00402nas a2200133 4500008004100000245006400041210006200105260001600167300001400183490000600197100002100203700001800224856002600242 2022 eng d00aLymphatic-to-blood vessel transdifferentiation in zebrafish0 aLymphatictoblood vessel transdifferentiation in zebrafish cJan-06-2022 a539 - 5410 v11 aJeltsch, Michael1 aAlitalo, Kari uhttps://rdcu.be/cOjJ000624nas a2200109 4500008004100000245006800041210006700109260001600176300003100192520016400223856012700387 2020 eng d00aVEGF-C Protects the Integrity of Bone Marrow Perivascular Niche0 aVEGFC Protects the Integrity of Bone Marrow Perivascular Niche c2020/07/31/ aaccepted - for publication3 aKey Points. Vegfc deletion in endothelial or LepR+ cells compromises the bone marrow perivascular niche and hematopoietic stem cell maintenance.Exogenous admin uhttps://ashpublications.org/blood/article/doi/10.1182/blood.2020005699/463465/VEGF-C-Protects-the-Integrity-of-Bone-Marrow01961nas a2200337 4500008004100000020001400041245005600055210005300111260001600164300001100180490000600191520106000197653001901257653001601276653003401292653001301326653002201339653001001361653001101371653001101382100001801393700002201411700002001433700002001453700002101473700002101494700002401515700001801539700002101557856004501578 2019 eng d a2050-084X00aKLK3/PSA and cathepsin D activate VEGF-C and VEGF-D0 aKLK3PSA and cathepsin D activate VEGFC and VEGFD c2019/05/17/ ae444780 v83 aVascular 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.10acancer biology10aCathepsin D10akallikrein-related peptidases10aKLK3/PSA10aLymphangiogenesis10amouse10aVEGF-C10aVEGF-D1 aJha, Sawan, K1 aRauniyar, Khushbu1 aChronowska, Ewa1 aMattonet, Kenny1 aMaina, Eunice, W1 aKoistinen, Hannu1 aStenman, Ulf-Håkan1 aAlitalo, Kari1 aJeltsch, Michael uhttps://elifesciences.org/articles/4447800451nas a2200109 4500008004100000245014500041210006900186260001600255300000900271490000600280856005500286 2017 eng d00aEfficient activation of the lymphangiogenic growth factor VEGF-C requires the C-terminal domain of VEGF-C and the N-terminal domain of CCBE10 aEfficient activation of the lymphangiogenic growth factor VEGFC  c2017/07/07/ a49160 v7 uhttps://www.nature.com/articles/s41598-017-04982-102454nas a2200121 4500008004100000245008800041210006900129260001200198300001200210490000800222520202000230856008202250 2016 eng d00aFunctional Importance of a Proteoglycan Co-Receptor in Pathologic Lymphangiogenesis0 aFunctional Importance of a Proteoglycan CoReceptor in Pathologic c05/2016 a210-2210 v1193 aRationale: 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. uhttp://circres.ahajournals.org/content/early/2016/05/25/CIRCRESAHA.116.30850402281nas a2200133 4500008004100000022001400041245010200055210006900157260001500226300001400241490000800255520182400263856006002087 2015 eng d a1524-457100aFunctional Dissection of the CCBE1 Protein: A Crucial Requirement for the Collagen Repeat Domain.0 aFunctional Dissection of the CCBE1 Protein A Crucial Requirement c2015 May 8 a1660-16690 v1163 a

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.

 uhttp://circres.ahajournals.org/content/116/10/1660.long00352nas a2200109 4500008004100000022002200041245002800063210002400091260003800115300001200153856007700165 2015 eng d a978-3-319-11888-800aThe TIE Receptor Family0 aTIE Receptor Family bSpringer International Publishing a743-775 uhttps://link.springer.com/content/pdf/10.1007%2F978-3-319-11888-8_16.pdf01918nas a2200109 4500008004100000245007600041210006900117260001200186490000800198520151000206856009201716 2014 eng d00aCCBE1 enhances lymphangiogenesis via ADAMTS3-mediated VEGF-C activation0 aCCBE1 enhances lymphangiogenesis via ADAMTS3mediated VEGFC activ c05/20140 v1293 aBackground—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. uhttp://circ.ahajournals.org/content/early/2014/02/19/CIRCULATIONAHA.113.002779.abstract01477nas a2200133 4500008004100000022001400041245011000055210006900165260000900234300000900243490000600252520103100258856005401289 2013 eng d a1937-914500aThe basis for the distinct biological activities of vascular endothelial growth factor receptor-1 ligands0 abasis for the distinct biological activities of vascular endothe c2013 ara520 v63 a

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.

 uhttps://www.jeltsch.org/Anisimov2013b?language=de01315nas a2200121 4500008004100000020001600041245005100057210005000108260000900158490000600167520096300173856005701136 2013 eng d a, 1943-026400aReceptor Tyrosine Kinase-Mediated Angiogenesis0 aReceptor Tyrosine KinaseMediated Angiogenesis c20130 v53 aThe 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. uhttp://cshperspectives.cshlp.org/content/5/9/a00918302006nas a2200133 4500008004100000020001400041245009100055210006900146260001200215300001800227490000800245520156900253856005001822 2013 eng d a1091-649000aStructural and mechanistic insights into VEGF receptor 3 ligand binding and activation0 aStructural and mechanistic insights into VEGF receptor 3 ligand  c08/2013 a12960 - 129650 v1103 aVascular 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. uhttp://www.pnas.org/content/110/32/12960.long01766nas a2200133 4500008004100000022001400041245014300055210006900198260001300267300001300280490000800293520127600301856005501577 2013 eng d a1477-912900aA truncation allele in vascular endothelial growth factor c reveals distinct modes of signaling during lymphatic and vascular development.0 atruncation allele in vascular endothelial growth factor c reveal c2013 Apr a1497-5060 v1403 a

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.

 uhttps://www.jeltsch.org/Villefranc2013?language=de02103nas a2200241 4500008004100000245010100041210006900142260001400211300001400225490000800239520135400247100002601601700002101627700002101648700002001669700001501689700002101704700001901725700002601744700002401770700001801794856004901812 2011 eng d00aStructural determinants of vascular endothelial growth factor-D receptor binding and specificity0 aStructural determinants of vascular endothelial growth factorD r c2011/Feb/ a1507 - 150 v1173 aVascular 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.1 aLeppänen, Veli-Matti1 aJeltsch, Michael1 aAnisimov, Andrey1 aTvorogov, Denis1 aAho, Kukka1 aKalkkinen, Nisse1 aToivanen, Pyry1 aYlä-Herttuala, Seppo1 aBallmer-Hofer, Kurt1 aAlitalo, Kari uhttp://view.ncbi.nlm.nih.gov/pubmed/2114808501603nas a2200265 4500008004100000245011900041210006900160260001400229300001300243490000700256520077800263100002101041700002001062700002001082700002101102700002201123700002101145700002301166700002301189700001601212700002101228700002101249700001801270856004901288 2010 eng d00aClaudin-like protein 24 interacts with the VEGFR-2 and VEGFR-3 pathways and regulates lymphatic vessel development0 aClaudinlike protein 24 interacts with the VEGFR2 and VEGFR3 path c2010/May/ a875 - 800 v243 aThe 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.1 aSaharinen, Pipsa1 aHelotera, Hanna1 aMiettinen, Juho1 aNorrmen, Camilla1 aD'Amico, Gabriela1 aJeltsch, Michael1 aLangenberg, Tobias1 aVandevelde, Wouter1 aNy, Annelii1 aDewerchin, Mieke1 aCarmeliet, Peter1 aAlitalo, Kari uhttp://view.ncbi.nlm.nih.gov/pubmed/2043942801838nas a2200289 4500008004100000245014100041210006900182260001400251300001300265490000700278520091500285100002001200700002101220700001501241700002601256700002001282700002701302700002501329700002101354700002201375700002101397700002101418700002101439700002101460700001801481856004901499 2010 eng d00aEffective suppression of vascular network formation by combination of antibodies blocking VEGFR ligand binding and receptor dimerization0 aEffective suppression of vascular network formation by combinati c2010/Dec/ a630 - 400 v183 aAntibodies 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.1 aTvorogov, Denis1 aAnisimov, Andrey1 aZheng, Wei1 aLeppänen, Veli-Matti1 aTammela, Tuomas1 aLaurinavicius, Simonas1 aHolnthoner, Wolfgang1 aHeloterä, Hanna1 aHolopainen, Tanja1 aJeltsch, Michael1 aKalkkinen, Nisse1 aLankinen, Hilkka1 aOjala, Päivi, M1 aAlitalo, Kari uhttp://view.ncbi.nlm.nih.gov/pubmed/2113004302522nas a2200241 4500008004100000245008800041210006900129260001200198300001400210490000800224520178600232100002602018700002102044700002102065700002102086700002102107700002002128700002102148700002002169700002402189700001802213856004902231 2010 eng d00aStructural determinants of growth factor binding and specificity by VEGF receptor 20 aStructural determinants of growth factor binding and specificity c02/2010 a2425 - 300 v1073 aVascular 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.1 aLeppänen, Veli-Matti1 aProta, Andrea, E1 aJeltsch, Michael1 aAnisimov, Andrey1 aKalkkinen, Nisse1 aStrandin, Tomas1 aLankinen, Hilkka1 aGoldman, Adrian1 aBallmer-Hofer, Kurt1 aAlitalo, Kari uhttp://view.ncbi.nlm.nih.gov/pubmed/2014511602377nas a2200265 4500008004100000245014000041210006900181260001100250300001100261490000600272520152800278100001901806700002101825700002401846700002001870700002301890700002501913700002001938700002101958700001801979700001801997700002502015700002202040856004902062 2010 eng d00aSuppressive effects of vascular endothelial growth factor-B on tumor growth in a mouse model of pancreatic neuroendocrine tumorigenesis0 aSuppressive effects of vascular endothelial growth factorB on tu c2010// ae141090 v53 aBACKGROUND: 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.1 aAlbrecht, Imke1 aKopfstein, Lucie1 aStrittmatter, Karin1 aSchomber, Tibor1 aFalkevall, Annelie1 aHagberg, Carolina, E1 aLorentz, Pascal1 aJeltsch, Michael1 aAlitalo, Kari1 aEriksson, Ulf1 aChristofori, Gerhard1 aPietras, Kristian uhttp://view.ncbi.nlm.nih.gov/pubmed/2112484102729nas a2200349 4500008004100000245015500041210006900196260001400265300001400279490000800293520163900301100001501940700002001955700002201975700002101997700002002018700002002038700002102058700001902079700002102098700002102119700002102140700002302161700002202184700002602206700001902232700001902251700002302270700001902293700001802312856004902330 2010 eng d00aVascular endothelial growth factor-B acts as a coronary growth factor in transgenic rats without inducing angiogenesis, vascular leak, or inflammation0 aVascular endothelial growth factorB acts as a coronary growth fa c2010/Oct/ a1725 - 330 v1223 aBACKGROUND: 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.1 aBry, Maija1 aKivelä, Riikka1 aHolopainen, Tanja1 aAnisimov, Andrey1 aTammela, Tuomas1 aSoronen, Jarkko1 aSilvola, Johanna1 aSaraste, Antti1 aJeltsch, Michael1 aKorpisalo, Petra1 aCarmeliet, Peter1 aLemström, Karl, B1 aShibuya, Masabumi1 aYlä-Herttuala, Seppo1 aAlhonen, Leena1 aMervaala, Eero1 aAndersson, Leif, C1 aKnuuti, Juhani1 aAlitalo, Kari uhttp://view.ncbi.nlm.nih.gov/pubmed/2093797402406nas a2200229 4500008004100000245009500041210006900136260001400205300001400219490000800233520168800241100002101929700002201950700002101972700002001993700002302013700002602036700002102062700002602083700001802109856004902127 2009 eng d00aActivated forms of VEGF-C and VEGF-D provide improved vascular function in skeletal muscle0 aActivated forms of VEGFC and VEGFD provide improved vascular fun c2009/Jun/ a1302 - 120 v1043 aThe 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.1 aAnisimov, Andrey1 aAlitalo, Annamari1 aKorpisalo, Petra1 aSoronen, Jarkko1 aKaijalainen, Seppo1 aLeppänen, Veli-Matti1 aJeltsch, Michael1 aYlä-Herttuala, Seppo1 aAlitalo, Kari uhttp://view.ncbi.nlm.nih.gov/pubmed/1944383502437nas a2200325 4500008004100000245014100041210006900182260001400251300001400265490000800279520142200287100002001709700001501729700002101744700003101765700002001796700002001816700002001836700002001856700001901876700002101895700002001916700002301936700001901959700002201978700002602000700001802026700001802044856004902062 2008 eng d00aOverexpression of vascular endothelial growth factor-B in mouse heart alters cardiac lipid metabolism and induces myocardial hypertrophy0 aOverexpression of vascular endothelial growth factorB in mouse h c2008/Oct/ a1018 - 260 v1033 aVascular 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.1 aKarpanen, Terhi1 aBry, Maija1 aOllila, Hanna, M1 aSeppänen-Laakso, Tuulikki1 aLiimatta, Erkki1 aLeskinen, Hanna1 aKivelä, Riikka1 aHelkamaa, Teemu1 aMerentie, Mari1 aJeltsch, Michael1 aPaavonen, Karri1 aAndersson, Leif, C1 aMervaala, Eero1 aHassinen, Ilmo, E1 aYlä-Herttuala, Seppo1 aOresic, Matej1 aAlitalo, Kari uhttp://view.ncbi.nlm.nih.gov/pubmed/1875782702098nas a2200385 4500008004100000245011800041210006900159260001400228300001400242490000700256520095200263100001301215700001501228700001901243700001901262700001601281700002001297700002101317700002001338700002401358700002901382700002001411700002901431700001601460700002501476700002501501700002301526700002101549700001901570700001801589700001801607700002101625700001701646856004901663 2008 eng d00aReevaluation of the role of VEGF-B suggests a restricted role in the revascularization of the ischemic myocardium0 aReevaluation of the role of VEGFB suggests a restricted role in  c2008/Sep/ a1614 - 200 v283 aOBJECTIVE: 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.1 aLi, Xuri1 aTjwa, Marc1 aVan Hove, Inge1 aEnholm, Berndt1 aNeven, Elke1 aPaavonen, Karri1 aJeltsch, Michael1 aJuan, Toni Diez1 aSievers, Richard, E1 aChorianopoulos, Emmanuel1 aWada, Hiromichi1 aVanwildemeersch, Maarten1 aNoel, Agnes1 aFoidart, Jean-Michel1 aSpringer, Matthew, L1 aDegenfeld, Georges1 aDewerchin, Mieke1 aBlau, Helen, M1 aAlitalo, Kari1 aEriksson, Ulf1 aCarmeliet, Peter1 aMoons, Lieve uhttp://view.ncbi.nlm.nih.gov/pubmed/1851169902705nas a2200229 4500008004100000245014300041210006900184260001400253300001400267490000700281520193200288100002502220700002202245700002102267700002102288700002102309700002602330700002202356700003002378700001802408856004902426 2008 eng d00aThe tyrosine kinase inhibitor cediranib blocks ligand-induced vascular endothelial growth factor receptor-3 activity and lymphangiogenesis0 atyrosine kinase inhibitor cediranib blocks ligandinduced vascula c2008/Jun/ a4754 - 620 v683 aSolid 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