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Cell Biology International (2011) 35, 11771187 (Printed in Great Britain)
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 email@example.com).
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.
3.1.1. Growth factor receptors
220.127.116.11. 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).
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).
18.104.22.168. 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).
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.
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).
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.
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.
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).
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).
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).
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).
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.
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).
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).
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.
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.
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.
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.
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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)