%0 Journal Article %J Molecular Genetics & Genomic Medicine %D 2020 %T Investigation on the role of biallelic variants in VEGF‐C found in a patient affected by Milroy‐like lymphedema %A Mukenge, Sylvain %A Jha, Sawan K. %A Catena, Marco %A Manara, Elena %A Leppänen, Veli‐Matti %A Lenti, Elisa %A Negrini, Daniela %A Bertelli, Matteo %A Brendolan, Andrea %A Jeltsch, Michael %A Aldrighetti, Luca %B Molecular Genetics & Genomic Medicine %V 00 %P e1389 %8 Feb-06-2022 %G eng %U https://onlinelibrary.wiley.com/doi/abs/10.1002/mgg3.1389 %! Mol Genet Genomic Med %R 10.1002/mgg3.1389 %0 Journal Article %J Blood %D 2020 %T VEGF-C Protects the Integrity of Bone Marrow Perivascular Niche %A Fang, Shentong %A Chen, Shuo %A Nurmi, Harri %A Leppänen, Veli-Matti %A Jeltsch, Michael %A Scadden, David T. %A Silberstein, Lev %A Mikkola, Hanna %A Alitalo, Kari %X Key Points. Vegfc deletion in endothelial or LepR+ cells compromises the bone marrow perivascular niche and hematopoietic stem cell maintenance.Exogenous admin %B Blood %P accepted - for publication %8 2020/07/31/ %G eng %U https://ashpublications.org/blood/article/doi/10.1182/blood.2020005699/463465/VEGF-C-Protects-the-Integrity-of-Bone-Marrow %! Blood %R 10.1182/blood.2020005699 %0 Journal Article %J eLife %D 2019 %T KLK3/PSA and cathepsin D activate VEGF-C and VEGF-D %A Jha, Sawan Kumar %A Rauniyar, Khushbu %A Chronowska, Ewa %A Mattonet, Kenny %A Maina, Eunice Wairimu %A Koistinen, Hannu %A Stenman, Ulf-Håkan %A Alitalo, Kari %A Jeltsch, Michael %K cancer biology %K Cathepsin D %K kallikrein-related peptidases %K KLK3/PSA %K Lymphangiogenesis %K mouse %K VEGF-C %K VEGF-D %X Vascular endothelial growth factor-C (VEGF-C) acts primarily on endothelial cells, but also on non-vascular targets, e.g. in the CNS and immune system. Here we describe a novel, unique VEGF-C form in the human reproductive system produced via cleavage by kallikrein-related peptidase 3 (KLK3), aka prostate-specific antigen (PSA). KLK3 activated VEGF-C specifically and efficiently through cleavage at a novel N-terminal site. We detected VEGF-C in seminal plasma, and sperm liquefaction occurred concurrently with VEGF-C activation, which was enhanced by collagen and calcium binding EGF domains 1 (CCBE1). After plasmin and ADAMTS3, KLK3 is the third protease shown to activate VEGF-C. Since differently activated VEGF-Cs are characterized by successively shorter N-terminal helices, we created an even shorter hypothetical form, which showed preferential binding to VEGFR-3. Using mass spectrometric analysis of the isolated VEGF-C-cleaving activity from human saliva, we identified cathepsin D as a protease that can activate VEGF-C as well as VEGF-D. %B eLife %V 8 %P e44478 %8 2019/05/17/ %@ 2050-084X %G eng %U https://elifesciences.org/articles/44478 %! eLife %0 Journal Article %J Scientific Reports %D 2017 %T Efficient activation of the lymphangiogenic growth factor VEGF-C requires the C-terminal domain of VEGF-C and the N-terminal domain of CCBE1 %A Jha, SK %A Rauniyar, Khushbu %A Kärpänen, Terhi %A Leppänen, Veli-Matti %A Brouillard, Pascal %A Vikkula, Miikka %A Alitalo, Kari %A Jeltsch, Michael %B Scientific Reports %V 7 %P 4916 %8 2017/07/07/ %G eng %U https://www.nature.com/articles/s41598-017-04982-1 %N 1 %R 10.1038/s41598-017-04982-1 %0 Journal Article %J Circulation Research %D 2016 %T Functional Importance of a Proteoglycan Co-Receptor in Pathologic Lymphangiogenesis %A Johns, Scott C. %A Yin, Xin %A Jeltsch, Michael %A Bishop, Joseph R. %A Schuksz, Manuela %A Ghazal, Roland El %A Wilcox-Adelman, Sarah A. %A Alitalo, Kari %A Fuster, Mark M. %K endothelial cell growth %K glycosaminoglycan %K lymphatic capillary %K Proteoglycan %K vascular endothelial growth factor receptor %X Rationale: Lymphatic vessel growth is mediated by major pro-lymphangiogenic factors such as VEGF-C and -D, among other endothelial effectors. Heparan sulfate is a linear polysaccharide expressed on proteoglycan core proteins on cell-membranes and matrix, playing roles in angiogenesis, although little is known regarding any function(s) in lymphatic remodeling in vivo. Objective: To explore the genetic basis and mechanisms whereby heparan sulfate proteoglycans mediate pathologic lymphatic remodeling. Methods and Results: Lymphatic endothelial deficiency in the major heparan sulfate biosynthetic enzyme N-deacetylase/N-sulfotransferase-1 (Ndst1; involved in glycan-chain sulfation) was associated with reduced lymphangiogenesis in pathologic models, including spontaneous neoplasia. Mouse mutants demonstrated tumor-associated lymphatic vessels with apoptotic nuclei. Mutant lymphatic endothelia demonstrated impaired mitogen (Erk) and survival (Akt) pathway signaling as well as reduced VEGF-C mediated protection from starvation-induced apoptosis. Lymphatic endothelial specific Ndst1 deficiency (in Ndst1f/fProx1+/CreERT2 mice) was sufficient to inhibit VEGF-C dependent lymphangiogenesis. Lymphatic heparan sulfate deficiency reduced phosphorylation of the major lymphatic growth receptor VEGFR-3 in response to multiple VEGF-C species. Syndecan-4 was the dominantly expressed heparan sulfate proteoglycan in mouse lymphatic endothelia, and pathologic lymphangiogenesis was impaired in Sdc4(-/-) mice. On the lymphatic cell surface, VEGF-C induced robust association between syndecan-4 and VEGFR-3 which was sensitive to glycan disruption. Moreover, VEGFR-3 mitogen and survival signaling was reduced in the setting of Ndst1 or Sdc4 deficiency. Conclusions: These findings demonstrate the genetic importance of heparan sulfate and the major lymphatic proteoglycan syndecan-4 in pathologic lymphatic remodeling. This may introduce novel future strategies to alter pathologic lymphatic-vascular remodeling. %B Circulation Research %V 119 %P 210-221 %8 05/2016 %G eng %U http://circres.ahajournals.org/content/early/2016/05/25/CIRCRESAHA.116.308504 %N 2 %R 10.1161/CIRCRESAHA.116.308504 %0 Journal Article %J Circ Res %D 2015 %T Functional Dissection of the CCBE1 Protein: A Crucial Requirement for the Collagen Repeat Domain. %A Roukens, M Guy %A Peterson-Maduro, Josi %A Padberg, Yvonne %A Jeltsch, Michael %A Leppänen, Veli-Matti %A Bos, Frank L %A Alitalo, Kari %A Schulte-Merker, Stefan %A Schulte, Dörte %K Animals %K Binding Sites %K Calcium-Binding Proteins %K Collagen %K Craniofacial Abnormalities %K Endothelial Cells %K Epidermal Growth Factor %K Gene Expression Regulation, Developmental %K Gene Knock-In Techniques %K Genital Diseases, Male %K Genotype %K Gestational Age %K HEK293 Cells %K Humans %K Lymphangiectasis, Intestinal %K Lymphatic Vessels %K lymphedema %K Mice %K Mice, Transgenic %K Mutation %K Phenotype %K Protein Binding %K Protein Interaction Domains and Motifs %K Signal Transduction %K Transfection %K Tumor Suppressor Proteins %K Vascular Endothelial Growth Factor C %K Zebrafish %K Zebrafish Proteins %X

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.

%B Circ Res %V 116 %P 1660-1669 %8 2015 May 8 %G eng %U http://circres.ahajournals.org/content/116/10/1660.long %N 10 %R 10.1161/CIRCRESAHA.116.304949 %0 Journal Article %J American Journal of Transplantation %D 2015 %T Ischemia-Reperfusion Injury Enhances Lymphatic Endothelial VEGFR3 and Rejection in Cardiac Allografts %A Dashkevich, A. %A Raissadati, A. %A Syrjälä, S. O. %A Zarkada, G. %A Keränen, M. A. I. %A Tuuminen, R. %A Krebs, R. %A Anisimov, A. %A Jeltsch, M. %A Leppänen, V.-M. %A Alitalo, K. %A Nykänen, A. I. %A Lemström, K. B. %X Organ damage and innate immunity during heart transplantation may evoke adaptive immunity with serious consequences. Because lymphatic vessels bridge innate and adaptive immunity, they are critical in immune surveillance; however, their role in ischemia–reperfusion injury (IRI) in allotransplantation remains unknown. We investigated whether the lymphangiogenic VEGF-C/VEGFR3 pathway during cardiac allograft IRI regulates organ damage and subsequent interplay between innate and adaptive immunity. We found that cardiac allograft IRI, within hours, increased graft VEGF-C expression and lymphatic vessel activation in the form of increased lymphatic VEGFR3 and adhesion protein expression. Pharmacological VEGF-C/VEGFR3 stimulation resulted in early lymphatic activation and later increase in allograft inflammation. In contrast, pharmacological VEGF-C/VEGFR3 inhibition during cardiac allograft IRI decreased early lymphatic vessel activation with subsequent dampening of acute and chronic rejection. Genetic deletion of VEGFR3 specifically in the lymphatics of the transplanted heart recapitulated the survival effect achieved by pharmacological VEGF-C/VEGFR3 inhibition. Our results suggest that tissue damage rapidly changes lymphatic vessel phenotype, which, in turn, may shape the interplay of innate and adaptive immunity. Importantly, VEGF-C/VEGFR3 inhibition during solid organ transplant IRI could be used as lymphatic-targeted immunomodulatory therapy to prevent acute and chronic rejection. %B American Journal of Transplantation %V 16 %P 1160-1172 %8 12/2015 %G eng %U http://onlinelibrary.wiley.com/doi/10.1111/ajt.13564/abstract %N 4 %R 10.1111/ajt.13564 %0 Book Section %B Receptor Tyrosine Kinases: Family and Subfamilies %D 2015 %T The TIE Receptor Family %A Saharinen, Pipsa %A Jeltsch, Michael %A Santoyo, MayteM. %A Leppänen, Veli-Matti %A Alitalo, Kari %E Wheeler, Deric L. %E Yarden, Yosef %K ANG %K Angiopoietin %K ANGPT %K Endothelial cell %K Lymphatic vessel %K Neovascularization %K TIE1 %K TIE2 %K Vascular dysfunction %B Receptor Tyrosine Kinases: Family and Subfamilies %I Springer International Publishing %P 743-775 %G eng %U https://link.springer.com/content/pdf/10.1007%2F978-3-319-11888-8_16.pdf %& 16 %R 10.1007/978-3-319-11888-8_16 %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 Cold Spring Harbor Perspectives in Biology %D 2013 %T Receptor Tyrosine Kinase-Mediated Angiogenesis %A Jeltsch, Michael %A Leppänen, Veli-Matti %A Saharinen, Pipsa %A Alitalo, Kari %X 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. %B Cold Spring Harbor Perspectives in Biology %V 5 %8 2013 %@ , 1943-0264 %G eng %U http://cshperspectives.cshlp.org/content/5/9/a009183 %N 9 %! Cold Spring Harb Perspect Biol %R 10.1101/cshperspect.a009183 %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 Development %D 2013 %T A truncation allele in vascular endothelial growth factor c reveals distinct modes of signaling during lymphatic and vascular development. %A Villefranc, Jacques A %A Nicoli, Stefania %A Bentley, Katie %A Jeltsch, Michael %A Zarkada, Georgia %A Moore, John C %A Gerhardt, Holger %A Alitalo, Kari %A Lawson, Nathan D %K Alleles %K Animals %K Animals, Genetically Modified %K Autocrine Communication %K Blood Vessels %K Cell Movement %K Codon, Nonsense %K Embryo, Nonmammalian %K Female %K Lymphatic System %K Mice %K Mice, Knockout %K Neovascularization, Physiologic %K Paracrine Communication %K Protein Isoforms %K Signal Transduction %K Vascular Endothelial Growth Factor C %K Zebrafish %K Zebrafish Proteins %X

Vascular endothelial growth factor C (Vegfc) is a secreted protein that guides lymphatic development in vertebrate embryos. However, its role during developmental angiogenesis is not well characterized. Here, we identify a mutation in zebrafish vegfc that severely affects lymphatic development and leads to angiogenesis defects on sensitized genetic backgrounds. The um18 mutation prematurely truncated Vegfc, blocking its secretion and paracrine activity but not its ability to activate its receptor Flt4. When expressed in endothelial cells, vegfc(um18) could not rescue lymphatic defects in mutant embryos, but induced ectopic blood vessel branching. Furthermore, vegfc-deficient endothelial cells did not efficiently contribute to tip cell positions in developing sprouts. Computational modeling together with assessment of endothelial cell dynamics by time-lapse analysis suggested that an autocrine Vegfc/Flt4 loop plays an important role in migratory persistence and filopodia stability during sprouting. Our results suggest that Vegfc acts in two distinct modes during development: as a paracrine factor secreted from arteries to guide closely associated lymphatic vasculature and as an autocrine factor to drive migratory persistence during angiogenesis.

%B Development %V 140 %P 1497-506 %8 2013 Apr %G eng %N 7 %R 10.1242/dev.084152 %0 Journal Article %J Circulation %D 2013 %T Vascular endothelial growth factor-angiopoietin chimera with improved properties for therapeutic angiogenesis %A Andrey Anisimov %A Denis Tvorogov %A Annamari Alitalo %A Veli-Matti Leppänen %A Y An %A EC Han %A F Orsenigo %A EI Gaál %A Tanja Holopainen %A YJ Koh %A Tuomas Tammela %A P Korpisalo %A Salla Keskitalo %A Michael Jeltsch %A Seppo Ylä-Herttuala %A Elisabetta Dejana %A GY Koh %A C Choi %A Pipsa Saharinen %A Kari Alitalo %X BACKGROUND: There is an unmet need for proangiogenic therapeutic molecules for the treatment of tissue ischemia in cardiovascular diseases. However, major inducers of angiogenesis such as vascular endothelial growth factor (VEGF/VEGF-A) have side effects that limit their therapeutic utility in vivo, especially at high concentrations. Angiopoietin-1 has been considered to be a blood vessel stabilization factor that can inhibit the intrinsic property of VEGF to promote vessel leakiness. In this study, we have designed and tested the angiogenic properties of chimeric molecules consisting of receptor-binding parts of VEGF and angiopoietin-1. We aimed at combining the activities of both factors into 1 molecule for easy delivery and expression in target tissues. METHODS AND RESULTS: The VEGF-angiopoietin-1 (VA1) chimeric protein bound to both VEGF receptor-2 and Tie2 and induced the activation of both receptors. Detailed analysis of VA1 versus VEGF revealed differences in the kinetics of VEGF receptor-2 activation and endocytosis, downstream kinase activation, and VE-cadherin internalization. The delivery of a VA1 transgene into mouse skeletal muscle led to increased blood flow and enhanced angiogenesis. VA1 was also very efficient in rescuing ischemic limb perfusion. However, VA1 induced less plasma protein leakage and myeloid inflammatory cell recruitment than VEGF. Furthermore, angioma-like structures associated with VEGF expression were not observed with VA1. CONCLUSIONS: The VEGF-angiopoietin-1 chimera is a potent angiogenic factor that triggers a novel mode of VEGF receptor-2 activation, promoting less vessel leakiness, less tissue inflammation, and better perfusion in ischemic muscle than VEGF. These properties of VA1 make it an attractive therapeutic tool. %B Circulation %V 127 %P 424-434 %8 01/2013 %G eng %N 4 %& 424 %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 Genes Dev %D 2010 %T Claudin-like protein 24 interacts with the VEGFR-2 and VEGFR-3 pathways and regulates lymphatic vessel development %A Saharinen, Pipsa %A Helotera, Hanna %A Miettinen, Juho %A Norrmen, Camilla %A D'Amico, Gabriela %A Jeltsch, Michael %A Langenberg, Tobias %A Vandevelde, Wouter %A Ny, Annelii %A Dewerchin, Mieke %A Carmeliet, Peter %A Alitalo, Kari %X The Claudin-like protein of 24 kDa (CLP24) is a hypoxia-regulated transmembrane protein of unknown function. We show here that clp24 knockdown in Danio rerio and Xenopus laevis results in defective lymphatic development. Targeted disruption of Clp24 in mice led to enlarged lymphatic vessels having an abnormal smooth muscle cell coating. We also show that the Clp24(-/-) phenotype was further aggravated in the Vegfr2(+/LacZ) or Vegfr3(+/LacZ) backgrounds and that CLP24 interacts with vascular endothelial growth factor receptor-2 (VEGFR-2) and VEGFR-3 and attenuates the transcription factor CREB phosphorylation via these receptors. Our results indicate that CLP24 is a novel regulator of VEGFR-2 and VEGFR-3 signaling pathways and of normal lymphatic vessel structure. %B Genes Dev %V 24 %P 875 - 80 %8 2010/May/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/20439428 %N 9 %! Genes & Development %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 %0 Journal Article %J Proceedings of the National Academy of Sciences of the United States of America %D 2010 %T Structural determinants of growth factor binding and specificity by VEGF receptor 2 %A Leppänen, Veli-Matti %A Prota, Andrea E %A Jeltsch, Michael %A Anisimov, Andrey %A Kalkkinen, Nisse %A Strandin, Tomas %A Lankinen, Hilkka %A Goldman, Adrian %A Ballmer-Hofer, Kurt %A Alitalo, Kari %X 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. %B Proceedings of the National Academy of Sciences of the United States of America %V 107 %P 2425 - 30 %8 02/2010 %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/20145116 %N 6 %0 Journal Article %J PLoS ONE %D 2010 %T Suppressive effects of vascular endothelial growth factor-B on tumor growth in a mouse model of pancreatic neuroendocrine tumorigenesis %A Albrecht, Imke %A Kopfstein, Lucie %A Strittmatter, Karin %A Schomber, Tibor %A Falkevall, Annelie %A Hagberg, Carolina E %A Lorentz, Pascal %A Jeltsch, Michael %A Alitalo, Kari %A Eriksson, Ulf %A Christofori, Gerhard %A Pietras, Kristian %X BACKGROUND: The family of vascular endothelial growth factors (VEGF) contains key regulators of blood and lymph vessel development, including VEGF-A, -B, -C, -D, and placental growth factor. The role of VEGF-B during physiological or pathological angiogenesis has not yet been conclusively delineated. Herein, we investigate the function of VEGF-B by the generation of mouse models of cancer with transgenic expression of VEGF-B or homozygous deletion of Vegfb. METHODOLOGY/PRINCIPAL FINDINGS: Ectopic expression of VEGF-B in the insulin-producing β-cells of the pancreas did not alter the abundance or architecture of the islets of Langerhans. The vasculature from transgenic mice exhibited a dilated morphology, but was of similar density as that of wildtype mice. Unexpectedly, we found that transgenic expression of VEGF-B in the RIP1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis retarded tumor growth. Conversely, RIP1-Tag2 mice deficient for Vegfb presented with larger tumors. No differences in vascular density, perfusion or immune cell infiltration upon altered Vegfb gene dosage were noted. However, VEGF-B acted to increase blood vessel diameter both in normal pancreatic islets and in RIP1-Tag2 tumors. CONCLUSIONS/SIGNIFICANCE: Taken together, our results illustrate the differences in biological function between members of the VEGF family, and highlight the necessity of in-depth functional studies of VEGF-B to fully understand the effects of VEGFR-1 inhibitors currently used in the clinic. %B PLoS ONE %V 5 %P e14109 %8 2010// %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/21124841 %N 11 %! PLoS ONE %0 Journal Article %J Circulation %D 2010 %T Vascular endothelial growth factor-B acts as a coronary growth factor in transgenic rats without inducing angiogenesis, vascular leak, or inflammation %A Bry, Maija %A Kivelä, Riikka %A Holopainen, Tanja %A Anisimov, Andrey %A Tammela, Tuomas %A Soronen, Jarkko %A Silvola, Johanna %A Saraste, Antti %A Jeltsch, Michael %A Korpisalo, Petra %A Carmeliet, Peter %A Lemström, Karl B %A Shibuya, Masabumi %A Ylä-Herttuala, Seppo %A Alhonen, Leena %A Mervaala, Eero %A Andersson, Leif C %A Knuuti, Juhani %A Alitalo, Kari %X BACKGROUND: Vascular endothelial growth factor-B (VEGF-B) binds to VEGF receptor-1 and neuropilin-1 and is abundantly expressed in the heart, skeletal muscle, and brown fat. The biological function of VEGF-B is incompletely understood. METHODS AND RESULTS: Unlike placenta growth factor, which binds to the same receptors, adeno-associated viral delivery of VEGF-B to mouse skeletal or heart muscle induced very little angiogenesis, vascular permeability, or inflammation. As previously reported for the VEGF-B(167) isoform, transgenic mice and rats expressing both isoforms of VEGF-B in the myocardium developed cardiac hypertrophy yet maintained systolic function. Deletion of the VEGF receptor-1 tyrosine kinase domain or the arterial endothelial Bmx tyrosine kinase inhibited hypertrophy, whereas loss of VEGF-B interaction with neuropilin-1 had no effect. Surprisingly, in rats, the heart-specific VEGF-B transgene induced impressive growth of the epicardial coronary vessels and their branches, with large arteries also seen deep inside the subendocardial myocardium. However, VEGF-B, unlike other VEGF family members, did not induce significant capillary angiogenesis, increased permeability, or inflammatory cell recruitment. CONCLUSIONS: VEGF-B appears to be a coronary growth factor in rats but not in mice. The signals for the VEGF-B-induced cardiac hypertrophy are mediated at least in part via the endothelium. Because cardiomyocyte damage in myocardial ischemia begins in the subendocardial myocardium, the VEGF-B-induced increased arterial supply to this area could have therapeutic potential in ischemic heart disease. %B Circulation %V 122 %P 1725 - 33 %8 2010/Oct/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/20937974 %N 17 %! Circulation %0 Journal Article %J Circ Res %D 2009 %T Activated forms of VEGF-C and VEGF-D provide improved vascular function in skeletal muscle %A Anisimov, Andrey %A Alitalo, Annamari %A Korpisalo, Petra %A Soronen, Jarkko %A Kaijalainen, Seppo %A Leppänen, Veli-Matti %A Jeltsch, Michael %A Ylä-Herttuala, Seppo %A Alitalo, Kari %X 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. %B Circ Res %V 104 %P 1302 - 12 %8 2009/Jun/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/19443835 %N 11 %! Circulation Research %0 Journal Article %J Circ Res %D 2008 %T Overexpression of vascular endothelial growth factor-B in mouse heart alters cardiac lipid metabolism and induces myocardial hypertrophy %A Karpanen, Terhi %A Bry, Maija %A Ollila, Hanna M %A Seppänen-Laakso, Tuulikki %A Liimatta, Erkki %A Leskinen, Hanna %A Kivelä, Riikka %A Helkamaa, Teemu %A Merentie, Mari %A Jeltsch, Michael %A Paavonen, Karri %A Andersson, Leif C %A Mervaala, Eero %A Hassinen, Ilmo E %A Ylä-Herttuala, Seppo %A Oresic, Matej %A Alitalo, Kari %X Vascular endothelial growth factor (VEGF)-B is poorly angiogenic but prominently expressed in metabolically highly active tissues, including the heart. We produced mice expressing a cardiac-specific VEGF-B transgene via the alpha-myosin heavy chain promoter. Surprisingly, the hearts of the VEGF-B transgenic mice showed concentric cardiac hypertrophy without significant changes in heart function. The cardiac hypertrophy was attributable to an increased size of the cardiomyocytes. Blood capillary size was increased, whereas the number of blood vessels per cell nucleus remained unchanged. Despite the cardiac hypertrophy, the transgenic mice had lower heart rate and blood pressure than their littermates, and they responded similarly to angiotensin II-induced hypertension, confirming that the hypertrophy does not compromise heart function. Interestingly, the isolated transgenic hearts had less cardiomyocyte damage after ischemia. Significantly increased ceramide and decreased triglyceride levels were found in the transgenic hearts. This was associated with structural changes and eventual lysis of mitochondria, resulting in accumulation of intracellular vacuoles in cardiomyocytes and increased death of the transgenic mice, apparently because of mitochondrial lipotoxicity in the heart. These results suggest that VEGF-B regulates lipid metabolism, an unexpected function for an angiogenic growth factor. %B Circ Res %V 103 %P 1018 - 26 %8 2008/Oct/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/18757827 %N 9 %! Circulation Research %0 Journal Article %J Arterioscler Thromb Vasc Biol %D 2008 %T Reevaluation of the role of VEGF-B suggests a restricted role in the revascularization of the ischemic myocardium %A Li, Xuri %A Tjwa, Marc %A Van Hove, Inge %A Enholm, Berndt %A Neven, Elke %A Paavonen, Karri %A Jeltsch, Michael %A Juan, Toni Diez %A Sievers, Richard E %A Chorianopoulos, Emmanuel %A Wada, Hiromichi %A Vanwildemeersch, Maarten %A Noel, Agnes %A Foidart, Jean-Michel %A Springer, Matthew L %A von Degenfeld, Georges %A Dewerchin, Mieke %A Blau, Helen M %A Alitalo, Kari %A Eriksson, Ulf %A Carmeliet, Peter %A Moons, Lieve %X OBJECTIVE: The endogenous role of the VEGF family member vascular endothelial growth factor-B (VEGF-B) in pathological angiogenesis remains unclear. METHODS AND RESULTS: We studied the role of VEGF-B in various models of pathological angiogenesis using mice lacking VEGF-B (VEGF-B(-/-)) or overexpressing VEGF-B(167). After occlusion of the left coronary artery, VEGF-B deficiency impaired vessel growth in the ischemic myocardium whereas, in wild-type mice, VEGF-B(167) overexpression enhanced revascularization of the infarct and ischemic border zone. By contrast, VEGF-B deficiency did not affect vessel growth in the wounded skin, hypoxic lung, ischemic retina, or ischemic limb. Moreover, VEGF-B(167) overexpression failed to enhance vascular growth in the skin or ischemic limb. CONCLUSIONS: VEGF-B appears to have a relatively restricted angiogenic activity in the ischemic heart. These insights might offer novel therapeutic opportunities. %B Arterioscler Thromb Vasc Biol %V 28 %P 1614 - 20 %8 2008/Sep/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/18511699 %N 9 %! Arteriosclerosis, Thrombosis, and Vascular Biology %0 Journal Article %J Cancer Res %D 2008 %T The tyrosine kinase inhibitor cediranib blocks ligand-induced vascular endothelial growth factor receptor-3 activity and lymphangiogenesis %A Heckman, Caroline A %A Holopainen, Tanja %A Wirzenius, Maria %A Keskitalo, Salla %A Jeltsch, Michael %A Ylä-Herttuala, Seppo %A Wedge, Stephen R %A Jürgensmeier, Juliane M %A Alitalo, Kari %X Solid tumors express a range of factors required to sustain their growth and promote their dissemination. Among these are vascular endothelial growth factor-A (VEGF-A), the key angiogenic stimulant, and VEGF-C, a primary mediator of lymphangiogenesis. Small molecule tyrosine kinase inhibitors offer the potential to inhibit more than one kinase and impede tumor growth by multiple mechanisms. However, their potency toward individual targets can vary. Cediranib (RECENTIN; AZD2171) is an inhibitor of VEGF signaling that has been shown in experimental models to prevent VEGF-A-induced angiogenesis and primary tumor growth, yet the effects of cediranib on VEGF receptor (VEGFR)-3-mediated endothelial cell function and lymphangiogenesis are unknown. To better understand the activity of cediranib against VEGFR-3 and its associated signaling events compared with its activity against VEGFR-2, we used the receptor-specific ligands VEGF-E and VEGF-C156S. In human endothelial cells, cediranib inhibited VEGF-E-induced phosphorylation of VEGFR-2 and VEGF-C156S-induced phosphorylation of VEGFR-3 at concentrations of