%0 Journal Article %J Circulation %D 2014 %T CCBE1 enhances lymphangiogenesis via ADAMTS3-mediated VEGF-C activation %A Jeltsch, Michael %A Jha, Sawan Kumar %A Tvorogov, Denis %A Anisimov, Andrey %A Leppänen, Veli-Matti %A Holopainen, Tanja %A Kivelä, Riikka %A Ortega, Sagrario %A Kärpänen, Terhi %A Alitalo, Kari %K ADAMTS3 %K angiogenesis %K CCBE1 %K endothelium %K growth factors and cytokines %K Hennekam Syndrome %K metalloproteinase %K vasculature %K VEGF-C %X Background—Hennekam lymphangiectasia-lymphedema syndrome (OMIM 235510) is a rare autosomal recessive disease, which is associated with mutations in the collagen- and calcium-binding EGF domains 1 (CCBE1) gene. Because of the striking phenotypic similarity of embryos lacking either the Ccbe1 gene or the lymphangiogenic growth factor Vegfc gene, we searched for CCBE1 interactions with the VEGF-C growth factor signaling pathway, which is critical in embryonic and adult lymphangiogenesis. Methods and Results—By analyzing VEGF-C produced by CCBE1-transfected cells, we found that while CCBE1 itself does not process VEGF-C, it promotes proteolytic cleavage of the otherwise poorly active 29/31-kDa form of VEGF-C by the A disintegrin and metalloprotease with thrombospondin motifs-3 (ADAMTS3) protease, resulting in the mature 21/23-kDa form of VEGF-C, which induces increased VEGF-C receptor signaling. Adeno-associated viral vector (AAV) mediated transduction of CCBE1 into mouse skeletal muscle enhanced lymphangiogenesis and angiogenesis induced by AAV-VEGF-C. Conclusions—These results identify ADAMTS3 as a VEGF-C activating protease and reveal a novel type of regulation of a vascular growth factor by a protein that enhances its proteolytic cleavage and activation. The results suggest CCBE1 is a potential therapeutic tool for the modulation of lymphangiogenesis and angiogenesis in a variety of diseases that involve the lymphatic system, such as lymphedema or lymphatic metastasis. %B Circulation %V 129 %8 05/2014 %G eng %U http://circ.ahajournals.org/content/early/2014/02/19/CIRCULATIONAHA.113.002779.abstract %N 19 %& 1962-1971 %R http://dx.doi.org/10.1161/CIRCULATIONAHA.113.002779 %0 Journal Article %J Sci Signal %D 2013 %T The basis for the distinct biological activities of vascular endothelial growth factor receptor-1 ligands %A Anisimov, Andrey %A Leppänen, Veli-Matti %A Tvorogov, Denis %A Zarkada, Georgia %A Jeltsch, Michael %A Holopainen, Tanja %A Kaijalainen, Seppo %A Alitalo, Kari %K PlGF %K receptor tyrosine kinase %K Signal Transduction %K VEGF-B %K VEGFR-1 %X

Vascular endothelial growth factors (VEGFs) regulate blood and lymphatic vessel development through VEGF receptors (VEGFRs). The VEGFR immunoglobulin homology domain 2 (D2) is critical for ligand binding, and D3 provides additional interaction sites. VEGF-B and placenta growth factor (PlGF) bind to VEGFR-1 with high affinity, but only PlGF is angiogenic in most tissues. We show that VEGF-B, unlike other VEGFs, did not require D3 interactions for high-affinity binding. VEGF-B with a PlGF-derived L1 loop (B-L1(P)) stimulated VEGFR-1 activity, whereas PlGF with a VEGF-B-derived L1 loop (P-L1(B)) did not. Unlike P-L1(B) and VEGF-B, B-L1(P) and PlGF were also angiogenic in mouse skeletal muscle. Furthermore, B-L1(P) also bound to VEGFR-2 and activated downstream signaling. These results establish a role for L1-mediated D3 interactions in VEGFR activation in endothelial cells and indicate that VEGF-B is a high-affinity VEGFR-1 ligand that, unlike PlGF, cannot efficiently induce signaling downstream of VEGFR-1.

%B Sci Signal %V 6 %P ra52 %8 2013 %G eng %N 282 %R 10.1126/scisignal.2003905 %0 Journal Article %J Proceedings of the National Academy of Sciences of the United States of America %D 2013 %T Structural and mechanistic insights into VEGF receptor 3 ligand binding and activation %A Leppänen, Veli-Matti %A Tvorogov, Denis %A Kisko, Kaisa %A Prota, Andrea E %A Jeltsch, Michael %A Anisimov, Andrey %A Markovic-Mueller, Sandra %A Stuttfeld, Edward %A Goldie, Kenneth N %A Ballmer-Hofer, Kurt %A Alitalo, Kari %K Amino Acid Sequence %K Binding Sites %K Binding, Competitive %K Crystallography, X-Ray %K Electrophoresis, Polyacrylamide Gel %K Humans %K Ligands %K Microscopy, Electron %K Models, Molecular %K Molecular Sequence Data %K Multiprotein Complexes %K Mutation %K Protein Binding %K Protein Multimerization %K Protein Structure, Tertiary %K Scattering, Small Angle %K Sequence Homology, Amino Acid %K Thermodynamics %K Vascular Endothelial Growth Factor C %K Vascular Endothelial Growth Factor Receptor-3 %K X-Ray Diffraction %X Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key drivers of blood and lymph vessel formation in development, but also in several pathological processes. VEGF-C signaling through VEGFR-3 promotes lymphangiogenesis, which is a clinically relevant target for treating lymphatic insufficiency and for blocking tumor angiogenesis and metastasis. The extracellular domain of VEGFRs consists of seven Ig homology domains; domains 1-3 (D1-3) are responsible for ligand binding, and the membrane-proximal domains 4-7 (D4-7) are involved in structural rearrangements essential for receptor dimerization and activation. Here we analyzed the crystal structures of VEGF-C in complex with VEGFR-3 domains D1-2 and of the VEGFR-3 D4-5 homodimer. The structures revealed a conserved ligand-binding interface in D2 and a unique mechanism for VEGFR dimerization and activation, with homotypic interactions in D5. Mutation of the conserved residues mediating the D5 interaction (Thr446 and Lys516) and the D7 interaction (Arg737) compromised VEGF-C induced VEGFR-3 activation. A thermodynamic analysis of VEGFR-3 deletion mutants showed that D3, D4-5, and D6-7 all contribute to ligand binding. A structural model of the VEGF-C/VEGFR-3 D1-7 complex derived from small-angle X-ray scattering data is consistent with the homotypic interactions in D5 and D7. Taken together, our data show that ligand-dependent homotypic interactions in D5 and D7 are essential for VEGFR activation, opening promising possibilities for the design of VEGFR-specific drugs. %B Proceedings of the National Academy of Sciences of the United States of America %V 110 %P 12960 - 12965 %8 08/2013 %@ 1091-6490 %G eng %U http://www.pnas.org/content/110/32/12960.long %N 32 %! Proc. Natl. Acad. Sci. U.S.A. %R 10.1073/pnas.1301415110 %0 Journal Article %J Blood %D 2011 %T Structural determinants of vascular endothelial growth factor-D receptor binding and specificity %A Leppänen, Veli-Matti %A Jeltsch, Michael %A Anisimov, Andrey %A Tvorogov, Denis %A Aho, Kukka %A Kalkkinen, Nisse %A Toivanen, Pyry %A Ylä-Herttuala, Seppo %A Ballmer-Hofer, Kurt %A Alitalo, Kari %X Vascular endothelial growth factors (VEGFs) and their tyrosine kinase receptors (VEGFR-1-3) are central mediators of angiogenesis and lymphangiogenesis. VEGFR-3 ligands VEGF-C and VEGF-D are produced as precursor proteins with long N- and C-terminal propeptides and show enhanced VEGFR-2 and VEGFR-3 binding on proteolytic removal of the propeptides. Two different proteolytic cleavage sites have been reported in the VEGF-D N-terminus. We report here the crystal structure of the human VEGF-D Cys117Ala mutant at 2.9 Å resolution. Comparison of the VEGF-D and VEGF-C structures shows similar extended N-terminal helices, conserved overall folds, and VEGFR-2 interacting residues. Consistent with this, the affinity and the thermodynamic parameters for VEGFR-2 binding are very similar. In comparison with VEGF-C structures, however, the VEGF-D N-terminal helix was extended by 2 more turns because of a better resolution. Both receptor binding and functional assays of N-terminally truncated VEGF-D polypeptides indicated that the residues between the reported proteolytic cleavage sites are important for VEGF-D binding and activation of VEGFR-3, but not of VEGFR-2. Thus, we define here a VEGFR-2-specific form of VEGF-D that is angiogenic but not lymphangiogenic. These results provide important new insights into VEGF-D structure and function. %B Blood %V 117 %P 1507 - 15 %8 2011/Feb/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/21148085 %N 5 %! Blood %0 Journal Article %J Cancer Cell %D 2010 %T Effective suppression of vascular network formation by combination of antibodies blocking VEGFR ligand binding and receptor dimerization %A Tvorogov, Denis %A Anisimov, Andrey %A Zheng, Wei %A Leppänen, Veli-Matti %A Tammela, Tuomas %A Laurinavicius, Simonas %A Holnthoner, Wolfgang %A Heloterä, Hanna %A Holopainen, Tanja %A Jeltsch, Michael %A Kalkkinen, Nisse %A Lankinen, Hilkka %A Ojala, Päivi M %A Alitalo, Kari %X Antibodies that block vascular endothelial growth factor (VEGF) have become an integral part of antiangiogenic tumor therapy, and antibodies targeting other VEGFs and receptors (VEGFRs) are in clinical trials. Typically receptor-blocking antibodies are targeted to the VEGFR ligand-binding site. Here we describe a monoclonal antibody that inhibits VEGFR-3 homodimer and VEGFR-3/VEGFR-2 heterodimer formation, signal transduction, as well as ligand-induced migration and sprouting of microvascular endothelial cells. Importantly, we show that combined use of antibodies blocking ligand binding and receptor dimerization improves VEGFR inhibition and results in stronger inhibition of endothelial sprouting and vascular network formation in vivo. These results suggest that receptor dimerization inhibitors could be used to enhance antiangiogenic activity of antibodies blocking ligand binding in tumor therapy. %B Cancer Cell %V 18 %P 630 - 40 %8 2010/Dec/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/21130043 %N 6 %! Cancer Cell