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Keywords:

  • endothelial cells;
  • growth factors;
  • lymphangiogenesis;
  • tumour progression

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Lymphangiogenesis
  5. Embryo lymphangiogenesis
  6. Structural features of the lymphatic system
  7. Lymphangiogenesis in vivo
  8. Markers of lymphatic endothelial cells
  9. Experimental models of lymphangiogenesis
  10. Molecular regulation of lymphangiogenesis
  11. Concluding remarks
  12. Acknowledgements
  13. References

The lymphatic system is implicated in interstitial fluid balance regulation, immune cell trafficking, oedema and cancer metastasis. However, the sequence of events that initiate and coordinate lymphatic vessel development (lymphangiogenesis) remains obscure. In effect, the understanding of physiological regulation of lymphatic vasculature has been overshadowed by the greater emphasis focused on angiogenesis, and delayed by a lack of specific markers, thereby limiting this field to no more than a descriptive characterization. Recently, new insights into lymphangiogenesis research have been due to the discovery of lymphatic-specific markers and growth factors of vascular endothelial growth factor (VEGF) family, such as VEGF-C and VEGF-D. Studies using transgenic mice overexpressing VEGF-C and VEGF-D have demonstrated a crucial role for these factors in tumour lymphangiogenesis. Knowledge of lymphatic development has now been redefined at the molecular level, providing an interesting target for innovative therapies. This review highlights the recent insights and advances into the field of lymphatic vascular research, outlining the most important aspects of the embryo development, structure, specific markers and methods applied for studying lymphangiogenesis. Finally, molecular mechanisms involved in the regulation of lymphangiogenesis are described.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Lymphangiogenesis
  5. Embryo lymphangiogenesis
  6. Structural features of the lymphatic system
  7. Lymphangiogenesis in vivo
  8. Markers of lymphatic endothelial cells
  9. Experimental models of lymphangiogenesis
  10. Molecular regulation of lymphangiogenesis
  11. Concluding remarks
  12. Acknowledgements
  13. References

Embryo vascular development involves a complex series of events during which endothelial cells differentiate, proliferate, migrate and undergo maturation into an organized vascular network (Risau & Flamme, 1995; Risau, 1997). The first step in the vessel development is called vasculogenesis, in which mesoderm-derived angioblasts proliferate and organize into the primitive vascular plexus, consisting of the major vessels. Remodelling and expansion of these primary vessels through both pruning and vessel enlargement, which result in a closely interconnecting branching pattern, is called angiogenesis, i.e. sprouting of new vessels from existing ones. The loops between vessels can also form via another mechanism called intussusceptive growth, a type of angiogenesis involving the in situ remodelling of vessels by protruding interstitial tissue columns. In this process, a large sinusoidal capillary is divided into smaller capillaries, which then grow separately (Risau, 1997).

The maturation of newly formed vessels into stable and functional vessels involves the synthesis of a new basement membrane, and recruitment of pericytes and smooth muscle cells to support the abluminal side of the vessel (Hellstrom et al. 2001). The vascular system is a highly heterogeneous and non-uniform organ system (Yancopoulos et al. 1998; Gale & Yancopoulos, 1999; Ribatti et al. 2002). It is widely accepted that the organotypic differentiation of endothelial cells is dependent on interactions with stromal parenchymal cells in the target tissues.

Lymphangiogenesis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Lymphangiogenesis
  5. Embryo lymphangiogenesis
  6. Structural features of the lymphatic system
  7. Lymphangiogenesis in vivo
  8. Markers of lymphatic endothelial cells
  9. Experimental models of lymphangiogenesis
  10. Molecular regulation of lymphangiogenesis
  11. Concluding remarks
  12. Acknowledgements
  13. References

The term ‘lymphangiogenesis’ describes any growth-inducing events of lymphatics, such as proliferation and tube formation on plastic (Leak & Jones, 1994), and invasion into collagen gels (Tan, 1998). Several excellent reviews have been published recently on this topic (Alitalo & Carmeliet, 2002; Stacker et al. 2002; Pepper & Skobe, 2003; Oliver, 2004).

The lymphatic system includes a wide network of capillaries, collecting vessels and ducts that permeate most of the organs (Ryan et al. 1986). Unlike the blood vasculature, which forms a continuous loop, the lymphatic system is an open-ended, one-way transit system. It assists in maintaining the blood volume, and carries cells, interstitial fluid components and metabolites that leak from capillaries and returns them to the venous circulation via the thoracic duct.

Furthermore, it entails part of the immune system by continuously circulating the white blood cells within the lymphoid organs and bone marrow and transporting antigen-presenting cells. Endothelial receptors and binding proteins are involved in this trafficking. Like developing blood vessels, the first lymphatics consist only of endothelial cells. The origin of embryo lymphatic endothelial cells is as yet unknown.

Embryo lymphangiogenesis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Lymphangiogenesis
  5. Embryo lymphangiogenesis
  6. Structural features of the lymphatic system
  7. Lymphangiogenesis in vivo
  8. Markers of lymphatic endothelial cells
  9. Experimental models of lymphangiogenesis
  10. Molecular regulation of lymphangiogenesis
  11. Concluding remarks
  12. Acknowledgements
  13. References

The development of the embryo lymphatic system has been studied descriptively, but experimental studies have not been performed until recently (Schneider et al. 1999; Wilting et al. 2001; Wigle et al. 2002). Studies on living animals through serial sectioning and injection methods have shown that early anlagen of the lymphatics of birds, mammals and humans are the lymph sacs, which develop in close association with the venous system (Ranvier, 1895; van der Putte, 1975). Due to their specific location, these are the jugular, subclavian, posterior and retroperitoneal (mesenterial) lymph sacs and the cisterna chyli. The lymph sacs derive by fusion of lymphatic capillaries filled with stagnant blood, which has prompted the speculation that the lymph sacs derive from veins (Lewis, 1905; Miller, 1912).

However, different theories regarding the origin of the lymphatics have been proposed. Historically, the best accepted theory is that of Sabin (1902, 1904). He suggested that early in fetal development, isolated primitive lymph sacs originate by endothelial cell budding from embryo veins. Thus the peripheral lymphatic vessels spread from these primary lymph sacs by endothelial sprouting into surrounding tissues and organs where local lymphatic capillaries form. Accordingly, lymphatic endothelial cells are derived exclusively from the endothelium of the venous system. This view seems to be supported by Wigle et al. (2002).

By contrast, Huntington (1908), McClure (1908) and Kampmeier (1912) suggested that the primitive lymphatic vessels arise in the mesenchyme from putative lymphangioblasts (independent on the veins) by confluence of ‘lymphatic clefts’, fuse with the lymph sacs by centripetal growth and secondarily established venous connections. This theory is in line with findings by Schneider et al. (1999), who demonstrated through the quail-chick chimera system (Le Douarin, 1969) that lymphangioblasts are present in the avian wing bud before the emergence of the jugulo-axillary lymph sacs. They grafted paraxial mesoderm of 2-day-old quail embryo into the shoulder region of 3-day-old chick embryo, and studied the integration of lymphangioblasts into the jugulo-axillary lymph sac. Then, the host embryo was re-incubated until day 6.5 (grafting experiments). From their morphological and molecular characteristics (QH1 staining, a specific marker of quail – but not of chick – endothelial cells) (Pardanaud et al. 1987), the jugular lymph sac was clearly distinguished from the jugular vein. In all specimens quail endothelial cells integrating into the endothelial lining of chick vessels were observed. Specifically, two specimens showed quail cells being both in jugular vein and in lymph sacs without the possibility of estimating whether lymphangioblasts had directly integrated into the lymph sac, or whether cells had been first integrated into the jugular vein and then given rise to the jugular lymph sac. However, the serial sectioning of one specimen revealed that no quail cells had integrated into the jugular vein, and that these cells were in the jugular lymph sac (at a great distance from the jugular vein), indicating that they had been integrated directly into the lymph sacs. Therefore, the early lymph sacs may arise as sprouts from adjacent veins but, additionally, mesenchymal lymphangioblasts contribute to the growing lymph sacs.

To determine the origin of the lymphatic endothelium the distal wing buds of 3.5-day-old chick embryos were grafted homotopically into 3–3.5-day-old quail embryos. The vascular endothelial growth factor receptor-3 (VEGFR-3) and QH1 double staining of the 10-old-day chimeric wings revealed that lymphatics were formed by both chick and quail endothelial cells (Schneider et al. 1999).

Papoutsi et al. (2001) studied the lymphangiogenic power of the splanchnic mesoderm of the avian allantoic bud of 3-day-old quail embryos grafted on to the chick embryo chorioallantoic membrane (CAM). The chick CAM contained areas where the endothelium of blood vessels and lymphatics were of quail origin (QH1 and VEGFR-3 double positive). The lymphatics were located in their typical position around arteries and veins, demonstrating lymphangiogenic potential of the allantoic mesoderm long before the development of the posterior lymph sacs.

Structural features of the lymphatic system

  1. Top of page
  2. Abstract
  3. Introduction
  4. Lymphangiogenesis
  5. Embryo lymphangiogenesis
  6. Structural features of the lymphatic system
  7. Lymphangiogenesis in vivo
  8. Markers of lymphatic endothelial cells
  9. Experimental models of lymphangiogenesis
  10. Molecular regulation of lymphangiogenesis
  11. Concluding remarks
  12. Acknowledgements
  13. References

The initial lymphatic vessels (also called ‘absorbing lymphatic vessels’ and, incorrectly, ‘lymphatic capillaries’) are blind-ended structures that optimally suit fluid and particle uptake. Like blood capillaries they are formed by a single non-fenestrated endothelial cell layer, but they differ in that: (1) initial lymphatic vessels usually possess a more irregular and wider lumen (10–60 µm in diameter); (2) their endothelium has an extremely attenuated cytoplasm, except in the perinuclear region (Fig. 1); (3) they are not encircled by pericytes and have absent or poorly developed basal lamina; (4) their tight junctions and adherens junctions, which are the major intercellular junctions in blood vessels, are not as frequently seen; (5) these junctions differ from those of blood vessels, which are typically involved in maintaining firm cell–cell adhesion to connect adjacent endothelial cells over entire cell boundaries, and represent focal points of adhesion (Leak, 1970; Schmelz et al. 1994); (6) their intimate association with the adjacent interstitial areas. In effect, lymphatic endothelial cells are closely linked to surrounding connective tissue by fine (10–12 nm) ‘anchoring filaments’ (Gerli et al. 1991) (Fig. 2). These filaments are attached to the cell abluminal surface and extended deeply into the connective tissue, firmly attaching endothelium to extracellular matrix fibres, which is thus highly sensitive to interstitial stresses. It has long been thought that an increase in the interstitial fluid volume causes the filaments to exert radial tension on the initial lymphatic vessel and pull open overlapping intercellular junctions (formed by extensive superimposition of adjacent endothelial cells), hence favouring interstitial drainage (Aukland & Reed, 1993). This mechanism creates a ‘tissue pump’ or a slight and temporary pressure gradient that enables lymph formation (Ikomi & Schmid-Schonbein, 1996).

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Figure 1. Initial lymphatic vessels (arrows) in the submucosa of human colon; the largest contains a well-formed valve. Surrounding blood microvessels (double arrows) are much smaller. Original magnification: ×200.

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Figure 2. Anchoring filaments (AF) in a precollector of the human thigh. Original magnification: ×20 000.

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Anchoring filaments are made of fibrillin (Solito et al. 1997; Gerli et al. 2000), a large glycoprotein that contains an Arg-Gly-Asp (RGD) motif capable of binding αvβ3 integrins. The latter are transmembrane glycoproteins that cluster at focal adhesion plaques. Extracellular matrix stimuli transmitted by the cytoplasmic tail of integrins into the cell may trigger several signalling proteins including Focal Adhesion Kinase (FAK). Upon FAK activation actin-mediated cytoskeletal rearrangements occur and permeability is also probably affected. Thus the lymphatic endothelium through fibrillin-containing anchoring filaments may play a much more complex role in lymph formation than is currently supposed (Weber et al. 2002). Finally, cultured lymphatic endothelial cells are able to produce an extensive network of fibrillin-containing microfibrils into the underlying matrix (Fig. 3).

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Figure 3. Immunofluorescence staining of cultured lymphatic endothelial cells deposit fibrillin, the major component of anchoring filaments, in the underlying extracellular matrix. Original magnification: ×400.

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In light of their importance in lymphatic function, the composition and architecture of extracellular matrix are likely to play a critical role in lymphangiogenesis. This agrees with the evidence that extensive and chronic degradation of the extracellular matrix renders lymphatics non-responsive to the changes in the interstitium and therefore causes dysfunction (Negrini et al. 1996).

Initial lymphatic vessels merge into larger vessels called ‘precollectors’ (Sacchi et al. 1997), which represent the initial drainage routes of lymph. Precollectors are structurally characterized by the alternation of areas with the same structural simplicity as initial lymphatic vessels and areas with a well-developed muscular coat (Fig. 4). The former probably have an absorbing function whereas the latter may be involved in lymph propulsion. Interestingly, the ultrastructural features of these different portions do not differ, and anchoring filaments are present in both. Retrograde flow is prevented by thin but well-formed valves, the only site where the lymphatic endothelium is underlined by a continuous basement membrane.

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Figure 4. A precollector of the human thigh (P, on the right) and blood vessels (on the left). The precollector wall has no muscular coat on the left side. Original magnification: ×200.

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Precollectors merge into collecting vessels (Fig. 5) that have a thick wall comprising a continuous layer of smooth muscle cells and may thus support a circumferential hoop stress. They also contain one-way valves that aid in lymph propulsion by preventing retrograde flow. Collecting vessels are interrupted by lymph nodes where lymph is filtered and can thus be distinguished in pre- and post-nodal collecting vessels. They eventually drain, through larger trunks, into the thoracic duct and the right lymphatic duct that discharge lymph into the large veins at the base of the neck.

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Figure 5. A collecting vessel (arrow) of the human thigh surrounded by several blood microvessels (double arrows). A continuous muscular coat enables distinction from precollectors. Original magnification: ×200.

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Lymphangiogenesis in vivo

  1. Top of page
  2. Abstract
  3. Introduction
  4. Lymphangiogenesis
  5. Embryo lymphangiogenesis
  6. Structural features of the lymphatic system
  7. Lymphangiogenesis in vivo
  8. Markers of lymphatic endothelial cells
  9. Experimental models of lymphangiogenesis
  10. Molecular regulation of lymphangiogenesis
  11. Concluding remarks
  12. Acknowledgements
  13. References

Detailed descriptive studies revealed the mechanisms of lymphangiogenesis in different animal and human tissues. Clark & Clark (1932) documented the extension of lymphatic capillaries by outgrowth from existing lymph vessels in rabbit ear transparent chambers. Bellman & Odén (1959) documented via contrast microlymphangiography the time course and extent of newly formed lymphatics in circumferential wounds of the rabbit ear, including lymphatic bridging through newly formed scar tissue (Odén, 1960). Subsequent studies documented restoration of distinctive ultrastructural features in newly regenerated lymphatic vessels, including the characteristic overlapping junctional complexes and Weibel–Palade bodies (storage depots for von Willebrand Factor – vWF) (Magari & Asano, 1978; Magari, 1987).

Lymphatics are less labile than blood capillaries; they send out fewer sprouts, anastomose less frequently and show much less tendency to retract or change in size or shape.

Although temporary lymphoedema occurs after lymphatic disruption, it usually resolves owing to spontaneous regeneration or reconnection of lymphatics (Reichert, 1926; Slavin et al. 1999).

Markers of lymphatic endothelial cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. Lymphangiogenesis
  5. Embryo lymphangiogenesis
  6. Structural features of the lymphatic system
  7. Lymphangiogenesis in vivo
  8. Markers of lymphatic endothelial cells
  9. Experimental models of lymphangiogenesis
  10. Molecular regulation of lymphangiogenesis
  11. Concluding remarks
  12. Acknowledgements
  13. References

Molecular markers that unequivocally distinguish lymphatics from blood vessels are critical to a better understanding of lymphatic vessel formation and function (Table 1). Several methods have been suggested to discriminate blood and lymphatic vessels in histological sections. Of these, some are more reliable and well-characterized than others.

Table 1.  Markers of lymphatic endothelial cells
AntigenECProtein classBiological effect
  1. EC, endothelial cell expression; P, pan-endothelial; B, blood endothelial vessel; L, lymphatic and vascular endothelial cells and other cells, as described in the text; LEC, lymphatic endothelial cell; HA, hyaluronan.

PECAM/CD31Pintegral membrane proteinadhesion molecule for transendothelial migration of haematopoietic progenitor and leukocytes
CD34B, Ltransmembrane cell surface glycoproteinleukocyte trafficking; regulation of haematopoiesis
Von Willebrand FactorB, Lcoagulating factor contained in Weibel–Palade bodiesplatelet aggregation and adhesion to the cell walls of injuredvessels
VEGFR-3Lreceptor tyrosine kinase on endothelial celllymphangiogenesis; survival of LEC
LYVE-1Lreceptor for extracellular matrix glycosaminoglycanstransport of HA from tissues to lymph nodes
PROX-1Lhomeobox transcription factordevelopmental lymphangiogenesis
PodoplaninLintegral membrane proteinunknown
β-Chemokine receptor D6Lchemokine receptor in afferent lymphaticsleukocyte trafficking
Macrophage mannose receptorLendocytotic receptor in macrophagesphagocytosis of bacteria, host-derived glycoproteins and viral endocytosis
DesmoplakinLcomponent of intercellular adhering junctionadhesion of LECs

The lymphatic endothelium has been characterized by using 5′-nucleotidase (Nase) – alkaline phosphatase (ALPase) double staining (Kato, 1990). The lymphatic endothelium is marked by strong 5′-Nase activity (Fig. 6) that is significantly lower or absent in blood vessels.

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Figure 6. The luminal membrane and pinocytotic vesicles of this bovine mesenterial collecting vessel are stained by the histochemical lymphatic marker 5′ Nase. Transmission electron microscopy; original magnification: ×45 000.

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Other techniques are toluidine blue staining following arterial perfusion–fixation and staining of basement membrane components, such as laminin and collagen type IV (Kubo et al. 1990). PAL-E does not stain the endothelium of large, medium-sized and small arteries, arterioles and large veins, and does not stain the endothelial lining of lymphatic vessels and sinus histiocytes (Schlingemann et al. 1985).

PECAM-1/CD31 and vWF are molecules widely expressed in all endothelial cells (Sleeman et al. 2001; Table 1). However, CD31/PAL-E double staining has been used for the detection of lymphatic endothelium (de Waal et al. 1997).

In summary, thus far none of the so-called lymphatic endothelium-specific markers has been shown to be absolutely specific for lymphatic vessels. The main proposed lymphatic markers are described below.

Lymphatic vessel endothelial hyaluronan receptor (LYVE-1)

LYVE-1, a homologue of the CD44 hyaluronan (HA) receptor, is a new member of the Link protein superfamily. It is a type I integral membrane polypeptide whose extracellular domain encodes a single cartilage Link module, the prototypic HA-binding domain conserved within all members of the Link or hyaladherin superfamily (Toole, 1990). The central core of the LYVE-1 Link module (C2–C3) is 57% identical to that of the human CD44 HA receptor, the only Link superfamily HA receptor described to date and the closest homologue of LYVE-1 (Banerji et al. 1999). These authors first described a restricted pattern of tissue expression by immunoperoxidase that revealed LYVE-1 lining of lymphatics in virtually every tissue. The greatest LYVE-1 expression was detected in submucosal lymph vessels underlying smooth muscle in the colon and in the lacteal vessels of intestinal villi (Banerji et al. 1999).

LYVE-1 is probably involved in the transport of HA across lymphatic endothelium, specifically in the movement of tissue HA from interstitium to lymph (Prevo et al. 2001). This is supported by the finding that LYVE-1 is present on both the luminal and the abluminal sides of lymphatic capillaries, suggesting shuttling across the endothelium or transcytosis (Nicosia, 1987). Furthermore, LYVE-1 probably regulates the entry of leukocytes or tumour cells into the lumen of afferent lymphatic capillaries, promoting dissemination to regional lymph nodes (Jackson et al. 2001). The role of LYVE-1 in transcytosis of HA or in the regulation of leukocyte or tumour cell entry is largely hypothetical at present.

Mouta Carreira et al. (2001) have shown that LYVE-1 is not exclusive to the lymphatic vessels, being expressed by normal hepatic blood sinusoidal endothelial cells in mice and humans. LYVE-1 is not expressed by angiogenic blood vessels of human liver tumours and is weakly present on microvessels of regenerative hepatic nodules in cirrhosis, though both vessels are largely derived from the liver sinusoids. In addition, lymphatic vessels were found within the parenchymal fibrous areas that develop de novo in cirrhosis, suggesting that cirrhosis is accompanied by lymphangiogenesis. Moreover, in human hepatocellular carcinoma and liver metastases, lymphatic vessels were restricted to the tumour margin and surrounding liver, whereas no lymphatics were observed in the tumour parenchyma or between tumour nodules.

Podoplanin

Podoplanin (also known as OTS-8, T1 alpha or E11 antigen) is a 43-kDa integral plasma membrane protein primarily found on the surface of rat glomerular epithelial cells (podocytes) and linked to flattening of foot processes that occurs in glomerular diseases (Matsui et al. 1999).

The biological function of podoplanin can be inferred from knock-out studies (Schacht et al. 2003). Podoplanin null mice die at birth due to respiratory failure and have defects in lymphatic but not in blood vessel pattern formation. These defects are associated with diminished lymphatic transport, congenital lymphoedema and dilation of lymphatic vessels. These data identify podoplanin as a novel critical player that regulates different key aspects of lymphatic vasculature formation.

Breiteneder-Geleff et al. (1999) studied a panel of vascular tumours with anti-podoplanin and anti-VEGFR-3 antibodies and showed overlapping patterns for these two markers in that: (1) affinity-purified rabbit anti-podoplanin IgG specifically and exclusively immunolabelled endothelial cells of vessels that were clearly distinct from PAL-E-labelled blood vessels; (2) podoplanin-positive vessels were also selectively stained with anti-VEGFR-3 IgG in double labelling experiments predominantly at the luminal surface; (3) dermal lymphatic capillaries identified by ultrastructural morphological criteria and immunoelectron microscopy were selectively labelled by anti-podoplanin antibodies; (4) in benign lymphatic tumours, podoplanin localized in endothelial cells; (5) immunoblotting of lymphangiomas with anti-podoplanin IgG revealed a ∼38-kDa protein that was identical to podoplanin in human lung and isolated glomeruli.

Prox-1

This is a marker for the subpopulation of endothelial cells that sprout to give rise to the lymphatic sacs during development (Wigle et al. 2002). Targeted deletion of the Prox-1 gene does not affect development of the vascular system, but the sprouting of the developing lymphatics is specifically ablated.

Wigle et al. (2002) suggested that blood endothelial cell differentiation is independent of Prox-1 and that it is mandatory for establishment of lymphatic endothelial cell identity. In contrast to the wild-type embryos, in Prox-1 null embryos the endothelial cells sprouting from the cardinal vein did not express the lymphatic vascular markers VEGFR-3, LYVE-1 or SLC chemokine. Instead, the mutant cells expressed the blood vascular markers laminin and CD34, indicating that these cells were not committed to the lymphatic endothelial cell lineage in the absence of Prox-1. Overall results suggest that Prox-1 activity is required for both maintenance of the sprouting of venous endothelial cells and differentiation towards the lymphatic phenotype. Accordingly, it has been proposed that a blood vascular phenotype is the default fate of sprouting embryo venous endothelial cells; upon expression of Prox-1, these sprouting cells adopt a lymphatic vasculature phenotype.

Hong et al. (2002) identified Prox-1 as a master switch in the program specifying lymphatic endothelial cell fate, showing that Prox-1 expression up-regulated the lymphatic markers podoplanin and VEGFR-3. Conversely, genes such as laminin, VEGF-C, neuropilin-1 (NRP-1) and intercellular adhesion molecule-1, whose expression parallel the blood endothelial cell phenotype, were down-regulated.

β-Chemokine receptor D6

This is a human chemokine receptor that binds with high affinity to a wide array of pro-inflammatory β-chemokines, including RANTES, MCP-1, and MCP-3, but not human MIP-1α (Nibbs et al. 1997). D6 was found on endothelial cells lining the lymphatic system in human dermis (Hub & Rot, 1998). D6 immunoreactivity was shown in the mucosa and other wall layers of the gut, in afferent lymphatics and subcapsular and medullary sinuses of lymph nodes, but not in blood vessel endothelial cells. D6 was also revealed in some malignant, highly vascularized tumours, suggesting their origin from lymphatic endothelial cells (Nibbs et al. 2001).

The pattern of D6 expression suggests a role for this molecule intervening in the regulation of chemokine-driven trafficking of leukocytes across lymphatics, or development and growth of lymphatics themselves. Moreover, the expression of this receptor on only a subset of lymphatics suggests a functional heterogeneity within the lymphatic vasculature. However, its role in the migration of tumour cells to regional lymph nodes needs further investigation.

Desmoplakin

This is a protein of the junction system connecting the very flat endothelial cells of lymphatic vessels (Schmelz et al. 1994). It is a marker for small lymphatic vessels (Sawa et al. 1999), and is not expressed by larger lymphatic collecting ducts, including the thoracic duct (Schmelz et al. 1994). However, desmoplakin can be detected in the junctions between cultered blood vessel endothelial cells (Kowalczyk et al. 1998). It thus remains to be determined whether desmoplakin is exclusively expressed in lymphatic capillaries, even if the lymphatic specificity of this marker has been established (Ebata et al. 2001).

Macrophage mannose receptor

The 180-kDa mannose receptor is mainly expressed on cells of the macrophage lineage where it mediates the uptake of micro-organisms, host-derived glycoproteins and viral endocytosis (Reading et al. 2000). The receptor is also expressed by mouse lymphatic endothelia whereas, by contrast, the expression in human lymphatic vessels needs to be further defined (Linehan et al. 2001). However, the biological role of this receptor in lymphatic vessels is unknown: perhaps it plays a role in antigen capture and clearing in inflammation and immune-mediated processes (Groger et al. 2000).

In conclusion, some of the putative highly lymphatic-specific markers have not as yet been confirmed. In this context, definitive identification of lymphatics should combine characteristic morphological features with consistent immunohistochemical profiles and leave open the question that, particularly in pathological states, lymphatics and blood vessels in tissue sections may become indistinguishable and need to be visualized as part of a functioning, communicating vascular network.

Experimental models of lymphangiogenesis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Lymphangiogenesis
  5. Embryo lymphangiogenesis
  6. Structural features of the lymphatic system
  7. Lymphangiogenesis in vivo
  8. Markers of lymphatic endothelial cells
  9. Experimental models of lymphangiogenesis
  10. Molecular regulation of lymphangiogenesis
  11. Concluding remarks
  12. Acknowledgements
  13. References

A great variety of experimental models have shed light on the mechanisms of lymphangiogenesis: (i) dye injection and mouse tail models; (ii) wound healing; (iii) the chick CAM; (iv) transgenic mouse models; (v) culturing and purification of lymphatic endothelial cells.

Dye injection and mouse tail model

Fluorescence microlymphography enables the visualization of the initial lymphatic network in human dermis in vivo (Stanton et al. 1997). This technique was used to stain the lymph capillaries in the superficial layer of the skin of the mouse tail and together with densitometric image analysis, flow velocity along the tail was measured (Leu et al. 1994).

Boardman & Swartz (2003) used the same experimental model to investigate the hypothesis that interstitial flow may initiate matrix remodelling and cell organization. They evaluated lymphatic function (fluid transport) and fluid channel architecture in situ with fluorescence microlymphangiography, and observed the formation of functional channels in 2–3 weeks within an initially acellular gel. Immunohistochemical staining confirmed that these channels were lined with lymphatic endothelial cells.

Wound healing

During wound healing lymphatic capillaries grow by sprouting from existing lymphatics, much in the same way as new blood capillaries sprout from existing capillaries or post-capillary veins during angiogenesis. The appearance of new lymphatic capillaries is always secondary to that of blood capillaries, although linear growth occurs at comparable speed (Clark, 1922; Clark & Clark, 1932).

Paavonen et al. (2000) studied VEGFR-3 expression in experimental wounds made in dorsal skin of pigs. VEGFR-3-positive vessels were observed in the granulation tissue from 5 days onward and very few VEGFR-3-positive lymphatic vessels persisted on day 9 and none on day 14. These results suggest that transient lymphangiogenesis occurs parallel with angiogenesis in healing wounds.

Witmer et al. (2001) demonstrated that in granulation tissue VEGFR-3 staining was observed in the proliferative superficial zone in plump blood vessel sprouts, in the intermediate zone in blood vessels and long lymphatic sprouts, and in the deeper fibrous zone in large lymphatics, in a pattern demonstrating that lymphangiogenesis follows behind blood vessel angiogenesis.

The chick CAM

The chick CAM is an extra-embryonic membrane, which serves as a gas exchange surface and its function is supported by a dense capillary network. Because of its extensive vascularization, the CAM has been broadly used to investigate the action of angiogenic and anti-angiogenic molecules (Ribatti et al. 2001). Moreover, it contains a dense network of lymphatics accompanying the arteries and veins.

Oh et al. (1997) studied the morphology and pattern of the lymphatics of normal CAM using semithin and ultrathin sectioning, immunohistochemical staining with anti-α-smooth muscle actin and fibronectin antibodies, in situ hybridization with VEGFR-2 and VEGFR-3 probes, and Mercox injection. They described a regular pattern of lymphatic vessels along all arteries, arterioles and veins, and observed an increasing number of lymphatic capillaries interconnecting larger lymphatic vessels in proportion to increasing size of the arteries. The lymphatics of CAM showed characteristics previously described for normal lymphatics such as huge diameter, porous and very thin endothelial lining and absence of poor basal lamina. Proliferation studies revealed a large number of BrdU-labelled nuclei of lymphatic endothelial cells after 1 and 2 days of VEGF-C application. Finally, VEGFR-3 mRNA expression was shown to be restricted to the lymphatic endothelial cells of the differentiated CAM.

Transgenic mouse models (Table 2)

Table 2.  Experimental animal models applied to investigate the role of VEGF-C and VEGF-D in the induction of lymphangiogenesis
Gene mouse modelProtein formResponseReference
VEGF-C transfection
 Adenovirus injection into mouse ear skinFull-lengthLymphangiogenesisEnholm et al. (2001)
 Expression in mouse skin (K-14 promoter)Full-lengthLymphangiogenesisJeltsch et al. (1997)
 Expression in mouse skin (K-14 promoter)Full-length form of VEGF-C156S mutantLymphangiogenesisVeikkola et al. (2001)
 Expression in mouse pancreas (rat insulin promoter)Full-lengthLymphangiogenesisMandriota et al. (2001)
 Tumour model, overexpression in MBA-MD-435 cells 7Full-lengthLymphangiogenesisSkobe et al. (2001a,b)
 Tumour model, overexpression in MCF-7 cellsFull-lengthLymphangiogenesisKarpanen et al. (2001)
 Tumour model, overexpression in AZ521 cellsFull-lengthLymphangiogenesisYanai et al. (2001)
 Tumour model, overexpression in MeWo cellsFull-lengthAngiogenesis LymphangiogenesisSkobe et al. (2001a,b)
VEGF-D transfection
 Expression in mouse skin (K-14 promoter)Full-lengthLymphangiogenesisVeikkola et al. (2001)
 Adenovirus injection into rat epigastric skinMatureAngiogenesis LymphangiogenesisByzova et al. (2002)
 Adenovirus injection into rat cremasteric muscleMatureAngiogenesisByzova et al. (2002)
 Tumour model, overexpression in 293EBNA cellsFull-lengthAngiogenesis LymphangiogenesisStacker et al. (2001)

Overexpression of VEGF-C in the skin of transgenic mice resulted in lymphatic, but not vascular endothelial proliferation and vessel enlargement (Jeltsch et al. 1997). Transgenic mice overexpressing VEGF-C under the control of the rat insulin promoter (Rip) developed extensive lymphatic vessels around the islets of Langerhans. When crossed with Rip1Tag2 transgenic mice that normally develop benign β-cell tumours, enlarged peritumoural lymphatics and a high rate of metastasis to pancreatic lymph nodes were observed (Mandriota et al. 2001). These findings demonstrate that VEGF-C-induced lymphangiogenesis mediates tumor cell dissemination and the formation of lymph node metastases.

Veikkola et al. (2001) created transgenic mice overexpressing a VEGFR-3-specific mutant of VEGF-C (VEGF-C156S) or VEGF-D in epidermal keratinocytes under the keratin 14 promoter. Both transgens induced the growth of lymphatic vessels in skin, whereas the blood vessel architecture was not affected. These results indicate that stimulation of the VEGFR-3 signal tranduction pathway is sufficient to induce specifically lymphangiogenesis in vivo.

Makinen et al. (2001a) showed that a soluble form of VEGFR-3 is a potent inhibitor of VEGF-C/VEGF-D signalling, and when expressed in the skin of transgenic mice, it inhibits fetal lymphangiogenesis and induces a regression of already formed lymphatic vessels, though the blood vasculature remains normal.

Karkkainen et al. (2001) described lymphoedema (Chy) mice with an inactivating VEGFR-3 mutation in their germ line, and swelling of the limbs because of hypoplastic cutaneous, but not visceral, lymphatic vessels. By using virus-mediated VEGF-C gene therapy, they were able to generate functional lymphatic vessels in the Chy mice, suggesting that growth factor gene therapy is applicable to human lymphoedema.

MBA-MD-435 breast cancer cells overexpressing VEGF-C were injected into nude mice and developed tumours with enlarged peritumoural lymphatics and a high number of intratumoural lymphatics compared with control tumours. In addition, MBA-MD-435 tumours exhibited enhanced rate of metastasis to lymph nodes and lung (Skobe et al. 2001a,b). Overexpression of VEGF-C in the MCF-7 human breast carcinoma cell line resulted in increased tumour growth, in the absence of angiogenesis, and parallel to lymphangiogenesis, particularly at the tumour periphery (Karpanen et al. 2001; Mattila et al. 2002). Moreover, a high rate of metastatic spread to regional lymph nodes was observed in contrast to mice injected with cells transfected with empty vector, and lymphangiogenesis was blocked by a soluble form of VEGFR-3 (VEGFR-3-Ig) (Mattila et al. 2002). Tumours derived from a VEGF-C-overexpressing melanoma cell line gave increased blood and lymphatic vessels and tumour-associated macrophages compared with control tumours (Skobe et al. 2001a,b). Overexpression of VEGF-C in the AZ521 human gastric carcinoma cell line also led to both increased growth of intratumoural lymphatic vessels and metastatic potential of cells when injected into nude mice (Yanai et al. 2001).

Overexpression of VEGF-D induced angiogenesis and increased tumour growth compared with controls transfected with the empty vector. VEGF-D also induced extensive lymphangiogenesis and metastatic spread via the lymphatics to draining lymph nodes (Stacker et al. 2001).

Culturing and purifying of lymphatic endothelial cells

Beginning in 1984, pure cultures of lymphatic endothelium were first isolated by Johnston & Walker from bovine mesenteric lymphatic collecting vessels (Johnston and Walker, 1984) and by Witte et al. from a patient with a massive cervicomediastinal lymphangioma and another patient with a large retroperitoneal chyle-containing lymphangioma (reviewed in Way et al. 1987). Lymphatic endothelium was isolated from thoracic duct (Gnepp & Chandler, 1985; Weber et al. 1994a). This vessel is immersed in the periadventitial fat of aorta and can be visualized after injection of Evans blue into its caudal end (Fig. 7). Other authors cultured bovine, ovine, rat and mouse lymphatic endothelium through multiple passages (Yong & Jones, 1991; Leak & Jones, 1993; Borron & Hay, 1994; Weber et al. 1994b). Lymphatic endothelial cells have been grown as a monolayer and on microcarrier beads using fetal bovine serum with and without heparin and endothelial cell growth supplement. In each instance, endothelium grew slowly in sheets with a cobblestone morphology, showing characteristic lymphatic-like overlapping cell junctions. Proliferation rate depended on varying growth conditions in primary and passage levels and there are reports of doubling times as high as 36–48 h (Yong & Jones, 1991).

image

Figure 7. Caudal end of bovine thoracic duct stained with Evans blue (arrow) in the periadventitial fat of the thoracic aorta (A).

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Lymphatic explant proliferation and sprouting has also been documented (Nicosia, 1987). Haemangiogenic growth factors, such as VEGF-A and fibroblast growth factor-2 (FGF-2), are capable of inducing lymphangiogenic activity in cultures, i.e. lymphatic endothelial cells invade collagen or fibrin gels and form tubules with lumens (Pepper et al. 1994). Indeed, lymphatic endothelial cells may remain pluripotent in vitro and dedifferentiate or transdifferentiate by modifying gene expression common to lymphatic and blood vascular endothelial cells. These findings imply that relatively pure endothelial populations can be isolated from lymphatic vascular tumours and lymphatic collecting vessels and propagated for long periods in vitro while retaining morphological characteristics similar to those of blood vascular endothelium.

Makinen et al. (2001b) isolated and cultured stable lineages of blood vascular and lymphatic endothelial cells from human dermal microvascular endothelium by using antibodies against the extracellular domain of VEGFR-3. They showed that VEGFR-3 stimulation alone protects the lymphatic endothelial cells from serum deprivation-induced apoptosis and induces their growth and migration.

Molecular regulation of lymphangiogenesis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Lymphangiogenesis
  5. Embryo lymphangiogenesis
  6. Structural features of the lymphatic system
  7. Lymphangiogenesis in vivo
  8. Markers of lymphatic endothelial cells
  9. Experimental models of lymphangiogenesis
  10. Molecular regulation of lymphangiogenesis
  11. Concluding remarks
  12. Acknowledgements
  13. References

Major advances in our understanding of the development and growth of lymphatic vessels stemmed from the discovery of a wide array of molecular mediators. The VEGF-C/VEGF-D/VEGFR-3 signalling system is currently thought to play a central role in the control of these processes in embryogenesis and other conditions.

VEGF-C

VEGF-C is a VEGF isoform closely related to VEGF-D, characterized by the presence of unique amino- and carboxy-terminal extensions flanking the common VEGF-homology domain (Joukov et al. 1996). VEGF-C is synthesized as a precursor protein that undergoes subsequent proteolytic processing reminiscent of the PDGF-A and PDGF-B chain processing, which suggests an evolutionary relationship (Heldin et al. 1993). The proteolytic processing probably provides a regulatory mechanism that allows fine tuning of the biological functions of VEGF-C.

Unprocessed VEGF-C binds to VEGFR-3 and the stepwise proteolytic processing of VEGF-C generates several VEGF-C forms with increased affinity for VEGFR-3, but only the fully processed VEGF-C is able to activate VEGFR-2 (Joukov et al. 1997). Because VEGFR-2 is present in many types of endothelia and VEGF-C is broadly expressed, it may well be that the biosynthesis of VEGF-C as a precursor prevents unnecessary angiogenesis elicited via VEGFR-2, and allows VEGF-C to signal preferentially via VEGFR-3, which is restricted to the venous endothelia during early stages of development and to the lymphatic endothelium during later stages. In certain circumstances, proteolytic processing would release mature VEGF-C, which signals via both VEGFR-3 and VEGFR-2, whose consensual activation may be necessary for full biological response to VEGF-C (Joukov et al. 1997).

VEGF-C may play several functions in the organization of the vascular tree. In VEGF-C-deficient mice, endothelial cells commit to the lymphatic lineage but do not sprout to form lymphatic vessels (Karkkainen et al. 2004). Sprouting was rescued by VEGF-C and VEGF-D but not VEGF-A, indicating VEGFR-3 specificity. The lack of lymphatic vessels resulted in prenatal death due to fluid accumulation in tissues. VEGFR-2-deficient mice die at an earlier stage than VEGF-A-deficient mice (Dumont et al. 1998). It is thus possible that other VEGFR-2 ligands, including VEGF-C, may compensate for the loss of VEGF-A.

VEGF-C induces lymphangiogenesis in the ears of mice and in the CAM (Oh et al. 1997; Enholm et al. 2001) and lymphatic vessel enlargement in the skin (Jeltsch et al. 1997). VEGF-C also has potent effects on blood vessels because its fully processed form also binds to VEGFR-2 of blood vessels and stimulates angiogenesis (Cao et al. 1998). Furthermore, VEGFR-3 is also expressed on endothelial cells of tumour blood vessels, and is thought to play an angiogenic role (Partanen et al. 1999).

Compared with VEGF-A, VEGF-C is 4–5 times less potent in the vascular permeability assay (Joukov et al. 1997). VEGF-C mRNA levels are increased by serum and its growth factors PDGF, epidermal growth factor (EGF) and transforming growth factor-β (TGF-β), and by the tumour promoter phorbol myristate 12,13-acetate (PMA) (Enholm et al. 2001). Conversely, hypoxia, Ras oncoprotein and mutant p53 tumour suppressor do not influence VEGF-C mRNA levels. Interleukin-1β (IL-1β) and the tumour necrosis factor-α (TNF-α) stimulate VEGF-C expression in human lung fibroblasts and human umbilical vein endothelial cells (Ristimaki et al. 1998). Furthermore, the anti-inflammatory dexamethasone inhibits IL-1β-induced VEGF-C mRNA expression. Hence, VEGF-C seems to be also a mediator of inflammatory reactions (Narko et al. 1999).

However, the biological effects of VEGF-C are tissue-specific and dependent on the abundance of blood vessels and lymphatics expressing its receptors in a given tissue. Saaristo et al. (2002) characterized the in vivo effects of VEGF-C on blood and lymphatic vessels in the skin and respiratory tract of nude mice: VEGF-C gave a dose-dependent enlargement and tortuosity of veins, which along with the collecting lymphatic vessels, were found to express VEGFR-2. Expression of angiopoietin-1 (Ang1) blocked the increased leakiness of the blood vessels induced by VEGF-C, whereas vessel enlargement and lymphangiogenesis were not affected. However, angiogenic sprouting was not observed.

VEGF-D

VEGF-D was first isolated from a differential display screening of murine fibroblast genes from mice carrying a targeted inactivation of the c-fos gene (Orlandini et al. 1996). The identified protein was first named ‘c-fos-induced growth factor’, but later renamed VEGF-D. After or during secretion, VEGF-D can be proteolytically cleaved at the N- and C-terminal regions of the VHD (Stacker et al. 1999). The processing of VEGF-D is required to produce a growth factor that binds both VEGFR-2 and VEGFR-3 with high affinity. The fully processed VEGF-D binds VEGFR-2 and VEGFR-3 with greater affinity than does unprocessed VEGF-D. The identification of the protease(s) responsible for VEGF-D processing will be important for determining the biological context of the regulation of the receptor affinity and specificity of VEGF-D.

VEGF-D is angiogenic in the rabbit cornea assay (Marconcini et al. 1999). In a mouse tumour model, VEGF-D promoted lymphangiogenesis (Achen et al. 1998) and metastatic spread via the lymphatics (Stacker et al. 2001). Lymphatic spread was blocked by a VEGF-D-specific antibody. Achen et al. (2002) analysed VEGF-D activity in human tumours and a mouse model of metastasis. Tumour vessels positive for VEGF-D were also positive for VEGFR-2 and/or VEGFR-3 but negative for VEGF-D mRNA, indicating that VEGF-D is secreted by tumour cells and subsequently associates with endothelium via receptor-mediated uptake. In the mouse model, VEGF-D synthesized in tumour cells became localized on the endothelium and thereby promoted metastatic spread. Overall data indicate that VEGF-D promotes tumour angiogenesis, lymphangiogenesis and metastatic spread by a paracrine mechanism.

Byzova et al. (2002) demonstrated that an adenovirus encoding the mature form of human VEGF-D induced predominantly angiogenesis in the rat cremaster muscle, and both angiogenesis and lymphangiogenesis when injected into the epigastric skin. Immunohistochemical analysis of the cremaster muscle demonstrated that adenovirus-induced neovascularization was composed primarily of laminin and VEGFR-2-positive vessels containing red blood cells, thus indicating a predominantly angiogenic response. In the skin model, the adenovirus induced angiogenesis and lymphangiogenesis, as indicated by staining with laminin, VEGFR-2 and VEGFR-3.

These data suggest that the absolute and relative expression levels of VEGFR-2 and VEGFR-3 in endothelium may influence whether VEGF-C/VEGF-D elicit angiogenic or lymphangiogenic effects. A strict demarcation of the effects of VEGFR-2 and VEGFR-3 activation in terms of angiogenesis and lymphangiogenesis is complicated by observations that VEGFR-3 is involved in maintenance of tumour blood vessels in a mouse model (Kubo et al. 2000) and humans (Partanen et al. 1999), and that VEGFR-2 expression is detectable on lymphatic endothelial cells (Kriehuber et al. 2001). Furthermore, the influence of other positive and negative regulators of angiogenesis and lymphangiogenesis may also modulate the biological consequences of VEGF-C and VEGF-D expression.

Vascular endothelial growth factor receptor-3 (VEGFR-3/Flt4)

VEGFR-3/Flt4 is a highly glycosilated, relatively stable surface tyrosine kinase of approximately 180 kDa. Its cDNA was cloned from human erythroleukaemia cells and placental libraries (Aprelikova et al. 1992).

Like VEGFR-1 and VEGFR-2, it is a member of a subfamily of receptor protein tyrosine kinases that are characterized by an extracellular region containing seven immunoglobulin-related domains and an intracellular domain with homology to the platelet-derived growth factor receptor (PDGFR) subfamily. Two alternatively spliced isoforms of VEGFR-3 have been described, which differ in the length of their cytoplasmic domains and probably in different signalling properties (Borg et al. 1995).

In addition to being expressed on lymphatic endothelial cells, VEGFR-3 has also been detected on cells of the haematopoietic system (Fournier et al. 1995), perhaps reflecting a common stem cell origin for haematopoietic and endothelial cells. Targeted inactivation of the VEGFR-3 gene is lethal in embryo mice (Dumont et al. 1998). The embryo dies because of defective blood vessel and heart development. This occurs at day 9.5, before the development of the lymphatic system, which emphasizes that VEGFR-3 is expressed in developing blood vessel in embryogenesis and only later becomes sufficiently restricted to the lymphatic system (Kaipainen et al. 1995). Moreover, VEGFR-3 can also be expressed on blood capillaries during tumour angiogenesis (Kubo et al. 2000). Overall, VEGFR-3 is still one of the best and most widely used markers of lymphatic endothelium.

Other regulators of lymphangiogenesis

Other molecules have been implicated in the development and/or maintenance of the lymphatic system and presumably of lymphangiogenesis. Transgenic mice lacking the Ets DNA-binding domain of the transcription factor Net die following birth due to the accumulation of fluid within the thoracic cavity (chylothorax) that is associated with dilated lymphatic vessels (Ayadi et al. 2001). Mice lacking the α9 integrin subunit also succumb to chylothorax (Huang et al. 2000) and mice deficient in angiopoietin-2 (Ang2) have abnormal lymphatic vessels, rescued by Ang1 (Gale et al. 2002). A protein expressed by lymphatic endothelium, for which a role in lymphatic development has yet to be defined, is neuropilin 2 (NRP-2). This is a non-tyrosine kinase receptor that binds the collapsin/semaphorin family of axon-guidance molecules and certain members of the VEGF family. It is suggested that NRP-2 is involved in the VEGFR-3-mediated signal transduction at sites where the VEGFR-2 and VEGFR-3 are coexpressed (Karkkainen et al. 2001).

Yuan et al. (2002) showed a selective requirement for NRP-2 in the formation of lymphatic vessels. Loss of NRP-2 function resulted in the absence or severe reduction in the number of small lymphatic vessels and capillaries in all tissues examined. Lymphatic vessel number was decreased as soon as these vessels developed in the embryo, and remained reduced or absent until after birth. The lymphatic vessels present in the skin of mutant animals were also located abnormally and sometimes enlarged, suggesting that guidance defects may also occur in the absence of NRP-2 function. Veins, which express lower levels of NRP-2, developed normally in the NRP-2 null animals suggesting that the NRP-2 mutation selectively affected the lymphatic compartment.

Abtahian et al. (2003) identified a failure to separate emerging lymphatic vessels from blood vessels in mice lacking the haematopoietic signal protein SLP-76 or SyK. Blood lymphatic connections lead to embryonic haemorrhage and arteriovenous shunting. Expression of SLP-76 could not be detected in endothelial cells, and blood-filled lymphatics also arose in wild-type mice reconstituted with SLP-76-deficient bone marrow.

Secondary lymphoid organ chemokine (SLC) is expressed in high endothelial venules and in T-cell zones of spleen and lymph node. Gunn et al. (1999) demonstrated that expression of SLC is undetectable in mice homozygous for the paucity of lymph node T-cell mutation.

Fang et al. (2000) demonstrated that FOXC2 knockout mice display cardiovascular, craniofacial and vertebral abnormalities similar to those seen in lymph-edema-distichiasis, and autosomal dominant disorder characterized by lymphoedema of the limbs and double rows of cyclashes (distichiasis). FOXC2 represents the second known gene to result in hereditary lymphoedema.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Lymphangiogenesis
  5. Embryo lymphangiogenesis
  6. Structural features of the lymphatic system
  7. Lymphangiogenesis in vivo
  8. Markers of lymphatic endothelial cells
  9. Experimental models of lymphangiogenesis
  10. Molecular regulation of lymphangiogenesis
  11. Concluding remarks
  12. Acknowledgements
  13. References

Until recently, the lymphatic vessels have received much less attention than blood vessels, despite their importance in medicine. Recent advances in understanding the development of the lymphatic system and the growth of lymphatic vessels have clearly demonstrated that it is mainly regulated by the VEGF-C/VEGF-D/VEGFR-3 signalling system (Fig. 8). Nevertheless, very little is known about the mechanisms and mediators of lymphangiogenesis.

image

Figure 8. Schematic representation of the VEGF-C/VEGF-D/VEGFR-3 signalling system.

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Identification of other molecules involved in the growth and development of the lymphatic vasculature will delineate more approaches for molecular manipulation of the lymphatics. Moreover, identification of genes that are differentially expressed between lymphatic and blood endothelial cells would shed more light on the different biological functions of these endothelia and would confirm that these two vascular systems work in a tightly regulated manner.

This research will provide an opportunity for the development of new and innovative therapeutic strategies for cancer and other human diseases.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Lymphangiogenesis
  5. Embryo lymphangiogenesis
  6. Structural features of the lymphatic system
  7. Lymphangiogenesis in vivo
  8. Markers of lymphatic endothelial cells
  9. Experimental models of lymphangiogenesis
  10. Molecular regulation of lymphangiogenesis
  11. Concluding remarks
  12. Acknowledgements
  13. References

This work was supported in part by Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan, and the Ministry for Education, the Universities and Research (MIUR, Molecular Engineering – C03 funds, Molecular Preclinical Therapy in Oncology – SP4 funds, and Interuniversity Funds for Basic Research – FIRB), Rome, Italy. The technical assistance of Ms Milena Rizzi is greatly acknowledged.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Lymphangiogenesis
  5. Embryo lymphangiogenesis
  6. Structural features of the lymphatic system
  7. Lymphangiogenesis in vivo
  8. Markers of lymphatic endothelial cells
  9. Experimental models of lymphangiogenesis
  10. Molecular regulation of lymphangiogenesis
  11. Concluding remarks
  12. Acknowledgements
  13. References
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