Brought to you by Portland Press Ltd.
Published on behalf of the International Federation for Cell Biology
Cancer Cell death Cell cycle Cytoskeleton Exo/endocytosis Differentiation Division Organelles Signalling Stem cells Trafficking
Cell Biology International (2011) 35, 1177–1187 (Printed in Great Britain)
Review article
Gene expression profile of lymphatic endothelial cells
Peiliang Wang* and Yongbo Cheng†1
*Center of Oncology, The Fifth Affiliated Hospital of Xinjiang Medical University, 118 Henan Road, Xinshi District, Urumqi 830011, Peoples Republic of China, and †Department of Medical Records, The 309th Hospital of Chinese Peoples Liberation Army, 17 Heishanhu Road, Haidian District, Beijing 100091, Peoples Republic of China


The lymphatic system was first described at around the same time as the blood circulation centuries ago, but the biological function elucidation of LECs (lymphatic endothelial cells) is far less than that of BVECs (blood vascular endothelial cells). Since the discovery of molecular markers for LECs and exploration of lymphatic role in tumour metastasis, more attention has been given to basic lymphatic research. Approx. 150 known genes were found to be expressed at the mRNA and protein levels by LECs. These molecules play an important role in lymphangiogenesis, signalling, tumour metastasis, immune function and fluid transport. This review provides a brief outline of gene expression profile of LECs and the molecular biological function, which will give the reader a better understanding about the mechanics of lymphatic function and some pathologies related to the lymphatic system such as lymphoedema, and facilitate advanced scientific research into lymphatic biology.


Key words: biological function, expression file, lymphatic endothelial cell, ultrastructure

Abbreviations: Ach, acetylcholine, AM, adrenomedullin, Ang2, angiopoietin-2, Aspp1, apoptosis stimulating protein of p53, bFGF, basic fibroblast growth factor, BVEC, blood vascular endothelial cell, CAR, coxsackie B virus and adenovirus receptor, CCL21, CC chemokine ligand 21, CXCL12, CXC chemokine ligand 12, DPPIV, dipeptidyl peptidase IV, ECM, extracellular matrix, FGFR, fibroblast growth factor receptor, Foxc2, forkhead box factor C2, FVIIIRA, factor VIII-related antigen, HA, hyaluronan, HGF, hepatocyte growth factor, ICAM, intercellular adhesion molecule, IGFR, insulin-like growth factor receptor, IL, interleukin, JAM, junctional adhesion molecule, LEC, lymphatic endothelial cell, LYVE-1, lymphatic vessel endothelial receptor-1, NFATc1, nuclear factor of activated T-cells cytoplasmic 1, NO, nitric oxide, Nrp2, neuropilin-2, PA, plasminogen activator, PAI-1, plasminogen activator inhibitor type 1, PDGFR, platelet-derived growth factor receptor, PECAM-1, platelet endothelial cellular adhesion molecule-1, Prox1, prospero-related homeobox gene-1, RANTES, regulated upon activation, normal T-cell expressed and secreted, SLC, secondary lymphoid tissue chemokine, SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor, SOX18, sex determining region Y box 18, TGFβ, transforming growth factor β, TGFBI, transforming growth factor BI, Thy1, thymus cell antigen 1, TLR, Toll-like receptor, TNF, tumour necrosis factor, tPA, tissue plasminogen activator, VEGF, vascular endothelial growth factor, VEGFR, VEGF receptor, WPB, Weibel–Palade body

1To whom correspondence should be addressed (email chengyb2001@yahoo.com.cn).


1. Introduction

The function of lymphatic vasculature as a conduit for immune cells and excess tissue fluid has been known for over a century (Tammela et al., 2005), but the knowledge of biology of LECs (lymphatic endothelial cells), which line lymphatic vessel wall throughout the body, has been less well characterized. Over the past decade, the discovery of molecular markers, the establishment of culture in vitro and numerous explorations of its role in tumour metastasis (Kaipainen et al., 1995; Banerji et al., 1999; Kriehuber et al., 2001; Swartz and Skobe, 2001; Ran et al., 2010) have incited radical increasing interest in basic LEC research. As well as BVECs (blood vascular endothelial cells), LECs not only compose a diffusion barrier between the intravascular and extravascular space of lymph vessel regulating permeability but also synthesize, release various autacoids that activate some signalling pathways such as growth, ontogeny, tumour metastasis and so on (Zwaans and Bielenberg, 2007; Nonaka et al., 2008; Ribatti, 2008). Here, we review the literature on ultrastructure and gene expression in LECs, especially focusing on expression molecules, which will expand our understanding about the mechanics of lymphatic function and some pathologies related to the lymphatic system such as lymphoedema, and facilitate advanced scientific researches into lymphatic biology.

2. Ultrastructure of LECs

sparsely LECs display ultrastructural features similar to those described for cultured BVECs. In LECs, transmission electron microscopy revealed the presence of abundant cellular organelles such as the Golgi apparatus, smooth endoplasmic reticulum, free ribosomes, numerous vesicles and membrane invaginations (Leak, 1972,1976; Jones and Yong, 1987). Especially, some researchers clearly demonstrated that WPBs (Weibel–Palade bodies) were present in cultured LECs in vitro and in lymphatic endothelium in vivo (Tabuchi and Yamamoto, 1974; Jones and Yong, 1987) (Figure 1). WPB is secretory organelle in endothelial cells, containing a variety of biologically active molecules. These molecules are released rapidly by stimulation and involved in regulating haemostasis and angiogenesis (Michaux and Cutler, 2004; Rondaij et al., 2006). The intracellular vesicles and membrane invaginations indicate the existence of a robust vesicular transport system, correlating with selectively and abundantly expressed proteins.

The intercellular contacts between LECs were found to be tight and gap junctions distributed sparsely in lymphatics (Azzali et al., 2002). Three types of contacts were observed, simple overlapping or end-to-end apposition, complex interdigitations between cell membranes and cytoplasmic processes and discrete deposition of electron-dense material between adjacent cell membranes (Jones and Yong, 1987).

3. Gene expression in LECs

Approx. 150 known genes, a small percentage of all genes, were identified to be expressed in LECs. They contribute to the normal function of the lymphatic system and reaction for lymphatic pathological conditions (Figure 2). A review of these molecules is presented below.

3.1. Receptors

3.1.1. Growth factor receptors

3.1.1.1. VEGFR [VEGF (vascular endothelial growth factor) receptor]

The VEGFR family includes three isotypes of VEGFR-1, VEGFR-2 and VEGFR-3 (Kato et al., 2006). Only two members, VEGFR-2 and VEGFR-3, are expressed in LECs. Among the VEGFR family, most research has focused on VEGFR-3. VEGFR-3 is the first specific antigenic marker for the lymphatic endothelium. It is expressed in blood vessels and mesenchymal tissues in the early fetal period, is localized in BVECs and LECs in the middle fetal period and in the adult becomes restricted to the lymphatic endothelium (Aprelikova et al., 1992; Kaipainen et al., 1995; Enholm et al., 1998). It is well known that the VEGFR family contributes to lymphangiogenesis. VEGFR-2 and VEGFR-3 co-operate in lymphatic vessel sprouting, the former being activated by VEGF-A and the latter by VEGF-C and VEGF-D. Between them, the more important regulator for lymphangiogenesis is VEGFR-3. It plays a central role in the molecular mechanism of the proliferation of LECs and the formation of lymphatic vessels (Kukk et al., 1996; Kato et al., 2006). As a mediator of lymphatic growth, VEGFR-3 also promotes tumour metastasis. It was demonstrated to be correlated to lymph node metastases (Zeng et al., 2004). Systemic administration of an anti-VEGFR-3 antibody may reduce cancer metastasis without affecting physiological lymphatic flow (Kato et al., 2006). VEGFR-3 is also expressed in non-endothelial compartments such as osteoblasts, neuronal progenitors and macrophages (Le Bras et al., 2006; Orlandini et al., 2006; Schmeisser et al., 2006). The potential expression of VEGFR-3 on tumour cells in vitro and in vivo is disputed (Petrova et al., 2008; Su et al., 2008).

3.1.1.2. Tie2

Tie2 is a specific receptor whose characterized ligands are Angs (angiopoietins). LECs displayed an intense immunoreaction for Ang2, and simultaneously exhibit immunoreactivity to Tie2, ranging from lymphatic capillaries to collectors, throughout embryonic and neonatal development. Via Ang2–Tie2 signalling, Tie2 joins the regulation of lymphatic development at the earliest stage of lymphovasculogenesis (Jila et al., 2007; Shimoda, 2009).

3.1.1.3. Nrp2 (neuropilin-2)

Nrp2 is a non-kinase receptor for class III semaphorins, regulating chemorepulsive guidance of developing axons. In the vasculature, it is firstly expressed in embryonic veins and later in the lymphatic endothelium, co-operating with VEGFR-3 to mediate VEGF-C-dependent LEC proliferation and lymphangiogenesis. Nrp2 knockout mice showed severe hypoplasia of lymphatic capillaries and some larger lymphatic vessels, whereas the collecting vessels such as the thoracic duct were not affected (Marika et al., 2002; Yuan et al., 2002).

3.1.2. CAR (coxsackie B virus and adenovirus receptor)

CAR is originally identified as the primary attachment protein for the entry of coxsackie B virus and adenoviruses into cells. It was found to be expressed in epithelial cells in the trachea, intestine and skin during adulthood (Raschperger et al., 2006). Recently, Vigl et al. (2009) found that CAR was more highly expressed in cultured LECs derived from human skin. Functional assays revealed that it was involved in LEC adhesion and migration, lymphatic vessel formation and the vascular permeability control. In short, the role of CAR lies in supporting the structural integrity of lymphatic vessels.

3.1.3. TLRs (Toll-like receptors)

TLRs are a part of the innate immune system sensing pathogen-associated molecular patterns. The reaction of antibodies to TLR2 and TLR4 were observed on lymphatic capillaries in the lamina propria mucosae of the small intestinal tissue. Through the expression of TLRs, the lymphatic endothelium contributes to allow dendritic cells to home into secondary lymphoid tissue, which results in the induction of CCL21 (CC chemokine ligand 21) chemokines and other cytokines (Kuroshima et al., 2004).

3.1.4. FGFRs (fibroblast growth factor receptors)

Matsuoa et al. (2007) reported TR-LE cells, a conditionally immortalized rat lymphatic endothelial cell line, expressed a high level of FGFR-1, and levels of FGFR-2, FGFR-3 and FGFR-4 were not detected. Shin et al. (2006) revealed that FGFR-3 was strongly and specifically expressed in the newly formed lymphatic vessels. Among the FGFR family, FGFR-2 was thought to be involved in tube-like formation of TR-LE cells (Matsuoa et al., 2007).

3.1.5. Endothelin-B receptors

There are endothelin-B receptors on the plasma membrane of LECs in collecting lymph vessels. The stimulation of endothelin-B receptors can release NO (nitric oxide) from the endothelial cells, which results in negative chrono- and ino-tropic effects on the rhythmic pump activity (Sakai et al., 1999).

3.1.6. Others

IGFR-1 (insulin-like growth factor receptor-1) and IGFR-2 were detected in primary LECs, and mediated LEC proliferation and migration, contributing to lymphangiogenesis in vivo (Bjorndahl et al., 2005). Using a combination of in situ RNA hybridization and antibody staining, Cao et al. (2004) demonstrated that primary mouse dermal LECs expressed PDGFR-α (platelet-derived growth factor receptor-α) and PDGFR-β, two tyrosine kinase receptors. Additionally, HGF (hepatocyte growth factor) receptor is expressed in LECs and relates to lymphangiogenesis (Jurisic and Detmar, 2009). With regard to the inflammatory cytokine receptor, three IL-20 (interleukin-20) receptor subunits, IL-20Rα, IL-20Rβ and IL-22Rα, were all detected in human LECs. They mediated IL-20 to play a potential role in activating and modulating the formation of lymphatic vessels (Hammer et al., 2009).

3.2. Adhesion molecules

LECs express several cell adhesion molecules that constitute adherens junctions. These junctions may be particularly important for lymph vessel formation, leucocyte trafficking and tumour metastasis.

3.2.1. Integrins

Integrin subunits, such as α1 and α9, are expressed in LECs (Petrova et al., 2002). They are involved in focal adhesions to bind externally to ECM (extracellular matrix) proteins and internally to several specialized cytoplasmic proteins, which form a complex network of specialized signal-transduction molecules. Since integrins have no enzyme activity, they link to some kinases such as focal adhesion kinase pp125FAK which may phosphorylate tyrosine residues of several cytoplasmic proteins (Weber et al., 2002). In concert with VEGFR-3, integrins participate in normal development of the lymphatic system (Marika et al., 2002). Mice lacking integrins developed fatal bilateral chylothorax, lymphoedema and lymphocytic infiltration in the chest wall (Huang et al., 2000).

3.2.2. JAMs (junctional adhesion molecules)

JAM-1, JAM-2 and JAM-3 belong to the glycoprotein family of adhesion molecules on human lymphatic endothelium. They were all observed in collecting lymphatic vessels of gingival tissue, inflamed tongue and uninflamed small intestine. JAM-1 and JAM-3, but not JAM-2, were detected in cultured human neonatal dermal LECs. It was thought that JAM-2 could be produced in mature vascular endothelium but not in cultured cells. The expression of the three JAMs on the lymphatic endothelium may contribute to seal the cell–cell contacts at interendothelial junctions. These junctions can allow lymphocytes to transmigrate into lymphatic vessels from tissue (Ueki et al., 2008).

3.2.3. PECAM-1 (platelet endothelial cellular adhesion molecule-1)

It is well known that PECAM-1 is a multifunctional integral membrane protein expressed in the intercellular junctions of BVECs and on most leucocytes. Some studies have clearly demonstrated that it also expressed on lymphatic capillaries in the lamina propria, mucosal muscle layer, submucosal connective tissue of the normal human small intestine. Cultured LECs also have the ability to express PECAM-1, which contributes to the adherence of lymphocytes to lymphatic endothelium (Sawa et al., 1999a, 2007; Zhang et al., 2005).

3.2.4. ICAM-1 (intercellular adhesion molecule-1)

There is no lymphatic capillary expressing detectable amounts of ICAM-1 in the small intestine, but cultured LECs and lymphatic capillary in the capsule of the lymph node can express it (Zhang et al., 2005). ICAMs can bind to integrins expressed on leucocytes and contribute to the adherence of lymphocytes to the lymphatic endothelium (Sawa et al., 1999a, 2007).

3.2.5. Galectin-8

Galectin-8 is a soluble, 35 kDa protein belonging to the subclass of tandem repeat-type galectins. It is a novel, glycosylation-dependent interaction partner of podoplanin, and, as well as podoplanin, it is more highly expressed by LECs. Co-operating with podoplanin, galectin-8 plays a role in supporting the connection of the lymphatic endothelium to the surrounding ECM (Cueni and Detmar, 2009). Additionally, the interaction between galectin-8 and certain integrins such as α3β1 and α6β1 triggers integrin-mediated signalling cascades and cytoskeletal reorganization (Levy et al., 2003). And, on secretion, it is retained at the cell surface and promotes LEC adhesion as well as haptotactic migration.

3.3. Chemokines

It was demonstrated that CCL21/SLC (secondary lymphoid tissue chemokine), CCL2/JE, CXCL12 (CXC chemokine ligand 12)/SDF-1α (stromal-cell-derived factor 1α), RANTES (regulated upon activation, normal T-cell expressed and secreted) were secreted by LECs (Gunn et al., 1998; Muller et al., 2001; Podgrabinska et al., 2002; Zlotnik, 2004; Ji et al., 2006; Sironi et al., 2006). Among these chemokines, soluble factors such as CXCL12 attract tumour cells into the lymphatics, which are facilitated by chemokine receptors such as CXCR4 (CXC chemokine receptor 4) and CCR7 (CC chemokine receptor 7). This chemokine/receptor system plays a critical role in mediating tumour cell homing. RANTES is a chemokine for T-cells and monocytes and exhibits specific, saturable in situ binding to the afferent lymphatic vessels in the dermis of human skin (Hub and Rot, 1998). CCL21/SLC was reported to be expressed in high endothelial venules of lymph nodes, cultured human lymphatic endothelium established from skin, the central lacteals of villi and lymphatic capillaries in the lamina propria mucosae of small intestinal. It played a critical role in allowing dendritic cells, naive T-cells, and central memory T-cells to home to lymphoid tissue (Dieu et al., 1998; Mashino et al., 2002; Kuroshima et al., 2004). Taken together, the chemokines and their receptors are involved in the process of lymphocyte, dendritic cell, Langerhans cell and even tumour cell entry into the lymphatic vessels.

3.4. Ang2

Ang2, one of three members of the Ang family, is ligand for the Tie2 and is expressed in LECs throughout early and later development. It is involved in autoregulation of lymphatic development via Ang2–Tie2 signalling. At the earliest stage of lymphovasculogenesis, Ang2 adjusts lymphatic specification and sprouting from the veins under the control of Prox1 (prospero-related homeobox gene-1). Thus Ang2 is constantly expressed in Prox1- and/or LYVE-1 (lymphatic vessel endothelial receptor-1)-immunopositive endothelial cells of lymphatic sacs and vessels. Ang2 also facilitates the developing LECs to form lymphatic vascular organization. Mice deficient in Ang2 displayed disorganization and hypoplasia of lymphatic capillaries, showed incorrect growth of smooth muscle in collecting lymphatic vessels and finally developed severe lymphoedema (Shimoda, 2009).

3.5. Prox1

Prox1 is the homologue of the Drosophila homeobox gene prospero. As a marker, it is exclusively detected in cultured LECs of human and tissues of other species (Rodriguez-Niedenführ et al., 2001). Prox1 is essential and sufficient for specification of the fate of LECs. In wild-type embryos, its expression is initiated at approx. E9.5 (embryonic day 9.5) in a subpopulation of cardinal vein endothelial cells. These lymphatic progenitor cells subsequently bud, proliferate and migrate to form the embryonic lymph sacs and vascular network (Oliver, 2004). Targeted deletion of the Prox1 gene affected development of the lymphatic vascular system: the budding and sprouting of the developing lymphatics were arrested, chylous fluid accumulated in the intestine and death occurred around midgestation (Marika et al., 2002).

3.6. Podoplanin

Podoplanin, a 38-kDa mucin-type transmembrane glycoprotein, was first cloned from normal kidney podocytes. It is highly expressed by lymphatic vasculature in various tissues such as skin, kidney, salivary glands and lung. Furthermore, it usually co-localizes with VEGFR-3 (Marika et al., 2002; Schacht et al., 2003). Podoplanin contributes to LEC adhesion, migration and the formation of connecting lymphatics between superficial and deep lymphatic plexi. At the same time, it is involved in regulating the permeability of lymphatic vessels or perhaps in maintaining their integrity. Podoplanin-null mice displayed obvious dilations of the cutaneous and submucosal intestinal lymphatic vasculature, which led to lymphoedema (Schacht et al., 2003; Al-Rawi et al., 2005).

3.7. LYVE-1

LYVE-1, CD44-related transmembrane glycoprotein, is expressed in cultured LECs and normal or pathological lymphatic tissues (Al-Rawi et al., 2005; Kato et al., 2006). There is evidence to suggest that LYVE-1 plays a role in transporting HA (hyaluronan) across the lymphatic vessel wall. It shuttles across the lymphatic endothelium and transports HA from tissue to lymph by transcytosis (Jackson et al., 2001). In addition, there were reports that LYVE-1 was involved in HA metabolism or HA-mediated cell migration, but this theory is disputable (Marika et al., 2002; Jackson, 2003; Al-Rawi et al., 2005).

3.8. Fibrillins

Fibrillins, a family of ECM glycoproteins, are the main component of anchoring filaments. Several reports showed immunohistochemical evidence that they were expressed in LECs (Sakai et al., 1986; Podgrabinska et al., 2002; Weber et al., 2002). Fibrillins can respond to ECM stimulation and mediate cell–matrix adhesions. By these connections, mechanical signals from the ECM transform into biochemical signals in endothelial cells. In addition, the complex anchoring filaments–focal adhesions control the permeability of lymphatic endothelium and finely adjust lymph formation (Weber et al., 2002).

3.9. TGFβ (transforming growth factor β)-induced protein [TGFBI (transforming growth factor BI)]

As the only integrin–ECM adaptor molecule, TGFBI is expressed in hypoxia-exposed LECs. Furthermore, this expression is dependent on TGFβ production by LECs. By interaction with integrin, TGFBI was demonstrated to contribute to LEC adhesion to and migration through the ECM (Irigoyen et al., 2007, 2008).

3.10. DPPIV (dipeptidyl peptidase IV)

As a membrane glycoprotein, DPPIV is specifically expressed by cultured LECs and identified as a novel lymphatic marker. It is involved in diverse biological processes, including cell differentiation, apoptosis and neoplastic transformation control. In addition, it promotes LEC binding to collagen, fibronectin and gelatin. siRNA (small interfering RNA)-mediated DPPIV knockdown inhibited LEC adhesion to collagen type I and fibronectin, which reduced cell migration and formation of tube-like structures (Ghersi et al., 2002; Boonacker and Van Noorden, 2003; Shin et al., 2008).

3.11. Foxc2 (forkhead box factor C2)

The forkhead transcription factor Foxc2 is highly expressed in all developing lymphatic vessels and endothelial cells of lymphatic valves. This expression often overlaps with that of Foxc1. Foxc2 regulates sprouting of LECs, controls formation and maintenance of the valves, controls formation and maturation of lymphatic collecting vessels and establishes the pericyte-free lymphatic capillary. Compound Foxc mutants showed a defect in the early sprouting, agenesis of valves or lack valves and increased pericyte invasion of lymphatic vessels, which resulted in lymphatic dysfunction (Dagenais et al., 2004; Petrova et al., 2004; Kato et al., 2006; Seo et al., 2006).

3.12. Aspp1 (apoptosis stimulating protein of p53)

LECs express Aspp1, which regulates cell–cell adhesion, migration or cytoskeletal changes. Aspp1 knockout mice showed subcutaneous oedema and disorganized lymphatic vasculature. Lymphangiography by injecting dye subcutaneously into the embryonic forelimb showed defective lymphatic drainage function and abnormal patterns in collecting lymphatic vessels (Hirashima et al., 2008).

3.13. SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) family

The SNARE family includes syntaxins and YKT6, and are all expressed in LECs. They play a central role in vesicular trafficking (Podgrabinska et al., 2002).

3.14. NFATc1 (nuclear factor of activated T-cells cytoplasmic 1)

NFATc1 is expressed on neoformative, developing and mature lymphatic vasculature. Throughout lymphatic development, it is co-expressed along with Prox1. Analysis of trunk, pulmonary and dermal lymphatics at E14.5 revealed that it was also co-expressed with VEGFR-3 and podoplanin. The location to nuclei at each stage of lymphatic development suggested that NFATc1 was in its active dephosphorylated form (Ranger et al., 1998). NFATc1 is thought to participate in patterning and maintenance of lymphatic vasculature (Kulkarni et al., 2009).

3.15. Thy1 (thymus cell antigen 1)

Thy1 is strongly expressed in LECs isolated from mouse intestine. Blockade of Thy1 inhibited tumour cell adhesion to cultured mouse LECs. Moreover, adhesion of human polymorphonuclear and mononuclear leucocytes to human LECs was Thy1-dependent (Jurisic et al., 2010). These results suggest that Thy1 plays a potential role in the cell adhesion processes required for inflammation and tumour progression.

3.16. NO

In addition to arterial and venous endothelial cells, LECs have the potential to generate of endogenous NO (von der Weid et al., 2001). NO is not only important mediator of lymphatic vasomotion but also links to carcinogenesis during chronic inflammation. Furthermore, the eNOS (endothelial nitric oxide synthase)/NO signalling pathway is involved in a range of lymphangiogenic processes such as proliferation and migration. Additionally, NO participates in regulation of lymphatic permeability (Hagendoorn et al., 2005; Kajiya et al., 2008; Massi et al., 2009).

3.17. FVIIIRA (Factor VIII-related antigen)

Johnston and Walker (1984) reported that approximately one-third of LECs express FVIIIRA, whereas Jones and Yong (1987) reported that all LECs express FVIIIRA. Additionally, frozen sections of bovine lymphatic and thoracic duct vessels displayed moderate-to-intense granular perinuclear fluorescence for FVIIIRA. Endothelial cells of lymphatic collecting vessels, lymphatic capillaries and sinusoids of lymph nodes were all found to contain FVIIIRA (Johnston and Walker, 1984; Jones and Yong, 1987).

3.18. tPA (tissue plasminogen activator) and PAI-1 (plasminogen activator inhibitor type 1)

It was demonstrated that tPA and PAI-1 were produced and secreted by LEC monolayer cultures and are also present in lymph. These findings provide additional evidence that the lymphatic endothelium not only line lymphatic vessels, but also contribute to the production of lymph components. The two proteins play a major role in the regulation of the fluidity of venous blood that is continuously returned to the heart (Leak et al., 2000).

3.19. Miscellaneous

The chemokine receptor D6 is another lymphatic endothelial molecule. Its identification has given a novel tool for examining tumour lymphatic structures (Irjala et al., 2003). At the protein level, the genes Sema4C and C4orf7 are expressed specifically in LECs and not in BVECs. They play an important role in oncogenesis and are considered potential lymphangiogenesis targets for cancer therapy (Wu et al., 2010). The LECs can produce and release TXA2 (thromboxane A2), which regulates lymphatic vasomotion (Gao et al., 1999). Other studies found weak expression of CD34, UEA-1 (Ulex europaeus agglutinin-1), ephrin B2, angiotonin E, 5′-nucleotidase, desmoplakin in cultured LECs, paraffin-embedded lymphatic tissues and frozen sections from lymphatic vessels (Jones and Yong, 1987; Borron and Hay, 1994; Kriehuber et al., 2001; Hirakawa et al., 2003; Ji and Kato, 2003).

4. Factors influencing LEC expression

4.1. SOX18 (sex-determining region Y box 18)

SOX18 is a transcription factor that induces differentiation of LECs. It directly activates Prox1 transcription by binding to its proximal promoter. Some other lymphatic endothelial markers are also induced by an overexpression of SOX18 (Francois et al., 2008).

4.2. Ach (acetylcholine)

Ohhashi and Takahashi (1991) demonstrated that Ach induced LECs to release some transferable substance that activated the relaxation of lymphatic smooth muscle cells. The transferable substance was strongly suggested to be NO or its related compound, which diffused into the smooth muscle cells and resulted in the relaxation of lymphatic ducts (Ohhashi and Takahashi, 1991).

4.3. bFGF (basic fibroblast growth factor)

bFGF can increase tissue-type PA (plasminogen activator) expression in LECs. This is accompanied by an increase in PAI-1, which is thought to play an important permissive role in angiogenesis by protecting the ECM against excessive proteolytic degradation (Pepper et al., 1994). Besides the effects in vivo, it was demonstrated that bFGF facilitated lymphangiogenesis of the cultured LECs in vitro (Ohhashi, 2004).

4.4. VEGF

VEGF-A is well known to be an angiogenesis and lymphangiogenesis inductor and promoter in normal and pathological conditions. By using the chick embryo chorioallantoic membrane assay as an experimental in vivo model, Cimpean et al. (2010) demonstrated that the combination of VEGF-A/HGF induced a strong expression of Prox1 in LECs. VEGF-C was found to induce Ang2 expression in cultured LECs, which indicated another possible connection between VEGF and Ang growth factor during lymphangiogenesis.

4.5. Prox1

Prox1, the transcription factor, can up-regulate expression of VEGFR-3, LYVE-1 and other lymphatic endothelial-specific molecules. It is a master gene that specifies LEC characteristics (Hong and Detmar, 2003).

4.6. TNFα (tumour necrosis factor α)

TNFα modulates the production of PAs and their PAIs in cultured LECs. The treatment of the cells with recombinant human TNFα for 24 h resulted in a 3- to 7-fold increase in the amount of PAI (Laschinger et al., 1990). TNFα also strongly increases the production of IL-6, CCL2/JE and keratin complex (Sironi et al., 2006).

4.7. AM (adrenomedullin)

Jin et al. (2011) reported that AM profoundly suppressed the gene expression of cell adhesion receptors and inflammatory factors in LECs, such as ICAM-1, VCAM-1 (vascular adhesion molecule-1), endothelial adhesion molecule-1 (E-selectin), IL-8 and chemokines. The suppression is thought to possibly occur via a cAMP/NF-κB (nuclear factor κB)-dependent pathway.

4.8. Miscellaneous

By up-regulating the expression of VEGFR-3, Ang1 stimulates LEC proliferation, promotes vessel enlargement and new sprout generation. This suggests a cross-talk between VEGF and Ang during lymphatic development (Karpanena and Mäkinen, 2006). IL-7 has been identified as a strong lymphangiogenic factor. It specifically increases the expression of lymphatic markers such as LYVE-1, podoplanin, Prox1 and induces the formation of lymphatic vessels in vivo (Al-Rawi et al., 2005). The production of TGFβ contributes to the increased TGFBI expression in LECs (Irigoyen et al., 2008). Heparin can increase the production of NO by LECs resulting in a decrease in the amplitude and frequency of contraction of smooth muscle cells in lymphatic vessels (Lobov and Pan'kova, 2010).

5. Expression comparison between LEC and BVEC

Immunohistochemistry and RT–PCR (reverse transcription–PCR) have provided techniques for a comparison of expression profiles of LEC and BVEC at protein and mRNA levels. The expression of many in vivo markers of the lymphatic vasculature, such as desmoplakin, podoplanin, Prox1, mannose receptor 1, VEGFR-3 and LYVE-1, are confined to LECs or significantly higher in LECs than in BVECs (Hirakawa et al., 2003; Farnsworth et al., 2006). LECs secrete a broader range of cytokines than that in BVECs. Consistent with the absence of a basement membrane in vivo, LECs secrete very little matrix in comparison with BVECs, but selectively produce collagen type XVIII and EMILIN, the component of anchoring filaments (Podgrabinska et al., 2002). Differential mRNA and protein expression between LECs and BVECs have been investigated and are reviewed in detail elsewhere (Hirakawa et al., 2003; Farnsworth et al., 2006; Jurisic and Detmar, 2009).

6. Application of LEC expression

In addition to BVECs, LECs express a broad range of biological molecules. They play an important role in the formation and function of the lymphatic system, which has permitted studies into lymphangiogenesis, signalling, tumour metastasis, immune function and fluid transport. To date, they are frequently used as lymphatic endothelial markers for identification of the isolated primary LECs or lymphatic vessels from tissues, facilitating a detailed analysis of pathogenesis. For example, how do leucocytes and tumour cells enter lymph vessels and traffic to lymph nodes? The knowledge will make us better understand lymphatic vessel invasion so as to direct therapy for and predict survival of cancer patients (Kato et al., 2006; Ito et al., 2007; Williams et al., 2007). Additionally, the studies of inhibition of growth factors in preventing tumour cell spread through lymphatic vessels in animal models are encouraging. The identification of novel functions for the lymphangiogenic factors may yield additional therapeutic uses in inflammatory diseases.

Commonly used lymphatic endothelial markers are shown in detail in Table 1. The only nuclear protein is Prox1. The membrane proteins include VEGFR-3, LYVE-1, podoplanin and DPPIV. The samples studied are from cultured LECs and tissues from human, bovine, mice, canine, rabbit, zebrafish and so on. Positive expression of podoplanin and VEGFR-3 after cellular cryopreservation and thaw are found similar to fresh LECs (Jiang et al., 2010). Notably, there are no absolute specific markers for the lymphatic system; for example, VEGFR-3 is also expressed in vessels in tumours, in the epithelial cell in retina and in myoepithelial cells (Achen et al., 2006). LYVE-1 is also expressed in endothelial cells in normal liver blood sinusoids, epithelial cells in kidney, adrenal gland, thyroid and pancreas (Carreira et al., 2001). D2–40 immunohistochemistry staining is observed in breast myoepithelium (Rabban and Chen, 2008).


Table 1 Lymphatic markers

Note: There are some specific markers, such as 5′-nucleotidase, that have been used in the past to distinguish between the blood vascular and lymphatic endothelia. Other markers, such as desmoplakin, CCL21/SLC, CCL20/MIP-3α, Nrp2, DPPIV, macrophage mannose receptor, integrin α9, β-chemokine receptor D6, are less commonly used.

Markers Cellular sites of expression Samples Species References
Prox1 Cell nucleus Various tissues and cultured LECs Human, chick, zebrafish, horse and mouse Rodriguez-Niedenführ et al. (2001), Odaka et al. (2006) and Junginger et al. (2010)
Podoplanin Cell membrane Various tissues and cultured LECs Human and mouse Hata et al. (2008), Cueni and Detmar (2009) and Ji et al. (2010)
VEGFR-3 Cell membrane Various tissues and cultured LECs Human and mouse Aprelikova et al. (1992), Kato et al. (2006) and Odaka et al. (2006),
LYVE-1 Cell membrane Various tissues and cultured LECs Mouse, human and sheep Banerji et al. (1999), Jackson et al. (2001), Jila et al. (2007),Yücel et al. (2009) and Ji et al. (2010),
D2–40 Antibody reactive against transmembrane sialoglycoprotein Various tissues Human Rogers et al. (2008), Dubina and Goldenberg (2009), Petitt et al. (2009) and Yücel et al. (2009)
5-Nucleotidase Cell membrane Tissue Monkey Sleeman et al. (2001) and Ji and Kato (2003)
Desmoplakin Cytoplasmic domain Various tissues Human and mouse Ebata et al. (2001), Petrova et al. (2002) and Hirakawa et al. (2003)
DPPIV Cell membrane Various tissues and cultured LECs Human Shin et al. (2008)
CCL21/SLC Various tissues and cultured LECs Human, mouse Hirakawa et al. (2003) and Odaka et al. (2006)
CCL20/MIP-3α (macrophage inflammatory protein 3α) Normal human skin Human Hirakawa et al. (2003)
Nrp2 Various tissues Mouse Yuan et al. (2002) and Odaka et al. (2006)
Macrophage mannose receptor Normal human skin Human Hirakawa et al. (2003)
Integrin α9 Tissue Mouse Petrova et al. (2002)
β-Chemokine receptor D6 Tissue Human Nibbs et al. (2001)



7. Concluding remarks

The anatomy of the lymphatic system was almost completely characterized by the early 19th century (Swartz, 2001), but the biology of LECs that line lymphatic vessels has remained unclear for a long time. Since the identification of lymphatic-specific markers and the isolation of pure LECs, marked progress has been made in understanding the functional expression of LECs. Up to now, approx. 150 known genes have been reported to be expressed in LECs (Sironi et al., 2006), the majority expressed at the mRNA level and a few at the protein level.

The molecular expression in LECs is dynamic and plastic in different physiological and pathological conditions. It can alter in different stages of ontogeny. LYVE-1 is highly expressed during early development but becomes down-regulated in the adult stage. The molecules are also expressed differently in different organs or in different states in the same organ. Nrp2 staining is detected in the intestinal lymphatic endothelium but not in the lymphatic vessels of the skin, which is the same for several adhesion molecules (Sawa et al., 1999a; Marika et al., 2002; Zhang et al., 2005; Tan et al., 2006). The expression of CAR, LYVE-1 and CCL21 can be modulated by pathological conditions of LECs. In inflamed intestine, PECAM-1, ICAM-3 and VCAM-1 were all detected, whereas in normal intestine only PECAM-1 was detected (Sawa et al., 1999a, 1999b). Notably, the mannose receptor expression in intratumoral lymphatic vessels is associated with increased lymph node metastasis in breast cancer (Irjala et al., 2003).

Although some molecular players influencing the development of the lymphatic system have been extensively used for studies on lymphangiogenesis, signalling and so on; the knowledge about how these molecules work in LECs and interact with their surrounding microenvironment are superficial. The differentiation of LECs from haemopoietic stem cells, control of lymphangiogenesis, functional expression of LECs, direction of growth of the network of lymphatic vessels and molecular mechanism of the pathological process are needed to make further investigation. These studies may open a new door to therapy for lymphoedema and nodal metastasis of cancer.

REFERENCES

Achen, MG, Mann, GB and Stacker, SA (2006) Targeting lymphangiogenesis to prevent tumour metastasis. Br J Cancer 94, 1355-60
Crossref   Medline   1st Citation  

Al-Rawi, MAA, Mansel, RE and Jiang, WG (2005) Molecular and cellular mechanisms of lymphangiogenesis. Eur J Surg Oncol 31, 117-21
Crossref   Medline   1st Citation   2nd   3rd   4th  

Aprelikova, O, Pajusola, K, Partanen, J, Armstrong, E, Alitalo, R and Bailey, SK (1992) FLT4, a novel class III receptor tyrosine kinase in chromosome 5q33-qter. Cancer Res 52, 746-8
Medline   1st Citation   2nd  

Azzali, G, Vitale, M and Arcari, ML (2002) Ultrastructure of absorbing peripheral lymphatic vessel (ALPA) in guinea pig Peyer's patches. Microvasc Res 64, 289-301
Crossref   Medline   1st Citation  

Banerji, S, Ni, J, Wang, SX, Clasper, S, Su, J and Tammi, R (1999) LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J Cell Biol 144, 789-801
Crossref   Medline   1st Citation   2nd  

Bjorndahl, M, Cao, R, Nissen, LJ, Clasper, S, Johnson, LA and Xue, Y (2005) Insulin-like growth factors 1 and 2 induce lymphangiogenesis in vivo. Proc Natl Acad Sci USA 102, 15593-8
Crossref   Medline   1st Citation  

Boonacker, E and Van Noorden, CJ (2003) The multifunctional or moonlighting protein CD26/DPPIV. Eur J Cell Biol 82, 53-73
Crossref   Medline   1st Citation  

Borron, P and Hay, J (1994) Characterization of ovine lymphatic endothelial cells and their interactions with lymphocytes. Lymphology 27, 6-13
Medline   1st Citation  

Cao, RH, Björndahl, MA, Religa, P, Clasper, S, Garvin, S and Galter, D (2004) PDGF-BB induces intratumoral lymphangiogenesis and promotes lymphatic metastasis. Cancer Cell 6, 333-45
Crossref   Medline   1st Citation  

Carreira, CM, Nasser, SM, Tomaso, E, Padera, TP, Boucher, Y and Tomarev, SI (2001) LYVE-1 is not restricted to the lymph vessels: expression in normal liver blood sinusoids and down-regulation in human liver cancer and cirrhosis. Cancer Res 61, 8079-84
Medline   1st Citation  

Cimpean, AM, Seclaman, E, Ceausu, R, Gaje, P, Feflea, F and Anghel, A (2010) VEGF-A/HGF induce Prox-1 expression in the chick embryo chorioallantoic membrane lymphatic vasculature. Clin Exp Med 10, 169-72
Crossref   Medline   1st Citation  

Cueni, LN and Detmar, M (2009) Galectin-8 interacts with podoplanin and modulates lymphatic endothelial cell functions. Exp Cell Res 315, 1715-23
Crossref   Medline   1st Citation   2nd  

Dagenais, SL, Hartsough, RL, Erickson, RP, Witte, MH, Butler, MG and Glover, TW (2004) Foxc2 is expressed in developing lymphatic vessels and other tissues associated with lymphedema-distichiasis syndrome. Gene Expr Patterns 4, 611-9
Crossref   Medline   1st Citation  

Dieu, MC, Vanbervliet, B, Vicari, A, Bridon, JM, Oldham, E and Aït-Yahia, S (1998) Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. J Exp Med 188, 373-86
Crossref   Medline   1st Citation  

Dubina, M and Goldenberg, G (2009) Positive staining of tumor-stage Kaposi sarcoma with lymphatic marker D2–40. J Am Acad Dermatol 61, 276-80
Crossref   Medline   1st Citation  

Ebata, N, Nodasaka, Y, Sawa, Y, Makino, S, Totsuka, Y and Yoshida, S (2001) Desmoplakin as a specific marker of lymphatic vessels. Microvasc Res 61, 40-8
Crossref   Medline   1st Citation  

Enholm, B, Jussila, L, Karkkainen, M and Alitalo, K (1998) Vascular endothelial growth factor-C: a growth factor for lymphatic and blood vascular endothelial cells. Trends Cardiovasc Med 8, 292-7
Crossref   Medline   1st Citation  

Farnsworth, RH, Achen, MG and Stacker, SA (2006) Lymphatic endothelium: an important interactive surface for malignant cells. Pulm Pharmacol Ther 19, 51-60
Crossref   Medline   1st Citation   2nd  

Francois, M, Caprini, A, Hosking, B, Orsenigo, F, Wilhelm, D and Browne, C (2008) Sox18 induces development of the lymphatic vasculature in mice. Nature 456, 643-7
Crossref   Medline   1st Citation  

Gao, J, Zhao, J, Rayner, SE and Van Helden, DF (1999) Evidence that the ATP-induced increase in vasomotion of guinea-pig mesenteric lymphatics involves an endothelium-dependent release of thromboxane A2. Br J Pharmacol 127, 1597-602
Crossref   Medline   1st Citation  

Ghersi, G, Dong, H, Goldstein, LA, Yeh, Y, Hakkinen, L and Larjava, HS (2002) Regulation of fibroblast migration on collagenous matrix by a cell surface peptidase complex. J Biol Chem 277, 29231-41
Crossref   Medline   1st Citation  

Gunn, MD, Tangemann, K, Tam, C, Cyster, JG, Rosen, SD and Williams, LT (1998) A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes. Proc Natl Acad Sci USA 95, 258-63
Crossref   Medline   1st Citation  

Hagendoorn, J, Padera, TP, Fukumura, D and Jain, RK (2005) Molecular regulation of microlymphatic formation and function: role of nitric oxide. Trends Cardiovasc Med 15, 169-73
Crossref   Medline   1st Citation  

Hammer, T, Tritsaris, K, Hübschmann, MV, Gibson, J, Nisato, RE and Pepper, MS (2009) IL-20 activates human lymphatic endothelial cells causing cell signalling and tube formation. Microvasc Res 78, 25-32
Crossref   Medline   1st Citation  

Hata, M, Ueki, T, Sato, A, Kojima, H and Sawa, Y (2008) Expression of podoplanin in the mouse salivary glands. Arch Oral Biol 53, 835-41
Crossref   Medline   1st Citation  

Hirakawa, S, Hong, YK, Harvey, N, Schacht, V, Matsuda, K and Libermann, T (2003) Identification of vascular lineage-specific genes by transcriptional profiling of isolated blood vascular and lymphatic endothelial cells. Am J Pathol 162, 575-586
Crossref   Medline   

Hirashima, M, Sano, K, Morisada, T, Rossant, KMJ and Suda, T (2008) Lymphatic vessel assembly is impaired in Aspp1-deficient mouse embryos. Dev Biol 316, 149-59
Crossref   Medline   1st Citation  

Hong, YK and Detmar, M (2003) Prox1, master regulator of the lymphatic vasculature phenotype. Cell Tissue Res 314, 85-92
Crossref   Medline   1st Citation  

Huang, XZ, Wu, JF, Ferrando, R, Lee, JH, Wang, YL and Farese, JR (2000) Fatal bilateral chylothorax in mice lacking the integrin alpha9beta1. Mol Cell Biol 20, 5208-15
Crossref   Medline   1st Citation  

Hub, E and Rot, A (1998) Binding of RANTES, MCP-1, MCP-3, and MIP-1alpha to cells in human skin. Am J Pathol 152, 749
Medline   1st Citation  

Irigoyen, M, Ansó, E, Martínez, E, Garayoa, M, Martínez-Irujo, JJ and Rouzaut, A (2007) Hypoxia alters the adhesive properties of lymphatic endothelial cells. A transcriptional and functional study. Biochim Biophys Acta 1773, 880-90
1st Citation  

Irigoyen, M, Ansó, E, Salvo, E, de las Herrerías, JD, Martínez-Irujo, JJ and Rouzaut, A (2008) TGFβ-induced protein mediates lymphatic endothelial cell adhesion to the extracellular matrix under low oxygen conditions. Cell Mol Life Sci 65, 2244-55
Crossref   Medline   1st Citation   2nd  

Irjala, H, Alanen, K, Grénman, R, Heikkilä, P, Joensuu, H and Jalkanen, S (2003) Mannose receptor (MR) and common lymphaticendothelial and vascular endothelial receptor (CLEVER)-1 direct the binding of cancer cells to the lymph vessel endothelium. Cancer Res 63, 4671-76
Medline   1st Citation   2nd  

Ito, M, Moriya, T, Ishida, T, Usami, S, Kasajima, A and Sasano, H (2007) Significance of pathological evaluation for lymphatic vessel invasion in invasive breast cancer. Breast Cancer 14, 381-7
Crossref   Medline   1st Citation  

Jackson, DG, Prevo, R, Clasper, S and Banerji, S (2001) LYVE-1, the lymphatic system and tumor lymphangiogenesis. Trends Immunol 22, 317-21
Crossref   Medline   1st Citation   2nd  

Jackson, DG (2003) The lymphatics revisited new perspectives from the hyaluronan receptor LYVE-1. Trends Cardiovasc Med 13, 1-7
Crossref   Medline   1st Citation  

Ji, RC and Kato, S (2003) Lymphatic network and lymphangiogenesis in the gastric wall. J Histochem Cytochem 51, 331-8
Crossref   Medline   1st Citation   2nd  

Ji, RC, Kurihara, K and Kato, S (2006) Lymphatic vascular endothelial hyaluronan receptor (LYVE)-1- and CCL21-positive lymphatic compartments in the diabetic thymus. Anat Sci Int 81, 201-9
Crossref   Medline   1st Citation  

Ji, RC, Eshita, Y, Xing, LP and Miura, M (2010) Multiple expressions of lymphatic markers and morphological evolution of newly formed lymphatics in lymphangioma and lymph node lymphangiogenesis. Microvasc Res 80, 195-201
Crossref   Medline   1st Citation   2nd  

Jiang, ZH, Hu, XQ, Kretlow, JD and Liu, NF (2010) Harvesting and cryopreservation of lymphatic endothelial cells for lymphatic tissue engineering. Cryobiology 60, 177-83
Crossref   Medline   1st Citation  

Jila, A, Kim, H, Nguyen, V, Dumont, DJ, Semple, J and Armstrong, D (2007) Lymphangiogenesis following obstruction of large postnodal lymphatics in sheep. Microvasc Res 73, 214-23
Crossref   Medline   1st Citation   2nd  

Jin, DH, Otani, K, Yamahara, K, Ikeda, T, Nagaya, N and Kangawa, K (2011) Adrenomedullin reduces expression of adhesion molecules on lymphatic endothelial cells. Regul Pept 166, 21-27
Crossref   Medline   1st Citation  

Johnston, MG and Walker, MA (1984) Lymphatic endothelial and smooth-muscle cells in tissue culture. In Vitro 20, 566-72
Crossref   Medline   1st Citation   2nd  

Jones, BE and Yong, LCJ (1987) Culture and characterization of bovine mesenteric lymphatic endothelium. In Vitro 23, 698-706
1st Citation   2nd   3rd   4th   5th   6th  

Junginger, J, Rötting, A, Staszyk, C, Kramer, K and Hewicker-Trautwein, M (2010) Identification of equine cutaneous lymphangioma by application of a lymphatic endothelial cell marker. J Comp Path 143, 57-60
Crossref   Medline   1st Citation  

Jurisic, G and Detmar, M (2009) Lymphatic endothelium in health and disease. Cell Tissue Res 335, 97-108
Crossref   Medline   1st Citation   2nd  

Jurisic, G, Iolyeva, M, Proulx, ST, Halin, C and Detmar, M (2010) Thymus cell antigen 1 (Thy1, CD90) is expressed by lymphatic vessels and mediates cell adhesion to lymphatic endothelium. Exp Cell Res 316, 2982-92
Crossref   Medline   1st Citation  

Kaipainen, A, Korhonen, J, Mustonen, T, van Hinsbergh, VWM, Fang, GH and Dumont, D (1995) Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc Natl Acad Sci USA 92, 3566-70
Crossref   Medline   1st Citation   2nd  

Kajiya, K, Huggenberger, R, Drinnenberg, I, Ma, B and Detmar, M (2008) Nitric oxide mediates lymphatic vessel activation via soluble guanylate cyclase alpha1beta1-impact on inflammation. FASEB J 22, 530-7
Medline   1st Citation  

Karpanena, T and Mäkinen, T (2006) Regulation of lymphangiogenesis from cell fate determination to vessel remodeling. Exp Cell Res 312, 575-83
Crossref   Medline   1st Citation  

Kato, S, Shimoda, H, Ji, RC and Miura, M (2006) Lymphangiogenesis and expression of specific molecules as lymphatic endothelial cell markers. Anat Sci Int 81, 71-83
Crossref   Medline   1st Citation   2nd   3rd   4th   5th   6th   7th  

Kriehuber, E, Breiteneder-Geleff, S, Groeger, M, Soleiman, A, Schoppmann, SF and Stingl, G (2001) Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized cell lineages. J Exp Med 194, 797-808
Crossref   Medline   1st Citation   2nd  

Kukk, E, Lymboussaki, A, Taira, S, Kaipainen, A, Jeltsch, M and Joukov, V (1996) VEGF-C receptor binding and pattern of expression with VEGFR-3 suggests a role in lymphatic vascular development. Development 122, 3829-37
Medline   1st Citation  

Kulkarni, RM, Greenberg, JM and Akeson, AL (2009) NFATc1 regulates lymphatic endothelial development. Mech Dev 126, 350-65
Crossref   Medline   1st Citation  

Kuroshima, S, Sawa, Y, Kawamoto, T, Yamaoka, Y, Notani, K and Yoshida, S (2004) Expression of Toll-like receptors 2 and 4 on human intestinal lymphatic vessels. Microvasc Res 67, 90-5
Crossref   Medline   1st Citation   2nd  

Laschinger, CA, Johnston, MG, Hay, JB and Wasi, S (1990) Production of plasminogen activator and plasminogen activator inhibitor by bovine lymphatic endothelial cells: modulation by TNF-alpha. Thromb Res 59, 567-79
Crossref   Medline   1st Citation  

Leak, LV (1972) The transport of exogenous peroxidase across the blood-tissue lymph interface. J Ultrastruct Res 39, 24-42
Crossref   Medline   1st Citation  

Leak, LV (1976) The structure of lymphatic capillaries in lymph formation. Fed Proc 35, 1863-71
Medline   1st Citation  

Leak, LV, Saunders, M, Day, AA and Jones, M (2000) Stimulation of plasminogen activator and inhibitor in the lymphatic endothelium. Microvasc Res 60, 201-11
Crossref   Medline   1st Citation  

Le Bras, B, Barallobre, MJ, Homman-Ludiye, J, Ny, A, Wyns, S and Tammela, T (2006) VEGF-C is a trophic factor for neural progenitors in the vertebrate embryonic brain. Nat Neurosci 9, 340-8
Crossref   Medline   1st Citation  

Levy, Y, Ronen, D, Bershadsky, AD and Zick, Y (2003) Sustained induction of ERK, protein kinase B, and p70 S6 kinase regulates cell spreading and formation of F-actin microspikes upon ligation of integrins by galectin-8, a mammalian lectin. J Biol Chem 278, 14533-42
Crossref   Medline   1st Citation  

Lobov, GI and Pan'kova, MN (2010) Heparin inhibits contraction of smooth muscle cells in lymphatic vessels. Bull Exp Biol Med 149, 4-6
Crossref   Medline   1st Citation  

Marika, J and Karkkainen, Alitalo, K (2002) Lymphatic endothelial regulation, lymphoedema, and lymph node metastasis. Semin Cell Dev Biol 13, 9-18
Crossref   Medline   1st Citation   2nd   3rd   4th   5th   6th  

Mashino, K, Sadanaga, N, Yamaguchi, H, Tanaka, F, Ohta, M and Shibuta, K (2002) Expression of chemokine receptor CCR7 is associated with lymph node metastasis of gastric carcinoma. Cancer Res 62, 2937-41
Medline   1st Citation  

Massi, D, De Nisi, MC, Franchi, A, Mourmouras, V, Baroni, G and Panelos, J (2009) Inducible nitric oxide synthase expression in melanoma: implications in lymphangiogenesis. Mod Pathol 22, 21-30
Crossref   Medline   1st Citation  

Matsuoa, M, Yamadab, S, Koizumib, K, Sakuraib, H and Saikib, L (2007) Tumour-derived fibroblast growth factor-2 exerts lymphangiogenic effects through Akt/mTOR/p70S6 kinase pathway in rat lymphatic endothelial cells. Eur J Cancer 43, 1748-54
Crossref   Medline   1st Citation   2nd  

Michaux, G and Cutler, DF (2004) How to roll an endothelial cigar: the biogenesis of Weibel–Palade bodies. Traffic 5, 69-78
Crossref   Medline   1st Citation  

Muller, A, Homey, B, Soto, H, Ge, N, Catron, D and Buchanan, ME (2001) Involvement of chemokine receptors in breast cancer metastasis. Nature 410, 50-6
Crossref   Medline   1st Citation  

Nonaka, H, Watabe, T, Saito, S, Miyazono, K and Miyajima, A (2008) Development of stabilin2+ endothelial cells from mouse embryonic stem cells by inhibition of TGFβ/activin signaling. Biochem Biophys Res Commun 375, 256-60
Crossref   Medline   1st Citation  

Nibbs, RJ, Kriehuber, E, Ponath, PD, Parent, D, Qin, S and Campbell, JD (2001) The beta chemokine receptor D6 is expressed by lymphatic endothelium and a subset of vascular tumors. Am J Pathol 158, 867-77
Crossref   Medline   1st Citation  

Odaka, C, Morisada, T, Oike, Y and Suda, T (2006) Distribution of lymphatic vessels in mouse thymus: immunofluorescence analysis. Cell Tissue Res 325, 13-22
Crossref   Medline   1st Citation   2nd   3rd   4th  

Ohhashi, T (2004) Lymphodynamic properties governing sentinel lymph nodes. Ann Surg Oncol 11, S275-8
1st Citation  

Ohhashi, T and Takahashi, N (1991) Acetylcholine-induced release of endothelium-derived relaxing factor from lymphatic endothelial cells. Am J Physiol 260, H1172-8
Medline   1st Citation   2nd  

Oliver, G (2004) Lymphatic vasculature development. Nat Rev Immunol 4, 35-45
Crossref   Medline   1st Citation  

Orlandini, M, Spreafico, A, Bardelli, M, Rocchigiani, M, Salameh, A and Nucciotti, S (2006) Vascular endothelial growth factor-D activates VEGFR-3 expressed in osteoblasts inducing their differentiation. J Biol Chem 281, 17961-7
Crossref   Medline   1st Citation  

Pepper, MS, Wasi, S, Ferrara, N, Orci, L and Montesano, R (1994) In vitro angiogenic and proteolytic properties of bovine lymphatic endothelial cells. Exp Cell Res 210, 298-305
Crossref   Medline   1st Citation  

Petitt, M, Allison, A, Shimoni, T, Uchida, T, Raimer, S and Kelly, B (2009) Lymphatic invasion detected by D2–40/S-100 dual immunohistochemistry does not predict sentinel lymph node status in melanoma. J Am Acad Dermatol 61, 819-28
Crossref   Medline   1st Citation  

Petrova, TV, Mäkinen, T, Mäkelä, TP, Saarela, J, Virtaner, I and Ferrell, RE (2002) Lymphatic endothelial reprogramming of vascular endothelial cells by the Prox-1 homeobox transcription factor. EMBO J 21, 4593-9
Crossref   Medline   1st Citation   2nd   3rd  

Petrova, TV, Karpanena, T, Norrmén, C, Mellor, R, Tamakoshi, T and Fineqold, D (2004) Defective valves and abnormal mural cell recruitment underlie lymphatic vascular failure in lymphedema distichiasis. Nat Med 10, 974-81
Crossref   Medline   1st Citation  

Petrova, TV, Bono, P, Holnthoner, W, Chesnes, J, Pytowski, B and Sihto, H (2008) VEGFR-3 expression is restricted to blood and lymphatic vessels in solid tumors. Cancer Cell 13, 554-6
Crossref   Medline   1st Citation  

Podgrabinska, S, Braun, P, Velasco, P, Kloos, B, Pepper, MS and Jackson, DG (2002) Molecular characterization of lymphatic endothelial cells. Proc Natl Acad Sci USA 99, 16069-74
Crossref   Medline   1st Citation   2nd   3rd   4th  

Rabban, JT and Chen, YY (2008) D2–40 expression by breast myoepithelium: potential pitfalls in distinguishing intralymphatic carcinoma from in situ carcinoma. Hum Pathol 39, 175-83
Crossref   Medline   1st Citation  

Ran, S, Volk, L, Hall, K and Flister, MJ (2010) Lymphangiogenesis and lymphatic metastasis in breast cancer. Pathophysiology 17, 229-51
Crossref   Medline   1st Citation  

Ranger, AM, Oukka, M, Rengarajan, J and Glimcher, LH (1998) Inhibitory function of two NFAT family members in lymphoid homeostasis and Th2 development. Immunity 9, 627-35
Crossref   Medline   1st Citation  

Raschperger, E, Thyberg, J, Pettersson, S, Philipson, L, Fuxe, J and Pettersson, RF (2006) The coxsackie- and adenovirus receptor (CAR) is an in vivo marker for epithelial tight junctions, with a potential role in regulating permeability and tissue homeostasis. Exp Cell Res 312, 1566-80
Crossref   Medline   1st Citation  

Ribatti, D (2008) Transgenic mouse models of angiogenesis and lymphangiogenesis. Int Rev Cell Mol Biol 266, 1-35
Medline   1st Citation  

Rodriguez-Niedenführ, M, Papoutsi, M, Christ, B, Nicolaides, KH, von Kaisenberg, CS and Tomarev, SI (2001) Prox1 is a marker of ectodermal placodes, endodermal compartments, lymphatic endothelium and lymphangioblasts. Anat Embryol 204, 399-406
Crossref   Medline   1st Citation   2nd  

Rogers, PA, Donoghue, JF and Girling, JE (2008) Endometrial lymphangiogenesis. Placenta 29, S48-54
Crossref   Medline   1st Citation  

Rondaij, MG, Bierings, R, Kragt, A, van Mourik, JA and Voorberg, J (2006) Dynamics and plasticity of Weibel–Palade bodies in endothelial cells. Arterioscler Thromb Vasc Biol 26, 1002-7
Crossref   Medline   1st Citation  

Sakai, LY, Keene, DR and Engvall, E (1986) Fibrillin, a new 350-kD glycoprotein, is a component of extracellular microfibrils. J Cell Biol 103, 2499-509
Crossref   Medline   1st Citation  

Sakai, H, Ikomi, F and Ohhashi, T (1999) Effects of endothelin on spontaneous contractions in lymph vessels. Am J Physiol Heart Circ Physiol 277, H459-66
1st Citation  

Sawa, Y, Yamaoka, YJ, Ebata, N, Ashikaga, Y, Kim, T, Suzuki, M and Yoshida, S (1999a) Immunohistochemical study on leukocyte adhesion molecules expressed on lymphatic endothelium. Microvasc Res 57, 292-7
Crossref   Medline   1st Citation   2nd   3rd   4th  

Sawa, Y, Shibata, K, Braithwaite, MW, Suzuki, M and Yoshida, S (1999b) Expression of immunoglobulin superfamily members on the lymphatic endothelium of inflamed human small intestine. Microvasc Res 57, 100-6
Crossref   Medline   1st Citation  

Sawa, Y, Sugimoto, Y, Ueki, T, Ishikawa, H, Sato, A and Nagato, T (2007) Effects of TNF-alpha on leukocyte adhesion molecule expressions in cultured human lymphatic endothelium. J Histochem Cytochem 55, 721-33
Crossref   Medline   1st Citation   2nd  

Schacht, V, Ramirez, MI, Hong, YK, Hirakawa, S, Feng, D and Harvey, N (2003) T1alpha/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema. EMBO J 22, 3546-56
Crossref   Medline   1st Citation   2nd   3rd   4th   5th   6th   7th   8th   9th  

Schmeisser, A, Christoph, M, Augstein, A, Marquetant, R, Kasper, M and Braun-Dullaeus, RC (2006) Apoptosis of human macrophages by Flt-4 signaling: implications for atherosclerotic plaque pathology. Cardiovasc Res 71, 774-84
Crossref   Medline   1st Citation  

Seo, S, Fujita, H, Nakano, A, Kang, M, Duarte, A and Kume, T (2006) The forkhead transcription factors, Foxc1 and Foxc2, are required for arterial specification and lymphatic sprouting during vascular development. Dev Biol 294, 58-70
1st Citation  

Shimoda, H (2009) Immunohistochemical demonstration of angiopoietin-2 in lymphatic vascular development. Histochem Cell Biol 31, 231-8
1st Citation   2nd  

Shin, JW, Min, M, Larrieu-Lahargue, F, Canron, X, Kunstfeld, R and Nguyen, L (2006) Prox1 promotes lineage-specific expression of fibroblast growth factor (FGF) receptor-3 in lymphatic endothelium: a role for FGF signaling in lymphangiogenesis. Mol Biol Cell 17, 576-84
Medline   1st Citation  

Shin, JW, Jurisic, G and Detmar, M (2008) Lymphatic-specific expression of dipeptidyl peptidase IV and its dual role in lymphatic endothelial function. Exp Cell Res 314, 3048-56
Crossref   Medline   1st Citation   2nd  

Sironi, M, Conti, A, Bernasconi, S, Fra, AM, Pasqualini, F and Nebuloni, M (2006) Generation and characterization of a mouse lymphatic endothelial cell line. Cell Tissue Res 325, 91-100
Crossref   Medline   1st Citation   2nd   3rd  

Sleeman, JP, Krishnan, J, Kirkin, V and Baumann, P (2001) Markers for the lymphatic endothelium: in search of the holy grail? Microsc Res Tech 55, 61-9
Crossref   Medline   1st Citation  

Su, JL, Chen, PS, Chien, MH, Chen, PB, Chen, YH and Lai, CC (2008) Further evidence for expression and function of the VEGF-C/VEGFR-3 axis in cancer cells. Cancer Cell 13, 557-60
Crossref   Medline   1st Citation  

Swartz, MA and Skobe, M (2001) Lymphatic function, lymphangiogenesis, and cancer metastasis. Microsc Res Tech 55, 92-9
Crossref   Medline   1st Citation  

Swartz, MA (2001) The physiology of the lymphatic system. Adv Drug Deliv Rev 50, 3-20
Crossref   Medline   1st Citation  

Tabuchi, H and Yamamoto, T (1974) Specific granules in the endothelia of blood and lymphatic vessels in the cardiac valves of dogs. Arch Histol Jpn 37, 217-25
Medline   1st Citation  

Tammela, T, Petrova, TV and Alitalo, K (2005) Molecular lymphangiogenesis: new players. Trends Cell Biol 15, 434-41
Crossref   Medline   1st Citation  

Tan, YZ, Wang, HJ, Zhang, WC and Li, HS (2006) Expression of adhesion molecules of immunoglobulin super family on lymphatic endothelial cells and different vascular endothelial cells. Acta Anat Sin 37, 52-6
1st Citation  

Ueki, T, Iwasawa, K, Ishikawa, H and Sawa, Y (2008) Expression of junctional adhesion molecules on the human lymphatic endothelium. Microvasc Res 75, 269-78
Crossref   Medline   1st Citation  

von der Weid, PY, Zhao, J and Van Helden, DF (2001) Nitric oxide decreases pacemaker activity in lymphatic vessels of guinea pig mesentery. Am J Physiol Heart Circ Physiol 280, H2707-16
Medline   1st Citation  

Vigl, B, Zgraggen, C, Rehman, N, Banziger-Tobler, NE, Detmar, M and Halin, C (2009) Coxsackie- and adenovirus receptor (CAR) is expressed in lymphatic vessels in human skin and affects lymphatic endothelial cell function in vitro. Exp Cell Res 315, 336-47
Crossref   Medline   1st Citation  

Weber, E, Rossi, A, Solito, R, Sacchi, G, Agliano, M and Gerli, R (2002) Focal adhesion molecules expression and fibrillin deposition by lymphatic and blood vessel endothelial cells in culture. Microvasc Res 64, 47-55
Crossref   Medline   1st Citation   2nd   3rd  

Williams, K, Flanagan, A, Folpe, A, Thakker, R and Athanasou, NA (2007) Lymphatic vessels are present in phosphaturic mesenchymal tumours. Virchows Arch 451, 871-5
Crossref   Medline   1st Citation  

Wu, MF, Han, LF, Shi, YY, Xu, G, Wei, JC and You, LY (2010) Development and characterization of a novel method for the analysis of gene expression patterns in lymphatic endothelial cells derived from primary breast tissues. J Cancer Res Clin Oncol 136, 863-72
Crossref   Medline   1st Citation  

Yuan, L, Moyon, D, Pardanaud, L, Breant, C, Karkkainen, MJ and Alitalo, K (2002) Abnormal lymphatic vessel development in neuropilin 2 mutant mice. Development 129, 4797-806
Medline   1st Citation   2nd  

Yücel, YH, Johnston, MG, Ly, T, Patel, M, Drake, B and Gümüs, E (2009) Identification of lymphatics in the ciliary body of the human eye: A novel ‘uveolymphatic’ outflow pathway. Exp Eye Res 89, 810-9
Crossref   Medline   1st Citation   2nd  

Zeng, Y, Opeskin, K, Baldwin, ME, Horvath, LG, Achen, MG and Stacker, SA (2004) Expression of vascular endothelial growth factor receptor-3 by lymphatic endothelial cells is associated with lymph node metastasis in prostate cancer. Clin Cancer Res 10, 5137-44
Crossref   Medline   1st Citation  

Zhang, WC, Wang, HJ and Tan, YZ (2005) Expression of adhesion molecules in both tissular and cultured lymphatic endothelial cells. Chin J Histochem Cytochem 14, 11-5
1st Citation   2nd   3rd  

Zlotnik, A (2004) Chemokines in neoplastic progression. Semin Cancer Biol 14, 181-5
Crossref   Medline   1st Citation  

Zwaans, BMM and Bielenberg, DR (2007) Potential therapeutic strategies for lymphatic metastasis. Microvasc Res 74, 145-58
Crossref   Medline   1st Citation  


Received 6 December 2010/22 April 2011; accepted 31 May 2011

Published online 1 November 2011, doi:10.1042/CBI20100871


© The Author(s) Journal compilation © 2011 Portland Press Limited


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)