TY - JOUR T1 - VEGF-C Protects the Integrity of Bone Marrow Perivascular Niche JF - Blood Y1 - 2020 AB - Key Points. Vegfc deletion in endothelial or LepR+ cells compromises the bone marrow perivascular niche and hematopoietic stem cell maintenance.Exogenous admin UR - https://ashpublications.org/blood/article/doi/10.1182/blood.2020005699/463465/VEGF-C-Protects-the-Integrity-of-Bone-Marrow JO - Blood ER - TY - JOUR T1 - KLK3/PSA and cathepsin D activate VEGF-C and VEGF-D JF - eLife Y1 - 2019 A1 - Jha, Sawan Kumar A1 - Rauniyar, Khushbu A1 - Chronowska, Ewa A1 - Mattonet, Kenny A1 - Maina, Eunice Wairimu A1 - Koistinen, Hannu A1 - Stenman, Ulf-Håkan A1 - Alitalo, Kari A1 - Jeltsch, Michael KW - cancer biology KW - Cathepsin D KW - kallikrein-related peptidases KW - KLK3/PSA KW - Lymphangiogenesis KW - mouse KW - VEGF-C KW - VEGF-D AB - 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. VL - 8 SN - 2050-084X UR - https://elifesciences.org/articles/44478 JO - eLife ER - TY - JOUR T1 - Efficient activation of the lymphangiogenic growth factor VEGF-C requires the C-terminal domain of VEGF-C and the N-terminal domain of CCBE1 JF - Scientific Reports Y1 - 2017 VL - 7 UR - https://www.nature.com/articles/s41598-017-04982-1 IS - 1 ER - TY - JOUR T1 - Functional Importance of a Proteoglycan Co-Receptor in Pathologic Lymphangiogenesis JF - Circulation Research Y1 - 2016 AB - Rationale: Lymphatic vessel growth is mediated by major pro-lymphangiogenic factors such as VEGF-C and -D, among other endothelial effectors. Heparan sulfate is a linear polysaccharide expressed on proteoglycan core proteins on cell-membranes and matrix, playing roles in angiogenesis, although little is known regarding any function(s) in lymphatic remodeling in vivo. Objective: To explore the genetic basis and mechanisms whereby heparan sulfate proteoglycans mediate pathologic lymphatic remodeling. Methods and Results: Lymphatic endothelial deficiency in the major heparan sulfate biosynthetic enzyme N-deacetylase/N-sulfotransferase-1 (Ndst1; involved in glycan-chain sulfation) was associated with reduced lymphangiogenesis in pathologic models, including spontaneous neoplasia. Mouse mutants demonstrated tumor-associated lymphatic vessels with apoptotic nuclei. Mutant lymphatic endothelia demonstrated impaired mitogen (Erk) and survival (Akt) pathway signaling as well as reduced VEGF-C mediated protection from starvation-induced apoptosis. Lymphatic endothelial specific Ndst1 deficiency (in Ndst1f/fProx1+/CreERT2 mice) was sufficient to inhibit VEGF-C dependent lymphangiogenesis. Lymphatic heparan sulfate deficiency reduced phosphorylation of the major lymphatic growth receptor VEGFR-3 in response to multiple VEGF-C species. Syndecan-4 was the dominantly expressed heparan sulfate proteoglycan in mouse lymphatic endothelia, and pathologic lymphangiogenesis was impaired in Sdc4(-/-) mice. On the lymphatic cell surface, VEGF-C induced robust association between syndecan-4 and VEGFR-3 which was sensitive to glycan disruption. Moreover, VEGFR-3 mitogen and survival signaling was reduced in the setting of Ndst1 or Sdc4 deficiency. Conclusions: These findings demonstrate the genetic importance of heparan sulfate and the major lymphatic proteoglycan syndecan-4 in pathologic lymphatic remodeling. This may introduce novel future strategies to alter pathologic lymphatic-vascular remodeling. VL - 119 UR - http://circres.ahajournals.org/content/early/2016/05/25/CIRCRESAHA.116.308504 IS - 2 ER - TY - JOUR T1 - Functional Dissection of the CCBE1 Protein: A Crucial Requirement for the Collagen Repeat Domain. JF - Circ Res Y1 - 2015 AB -
RATIONALE: Collagen- and calcium-binding EGF domain-containing protein 1 (CCBE1) is essential for lymphangiogenesis in vertebrates and has been associated with Hennekam syndrome. Recently, CCBE1 has emerged as a crucial regulator of vascular endothelial growth factor-C (VEGFC) signaling.
OBJECTIVE: CCBE1 is a secreted protein characterized by 2 EGF domains and 2 collagen repeats. The functional role of the different CCBE1 protein domains is completely unknown. Here, we analyzed the functional role of the different CCBE1 domains in vivo and in vitro.
METHODS AND RESULTS: We analyzed the functionality of several CCBE1 deletion mutants by generating knock-in mice expressing these mutants, by analyzing their ability to enhance Vegfc signaling in vivo in zebrafish, and by testing their ability to induce VEGFC processing in vitro. We found that deleting the collagen domains of CCBE1 has a much stronger effect on CCBE1 activity than deleting the EGF domains. First, although CCBE1ΔCollagen mice fully phenocopy CCBE1 knock-out mice, CCBE1ΔEGF knock-in embryos still form rudimentary lymphatics. Second, Ccbe1ΔEGF, but not Ccbe1ΔCollagen, could partially substitute for Ccbe1 to enhance Vegfc signaling in zebrafish. Third, CCBE1ΔEGF, similarly to CCBE1, but not CCBE1ΔCollagen could activate VEGFC processing in vitro. Furthermore, a Hennekam syndrome mutation within the collagen domain has a stronger effect than a Hennekam syndrome mutation within the EGF domain.
CONCLUSIONS: We propose that the collagen domains of CCBE1 are crucial for the activation of VEGFC in vitro and in vivo. The EGF domains of CCBE1 are dispensable for regulation of VEGFC processing in vitro, however, they are necessary for full lymphangiogenic activity of CCBE1 in vivo.
VL - 116 UR - http://circres.ahajournals.org/content/116/10/1660.long IS - 10 ER - TY - CHAP T1 - The TIE Receptor Family T2 - Receptor Tyrosine Kinases: Family and Subfamilies Y1 - 2015 JF - Receptor Tyrosine Kinases: Family and Subfamilies PB - Springer International Publishing UR - https://link.springer.com/content/pdf/10.1007%2F978-3-319-11888-8_16.pdf ER - TY - JOUR T1 - CCBE1 enhances lymphangiogenesis via ADAMTS3-mediated VEGF-C activation JF - Circulation Y1 - 2014 AB - Background—Hennekam lymphangiectasia-lymphedema syndrome (OMIM 235510) is a rare autosomal recessive disease, which is associated with mutations in the collagen- and calcium-binding EGF domains 1 (CCBE1) gene. Because of the striking phenotypic similarity of embryos lacking either the Ccbe1 gene or the lymphangiogenic growth factor Vegfc gene, we searched for CCBE1 interactions with the VEGF-C growth factor signaling pathway, which is critical in embryonic and adult lymphangiogenesis. Methods and Results—By analyzing VEGF-C produced by CCBE1-transfected cells, we found that while CCBE1 itself does not process VEGF-C, it promotes proteolytic cleavage of the otherwise poorly active 29/31-kDa form of VEGF-C by the A disintegrin and metalloprotease with thrombospondin motifs-3 (ADAMTS3) protease, resulting in the mature 21/23-kDa form of VEGF-C, which induces increased VEGF-C receptor signaling. Adeno-associated viral vector (AAV) mediated transduction of CCBE1 into mouse skeletal muscle enhanced lymphangiogenesis and angiogenesis induced by AAV-VEGF-C. Conclusions—These results identify ADAMTS3 as a VEGF-C activating protease and reveal a novel type of regulation of a vascular growth factor by a protein that enhances its proteolytic cleavage and activation. The results suggest CCBE1 is a potential therapeutic tool for the modulation of lymphangiogenesis and angiogenesis in a variety of diseases that involve the lymphatic system, such as lymphedema or lymphatic metastasis. VL - 129 UR - http://circ.ahajournals.org/content/early/2014/02/19/CIRCULATIONAHA.113.002779.abstract IS - 19 ER - TY - JOUR T1 - The basis for the distinct biological activities of vascular endothelial growth factor receptor-1 ligands JF - Sci Signal Y1 - 2013 AB -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.
VL - 6 IS - 282 ER - TY - JOUR T1 - Receptor Tyrosine Kinase-Mediated Angiogenesis JF - Cold Spring Harbor Perspectives in Biology Y1 - 2013 AB - The endothelial cell is the essential cell type forming the inner layer of the vasculature. Two families of receptor tyrosine kinases (RTKs) are almost completely endothelial cell specific: the vascular endothelial growth factor (VEGF) receptors (VEGFR1-3) and the Tie receptors (Tie1 and Tie2). Both are key players governing the generation of blood and lymphatic vessels during embryonic development. Because the growth of new blood and lymphatic vessels (or the lack thereof) is a central element in many diseases, the VEGF and the Tie receptors provide attractive therapeutic targets in various diseases. Indeed, several drugs directed to these RTK signaling pathways are already on the market, whereas many are in clinical trials. Here we review the VEGFR and Tie families, their involvement in developmental and pathological angiogenesis, and the different possibilities for targeting them to either block or enhance angiogenesis and lymphangiogenesis. VL - 5 SN - , 1943-0264 UR - http://cshperspectives.cshlp.org/content/5/9/a009183 IS - 9 JO - Cold Spring Harb Perspect Biol ER - TY - JOUR T1 - Structural and mechanistic insights into VEGF receptor 3 ligand binding and activation JF - Proceedings of the National Academy of Sciences of the United States of America Y1 - 2013 AB - 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. VL - 110 SN - 1091-6490 UR - http://www.pnas.org/content/110/32/12960.long IS - 32 JO - Proc. Natl. Acad. Sci. U.S.A. ER - TY - JOUR T1 - A truncation allele in vascular endothelial growth factor c reveals distinct modes of signaling during lymphatic and vascular development. JF - Development Y1 - 2013 AB -Vascular endothelial growth factor C (Vegfc) is a secreted protein that guides lymphatic development in vertebrate embryos. However, its role during developmental angiogenesis is not well characterized. Here, we identify a mutation in zebrafish vegfc that severely affects lymphatic development and leads to angiogenesis defects on sensitized genetic backgrounds. The um18 mutation prematurely truncated Vegfc, blocking its secretion and paracrine activity but not its ability to activate its receptor Flt4. When expressed in endothelial cells, vegfc(um18) could not rescue lymphatic defects in mutant embryos, but induced ectopic blood vessel branching. Furthermore, vegfc-deficient endothelial cells did not efficiently contribute to tip cell positions in developing sprouts. Computational modeling together with assessment of endothelial cell dynamics by time-lapse analysis suggested that an autocrine Vegfc/Flt4 loop plays an important role in migratory persistence and filopodia stability during sprouting. Our results suggest that Vegfc acts in two distinct modes during development: as a paracrine factor secreted from arteries to guide closely associated lymphatic vasculature and as an autocrine factor to drive migratory persistence during angiogenesis.
VL - 140 IS - 7 ER - TY - JOUR T1 - Structural determinants of vascular endothelial growth factor-D receptor binding and specificity JF - Blood Y1 - 2011 A1 - Leppänen, Veli-Matti A1 - Jeltsch, Michael A1 - Anisimov, Andrey A1 - Tvorogov, Denis A1 - Aho, Kukka A1 - Kalkkinen, Nisse A1 - Toivanen, Pyry A1 - Ylä-Herttuala, Seppo A1 - Ballmer-Hofer, Kurt A1 - Alitalo, Kari AB - 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. VL - 117 UR - http://view.ncbi.nlm.nih.gov/pubmed/21148085 IS - 5 JO - Blood ER - TY - JOUR T1 - Claudin-like protein 24 interacts with the VEGFR-2 and VEGFR-3 pathways and regulates lymphatic vessel development JF - Genes Dev Y1 - 2010 A1 - Saharinen, Pipsa A1 - Helotera, Hanna A1 - Miettinen, Juho A1 - Norrmen, Camilla A1 - D'Amico, Gabriela A1 - Jeltsch, Michael A1 - Langenberg, Tobias A1 - Vandevelde, Wouter A1 - Ny, Annelii A1 - Dewerchin, Mieke A1 - Carmeliet, Peter A1 - Alitalo, Kari AB - 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. VL - 24 UR - http://view.ncbi.nlm.nih.gov/pubmed/20439428 IS - 9 JO - Genes & Development ER - TY - JOUR T1 - Effective suppression of vascular network formation by combination of antibodies blocking VEGFR ligand binding and receptor dimerization JF - Cancer Cell Y1 - 2010 A1 - Tvorogov, Denis A1 - Anisimov, Andrey A1 - Zheng, Wei A1 - Leppänen, Veli-Matti A1 - Tammela, Tuomas A1 - Laurinavicius, Simonas A1 - Holnthoner, Wolfgang A1 - Heloterä, Hanna A1 - Holopainen, Tanja A1 - Jeltsch, Michael A1 - Kalkkinen, Nisse A1 - Lankinen, Hilkka A1 - Ojala, Päivi M A1 - Alitalo, Kari AB - 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. VL - 18 UR - http://view.ncbi.nlm.nih.gov/pubmed/21130043 IS - 6 JO - Cancer Cell ER - TY - JOUR T1 - Structural determinants of growth factor binding and specificity by VEGF receptor 2 JF - Proceedings of the National Academy of Sciences of the United States of America Y1 - 2010 A1 - Leppänen, Veli-Matti A1 - Prota, Andrea E A1 - Jeltsch, Michael A1 - Anisimov, Andrey A1 - Kalkkinen, Nisse A1 - Strandin, Tomas A1 - Lankinen, Hilkka A1 - Goldman, Adrian A1 - Ballmer-Hofer, Kurt A1 - Alitalo, Kari AB - 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. VL - 107 UR - http://view.ncbi.nlm.nih.gov/pubmed/20145116 IS - 6 ER - TY - JOUR T1 - Suppressive effects of vascular endothelial growth factor-B on tumor growth in a mouse model of pancreatic neuroendocrine tumorigenesis JF - PLoS ONE Y1 - 2010 A1 - Albrecht, Imke A1 - Kopfstein, Lucie A1 - Strittmatter, Karin A1 - Schomber, Tibor A1 - Falkevall, Annelie A1 - Hagberg, Carolina E A1 - Lorentz, Pascal A1 - Jeltsch, Michael A1 - Alitalo, Kari A1 - Eriksson, Ulf A1 - Christofori, Gerhard A1 - Pietras, Kristian AB - 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. VL - 5 UR - http://view.ncbi.nlm.nih.gov/pubmed/21124841 IS - 11 JO - PLoS ONE ER - TY - JOUR T1 - Vascular endothelial growth factor-B acts as a coronary growth factor in transgenic rats without inducing angiogenesis, vascular leak, or inflammation JF - Circulation Y1 - 2010 A1 - Bry, Maija A1 - Kivelä, Riikka A1 - Holopainen, Tanja A1 - Anisimov, Andrey A1 - Tammela, Tuomas A1 - Soronen, Jarkko A1 - Silvola, Johanna A1 - Saraste, Antti A1 - Jeltsch, Michael A1 - Korpisalo, Petra A1 - Carmeliet, Peter A1 - Lemström, Karl B A1 - Shibuya, Masabumi A1 - Ylä-Herttuala, Seppo A1 - Alhonen, Leena A1 - Mervaala, Eero A1 - Andersson, Leif C A1 - Knuuti, Juhani A1 - Alitalo, Kari AB - 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. VL - 122 UR - http://view.ncbi.nlm.nih.gov/pubmed/20937974 IS - 17 JO - Circulation ER - TY - JOUR T1 - Activated forms of VEGF-C and VEGF-D provide improved vascular function in skeletal muscle JF - Circ Res Y1 - 2009 A1 - Anisimov, Andrey A1 - Alitalo, Annamari A1 - Korpisalo, Petra A1 - Soronen, Jarkko A1 - Kaijalainen, Seppo A1 - Leppänen, Veli-Matti A1 - Jeltsch, Michael A1 - Ylä-Herttuala, Seppo A1 - Alitalo, Kari AB - 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. VL - 104 UR - http://view.ncbi.nlm.nih.gov/pubmed/19443835 IS - 11 JO - Circulation Research ER - TY - JOUR T1 - Overexpression of vascular endothelial growth factor-B in mouse heart alters cardiac lipid metabolism and induces myocardial hypertrophy JF - Circ Res Y1 - 2008 A1 - Karpanen, Terhi A1 - Bry, Maija A1 - Ollila, Hanna M A1 - Seppänen-Laakso, Tuulikki A1 - Liimatta, Erkki A1 - Leskinen, Hanna A1 - Kivelä, Riikka A1 - Helkamaa, Teemu A1 - Merentie, Mari A1 - Jeltsch, Michael A1 - Paavonen, Karri A1 - Andersson, Leif C A1 - Mervaala, Eero A1 - Hassinen, Ilmo E A1 - Ylä-Herttuala, Seppo A1 - Oresic, Matej A1 - Alitalo, Kari AB - 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. VL - 103 UR - http://view.ncbi.nlm.nih.gov/pubmed/18757827 IS - 9 JO - Circulation Research ER - TY - JOUR T1 - Reevaluation of the role of VEGF-B suggests a restricted role in the revascularization of the ischemic myocardium JF - Arterioscler Thromb Vasc Biol Y1 - 2008 A1 - Li, Xuri A1 - Tjwa, Marc A1 - Van Hove, Inge A1 - Enholm, Berndt A1 - Neven, Elke A1 - Paavonen, Karri A1 - Jeltsch, Michael A1 - Juan, Toni Diez A1 - Sievers, Richard E A1 - Chorianopoulos, Emmanuel A1 - Wada, Hiromichi A1 - Vanwildemeersch, Maarten A1 - Noel, Agnes A1 - Foidart, Jean-Michel A1 - Springer, Matthew L A1 - von Degenfeld, Georges A1 - Dewerchin, Mieke A1 - Blau, Helen M A1 - Alitalo, Kari A1 - Eriksson, Ulf A1 - Carmeliet, Peter A1 - Moons, Lieve AB - 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. VL - 28 UR - http://view.ncbi.nlm.nih.gov/pubmed/18511699 IS - 9 JO - Arteriosclerosis, Thrombosis, and Vascular Biology ER - TY - JOUR T1 - The tyrosine kinase inhibitor cediranib blocks ligand-induced vascular endothelial growth factor receptor-3 activity and lymphangiogenesis JF - Cancer Res Y1 - 2008 A1 - Heckman, Caroline A A1 - Holopainen, Tanja A1 - Wirzenius, Maria A1 - Keskitalo, Salla A1 - Jeltsch, Michael A1 - Ylä-Herttuala, Seppo A1 - Wedge, Stephen R A1 - Jürgensmeier, Juliane M A1 - Alitalo, Kari AB - 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. VL - 68 UR - http://view.ncbi.nlm.nih.gov/pubmed/18559522 IS - 12 JO - Cancer Research ER - TY - JOUR T1 - Distinct architecture of lymphatic vessels induced by chimeric vascular endothelial growth factor-C/vascular endothelial growth factor heparin-binding domain fusion proteins JF - Circ Res Y1 - 2007 A1 - Tammela, Tuomas A1 - He, Yulong A1 - Lyytikkä, Johannes A1 - Jeltsch, Michael A1 - Markkanen, Johanna A1 - Pajusola, Katri A1 - Ylä-Herttuala, Seppo A1 - Alitalo, Kari AB - 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. VL - 100 UR - http://view.ncbi.nlm.nih.gov/pubmed/17478733 IS - 10 JO - Circulation Research ER - TY - JOUR T1 - Enhanced capillary formation stimulated by a chimeric vascular endothelial growth factor/vascular endothelial growth factor-C silk domain fusion protein JF - Circ Res Y1 - 2007 A1 - Keskitalo, Salla A1 - Tammela, Tuomas A1 - Lyytikka, Johannes A1 - Karpanen, Terhi A1 - Jeltsch, Michael A1 - Markkanen, Johanna A1 - Yla-Herttuala, Seppo A1 - Alitalo, Kari AB - 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. VL - 100 UR - http://view.ncbi.nlm.nih.gov/pubmed/17478734 IS - 10 JO - Circulation Research ER - TY - JOUR T1 - Functional interaction of VEGF-C and VEGF-D with neuropilin receptors JF - FASEB J Y1 - 2006 A1 - Kärpänen, Terhi A1 - Heckman, Caroline A A1 - Keskitalo, Salla A1 - Jeltsch, Michael A1 - Ollila, Hanna A1 - Neufeld, Gera A1 - Tamagnone, Luca A1 - Alitalo, Kari AB - 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. VL - 20 UR - http://view.ncbi.nlm.nih.gov/pubmed/16816121 IS - 9 JO - The FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology ER - TY - JOUR T1 - Vascular endothelial growth factor (VEGF)/VEGF-C mosaic molecules reveal specificity determinants and feature novel receptor binding patterns JF - J Biol Chem Y1 - 2006 A1 - Jeltsch, Michael A1 - Karpanen, Terhi A1 - Strandin, Tomas A1 - Aho, Kukka A1 - Lankinen, Hilkka A1 - Alitalo, Kari AB - 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. VL - 281 UR - http://view.ncbi.nlm.nih.gov/pubmed/16505489 IS - 17 JO - The Journal of Biological Chemistry ER - TY - CHAP T1 - VEGF Receptors T2 - Sigma-RBI Handbook of Receptor Classification and Signal Transduction Y1 - 2006 JF - Sigma-RBI Handbook of Receptor Classification and Signal Transduction PB - Sigma UR - http://www.sigmaaldrich.com/technical-documents/articles/biology/rbi-handbook.html ER - TY - JOUR T1 - Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation JF - J Clin Invest Y1 - 2005 A1 - Baluk, Peter A1 - Tammela, Tuomas A1 - Ator, Erin A1 - Lyubynska, Natalya A1 - Achen, Marc G A1 - Hicklin, Daniel J A1 - Jeltsch, Michael A1 - Petrova, Tatiana V A1 - Pytowski, Bronislaw A1 - Stacker, Steven A A1 - Ylä-Herttuala, Seppo A1 - Jackson, David G A1 - Alitalo, Kari A1 - McDonald, Donald M AB - 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. VL - 115 UR - http://view.ncbi.nlm.nih.gov/pubmed/15668734 IS - 2 JO - The Journal of Clinical Investigation ER - TY - JOUR T1 - Vascular endothelial cell growth factor receptor 3-mediated activation of lymphatic endothelium is crucial for tumor cell entry and spread via lymphatic vessels JF - Cancer Res Y1 - 2005 A1 - He, Yulong A1 - Rajantie, Iiro A1 - Pajusola, Katri A1 - Jeltsch, Michael A1 - Holopainen, Tanja A1 - Yla-Herttuala, Seppo A1 - Harding, Thomas A1 - Jooss, Karin A1 - Takahashi, Takashi A1 - Alitalo, Kari AB - 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. VL - 65 UR - http://view.ncbi.nlm.nih.gov/pubmed/15930292 IS - 11 JO - Cancer Research ER - TY - JOUR T1 - Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins JF - Nat Immunol Y1 - 2004 A1 - Karkkainen, Marika J A1 - Haiko, Paula A1 - Sainio, Kirsi A1 - Partanen, Juha A1 - Taipale, Jussi A1 - Petrova, Tatiana V A1 - Jeltsch, Michael A1 - Jackson, David G A1 - Talikka, Marja A1 - Rauvala, Heikki A1 - Betsholtz, Christer A1 - Alitalo, Kari AB - 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. VL - 5 UR - http://view.ncbi.nlm.nih.gov/pubmed/14634646 IS - 1 JO - Nature Immunology ER - TY - JOUR T1 - Genesis and pathogenesis of lymphatic vessels JF - Cell Tissue Res Y1 - 2003 A1 - Jeltsch, Michael A1 - Tammela, Tuomas A1 - Alitalo, Kari A1 - Wilting, Jörg AB - 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. VL - 314 UR - http://view.ncbi.nlm.nih.gov/pubmed/12942362 IS - 1 JO - Cell and Tissue Research ER - TY - JOUR T1 - Intrinsic versus microenvironmental regulation of lymphatic endothelial cell phenotype and function JF - FASEB J Y1 - 2003 A1 - Veikkola, Tanja A1 - Lohela, Marja A1 - Ikenberg, Kristian A1 - Mäkinen, Taija A1 - Korff, Thomas A1 - Saaristo, Anne A1 - Petrova, Tatania A1 - Jeltsch, Michael A1 - Augustin, Hellmut G A1 - Alitalo, Kari AB - 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. VL - 17 UR - http://view.ncbi.nlm.nih.gov/pubmed/14597670 IS - 14 JO - The FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology ER - TY - JOUR T1 - VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia JF - J Cell Biol Y1 - 2003 A1 - Gerhardt, Holger A1 - Golding, Matthew A1 - Fruttiger, Marcus A1 - Ruhrberg, Christiana A1 - Lundkvist, Andrea A1 - Abramsson, Alexandra A1 - Jeltsch, Michael A1 - Mitchell, Christopher A1 - Alitalo, Kari A1 - Shima, David A1 - Betsholtz, Christer AB - 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. VL - 161 UR - http://view.ncbi.nlm.nih.gov/pubmed/12810700 IS - 6 JO - The Journal of Cell Biology ER - TY - JOUR T1 - Adenoviral VEGF-C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes JF - FASEB J Y1 - 2002 A1 - Saaristo, Anne A1 - Veikkola, Tanja A1 - Enholm, Berndt A1 - Hytönen, Maija A1 - Arola, Johanna A1 - Pajusola, Katri A1 - Turunen, Païvi A1 - Jeltsch, Michael A1 - Karkkainen, Marika J A1 - Kerjaschki, Dontscho A1 - Bueler, Hansruedi A1 - Ylä-Herttuala, Seppo A1 - Alitalo, Kari AB - 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. VL - 16 UR - http://view.ncbi.nlm.nih.gov/pubmed/12087065 IS - 9 JO - The FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology ER -