@article {708, title = {VEGF-C Protects the Integrity of Bone Marrow Perivascular Niche}, journal = {Blood}, year = {2020}, month = {2020/07/31/}, pages = {accepted - for publication}, abstract = {Key Points. Vegfc deletion in endothelial or LepR+ cells compromises the bone marrow perivascular niche and hematopoietic stem cell maintenance.Exogenous admin}, doi = {10.1182/blood.2020005699}, url = {https://ashpublications.org/blood/article/doi/10.1182/blood.2020005699/463465/VEGF-C-Protects-the-Integrity-of-Bone-Marrow} } @article {589, title = {Efficient activation of the lymphangiogenic growth factor VEGF-C requires the C-terminal domain of VEGF-C and the N-terminal domain of CCBE1}, journal = {Scientific Reports}, volume = {7}, year = {2017}, month = {2017/07/07/}, pages = {4916}, doi = {10.1038/s41598-017-04982-1}, url = {https://www.nature.com/articles/s41598-017-04982-1} } @article {502, title = {Functional Dissection of the CCBE1 Protein: A Crucial Requirement for the Collagen Repeat Domain.}, journal = {Circ Res}, volume = {116}, year = {2015}, month = {2015 May 8}, pages = {1660-1669}, abstract = {

RATIONALE: Collagen- and calcium-binding EGF domain-containing protein 1 (CCBE1) is essential for lymphangiogenesis in vertebrates and has been associated with Hennekam syndrome. Recently, CCBE1 has emerged as a crucial regulator of vascular endothelial growth factor-C (VEGFC) signaling.

OBJECTIVE: CCBE1 is a secreted protein characterized by 2 EGF domains and 2 collagen repeats. The functional role of the different CCBE1 protein domains is completely unknown. Here, we analyzed the functional role of the different CCBE1 domains in vivo and in vitro.

METHODS AND RESULTS: We analyzed the functionality of several CCBE1 deletion mutants by generating knock-in mice expressing these mutants, by analyzing their ability to enhance Vegfc signaling in vivo in zebrafish, and by testing their ability to induce VEGFC processing in vitro. We found that deleting the collagen domains of CCBE1 has a much stronger effect on CCBE1 activity than deleting the EGF domains. First, although CCBE1ΔCollagen mice fully phenocopy CCBE1 knock-out mice, CCBE1ΔEGF knock-in embryos still form rudimentary lymphatics. Second, Ccbe1ΔEGF, but not Ccbe1ΔCollagen, could partially substitute for Ccbe1 to enhance Vegfc signaling in zebrafish. Third, CCBE1ΔEGF, similarly to CCBE1, but not CCBE1ΔCollagen could activate VEGFC processing in vitro. Furthermore, a Hennekam syndrome mutation within the collagen domain has a stronger effect than a Hennekam syndrome mutation within the EGF domain.

CONCLUSIONS: We propose that the collagen domains of CCBE1 are crucial for the activation of VEGFC in vitro and in vivo. The EGF domains of CCBE1 are dispensable for regulation of VEGFC processing in vitro, however, they are necessary for full lymphangiogenic activity of CCBE1 in vivo.

}, issn = {1524-4571}, doi = {10.1161/CIRCRESAHA.116.304949}, url = {http://circres.ahajournals.org/content/116/10/1660.long} } @inbook {503, title = {The TIE Receptor Family}, booktitle = {Receptor Tyrosine Kinases: Family and Subfamilies}, year = {2015}, pages = {743-775}, publisher = {Springer International Publishing}, organization = {Springer International Publishing}, chapter = {16}, issn = {978-3-319-11888-8}, doi = {10.1007/978-3-319-11888-8_16}, url = {https://link.springer.com/content/pdf/10.1007\%2F978-3-319-11888-8_16.pdf} } @article {441, title = {CCBE1 enhances lymphangiogenesis via ADAMTS3-mediated VEGF-C activation}, journal = {Circulation}, volume = {129}, year = {2014}, month = {05/2014}, chapter = {1962-1971}, abstract = {Background{\textemdash}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{\textemdash}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{\textemdash}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.}, doi = {http://dx.doi.org/10.1161/CIRCULATIONAHA.113.002779}, url = {http://circ.ahajournals.org/content/early/2014/02/19/CIRCULATIONAHA.113.002779.abstract} } @article {426, title = {The basis for the distinct biological activities of vascular endothelial growth factor receptor-1 ligands}, journal = {Sci Signal}, volume = {6}, year = {2013}, month = {2013}, pages = {ra52}, abstract = {

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.

}, issn = {1937-9145}, doi = {10.1126/scisignal.2003905} } @article {445, title = {Receptor Tyrosine Kinase-Mediated Angiogenesis}, journal = {Cold Spring Harbor Perspectives in Biology}, volume = {5}, year = {2013}, month = {2013}, abstract = {The endothelial cell is the essential cell type forming the inner layer of the vasculature. Two families of receptor tyrosine kinases (RTKs) are almost completely endothelial cell specific: the vascular endothelial growth factor (VEGF) receptors (VEGFR1-3) and the Tie receptors (Tie1 and Tie2). Both are key players governing the generation of blood and lymphatic vessels during embryonic development. Because the growth of new blood and lymphatic vessels (or the lack thereof) is a central element in many diseases, the VEGF and the Tie receptors provide attractive therapeutic targets in various diseases. Indeed, several drugs directed to these RTK signaling pathways are already on the market, whereas many are in clinical trials. Here we review the VEGFR and Tie families, their involvement in developmental and pathological angiogenesis, and the different possibilities for targeting them to either block or enhance angiogenesis and lymphangiogenesis.}, isbn = {, 1943-0264}, doi = {10.1101/cshperspect.a009183}, url = {http://cshperspectives.cshlp.org/content/5/9/a009183} } @article {442, title = {Structural and mechanistic insights into VEGF receptor 3 ligand binding and activation}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {110}, year = {2013}, month = {08/2013}, pages = {12960 - 12965}, abstract = {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.}, isbn = {1091-6490}, doi = {10.1073/pnas.1301415110}, url = {http://www.pnas.org/content/110/32/12960.long} } @article {45, title = {Structural determinants of vascular endothelial growth factor-D receptor binding and specificity}, journal = {Blood}, volume = {117}, year = {2011}, month = {2011/Feb/}, pages = {1507 - 15}, abstract = {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 {\r A} 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.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/21148085}, author = {Lepp{\"a}nen, Veli-Matti and Jeltsch, Michael and Anisimov, Andrey and Tvorogov, Denis and Aho, Kukka and Kalkkinen, Nisse and Toivanen, Pyry and Yl{\"a}-Herttuala, Seppo and Ballmer-Hofer, Kurt and Alitalo, Kari} } @article {42, title = {Effective suppression of vascular network formation by combination of antibodies blocking VEGFR ligand binding and receptor dimerization}, journal = {Cancer Cell}, volume = {18}, year = {2010}, month = {2010/Dec/}, pages = {630 - 40}, abstract = {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.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/21130043}, author = {Tvorogov, Denis and Anisimov, Andrey and Zheng, Wei and Lepp{\"a}nen, Veli-Matti and Tammela, Tuomas and Laurinavicius, Simonas and Holnthoner, Wolfgang and Heloter{\"a}, Hanna and Holopainen, Tanja and Jeltsch, Michael and Kalkkinen, Nisse and Lankinen, Hilkka and Ojala, P{\"a}ivi M and Alitalo, Kari} } @article {40, title = {Structural determinants of growth factor binding and specificity by VEGF receptor 2}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {107}, year = {2010}, month = {02/2010}, pages = {2425 - 30}, abstract = {Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel formation through activation of three receptor tyrosine kinases, VEGFR-1, -2, and -3. The extracellular domain of VEGF receptors consists of seven immunoglobulin homology domains, which, upon ligand binding, promote receptor dimerization. Dimerization initiates transmembrane signaling, which activates the intracellular tyrosine kinase domain of the receptor. VEGF-C stimulates lymphangiogenesis and contributes to pathological angiogenesis via VEGFR-3. However, proteolytically processed VEGF-C also stimulates VEGFR-2, the predominant transducer of signals required for physiological and pathological angiogenesis. Here we present the crystal structure of VEGF-C bound to the VEGFR-2 high-affinity-binding site, which consists of immunoglobulin homology domains D2 and D3. This structure reveals a symmetrical 22 complex, in which left-handed twisted receptor domains wrap around the 2-fold axis of VEGF-C. In the VEGFs, receptor specificity is determined by an N-terminal alpha helix and three peptide loops. Our structure shows that two of these loops in VEGF-C bind to VEGFR-2 subdomains D2 and D3, while one interacts primarily with D3. Additionally, the N-terminal helix of VEGF-C interacts with D2, and the groove separating the two VEGF-C monomers binds to the D2/D3 linker. VEGF-C, unlike VEGF-A, does not bind VEGFR-1. We therefore created VEGFR-1/VEGFR-2 chimeric proteins to further study receptor specificity. This biochemical analysis, together with our structural data, defined VEGFR-2 residues critical for the binding of VEGF-A and VEGF-C. Our results provide significant insights into the structural features that determine the high affinity and specificity of VEGF/VEGFR interactions.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/20145116}, author = {Lepp{\"a}nen, Veli-Matti and Prota, Andrea E and Jeltsch, Michael and Anisimov, Andrey and Kalkkinen, Nisse and Strandin, Tomas and Lankinen, Hilkka and Goldman, Adrian and Ballmer-Hofer, Kurt and Alitalo, Kari} } @article {38, title = {Activated forms of VEGF-C and VEGF-D provide improved vascular function in skeletal muscle}, journal = {Circ Res}, volume = {104}, year = {2009}, month = {2009/Jun/}, pages = {1302 - 12}, abstract = {The therapeutic potential of vascular endothelial growth factor (VEGF)-C and VEGF-D in skeletal muscle has been of considerable interest as these factors have both angiogenic and lymphangiogenic activities. Previous studies have mainly used adenoviral gene delivery for short-term expression of VEGF-C and VEGF-D in pig, rabbit, and mouse skeletal muscles. Here we have used the activated mature forms of VEGF-C and VEGF-D expressed via recombinant adeno-associated virus (rAAV), which provides stable, long-lasting transgene expression in various tissues including skeletal muscle. Mouse tibialis anterior muscle was transduced with rAAV encoding human or mouse VEGF-C or VEGF-D. Two weeks later, immunohistochemical analysis showed increased numbers of both blood and lymph vessels, and Doppler ultrasound analysis indicated increased blood vessel perfusion. The lymphatic vessels further increased at the 4-week time point were functional, as shown by FITC-lectin uptake and transport. Furthermore, receptor activation and arteriogenic activity were increased by an alanine substitution mutant of human VEGF-C (C137A) having an increased dimer stability and by a chimeric CAC growth factor that contained the VEGF receptor-binding domain flanked by VEGF-C propeptides, but only the latter promoted significantly more blood vessel perfusion when compared to the other growth factors studied. We conclude that long-term expression of VEGF-C and VEGF-D in skeletal muscle results in the generation of new functional blood and lymphatic vessels. The therapeutic value of intramuscular lymph vessels in draining tissue edema and lymphedema can now be evaluated using this model system.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/19443835}, author = {Anisimov, Andrey and Alitalo, Annamari and Korpisalo, Petra and Soronen, Jarkko and Kaijalainen, Seppo and Lepp{\"a}nen, Veli-Matti and Jeltsch, Michael and Yl{\"a}-Herttuala, Seppo and Alitalo, Kari} }