@article {694, title = {Lymphatics and the eye. [Finnish]}, journal = {Duodecim L{\"a}{\"a}ketieteellinen Aikakauskirja}, volume = {136}, year = {2020}, month = {2020/02/10/}, pages = {1777-1788}, type = {review}, chapter = {1777}, issn = {2242-3281}, doi = {10.5281/zenodo.4005517}, url = {https://www.duodecimlehti.fi/lehti/2020/16/duo15739}, author = {Gucciardo, Erika and Lehti, Timo A. and Korhonen, Ani and Salv{\'e}n, Petri and Lehti, Kaisa and Jeltsch, Michael and Loukovaara, Sirpa} } @article {693, title = {KLK3/PSA and cathepsin D activate VEGF-C and VEGF-D}, journal = {eLife}, volume = {8}, year = {2019}, month = {2019/05/17/}, pages = {e44478}, abstract = {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.}, keywords = {cancer biology, Cathepsin D, kallikrein-related peptidases, KLK3/PSA, Lymphangiogenesis, mouse, VEGF-C, VEGF-D}, isbn = {2050-084X}, url = {https://elifesciences.org/articles/44478}, author = {Jha, Sawan Kumar and Rauniyar, Khushbu and Chronowska, Ewa and Mattonet, Kenny and Maina, Eunice Wairimu and Koistinen, Hannu and Stenman, Ulf-H{\r a}kan and Alitalo, Kari and Jeltsch, Michael} } @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}, author = {Jha, SK and Rauniyar, Khushbu and K{\"a}rp{\"a}nen, Terhi and Lepp{\"a}nen, Veli-Matti and Brouillard, Pascal and Vikkula, Miikka and Alitalo, Kari and Jeltsch, Michael} } @article {505, title = {Ischemia-Reperfusion Injury Enhances Lymphatic Endothelial VEGFR3 and Rejection in Cardiac Allografts}, journal = {American Journal of Transplantation}, volume = {16}, year = {2015}, month = {12/2015}, pages = {1160-1172}, abstract = {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{\textendash}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.}, doi = {10.1111/ajt.13564}, url = {http://onlinelibrary.wiley.com/doi/10.1111/ajt.13564/abstract}, author = {Dashkevich, A. and Raissadati, A. and Syrj{\"a}l{\"a}, S. O. and Zarkada, G. and Ker{\"a}nen, M. A. I. and Tuuminen, R. and Krebs, R. and Anisimov, A. and Jeltsch, M. and Lepp{\"a}nen, V.-M. and Alitalo, K. and Nyk{\"a}nen, A. I. and Lemstr{\"o}m, K. B.} } @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.}, keywords = {ADAMTS3, angiogenesis, CCBE1, endothelium, growth factors and cytokines, Hennekam Syndrome, metalloproteinase, vasculature, VEGF-C}, doi = {http://dx.doi.org/10.1161/CIRCULATIONAHA.113.002779}, url = {http://circ.ahajournals.org/content/early/2014/02/19/CIRCULATIONAHA.113.002779.abstract}, author = {Jeltsch, Michael and Jha, Sawan Kumar and Tvorogov, Denis and Anisimov, Andrey and Lepp{\"a}nen, Veli-Matti and Holopainen, Tanja and Kivel{\"a}, Riikka and Ortega, Sagrario and K{\"a}rp{\"a}nen, Terhi and Alitalo, Kari} } @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.
}, keywords = {PlGF, receptor tyrosine kinase, Signal Transduction, VEGF-B, VEGFR-1}, issn = {1937-9145}, doi = {10.1126/scisignal.2003905}, author = {Anisimov, Andrey and Lepp{\"a}nen, Veli-Matti and Tvorogov, Denis and Zarkada, Georgia and Jeltsch, Michael and Holopainen, Tanja and Kaijalainen, Seppo and Alitalo, Kari} } @article {443, title = {Die lymphangiogenic growth factors VEGF-C and VEGF-D. Part 2: The role of VEGF-C and VEGF-D in diseases of the lymphatic system. [bilingual: English, German].}, journal = {Lymphologie in Forschung und Praxis}, volume = {17}, year = {2013}, month = {11/2013}, pages = {96 - 104}, abstract = {VEGF-C and VEGF-D are the two central signaling molecules that stimulate the develop- ment and growth of the lymphatic system. Both belong to the vascular endothelial growth factor (VEGF) protein family, which plays important roles in the growth of blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis). In mammals, the VEGF family comprises five members: VEGF-A, PlGF, VEGF-B, VEGF-C and VEGF-D. The family was named after VEGF-A, the first member to be discovered. VEGF-C and VEGF-D form a subgroup within this family in terms of function and structure. Their distinctive biosynthesis differentiates them from the other VEGFs: they are produced as inactive precursors and need to be activated by proteolytic removal of their long N- and C-terminal propeptides. Unlike the other VEGFs, VEGF-C and VEGF-D are direct stimulators of lymphatic vessel growth. They exert their lymphangiogenic function via VEGF receptor 3, which is expressed in the adult organism almost exclusively on lymphatic endothelial cells. In this review, we provide an overview of the VEGF protein family and their receptors. We focus on the lymphangiogenic VEGF-C and VEGF-D, discussing their biosynthesis and their role in embryonic lymphangiogenesis.}, keywords = {growth factors, Lymphangiogenesis, lymphedema, lymphogenic metastasis, VEGF-C, VEGF-D}, url = {http://jeltsch.org/sites/jeltsch.org/files/JeltschMichael_Lymphforsch2013_96.pdf}, author = {Krebs, Rainer and Jeltsch, Michael} } @article {444, title = {The lymphangiogenic growth factors VEGF-C and VEGF-D. Part 1: Basic principles and embryonic development. [bilingual: English, German].}, journal = {Lymphologie in Forschung und Praxis}, volume = {17}, year = {2013}, month = {05/2013}, pages = {30 - 37}, abstract = {VEGF-C and VEGF-D are the two central signaling molecules that stimulate the develop- ment and growth of the lymphatic system. Both belong to the vascular endothelial growth factor (VEGF) protein family, which plays important roles in the growth of blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis). In mammals, the VEGF family comprises five members: VEGF-A, PlGF, VEGF-B, VEGF-C and VEGF-D. The family was named after VEGF-A, the first member to be discovered. VEGF-C and VEGF-D form a subgroup within this family in terms of function and structure. Their distinctive biosynthesis differentiates them from the other VEGFs: they are produced as inactive precursors and need to be activated by proteolytic removal of their long N- and C-terminal propeptides. Unlike the other VEGFs, VEGF-C and VEGF-D are direct stimulators of lymphatic vessel growth. They exert their lymphangiogenic function via VEGF receptor 3, which is expressed in the adult organism almost exclusively on lymphatic endothelial cells. In this review, we provide an overview of the VEGF protein family and their receptors. We focus on the lymphangiogenic VEGF-C and VEGF-D, discussing their biosynthesis and their role in embryonic lymphangiogenesis.}, keywords = {growth factors, Lymphangiogenesis, VEGF-C, VEGF-D}, url = {http://jeltsch.org/sites/jeltsch.org/files/JeltschMichael_Lymphforsch2013_30.pdf}, author = {Krebs, Rainer and Jeltsch, Michael} } @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.}, keywords = {Amino Acid Sequence, Binding Sites, Binding, Competitive, Crystallography, X-Ray, Electrophoresis, Polyacrylamide Gel, Humans, Ligands, Microscopy, Electron, Models, Molecular, Molecular Sequence Data, Multiprotein Complexes, Mutation, Protein Binding, Protein Multimerization, Protein Structure, Tertiary, Scattering, Small Angle, Sequence Homology, Amino Acid, Thermodynamics, Vascular Endothelial Growth Factor C, Vascular Endothelial Growth Factor Receptor-3, X-Ray Diffraction}, isbn = {1091-6490}, doi = {10.1073/pnas.1301415110}, url = {http://www.pnas.org/content/110/32/12960.long}, author = {Lepp{\"a}nen, Veli-Matti and Tvorogov, Denis and Kisko, Kaisa and Prota, Andrea E and Jeltsch, Michael and Anisimov, Andrey and Markovic-Mueller, Sandra and Stuttfeld, Edward and Goldie, Kenneth N and Ballmer-Hofer, Kurt and Alitalo, Kari} } @article {424, title = {Vascular endothelial growth factor-angiopoietin chimera with improved properties for therapeutic angiogenesis}, journal = {Circulation}, volume = {127}, year = {2013}, month = {01/2013}, pages = {424-434}, chapter = {424}, abstract = {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.}, author = {Andrey Anisimov and Denis Tvorogov and Annamari Alitalo and Veli-Matti Lepp{\"a}nen and Y An and EC Han and F Orsenigo and EI Ga{\'a}l and Tanja Holopainen and YJ Koh and Tuomas Tammela and P Korpisalo and Salla Keskitalo and Michael Jeltsch and Seppo Yl{\"a}-Herttuala and Elisabetta Dejana and GY Koh and C Choi and Pipsa Saharinen and Kari Alitalo} } @article {427, title = {Critical role of VEGF-C/VEGFR-3 signaling in innate and adaptive immune responses in experimental obliterative bronchiolitis.}, journal = {Am J Pathol}, volume = {181}, year = {2012}, month = {2012 Nov}, pages = {1607-20}, abstract = {Chronic inflammation, a hallmark of obliterative bronchiolitis, is known to induce lymphangiogenesis. We therefore studied the role of lymphangiogenic vascular endothelial growth factor C (VEGF-C), its receptor VEGFR-3, and lymphangiogenesis during development of experimental obliterative bronchiolitis [ie, obliterative airway disease (OAD)] in rat tracheal allografts. The functional importance of VEGF-C was investigated by adenovirus-mediated overexpression of VEGF-C (AdVEGF-C), and by inhibition of VEGF-C activity with VEGFR-3-Ig (AdVEGFR-3-Ig). Analyses included histology, immunohistochemistry, and real-time RT-PCR 10 and 30 days after transplantation. In the course of OAD development, lymphangiogenesis was induced in the airway wall during the alloimmune response, which was reversed by cyclosporine A in a dose-dependent fashion. VEGF-C overexpression in tracheal allografts induced epithelial activation, neutrophil chemotaxis, and a shift toward a Th17 adaptive immune response, followed by enhanced lymphangiogenesis and the development of OAD. In contrast, inhibition of VEGF-C activity with VEGFR-3-Ig inhibited lymphangiogenesis and angiogenesis and reduced infiltration of CD4(+) T cells and the development of OAD. Lymphangiogenesis was linked to T-cell responses during the development of OAD, and VEGF-C/VEGFR-3 signaling modulated innate and adaptive immune responses in the development of OAD in rat tracheal allografts. Our results thus suggest VEGFR-3-signaling as a novel strategy to regulate T-cell responses in the development of obliterative bronchiolitis after lung transplantation.
}, keywords = {Adaptive Immunity, Animals, Bronchiolitis Obliterans, Chemotaxis, Cyclosporine, Dendritic Cells, Dose-Response Relationship, Drug, Down-Regulation, Epithelial Cells, Epithelium, Graft Rejection, Immunity, Innate, Immunoglobulins, Inflammation, Lymphangiogenesis, Macrophages, Neutrophils, Rats, Signal Transduction, Th17 Cells, Trachea, Transplantation, Homologous, Up-Regulation, Vascular Endothelial Growth Factor C, Vascular Endothelial Growth Factor Receptor-3}, issn = {1525-2191}, doi = {10.1016/j.ajpath.2012.07.021}, author = {Krebs, Rainer and Tikkanen, Jussi M and Ropponen, Jussi O and Jeltsch, Michael and Jokinen, Janne J and Yl{\"a}-Herttuala, Seppo and Nyk{\"a}nen, Antti I and Lemstr{\"o}m, Karl B} } @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 {44, title = {VEGF-C/VEGFR-3 Signaling Regulates Inflammatory Response in Development of Obliterative Airway Disease}, journal = {Journal of Heart and Lung Transplantation}, volume = {30}, year = {2011}, month = {2011//}, pages = {S118 - S118}, author = {Krebs, R and Tikkanen, JM and Ropponen, JO and Jeltsch, M and Jokinen, JJ and Yl{\"a}-Herttuala, S and Koskinen, PK and Nyk{\"a}nen, AI and Lemstr{\"o}m, KB} } @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 {41, title = {Suppressive effects of vascular endothelial growth factor-B on tumor growth in a mouse model of pancreatic neuroendocrine tumorigenesis}, journal = {PLoS ONE}, volume = {5}, year = {2010}, month = {2010//}, pages = {e14109}, abstract = {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.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/21124841}, author = {Albrecht, Imke and Kopfstein, Lucie and Strittmatter, Karin and Schomber, Tibor and Falkevall, Annelie and Hagberg, Carolina E and Lorentz, Pascal and Jeltsch, Michael and Alitalo, Kari and Eriksson, Ulf and Christofori, Gerhard and Pietras, Kristian} } @article {43, title = {Vascular endothelial growth factor-B acts as a coronary growth factor in transgenic rats without inducing angiogenesis, vascular leak, or inflammation}, journal = {Circulation}, volume = {122}, year = {2010}, month = {2010/Oct/}, pages = {1725 - 33}, abstract = {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.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/20937974}, author = {Bry, Maija and Kivel{\"a}, Riikka and Holopainen, Tanja and Anisimov, Andrey and Tammela, Tuomas and Soronen, Jarkko and Silvola, Johanna and Saraste, Antti and Jeltsch, Michael and Korpisalo, Petra and Carmeliet, Peter and Lemstr{\"o}m, Karl B and Shibuya, Masabumi and Yl{\"a}-Herttuala, Seppo and Alhonen, Leena and Mervaala, Eero and Andersson, Leif C and Knuuti, Juhani 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} } @article {37, title = {Overexpression of vascular endothelial growth factor-B in mouse heart alters cardiac lipid metabolism and induces myocardial hypertrophy}, journal = {Circ Res}, volume = {103}, year = {2008}, month = {2008/Oct/}, pages = {1018 - 26}, abstract = {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.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/18757827}, author = {Karpanen, Terhi and Bry, Maija and Ollila, Hanna M and Sepp{\"a}nen-Laakso, Tuulikki and Liimatta, Erkki and Leskinen, Hanna and Kivel{\"a}, Riikka and Helkamaa, Teemu and Merentie, Mari and Jeltsch, Michael and Paavonen, Karri and Andersson, Leif C and Mervaala, Eero and Hassinen, Ilmo E and Yl{\"a}-Herttuala, Seppo and Oresic, Matej and Alitalo, Kari} } @article {35, title = {The tyrosine kinase inhibitor cediranib blocks ligand-induced vascular endothelial growth factor receptor-3 activity and lymphangiogenesis}, journal = {Cancer Res}, volume = {68}, year = {2008}, month = {2008/Jun/}, pages = {4754 - 62}, abstract = {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 =1nmol/L and inhibited activation of downstream signaling molecules. Additionally, cediranib blocked VEGF-C156S-induced and VEGF-E-induced proliferation, survival, and migration of lymphatic and blood vascular endothelial cells. In vivo, cediranib (6 mg/kg/d) prevented angiogenesis and lymphangiogenesis induced by VEGF-E-expressing and VEGF-C156S-expressing adenoviruses, respectively. Cediranib (6 mg/kg/day) also blocked angiogenesis and lymphangiogenesis induced by adenoviruses expressing VEGF-A or VEGF-C and compromised the blood and lymphatic vasculatures of VEGF-C-expressing tumors. Cediranib may, therefore, be an effective means of preventing tumor progression, not only by inhibiting VEGFR-2 activity and angiogenesis, but also by concomitantly inhibiting VEGFR-3 activity and lymphangiogenesis.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/18559522}, author = {Heckman, Caroline A and Holopainen, Tanja and Wirzenius, Maria and Keskitalo, Salla and Jeltsch, Michael and Yl{\"a}-Herttuala, Seppo and Wedge, Stephen R and J{\"u}rgensmeier, Juliane M and Alitalo, Kari} } @article {34, title = {Enhanced capillary formation stimulated by a chimeric vascular endothelial growth factor/vascular endothelial growth factor-C silk domain fusion protein}, journal = {Circ Res}, volume = {100}, year = {2007}, month = {2007/May/}, pages = {1460 - 7}, abstract = {Vascular endothelial growth factor (VEGF)-C and VEGF-D require proteolytic cleavage of the carboxy terminal silk-homology domain for activation. To study the functions of the VEGF-C propeptides, we engineered a chimeric growth factor protein, VEGF-CAC, composed of the amino- and carboxy-terminal propeptides of VEGF-C fused to the receptor-activating core domain of VEGF. Like VEGF-C, VEGF-CAC underwent proteolytic cleavage, and like VEGF, it bound to and activated VEGF receptor-1 and VEGF receptor-2, but not the VEGF-C receptor VEGF receptor-3. VEGF-CAC also bound to neuropilins in a heparin-dependent manner. Strikingly, when VEGF-CAC was expressed via an adenovirus vector in the ear skin of immunodeficient mice, it proved to be a more potent inducer of capillary angiogenesis than VEGF. The VEGF-CAC-induced vessels differed greatly from those induced by VEGF, as they formed a very dense and fine network of pericyte and basement membrane-covered capillaries that were functional, as shown by lectin perfusion experiments. VEGF-CAC could prove useful in proangiogenic therapies in patients experiencing tissue ischemia.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/17478734}, author = {Keskitalo, Salla and Tammela, Tuomas and Lyytikka, Johannes and Karpanen, Terhi and Jeltsch, Michael and Markkanen, Johanna and Yla-Herttuala, Seppo and Alitalo, Kari} } @conference {625, title = {Inhibiton of VEGF-C-induced VEGFR-3 activity and lymphatic endothelial cell function by the tyrosine kinase inhibitor AZD2171}, booktitle = {98th Annual Meeting of the American-Association-for-Cancer-Research}, year = {2007}, month = {2007///}, publisher = {American Association for Cancer Research}, organization = {American Association for Cancer Research}, address = {Los Angeles, CA}, abstract = {Solid tumors express a range of growth factors required to sustain their growth and promote their dissemination. Among these factors is vascular endothelial growth factor-A (VEGF-A), the key angiogenic stimulant, and VEGF-C, a primary mediator of lymphangiogenesis. Small molecule tyrosine kinase inhibitors can prevent VEGF signaling activity by targeting the VEGF receptors and are an effective approach to impede tumor progression. The indole-ether quinazoline AZD2171 is a highly potent ATP-competitive inhibitor of VEGFR-2 (KDR) kinase, with additional activity against VEGFR-1 (Flt-1) and -3 (Flt-4), that has been shown in experimental models to prevent VEGF-A-induced angiogenesis and primary tumor growth (Wedge et al. Cancer Res 2005;65:4389-4400). For these studies we wished to further assess the ability of AZD2171 to inhibit VEGFR-3 and its associated functions. Upon binding its ligands VEGF-C or -D, VEGFR-3 becomes activated with the resulting signaling cascade eventually translated into increased proliferation, survival and migration of lymphatic and blood vascular endothelial cells. At concentrations of <=1 nM AZD2171 inhibited VEGFR-3 phosphorylation in porcine aortic endothelial cells selectively expressing the human receptor, and in human dermal microvascular endothelial cells (HDMVECs). In HDMVECs, AZD2171 prevented phosphorylation of signaling molecules downstream of VEGFR-2 and -3, ERK1/2, Akt and CREB, induced by the VEGFR-2 and -3-specific ligands VEGF-E and -C156S, respectively. Additionally, AZD2171 blocked VEGF-E- and -C156S-induced proliferation of both lymphatic and blood vascular endothelial cells at similar concentrations, and prevented ligand-induced endothelial cell cord formation in a Matrigel assay. The effects of AZD2171 on VEGF-C-induced lymphangiogenesis are currently being assessed in vivo. These studies, together with previous results, not only demonstrate that AZD2171 may be an effective means of preventing tumor progression by inhibition of VEGFR-2 activity and angiogenesis, but may also prevent further tumor spread by inhibiting VEGFR-3 activity}, url = {http://dx.doi.org/10.1158/0008-5472.CAN-07-5809}, author = {Heckman, Caroline A. and Holopainen, Tanja and Wirzenius, Maria and Keskitalo, Salla and Jeltsch, Michael and Wedge, Stephen R. and Jurgensmeier, Juliane M.} } @article {31, title = {Functional interaction of VEGF-C and VEGF-D with neuropilin receptors}, journal = {FASEB J}, volume = {20}, year = {2006}, month = {2006/Jul/}, pages = {1462 - 72}, abstract = {Lymphatic vascular development is regulated by vascular endothelial growth factor receptor-3 (VEGFR-3), which is activated by its ligands VEGF-C and VEGF-D. Neuropilin-2 (NP2), known to be involved in neuronal development, has also been implicated to play a role in lymphangiogenesis. We aimed to elucidate the mechanism by which NP2 is involved in lymphatic endothelial cell signaling. By in vitro binding studies we found that both VEGF-C and VEGF-D interact with NP2, VEGF-C in a heparin-independent and VEGF-D in a heparin-dependent manner. We also mapped the domains of VEGF-C and NP2 required for their binding. The functional importance of the interaction of NP2 with the lymphangiogenic growth factors was demonstrated by cointernalization of NP2 along with VEGFR-3 in endocytic vesicles of lymphatic endothelial cells upon stimulation with VEGF-C or VEGF-D. NP2 also interacted with VEGFR-3 in coprecipitation studies. Our results show that NP2 is directly involved in an active signaling complex with the key regulators of lymphangiogenesis and thus suggest a mechanism by which NP2 functions in the development of the lymphatic vasculature.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/16816121}, author = {K{\"a}rp{\"a}nen, Terhi and Heckman, Caroline A and Keskitalo, Salla and Jeltsch, Michael and Ollila, Hanna and Neufeld, Gera and Tamagnone, Luca and Alitalo, Kari} } @article {32, title = {Vascular endothelial growth factor (VEGF)/VEGF-C mosaic molecules reveal specificity determinants and feature novel receptor binding patterns}, journal = {J Biol Chem}, volume = {281}, year = {2006}, month = {2006/Apr/}, pages = {12187 - 95}, abstract = {Vascular endothelial growth factors (VEGFs) and their receptors play key roles in angiogenesis and lymphangiogenesis. VEGF activates VEGF receptor-1 (VEGFR-1) and VEGFR-2, whereas VEGF-C activates VEGFR-2 and VEGFR-3. We have created a library of VEGF/VEGF-C mosaic molecules that contains factors with novel receptor binding profiles, notably proteins binding to all three VEGF receptors ("super-VEGFs"). The analyzed super-VEGFs show both angiogenic and lymphangiogenic effects in vivo, although weaker than the parental molecules. The composition of the VEGFR-3 binding molecules and scanning mutagenesis revealed determinants of receptor binding and specificity. VEGFR-2 and VEGFR-3 showed striking differences in their requirements for VEGF-C binding; extracellular domain 2 of VEGFR-2 was sufficient, whereas in VEGFR-3, both domains 1 and 2 were necessary.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/16505489}, author = {Jeltsch, Michael and Karpanen, Terhi and Strandin, Tomas and Aho, Kukka and Lankinen, Hilkka and Alitalo, Kari} } @article {28, title = {Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins}, journal = {Nat Immunol}, volume = {5}, year = {2004}, month = {2004/Jan/}, pages = {74 - 80}, abstract = {Lymphatic vessels are essential for immune surveillance, tissue fluid homeostasis and fat absorption. Defects in lymphatic vessel formation or function cause lymphedema. Here we show that the vascular endothelial growth factor C (VEGF-C) is required for the initial steps in lymphatic development. In Vegfc-/- mice, endothelial cells commit to the lymphatic lineage but do not sprout to form lymph vessels. Sprouting was rescued by VEGF-C and VEGF-D but not by VEGF, indicating VEGF receptor 3 specificity. The lack of lymphatic vessels resulted in prenatal death due to fluid accumulation in tissues, and Vegfc+/- mice developed cutaneous lymphatic hypoplasia and lymphedema. Our results indicate that VEGF-C is the paracrine factor essential for lymphangiogenesis, and show that both Vegfc alleles are required for normal lymphatic development.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/14634646}, author = {Karkkainen, Marika J and Haiko, Paula and Sainio, Kirsi and Partanen, Juha and Taipale, Jussi and Petrova, Tatiana V and Jeltsch, Michael and Jackson, David G and Talikka, Marja and Rauvala, Heikki and Betsholtz, Christer and Alitalo, Kari} } @article {27, title = {Intrinsic versus microenvironmental regulation of lymphatic endothelial cell phenotype and function}, journal = {FASEB J}, volume = {17}, year = {2003}, month = {2003/Nov/}, pages = {2006 - 13}, abstract = {Vascular endothelial cells are characterized by a high degree of functional and phenotypic plasticity, which is controlled both by their pericellular microenvironment and their intracellular gene expression programs. To gain further insight into the mechanisms regulating the endothelial cell phenotype, we have compared the responses of lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BECs) to vascular endothelial growth factors (VEGFs). VEGFR-3-specific signals are sufficient for LEC but not BEC proliferation, as shown by the ability of the specific ligand VEGF-C156S to stimulate cell cycle entry only in LECs. On the other hand, we found that VEGFR-3 stimulation did not induce LEC cell shape changes typical of VEGFR-2-stimulated LECs, indicating receptor-specific differences in the cytoskeletal responses. Genes induced via VEGFR-2 also differed between BECs and LECs: angiopoietin-2 (Ang-2) was induced via VEGFR-2 in BECs and LECs, but the smooth muscle cell (SMC) chemoattractant BMP-2 was induced only in BECs. Both BECs and LECs were able to promote SMC chemotaxis, but contact with SMCs led to down-regulation of VEGFR-3 expression in BECs in a 3-dimensional coculture system. This was consistent with the finding that VEGFR-3 is down-regulated in vivo at sites of endothelial cell-pericyte/smooth muscle cell contacts. Collectively, these data show intrinsic cell-specific differences of BEC and LEC responses to VEGFs and identify a pericellular regulatory mechanism for VEGFR-3 down-regulation in endothelial cells.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/14597670}, author = {Veikkola, Tanja and Lohela, Marja and Ikenberg, Kristian and M{\"a}kinen, Taija and Korff, Thomas and Saaristo, Anne and Petrova, Tatania and Jeltsch, Michael and Augustin, Hellmut G and Alitalo, Kari} } @article {24, title = {Adenoviral VEGF-C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes}, journal = {FASEB J}, volume = {16}, year = {2002}, month = {2002/Jul/}, pages = {1041 - 9}, abstract = {Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are important regulators of blood and lymphatic vessel growth and vascular permeability. The VEGF-C/VEGFR-3 signaling pathway is crucial for lymphangiogenesis, and heterozygous inactivating missense mutations of the VEGFR-3 gene are associated with hereditary lymphedema. However, VEGF-C can have potent effects on blood vessels because its receptor VEGFR-3 is expressed in certain blood vessels and because the fully processed form of VEGF-C also binds to the VEGFR-2 of blood vessels. To characterize the in vivo effects of VEGF-C on blood and lymphatic vessels, we have overexpressed VEGF-C via adenovirus- and adeno-associated virus-mediated transfection in the skin and respiratory tract of athymic nude mice. This resulted in dose-dependent enlargement and tortuosity of veins, which, along with the collecting lymphatic vessels were found to express VEGFR-2. Expression of angiopoietin 1 blocked the increased leakiness of the blood vessels induced by VEGF-C whereas vessel enlargement and lymphangiogenesis were not affected. However, angiogenic sprouting of new blood vessels was not observed in response to AdVEGF-C or AAV-VEGF-C. These results show that virally produced VEGF-C induces blood vessel changes, including vascular leak, but its angiogenic potency is much reduced compared with VEGF in normal skin.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/12087065}, author = {Saaristo, Anne and Veikkola, Tanja and Enholm, Berndt and Hyt{\"o}nen, Maija and Arola, Johanna and Pajusola, Katri and Turunen, Pa{\"\i}vi and Jeltsch, Michael and Karkkainen, Marika J and Kerjaschki, Dontscho and Bueler, Hansruedi and Yl{\"a}-Herttuala, Seppo and Alitalo, Kari} } @article {21, title = {Adenoviral expression of vascular endothelial growth factor-C induces lymphangiogenesis in the skin}, journal = {Circ Res}, volume = {88}, year = {2001}, month = {2001/Mar/}, pages = {623 - 9}, abstract = {The growth of blood and lymphatic vasculature is mediated in part by secreted polypeptides of the vascular endothelial growth factor (VEGF) family. The prototype VEGF binds VEGF receptor (VEGFR)-1 and VEGFR-2 and is angiogenic, whereas VEGF-C, which binds to VEGFR-2 and VEGFR-3, is either angiogenic or lymphangiogenic in different assays. We used an adenoviral gene transfer approach to compare the effects of these growth factors in adult mice. Recombinant adenoviruses encoding human VEGF-C or VEGF were injected subcutaneously into C57Bl6 mice or into the ears of nude mice. Immunohistochemical analysis showed that VEGF-C upregulated VEGFR-2 and VEGFR-3 expression and VEGF upregulated VEGFR-2 expression at 4 days after injection. After 2 weeks, histochemical and immunohistochemical analysis, including staining for the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), the vascular endothelial marker platelet-endothelial cell adhesion molecule-1 (PECAM-1), and the proliferating cell nuclear antigen (PCNA) revealed that VEGF-C induced mainly lymphangiogenesis in contrast to VEGF, which induced only angiogenesis. These results have significant implications in the planning of gene therapy using these growth factors.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/11282897}, author = {Enholm, B and Karpanen, T and Jeltsch, M and Kubo, H and Stenback, F and Prevo, R and Jackson, D G and Yla-Herttuala, S and Alitalo, K} } @article {22, title = {Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice}, journal = {EMBO J}, volume = {20}, year = {2001}, month = {2001/Mar/}, pages = {1223 - 31}, abstract = {Vascular endothelial growth factor receptor-3 (VEGFR-3) has an essential role in the development of embryonic blood vessels; however, after midgestation its expression becomes restricted mainly to the developing lymphatic vessels. The VEGFR-3 ligand VEGF-C stimulates lymphangiogenesis in transgenic mice and in chick chorioallantoic membrane. As VEGF-C also binds VEGFR-2, which is expressed in lymphatic endothelia, it is not clear which receptors are responsible for the lymphangiogenic effects of VEGF-C. VEGF-D, which binds to the same receptors, has been reported to induce angiogenesis, but its lymphangiogenic potential is not known. In order to define the lymphangiogenic signalling pathway we have created transgenic mice overexpressing a VEGFR-3-specific mutant of VEGF-C (VEGF-C156S) or VEGF-D in epidermal keratinocytes under the keratin 14 promoter. Both transgenes induced the growth of lymphatic vessels in the skin, whereas the blood vessel architecture was not affected. Evidence was also obtained that these growth factors act in a paracrine manner in vivo. These results demonstrate that stimulation of the VEGFR-3 signal transduction pathway is sufficient to induce specifically lymphangiogenesis in vivo.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/11250889}, author = {Veikkola, T and Jussila, L and Makinen, T and Karpanen, T and Jeltsch, M and Petrova, T V and Kubo, H and Thurston, G and McDonald, D M and Achen, M G and Stacker, S A and Alitalo, K} } @article {20, title = {Intravascular adenovirus-mediated VEGF-C gene transfer reduces neointima formation in balloon-denuded rabbit aorta}, journal = {Circulation}, volume = {102}, year = {2000}, month = {2000/Oct/}, pages = {2262 - 8}, abstract = {BACKGROUND: Gene transfer to the vessel wall may provide new possibilities for the treatment of vascular disorders, such as postangioplasty restenosis. In this study, we analyzed the effects of adenovirus-mediated vascular endothelial growth factor (VEGF)-C gene transfer on neointima formation after endothelial denudation in rabbits. For comparison, a second group was treated with VEGF-A adenovirus and a third group with lacZ adenovirus. Clinical-grade adenoviruses were used for the study. METHODS AND RESULTS: Aortas of cholesterol-fed New Zealand White rabbits were balloon-denuded, and gene transfer was performed 3 days later. Animals were euthanized 2 and 4 weeks after the gene transfer, and intima/media ratio (I/M), histology, and cell proliferation were analyzed. Two weeks after the gene transfer, I/M in the lacZ-transfected control group was 0. 57+/-0.04. VEGF-C gene transfer reduced I/M to 0.38+/-0.02 (P:<0.05 versus lacZ group). I/M in VEGF-A-treated animals was 0.49+/-0.17 (P:=NS). The tendency that both VEGF groups had smaller I/M persisted at the 4-week time point, when the lacZ group had an I/M of 0.73+/-0.16, the VEGF-C group 0.44+/-0.14, and the VEGF-A group 0. 63+/-0.21 (P:=NS). Expression of VEGF receptors 1, 2, and 3 was detected in the vessel wall by immunocytochemistry and in situ hybridization. As an additional control, the effect of adenovirus on cell proliferation was analyzed by performing gene transfer to intact aorta without endothelial denudation. No differences were seen in smooth muscle cell proliferation or I/M between lacZ adenovirus and 0.9\% saline-treated animals. CONCLUSIONS: Adenovirus-mediated VEGF-C gene transfer may be useful for the treatment of postangioplasty restenosis and vessel wall thickening after vascular manipulations.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/11056103}, author = {Hiltunen, M O and Laitinen, M and Turunen, M P and Jeltsch, M and Hartikainen, J and Rissanen, T T and Laukkanen, J and Niemi, M and Kossila, M and H{\"a}kkinen, T P and Kivel{\"a}, A and Enholm, B and Mansukoski, H and Turunen, A M and Alitalo, K and Yl{\"a}-Herttuala, S} } @article {17, title = {Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells}, journal = {Proc Natl Acad Sci U S A}, volume = {95}, year = {1998}, month = {1998/Sep/}, pages = {11709 - 14}, abstract = {The vascular endothelial growth factor (VEGF) family has recently expanded by the identification and cloning of three additional members, namely VEGF-B, VEGF-C, and VEGF-D. In this study we demonstrate that VEGF-B binds selectively to VEGF receptor-1/Flt-1. This binding can be blocked by excess VEGF, indicating that the interaction sites on the receptor are at least partially overlapping. Mutating the putative VEGF receptor-1/Flt-1 binding determinants Asp63, Asp64, and Glu67 to alanine residues in VEGF-B reduced the affinity to VEGF receptor-1 but did not abolish binding. Mutational analysis of conserved cysteines contributing to VEGF-B dimer formation suggest a structural conservation with VEGF and platelet-derived growth factor. Proteolytic processing of the 60-kDa VEGF-B186 dimer results in a 34-kDa dimer containing the receptor-binding epitopes. The binding of VEGF-B to its receptor on endothelial cells leads to increased expression and activity of urokinase type plasminogen activator and plasminogen activator inhibitor 1, suggesting a role for VEGF-B in the regulation of extracellular matrix degradation, cell adhesion, and migration.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/9751730}, author = {Olofsson, B and Korpelainen, E and Pepper, M S and Mandriota, S J and Aase, K and Kumar, V and Gunji, Y and Jeltsch, M M and Shibuya, M and Alitalo, K and Eriksson, U} } @article {16, title = {Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4)}, journal = {Proc Natl Acad Sci U S A}, volume = {95}, year = {1998}, month = {1998/Jan/}, pages = {548 - 53}, abstract = {We have identified a member of the VEGF family by computer-based homology searching and have designated it VEGF-D. VEGF-D is most closely related to VEGF-C by virtue of the presence of N- and C-terminal extensions that are not found in other VEGF family members. In adult human tissues, VEGF-D mRNA is most abundant in heart, lung, skeletal muscle, colon, and small intestine. Analyses of VEGF-D receptor specificity revealed that VEGF-D is a ligand for both VEGF receptors (VEGFRs) VEGFR-2 (Flk1) and VEGFR-3 (Flt4) and can activate these receptors. However. VEGF-D does not bind to VEGFR-1. Expression of a truncated derivative of VEGF-D demonstrated that the receptor-binding capacities reside in the portion of the molecule that is most closely related in primary structure to other VEGF family members and that corresponds to the mature form of VEGF-C. In addition, VEGF-D is a mitogen for endothelial cells. The structural and functional similarities between VEGF-D and VEGF-C define a subfamily of the VEGFs.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/9435229}, author = {Achen, M G and Jeltsch, M and Kukk, E and M{\"a}kinen, T and Vitali, A and Wilks, A F and Alitalo, K and Stacker, S A} } @article {15, title = {Vascular endothelial growth factor (VEGF)-C synergizes with basic fibroblast growth factor and VEGF in the induction of angiogenesis in vitro and alters endothelial cell extracellular proteolytic activity}, journal = {J Cell Physiol}, volume = {177}, year = {1998}, month = {1998/Dec/}, pages = {439 - 52}, abstract = {Vascular endothelial growth factor-C (VEGF-C) is a recently characterized member of the VEGF family of angiogenic polypeptides. We demonstrate here that VEGF-C is angiogenic in vitro when added to bovine aortic or lymphatic endothelial (BAE and BLE) cells but has little or no effect on bovine microvascular endothelial (BME) cells. As reported previously for VEGF, VEGF-C and basic fibroblast growth factor (bFGF) induced a synergistic in vitro angiogenic response in all three cells lines. Unexpectedly, VEGF and VEGF-C also synergized in the in vitro angiogenic response when assessed on BAE cells. Characterization of VEGF receptor (VEGFR) expression revealed that BME, BAE, and BLE cell lines express VEGFR-1 and -2, whereas of the three cell lines assessed, only BAE cells express VEGFR-3. We also demonstrate that VEGF-C increases plasminogen activator (PA) activity in the three bovine endothelial cell lines and that this is accompanied by a concomitant increase in PA inhibitor-1. Addition of alpha2-antiplasmin to BME cells co-treated with bFGF and VEGF-C partially inhibited collagen gel invasion. These results demonstrate, first, that by acting in concert with bFGF or VEGF, VEGF-C has a potent synergistic effect on the induction of angiogenesis in vitro and, second, that like VEGF and bFGF, VEGF-C is capable of altering endothelial cell extracellular proteolytic activity. These observations also highlight the notion of context, i.e., that the activity of an angiogenesis-regulating cytokine depends on the presence and concentration of other cytokines in the pericellular environment of the responding endothelial cell.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/9808152}, author = {Pepper, M S and Mandriota, S J and Jeltsch, M and Kumar, V and Alitalo, K} } @article {13, title = {Genomic organization of human and mouse genes for vascular endothelial growth factor C}, journal = {J Biol Chem}, volume = {272}, year = {1997}, month = {1997/Oct/}, pages = {25176 - 83}, abstract = {We report here the cloning and characterization of human and mouse genes for vascular endothelial growth factor C (VEGF-C), a newly isolated member of the vascular endothelial growth factor/platelet-derived growth factor (VEGF/PDGF) family. Both VEGF-C genes comprise over 40 kilobase pairs of genomic DNA and consist of seven exons, all containing coding sequences. The VEGF homology domain of VEGF-C is encoded by exons 3 and 4. Exons 5 and 7 encode cysteine-rich motifs of the type C6C10CRC, and exon 6 encodes additional C10CXCXC motifs typical of a silk protein. A putative alternatively spliced rare RNA form lacking exon 4 was identified in human fibrosarcoma cells, and a major transcription start site was located in the human VEGF-C gene 523 base pairs upstream of the translation initiation codon. The upstream promoter sequences contain conserved putative binding sites for Sp-1, AP-2, and NF-kappaB transcription factors but no TATA box, and they show promoter activity when transfected into cells. The VEGF-C gene structure is thus assembled from exons encoding propeptides and distinct cysteine-rich domains in addition to the VEGF homology domain, and it shows both similarities and distinct differences in comparison with other members of the VEGF/PDGF gene family.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/9312130}, author = {Chilov, D and Kukk, E and Taira, S and Jeltsch, M and Kaukonen, J and Palotie, A and Joukov, V and Alitalo, K} } @article {10, title = {Hyperplasia of lymphatic vessels in VEGF-C transgenic mice}, journal = {Science (80- )}, volume = {276}, year = {1997}, month = {1997/May/}, pages = {1423 - 5}, abstract = {No growth factors specific for the lymphatic vascular system have yet been described. Vascular endothelial growth factor (VEGF) regulates vascular permeability and angiogenesis, but does not promote lymphangiogenesis. Overexpression of VEGF-C, a ligand of the VEGF receptors VEGFR-3 and VEGFR-2, in the skin of transgenic mice resulted in lymphatic, but not vascular, endothelial proliferation and vessel enlargement. Thus, VEGF-C induces selective hyperplasia of the lymphatic vasculature, which is involved in the draining of interstitial fluid and in immune function, inflammation, and tumor metastasis. VEGF-C may play a role in disorders involving the lymphatic system and may be of potential use in therapeutic lymphangiogenesis.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/9162011}, author = {Jeltsch, M and Kaipainen, A and Joukov, V and Meng, X and Lakso, M and Rauvala, H and Swartz, M and Fukumura, D and Jain, R K and Alitalo, K} } @article {14, title = {Proteolytic processing regulates receptor specificity and activity of VEGF-C}, journal = {EMBO J}, volume = {16}, year = {1997}, month = {1997/Jul/}, pages = {3898 - 911}, abstract = {The recently identified vascular endothelial growth factor C (VEGF-C) belongs to the platelet-derived growth factor (PDGF)/VEGF family of growth factors and is a ligand for the endothelial-specific receptor tyrosine kinases VEGFR-3 and VEGFR-2. The VEGF homology domain spans only about one-third of the cysteine-rich VEGF-C precursor. Here we have analysed the role of post-translational processing in VEGF-C secretion and function, as well as the structure of the mature VEGF-C. The stepwise proteolytic processing of VEGF-C generated several VEGF-C forms with increased activity towards VEGFR-3, but only the fully processed VEGF-C could activate VEGFR-2. Recombinant {\textquoteright}mature{\textquoteright} VEGF-C made in yeast bound VEGFR-3 (K[D] = 135 pM) and VEGFR-2 (K[D] = 410 pM) and activated these receptors. Like VEGF, mature VEGF-C increased vascular permeability, as well as the migration and proliferation of endothelial cells. Unlike other members of the PDGF/VEGF family, mature VEGF-C formed mostly non-covalent homodimers. These data implicate proteolytic processing as a regulator of VEGF-C activity, and reveal novel structure-function relationships in the PDGF/VEGF family.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/9233800}, author = {Joukov, V and Sorsa, T and Kumar, V and Jeltsch, M and Claesson-Welsh, L and Cao, Y and Saksela, O and Kalkkinen, N and Alitalo, K} } @article {12, title = {Vascular endothelial growth factors VEGF-B and VEGF-C}, journal = {J Cell Physiol}, volume = {173}, year = {1997}, month = {1997/Nov/}, pages = {211 - 5}, url = {http://view.ncbi.nlm.nih.gov/pubmed/9365524}, author = {Joukov, V and Kaipainen, A and Jeltsch, M and Pajusola, K and Olofsson, B and Kumar, V and Eriksson, U and Alitalo, K} } @article {9, title = {VEGF-C receptor binding and pattern of expression with VEGFR-3 suggests a role in lymphatic vascular development}, journal = {Development}, volume = {122}, year = {1996}, month = {1996/Dec/}, pages = {3829 - 37}, abstract = {The vascular endothelial growth factor family has recently been expanded by the isolation of two new VEGF-related factors, VEGF-B and VEGF-C. The physiological functions of these factors are largely unknown. Here we report the cloning and characterization of mouse VEGF-C, which is produced as a disulfide-linked dimer of 415 amino acid residue polypeptides, sharing an 85\% identity with the human VEGF-C amino acid sequence. The recombinant mouse VEGF-C protein was secreted from transfected cells as VEGFR-3 (Flt4) binding polypeptides of 30-32x10(3) Mr and 22-23x10(3) Mr which preferentially stimulated the autophosphorylation of VEGFR-3 in comparison with VEGFR-2 (KDR). In in situ hybridization, mouse VEGF-C mRNA expression was detected in mesenchymal cells of postimplantation mouse embryos, particularly in the regions where the lymphatic vessels undergo sprouting from embryonic veins, such as the perimetanephric, axillary and jugular regions. In addition, the developing mesenterium, which is rich in lymphatic vessels, showed strong VEGF-C expression. VEGF-C was also highly expressed in adult mouse lung, heart and kidney, where VEGFR-3 was also prominent. The pattern of expression of VEGF-C in relation to its major receptor VEGFR-3 during the sprouting of the lymphatic endothelium in embryos suggests a paracrine mode of action and that one of the functions of VEGF-C may be in the regulation of angiogenesis of the lymphatic vasculature.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/9012504}, author = {Kukk, E and Lymboussaki, A and Taira, S and Kaipainen, A and Jeltsch, M and Joukov, V and Alitalo, K} }