%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 Journal of Heart and Lung Transplantation %D 2011 %T VEGF-C/VEGFR-3 Signaling Regulates Inflammatory Response in Development of Obliterative Airway Disease %A Krebs, R %A Tikkanen, JM %A Ropponen, JO %A Jeltsch, M %A Jokinen, JJ %A Ylä-Herttuala, S %A Koskinen, PK %A Nykänen, AI %A Lemström, KB %B Journal of Heart and Lung Transplantation %V 30 %P S118 - S118 %8 2011// %G eng %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 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 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 Circ Res %D 2007 %T Distinct architecture of lymphatic vessels induced by chimeric vascular endothelial growth factor-C/vascular endothelial growth factor heparin-binding domain fusion proteins %A Tammela, Tuomas %A He, Yulong %A Lyytikkä, Johannes %A Jeltsch, Michael %A Markkanen, Johanna %A Pajusola, Katri %A Ylä-Herttuala, Seppo %A Alitalo, Kari %X Vascular endothelial growth factor (VEGF)-C and VEGF-D are composed of the receptor-binding VEGF homology domain and a carboxy-terminal silk homology domain that requires proteolytic cleavage for growth factor activation. Here, we explored whether the C-terminal heparin-binding domain of the VEGF(165) or VEGF(189) isoform also containing neuropilin-binding sequences could substitute for the silk homology domain of VEGF-C. Such VEGF-C/VEGF-heparin-binding domain chimeras were produced and shown to activate VEGF-C receptors, and, when expressed in tissues via adenovirus or adeno-associated virus vectors, stimulated lymphangiogenesis in vivo. However, both chimeras induced a distinctly different pattern of lymphatic vessels when compared with VEGF-C. Whereas VEGF-C-induced vessels were initially a dense network of small diameter vessels, the lymphatic vessels induced by the chimeric growth factors tended to form directly along tissue borders, along basement membranes that are rich in heparan sulfate. For example, in skeletal muscle, the chimeras induced formation of lumenized lymphatic vessels more efficiently than wild-type VEGF-C. We conclude that the matrix-binding domain of VEGF can target VEGF-C activity to heparin-rich basement membrane structures. These properties may prove useful for tissue engineering and attempts to regenerate lymphatic vessels in lymphedema patients. %B Circ Res %V 100 %P 1468 - 75 %8 2007/May/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/17478733 %N 10 %! Circulation Research %0 Journal Article %J Circ Res %D 2007 %T Enhanced capillary formation stimulated by a chimeric vascular endothelial growth factor/vascular endothelial growth factor-C silk domain fusion protein %A Keskitalo, Salla %A Tammela, Tuomas %A Lyytikka, Johannes %A Karpanen, Terhi %A Jeltsch, Michael %A Markkanen, Johanna %A Yla-Herttuala, Seppo %A Alitalo, Kari %X 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. %B Circ Res %V 100 %P 1460 - 7 %8 2007/May/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/17478734 %N 10 %! Circulation Research %0 Journal Article %J FASEB J %D 2006 %T Functional interaction of VEGF-C and VEGF-D with neuropilin receptors %A Kärpänen, Terhi %A Heckman, Caroline A %A Keskitalo, Salla %A Jeltsch, Michael %A Ollila, Hanna %A Neufeld, Gera %A Tamagnone, Luca %A Alitalo, Kari %X 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. %B FASEB J %V 20 %P 1462 - 72 %8 2006/Jul/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/16816121 %N 9 %! The FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology %0 Journal Article %J J Clin Invest %D 2005 %T Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation %A Baluk, Peter %A Tammela, Tuomas %A Ator, Erin %A Lyubynska, Natalya %A Achen, Marc G %A Hicklin, Daniel J %A Jeltsch, Michael %A Petrova, Tatiana V %A Pytowski, Bronislaw %A Stacker, Steven A %A Ylä-Herttuala, Seppo %A Jackson, David G %A Alitalo, Kari %A McDonald, Donald M %X Edema occurs in asthma and other inflammatory diseases when the rate of plasma leakage from blood vessels exceeds the drainage through lymphatic vessels and other routes. It is unclear to what extent lymphatic vessels grow to compensate for increased leakage during inflammation and what drives the lymphangiogenesis that does occur. We addressed these issues in mouse models of (a) chronic respiratory tract infection with Mycoplasma pulmonis and (b) adenoviral transduction of airway epithelium with VEGF family growth factors. Blood vessel remodeling and lymphangiogenesis were both robust in infected airways. Inhibition of VEGFR-3 signaling completely prevented the growth of lymphatic vessels but not blood vessels. Lack of lymphatic growth exaggerated mucosal edema and reduced the hypertrophy of draining lymph nodes. Airway dendritic cells, macrophages, neutrophils, and epithelial cells expressed the VEGFR-3 ligands VEGF-C or VEGF-D. Adenoviral delivery of either VEGF-C or VEGF-D evoked lymphangiogenesis without angiogenesis, whereas adenoviral VEGF had the opposite effect. After antibiotic treatment of the infection, inflammation and remodeling of blood vessels quickly subsided, but lymphatic vessels persisted. Together, these findings suggest that when lymphangiogenesis is impaired, airway inflammation may lead to bronchial lymphedema and exaggerated airflow obstruction. Correction of defective lymphangiogenesis may benefit the treatment of asthma and other inflammatory airway diseases. %B J Clin Invest %V 115 %P 247 - 57 %8 2005/Feb/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/15668734 %N 2 %! The Journal of Clinical Investigation %0 Journal Article %J Cancer Res %D 2005 %T Vascular endothelial cell growth factor receptor 3-mediated activation of lymphatic endothelium is crucial for tumor cell entry and spread via lymphatic vessels %A He, Yulong %A Rajantie, Iiro %A Pajusola, Katri %A Jeltsch, Michael %A Holopainen, Tanja %A Yla-Herttuala, Seppo %A Harding, Thomas %A Jooss, Karin %A Takahashi, Takashi %A Alitalo, Kari %X Lymphangiogenic growth factors vascular endothelial growth factor (VEGF)-C and VEGF-D have been shown to promote lymphatic metastasis by inducing tumor-associated lymphangiogenesis. In this study, we have investigated how tumor cells gain access into lymphatic vessels and at what stage tumor cells initiate metastasis. We show that VEGF-C produced by tumor cells induced extensive lymphatic sprouting towards the tumor cells as well as dilation of the draining lymphatic vessels, suggesting an active role of lymphatic endothelial cells in lymphatic metastasis. A significant increase in lymphatic vessel growth occurred between 2 and 3 weeks after tumor xenotransplantation, and lymph node metastasis occurred at the same stage. These processes were blocked dose-dependently by inhibition of VEGF receptor 3 (VEGFR-3) signaling by systemic delivery of a soluble VEGFR-3-immunoglobulin (Ig) fusion protein via adenoviral or adeno-associated viral vectors. However, VEGFR-3-Ig did not suppress lymph node metastasis when the treatment was started at a later stage after the tumor cells had already spread out, suggesting that tumor cell entry into lymphatic vessels is a key step during tumor dissemination via the lymphatics. Whereas lymphangiogenesis and lymph node metastasis were significantly inhibited by VEGFR-3-Ig, some tumor cells were still detected in the lymph nodes in some of the treated mice. This indicates that complete blockade of lymphatic metastasis may require the targeting of both tumor lymphangiogenesis and tumor cell invasion. %B Cancer Res %V 65 %P 4739 - 46 %8 2005/Jun/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/15930292 %N 11 %! Cancer Research %0 Journal Article %J Nat Immunol %D 2004 %T Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins %A Karkkainen, Marika J %A Haiko, Paula %A Sainio, Kirsi %A Partanen, Juha %A Taipale, Jussi %A Petrova, Tatiana V %A Jeltsch, Michael %A Jackson, David G %A Talikka, Marja %A Rauvala, Heikki %A Betsholtz, Christer %A Alitalo, Kari %X 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. %B Nat Immunol %V 5 %P 74 - 80 %8 2004/Jan/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/14634646 %N 1 %! Nature Immunology %0 Journal Article %J Cell Tissue Res %D 2003 %T Genesis and pathogenesis of lymphatic vessels %A Jeltsch, Michael %A Tammela, Tuomas %A Alitalo, Kari %A Wilting, Jörg %X The lymphatic system is generally regarded as supplementary to the blood vascular system, in that it transports interstitial fluid, macromolecules, and immune cells back into the blood. However, in insects, the open hemolymphatic (or lymphohematic) system ensures the circulation of immune cells and interstitial fluid through the body. The Drosophila homolog of the mammalian vascular endothelial growth factor receptor (VEGFR) gene family is expressed in hemocytes, suggesting a close relationship to the endothelium that develops later in phylogeny. Lymph hearts are typical organs for the propulsion of lymph in lower vertebrates and are still transiently present in birds. The lymphatic endothelial marker VEGFR-3 is transiently expressed in embryonic blood vessels and is crucial for their development. We therefore regard the question of whether the blood vascular system or the lymphatic system is primary or secondary as open. Future molecular comparisons should be performed without any bias based on the current prevalence of the blood vascular system over the lymphatic system. Here, we give an overview of the structure, function, and development of the lymphatics, with special emphasis on the recently discovered lymphangiogenic growth factors. %B Cell Tissue Res %V 314 %P 69 - 84 %8 2003/Oct/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/12942362 %N 1 %! Cell and Tissue Research %0 Journal Article %J FASEB J %D 2002 %T Adenoviral VEGF-C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes %A Saaristo, Anne %A Veikkola, Tanja %A Enholm, Berndt %A Hytönen, Maija %A Arola, Johanna %A Pajusola, Katri %A Turunen, Païvi %A Jeltsch, Michael %A Karkkainen, Marika J %A Kerjaschki, Dontscho %A Bueler, Hansruedi %A Ylä-Herttuala, Seppo %A Alitalo, Kari %X 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. %B FASEB J %V 16 %P 1041 - 9 %8 2002/Jul/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/12087065 %N 9 %! The FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology %0 Journal Article %J EMBO J %D 2001 %T Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice %A Veikkola, T %A Jussila, L %A Makinen, T %A Karpanen, T %A Jeltsch, M %A Petrova, T V %A Kubo, H %A Thurston, G %A McDonald, D M %A Achen, M G %A Stacker, S A %A Alitalo, K %X 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. %B EMBO J %V 20 %P 1223 - 31 %8 2001/Mar/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/11250889 %N 6 %! The EMBO Journal %0 Conference Paper %B Molecular Targets and Cancer Therapeutics: Discovery, Biology, and Clinical Applications %D 2001 %T Signalling via VEGFR-3 is sufficient for lymphangiogenesis in transgenic mice. %A Jussila, L. %A Veikkola, T. %A Jeltsch, M. %A Thurston, G. %A McDonald, D. %A Achen, M. %A Stacker, S. %A Alitalo, K. %B Molecular Targets and Cancer Therapeutics: Discovery, Biology, and Clinical Applications %C Miami Beach, Florida %8 2001/10/29/ %G eng %0 Journal Article %J Circulation %D 2000 %T Intravascular adenovirus-mediated VEGF-C gene transfer reduces neointima formation in balloon-denuded rabbit aorta %A Hiltunen, M O %A Laitinen, M %A Turunen, M P %A Jeltsch, M %A Hartikainen, J %A Rissanen, T T %A Laukkanen, J %A Niemi, M %A Kossila, M %A Häkkinen, T P %A Kivelä, A %A Enholm, B %A Mansukoski, H %A Turunen, A M %A Alitalo, K %A Ylä-Herttuala, S %X 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. %B Circulation %V 102 %P 2262 - 8 %8 2000/Oct/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/11056103 %N 18 %! Circulation %0 Journal Article %J J Biol Chem %D 1997 %T Genomic organization of human and mouse genes for vascular endothelial growth factor C %A Chilov, D %A Kukk, E %A Taira, S %A Jeltsch, M %A Kaukonen, J %A Palotie, A %A Joukov, V %A Alitalo, K %X 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. %B J Biol Chem %V 272 %P 25176 - 83 %8 1997/Oct/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/9312130 %N 40 %! The Journal of Biological Chemistry %0 Journal Article %J Development %D 1996 %T VEGF-C receptor binding and pattern of expression with VEGFR-3 suggests a role in lymphatic vascular development %A Kukk, E %A Lymboussaki, A %A Taira, S %A Kaipainen, A %A Jeltsch, M %A Joukov, V %A Alitalo, K %X 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. %B Development %V 122 %P 3829 - 37 %8 1996/Dec/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/9012504 %N 12 %! Development (Cambridge, England)