Molecular regulation of lymphangiogenesis and targets for tissue oedema

18

Review

TRENDS in Molecular Medicine Vol.7 No.1 January 2001

Molecular regulation of lymphangiogenesis and targets for tissue oedema Marika J. Karkkainen, Lotta Jussila, Robert E. Ferrell, David N. Finegold and Kari Alitalo New insight has recently been obtained into the molecular mechanisms regulating the function of lymphatic endothelial cells.Vascular endothelial growth factors-C and -D have been shown to stimulate lymphangiogenesis, and their receptor VEGFR-3 has been linked to human hereditary lymphoedema, although there is evidence that other genes are also involved. These data suggest that it may become possible to stimulate lymphatic growth and function and to treat tissue oedema involved in many diseases. Molecular regulators of lymphatic vessels

Marika J. Karkkainen* Lotta Jussila Kari Alitalo Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research Haartman Institute, University of Helsinki P.O.B. 21 (Haartmaninkatu 3), 00014 Helsinki, Finland. *e-mail: Marika.Karkkainen@ Helsinki.FI Robert E. Ferrell Dept of Human Genetics, Graduate School, Public Health, University of Pittsburgh, 130 DeSoto Street, Pittsburgh, PA 15261, USA. David N. Finegold Dept of Human Genetics, University of Pittsburgh, 130 DeSoto Street, Pittsburgh, PA 15261, USA, and Department of Pediatrics, University of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213, USA.

The lymphatic system transports tissue fluid, extravasated plasma proteins and cells back into the blood circulation. A set of coordinated structures, including ENDOTHELIAL CELLS (see GLOSSARY), form the initial lymphatic sinuses, which drain into the lymphatic capillaries. Fluid and macromolecules from the stromal compartment readily enter these sinuses and lymphatic capillaries, which consist of a single endothelial cell layer surrounded by an incomplete basement membrane. From the initial lymphatics, the fluid is transferred to the collecting lymphatics, consisting of endothelial, muscular and adventitial layers, and ultimately into the venous circulation via the thoracic duct. The lymphatic system is organized in a parallel fashion to the circulatory system and it pumps lymph fluid using the intrinsic contractility of the smooth muscle layer of the larger lymphatic vessels. When a collecting lymphatic vessel becomes stretched with fluid, the smooth muscle cells in the wall of the vessel increase their contractility. In addition to this pumping caused by the intrinsic contraction, external factors such as skeletal muscle movements and arterial pulsation that compress the lymph vessels, can increase the efficiency of pumping1. The lymphatic endothelial cells are attached to the surrounding connective tissue by anchoring filaments and, furthermore, the luminal side of the lymphatic vessels is segmented by valves, which prevent lymphatic backflow. The lymphatic system also contains several lymphoid organs (spleen, lymph nodes, tonsils, and thymus), that are essential in immune responses. Unique proteins have been shown to mediate the growth and/or embryonic development of the lymphatic endothelium. One such protein is the receptor tyrosine kinase designated VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 3 (VEGFR-3, also known as fms-like tyrosine kinase 4, FLT4)2,3. Targeted disruption of the Vegfr3 gene in mice

demonstrates the essential role of VEGFR-3 in the development of the cardiovascular system4. However, later in development Vegfr3 expression becomes restricted mainly to the lymphatic vessels5 (Fig. 1). Two members of the VEGF family, VEGF-C and VEGF-D, have been found to bind to and activate VEGFR-3 (Refs 6,7). During development, VEGF-C mRNA is abundantly expressed in mesenchymal cells adjacent to the VEGFR-3 positive endothelia, suggesting paracrine ligand–receptor signaling8. Accordingly, when VEGF-C was transgenically overexpressed in mouse skin, a hyperplastic lymphatic vessel network developed in the dermis9. VEGF-C is also mitogenic towards lymphatic endothelial cells and it shows a selective LYMPHANGIOGENIC response in differentiated avian chorioallantoic membrane10. Recent data suggest that VEGF-D, and a VEGFR-3 specific mutant of VEGF-C, in which the cysteine residue (156) is substituted by a serine residue (C156S), resulting in loss of VEGFR-2 binding ability11, are also lymphangiogenic when overexpressed in the skin of transgenic mice. However, this effect is somewhat weaker than the one obtained with VEGF-C (T. Veikkola and L. Jussila, unpublished). By contrast, inhibition of lymphatic growth is obtained when a secreted soluble form of the VEGFR-3 extracelluluar domain is expressed in a similar transgenic mouse model (T. Makinen, unpublished). These results, combined with the expression patterns of VEGF-C and VEGFR-3 in the lymphatic vasculature, suggest that lymphatic growth is induced by VEGF-C and mediated via VEGFR-3. Recent studies have shown that VEGFR-3 is also expressed in some fenestrated blood vascular endothelial cells and in angiogenic blood vessels in tumors12,13. Notwithstanding these insights involving one lymphatic endothelial cell receptor and its two ligands, the developmental regulation of the lymphatic system has not yet been intensely studied. In addition to VEGFR-3, three other genes have been shown to be relatively specifically expressed in lymphatic endothelial cells. The homeobox-containing gene Prox1 is expressed in a subpopulation of endothelial cells that give rise to the lymphatic system14. In Prox1 knockout mice, the formation of blood vasculature is unaffected, whereas the budding and sprouting of the lymphatic endothelial cells are arrested. These results suggest that Prox1 is required

http://tmm.trends.com 1471-4914/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1471-4914(00)01864-5

Review

(a)

TRENDS in Molecular Medicine Vol.7 No.1 January 2001

(b)

19

Glossary Chylous ascites: A large collection of lymphatic fluid in the abdominal cavity, secondary to blockage of or injury to the main lymph duct. Endothelial cell: A cell lining the inner surface of blood or lymphatic vessels.

(c)

Lymphangiogenesis: The development and growth of lympatic vasculature. Lymphoedema: A chronic swelling of the extremities due to the accumulation of lymph into the interstitial tissue. Primary lymphoedema: Lymphoedema with no identifiable antecedent cause, that can be present at birth (congenital), at puberty (praecox) or, more rarely, at adulthood (tarda). Secondary lymphoedema: Acquired lymphoedema that develops when the lymph vessels are damaged or lymph nodes removed. Vascular endothelial growth factor c (VEGF-C): A dimeric growth factor homologous to VEGF, that binds to VEGFR-2 and -3.

Fig. 1. The lymphatic vasculature in the Vegfr3+/-/LacZ knock-in model4. The LacZ marker gene was inserted in into the Vegfr3 locus and the lymphatic vessels were visualized by ß-gal staining of the embryo. (a) The lymphatic vessel network in an embryonic day (E)15.5 mouse embryo. (b) A higher magnification of the lymphatics in the head of the embryo. (c) The lymphatic vessels in a section from adult mouse heart. The thin-walled lymphatic vessels are stained blue for βgalactosidase (blue arrows), whereas the large artery, surrounded by a thick smooth muscle cell layer, is not stained (red arrowheads). Scale bars: (a) 2 mm (b) 1 mm (c) 100 ␮m.

for lymphatic development, and that the blood vascular and lymphatic systems subsequently employ distinct regulatory proteins. Podoplanin, an integral membrane protein first found in glomerular epithelial cells, has also been shown to be specifically expressed in the endothelium of lymphatic capillaries, but not in the blood vasculature15. In the skin and kidney vasculature, podoplanin colocalized with VEGFR-3, and all endothelial cells of Kaposi’s sarcomas expressed both VEGFR-3 and podoplanin, thus mimicking cells of lymphatic endothelial origin15,16. LYVE-1, a CD44 homologue, has recently been identified as a receptor for the extracellular matrix glycosaminoglycan hyaluronan. LYVE-1 colocalizes with hyaluronan on the luminal surface of the lymph vessel wall and is completely absent from blood vessels17. Although the specific roles of Prox1, podoplanin and LYVE-1 in lymphatic endothelial cell functions are yet to be defined, these markers can now be used to study the lymphatic development and to explore the etiology of lymphatic diseases. Regulation of endothelial cell permeability

Liquid, macromolecules and migrating cells pass through the blood capillary endothelium into the surrounding tissues, and are gradually absorbed into the lymphatic system. Blood capillary permeability is tightly controlled, but several molecules can regulate it. Microvessels are hyper-permeable to plasma proteins and macromolecules at sites where VEGF and its receptors are overexpressed, for example in tumors, in wound healing, or at sites of inflammation, and in physiological processes, such as corpus luteum formation and ovulation. VEGF was discovered as a vascular permeability factor (VPF) because of its ability to increase the permeability of microvessels to circulating macromolecules18. The complex binding pattern of VEGF family members to their receptors now enables studies of the biological effects mediated by specific VEGFRs19,20. Several studies suggest VEGFR-2 as the primary receptor mediating the http://tmm.trends.com

Vascular endothelial growth factor receptor 3 (VEGFR-3): A transmembrane tyrosine kinase receptor expressed mainly in the lymphatic endothelial cells.

VEGF permeability effect. VEGF binds VEGFR-1 and VEGFR-2, and is a very potent inducer of vascular leakage, whereas both VEGF-B and placenta growth factor (PlGF) only bind VEGFR-1, and do not induce permeability (Fig. 2). It has also been shown that the VEGF-C (C156S)11 mutant, which only binds VEGFR3, is not capable of inducing the permeability response, further suggesting that this response is transduced via VEGFR-2. Furthermore, recently discovered viral VEGF homologues, VEGF-Es (for example, NZ7- and NZ2-VEGF) only bind VEGFR-2 and are strong inducers of vascular permeability21,22. These data imply that VEGFR-2 mediates the permeability response. Interestingly, the c-Src signaling molecule also seems to be a necessary mediator of the VEGFinduced permeability response, as adenoviral vectors expressing VEGF do not induce vascular permeability in c-Src knockout mice23. Recent data suggest that Angiopoietin-1 (Ang-1) can inhibit the vascular permeability. Angiopoietins (Angs) are ligands for Tie-2 (also called Tek), an endothelial cell-specific receptor tyrosine kinase that is important in the stabilization and maturation of the vascular network in embryos24. Ang-1 is an agonist for Tie-2, whereas in at least certain conditions the related growth factor Ang-2 functions as a naturally occurring antagonist for Ang-1 and Tie2. Transgenic overexpression of Ang-1 in mouse skin results in large vessels at normal density, whereas the dermal microvessels induced by VEGF in the same model were numerous, tortuous and leaky25,26. Surprisingly, mice coexpressing Ang-1 and VEGF in the skin showed enlarged and more numerous, but leakage-resistant vessels, suggesting that Ang-1 is capable of inhibiting the VEGF-induced permeability27. Similar results were also obtained when adenoviral VEGF and/or Ang-1 were

Review

20

TRENDS in Molecular Medicine Vol.7 No.1 January 2001

Blood vessels PLGF VEGF-B VEGF

VEGFR-1

ANG-1 VEGF-E

VEGFR-2

ANG-2? H2O proteins lymphocytes

VEGF-C VEGF-C C156S VEGF-D (VEGF)

VEGFR-3

VEGFR-2

Lymphatic vessels TRENDS in Molecular Medicine

Fig. 2. Growth factor regulation and functional dynamics of the lymphatic vessels. Fluid and macromolecules extravasated from blood vessels are taken up by lymphatic vessels. In most blood vessels, endothelial cells (pink) are surrounded by a continuous basement membrane (black line) and pericytes/smooth muscle cells (white). Blood vessel permeability is tightly regulated, for example, via VEGF/VEGFR-2 signaling, and Ang-1 can block the VEGF-induced increase of vessel permeability. The protein-rich fluid from the interstitial tissues enters the lymphatic capillaries (blue). Lymphatic vessels are characterized by an incomplete basement membrane, anchoring filaments and luminal valves, that prevent lymph backflow. VEGFR-3 in expressed on the surface of lymphatic endothelial cells, and VEGF-C as well as VEGF-D are important mediators of the lymphatic endothelial cell growth via this receptor.

administered intravenously to mice28. Surprisingly, recent data also suggest that Ang-2 has a role in lymphatic development. In Ang-2 knockout mice, the peritoneal cavity is filled with CHYLOUS ASCITES, the mouse develops severe peripheral edema, and the hindlimb and abdominal lymphatics are apparently not functional and perhaps disconnected (N. Gale, C. Suri, M. Witte and G. Yancopoulos, pers. commun.). These new results should be considered in designing gene therapy for various diseases in which endothelial cells and vessel permeability are involved. Lymphoedema

Lymphatic vessels play a central role in maintaining the fluid balance of the interstitial tissues. Use of the term lymphoedema is generally restricted to highprotein oedema due to an abnormality of lymph flow and transport, and does not refer to high-protein oedemas due to increased capillary permeability (due to burns, inflammation, etc.) or to tissue oedemas such as those due to heart failure and venous insufficiency. In lymphoedma, the transport capacity of lymphatic vessels is usually decreased, and protein-rich fluid collects in the tissues causing chronic swelling. Because of the anatomy of the lymphatic network and the positional hydrostatic pressure in the dependent parts of humans, lymphoedema mainly affects the lower extremities. http://tmm.trends.com

Lymphoedema is usually divided into two main categories. PRIMARY LYMPHOEDEMA is a condition with no identifiable antecedent cause, that can be present at birth (congenital), at puberty (praecox) or, more rarely, at adulthood (tarda). Approximately 35% of the primary lymphoedema patients have a positive family history of the disease29. Inherited primary lymphoedema can be congenital (Milroy’s disease, MIM 153100) or it can develop at the onset of puberty (Meige’s disease, MIM 153200)30. In Milroy’s disease, the superficial or subcutaneous lymphatic vessels are usually aplastic or hypoplastic, whereas the microlymphatic network in late onset lymphoedema is larger than in healthy controls31–33. In both of these cases, the lymphatics fail to transport the fluid into the venous circulation, resulting in a disabling and disfiguring swelling of the extremities. In contrast to the primary lymphoedema, SECONDARY LYMPHOEDEMA develops when the lymphatic vessels are damaged by infection or radiation therapy, or when lymph nodes are surgically removed. In both primary and secondary lymphoedema, the stagnant, protein-rich fluid causes tissue channels to increase in size and number, and also reduces oxygen availability in tissues. Its consequences include tissue fibrosis and adipose degeneration, interference with wound healing, and susceptibility to infections. VEGFR3 mutations in lymphoedema

Despite having been described over a century ago, little progress has been made in understanding the mechanisms that cause hereditary lymphoedema. However, several groups have recently shown linkage of the early onset primary lymphoedema to the VEGFR3 locus in distal chromosome 5q (Refs 34–36), confirming the importance of VEGFR-3 for normal lymphatic vascular function. In two very recent studies, six specific lymphoedema-linked missense mutations have been found in the VEGFR-3 tyrosine kinase domain (Refs 37,38 and A. Evans and A. Child, unpublished). In normal VEGFR-3 activation, ligand binding results in receptor dimerization and intracellular tyrosyl transphosphorylation, whereas all lymphoedema-associated mutant receptors (G857R, H1035R, R1041P, L1044P and P1114L) were incapable of tyrosyl phosphorylation (Fig. 3). The mutant VEGFR-3 proteins were poor activators of the downstream signaling cascades, suggesting that they fail to transduce physiological VEGF-C/VEGF-D signals into the nucleus. The kinase-inactive receptors were also degraded at a slower rate than the wild-type receptors after ligand binding, were thus more stable, and accumulated on the cell surface. This probably contributes to the development of lymphoedema by reducing the relative amount of ligand binding to the wild-type VEGFR-3 and the resulting signaling. These in vitro data suggest that inherited mutations, which lead to amino acid changes inactivating the VEGFR-3 kinase and interfering with its signaling function, lead to the development of lymphoedema.

Review

Fig. 3. The molecular mechanisms of hereditary lymphoedema caused by VEGFR3 mutations. Normally VEGF-C or VEGF-D binding to VEGFR-3 on the lymphatic endothelial cell surface results in receptor dimerization and phosphorylation of the intracellular tyrosine kinase domain of the receptor. In some cases of hereditary lymphoedema, the VEGFR-3 tyrosine kinase encoded by one of the two gene alleles is inactivated and this leads to reduced phosphorylation and signaling of the receptor and development of the lymphoedema phenotype.

TRENDS in Molecular Medicine Vol.7 No.1 January 2001

Normal

Lymphoedema

VEGF-C VEGF-D

VEGF-C VEGF-D

VEGFR-3 WT WT

VEGFR-3 Mutant WT

ss ss

ss ss

P

P

Lymphangiogenesis

G857R H1035R R1041P L1044P P1114L

P

Less signaling Mutant more stable accumulation of the mutant receptor on the cell surface Hypoplastic lymphatic vessels TRENDS in Molecular Medicine

Acknowledgements The authors’ own work cited here was supported by grants from the Finnish Cancer Organization, Finnish Cultural Foundation, Ida Montini Foundation, Emil Aaltonen Foundation, the Finnish Academy of Sciences, the Sigrid Juselius Foundation, the Novo Nordisk Foundation, the European Union Biomed Program, the European Union (Biomed grant no. PL963380), NIH grant no. HD35174, and a grant from the D.T. Watson Rehabilitation Hospital, Sewickley, PA.

In contrast to the missense mutations, VEGFR-3 haploinsufficiency probably does not lead to lymphoedema. In a review of the literature on chromosomal abnormalities involving deletion of the VEGFR3 interval, there is no mention of lymphoedema39,40. All affected individuals in our study had only one mutant allele, but no VEGFR3 deletions. This is compatible with the result that in mice, inactivation of both Vegfr3 alleles causes a lethal failure of cardiovascular development, whereas the heterozygotes have no obvious phenotypic abnormality4. However, heterozygous mice with targeted deletions of the Vegfr3 gene might be protected from the symptoms of lymphoedema due to genetic differences between mice and humans, and also because, unlike humans, mice do not develop a high hydrostatic pressure in their extremities.

of aortic arch patterning and skeletogenesis44. The disease locus in the cholestasis-lymphoedema syndrome (Aagenaes syndrome, MIM 214900), characterized by severe neonatal cholestasis and chronic severe lymphoedema, was mapped to chromosome 15q (Ref. 45). The features of Turner syndrome also include lymphoedema, and the loci responsible for the symptoms of this disease were mapped to a region Xp11.2–p22.1 (Ref. 46). Thus, other lymphoedema genes exist and they might encode, for example, proteins involved in VEGFR-3 downstream signaling pathways. Further studies could reveal new factors affecting lymphatic endothelial-cell regulation and help us to explain the genotypic and phenotypic heterogeneity of lymphoedema. It should also be noted that not all members of the primary lymphoedema families with inactivating VEGFR3 mutations are affected by the disease37. This suggests that additional genetic or environmental factors play a role in the development of lymphoedema. Future prospects for therapy of lymphoedema

The identification of mutations in high-risk members of lymphoedema families will facilitate the identification of environmental factors that influence the expression and severity of lymphoedema. As VEGFR-3 has been shown to be one important factor in lymphatic development, its signaling properties could be beneficial both in terms of diagnosis and possible therapy of lymphoedema. During embryogenesis, VEGF-C becomes restricted to the cells adjacent to VEGFR-3-expressing endothelia, suggesting paracrine VEGF-C/VEGFR-3 signaling as a mediator of lymphangiogenesis. Therefore, gene therapy for lymphoedema could involve the administration of VEGF-C or VEGF-D to the affected sites of the patients, or transfer of remaining lymphatic tissue from internal sites to affected skin, combined with growth factor administration. The increased paracrine secretion of the ligands could then stimulate enough VEGFR-3 signaling to overcome the lymphatic insufficiency. Use of the VEGFR-3 specific form VEGF-C (C156S)11 in gene therapy could prevent its possible effects on the blood vascular endothelium. Although administration of VEGF-C in quantities

Other genetic changes in hereditary lymphoedema

Although mutations inhibiting the biological activity of VEGFR-3 are one cause of primary lymphoedema34–36, there are several other primary lymphoedema families that are not linked to chromosome 5. In other types of lymphoedema, the linkage studies have indicated involvement of other genetic loci. The lymphoedemadistichiasis syndrome (MIM 153200), characterized by late onset lymphoedema and a double row of eyelashes, has been linked to the chromosomal region 16q24 (Refs 41,42). Very recent data suggest that mutations in the FOXC2 (MFH-1) gene, located in this region, are responsible for the lymphoedema-distichiasis syndrome43. FOXC2 is a forkhead/winged-helix family transcription factor that is involved in the development http://tmm.trends.com

21

Outstanding questions • •

• •

What are the cellular mechanisms leading to lymphoedema? What are the other genes involved in the development of hereditary lymphoedema, and does the genotype correspond to the phenotype? What is the optimal approach for the gene therapeutic treatments for lymphoedema? Can VEGF-C be used as a gene therapeutic agent to cure lymphoedema?

22

Review

TRENDS in Molecular Medicine Vol.7 No.1 January 2001

exceeding those usually found in interstitial fluids is expected to stimulate VEGFR-3 in patients who have insufficient VEGFR-3 signaling, this effect has yet to be analysed in the context of the dominant negative heterozygous mutations. Interesting recent evidence indicates that there may be endothelial-cell progenitors circulating in blood47, and lymphatic endothelial precursor cells have been detected in avian embryos48. Such possible cells could provide important tools for the further development of cell and gene based therapies for lymphoedema. In these studies, References 1 Guyton, A.C. (1991) The microcirculation and the lymphatic system: Capillary fluid exchange, interstitial fluid and lymph flow. In Textbook of Medical Physiology (Wonsiewich, M.J., ed.), pp. 170–184, W.B. Saunders. 2 Galland, F. et al. (1993) The FLT4 gene encodes a transmembrane tyrosine kinase related to the vascular endothelial growth factor receptor. Oncogene 8, 1233–1240 3 Pajusola, K. et al. (1992) FLT4 receptor tyrosine kinase contains seven immunoglobulin-like loops and is expressed in multiple human tissues and cell lines. Cancer Res. 52, 5738–5743 4 Dumont, D.J. et al. (1998) Cardiovascular failure in mouse embryos deficient in VEGF receptor-3. Science 282, 946–949 5 Kaipainen, A. et al. (1995) Expression of the fmslike tyrosine kinase FLT4 gene becomes restricted to lymphatic endothelium during development. Proc. Natl. Acad. Sci. U. S. A. 92, 3566–3570 6 Joukov, V. et al. (1996) A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases. EMBO J. 15, 290–298 7 Achen, M.G. et al. (1998) Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4). Proc. Natl. Acad. Sci. U. S. A. 95, 548–553 8 Kukk, E. et al. (1996) VEGF-C receptor binding and pattern of expression with VEGFR-3 suggest a role in lymphatic vascular development. Development 122, 3829–3837 9 Jeltsch, M. et al. (1997) Hyperplasia of lymphatic vessels in VEGF-C transgenic mice. Science 276, 1423–1425 10 Oh, S.J. et al. (1997) VEGF and VEGF-C: specific induction of angiogenesis and lymphangiogenesis in the differentiated avian chorioallantoic membrane. Dev. Biol. 188, 96–109 11 Joukov, V. et al. (1998) A recombinant mutant vascular endothelial growth factor-C that has lost vascular endothelial growth factor receptor-2 binding, activation, and vascular permeability activities. J. Biol. Chem. 273, 6599–6602 12 Partanen, T.A. et al. (2000) VEGF-C and VEGF-D expression in neuroendocrine cells and their receptor, VEGFR-3, in fenestrated blood vessels in human tissues. FASEB J. 14, 2087–2096 13 Valtola, R. et al. (1999) VEGFR-3 and its ligand VEGF-C are associated with angiogenesis in breast cancer. Am. J. Pathol. 154, 1381–1390 14 Wigle, J.T. and Oliver, G. (1999) Prox1 function is required for the development of the murine lymphatic system. Cell 98, 769–778 15 Breiteneder-Geleff, S. et al. (1999) Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific http://tmm.trends.com

16

17

18

19

20 21

22

23

24

25

26

27

28

29 30

genetic mouse models would be essential. By introducing some of the lymphoedema mutations into the mouse genome, the development and progression of lymphoedema could be studied, and also the possible treatments for the disease could be evaluated. Nevertheless, already at this point we have obtained important new data on the molecular mechanisms of lymphoedema, and the recent findings on gene alterations permit development of more targeted and effective counseling and therapeutic approaches to this difficult disease.

marker for lymphatic endothelium. Am. J. Pathol. 154, 385–394 Weninger, W. et al. (1999) Expression of vascular endothelial growth factor receptor-3 and podoplanin suggests a lymphatic endothelial cell origin of Kaposi’s sarcoma tumor cells. Lab. Invest. 79, 243–251 Banerji, S. et al. (1999) LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J. Cell Biol. 144, 789–801 Dvorak, H.F. et al. (1999) Vascular permeability factor/vascular endothelial growth factor and the significance of microvascular hyperpermeability in angiogenesis. Curr. Top. Microbiol. Immunol. 237, 97–132 Veikkola, T. et al. (2000) Regulation of angiogenesis via vascular endothelial growth factor receptors. Cancer Res. 60, 203–212 Carmeliet, P. (2000) Mechanisms of angiogenesis and arteriogenesis. Nat. Med. 6, 389–395 Ogawa, S. et al. (1998) A novel type of vascular endothelial growth factor, VEGF-E (NZ-7 VEGF), preferentially utilizes KDR/Flk-1 receptor and carries a potent mitotic activity without heparinbinding domain. J. Biol. Chem. 273, 31273–31282 Wise, L.M. et al. (1999) Vascular endothelial growth factor (VEGF)-like protein from orf virus NZ2 binds to VEGFR2 and neuropilin-1. Proc. Natl. Acad. Sci. U. S. A. 96, 3071–3076 Eliceiri, B.P. et al. (1999) Selective requirement for Src kinases during VEGF-induced angiogenesis and vascular permeability. Mol. Cell 4, 915–924 Gale, N.W. and Yancopoulos, G.D. (1999) Growth factors acting via endothelial cell-specific receptor tyrosine kinases: VEGFs, angiopoietins, and ephrins in vascular development. Genes Dev. 13, 1055–1066 Suri, C. et al. (1998) Increased vascularization in mice overexpressing angiopoietin-1. Science 282, 468–471 Detmar, M. et al. (1998) Increased microvascular density and enhanced leukocyte rolling and adhesion in the skin of VEGF transgenic mice. J. Invest. Dermatol. 111, 1–6 Thurston, G. et al. (1999) Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1. Science 286, 2511–2514 Thurston, G. et al. (2000) Angiopoietin-1 protects the adult vasculature against plasma leakage. Nat. Med. 6, 460–463 Dale, R.F. (1985) The inheritance of primary lymphoedema. J. Med. Genet. 22, 274–278 Witte, M.H. et al. (1997) Lymphangiogenesis: Mechanisms, significance and clinical implications. In Regulation of Angiogenesis (Goldberg, I.D. and Rosen, E.M., eds.), pp. 65–112, Birkhäuser

31 Bollinger, A. et al. (1983) Aplasia of superficial lymphatic capillaries in hereditary and connatal lymphedema (Milroy’s disease). Lymphology 16, 27–30 32 Pfister, G. et al. (1990) Diameters of lymphatic capillaries in patients with different forms of primary lymphedema. Lymphology 23, 140–144 33 Bollinger, A. (1993) Microlymphatics of human skin. Int. J. Microcirc. Clin. Exp. 12, 1–15 34 Ferrell, R.E. et al. (1998) Hereditary lymphedema: evidence for linkage and genetic heterogeneity. Hum. Mol. Genet. 7, 2073–2078 35 Witte, M.H. et al. (1998) Phenotypic and genotypic heterogeneity in familial Milroy lymphedema. Lymphology 31, 145–155 36 Evans, A.L. et al. (1999) Mapping of primary congenital lymphedema to the 5q35.3 region. Am. J. Hum. Genet. 64, 547–555 37 Karkkainen, M.J. et al. (2000) Missense mutations interfere with VEGFR-3 signaling in primary lymphoedema. Nat. Genet. 25, 153–159 38 Irrthum, A. et al. (2000) Congenital hereditary lymphedema caused by a mutation that inactivates VEGFR3 tyrosine kinase. Am. J. Hum. Genet. 67, 295–301 39 Groen, S.E. et al. (1998) Repeated unbalanced offspring due to a familial translocation involving chromosomes 5 and 6. Am. J. Med. Genet. 80, 448–453 40 Barber, J.C. et al. (1996) Unbalanced translocation in a mother and her son in one of two 5;10 translocation families. Am. J. Med. Genet. 62, 84–90 41 Mangion, J. et al. (1999) A gene for lymphedemadistichiasis maps to 16q24.3. Am. J. Hum. Genet. 65, 427–432 42 Bell, R. et al. (2000) Reduction of the genetic interval for lymphoedema-distichiasis to below 2 Mb. J. Med. Genet. 37, 725–726 43 Fang, J. et al. Mutations in FOXC2 (MFH-1), a forkhead family transcription factor, are responsible for the hereditary lymphedemadistichiasis syndrome. Am. J. Hum. Genet. (in press) 44 Iida, K., et al. (1997) Essential roles of the wingedhelix transcription factor MFH-1 in aortic arch patterning and skeletogenesis. Development 124, 4627–4638 45 Bull, L.N. et al. (2000) Mapping of the locus for cholestasis-lymphedema syndrome (Aagenaes syndrome) to a 6.6-cM interval on chromosome 15q. Am. J. Hum. Genet. 67, 994–999 46 Zinn, A.R. et al. (1998) Evidence for a Turner syndrome locus or loci at Xp11.2–p22.1. Am. J. Hum. Genet. 63, 1757–1766 47 Shi, Q. et al. (1998) Evidence for circulating bone marrow-derived endothelial cells. Blood 92, 362–367 48 Schneider, M. et al. (1999) Lymphangioblasts in the avian wing bud. Dev. Dyn. 216, 311–319