From the molecular biological foundations to causal treatment options for diseases of the lymphatic system

PD Dr Michael Jeltsch, University of Helsinki, Finland

Research into the molecular basis of lymphangiogenesis in embryonic development and pathological processes has led to a rapid expansion of our knowledge (Krebs and Jeltsch 2013a, 2013b). The molecular biology era of lymphatic research began with the discovery of VEGF growth factors and their receptors 25 years ago. This review therefore focuses on these molecules.

After it was demonstrated in 1995 that VEGFR-3 is specifically expressed on the endothelium of lymphatic vessels in the adult organism (Kaipainen et al. 1995), several laboratories attempted to identify the ligand or ligands that activate it. The first discovery was made by Kari Alitalo in 1996 with VEGF-C (Joukov et al. 1996). Shortly afterwards, Alitalo’s group was also able to demonstrate that VEGF-C is a specifically lymphangiogenic growth factor (Jeltsch et al. 1997; Oh et al. 1997) and elucidated the details of VEGF-C biosynthesis (Joukov et al. 1997). It was published in 2000 that mutations in the VEGFR-3 gene can cause hereditary lymphoedema (Karkkainen et al. 2000). However, it was not until twelve years later that it was demonstrated that VEGF-C mutations can also be a cause (Gordon et al. 2013). As early as 2001, a preclinical study showed that primary lymphoedema can be successfully treated with the growth factor VEGF-C (Karkkainen et al. 2001). Nevertheless, it is only this year that clinical trials will begin which (unlike the previously published treatment study) target not primary but secondary lymphoedema following breast cancer surgery. VEGF-C-producing adenoviral vectors (brand name Lymfactin™) are being used to increase the success rate of lymph node transplants. The trial is being conducted by Herantis Pharma in collaboration with several European partners (Honkonen et al. 2013; Herantis Pharma 2014).

The active role of VEGF-C in the lymphogenous metastasis of primary tumours, and thus the potential therapeutic efficacy of blocking the VEGF-C/VEGFR-3 signalling pathway, was first described in 2001 by two research groups (Karpanen et al. 2001; Mandriota et al. 2001). Two biopharmaceuticals that block VEGF-C signal transduction are now in clinical trials, though not for the inhibition of metastasis, but as an adjunct to the standard anti-angiogenic therapy bevacizumab (Martell et al. 2011; Falchook et al. 2013).

Of the latest, and in some cases controversial, (re)discoveries in lymphangiogenesis research, only three will be mentioned here that fall within the field of VEGF research:

• The lymph capillaries of the skin play an important role in osmoregulation and blood pressure regulation, specifically via the release of VEGF-C by salt-sensitive macrophages and the associated clearance of interstitial Na+ stores in the skin (Wiig et al. 2013).
• The Schlemm’s canal develops from venous structures into a vessel with typical lymphatic molecular markers and properties (Aspelund et al., in press).
• The CCBE1 protein acts in a pro-lymphangiogenic manner by concentrating inactive pro-VEGF-C on cell surfaces and extracellular structures, thereby enabling its activation by the immobilised protease ADAMTS3. When VEGF-C and CCBE1, a dramatic increase in the lymphangiogenic effect of VEGF-C was observed in vivo, suggesting the use of CCBE1 in pro-lymphangiogenic therapies (Jeltsch et al. 2014).

Bibliography

Krebs R, Jeltsch M. The lymphangiogenic growth factors VEGF-C and VEGF-D. Part 1. Fundamentals and embryonic development. Lymphforsch. 2013 a;17(1):30–7. Available from: https://doi.org/10.5281/zenodo.3611691 

Krebs R, Jeltsch M. The lymphangiogenic growth factors VEGF-C and VEGF-D. Part 2. The role of VEGF-C and VEGF-D in diseases of the lymphatic system. Lymphforsch. 2013 b;17(2):96–104. Available from: https://doi.org/10.5281/zenodo.4438655 

Kaipainen A, Korhonen J, Mustonen T, van Hinsbergh VW, Fang GH, Dumont D, Breitman M, Alitalo K. Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. *Proc Natl Acad Sci *USA. 11 April 1995;92(8):3566–70. Availabe from: https://doi.org/10.1073/pnas.92.8.3566 

Joukov V, Pajusola K, Kaipainen A, Chilov D, Lahtinen I, Kukk E, Saksela O, Kalkkinen N, 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. 1996;15(2):290–8. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC449944/ 

Jeltsch M, Kaipainen A, Joukov V, Meng X, Lakso M, Rauvala H, Swartz M, Fukumura D, Jain RK, Alitalo K. Hyperplasia of Lymphatic Vessels in VEGF-C Transgenic Mice. Science. 1997;276(5317):1423–5. Available from: https://www.sciencemag.org/content/276/5317/1423 

Oh S-J, Jeltsch MM, Birkenhäger R, McCarthy JEG, Weich HA, Christ B, Alitalo K, Wilting J. VEGF and VEGF-C: Specific Induction of Angiogenesis and Lymphangiogenesis in the Differentiated Avian Chorioallantoic Membrane. Developmental Biology. 1997;188(1):96–109. Available from: https://www.sciencedirect.com/science/article/pii/S0012160697986395 

Joukov V, Sorsa T, Kumar V, Jeltsch M, Claesson-Welsh L, Cao Y, Saksela O, Kalkkinen N, Alitalo K. Proteolytic processing regulates receptor specificity and activity of VEGF-C. The EMBO Journal. 1997;16(13):3898–911. Available from: https://www.nature.com/emboj/journal/v16/n13/full/7590367a.html 

Karkkainen MJ, Ferrell RE, Lawrence EC, Kimak MA, Levinson KL, McTigue MA, Alitalo K, Finegold DN. Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema. Nature Genetics. 2000;25(2):153–9. Available from: https://www.nature.com/ng/journal/v25/n2/full/ng0600_153.html 

Gordon K, Schulte D, Brice G, Simpson MA, Roukens MG, van Impel A, Connell F, Kalidas K, Jeffery S, Mortimer PS, Mansour S, Schulte-Merker S, Ostergaard P. Mutation in Vascular Endothelial Growth Factor-C, a Ligand for Vascular Endothelial Growth Factor Receptor-3, Is Associated With Autosomal Dominant Milroy-Like Primary Lymphoedema: Novelty and Significance. Circulation Research. 2013;112(6):956–60. Available from: https://circres.ahajournals.org/content/112/6/956 

Karkkainen MJ, Saaristo A, Jussila L, Karila KA, Lawrence EC, Pajusola K, Bueler H, Eichmann A, Kauppinen R, Kettunen MI, Ylä-Herttuala S, Finegold DN, Ferrell RE, Alitalo K. A model for gene therapy of human hereditary lymphoedema. PNAS. 2001;98(22):12677–82. Available from: https://www.pnas.org/content/98/22/12677 

Honkonen KM, Visuri MT, Tervala TV, Halonen PJ, Koivisto M, Lahteenvuo MT, Alitalo KK, Yla-Herttuala S, Saaristo AM. Lymph Node Transfer and Perinodal Lymphatic Growth Factor Treatment for Lymphoedema. Annals of Surgery. 2013;257(5):961–7. Available from: https://dx.doi.org/10.1097/SLA.0b013e31826ed043 

Herantis Pharma. Lymfactin® for lymphoedema

. 2014. Available from: https://web.archive.org/web/20140531191950/http://herantis.com/pipeline/lymfactin-for-lymphedema/ 

Karpanen T, Egeblad M, Karkkainen MJ, Kubo H, Ylä-Herttuala S, Jäättelä M, Alitalo K. Vascular Endothelial Growth Factor C Promotes Tumour Lymphangiogenesis and Intralymphatic Tumour Growth. Cancer Res. 2001;61(5):1786–90. Available from: https://cancerres.aacrjournals.org/content/61/5/1786 

Mandriota SJ, Jussila L, Jeltsch M, Compagni A, Baetens D, Prevo R, Banerji S, Huarte J, Montesano R, Jackson DG, Orci L, Alitalo K, Christofori G, Pepper MS. Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis. EMBO J. 2001;20(4):672–82. Available from: https://dx.doi.org/10.1093/emboj/20.4.672 

Martell RE, Chiorean EG, Youssoufian H, Rutstein M, Pytowski B, Benjamin L, Abad L, Patel J, Steele T, Dowd M, Qin A, Kukel C, Fox F, Dontabhaktuni A, Morozov A. Phase I study of the anti-VEGFR-3 monoclonal antibody IMC-3C5 in subjects with advanced solid tumours refractory to standard therapy or for which no standard therapy is available. 2011. Available from: https://meetinglibrary.asco.org/content/61178?format=posterImg 

Falchook GS, Desai J, Leitch IM, Goldman JW, Wheler JJ, Fu S, Kurzrok R, Baldwin ME, Rosen LS. Phase 1 Study of VGX-100, an anti-VEGF-C monoclonal antibody, with or without Bevacizumab in Patients with Advanced Solid Tumours. 2013. Available from: https://web.archive.org/web/20140305122346/https://www.circadian.com.au/sites/default/files/VGX-100%20Phase%201%20Oncology%20Trial%20Presented%20at%20ASCO%202013.pdf 

Wiig H, Schröder A, Neuhofer W, Jantsch J, Kopp C, Karlsen TV, Boschmann M, Goss J, Bry M, Rakova N, Dahlmann A, Brenner S, Tenstad O, Nurmi H, Mervaala E, Wagner H, Beck F-X, Müller DN, Kerjaschki D, et al. Immune cells control skin lymphatic electrolyte homeostasis and blood pressure. J Clin Invest. 2013;123(7):2803–15. Available from: https://www.jci.org/articles/view/60113 

Aspelund A, Tammela T, Antila S, Nurmi H, Leppänen V-M, Zarkada G, Stanczuk L, Francois M, Mäkinen T, Saharinen P, Immonen I, Alitalo K. Schlemm’s canal is a VEGF-C/VEGFR-3-responsive lymphatic-like vessel. Journal of Clinical Investigation. Available from: https://doi.org/10.1172/JCI75395 

Jeltsch M, Jha SK, Tvorogov D, Anisimov A, Leppänen V-M, Holopainen T, Kivelä R, Ortega S, Kärpanen T, Alitalo K. CCBE1 enhances lymphangiogenesis via a disintegrin and metalloprotease with thrombospondin motifs-3–mediated vascular endothelial growth factor-C activation. Circulation. 2014;129(19):1962–71. Available from: https://circ.ahajournals.org/content/129/19/1962