01918nas 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<p>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.</p>  uhttps://www.jeltsch.org/Anisimov2013b?language=de02006nas 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.long02103nas 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/2114808501838nas 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/2014511602729nas 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/19443835