%0 Journal Article %J Duodecim Lääketieteellinen Aikakauskirja %D 2020 %T Lymphatics and the eye. [Finnish] %A Gucciardo, Erika %A Lehti, Timo A. %A Korhonen, Ani %A Salvén, Petri %A Lehti, Kaisa %A Jeltsch, Michael %A Loukovaara, Sirpa %B Duodecim Lääketieteellinen Aikakauskirja %V 136 %P 1777-1788 %8 2020/02/10/ %G eng %U https://www.duodecimlehti.fi/lehti/2020/16/duo15739 %N 16 %9 review %! Duodecim %& 1777 %R 10.5281/zenodo.4005517 %0 Journal Article %J eLife %D 2019 %T KLK3/PSA and cathepsin D activate VEGF-C and VEGF-D %A Jha, Sawan Kumar %A Rauniyar, Khushbu %A Chronowska, Ewa %A Mattonet, Kenny %A Maina, Eunice Wairimu %A Koistinen, Hannu %A Stenman, Ulf-Håkan %A Alitalo, Kari %A Jeltsch, Michael %K cancer biology %K Cathepsin D %K kallikrein-related peptidases %K KLK3/PSA %K Lymphangiogenesis %K mouse %K VEGF-C %K VEGF-D %X Vascular endothelial growth factor-C (VEGF-C) acts primarily on endothelial cells, but also on non-vascular targets, e.g. in the CNS and immune system. Here we describe a novel, unique VEGF-C form in the human reproductive system produced via cleavage by kallikrein-related peptidase 3 (KLK3), aka prostate-specific antigen (PSA). KLK3 activated VEGF-C specifically and efficiently through cleavage at a novel N-terminal site. We detected VEGF-C in seminal plasma, and sperm liquefaction occurred concurrently with VEGF-C activation, which was enhanced by collagen and calcium binding EGF domains 1 (CCBE1). After plasmin and ADAMTS3, KLK3 is the third protease shown to activate VEGF-C. Since differently activated VEGF-Cs are characterized by successively shorter N-terminal helices, we created an even shorter hypothetical form, which showed preferential binding to VEGFR-3. Using mass spectrometric analysis of the isolated VEGF-C-cleaving activity from human saliva, we identified cathepsin D as a protease that can activate VEGF-C as well as VEGF-D. %B eLife %V 8 %P e44478 %8 2019/05/17/ %@ 2050-084X %G eng %U https://elifesciences.org/articles/44478 %! eLife %0 Journal Article %J Lymphologie in Forschung und Praxis %D 2019 %T The Proteolytic Activation of Vascular Endothelial Growth Factor-C %A Lackner, Marcel %A Schmotz, Constanze %A Jeltsch, Michael %K Lymphangiogenesis %K proteinases %K proteolysis %K VEGF-C %X The enzymatic cleavage of the protein backbone (proteolysis) is integral to many biological processes, e.g. for the break­down of proteins in the digestive system. Specific proteolytic cleavages are also used to turn on or off the activity of proteins. For example, the lymphangiogenic vascular endothelial growth factor-­C (VEGF-­C) is synthesized as a precursor molecule that must be converted to a mature form by the enzymatic removal of C-­ and N-­terminal propeptides before it can bind and activate its receptors. The constitutive C-­terminal cleavage is mediated by proprotein convertases such as furin. The subsequent ac­tivating cleavage can be mediated by at least four different proteases: by plasmin, ADAMTS3, prostate­-specific antigen (PSA) and cathepsin D. Processing by different proteases results in distinct forms of "ma­ture" VEGF­-C, that differ in their affinity and their receptor activation potential. This processing is tightly regulated by the CCBE1 protein. CCBE1 regulates the acti­vating cleavage of VEGF­C by ADAMTS3 and PSA, but not by plasmin. During embryonic development of the lymphatic system, VEGF-­C is activated primarily by the ADAMTS3 protease. In contrast, it is believed that plasmin is responsible for wound healing lymphangiogenesis and PSA for tumor-­associated pathological lym­phangiogenesis. Cathepsin D has also been implicated in tumor lymphangiogenesis. In addition, cathepsin D in saliva might activate latent VEGF­C upon wound licking, thereby accelerating wound healing. The molecular details of proteolytic activation of VEGF­-C are only recently extensively explored, and we likely do not know yet all activating proteases. It appears that the activity of VEGF­-C is regulated for different specific functions by different proteinases. Although VEGF­-C clearly plays a pivotal role for tumor progression and metastasis in experimental animal studies, the rele­vance of most correlative studies on the role of VEGF­-C in human cancers is quite limited until now, also due to the lack of methods to differentiate between inactive and active forms. %B Lymphologie in Forschung und Praxis %V 23 %P 88 - 98 %8 2019/12/18/ %G eng %U https://doi.org/10.5281/zenodo.3629263 %N 2 %9 Review %! LymphForsch %( Die proteolytische Aktivierung des Vaskulären Endothelzellwachstumsfaktors-C %R 10.5281/zenodo.3629263 %0 Journal Article %J Genes Dev %D 2010 %T Claudin-like protein 24 interacts with the VEGFR-2 and VEGFR-3 pathways and regulates lymphatic vessel development %A Saharinen, Pipsa %A Helotera, Hanna %A Miettinen, Juho %A Norrmen, Camilla %A D'Amico, Gabriela %A Jeltsch, Michael %A Langenberg, Tobias %A Vandevelde, Wouter %A Ny, Annelii %A Dewerchin, Mieke %A Carmeliet, Peter %A Alitalo, Kari %X The Claudin-like protein of 24 kDa (CLP24) is a hypoxia-regulated transmembrane protein of unknown function. We show here that clp24 knockdown in Danio rerio and Xenopus laevis results in defective lymphatic development. Targeted disruption of Clp24 in mice led to enlarged lymphatic vessels having an abnormal smooth muscle cell coating. We also show that the Clp24(-/-) phenotype was further aggravated in the Vegfr2(+/LacZ) or Vegfr3(+/LacZ) backgrounds and that CLP24 interacts with vascular endothelial growth factor receptor-2 (VEGFR-2) and VEGFR-3 and attenuates the transcription factor CREB phosphorylation via these receptors. Our results indicate that CLP24 is a novel regulator of VEGFR-2 and VEGFR-3 signaling pathways and of normal lymphatic vessel structure. %B Genes Dev %V 24 %P 875 - 80 %8 2010/May/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/20439428 %N 9 %! Genes & Development %0 Journal Article %J Proceedings of the National Academy of Sciences of the United States of America %D 2010 %T Structural determinants of growth factor binding and specificity by VEGF receptor 2 %A Leppänen, Veli-Matti %A Prota, Andrea E %A Jeltsch, Michael %A Anisimov, Andrey %A Kalkkinen, Nisse %A Strandin, Tomas %A Lankinen, Hilkka %A Goldman, Adrian %A Ballmer-Hofer, Kurt %A Alitalo, Kari %X Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel formation through activation of three receptor tyrosine kinases, VEGFR-1, -2, and -3. The extracellular domain of VEGF receptors consists of seven immunoglobulin homology domains, which, upon ligand binding, promote receptor dimerization. Dimerization initiates transmembrane signaling, which activates the intracellular tyrosine kinase domain of the receptor. VEGF-C stimulates lymphangiogenesis and contributes to pathological angiogenesis via VEGFR-3. However, proteolytically processed VEGF-C also stimulates VEGFR-2, the predominant transducer of signals required for physiological and pathological angiogenesis. Here we present the crystal structure of VEGF-C bound to the VEGFR-2 high-affinity-binding site, which consists of immunoglobulin homology domains D2 and D3. This structure reveals a symmetrical 22 complex, in which left-handed twisted receptor domains wrap around the 2-fold axis of VEGF-C. In the VEGFs, receptor specificity is determined by an N-terminal alpha helix and three peptide loops. Our structure shows that two of these loops in VEGF-C bind to VEGFR-2 subdomains D2 and D3, while one interacts primarily with D3. Additionally, the N-terminal helix of VEGF-C interacts with D2, and the groove separating the two VEGF-C monomers binds to the D2/D3 linker. VEGF-C, unlike VEGF-A, does not bind VEGFR-1. We therefore created VEGFR-1/VEGFR-2 chimeric proteins to further study receptor specificity. This biochemical analysis, together with our structural data, defined VEGFR-2 residues critical for the binding of VEGF-A and VEGF-C. Our results provide significant insights into the structural features that determine the high affinity and specificity of VEGF/VEGFR interactions. %B Proceedings of the National Academy of Sciences of the United States of America %V 107 %P 2425 - 30 %8 02/2010 %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/20145116 %N 6 %0 Journal Article %J PLoS ONE %D 2010 %T Suppressive effects of vascular endothelial growth factor-B on tumor growth in a mouse model of pancreatic neuroendocrine tumorigenesis %A Albrecht, Imke %A Kopfstein, Lucie %A Strittmatter, Karin %A Schomber, Tibor %A Falkevall, Annelie %A Hagberg, Carolina E %A Lorentz, Pascal %A Jeltsch, Michael %A Alitalo, Kari %A Eriksson, Ulf %A Christofori, Gerhard %A Pietras, Kristian %X BACKGROUND: The family of vascular endothelial growth factors (VEGF) contains key regulators of blood and lymph vessel development, including VEGF-A, -B, -C, -D, and placental growth factor. The role of VEGF-B during physiological or pathological angiogenesis has not yet been conclusively delineated. Herein, we investigate the function of VEGF-B by the generation of mouse models of cancer with transgenic expression of VEGF-B or homozygous deletion of Vegfb. METHODOLOGY/PRINCIPAL FINDINGS: Ectopic expression of VEGF-B in the insulin-producing β-cells of the pancreas did not alter the abundance or architecture of the islets of Langerhans. The vasculature from transgenic mice exhibited a dilated morphology, but was of similar density as that of wildtype mice. Unexpectedly, we found that transgenic expression of VEGF-B in the RIP1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis retarded tumor growth. Conversely, RIP1-Tag2 mice deficient for Vegfb presented with larger tumors. No differences in vascular density, perfusion or immune cell infiltration upon altered Vegfb gene dosage were noted. However, VEGF-B acted to increase blood vessel diameter both in normal pancreatic islets and in RIP1-Tag2 tumors. CONCLUSIONS/SIGNIFICANCE: Taken together, our results illustrate the differences in biological function between members of the VEGF family, and highlight the necessity of in-depth functional studies of VEGF-B to fully understand the effects of VEGFR-1 inhibitors currently used in the clinic. %B PLoS ONE %V 5 %P e14109 %8 2010// %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/21124841 %N 11 %! PLoS ONE %0 Journal Article %J Circulation %D 2010 %T Vascular endothelial growth factor-B acts as a coronary growth factor in transgenic rats without inducing angiogenesis, vascular leak, or inflammation %A Bry, Maija %A Kivelä, Riikka %A Holopainen, Tanja %A Anisimov, Andrey %A Tammela, Tuomas %A Soronen, Jarkko %A Silvola, Johanna %A Saraste, Antti %A Jeltsch, Michael %A Korpisalo, Petra %A Carmeliet, Peter %A Lemström, Karl B %A Shibuya, Masabumi %A Ylä-Herttuala, Seppo %A Alhonen, Leena %A Mervaala, Eero %A Andersson, Leif C %A Knuuti, Juhani %A Alitalo, Kari %X BACKGROUND: Vascular endothelial growth factor-B (VEGF-B) binds to VEGF receptor-1 and neuropilin-1 and is abundantly expressed in the heart, skeletal muscle, and brown fat. The biological function of VEGF-B is incompletely understood. METHODS AND RESULTS: Unlike placenta growth factor, which binds to the same receptors, adeno-associated viral delivery of VEGF-B to mouse skeletal or heart muscle induced very little angiogenesis, vascular permeability, or inflammation. As previously reported for the VEGF-B(167) isoform, transgenic mice and rats expressing both isoforms of VEGF-B in the myocardium developed cardiac hypertrophy yet maintained systolic function. Deletion of the VEGF receptor-1 tyrosine kinase domain or the arterial endothelial Bmx tyrosine kinase inhibited hypertrophy, whereas loss of VEGF-B interaction with neuropilin-1 had no effect. Surprisingly, in rats, the heart-specific VEGF-B transgene induced impressive growth of the epicardial coronary vessels and their branches, with large arteries also seen deep inside the subendocardial myocardium. However, VEGF-B, unlike other VEGF family members, did not induce significant capillary angiogenesis, increased permeability, or inflammatory cell recruitment. CONCLUSIONS: VEGF-B appears to be a coronary growth factor in rats but not in mice. The signals for the VEGF-B-induced cardiac hypertrophy are mediated at least in part via the endothelium. Because cardiomyocyte damage in myocardial ischemia begins in the subendocardial myocardium, the VEGF-B-induced increased arterial supply to this area could have therapeutic potential in ischemic heart disease. %B Circulation %V 122 %P 1725 - 33 %8 2010/Oct/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/20937974 %N 17 %! Circulation %0 Journal Article %J Circ Res %D 2009 %T Activated forms of VEGF-C and VEGF-D provide improved vascular function in skeletal muscle %A Anisimov, Andrey %A Alitalo, Annamari %A Korpisalo, Petra %A Soronen, Jarkko %A Kaijalainen, Seppo %A Leppänen, Veli-Matti %A Jeltsch, Michael %A Ylä-Herttuala, Seppo %A Alitalo, Kari %X The therapeutic potential of vascular endothelial growth factor (VEGF)-C and VEGF-D in skeletal muscle has been of considerable interest as these factors have both angiogenic and lymphangiogenic activities. Previous studies have mainly used adenoviral gene delivery for short-term expression of VEGF-C and VEGF-D in pig, rabbit, and mouse skeletal muscles. Here we have used the activated mature forms of VEGF-C and VEGF-D expressed via recombinant adeno-associated virus (rAAV), which provides stable, long-lasting transgene expression in various tissues including skeletal muscle. Mouse tibialis anterior muscle was transduced with rAAV encoding human or mouse VEGF-C or VEGF-D. Two weeks later, immunohistochemical analysis showed increased numbers of both blood and lymph vessels, and Doppler ultrasound analysis indicated increased blood vessel perfusion. The lymphatic vessels further increased at the 4-week time point were functional, as shown by FITC-lectin uptake and transport. Furthermore, receptor activation and arteriogenic activity were increased by an alanine substitution mutant of human VEGF-C (C137A) having an increased dimer stability and by a chimeric CAC growth factor that contained the VEGF receptor-binding domain flanked by VEGF-C propeptides, but only the latter promoted significantly more blood vessel perfusion when compared to the other growth factors studied. We conclude that long-term expression of VEGF-C and VEGF-D in skeletal muscle results in the generation of new functional blood and lymphatic vessels. The therapeutic value of intramuscular lymph vessels in draining tissue edema and lymphedema can now be evaluated using this model system. %B Circ Res %V 104 %P 1302 - 12 %8 2009/Jun/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/19443835 %N 11 %! Circulation Research %0 Journal Article %J Circ Res %D 2008 %T Overexpression of vascular endothelial growth factor-B in mouse heart alters cardiac lipid metabolism and induces myocardial hypertrophy %A Karpanen, Terhi %A Bry, Maija %A Ollila, Hanna M %A Seppänen-Laakso, Tuulikki %A Liimatta, Erkki %A Leskinen, Hanna %A Kivelä, Riikka %A Helkamaa, Teemu %A Merentie, Mari %A Jeltsch, Michael %A Paavonen, Karri %A Andersson, Leif C %A Mervaala, Eero %A Hassinen, Ilmo E %A Ylä-Herttuala, Seppo %A Oresic, Matej %A Alitalo, Kari %X Vascular endothelial growth factor (VEGF)-B is poorly angiogenic but prominently expressed in metabolically highly active tissues, including the heart. We produced mice expressing a cardiac-specific VEGF-B transgene via the alpha-myosin heavy chain promoter. Surprisingly, the hearts of the VEGF-B transgenic mice showed concentric cardiac hypertrophy without significant changes in heart function. The cardiac hypertrophy was attributable to an increased size of the cardiomyocytes. Blood capillary size was increased, whereas the number of blood vessels per cell nucleus remained unchanged. Despite the cardiac hypertrophy, the transgenic mice had lower heart rate and blood pressure than their littermates, and they responded similarly to angiotensin II-induced hypertension, confirming that the hypertrophy does not compromise heart function. Interestingly, the isolated transgenic hearts had less cardiomyocyte damage after ischemia. Significantly increased ceramide and decreased triglyceride levels were found in the transgenic hearts. This was associated with structural changes and eventual lysis of mitochondria, resulting in accumulation of intracellular vacuoles in cardiomyocytes and increased death of the transgenic mice, apparently because of mitochondrial lipotoxicity in the heart. These results suggest that VEGF-B regulates lipid metabolism, an unexpected function for an angiogenic growth factor. %B Circ Res %V 103 %P 1018 - 26 %8 2008/Oct/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/18757827 %N 9 %! Circulation Research %0 Journal Article %J Arterioscler Thromb Vasc Biol %D 2008 %T Reevaluation of the role of VEGF-B suggests a restricted role in the revascularization of the ischemic myocardium %A Li, Xuri %A Tjwa, Marc %A Van Hove, Inge %A Enholm, Berndt %A Neven, Elke %A Paavonen, Karri %A Jeltsch, Michael %A Juan, Toni Diez %A Sievers, Richard E %A Chorianopoulos, Emmanuel %A Wada, Hiromichi %A Vanwildemeersch, Maarten %A Noel, Agnes %A Foidart, Jean-Michel %A Springer, Matthew L %A von Degenfeld, Georges %A Dewerchin, Mieke %A Blau, Helen M %A Alitalo, Kari %A Eriksson, Ulf %A Carmeliet, Peter %A Moons, Lieve %X OBJECTIVE: The endogenous role of the VEGF family member vascular endothelial growth factor-B (VEGF-B) in pathological angiogenesis remains unclear. METHODS AND RESULTS: We studied the role of VEGF-B in various models of pathological angiogenesis using mice lacking VEGF-B (VEGF-B(-/-)) or overexpressing VEGF-B(167). After occlusion of the left coronary artery, VEGF-B deficiency impaired vessel growth in the ischemic myocardium whereas, in wild-type mice, VEGF-B(167) overexpression enhanced revascularization of the infarct and ischemic border zone. By contrast, VEGF-B deficiency did not affect vessel growth in the wounded skin, hypoxic lung, ischemic retina, or ischemic limb. Moreover, VEGF-B(167) overexpression failed to enhance vascular growth in the skin or ischemic limb. CONCLUSIONS: VEGF-B appears to have a relatively restricted angiogenic activity in the ischemic heart. These insights might offer novel therapeutic opportunities. %B Arterioscler Thromb Vasc Biol %V 28 %P 1614 - 20 %8 2008/Sep/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/18511699 %N 9 %! Arteriosclerosis, Thrombosis, and Vascular Biology %0 Journal Article %J J Biol Chem %D 2006 %T Vascular endothelial growth factor (VEGF)/VEGF-C mosaic molecules reveal specificity determinants and feature novel receptor binding patterns %A Jeltsch, Michael %A Karpanen, Terhi %A Strandin, Tomas %A Aho, Kukka %A Lankinen, Hilkka %A Alitalo, Kari %X 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. %B J Biol Chem %V 281 %P 12187 - 95 %8 2006/Apr/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/16505489 %N 17 %! The Journal of Biological Chemistry %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 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 FASEB J %D 2003 %T Intrinsic versus microenvironmental regulation of lymphatic endothelial cell phenotype and function %A Veikkola, Tanja %A Lohela, Marja %A Ikenberg, Kristian %A Mäkinen, Taija %A Korff, Thomas %A Saaristo, Anne %A Petrova, Tatania %A Jeltsch, Michael %A Augustin, Hellmut G %A Alitalo, Kari %X 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. %B FASEB J %V 17 %P 2006 - 13 %8 2003/Nov/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/14597670 %N 14 %! The FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology %0 Journal Article %J J Cell Biol %D 2003 %T VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia %A Gerhardt, Holger %A Golding, Matthew %A Fruttiger, Marcus %A Ruhrberg, Christiana %A Lundkvist, Andrea %A Abramsson, Alexandra %A Jeltsch, Michael %A Mitchell, Christopher %A Alitalo, Kari %A Shima, David %A Betsholtz, Christer %X Vascular endothelial growth factor (VEGF-A) is a major regulator of blood vessel formation and function. It controls several processes in endothelial cells, such as proliferation, survival, and migration, but it is not known how these are coordinately regulated to result in more complex morphogenetic events, such as tubular sprouting, fusion, and network formation. We show here that VEGF-A controls angiogenic sprouting in the early postnatal retina by guiding filopodial extension from specialized endothelial cells situated at the tips of the vascular sprouts. The tip cells respond to VEGF-A only by guided migration; the proliferative response to VEGF-A occurs in the sprout stalks. These two cellular responses are both mediated by agonistic activity of VEGF-A on VEGF receptor 2. Whereas tip cell migration depends on a gradient of VEGF-A, proliferation is regulated by its concentration. Thus, vessel patterning during retinal angiogenesis depends on the balance between two different qualities of the extracellular VEGF-A distribution, which regulate distinct cellular responses in defined populations of endothelial cells. %B J Cell Biol %V 161 %P 1163 - 77 %8 2003/Jun/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/12810700 %N 6 %! The Journal of Cell Biology %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 Circ Res %D 2001 %T Adenoviral expression of vascular endothelial growth factor-C induces lymphangiogenesis in the skin %A Enholm, B %A Karpanen, T %A Jeltsch, M %A Kubo, H %A Stenback, F %A Prevo, R %A Jackson, D G %A Yla-Herttuala, S %A Alitalo, K %X 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. %B Circ Res %V 88 %P 623 - 9 %8 2001/Mar/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/11282897 %N 6 %! Circulation Research %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 Proc Natl Acad Sci U S A %D 1998 %T Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells %A Olofsson, B %A Korpelainen, E %A Pepper, M S %A Mandriota, S J %A Aase, K %A Kumar, V %A Gunji, Y %A Jeltsch, M M %A Shibuya, M %A Alitalo, K %A Eriksson, U %X 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. %B Proc Natl Acad Sci U S A %V 95 %P 11709 - 14 %8 1998/Sep/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/9751730 %N 20 %! Proceedings of the National Academy of Sciences of the United States of America %0 Journal Article %J Proc Natl Acad Sci U S A %D 1998 %T Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4) %A Achen, M G %A Jeltsch, M %A Kukk, E %A Mäkinen, T %A Vitali, A %A Wilks, A F %A Alitalo, K %A Stacker, S A %X 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. %B Proc Natl Acad Sci U S A %V 95 %P 548 - 53 %8 1998/Jan/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/9435229 %N 2 %! Proceedings of the National Academy of Sciences of the United States of America %0 Journal Article %J Science (80- ) %D 1997 %T Hyperplasia of lymphatic vessels in VEGF-C transgenic mice %A Jeltsch, M %A Kaipainen, A %A Joukov, V %A Meng, X %A Lakso, M %A Rauvala, H %A Swartz, M %A Fukumura, D %A Jain, R K %A Alitalo, K %X 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. %B Science (80- ) %V 276 %P 1423 - 5 %8 1997/May/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/9162011 %N 5317 %! Science %0 Journal Article %J EMBO J %D 1997 %T Proteolytic processing regulates receptor specificity and activity of VEGF-C %A Joukov, V %A Sorsa, T %A Kumar, V %A Jeltsch, M %A Claesson-Welsh, L %A Cao, Y %A Saksela, O %A Kalkkinen, N %A Alitalo, K %X 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 'mature' 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. %B EMBO J %V 16 %P 3898 - 911 %8 1997/Jul/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/9233800 %N 13 %! The EMBO Journal