Mini-Review |
2 Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, NY 10029
Address correspondence to Mihaela Skobe, Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1130, New York, NY 10029. Tel.: (212) 659-5570. Fax: (212) 987-2240. email: mihaela.skobe{at}mssm.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Whereas the main function of large lymphatics is efficient transport of lymph back into the blood circulation, the lymphatic microvasculature is responsible for the uptake of components from the interstitium. Given their central role in regulating interstitial fluid pressure and cell trafficking, it is surprising that lymphatic endothelial cells (LECs) have until recently been poorly characterized, at least from a molecular point of view. This scenario is changing rapidly following the development of techniques for the isolation of pure LECs and the characterization of their molecular properties.
![]() |
Structurefunction relationships of the lymphatic capillary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
![]() |
Molecular regulation of lymphatic vessel formation and differentiation |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
VEGF-C also binds to a nonkinase receptor neuropilin-2 (NRP2) (Karkkainen et al., 2001), a classic receptor for class III semaphorins, which regulate chemorepulsive guidance of developing axons. Recent studies in NRP2-deficient mice demonstrated impeded development of lymphatic capillaries in most tissues, suggesting a role for NRP2 in LEC proliferation and, perhaps, guidance. NRP2 may cooperate with VEGFR-3 to mediate VEGF-Cdependent lymphangiogenesis (Yuan et al., 2002).
Finally, Ang2 is expressed by LECs (Petrova et al., 2002; Podgrabinska et al., 2002) and is required for the proper development of the lymphatic system (Gale et al., 2002). Mice deficient in Ang2 displayed disorganization and hypoplasia of lymphatic capillaries, and collecting lymphatic vessels were not properly invested by smooth muscle. As a result, Ang2 knockout mice developed severe lymphedema. Interestingly, the lymphatic phenotype caused by Ang2 deficiency was rescued by Ang1, suggesting redundant roles for these molecules in lymphatic development.
The homeobox transcription factor Prox-1 appears to be required for the commitment of endothelial cells to the lymphatic differentiation program (Wigle and Oliver, 1999; Wigle et al., 2002). Prox-1 expression in embryos localizes to a subpopulation of endothelial cells in embryonic veins, which are commited to the lymphatic pathway. Functional inactivation of Prox-1 in mice results in the arrest of lymphatic vessel development. In adult tissues, Prox-1 is expressed exclusively by LECs, and overexpression of Prox-1 in blood endothelial cells (BECs) down-regulated BEC-specific transcripts and up-regulated LEC-specific transcripts, thus conferring the lymphatic endothelial phenotype on these cells (Hong et al., 2002; Petrova et al., 2002). Most recent evidence suggested that the adaptor protein SLP76 and the tyrosine kinase syk, which are expressed primarily in hematopoietic cells, may also contribute to the anatomical separation of the blood and lymphatic vasculature (Abtahian et al., 2003).
![]() |
Isolation and molecular characterization of LECs |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The above studies demonstrated that LECs and BECs retain their differentiated phenotypes in culture. LECs were distinguished by their homotypic association, selective responsiveness to VEGF-C in terms of growth, survival and morphogenesis, differential ECM requirements, and the distinct gene expression profile. LECs established by the different methods, however, exhibited certain differences in gene expression that may be attributed to the different source of tissues employed, i.e., adult versus neonatal skin. Alternatively, the different isolation strategies may select for specific subpopulations of LECs. LECs isolated using VEGFR-3 antibodies may be partly contaminated with BECs, since VEGFR-3 can also be expressed by the blood vascular endothelium (Partanen et al., 1999). Finally, isolated LECs were propagated under different conditions, which may further account for the variations in phenotype. It remains to be determined which purification strategy and culture conditions allow for optimal preservation of the lymphatic endothelial phenotype in vitro.
The availability of microvascular LECs now permits analyses of their molecular and functional characteristics. The molecular signature of LECs appears to reflect their unique functional characteristics and provides novel insight into the molecular basis of lymphatic function (Petrova et al., 2002; Podgrabinska et al., 2002; Hirakawa et al., 2003). For example, LECs express remarkably high levels of genes implicated in protein metabolism, sorting and trafficking (Podgrabinska et al., 2002). Genes with particularly high representation were those encoding proteins that control specificity of vesicle targeting and fusion, such as members of the SNARE family, rab GTPases, AAA ATPases, and sec-related proteins (Mellman and Warren, 2000), indicating the existence of a robust vesicular transport system. The lymphatic endothelium is characterized by an abundance of membrane invaginations and cytoplasmic vesicles (Leak, 1972, 1976), yet their functional significance has not been established. Intercellular clefts are considered to be a major pathway for the movement of fluid and proteins into lymphatics (Schmid-Schönbein, 1990b). However, some early studies also demonstrated the presence of interstitially injected molecular tracers within intracellular vesicles of LECs (Leak, 1972, 1976). In agreement with these findings, the results of the gene profiling studies suggest that, in addition to intercellular transport, transendothelial pathways may also be used as a mechanism for entry of molecules into lymphatics (Podgrabinska et al., 2002). This raises the intriguing possibility that lymphatics may have the capacity to selectively remove molecules from the interstitium and thereby actively control the composition of lymph and interstitial fluid.
![]() |
Role of lymphatic vessels in tumor dissemination |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent studies, however, have demonstrated enlarged lymphatic vessels and lymphangiogenesis in peritumoral areas of several human tumors using lymphatic endothelial markers (Stacker et al., 2002; Pepper et al., 2003). The number of tumor-associated lymphatics has been correlated with lymph node metastases, yet intratumoral lymphatics have so far been observed only in human head and neck cancers and in melanoma. The relative importance of preexisting versus newly-formed lymphatic vessels to lymphogenous metastasis is not understood. Although preexisting peritumoral lymphatics are likely to be sufficient for tumor spread, recruitment of lymphatic vessels into the close proximity of a tumor may increase the propensity of tumors to metastasize. Increased lymphatic vessel density and/or presence of intratumoral lymphatics should therefore be regarded as an additional pathway rather than a necessity for metastasis.
Notably, a large number of studies demonstrated a striking correlation between the VEGF-C expression in human tumors and lymph node metastases (Stacker et al., 2002; Pepper et al., 2003). Moreover, recent experimental studies using VEGF-Coverexpressing tumor cells have provided direct evidence for the causal role of VEGF-C in tumor lymphangiogenesis and lymphogenous metastasis (Mandriota et al., 2001; Skobe et al., 2001). Although an increase in lymphatic vessel density may promote tumor spread simply by creating more opportunities for metastatic tumor cells to leave the primary tumor site, lymphatic vessels may also play a more active role in metastasis. For example, soluble factors constitutively expressed by LECs may facilitate tumor cell invasion of lymphatic vessels. Activation of LECs by VEGF-C or other factors produced by a tumor could promote release of chemokines, which may attract tumor cells into the lymphatics. As the migration of cancer cells to regional lymph nodes resembles physiological migration of leukocytes, it is conceivable that the chemokine-mediated normal mechanisms of lymphocyte homing may also be used for metastatic dissemination.
Thus far, the importance of two chemokine receptors (CCRs) in lymph node metastasis has been established: CXCR4 and CCR7. CXCR4 was found to be up-regulated in malignant melanoma and in breast cancer, whereas its ligand CXCL12 is highly expressed in lymph nodes and other target organs for breast cancer metastasis. A neutralizing antibody to CXCR4 inhibited metastases to lymph nodes and other organs, demonstrating a critical role for this chemokine/receptor system in mediating tumor cell homing (Muller et al., 2001). CCR7 and its ligands chemokines CCL19 and CCL21 are of crucial importance for the migration of lymphocytes and dendritic cells to lymph nodes. CCR7 was also found to be highly expressed by human malignant melanoma and breast cancer cells (Muller et al., 2001), and its expression has been associated with lymph node metastasis in gastric cancer (Mashino et al., 2002) and in nonsmall cell lung cancer (Takanami, 2003). Overexpression of CCR7 in a mouse model of melanoma enhanced metastases to lymph nodes, which could be blocked by neutralizing its ligand CCL21 (Wiley et al., 2001). CCL21 and several other chemokines are constitutively expressed by LECs (Kriehuber et al., 2001; Makinen et al., 2001; Podgrabinska et al., 2002), suggesting an active role for LECs in governing cell migration in normal physiology and in cancer. However, a direct role for lymphatic endothelium in the process still remains to be demonstrated.
Mechanisms mediating tumor cell transmigration across the lymphatic endothelium into this lymphatic vessels also remain obscure. The prevailing view has been that tumor cells passively enter lymphatics between intercellular junctions. Based on the differences in their structure, it has been assumed that the entry of cells into lymphatics is easier than into blood vessels. An alternative novel hypothesis is that tumor cell entry requires adhesive interactions with LECs. There is no direct experimental evidence in support of either concept.
Thus far, very few cell adhesion molecules expressed by LECs have been identified. Several genes encoding proteins that constitute adherens junctions, such as desmoplakin, plakoglobin, plakophillin 2, H-cadherin, and zona occludens 2, were identified in LECs by gene profiling (Petrova et al., 2002; Podgrabinska et al., 2002). Another junctional adhesion molecule belonging to the immunoglobulin superfamily, JAM-2, was found to be expressed in tight junctions of lymphatic vessels and was shown to facilitate lymphocyte transmigration (Aurrand-Lions et al., 2001; Johnson-Leger et al., 2002). The nature of the lymphatic endothelial junctions may indeed facilitate cell entry and the identification of adhesion molecules typical for lymphatic endothelium may be particularly important for the understanding of leukocyte trafficking and tumor metastasis via lymphatics.
In this regard, macrophage mannose receptor (MR) I expressed by LECs has been shown to mediate adhesion of lymphocytes to lymphatics in lymph nodes (Irjala et al., 2001). MR on LECs supports lymphocyte binding to lymphatic vessels in an L-selectindependent fashion, and this interaction has been suggested to control lymphocyte exit from the lymph nodes. MR was also found to be selectively expressed by cultured LECs (Petrova et al., 2002; Podgrabinska et al., 2002), and its presence on afferent lymphatics suggests its possible involvement also in lymphocyte exit from peripheral tissues. Common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1) is another recently identified adhesion molecule implicated in binding of lymphocytes to LECs in lymph nodes (Irjala et al., 2003b). Because CLEVER-1 is an inducible vascular adhesion molecule, it has been suggested to regulate migration of leukocytes to sites of inflammation. MR and CLEVER-1 expression have also been detected on peri- and intratumoral lymphatic vessels in human head and neck, and breast carcinomas (Irjala et al., 2003a). Notably, expression of MR on intratumoral lymphatic vessels was associated with increased lymph node metastases in breast cancer. These pioneering studies aid in shaping the new concept of a more active role of lymphatic vessels in cancer.
![]() |
Summary and perspectives |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Submitted: 18 August 2003
Accepted: 15 September 2003
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abtahian, F., A. Guerriero, E. Sebzda, M.M. Lu, R. Zhou, A. Mocsai, E.E. Myers, B. Huang, D.G. Jackson, V.A. Ferrari, et al. 2003. Regulation of blood and lymphatic vascular separation by signaling proteins SLP-76 and Syk. Science. 299:247251.
Achen, M.G., M. Jeltsch, E. Kukk, T. Makinen, A. Vitali, A.F. Wilks, K. Alitalo, and S.A. Stacker. 1998. Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (FLT-4). Proc. Natl. Acad. Sci. USA. 95:548553.
Alitalo, K., and P. Carmeliet. 2002. Molecular mechanisms of lymphangiogenesis in health and disease. Cancer Cell. 1:219227.[CrossRef][Medline]
Aukland, K., and R.K. Reed. 1993. Interstitial-lymphatic mechanisms in the control of extracellular fluid volume. Physiol. Rev. 73:178.
Aurrand-Lions, M., L. Duncan, C. Ballestrem, and B.A. Imhof. 2001. JAM-2, a novel immunoglobulin superfamily molecule, expressed by endothelial and lymphatic cells. J. Biol. Chem. 276:27332741.
Carmeliet, P., and R.K. Jain. 2000. Angiogenesis in cancer and other diseases. Nature. 407:249257.[CrossRef][Medline]
Gale, N.W., G. Thurston, S.F. Hackett, R. Renard, Q. Wang, J. McClain, C. Martin, C. Witte, M.H. Witte, D. Jackson, et al. 2002. Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by Angiopoietin-1. Dev. Cell. 3:411423.[Medline]
Gerli, R., L. Ibba, and C. Fruschelli. 1990. A fibrillar elastic apparatus around human lymph capillaries. Anat. Embryol. 181:281286.[Medline]
Hartveit, E. 1990. Attenuated cells in breast stroma: the missing lymphatic system of the breast. Histopathology. 16:533543.[Medline]
Hirakawa, S., Y.K. Hong, N. Harvey, V. Schacht, K. Matsuda, T. Libermann, and M. Detmar. 2003. Identification of vascular lineage-specific genes by transcriptional profiling of isolated blood vascular and lymphatic endothelial cells. Am. J. Pathol. 162:575586.
Hong, Y.K., N. Harvey, Y.H. Noh, V. Schacht, S. Hirakawa, M. Detmar, and G. Oliver. 2002. Prox1 is a master control gene in the program specifying lymphatic endothelial cell fate. Dev. Dyn. 225:351357.[CrossRef][Medline]
Ikomi, F., and G.W. Schmid-Schönbein. 1996. Lymph pump mechanics in the rabbit hind leg. Am. J. Physiol. 271:H173H183.[Medline]
Irjala, H., K. Alanen, R. Grenman, P. Heikkila, H. Joensuu, and S. Jalkanen. 2003a. Mannose receptor (MR) and common lymphatic endothelial and vascular endothelial receptor (CLEVER)-1 direct the binding of cancer cells to the lymph vessel endothelium. Cancer Res. 63:46714676.
Irjala, H., K. Elima, E.L. Johansson, M. Merinen, K. Kontula, K. Alanen, R. Grenman, M. Salmi, and S. Jalkanen. 2003b. The same endothelial receptor controls lymphocyte traffic both in vascular and lymphatic vessels. Eur. J. Immunol. 33:815824.[CrossRef][Medline]
Irjala, H., E.L. Johansson, R. Grenman, K. Alanen, M. Salmi, and S. Jalkanen. 2001. Mannose receptor is a novel ligand for L-selectin and mediates lymphocyte binding to lymphatic endothelium. J. Exp. Med. 194:10331042.
Johnson-Leger, C.A., M. Aurrand-Lions, N. Beltraminelli, N. Fasel, and B.A. Imhof. 2002. Junctional adhesion molecule-2 (JAM-2) promotes lymphocyte transendothelial migration. Blood. 100:24792486.
Joukov, V., K. Pajusola, A. Kaipainen, D. Chilov, I. Lahtinen, E. Kukk, O. Saksela, N. Kalkkinen, and K. Alitalo. 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. 15:290298.[Abstract]
Joukov, V., T. Sorsa, V. Kumar, M. Jeltsch, W.L. Claesson, Y. Cao, O. Saksela, N. Kalkkinen, and K. Alitalo. 1997. Proteolytic processing regulates receptor specificity and activity of VEGF-C. EMBO J. 16:38983911.
Karkkainen, M.J., A. Saaristo, L. Jussila, K.A. Karila, E.C. Lawrence, K. Pajusola, H. Bueler, A. Eichmann, R. Kauppinen, M.I. Kettunen, et al. 2001. A model for gene therapy of human hereditary lymphedema. Proc. Natl. Acad. Sci. USA. 98:1267712682.
Kriehuber, E., S. Breiteneder-Geleff, M. Groeger, A. Soleiman, S.F. Schoppmann, G. Stingl, D. Kerjaschki, and D. Maurer. 2001. Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized cell lineages. J. Exp. Med. 194:797808.
Leak, L.V. 1972. The transport of exogenous peroxidase across the blood-tissue-lymph interface. J. Ultrastruct. Res. 39:2442.[Medline]
Leak, L.V. 1976. The structure of lymphatic capillaries in lymph formation. Fed. Proc. 35:18631871.[Medline]
Leak, L.V., and J.F. Burke. 1966. Fine structure of the lymphatic capillary and the adjoining connective tissue area. Am. J. Anat. 118:785810.[Medline]
Lee, J., A. Gray, J. Yuan, S.M. Luoh, H. Avraham, and W.I. Wood. 1996. Vascular endothelial growth factor-related protein: a ligand and specific activator of the tyrosine kinase receptor Flt4. Proc. Natl. Acad. Sci. USA. 93:19881992.
Leu, A.J., D.A. Berk, A. Lymboussaki, K. Alitalo, and R.K. Jain. 2000. Absence of functional lymphatics within a murine sarcoma: a molecular and functional evaluation. Cancer Res. 60:43244327.
Makinen, T., T. Veikkola, S. Mustjoki, T. Karpanen, B. Catimel, E.C. Nice, L. Wise, A. Mercer, H. Kowalski, D. Kerjaschki, et al. 2001. Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3. EMBO J. 20:47624773.
Mandriota, S.J., L. Jussila, M. Jeltsch, A. Compagni, D. Baetens, R. Prevo, S. Banerji, J. Huarte, R. Montesano, D.G. Jackson, et al. 2001. Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis. EMBO J. 20:672682.
Mashino, K., N. Sadanaga, H. Yamaguchi, F. Tanaka, M. Ohta, K. Shibuta, H. Inoue, and M. Mori. 2002. Expression of chemokine receptor CCR7 is associated with lymph node metastasis of gastric carcinoma. Cancer Res. 62:29372941.
Mellman, I., and G. Warren. 2000. The road taken: past and future foundations of membrane traffic. Cell. 100:99112.[Medline]
Muller, A., B. Homey, H. Soto, N. Ge, D. Catron, M.E. Buchanan, T. McClanahan, E. Murphy, W. Yuan, S.N. Wagner, et al. 2001. Involvement of chemokine receptors in breast cancer metastasis. Nature. 410:5056.[CrossRef][Medline]
Padera, T.P., A. Kadambi, E. di Tomaso, C.M. Carreira, E.B. Brown, Y. Boucher, N.C. Choi, D. Mathisen, J. Wain, E.J. Mark, et al. 2002. Lymphatic metastasis in the absence of functional intratumor lymphatics. Science. 296:18831886.
Partanen, T.A., K. Alitalo, and M. Miettinen. 1999. Lack of lymphatic vascular specificity of vascular endothelial growth factor receptor 3 in 185 vascular tumors. Cancer. 86:24062412.[CrossRef][Medline]
Pepper, M.S. 2001. Lymphangiogenesis and tumor metastasis: myth or reality? Clin. Cancer Res. 7:462468.
Pepper, M.S., J.C. Tille, R. Nisato, and M. Skobe. 2003. Lymphangiogenesis and tumor metastasis. Cell Tissue Res. In press.
Petrova, T.V., T. Makinen, T.P. Makela, J. Saarela, I. Virtanen, R.E. Ferrell, D.N. Finegold, D. Kerjaschki, S. Yla-Herttuala, and K. Alitalo. 2002. Lymphatic endothelial reprogramming of vascular endothelial cells by the Prox-1 homeobox transcription factor. EMBO J. 21:45934599.
Podgrabinska, S., P. Braun, P. Velasco, B. Kloos, M.S. Pepper, D.G. Jackson, and M. Skobe. 2002. Molecular characterization of lymphatic endothelial cells. Proc. Natl. Acad. Sci. USA. 99:1606916074.
Schmid-Schönbein, G.W. 1990a. Mechanisms causing initial lymphatics to expand and compress to promote lymph flow. Arch. Histol. Cytol. 53(Suppl.):107114.[Medline]
Schmid-Schönbein, G.W. 1990b. Microlymphatics and lymph flow. Physiol. Rev. 70:9871028.
Skobe, M., T. Hawighorst, D.G. Jackson, R. Prevo, L. Janes, P. Velasco, L. Riccardi, K. Alitalo, K. Claffey, and M. Detmar. 2001. Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat. Med. 7:192198.[CrossRef][Medline]
Sleeman, J.P., J. Krishnan, V. Kirkin, and P. Baumann. 2001. Markers for the lymphatic endothelium: in search of the holy grail? Microsc. Res. Tech. 55:6169.[CrossRef][Medline]
Stacker, S.A., M.G. Achen, L. Jussila, M.E. Baldwin, and K. Alitalo. 2002. Lymphangiogenesis and cancer metastasis. Nat. Rev. Cancer. 2:573583.[CrossRef][Medline]
Takanami, I. 2003. Overexpression of CCR7 mRNA in nonsmall cell lung cancer: correlation with lymph node metastasis. Int. J. Cancer. 105:186189.[CrossRef][Medline]
Wigle, J.T., N. Harvey, M. Detmar, I. Lagutina, G. Grosveld, M.D. Gunn, D.G. Jackson, and G. Oliver. 2002. An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype. EMBO J. 21:15051513.
Wigle, J.T., and G. Oliver. 1999. Prox1 function is required for the development of the murine lymphatic system. Cell. 98:769778.[Medline]
Wiley, H.E., E.B. Gonzalez, W. Maki, M.T. Wu, and S.T. Hwang. 2001. Expression of CC chemokine receptor-7 and regional lymph node metastasis of B16 murine melanoma. J. Natl. Cancer Inst. 93:16381643.
Yuan, L., D. Moyon, L. Pardanaud, C. Breant, M.J. Karkkainen, K. Alitalo, and A. Eichmann. 2002. Abnormal lymphatic vessel development in neuropilin 2 mutant mice. Development. 129:47974806.[Medline]