Download Kari Alitalo - University of Miami Miller School of Medicine

Lymphangiogenesis in Development and Human Disease
Kari Alitalo and collaborators Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research,
Haartman Institute and Biomedicum Helsinki, P.O.B. 63 (Haartmaninkatu 8), 00014 University of Helsinki, Finland
Angiogenesis and permeability of blood vessels are regulated by vascular endothelial growth factor (VEGF) via its
two receptors VEGFR-1 and VEGFR-2. A decade ago we discovered VEGFR-3 that did not bind VEGF; its
expression became restricted mainly to lymphatic endothelia during development (1). This marker allowed us to
isolate and culture lymphatic endothelial cells and to show that the Prox1 transcription factor regulates lymphatic
endothelial specific genes (2,3). We generated homozygous VEGFR-3 targeted mice which died around midgestation
due to failure of cardiovascular development, suggesting a blood vascular function for VEGFR-3 (4). However,
inhibition of VEGFR-3 signal transduction later during development did not affect the blood vessels, while it led to
regression of the growing lymphatic vessels by endothelial cell apoptosis (5). We also purified and cloned the
VEGFR-3 ligand, VEGF-C (6). Transgenic mice expressing VEGF-C or its VEGFR-3 specific mutant showed
evidence of lymphangiogenesis and VEGF-C knockout mice had defective lymphatic vessels (7, 8, 9). The
proteolytically processed form of VEGF-C bound also to VEGFR-2 and was angiogenic (10). VEGF-D which is
closely related to VEGF-C, was similarly processed and bound to the same receptors (11). Thus VEGF-C and
VEGF-D appear to be both angiogenic and lymphangiogenic growth factors (12).
Another step in the angiogenic pathway involves the angiopoietins (Ang:s). Ang-1 activates the Tie-2 receptor of
endothelial cells, while the related Ang-2 is involved in the destabilization of blood vessels. We recently reported
that Ang-1 stimulates also the related "orphan" Tie-1 receptor (13, 14) and that it can induce lymphatic vessel
sprouting and hyperplasia (15). Lymphangiogenic factors, such as Ang-1 or fibroblast growth factor (FGF) act at
least partially via VEGF-C/D induction (15, 16). - Further maturation of the lymphatic vessels involves the FoxC2
transcription factor that inhibits smooth muscle cell recruitment and promotes the development of valves (17).
FoxC2 mutations and heterozygous missense point mutations inactivating VEGFR-3 tyrosine kinase function (18),
have been associated with human lymphedema. In a lymphedema mouse model with VEGFR-3 mutation, VEGF-C
gene therapy restored functional lymphatic vessels to the treated skin (19, 20), suggesting that gene therapy could be
tried also in human lymphedema. Other conditions where the VEGF-C/D/VEGFR-3 system can be targeted with
clinical benefit include inflammatory diseases and cancer (21, 22).
In human tumors, VEGF-C/D expression correlates with vascular invasion, lymphatic vessel and lymph node
involvement, distant metastasis, and, in some instances, poor clinical outcomes. Also studies of various tumor
models have shown that VEGF-C and VEGF-D overexpression can enhance lymphatic metastasis, while a soluble
VEGFR-3 fusion protein ("VEGF-C/D Trap") inhibited lymphatic metastasis (23,24). In some models lymphatic, but
not lung metastases were blocked with a VEGF-C/D trap while in others the treatment inhibited both lymph node
and lung metastases. Although these experiments provide strong support for the involvement of VEGF-C, VEGF-D,
and their receptor VEGFR-3 in the lymphatic spread of malignancy, the underlying mechanisms have only recently
been addressed.
Lymph vessel proliferation seen in tumor models overexpressing the lymphangiogenic factors may not be a
prominent feature in several human cancers, and may in fact not be needed for enhanced metastasis in most solid
tumors. While intratumoral lymphatic vessels have been detected in some solid tumors, such as melanomas and head
and neck carcinomas, at least in experimental tumors they may not be completely functional, because of their
collapse in conditions of high intratumoral pressure. On the other hand, the pressure gradient and lymph vessels at
the tumor margin may be more important in spreading tumor cells through the process of vessel sprouting stimulated
by tumor-secreted VEGF-C or VEGF-D. In this process the endothelial cells send long filopodia towards the growth
factor producing tumor cells and then form tumor-directed vessel sprouts, where the vessel lumen opens up and may
allow facilitated access of tumor cells to the lumen (25). Furthermore, the collecting lymphatic vessels draining fluid
from the tumor area are stimulated to dilate by intraluminal VEGF-C via the process of endothelial proliferation in
the vessel wall (25). Clumps of metastatic tumor cells could then undergo an easier transit in lymph flowing in the
dilated hyperplastic vessels. The VEGF-C/D trap inhibited sprouting and vessel dilation, and seemed to restore the
integrity of the vessel wall (25). Similarly, blocking monoclonal antibodies that target VEGF-C, VEGF-D or their
receptor(s) and small molecules that inhibit the tyrosine kinase catalytic domain of these receptors could be used for
the inhibition of experimental tumor metastasis. Further work should soon tell if these same molecules inhibit further
systemic metastasis or angiogenesis in tumor models.
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Kaipainen, A., Korhonen, J., Mustonen, T., van Hinsbergh, V.W.M., Fang, G.-H., Dumont, D., Breitman,
M. and Alitalo, K.: Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic
endothelium during development. Proc Natl Acad Sci. 92:3566-3570, 1995.
Mäkinen T, Veikkola T, Mustjoki S, Karpanen T, Wise L, Mercer A, Kerjaschki D, Catimel B, Nice EC,
Stacker SA, Achen MG, Alitalo K. Isolated lymphatic endothelial cells transduce growth, survival and
migration signals via the VEGF-C/D receptor VEGFR-3. EMBO J. 20:4762-4773, 2001.
Petrova TV, Makinen T, Makela TP, Saarela J, Virtanen I, Ferrell RE, Finegold DN, Kerjaschki D, YlaHerttuala S, Alitalo K. Lymphatic endothelial reprogramming of vascular endothelial cells by the Prox-1
homeobox transcription factor. EMBO J. 21:4593-9, 2002.
Dumont, D.J., Jussila, L., Taipale, J., Lymboussaki, A., Mustonen, T., Pajusola, K., Breitman, M. and
Alitalo, K. Cardiovascular failure in mouse embryos deficient in VEGF receptor-3. Science 282:946-949,
1998.
Mäkinen, T., Jussila, L., Veikkola, T., Kärpänen, T., Kettunen, M.I., Pulkkanen, K.J., Kauppinen, R.,
Jackson, D.G., Kubo, H., Nishikawa, S.-I., Ylä-Herttuala, S. and Alitalo, K.: Inhibition of
lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3.
Nature Medicine 7:199-205, 2001.
Joukov, V., Pajusola, K., Kaipainen, Chilov, D., A., Lahtinen, I., Kukk, E., Saksela, O., Kalkkinen, N. and
Alitalo, K.: 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, 1996.
Jeltsch, M., Kaipainen, A., Joukov, V., Meng, X., Lakso, M., Rauvala, H., Swartz, M., Fukumura, D.,
Rakesh, K.J. and Alitalo, K.: Hyperplasia of lymphatic vessels in VEGF-C transgenic mice. Science
276:1423-1425, 1997.
Veikkola, T., Jussila, L., Mäkinen, T., Karpanen, T., Jeltsch, M., Petrova, T.V., Kubo, H., Thurston, G.,
McDonald, D.M., Jackson, D.G., Achen, M.G., Stacker, S.A., and Alitalo, K.: Signalling via vascular
endothelial growth factor-3 is sufficient for lymphangiogenesis in transgenic mice. EMBO J. 20:1-9, 2001.
Karkkainen MJ, Haiko P, Sainio K, Partanen J, Taipale J, Petrova TV, Jeltsch M, Jackson DG, Talikka M,
Rauvala H, Betsholtz C, Alitalo K. Vascular endothelial growth factor C is required for sprouting of the first
lymphatic vessels from embryonic veins. Nature Immunology 5:74-80, 2004.
Cao, Y., Linden, P., Farnebo, J., Cao, R., Eriksson, A., Kumar, V., Qi, J.-H., Claesson-Welsh, L., Alitalo,
K.: Vascular endothelial growth factor-C induces angiogenesis in vivo. Proc Natl Acad Sci. 95:1438914394, 1998.
Achen, M.G., Jeltsch, M., Kukk, E., Mäkinen, T., Vitali, A., Wilks, A.F., Alitalo, K. and Stacker, S.A.:
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. 95:548-553, 1998.
Alitalo, K., Tammela, T. and Petrova, T. Lymphangiogenesis in development and human disease. Nature
438:946-53, 2005.
Partanen J., Armstrong E., Mäkelä T.P., Korhonen J., Sandberg M., Renkonen R., Knuutila S., Huebner K.
and Alitalo K.: A novel endothelial cel surface receptor tyrosine kinase with extracellular epidermal growth
factor homology domains. Mol. Cell Biol. 12:1698-1707, 1992.
Saharinen P, Kerkela K, Ekman N, Marron M, Brindle N, Lee GM, Augustin H, Koh GY, Alitalo K.
Multiple angiopoietin recombinant proteins activate the Tie1 receptor tyrosine kinase and promote its
interaction with Tie2. J Cell Biol. 169:239-43, 2005.
Tammela T, Saaristo A, Lohela M, Morisada T, Tornberg J, Norrmen C, Oike Y, Pajusola K, Thurston G,
Suda T, Yla-Herttuala S, Alitalo K. Angiopoietin-1 promotes lymphatic sprouting and hyperplasia. Blood
105:4642-8, 2005.
Kubo H, Cao R, Brakenhielm E, Makinen T, Cao Y, Alitalo K.: Blockade of vascular endothelial growth
factor receptor-3 signaling inhibits fibroblast growth factor-2-induced lymphangiogenesis in mouse cornea.
Proc Natl Acad Sci. 99:8868-73, 2002.
Petrova TV, Karpanen T, Norrmen C, Mellor R, Tamakoshi T, Finegold D, Ferrell R, Kerjaschki D,
Mortimer P, Yla-Herttuala S, Miura N, Alitalo K. Defective valves and abnormal mural cell recruitment
underlie lymphatic vascular failure in lymphedema distichiasis. Nature Medicine 10:974-81, 2004.
Karkkainen, M.J., Ferrell, R.E., Lawrence, E.C., Kimak, M.A., Levinson, K.L., McTigue, M.A., Alitalo, K.
and Finegold, D.N.: Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema.
Nature Genetics 25: 153-159, 2000.
Karkkainen MJ, Saaristo A, Jussila L, Karila KA, Lawrence EC, Pajusola K, Bueler H, Eichmann A,
Kauppinen R, Kettunen MI, Yla-Herttuala S, Finegold DN, Ferrell RE, Alitalo K. A model for gene therapy
of human hereditary lymphedema. Proc Natl Acad Sci. 98:12677-82, 2001.
20. Saaristo A, Veikkola T, Tammela T, Enholm B, Karkkainen MJ, Pajusola K, Bueler H, Yla-Herttuala S,
Alitalo K. Lymphangiogenic gene therapy with minimal blood vascular side effects. J Exp Med. 196: 71930, 2002.
21. Karpanen T, Egeblad M, Karkkainen MJ, Kubo H, Yla-Herttuala S, Jaattela M, Alitalo K. Vascular
endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth. Cancer
Res. 61:1786-90, 2001.
22. Baluk P, Tammela T, Ator E, Lyubynska N, Achen MG, Hicklin DJ, Jeltsch M, Petrova TV, Pytowski B,
Stacker SA, Yla-Herttuala S, Jackson DG, Alitalo K, McDonald DM. Pathogenesis of persistent lymphatic
vessel hyperplasia in chronic airway inflammation. J Clin Invest. 115:247-57, 2005.
23. Wang HW, Trotter MW, Lagos D, Bourboulia D, Henderson S, Makinen T, Elliman S, Flanagan AM,
Alitalo K, Boshoff C. Kaposi sarcoma herpesvirus-induced cellular reprogramming contributes to the
lymphatic endothelial gene expression in Kaposi sarcoma. Nature Genetics 36:687-93, 2004.
24. He Y, Kozaki K, Karpanen T, Koshikawa K, Yla-Herttuala S, Takahashi T, Alitalo K. Suppression of
tumor lymphangiogenesis and lymph node metastasis by blocking vascular endothelial growth factor
receptor 3 signaling. J Natl Cancer Inst. 94:819-25, 2002.
25. He Y, Rajantie I, Ilmonen M, Makinen T, Karkkainen MJ, Haiko P, Salven P, Alitalo K. Preexisting
lymphatic endothelium but not endothelial progenitor cells are essential for tumor lymphangiogenesis and
lymphatic metastasis. Cancer Res. 64:3737-40, 2004.