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VEGFR-3 Ligands and Lymphangiogenesis (1)

part one

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Angiogenesis blood vessel growth), lymphangiogenesis (lymph system growth) are all intrinsically connected with lymphedema and share many of the same genes. We have several pages on both processes.

Pat O'Connor

May 23, 2008

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VEGFR-3 Ligands and Lymphangiogenesis

Michael Jeltsch

Molecular/Cancer Biology Laboratory
Biomedicum Helsinki and Haartman Institute

Department of Biosciences, Division of Biochemistry
Faculty of Science

University of Helsinki
Finland

Academic dissertation

To be publicly discussed with the permission of the Faculty of Science of the University of Helsinki in the lecture hall 3, Biomedicum Helsinki, Haartmaninkatu 8, Helsinki, on November 29th, 2002, at 12:00.

Helsinki 2002

ISBN 952-10-0652-8 (HTML version)
University of Helsinki Online Publications
Helsinki 2002

ABSTRACT

Most of us have seen their own blood at one or the other time. The occasion might have been a small accident or in unfortunate cases a severe blood loss caused by a major injury. We also can feel our heart beating and the resulting pressure wave, the pulse. The existence of the cardiovascular system is obvious to us. Unlike the cardiovascular system, the lymphatic system has, until recently, escaped notable attention not only by the laymen, but also by the scientific community. It is unclear why the lymphatic system originally developed in higher vertebrates. Now, its main function seems to be to collect fluid that has leaked from the blood vessels and to return it into the cardiovascular system. Much of our knowledge about the development and structure of the lymphatic system is of considerable age, and it has been said that there has not been any progress in our understanding since the fine structure of the lymphatics was described with the introduction of the electron microscope.

Vascular endothelial growth factor (VEGF) is the principal direct inducer of blood vessel growth, but it does not promote the growth of lymphatic vessels. This study demonstrates for the first time specific lymphangiogenesis as a response to the VEGF homologue VEGF-C. Overexpression of full length VEGF-C under the keratin-14 promoter in the skin of transgenic mice caused a proliferation of the lymphatic endothelium and lymphatic vessel enlargement. In the chorioallantoic membrane assay, the mature form of VEGF-C was also largely specific for lymphatic endothelial cells. A newly discovered close homologue of VEGF-C, VEGF-D was then shown to have the same receptor-binding pattern as VEGF-C.

Contrary to the interaction of VEGF with its receptors, VEGF-C interaction with VEGFR-3 has not been analyzed at the molecular level. The structural determinants of VEGFR-3 binding were characterized in relation to VEGF using a non-random family shuffling approach with VEGF and VEGF-C as parent molecules. This approach led to the identification of VEGF/VEGF-C mosaic molecules that showed novel receptor binding profiles and a panel of these molecules was used to delineate the requirements of specific receptors in the induction of angiogenesis versus lymphangiogenesis.

1 The Two Vascular Systems

Large multicellular organisms with a high metabolic demand use carrier molecules to distribute oxygen within their bodies. In vertebrates a closed vascular system guides the transport of these carrier molecules. Therefore the vascular system has to be functional early in development. When the human embryo reaches the size of approximately 3 mm at embryonic day 22, its heart starts beating. Later, when the cardiovascular system is already functioning, a second vascular system develops: the lymphatic system. Unlike the cardiovascular system it is not a circulatory system. Lymphatic flow starts in blind-ended capillary networks that penetrate most of the body tissues. Collecting lymphatics drain the capillary networks and after collecting fluid from many tributaries the largest collecting lymphatic vessel (the thoracic duct) ultimately reaches the veins. A schematic view of the general setup of the lymphatic system is given in Figure 1.

Compared to our tremendous knowledge about the cardiovascular system the understanding of the lymphatic system is still very rudimentary. The lymphatic system has lagged behind the cardiovascular system both in its discovery and exploration, probably due to the cardiovascular system's visual prevalence and importance.

Retrospectively, three heydays have shaped our understanding of the lymphatic system. The first took place during the first years of the 20th century when researchers like Sabin, Kampmeier, Huntington and McClure were studying the ontogeny of the lymphatic system (reviewed by Wilting et al. 1999). The second boost of knowledge resulted from the use of the electron microscope during the 1960s to solve questions about the fine structure of the initial lymphatics and their functioning (reviewed by Leak 1970). At the moment, lymphatic research is experiencing an impressive comeback thanks to the arrival of molecular biology, genetics and, last but not least, by the discovery of markers specific for lymphatic endothelium (reviewed by Oliver and Detmar 2002).

2 The Cardiovascular System

During early embryogenesis, most of the blood vessels form by vasculogenesis, the in-situ formation of an immature network of endothelial channels by the differentiation of precursor cells (angioblasts). Vasculogenesis starts in extra-embryonic tissues where putative mesodermal precursor cells (hemangioblasts) aggregate into blood islands. Cells in the center of a blood island develop into hematopoietic stem cells, and cells at the periphery differentiate into angioblasts. In the embryo proper, vasculogenesis gives rise to the heart endocardium, the paired dorsal aortas and the primitive vasculature of endodermally derived organs (e.g. lung, spleen and pancreas; reviewed by Wilting and Christ 1996).

The primitive vascular network grows and remodels into a functional, hierarchical structure containing large caliber vessels for low-resistance fast flow and small capillaries optimized for diffusion. Different cellular and molecular mechanisms (splitting, fusion, sprouting, and intercalation) participate in the remodeling and expansion and they are collectively referred to as angiogenesis (reviewed by Risau 1998). While most organs become vascularized by a combination of vasculogenesis and angiogenesis, initially avascular ectodermal tissues such as the brain, become vascularized exclusively by angiogenic mechanisms (Plate 1999).

The endothelial cell layer (intima) of vessels larger than capillaries becomes wrapped by additional sheets: a cover of muscular tissue (media) and connective tissue (adventitia). Therefore, arteriogenesis requires the recruitment and organization of non-endothelial cells (reviewed by Carmeliet 2000a). Recent data shows that during embryonic development blood vessels not only fulfill transport functions but also play important roles in the induction and morphogenesis of organs (Lammert et al. 2001; Matsumoto et al. 2001). It has been thought that angiogenesis is the only way of neovascularization in adult organisms, but recently a population of progenitor cells able to differentiate into endothelial cells was isolated from circulating blood (Asahara et al. 1997) and identified as originating from bone marrow (Shi et al. 1998). Although the evidence for vasculogenesis from circulating endothelial precursors is convincing, the relative contribution of endothelial precursors to adult physiological or pathological angiogenesis is still unclear (Asahara et al. 1999; Crosby et al. 2000). In any case, there is reason to rigorously review these reports, since it has been shown that cell fusion events can produce experimental results that can be misinterpreted as differentiation events (Terada et al. 2002).

Quiescence is the default state of adult vasculature, and only few physiological processes in the adult involve endothelial cell proliferation, e.g. the female reproductive cycle (reviewed by Reynolds et al. 1992) or exercised-induced muscular hyperplasia (reviewed by Tomanek and Torry 1994). On the other hand, in disease angiogenesis frequently contributes to the pathological process (e.g. in tumorigenesis) and even may be the main pathological process itself (e.g. in wet age-related macular degeneration; reviewed by Carmeliet and Jain 2000).

Vascular endothelium is heterogeneous. Three distinct types of blood capillaries can be distinguished: continuous, fenestrated and discontinuous. Continuous endothelium has an uninterrupted basement membrane and most of the capillaries belong to this type. Fenestrated endothelium is characterized additionally by the presence of circular transcellular openings (fenestrae) with a diameter of 60 to 80 nm. Fenestrated endothelium is usually found in organs with high rates of fluid exchange (small intestine, kidney, salivary glands). Discontinuous endothelium has large intercellular gaps (up to 1 microm) and no basement membrane, and allows for almost unrestricted transport of molecules from interstitium to capillary lumen. Discontinuous endothelium is only found in specialized organs, e.g. in the liver, spleen and bone marrow (reviewed by Risau 1998).

3 Form and Function of the Lymphatic System

The lymphatic system consists of a network of thin-walled vessels that drain fluid and particular matter from the interstitial spaces. However, unlike blood vessels, the lymphatics do not form a circular system. The unidirectional lymph flow recovers fluid from the periphery and returns it to the cardiovascular system (see Figure 1).

Lymphatic flow begins in the capillary networks (initial or terminal lymphatics). Lymphatic capillaries consist of a single layer of non-fenestrated endothelial cells sitting on an incomplete basement membrane. Prenodal collecting vessels drain the capillary networks and transport lymph to the regional lymph nodes. Lymphatic capillaries are not invested with mural cells, but collecting vessels do posses a smooth muscle cell layer (Schmid-Schonbein 1990b). Postnodal collecting vessels carry lymph between successive sets of lymph nodes or to larger lymphatic collecting vessels. Eventually, the larger lymph-collecting vessels drain lymph from the final set of lymph nodes into the lymph ducts. Lymph from the intestinal, hepatic and lumbar region drains into the cisterna chyli. The cisterna chyli acts as a collecting reservoir at the posterior end of the thoracic duct. The thoracic duct ascends upwards from the cisterna chyli. On its way it receives lymph from the rest of the body except from the upper right quadrant. The thoracic duct empties directly into the venous blood at the junction of the left internal jugular vein and the left subclavian vein. The lymph of the upper right quadrant is collected into the right lymphatic duct and returned into the veins at the right jugulo-subclavian confluence (for a general review of the anatomy and the function of the lymphatic system see Foster 1996; Swartz 2001).

Figure 1. Schematic view of the lymphatic system (adapted from Klein 1990)
Figure 1

3.1 Drainage

The heart is a powerful pump, and when blood enters the capillary bed it is still under a pressure of about 4-5 kPa. Due to this pressure 20 to 30 liters of plasma leak each day from the capillaries and become interstitial fluid (Landis and Pappenheimer 1963). The orthodox view is that about 90% of this extravasated fluid is reabsorbed at the venous end of the capillaries and post-capillary venules, driven by osmotic forces (Starling 1895-96). The remaining 10% are drained by the lymphatic vessels and returned to the cardiovascular system. For this reason lymphatic capillary beds are found in most vascular tissues. It should be noted, however, that not all experimental data supports the reabsorption theory (Bates et al. 1994). Two to five liters of thoracic duct lymph are formed in humans each day (Bierman et al. 1953; Linder and Blomstrand 1958). Lymph contains most of the components of plasma, although the concentration of high molecular weight components is lower due to the capillary filtration effect. The total protein concentration is about two thirds of that of serum (Bergstrom and Werner 1966; Werner 1966).

3.2 Immune Defence

In addition to the drainage function, the lymphatics play multiple important roles in immune defence mechanisms. If body tissues are invaded by pathogens some are taken up together with the interstitial fluid by the lymphatics. The lymph passes through one or several lymph nodes before it enters the venous system. In collaboration with the antigen-presenting cells of the lymph nodes, T and B cells recognize non-self epitopes and mount an immune response. The activated immune cells proliferate in the lymph nodes and produce antibodies. Both cells and antibodies are delivered into the general circulation via the lymphatics. Not only the interstitial spaces but also the blood is screened via the lymph nodes, since about 50% of the plasma protein passes through the lymphatics each day (Klein 1990).

3.3 Intestinal Absorption

Another significant function of the lymphatics is the intestinal absorption and transport of long chain dietary triglycerides and lipophilic compounds such as fat-soluble vitamins or chlorinated organic compounds. After absorption from the gastrointestinal tract, blood and lymph capillaries compete for the transport of molecules to the systemic circulation. The majority of enterally administered compounds is absorbed into the portal blood since its throughput is about 500 times higher than that of the lymph. However, high molecular weight molecules and colloids are preferentially taken up by the lymphatics because of the highly permeable structure of the intestinal villous lymphatics (chylous vessels or lacteals). E.g. chylomicrons have a diameter of 200 to 800 nm and after being assembled and released by the enterocyte, gain access almost exclusively to chylous vessels. When pharmacological substances are absorbed by intestinal blood capillaries and transported via the portal blood to the liver, a large proportion can become inactivated (first-pass metabolic effect). Intestinal lymphatic absorption bypasses first-pass metabolic effects (Porter 1997).

3.4 Lymph Formation

The endothelial cells of the initial lymphatics lack tight junctions. Instead they are equipped with overlapping endothelial junctions, which function as mechanical flap valves. Filaments anchor the lymphatic endothelial cells into the surrounding connective tissue and are involved in the operation of the valves. Increased interstitial pressure forces these inter-endothelial valves to open and interstitial fluid and particulates gain access to the lymphatic lumen (Casley-Smith 1980).

In addition to fluid uptake via the valves, fluid transport by transcytosis and transendothelial channels plays a role (Leak 1971). Also both the hydrostatic and osmotic pressure gradient between lymphatic lumen and the interstitium have been suggested to contribute to lymph formation, although these forces cannot account for the removal of large proteins and particulate matter (Casley-Smith 1982a). The relative contributions of these mechanisms varies, and the mechanical valves seem to be especially important in situations of increased functional demand.

3.5 Lymph Propulsion

Lymphatic capillaries have no intrinsic contractility and depend entirely on extrinsic forces for lymph propulsion. The notable exception are the initial lymphatics in the bat wing, which do have their own contractile smooth muscle (Hogan and Unthank 1986). Alternating compression and dilation of the lymphatics by respiratory movement, contraction of skeletal and intestinal muscles, and the pressure pulse generated by adjacent arteries and arterioles propels lymph forward (Schmid-Schonbein 1990a). The directional flow of the lymph is maintained by funnel-shaped valves. The unit between two valves in the collecting lymphatics is termed lymphangion.

Contrary to the initial lymphatics, collecting lymphatics are contractile. Lymph flow from one lymphangion to the next is maintained against a pressure gradient and both extrinsic forces and contractions of the smooth muscle cell layer work together to pump the lymph against this gradient (Kinmonth and Taylor 1956; Olszewski and Engeset 1980). Median pressure in the initial lymphatics is close to atmospheric or interstitial values (Zweifach and Prather 1975). The pressure rises in the collecting lymphatics, and in the thoracic duct diastolic pressure ranges between -0.7 and 2.3 kPa and systolic pressure between 0.3 to 3 kPa (Kinnaert 1973).

3.6 Pre-lymphatic Channels

Interstitial transport happens both by diffusion and convection. Movement of large molecules and particles in the interstitium is not always uniform (Jain and Gerlowski 1986), suggesting the existence of preferred pathways ("pre-lymphatic tissue channels"; Casley-Smith 1980), but the significance of these channels and even their existence has been questioned (Casley-Smith 1982b). Drainage can be achieved via "pre-lymphatic channels" as seen in the central nervous system: there is little doubt about the quasi-lymphatic function of the "pre-lymphatic" perivascular spaces in the brain. These spaces (Virchow-Robin spaces) connect to the cervical lymph nodes (Casley-Smith et al. 1976), and this connection is important for both drainage and immune response to brain infections (reviewed by Esiri and Gay 1990; Weller et al. 1996). Based on these functional criteria these channels have been occasionally classified as lymphatic, although they are devoid of an endothelial lining. However, capillary filtration is greatly reduced in the brain due to the blood brain barrier, and the majority of drainage is accomplished via the cerebrospinal fluid by the arachnoid villi and the adjacent specialized venous sinuses of the dura (Weed 1922/1923). There are several other vascular structures whose classification based on immunohistochemical and functional criteria remains inconclusive. These structures include the canal of Schlemm in the eye (Foets et al. 1992) and the perivascular spaces of the liver. It has been shown that genes specific for lymphatic endothelial cells, e.g. the receptor tyrosine kinase VEGFR-3, can be upregulated in non-lymphatic endothelial cells that fulfill a lymphatic function (Partanen et al. 2000). In the liver both blood vascular endothelial cells and hepatocytes line the perivascular spaces of the discontinuous liver capillary endothelium (Spaces of Disse). Despite little evidence they are assumed to fulfill a draining function, especially since the liver lobules themselves do not contain lymphatics (Niiro and O'Morchoe 1986; Trutmann and Sasse 1994). Also non-endothelial cells with an endothelial function have been shown to express VEGFR-3, e.g. the trophoblast cells of the placenta (Dunk and Ahmed 2001).

The matrix-lined channels seen in some melanomas are reminiscent of pre-lymphatic tissue channels. It was suggested that these channels participate in the tumor circulation, and such a behavior has been termed vasculogenic mimicry (Maniotis et al. 1999; Folberg et al. 2000). Tumor cells are known to contribute to the intima of tumor vessels, but the absence of endothelial cells in vasculogenic mimicry as described by Maniotis et al. remains controversial (McDonald et al. 2000).

4 Development of the Lymphatic System

During the last 80 years developmental biology of the vascular system has focused on its cardiovascular part. Our knowledge about the development of the lymphatic system is based almost entirely on studies done at the beginning of the 20th century. The landmark studies of lymphatic development have been done in very diverse species such as pig, frog and chicken, which might explain some of the controversial findings. Despite being the standard model organism, the mouse is still one of the less well characterized species concerning embryonic lymphatic development, although the situation is rapidly changing. Among the nowadays frequently used model organisms only the mouse has a large number of lymph nodes as do humans. However, it is still unknown whether the mouse can serve as a truly good model for all aspects of lymphatic research, due to its small size (creating only a minute amount of hydrostatic pressure), differences in the lymphatico-venous connections and many differences at the molecular level.

In the mouse the development of the lymphatic system starts at E10.5 (corresponding to 6.5-7 weeks of human embryonic development and E4.5 in the chick). By that time the cardiovascular system is already fully functional (Clark and Clark 1920; van der Putte 1975a; b). A discrete population of endothelial cells expressing the lymphatic-specific transcription factor Prox-1 can be already identified at E9.5. They are located on one side of the anterior cardinal vein, and at E10.5 the first lymphatic outgrowths (lymphatic primordia) can be identified at that location (Wigle and Oliver 1999; Wigle et al. 2002). It is not understood what induces the outgrowth of these lymphatic primordia. The lymphatic primordia remodel and finally fuse into lymphatic plexuses (lymph sacs). There is considerable inter-species variance in the number and exact location of the lymphatic primordia and lymph sacs, although the jugular region seems always to be the main area of lymphatic induction. In mammalian embryos eight lymph sacs have been described: the paired jugular, subclavian and posterior lymph sacs, the unpaired retroperitoneal sac and the cisterna chyli (Sabin 1909; van der Putte 1975a). Two major contradicting theories have emerged about the events that follow the above-mentioned formation of the lymph sacs.

4.1 Centrifugal Sprouting

According to Sabin the peripheral lymphatic system develops from the embryonic lymph sacs exclusively by the sprouting of endothelial cells into the surrounding tissues and organs (Sabin 1902; Clark 1912). Most recent data favors this theory, including expression studies of lymphatic-specific markers (Kaipainen et al. 1995; Kukk et al. 1996) and the Prox-1 knock-out mouse (Wigle et al. 2002). However, there is no agreement among the advocates of the centrifugal sprouting theory about the relationship of the embryonic lymph sacs to the adult lymphatico-venous communications. According to Huntigton and McClure (1910) all lymph sacs lose their connections with the veins and the adult jugular lymphatico-venous communication is a secondary development. Alternatively, the jugular communication of the adult animal is a persisting embryonic communication and data from the mouse argues for this theory (van der Putte 1975b).

4.2 Centripetal Sprouting

McClure and Huntington proposed a vasculogenic mechanism for the establishment of the peripheral lymphatic system. In the mesenchyme lymphatic spaces would arise independently from the veins, fusing into a primitive lymphatic network, which subsequently would spread centripetally and connect to the venous system. The centripetally sprouting lymphatics would either integrate or replace the embryonic lymph sacs (Huntington and McClure 1910; Kampmeier 1912). It is true that the luminal continuum of the lymphatic primordia to the early veins is often not seen (van der Putte 1975a), but a venous origin does not in itself require such, as individual cells might migrate to form the lymphatic primordia.

A model that incorporates both sprouting from lymph sacs and in-situ differentiation of mesenchymal precursors was already proposed in 1932 by van der Jagt and support for this model has been recently gathered. The lymphatics of the avian chorioallantoic membrane (CAM) and perhaps also the wing are apparently not only sprouts from the lymph sacs but also in-situ derivations from mesenchyme. Homotopic grafting of Prox-1 negative, day 2 quail allantoic buds into chick hosts and day 3.5 chick wing buds into quail hosts resulted in lymphatics composed of both donor and host endothelial cells in the graft area (Papoutsi et al. 2001; Schneider et al. 1999).

The development of the lymphatics, just like the one of the blood vessels, is restrained by their evolutionary origin. Much the same as the transient aortic arches in mammalian development are the lymph hearts in birds, which are formed and functional during embryogenesis but disappear by adulthood. In the adult organism lymphatic endothelial cells are normally quiescent, but angiogenic processes - both pathological and physiological - are often accompanied by lymphangiogenesis (Clark and Clark 1932; Ohtani et al. 1998; Paavonen et al. 2000; Mimura et al. 2001).

5 Lymphatics in Different Vertebrates

Until recently there have been misconceptions about the existence of lymphatics in some vertebrate classes. Additionally, sparse data rules out any attempt to draw conclusions about the phylogeny of the lymphatic system; and almost all of the research on comparative anatomy was done during the 19th and early 20th century with the limitations of that period.

5.1 Fish

Already in 1919 Mayer tried to explain the contradictory findings about the piscine lymphatics by the existence of a secondary vascular system. However, only in 1981 could Vogel and Claviez experimentally prove this existence. The secondary vascular system constitutes a separate, parallel circulatory system and includes the vessels earlier assumed to be lymphatics (Hoyer 1934). It starts from the systemic arteries, forms its own capillary networks, which supply mainly the oral mucous membranes and the skin, and returns to the systemic venous system. It functions presumably in skin respiration, osmoregulation and immune defence. Based on its anatomical and functional characteristics it has been hypothesized that the secondary circulation might be an evolutionary predecessor of the lymphatic system (Steffensen and Lomholt 1992). There is evidence for a "true" lymphatic system in lungfish and it is thus reasonable to speculate that the first occurrence of a lymphatic system was associated with the transition from aquatic to terrestrial life (Laurent et al. 1978).

Growth factors and receptors that regulate lymphatic growth and development in higher vertebrates are present in fish, but their relevance for the secondary vascular system has not been analyzed (Stainier et al. 1995).

Molecules of the same growth factor family have also been identified in invertebrates, that lack endothelial cells altogether. In Drosophila these molecules direct embryonic blood cell migration. Probably only recently did these molecules assume their roles in blood vessel and lymphatic development, and it is conceivable that blood vessels evolved from blood cells (Duchek et al. 2001; Heino et al. 2001; Cho et al. 2002). In ontogenesis, however, the inverse can be observed (Ciau-Uitz et al. 2000; de Bruijn et al. 2000).

5.2 Amphibians and Reptiles

All amphibian orders are believed to have a lymphatic system. It is comparable to the mammalian system except in frogs and toads, where the superficial initial lymphatics fuse during metamorphosis to form cutaneous lymph sacs in the adult animal (Hoyer 1934; Kotani 1990). Characteristic for all amphibians are the lymph hearts, which are located at the entry points of lymph into the veins. Entry points of the lymph into the blood circulation can occur in vertebrates in three different areas: in the jugular, the lumbar and the caudal region. Most amphibian lymphatic systems communicate with the venous system in all three areas. Lymph hearts range in number from four to six in frogs to over two hundred in caecilians. Unlike their name suggests, the main function of lymph hearts is probably not the propulsion of the lymph, but rather maintaining the directionality of lymphatic flow and regulating the entry of lymph fluid into the circulation. In reptiles lymphatico-venous communications exist in the caudal and the jugular regions, but lymph hearts are found only in the caudal region (Hoyer 1934).

5.3 Birds and Mammals

Mammals and most birds do not possess lymph hearts. However, unlike other vertebrates, mammals and aquatic birds possess lymph nodes. Vertebrates differ significantly in the number of lymph nodes: in humans there are between 400 and 500 lymph nodes while ducks have only four of them (Weidenreich et al. 1934). In most mammals multiple lymphatico-venous communications are formed during development. Usually only the paired communication in the jugular region persists into adulthood. Several mammalian species maintain lumbar communications into the inferior caval vein and the renal vein, draining the lymphatics of the lower extremities and the mesentery (Silvester 1912; Job 1918).

There is substantial intra-species variability in the setup of the lymphatic system. Humans have usually a paired jugular communication, but additional lymphatico-venous communications at both central and peripheral locations are not uncommon (Wolfel 1965; Threefoot and Kossover 1966; Pressman et al. 1967; Aboul-Enein et al. 1984).

It is not known whether the need for drainage was the primary selection force for the development of the lymphatics. Apart from a high-pressure cardiovascular system other evolutionary triggers might have been involved. Two of them, the hydrostatic pressure associated with the transition of aquatic to terrestrial life and the transition from poikilothermic to homeothermic temperature regulation, are associated with an increase in blood pressure, while a third one, the development of an adaptive immune system, does not require a high-pressure cardiovascular system per se.

6 Pathology

6.1 Blood Vessels and Lymphatic Vessels in Disease

Angiogenesis or the lack thereof is a key event in the development and progression of major pathological conditions including diabetic retinopathy, psoriasis, rheumatoid arthritis, cardiovascular diseases, and tumor growth (reviewed by Carmeliet and Jain 2000). Much of the interest in angiogenesis research is driven by the idea of therapeutic intervention. Pro-angiogenic proteins like VEGF and VEGF-C have been used in clinical studies to boost the growth and perfusion of blood vessels after vascular injuries like myocardial infarction (Isner et al. 1996; Witzenbichler et al. 1998). Also, because of convincing evidence that solid tumors are angiogenesis-dependent, anti-angiogenic compounds have entered clinical trials (Hanahan and Folkman 1996). Similar to developing embryos, tumors can only grow beyond the limits of diffusion by establishing a vascular network for the distribution of oxygen and nutrients. An imbalance between pro-angiogenic and anti-angiogenic factors results in vessel ingrowth and is followed by rapid tumor expansion (Hanahan and Folkman 1996). Despite encouraging results obtained in several mouse models of anti-angiogenic tumor therapy and pro-angiogenic therapy of ischemia, success in preclinical and clinical trials is limited (Cao 2001; Hammond and McKirnan 2001). Caution has been recommended (Carmeliet 2000b) and also principal objections against the safety and feasibility of such therapies have been raised (Blagosklonny 2001). In developed countries the angiogenesis-related cardiovascular and neoplastic diseases are major health issues, whereas lymphatic disorders are relatively rare.

6.2 Lymphedema

Insufficiency of lymphatic transport can result in lymphedema, which can either be hereditary or with unknown etiology (primary lymphedema), or a consequence of a previous disease or trauma (secondary or acquired lymphedema). Iatrogenic lymphedema, and especially postmastectomy edema, represents probably the most common lymphatic condition in civilized countries. Its incidence has been estimated to between 6 to 30% after surgical treatment of mammary carcinoma (Petrek and Heelan 1998). Surgical damage to the lymphatics is commonly thought to be the cause of post-surgical edema, but also other reasons such as compromised venous return seem to be involved (reviewed by Pain and Purushotham 2000). The worldwide most common cause of lymphedema is, however, filariasis - mostly caused by infection with Wuchereria bancrofti or Brugia malayi. These parasitic nematodes are transmitted by mosquito bites. The parasite lives and reproduces in the lymphatic system causing a massive lymphatic dilatation in early stages of the disease. In in advanced disease, lymphatic transport is blocked leading to an extreme enlargement of the limbs or other areas of the body called elephantiasis (Rao et al. 1996; Dreyer et al. 2000).

In hereditary lymphedema lymphatic vessels can either be hypoplastic or hyperplastic, but non-functional. In addition to some broad-spectrum syndromes such as Ulrich-Turner and Noonan, that are associated with lymphedema, a large number of distinct lymphedema syndromes has been described. The current phenotypic classification seems inadequate, based on recent clinical and genetic data showing that the same genetic cause can give rise to several distinct phenotypes, and the same phenotype can be caused by distinct genetic alterations (Kääriäinen 1984; Ferrell et al. 1998; Finegold et al. 2001).

6.2.1 Hereditary Lymphedema Type I and II

Type I hereditary lymphedema (Milroy disease, OMIM 153100) is an early onset form of hereditary lymphedema. In these patients, the initial superficial lymphatics of edematous areas cannot be demonstrated by fluorescence microlymphography and are believed to be absent or highly hypoplastic. However, in non-edematous areas superficial lymphatics are present (Bollinger et al. 1983).

In some families inheritance is strongly linked to dominant missense mutations in the VEGFR-3 gene on chromosome 5 (Karkkainen et al. 2000). However, penetrance is incomplete or variable in these families. Other families showed additional linkage to multiple loci on chromosomes 3, 11 and 18, unrelated to any known target genes. Thus, oligogenic pattern of inheritance, modifier genes and environmental factors might be necessary to explain the hereditary patterns. Type II hereditary lymphedema (Meige disease, OMIM 153200) differs from type I by a later disease onset (around puberty), and its etiology appears even more complex with only 10% of the families showing dominant pattern of inheritance and a penetrance of 40% (Holberg et al. 2001).

6.2.2 Lymphedema-Distichiasis Syndrome

Dominant mutations in the transcription factor FOXC2 have been identified as the cause of lymphedema-distichiasis syndrome (OMIM 153400; Fang et al. 2000; Finegold et al. 2001). In addition to lymphedema distichiasis three other lymphedema syndromes co-segregated with FOXC2 mutations: type II hereditary lymphedema, lymphedema/ptosis (OMIM 153000) and lymphedema/yellow-nail syndrome (OMIM 153300). All indentified FOXC2 mutations result in a truncated protein and the observed phenotype is likely a result of haploinsufficiency.

6.2.3 Lymphangiectasia

Lymphangiectasia is a lymphatic disorder characterized by dilated dysfunctional lymphatics. The condition can be limited to a specific organ. The lungs are affected in hereditary pulmonary cystic lymphangiectasia (OMIM 265300) and the intestine in Hennekam lymphangiectasia-lymphedema syndrome (OMIM 235510; Hennekam et al. 1989; Gilewski et al. 1996). The causes of lymphangiectasia and lymphedema are thought to be similar and both conditions do occur jointly in syndromes such as Noonan Type I (OMIM 163950), Hennekam or hereditary intestinal lymphangiectasia (OMIM 152800). Similar to lymphedema, also acquired forms are known (Celis et al. 1999).

6.3 Lymphatic Neoplasms

Occasionally, neoplasms are derived from lymphatic endothelial cells. Lymphangiomas constitute approximately 5% of all benign lesions of infancy and childhood (Zadvinskis et al. 1992). Since lymphangiomas can present either as localized mass or as a diffuse tumor, it is questionable whether all lymphangiomas represent true neoplasms (Scalzetti et al. 1991). Unlike lymphangioma, lymphangiosarcoma is a true malignant lesion of lymphatic endothelial cells. Mostly it occurs as a complication of post-mastectomy edema (Stewart Treves syndrome; Janse et al. 1995).

6.4 Tumor Lymphangiogenesis

The lymphatic system serves as the primary pathway for metastatic spread of tumor cells to regional lymph nodes, and possibly also to distant organs. The prognostic value of lymph node metastasis was recognized long before the concept of lymphangiogenesis both within and adjacent to tumors became widely accepted (Fisher et al. 1969; Carter et al. 1989). Tumor lymphangiogenesis occurs in experimental models in mice with important implications for metastasis (Karpanen et al. 2001; Mandriota et al. 2001; Skobe et al. 2001; Stacker et al. 2001). It is, nevertheless, still unclear to what extent lymphangiogenesis occurs in human cancers, and what the consequences are for cell dissemination (Leu et al. 2000; Birner et al. 2001; Jackson et al. 2001; Schoppmann et al. 2001).

6.5 Therapeutic Lymphangiogenesis

The regeneration of lymphatic vessels was observed by Clark and Clark already in 1932. However, expertise in therapeutic lymphangiogenesis is just emerging (Karkkainen et al. 2001). Several recent papers have been shown that in angiogenic therapy the balance between harm and help is not trivial (Masaki et al. 2002) and the use of single molecules is likely to be insufficient (Richardson et al. 2001). Influencing the molecular decision makers such as Hypoxia-inducible factor-1 (HIF-1) instead of the effector molecules such as VEGF might be easier than the futile attempt to mimic the temporally and spatially complex growth factor cocktail of mother nature (Vincent et al. 2000; Elson et al. 2001). Whichever pro-angiogenic therapy, it is possible that pro-angiogenic or pro-lymphangiogenic therapy leads to accelerated cancer progression and metastasis.

So far no good animal model has been developed for aquired lymphedema, but a mouse model of hereditary lymphedema does exist: the Chy mouse. Chy mice carry a mutant Vegfr-3 allel coding for a kinase-dead receptor. In this model VEGF-C was used to compensate for insufficient VEGFR-3 signaling. This resulted in the growth of new functional lymphatics (Karkkainen et al. 2001). It is not clear why VEGF-C does induce lymphatic sprouting in these mice, but fails to do so in the keratin-14 VEGF-C transgenic mice. The VEGF-C transgenic mice were edematous, indicating that also too much VEGFR-3 signaling can impair lymphatic function (I). From the visceral lymphatics in the Chy mouse, only the lacteals are aplastic although the VEGFR-3 mutation is assumed to be dominant negative in all lymphatic endothelial cells and there is circumstantial evidence that lymphangiogenesis might occur independently of VEGFR-3 activation (Taija Makinen, personal communication). Thus many molecular players are still to be identified. In conclusion, therapeutic modulation of vascular growth in pathologic conditions represents the major challenge in the fields of both angiogenesis and lymphangiogenesis.

http://ethesis.helsinki.fi/julkaisut/mat/bioti/vk/jeltsch/vegfr3li.html

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Lymphedema People Angiogenesis Related Pages:

Angiogenesis

http://www.lymphedemapeople.com/thesite/angiogenesis.htm

Angiogenesis and Cancer

http://www.lymphedemapeople.com/thesite/angiogenesis_and_cancer.htm

Angiogenesis and Cancer Control

http://www.lymphedemapeople.com/thesite/angiogenesis_and_cancer_control.htm

Angiogenesis Inhibitors and Cancer

http://www.lymphedemapeople.com/thesite/angiogenesis_inhibitors_and_cancer.htm

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Lymphedema People Lymphangiogenesis Related Pages:

The Formation of Lymphatic Vessels and Its Importance in the Setting of Malignancy

http://www.lymphedemapeople.com/thesite/lymphangiogenesis_formation_of_lymphatic_vessels_and_malignancy.htm

Lymphangiogenesis Lymphedema and Cancer

http://www.lymphedemapeople.com/thesite/lymphangiogenesis_lymphedema_and_cancer.htm

Lymphangiogenesis and Gastric Cancer

http://www.lymphedemapeople.com/thesite/lymphangiogenesis_and_gastric_cancer.htm

Lymphangiogenesis in Head and Neck Cancer

http://www.lymphedemapeople.com/thesite/lymphangiogenesis_in_head_and_neck_cancer.htm

Lymphangiogenesis and Kaposi's Sarcoma VEGF-C

http://www.lymphedemapeople.com/thesite/lymphangiogenesis_and_kaposis_sarcoma_vegfc.htm

Lymphangiogenesis in Wound Healing

http://www.lymphedemapeople.com/thesite/lymphangiogenesis_in_wound_healing.htm

A model for gene therapy of human hereditary lymphedema

http://www.lymphedemapeople.com/thesite/a_model_for_gene_therapy_of_human_lymphedema.htm

VEGFR-3 Ligands and Lymphangiogenesis (1)

http://www.lymphedemapeople.com/thesite/vegfr3_ligands_lymphangiogenesis_1.htm

VEGFR-3 Ligands and Lymphangiogenesis (2)

http://www.lymphedemapeople.com/thesite/vegfr3_ligands_lymphangiogenesis_2.htm

VEGFR-3 Ligands and Lymphangiogenesis (3)

http://www.lymphedemapeople.com/thesite/vegfr3_ligands_lymphangiogenesis_3.htm

Vascular Endothelial Growth Factor; VEGF

http://www.lymphedemapeople/thesite/vascular_endothelial_growth_factor_VEGF.htm

VEGF-D is the strongest angiogenic and lymphangiogenic effector

http://www.lymphedemapeople.com/thesite/VEGFD_Angiogenic_Lymphangiogenic_Effector.htm

Inhibition of Lymphatic Regeneration by VEGFR3

http://www.lymphedemapeople.com/thesite/Inhibition_of_Lymphatic_Regeneration_by_VEGFR3.htm

VEGFR3 and Metastasis in Prostate Cancer

http://www.lymphedemapeople.com/thesite/VEGFR3_and_Metastasis_in_Prostate_Cancer.htm

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Lymphedema People Genetics, Research, Lymphangiogenesis, Angiogenesis Forum

http://www.lymphedemapeople.com/phpBB2/viewforum.php?f=16

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Join us as we work for lymphedema patients everywehere:

Advocates for Lymphedema

Dedicated to be an advocacy group for lymphedema patients. Working towards education, legal reform, changing insurance practices, promoting research, reaching for a cure.

http://health.groups.yahoo.com/group/AdvocatesforLymphedema/

Subscribe: AdvocatesforLymphedema-subscribe@yahoogroups.com

Pat O'Connor

Lymphedema People / Advocates for Lymphedema

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For information about Lymphedema

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema\

For Information about Lymphedema Complications

http://www.lymphedemapeople.com/wiki/doku.php?id=complications_of_lymphedema

For Lymphedema Personal Stories

http://www.lymphedemapeople.com/phpBB2/viewforum.php?f=3

For information about How to Treat a Lymphedema Wound

http://www.lymphedemapeople.com/wiki/doku.php?id=how_to_treat_a_lymphedema_wound

For information about Lymphedema Treatment 

http://www.lymphedemapeople.com/wiki/doku.php?id=treatment

For information about Exercises for Lymphedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=exercises_for_lymphedema

For information on Infections Associated with Lymphedema

http://www.lymphedemapeople.com/wiki/doku.php?id=infections_associated_with_lymphedema

For information on Lymphedema in Children

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_in_children

Lymphedema Glossary

http://www.lymphedemapeople.com/wiki/doku.php?id=glossary:listing

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Lymphedema People - Support Groups

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Children with Lymphedema

The time has come for families, parents, caregivers to have a support group of their own. Support group for parents, families and caregivers of chilren with lymphedema. Sharing information on coping, diagnosis, treatment and prognosis. Sponsored by Lymphedema People.

http://health.groups.yahoo.com/group/childrenwithlymphedema/

Subscribe: childrenwithlymphedema-subscribe@yahoogroups.com

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Lipedema Lipodema Lipoedema

No matter how you spell it, this is another very little understood and totally frustrating conditions out there. This will be a support group for those suffering with lipedema/lipodema. A place for information, sharing experiences, exploring treatment options and coping.

Come join, be a part of the family!

http://health.groups.yahoo.com/group/lipedema_lipodema_lipoedema/?yguid=209645515

Subscribe: lipedema_lipodema_lipoedema-subscribe@yahoogroups.com

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MEN WITH LYMPHEDEMA

If you are a man with lymphedema; a man with a loved one with lymphedema who you are trying to help and understand come join us and discover what it is to be the master instead of the sufferer of lymphedema.

http://health.groups.yahoo.com/group/menwithlymphedema/

Subscribe: menwithlymphedema-subscribe@yahoogroups.com

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All About Lymphangiectasia

Support group for parents, patients, children who suffer from all forms of lymphangiectasia. This condition is caused by dilation of the lymphatics. It can affect the intestinal tract, lungs and other critical body areas.

http://health.groups.yahoo.com/group/allaboutlymphangiectasia/

Subscribe: allaboutlymphangiectasia-subscribe@yahoogroups.com

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Lymphatic Disorders Support Group @ Yahoo Groups

While we have a number of support groups for lymphedema... there is nothing out there for other lymphatic disorders. Because we have one of the most comprehensive information sites on all lymphatic disorders, I thought perhaps, it is time that one be offered.

DISCRIPTION

Information and support for rare and unusual disorders affecting the lymph system. Includes lymphangiomas, lymphatic malformations, telangiectasia, hennekam's syndrome, distichiasis, Figueroa
syndrome, ptosis syndrome, plus many more. Extensive database of information available through sister site Lymphedema People.

http://health.groups.yahoo.com/group/lymphaticdisorders/

Subscribe: lymphaticdisorders-subscribe@yahoogroups.com

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Lymphedema People New Wiki Pages

Have you seen our new “Wiki” pages yet?  Listed below are just a sample of the more than 140 pages now listed in our Wiki section. We are also working on hundred more.  Come and take a stroll! 

Lymphedema Glossary 

http://www.lymphedemapeople.com/wiki/doku.php?id=glossary:listing 

Lymphedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema 

Arm Lymphedema  

http://www.lymphedemapeople.com/wiki/doku.php?id=arm_lymphedema 

Leg Lymphedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=leg_lymphedema 

Acute Lymphedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=acute_lymphedema 

The Lymphedema Diet 

http://www.lymphedemapeople.com/wiki/doku.php?id=the_lymphedema_diet 

Exercises for Lymphedema  

http://www.lymphedemapeople.com/wiki/doku.php?id=exercises_for_lymphedema 

Diuretics are not for Lymphedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=diuretics_are_not_for_lymphedema 

Lymphedema People Online Support Groups 

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_people_online_support_groups 

Lipedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=lipedema 

Treatment 

http://www.lymphedemapeople.com/wiki/doku.php?id=treatment 

Lymphedema and Pain Management 

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_and_pain_management 

Manual Lymphatic Drainage (MLD) and Complex Decongestive Therapy (CDT)

http://www.lymphedemapeople.com/wiki/doku.php?id=manual_lymphatic_drainage_mld_complex_decongestive_therapy_cdt 

Infections Associated with Lymphedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=infections_associated_with_lymphedema 

How to Treat a Lymphedema Wound 

http://www.lymphedemapeople.com/wiki/doku.php?id=how_to_treat_a_lymphedema_wound 

Fungal Infections Associated with Lymphedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=fungal_infections_associated_with_lymphedema 

Lymphedema in Children 

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_in_children 

Lymphoscintigraphy 

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphoscintigraphy 

Magnetic Resonance Imaging 

http://www.lymphedemapeople.com/wiki/doku.php?id=magnetic_resonance_imaging 

Extraperitoneal para-aortic lymph node dissection (EPLND) 

http://www.lymphedemapeople.com/wiki/doku.php?id=extraperitoneal_para-aortic_lymph_node_dissection_eplnd 

Axillary node biopsy 

http://www.lymphedemapeople.com/wiki/doku.php?id=axillary_node_biopsy

Sentinel Node Biopsy 

http://www.lymphedemapeople.com/wiki/doku.php?id=sentinel_node_biopsy

 Small Needle Biopsy - Fine Needle Aspiration 

http://www.lymphedemapeople.com/wiki/doku.php?id=small_needle_biopsy 

Magnetic Resonance Imaging 

http://www.lymphedemapeople.com/wiki/doku.php?id=magnetic_resonance_imaging 

Lymphedema Gene FOXC2

 http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_foxc2

 Lymphedema Gene VEGFC

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_vegfc

 Lymphedema Gene SOX18

 http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_sox18

 Lymphedema and Pregnancy

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_and_pregnancy

Home page: Lymphedema People

http://www.lymphedemapeople.com

Page Updated: Dec. 17, 2011