase-type plasminogen activator; VEGF-C, vascular endothelial growth factor-C.

Dysfunction of lymphatic vessels In lymphedema, the transport capacity of lymphatic vessels is decreased, and fluid accumulates in tissues causing chronic and disabling swelling, tissue fibrosis, adipose degeneration, poor immune function and susceptibility to infections, as well as impaired wound healing [45]. Primary lymphedemas are rare developmental disorders, which can manifest at birth (Milroy’s disease) or at the onset of puberty (Meige’s disease) [46]. It has been estimated that 1:6000 of newborns develop primary lymphedema [47]. In Milroy’s disease, the superficial or subcutaneous lymphatic vessels are usually aplastic or hypoplastic, whereas in other lymphedema syndromes, such as in lymphedema-distichiasis, the microlymphatic network is normal or larger than in healthy controls [48,49]. Secondary lymphedema, caused by damage to the lymphatic vessels due to, for example, radiation therapy, surgery or infections, has a prevalence of ,3-5 million people in the USA. It has been estimated that . 90 million people worldwide suffer from the most common form of lymphedema, caused by filariasis, which can lead to permanent disability due to massive edema and subsequent deformation of the limbs or genitals [45]. Different genetic loci are involved in primary lymphedema syndromes. Several heterozygous VEGFR3 missense mutations have been found in Milroy’s disease, resulting in the expression of an inactive tyrosine kinase [50,51]. Heterozygous Vegfr3 inactivation has been also found in Chy mice, which develop chylous ascites and lymphedema [28]. Mutations in the FOXC2 gene, coding www.sciencedirect.com

for a forkhead family transcription factor, are responsible for the hereditary lymphedema-distichiasis syndrome, and Foxc2 targeted mice have lymphatic abnormalities [52,53]. Dysfunction of SOX18, a transcription factor of the SOX family, has been identified as a cause for hypotrichosis-lymphedema-telangiectasia syndrome in humans [54]. Mutations in the RELN gene coding for reelin, an ECM protein guiding neuronal-cell migration, are associated with autosomal recessive lissencephaly with cerebellar hypoplasia (LCH). In addition to severe brain abnormalities, the affected children also have congenital lymphedema, and accumulation of chylous ascites has been reported [55]. Reelin is expressed by LECs of the dermal microvasculature, supporting a function for reelin outside the nervous system [36,38]. Currently, manual lymph drainage, compression bandages and stockings are used for treatment of lymphedema. VEGF-C gene therapy induced the growth of new lymphatic vessels in the skin of Chy mice, suggesting that some forms of lymphedema might be treated by similar approaches in humans [28]. In addition, recombinant VEGF-C protein induced lymphangiogenesis in a surgical model of lymphedema in the rabbit ear [56]. This improved lymphatic function and reversed the abnormalities in tissue architecture resulting from chronic lymphatic insufficiency. Similarly, use of VEGF-C plasmid in rabbit-ear and mouse-tail lymphedema models resulted in amelioration of lymphatic function and alleviation of the signs of lymphedema [57]. These results from preclinical models are encouraging for the development of lymphangiogenic

392

Review

TRENDS in Immunology

growth factors as therapeutics for the treatment of lymphedema. Furthermore, characterization of other genes involved in the regulation of lymphatic vessel growth and in the pathogenesis of lymphatic dysfunction should give us more insight into the molecular mechanisms of lymphedema. Lymphangiogenesis promotes tumor metastasis The capacity of malignant tumors to metastasize presents a difficult problem for cancer treatment. Lymphatic vessels provide one of the main routes for tumor metastasis, especially for tumors of the breast, lung and gastrointestinal tract, which frequently colonize draining regional lymph nodes. Compared to the blood vasculature, little is known about the biology of the lymphatic vessels in tumors, the regulation of tumor lymphangiogenesis or the mechanisms that determine the interactions of tumor cells with the lymphatic vessels. Although peritumoral lymphatic vessels contribute to tumor metastasis, opposite views exist as to whether intratumoral lymphatics have any role in tumor metastasis [58]. Many human tumors express VEGF-C, and increased VEGF-C expression correlates with lymph node metastasis in, for example, thyroid, prostate, gastric, colorectal and lung cancers [59,60]. In breast cancer, VEGF-C expression correlates with lymph node positive tumors, whereas VEGF-D showed expression predominantly in inflammatory breast carcinoma [61]. The mechanisms regulating VEGF-C or VEGF-D expression in tumors are not fully understood. Although VEGF-C is commonly expressed in cancer, it is not known to what extent tumor cells are directly responsible for the secretion of lymphangiogenic growth factors, such as VEGF-C and VEGF-D [62]. It has been suggested that VEGF-D is expressed during differentiation, but not in actively proliferating cells, because it is negatively regulated by b-catenin, a transcription factor of the Wnt pathway that stimulates cell proliferation [63]. It remains to be analyzed how much inflammatory cells, such as macrophages, contribute to peritumoral lymphangiogenesis. In two studies, tumorassociated macrophages have indeed been associated with tumor lymphangiogenesis [12,64]. Recent studies using various rodent models have provided evidence that tumor lymphangiogenesis facilitates lymphatic metastasis. In a transgenic mouse model, overexpression of VEGF-C in the b-cells of the pancreatic islets increased lymphangiogenesis around the primary tumor and enhanced tumor-cell spread to the draining lymph nodes [65]. Similarly, overexpression of VEGF-C or VEGF-D in orthotopic mouse tumors increased the number of peri- and/or intra-tumoral lymphatic vessels and enhanced metastasis to regional lymph nodes [66– 69]. The secreted, soluble VEGFR-3 – Ig fusion protein produced by transfected human breast or lung carcinoma cells that have a high VEGF-C expression, or delivered by a systemic route using adenoviruses, inhibits tumor lymphangiogenesis and lymph node metastasis in immunodeficient mice, further supporting the role of lymphatics in tumor development [67,70]. Similar results have been obtained using a syngeneic rat tumor model [71]. Tumor growth has also been inhibited to some degree in several www.sciencedirect.com

Vol.25 No.7 July 2004

studies using VEGFR-3 inhibition, suggesting an angiogenic role for VEGFR-3, which is induced in blood vessels in many tumors. VEGF-C and VEGF-D might enhance metastasis by increasing the number of lymphatic vessels, which increases the contact surface area between the invading cancer cells and the lymphatic endothelium. However, there is currently little evidence that lymphatic vessels would proliferate in the major forms of human cancer. Perhaps a more appealing hypothesis would be that activation of the lymphatic endothelium by tumor cellsecreted factors promotes tumor-cell– LEC interactions and increases lymph vessel size, thus facilitating the entry of tumor cells and aggregates into the lymphatics. VEGF-C and VEGF-D can also increase vascular leakage, and the increased tumor interstitial fluid pressure might promote tumor-cell entry to the lymphatics, as well as to the veins. Different tumors have been found to metastasize preferentially to different organs, suggesting that tumor spread is a guided process. For example, breast cancer frequently metastasizes to regional lymph nodes, bone marrow, lung and liver. In some studies, human breast cancer cells were found to express the chemokine receptors CXCR4 and CCR7, and their respective ligands, CXCL12 (SDF-1) and CCL21 (SLC) are highly expressed in the target organs of breast cancer metastasis [72]. Neutralization of the CXCL12– CXCR4 interactions impairs metastasis to regional lymph nodes and lung, indicating that chemokines and their receptors can have a crucial role in determining the metastatic fate of tumor cells [72]. Interestingly, isolated LECs express SDF-1 and SLC, suggesting that they can attract tumor cells through the secretion of chemokines [35,36]. The endothelial surfaces of tumor lymph vessels also seem to have distinct molecular features not shared by normal lymphatics [73]. Interestingly, Kaposi’s sarcoma (KS) tumor cells express lymphatic markers, such as VEGFR-3 and podoplanin, but lack BEC and haematopoietic markers, including PAL-E (Pathologishe Anatomie Leiden-endothelium antigen) and CD45, respectively, suggesting that the KS tumors might have a lymphatic endothelial origin [74,75]. Inflammation stimulates angiogenesis and lymphangiogenesis In adults, wound healing, cancer and inflammation are pathological conditions associated with rapid neovascularization. There is increasing evidence that inflammatory cells have an important role in pathological angiogenesis and lymphangiogenesis [76]. Activated leukocytes secrete several cytokines and other regulatory proteins, such as VEGF. The expression of VEGF is induced by hypoxia, which has an important role in tumor angiogenesis, and by proinflammatory cytokines [77– 79]. VEGF is chemotactic for monocytes and macrophages, which express VEGFR-1, and it directly upregulates adhesion molecules on the vascular endothelium, increasing leukocyte transendothelial migration [80,81]. Macrophages secrete many angiogenic and lymphangiogenic factors, including VEGF-C and VEGF-D [12]. VEGF-C is also chemotactic for macrophages, and its receptor VEGFR-3 is expressed by a fraction of peripheral blood monocytes and activated tissue

Review

TRENDS in Immunology

macrophages [12,64]. Thus, VEGFR-1, and perhaps also VEGFR-3, might have crucial roles in amplification of pathological lymphangiogenesis and angiogenesis. Furthermore, VEGF could indirectly support inflammatory lymphangiogenesis through such a pathway and through the vascular leak it induces. During wound healing the growth of lymphatic vessels lags behind that of blood vessels, and shortly after their formation, the lymphatic vessels tend to regress [82]. Lymphangiogenesis was not detected in chronic wounds, suggesting disturbed VEGFR-3 signaling in an abnormal wound healing process, which might be further impaired by the lack of lymphatic vessels [82]. However, in chronic wounds and in tumor vessels, VEGFR-3 is expressed by the BECs [82,83]. Recently, VEGFR-3 was found to be expressed in corneal DCs [11]. Inflammation increased the expression of VEGFR-3 and induced VEGF-C in DCs, possibly by the secretion of proinflammatory cytokines [11]. During inflammation, the normally avascular cornea is vascularized through the penetration of new blood and lymphatic vessels from the neighboring conjunctiva, and the numbers of corneal DCs increase as a result of infiltrating DCs from the surrounding vasculature [11]. The induction of lymphatic vessels into the cornea should enhance the delivery of DCs to the lymph nodes, which contributes to inflammation (e.g. in the rejection of corneal grafts) [84,85]. Such results suggest a potential role for VEGFR-3 signaling in immunity, by mediating APC trafficking through DC recruitment and/or by promoting the molecular interactions of APCs with LECs, thereby facilitating APC entry into the lymphatics [11]. Concluding remarks Since the discovery of the first LEC-specific molecules less than a decade ago, tremendous progress has been made in understanding the function and development of lymphatic vessels. Prox-1, VEGF-C, VEGF-D and VEGFR-3 are central regulators of lymphatic development. Prox-1 is a determinant of the commitment of LECs in the embryonic cardinal veins; however, it is not known what turns on the polarized Prox-1 expression in this subpopulation of venous ECs. Vegfc 2/2 embryos do not have an obviously abnormal blood vascular phenotype, although the processed form of VEGF-C binds to both VEGFR-3 and VEGFR-2, which are essential for blood vessel development [5]. Endogenous VEGF-D does not rescue the phenotype in Vegfc 2/2 embryos [5]. However, it is possible that VEGF-D is responsible for the activation of VEGFR-3 during vascular remodeling [5]. Recent studies have identified several new LEC-specific molecules, and based on knockout studies, many of these molecules are also functionally important for the lymphatic vasculature. However, recent results suggest that in addition to their role in lymphangiogenesis, VEGF-C, VEGF-D and VEGFR-3 might be involved in other processes, including adaptive immunity. Although VEGFR-3 and its ligands have been studied extensively during embryonic and growth factor-driven lymphangiogenesis, as well as in tumors, knowledge of the function of the www.sciencedirect.com

Vol.25 No.7 July 2004

393

VEGFR-3 signal transduction system in other physiological and pathological processes is only superficial. The studies of VEGF-C and VEGF-D in the treatment of lymphedema and inhibition of these growth factors in preventing tumor-cell spread through lymphatic vessels in animal models are encouraging. The identification of novel functions for the lymphangiogenic factors, for example, in inflammatory diseases and immunity, might yield additional therapeutic uses for these powerful factors. References 1 Witte, M.H. et al. (2001) Lymphangiogenesis and lymphangiodysplasia: from molecular to clinical lymphology. Microsc. Res. Tech. 55, 122 – 145 2 Oliver, G. and Detmar, M. (2002) The rediscovery of the lymphatic system: old and new insights into the development and biological function of the lymphatic vasculature. Genes Dev. 16, 773– 783 3 Sabin, F.R. (1902) On the origin of the lymphatic system from the veins and the development of the lymph hearts and thoracic duct in the pig. Am. J. Anat. 1, 367 – 391 4 Wigle, J.T. and Oliver, G. (1999) Prox1 function is required for the development of the murine lymphatic system. Cell 98, 769– 778 5 Karkkainen, M.J. et al. (2004) Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins. Nat. Immunol. 5, 74 – 80 6 Wigle, J.T. et al. (2002) An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype. EMBO J. 21, 1505 – 1513 7 Mebius, R.E. (2003) Organogenesis of lymphoid tissues. Nat. Rev. Immunol. 3, 292 – 303 8 Dumont, D.J. et al. (1998) Cardiovascular failure in mouse embryos deficient in VEGF receptor-3. Science 282, 946 – 949 9 Kaipainen, A. et al. (1995) Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc. Natl. Acad. Sci. U. S. A. 92, 3566 – 3570 10 Partanen, T.A. et al. (2000) VEGF-C and VEGF-D expression in neuroendocrine cells and their receptor, VEGFR-3, in fenestrated blood vessels in human tissues. FASEB J. 14, 2087– 2096 11 Hamrah, P. et al. (2003) Novel expression of vascular endothelial growth factor receptor (VEGFR)-3 and VEGF-C on corneal dendritic cells. Am. J. Pathol. 163, 57 – 68 12 Schoppmann, S.F. et al. (2002) Tumor-associated macrophages express lymphatic endothelial growth factors and are related to peritumoral lymphangiogenesis. Am. J. Pathol. 161, 947 – 956 13 Jeltsch, M. et al. (1997) Hyperplasia of lymphatic vessels in VEGF-C transgenic mice. Science 276, 1423 – 1425 14 Oh, S.J. et al. (1997) VEGF and VEGF-C: specific induction of angiogenesis and lymphangiogenesis in the differentiated avian chorioallantoic membrane. Dev. Biol. 188, 96– 109 15 Veikkola, T. et al. (2001) Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice. EMBO J. 20, 1223– 1231 16 Makinen, T. et al. (2001) Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3. Nat. Med. 7, 199 – 205 17 Joukov, V. et al. (1997) Proteolytic processing regulates receptor specificity and activity of VEGF-C. EMBO J. 16, 3898– 3911 18 Stacker, S.A. et al. (1999) Biosynthesis of vascular endothelial growth factor-D involves proteolytic processing which generates non-covalent homodimers. J. Biol. Chem. 274, 32127 – 32136 19 Kubo, H. et al. (2002) Blockade of vascular endothelial growth factor receptor-3 signaling inhibits fibroblast growth factor-2-induced lymphangiogenesis in mouse cornea. Proc. Natl. Acad. Sci. U. S. A. 99, 8868– 8873 20 Saaristo, A. et al. (2002) Adenoviral VEGF-C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes. FASEB J. 16, 1041– 1049 21 Rissanen, T.T. et al. (2003) VEGF-D is the strongest angiogenic and lymphangiogenic effector among VEGFs delivered into skeletal muscle via adenoviruses. Circ. Res. 92, 1098– 1106

394

Review

TRENDS in Immunology

22 Baldwin, M.E. (2001) The specificity of receptor binding by vascular endothelial growth factor-D is different in mouse and man. J. Biol. Chem. 276, 19166 – 19171 23 Nagy, J.A. et al. (2002) Vascular permeability factor/vascular endothelial growth factor induces lymphangiogenesis as well as angiogenesis. J. Exp. Med. 196, 1497– 1506 24 Makinen, T. et al. (2001) Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3. EMBO J. 20, 4762 – 4773 25 Cursiefen, C. et al. (2004) VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment. J. Clin. Invest. 113, 1040 – 1050 26 Gale, N.W. et al. (2002) Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by angiopoietin-1. Dev. Cell 3, 411 – 423 27 Veikkola, T. et al. (2003) Intrinsic versus microenvironmental regulation of lymphatic endothelial cell phenotype and function. FASEB J. 17, 2006– 2013 28 Karkkainen, M.J. et al. (2001) A model for gene therapy of human hereditary lymphedema. Proc. Natl. Acad. Sci. U. S. A. 98, 12677 – 12682 29 Yuan, L. et al. (2002) Abnormal lymphatic vessel development in neuropilin 2 mutant mice. Development 129, 4797 – 4806 30 Breiteneder-Geleff, S. et al. (1999) Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium. Am. J. Pathol. 154, 385 – 394 31 Schacht, V. et al. (2003) T1a/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema. EMBO J. 22, 3546 – 3556 32 Ayadi, A. et al. (2001) Net-targeted mutant mice develop a vascular phenotype and up-regulate egr-1. EMBO J. 20, 5139– 5152 33 Huang, X.Z. et al. (2000) Fatal bilateral chylothorax in mice lacking the integrin a9b1. Mol. Cell. Biol. 20, 5208 – 5215 34 Wang, J.F. et al. (2001) Stimulation of b1 integrin induces tyrosine phosphorylation of vascular endothelial growth factor receptor-3 and modulates cell migration. J. Biol. Chem. 276, 41950 – 41957 35 Kriehuber, E. et al. (2001) Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized cell lineages. J. Exp. Med. 194, 797– 808 36 Petrova, T.V. et al. (2002) Lymphatic endothelial reprogramming of vascular endothelial cells by the Prox-1 homeobox transcription factor. EMBO J. 21, 4593 – 4599 37 Podgrabinska, S. et al. (2002) Molecular characterization of lymphatic endothelial cells. Proc. Natl. Acad. Sci. U. S. A. 99, 16069 – 16074 38 Hirakawa, S. et al. (2003) Identification of vascular lineage-specific genes by transcriptional profiling of isolated blood vascular and lymphatic endothelial cells. Am. J. Pathol. 162, 575– 586 39 Joukov, V. et al. (1998) A recombinant mutant vascular endothelial growth factor-C that has lost vascular endothelial growth factor receptor-2 binding, activation, and vascular permeability activitities. J. Biol. Chem. 273, 6599– 6602 40 Cross, M.J. et al. (2003) VEGF-receptor signal transduction. Trends Biochem. Sci. 28, 488 – 494 41 Pajusola, K. et al. (1994) Signalling properties of FLT4, a proteolytically processed receptor tyrosine kinase related to two VEGF receptors. Oncogene 9, 3545– 3555 42 Dixelius, J. et al. (2003) Ligand-induced vascular endothelial growth factor receptor-3 (VEGFR-3) heterodimerization with VEGFR-2 in primary lymphatic endothelial cells regulates tyrosine phosphorylation sites. J. Biol. Chem. 278, 40973 – 40979 43 Hong, Y.K. et al. (2002) Prox1 is a master control gene in the program specifying lymphatic endothelial cell fate. Dev. Dyn. 225, 351 – 357 44 Mebius, R.E. et al. (1991) The influence of afferent lymphatic vessel interruption on vascular addressin expression. J. Cell Biol. 115, 85 – 95 45 Rockson, S.G. (2001) Lymphedema. Am. J. Med. 110, 288 – 295 46 Witte, M.H. et al. (1998) Phenotypic and genotypic heterogeneity in familial Milroy lymphedema. Lymphology 31, 145 – 155 47 Dale, R.F. (1985) The inheritance of primary lymphoedema. J. Med. Genet. 22, 274 – 278 48 Bollinger, A. et al. (1983) Aplasia of superficial lymphatic capillaries in hereditary and connatal lymphedema (Milroy’s disease). Lymphology 16, 27 – 30 www.sciencedirect.com

Vol.25 No.7 July 2004

49 Pfister, G. et al. (1990) Diameters of lymphatic capillaries in patients with different forms of primary lymphedema. Lymphology 23, 140 – 144 50 Karkkainen, M.J. et al. (2000) Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema. Nat. Genet. 25, 153–159 51 Irrthum, A. et al. (2000) Congenital hereditary lymphedema caused by a mutation that inactivates VEGFR3 tyrosine kinase. Am. J. Hum. Genet. 67, 295– 301 52 Fang, J. et al. (2000) Mutations in FOXC2 (MFH-1), a forkhead family transcription factor, are responsible for the hereditary lymphedemadistichiasis syndrome. Am. J. Hum. Genet. 67, 1382 – 1388 53 Kriederman, B.M. et al. (2003) FOXC2 haploinsufficient mice are a model for human autosomal dominant lymphedema-distichiasis syndrome. Hum. Mol. Genet. 12, 1179 – 1185 54 Irrthum, A. et al. (2003) Mutations in the transcription factor gene SOX18 underlie recessive and dominant forms of hypotrichosislymphedema-telangiectasia. Am. J. Hum. Genet. 72, 1470– 1478 55 Hong, S.E. et al. (2000) Autosomal recessive lissencephaly with cerebellar hypoplasia is associated with human RELN mutations. Nat. Genet. 26, 93 – 96 56 Szuba, A. et al. (2002) Therapeutic lymphangiogenesis with human recombinant VEGF-C. FASEB J. 16, 1985 – 1987 57 Yoon, Y.S. et al. (2003) VEGF-C gene therapy augments postnatal lymphangiogenesis and ameliorates secondary lymphedema. J. Clin. Invest. 111, 717 – 725 58 Stacker, S.A. et al. (2002) Lymphangiogenesis and cancer metastasis. Nat. Rev. Cancer 2, 573– 583 59 Akagi, K. et al. (2000) Vascular endothelial growth factor-C (VEGF-C) expression in human colorectal cancer tissues. Br. J. Cancer 83, 887– 891 60 Niki, T. et al. (2000) Expression of vascular endothelial growth factors A, B, C, and D and their relationships to lymph node status in lung adenocarcinoma. Clin. Cancer Res. 6, 2431– 2439 61 Kurebayashi, J. et al. (1999) Expression of vascular endothelial growth factor (VEGF) family members in breast cancer. Jpn. J. Cancer Res. 90, 977– 981 62 Salven, P. et al. (1998) Vascular endothelial growth factors VEGF-B and VEGF-C are expressed in human tumors. Am. J. Pathol. 153, 103– 108 63 Orlandini, M. et al. (2003) b-catenin inversely regulates vascular endothelial growth factor-D mRNA stability. J. Biol. Chem. 278, 44650 – 44656 64 Skobe, M. et al. (2001) Concurrent induction of lymphangiogenesis, angiogenesis, and macrophage recruitment by vascular endothelial growth factor-C in melanoma. Am. J. Pathol. 159, 893 – 903 65 Mandriota, S.J. et al. (2001) Vascular endothelial growth factor-Cmediated lymphangiogenesis promotes tumour metastasis. EMBO J. 20, 672 – 682 66 Skobe, M. et al. (2001) Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat. Med. 7, 192 – 198 67 Karpanen, T. et al. (2001) Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth. Cancer Res. 61, 1786– 1790 68 Stacker, S.A. et al. (2001) VEGF-D promotes the metastatic spread of tumor cells via the lymphatics. Nat. Med. 7, 186 – 191 69 Mattila, M.M. et al. (2002) VEGF-C induced lymphangiogenesis is associated with lymph node metastasis in orthotopic MCF-7 tumors. Int. J. Cancer 98, 946 – 951 70 He, Y. et al. (2002) Suppression of tumor lymphangiogenesis and lymph node metastasis by blocking vascular endothelial growth factor receptor 3 signaling. J. Natl. Cancer Inst 94, 819 – 825 71 Krishnan, J. et al. (2003) Differential in vivo and in vitro expression of vascular endothelial growth factor (VEGF)-C and VEGF-D in tumors and its relationship to lymphatic metastasis in immunocompetent rats. Cancer Res. 63, 713– 722 72 Muller, A. et al. (2001) Involvement of chemokine receptors in breast cancer metastasis. Nature 410, 50 – 56 73 Laakkonen, P. et al. (2002) A tumor-homing peptide with a targeting specificity related to lymphatic vessels. Nat. Med. 8, 751– 755 74 Marchio, S. et al. (1999) Vascular endothelial growth factor-C stimulates the migration and proliferation of Kaposi’s sarcoma cells. J. Biol. Chem. 274, 27617 – 27622 75 Weninger, W. et al. (1999) Expression of vascular endothelial growth

Review

76 77

78

79 80

81

82

TRENDS in Immunology

factor receptor-3 and podoplanin suggests a lymphatic endothelial cell origin of Kaposi’s sarcoma tumor cells. Lab. Invest. 79, 243 – 251 Coussens, L.M. and Werb, Z. (2002) Inflammation and cancer. Nature 420, 860 – 867 Plate, K.H. et al. (1992) Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature 359, 845 – 848 Shweiki, D. et al. (1992) Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 359, 843 – 845 Blouw, B. et al. (2003) The hypoxic response of tumors is dependent on their microenvironment. Cancer Cell 4, 133 – 146 Barleon, B. et al. (1996) Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. Blood 87, 3336 – 3343 Melder, R.J. et al. (1996) During angiogenesis, vascular endothelial growth factor and basic fibroblast growth factor regulate natural killer cell adhesion to tumor endothelium. Nat. Med. 2, 992 – 997 Paavonen, K. et al. (2000) Vascular endothelial growth factor receptor3 in lymphangiogenesis in wound healing. Am. J. Pathol. 156, 1499 – 1504

Vol.25 No.7 July 2004

395

83 Jussila, L. et al. (1998) Lymphatic endothelium and Kaposi’s sarcoma spindle cells detected by antibodies against the vascular endothelial growth factor receptor-3. Cancer Res. 58, 1599– 1604 84 Yamagami, S. and Dana, M.R. (2001) The critical role of lymph nodes in corneal alloimmunization and graft rejection. Invest. Ophthalmol. Vis. Sci. 42, 1293– 1298 85 Yamagami, S. et al. (2002) Draining lymph nodes play an essential role in alloimmunity generated in response to high-risk corneal transplantation. Cornea 21, 405 – 409 86 Enholm, B. et al. (1997) Comparison of VEGF, VEGF-B, VEGF-C and Ang-1 mRNA regulation by serum, growth factors, oncoproteins and hypoxia. Oncogene 14, 2475– 2483 87 Ristimaki, A. et al. (1998) Proinflammatory cytokines regulate expression of the lymphatic endothelial mitogen vascular endothelial growth factor-C. J. Biol. Chem. 273, 8413 – 8418 88 Irjala, H. et al. (2003) Mannose receptor (MR) and common lymphatic endothelial and vascular endothelial receptor (CLEVER)-1 direct the binding of cancer cells to the lymph vessel endothelium. Cancer Res. 63, 4671 – 4676

TI: making the most of your personal subscription High-quality printouts (from PDF files) Links to other articles, other journals and cited software and databases All you have to do is: Obtain your subscription key from the address label of your print subscription. Then go to http://www.trends.com, click on the Claim online access button and select Trends in Immunology. You will see a BioMedNet login screen. Enter your BioMedNet username and password. If you are not already a BioMedNet member, please click on the Register button. Once registered, you will be asked to enter your subscription key. Following confirmation, you will have full access to Trends in Immunology. If you obtain an error message please contact Customer Services ([email protected]) stating your subscription key and BioMedNet username and password. Please note that you only need to enter your subscription key once; BioMedNet ’remembers’ your subscription. Institutional online access is available at a premium. If your institute is interested in subscribing to online, please ask them to contact [email protected]. www.sciencedirect.com