@article {694, title = {Lymphatics and the eye. [Finnish]}, journal = {Duodecim L{\"a}{\"a}ketieteellinen Aikakauskirja}, volume = {136}, year = {2020}, month = {2020/02/10/}, pages = {1777-1788}, type = {review}, chapter = {1777}, issn = {2242-3281}, doi = {10.5281/zenodo.4005517}, url = {https://www.duodecimlehti.fi/lehti/2020/16/duo15739}, author = {Gucciardo, Erika and Lehti, Timo A. and Korhonen, Ani and Salv{\'e}n, Petri and Lehti, Kaisa and Jeltsch, Michael and Loukovaara, Sirpa} } @article {708, title = {VEGF-C Protects the Integrity of Bone Marrow Perivascular Niche}, journal = {Blood}, year = {2020}, month = {2020/07/31/}, pages = {accepted - for publication}, abstract = {Key Points. Vegfc deletion in endothelial or LepR+ cells compromises the bone marrow perivascular niche and hematopoietic stem cell maintenance.Exogenous admin}, doi = {10.1182/blood.2020005699}, url = {https://ashpublications.org/blood/article/doi/10.1182/blood.2020005699/463465/VEGF-C-Protects-the-Integrity-of-Bone-Marrow}, author = {Fang, Shentong and Chen, Shuo and Nurmi, Harri and Lepp{\"a}nen, Veli-Matti and Jeltsch, Michael and Scadden, David T. and Silberstein, Lev and Mikkola, Hanna and Alitalo, Kari} } @article {693, title = {KLK3/PSA and cathepsin D activate VEGF-C and VEGF-D}, journal = {eLife}, volume = {8}, year = {2019}, month = {2019/05/17/}, pages = {e44478}, abstract = {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.}, keywords = {cancer biology, Cathepsin D, kallikrein-related peptidases, KLK3/PSA, Lymphangiogenesis, mouse, VEGF-C, VEGF-D}, isbn = {2050-084X}, url = {https://elifesciences.org/articles/44478}, author = {Jha, Sawan Kumar and Rauniyar, Khushbu and Chronowska, Ewa and Mattonet, Kenny and Maina, Eunice Wairimu and Koistinen, Hannu and Stenman, Ulf-H{\r a}kan and Alitalo, Kari and Jeltsch, Michael} } @article {690, title = {The Proteolytic Activation of Vascular Endothelial Growth Factor-C}, journal = {Lymphologie in Forschung und Praxis}, volume = {23}, year = {2019}, month = {2019/12/18/}, pages = {88 - 98}, type = {Review}, abstract = {The enzymatic cleavage of the protein backbone (proteolysis) is integral to many biological processes, e.g. for the break-down of proteins in the digestive system. Specific proteolytic cleavages are also used to turn on or off the activity of proteins. For example, the lymphangiogenic vascular endothelial growth factor--C (VEGF--C) is synthesized as a precursor molecule that must be converted to a mature form by the enzymatic removal of C-- and N--terminal propeptides before it can bind and activate its receptors. The constitutive C--terminal cleavage is mediated by proprotein convertases such as furin. The subsequent ac-tivating cleavage can be mediated by at least four different proteases: by plasmin, ADAMTS3, prostate--specific antigen (PSA) and cathepsin D. Processing by different proteases results in distinct forms of "ma-ture" VEGF--C, that differ in their affinity and their receptor activation potential. This processing is tightly regulated by the CCBE1 protein. CCBE1 regulates the acti-vating cleavage of VEGF-C by ADAMTS3 and PSA, but not by plasmin. During embryonic development of the lymphatic system, VEGF--C is activated primarily by the ADAMTS3 protease. In contrast, it is believed that plasmin is responsible for wound healing lymphangiogenesis and PSA for tumor--associated pathological lym-phangiogenesis. Cathepsin D has also been implicated in tumor lymphangiogenesis. In addition, cathepsin D in saliva might activate latent VEGF-C upon wound licking, thereby accelerating wound healing. The molecular details of proteolytic activation of VEGF--C are only recently extensively explored, and we likely do not know yet all activating proteases. It appears that the activity of VEGF--C is regulated for different specific functions by different proteinases. Although VEGF--C clearly plays a pivotal role for tumor progression and metastasis in experimental animal studies, the rele-vance of most correlative studies on the role of VEGF--C in human cancers is quite limited until now, also due to the lack of methods to differentiate between inactive and active forms.}, keywords = {Lymphangiogenesis, proteinases, proteolysis, VEGF-C}, doi = {10.5281/zenodo.3629263}, url = {https://doi.org/10.5281/zenodo.3629263}, author = {Lackner, Marcel and Schmotz, Constanze and Jeltsch, Michael} } @article {588, title = {Factors regulating the substrate specificity of cytosolic phospholipase A2-alpha in vitro}, journal = {Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids}, volume = {1861}, year = {2016}, month = {2016/07/01/}, pages = {1597}, keywords = {Arachidonic acid, Bilayer, Catalytic site, Mass spectrometry, Micelle, Phospholipase A}, author = {Batchu, Krishna Chaithanya and H{\"a}nninen, Satu and Jha, Sawan Kumar and Jeltsch, Michael and Somerharju, Pentti} } @article {536, title = {Functional Importance of a Proteoglycan Co-Receptor in Pathologic Lymphangiogenesis}, journal = {Circulation Research}, volume = {119}, year = {2016}, month = {05/2016}, pages = {210-221}, abstract = {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.}, keywords = {endothelial cell growth, glycosaminoglycan, lymphatic capillary, Proteoglycan, vascular endothelial growth factor receptor}, doi = {10.1161/CIRCRESAHA.116.308504}, url = {http://circres.ahajournals.org/content/early/2016/05/25/CIRCRESAHA.116.308504}, author = {Johns, Scott C. and Yin, Xin and Jeltsch, Michael and Bishop, Joseph R. and Schuksz, Manuela and Ghazal, Roland El and Wilcox-Adelman, Sarah A. and Alitalo, Kari and Fuster, Mark M.} } @article {527, title = {Lymphatic Vessels in Regenerative Medicine and Tissue Engineering}, journal = {Tissue Engineering Part B}, volume = {22}, year = {2016}, month = {2016}, pages = {1-13}, type = {Review}, abstract = {Once a DOI is available for this article, the final publication will be available from Mary Ann Liebert, Inc., publishers at http://dx.doi.org/10.1089/TEN.TEB.2016.0034. The postprint manuscript is available from here and for the next 30 days also from the publisher via this bit.ly shortcut: http://bit.ly/1VKjjMk. }, doi = {10.1089/ten.TEB.2016.0034}, url = {http://online.liebertpub.com/doi/10.1089/ten.TEB.2016.0034}, author = {Schaupper, Mira V. and Jeltsch, Michael and Rohringer, Sabrina and Redl, Heinz and Holnthoner, Wolfgang} } @article {502, title = {Functional Dissection of the CCBE1 Protein: A Crucial Requirement for the Collagen Repeat Domain.}, journal = {Circ Res}, volume = {116}, year = {2015}, month = {2015 May 8}, pages = {1660-1669}, abstract = {

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.

}, keywords = {Animals, Binding Sites, Calcium-Binding Proteins, Collagen, Craniofacial Abnormalities, Endothelial Cells, Epidermal Growth Factor, Gene Expression Regulation, Developmental, Gene Knock-In Techniques, Genital Diseases, Male, Genotype, Gestational Age, HEK293 Cells, Humans, Lymphangiectasis, Intestinal, Lymphatic Vessels, lymphedema, Mice, Mice, Transgenic, Mutation, Phenotype, Protein Binding, Protein Interaction Domains and Motifs, Signal Transduction, Transfection, Tumor Suppressor Proteins, Vascular Endothelial Growth Factor C, Zebrafish, Zebrafish Proteins}, issn = {1524-4571}, doi = {10.1161/CIRCRESAHA.116.304949}, url = {http://circres.ahajournals.org/content/116/10/1660.long}, author = {Roukens, M Guy and Peterson-Maduro, Josi and Padberg, Yvonne and Jeltsch, Michael and Lepp{\"a}nen, Veli-Matti and Bos, Frank L and Alitalo, Kari and Schulte-Merker, Stefan and Schulte, D{\"o}rte} } @inbook {500, title = {The genetic causes of primary lymphedema.}, booktitle = {Erkrankungen des Lymphgef{\"a}{\ss}systems}, year = {2015}, pages = {210-229}, publisher = {Viavital Verlag}, organization = {Viavital Verlag}, edition = {6}, chapter = {5}, address = {Cologne}, abstract = {English: Primary lymphedema can be treated, but not cured. In addition, their diagnosis is due to heterogeneous phenotypes often ambiguous. However, these problems can be tackled by identifying the edema-causing genetic lesions to yield unambiguous diagnoses and by developing specific treatments that address the underlying, molecular cause. New developments in molecular biology are providing the necessary tools for these tasks and in the recent years the genetic causes of many forms of primary lymphedema have been identified, notably by exome sequencing. For a significant proportion of lymphatic disorders multifactorial genetic causes are suspected. This chapter provides an overview of the current knowledge on the genetic origin, the categorization as well as the molecular and biochemical causes of primary lymphedema. German: Prim{\"a}re Lymph{\"o}deme sind behandelbar, aber nicht heilbar. Zudem ist die Diagnostik aufgrund heterogener Ph{\"a}notypen oft nicht eindeutig. Um diese Probleme anzugehen, m{\"u}ssen die das {\"O}dem verursachenden genetischen Ursachen gefunden, diagnostiziert und gezielt behandelt werden. Die hierzu notwendigen Techniken liefern die neuen Entwicklungen in der Molekularbiologie. Insbesondere durch die Technik der Exom-Sequenzierung wurden in den letzten Jahren die genetischen Ursachen vieler prim{\"a}rer Lymph{\"o}deme identifiziert. F{\"u}r einen weiteren gro{\ss}en Anteil dieser Erkrankungen werden multifaktorielle genetische Dispositionen vermutet. Dieses Kapitel gibt einen {\"U}berblick {\"u}ber den derzeitigen Kenntnisstand der genetischen Ursachen, der Kategorisierung sowie der molekularbiologischen und biochemischen Grundlagen prim{\"a}rer Lymph{\"o}deme.}, isbn = {978-3-934371-53-8}, author = {Mattonet, Kenny and Wilting, J{\"o}rg and Jeltsch, Michael}, editor = {Weissleder, Horst and Schuchhardt, Christian} } @article {505, title = {Ischemia-Reperfusion Injury Enhances Lymphatic Endothelial VEGFR3 and Rejection in Cardiac Allografts}, journal = {American Journal of Transplantation}, volume = {16}, year = {2015}, month = {12/2015}, pages = {1160-1172}, abstract = {Organ damage and innate immunity during heart transplantation may evoke adaptive immunity with serious consequences. Because lymphatic vessels bridge innate and adaptive immunity, they are critical in immune surveillance; however, their role in ischemia{\textendash}reperfusion injury (IRI) in allotransplantation remains unknown. We investigated whether the lymphangiogenic VEGF-C/VEGFR3 pathway during cardiac allograft IRI regulates organ damage and subsequent interplay between innate and adaptive immunity. We found that cardiac allograft IRI, within hours, increased graft VEGF-C expression and lymphatic vessel activation in the form of increased lymphatic VEGFR3 and adhesion protein expression. Pharmacological VEGF-C/VEGFR3 stimulation resulted in early lymphatic activation and later increase in allograft inflammation. In contrast, pharmacological VEGF-C/VEGFR3 inhibition during cardiac allograft IRI decreased early lymphatic vessel activation with subsequent dampening of acute and chronic rejection. Genetic deletion of VEGFR3 specifically in the lymphatics of the transplanted heart recapitulated the survival effect achieved by pharmacological VEGF-C/VEGFR3 inhibition. Our results suggest that tissue damage rapidly changes lymphatic vessel phenotype, which, in turn, may shape the interplay of innate and adaptive immunity. Importantly, VEGF-C/VEGFR3 inhibition during solid organ transplant IRI could be used as lymphatic-targeted immunomodulatory therapy to prevent acute and chronic rejection.}, doi = {10.1111/ajt.13564}, url = {http://onlinelibrary.wiley.com/doi/10.1111/ajt.13564/abstract}, author = {Dashkevich, A. and Raissadati, A. and Syrj{\"a}l{\"a}, S. O. and Zarkada, G. and Ker{\"a}nen, M. A. I. and Tuuminen, R. and Krebs, R. and Anisimov, A. and Jeltsch, M. and Lepp{\"a}nen, V.-M. and Alitalo, K. and Nyk{\"a}nen, A. I. and Lemstr{\"o}m, K. B.} } @article {481, title = {Substrate efflux propensity is the key determinant of iPLA-β-mediated glycerophospholipid hydrolysised}, journal = {Journal of Biological Chemistry}, year = {2015}, month = {2015/02/23}, abstract = {A-type phospholipases (PLAs) are key players in glycerophospholipid (GPL)homeostasis and in mammalian cells, Ca2+-independent PLA-beta (iPLAβ) in particular has been implicated in this essential process.However, the regulation of this enzyme,which is necessary to avoid futile competition between synthesis and degradation, is not understood. Recently, we provided evidence that the efflux of the substrate molecules from the bilayer is the rate-limiting step in the hydrolysis of GPLs by some secretory nonhomeostatic) PLAs. To study if this is the case with iPLAβ as well a mass-spectrometric assay was employed to determine the rate of hydrolysis of multiple saturated and unsaturated GPL species in parallel using micelles or vesicle bilayers as the macrosubstrate. With micelles, the hydrolysis decreased with increasing acyl chain length independent of unsaturation and modest discrimination between acyl positional isomers was observed, presumably due to the differences in the structure of the sn1 and sn2 acyl binding sites of the protein. In striking contrast, no significant discrimination between positional isomers was observed with bilayers, and the rate of hydrolysis decreased with the acyl chain length logarithmically and far more than with micelles. These data provide compelling evidence that efflux of the substrate molecule from the bilayer, which also decreases monotonously with acyl chain length, is the rate-determining step in iPLAβ- mediated hydrolysis of GPLs in membranes. This finding is intriguing as it may help to understand how homeostatic PLAs are regulated and how degradation and biosynthesis are coordinated.}, doi = {10.1074/jbc.M115.642835}, url = {http://www.jbc.org/content/early/2015/02/23/jbc.M115.642835.abstract}, author = {Batchu, Krishna Chaithanya and Hokynar, Kati and Jeltsch, Michael and Mattonet, Kenny and Somerharju, Pentti} } @inbook {503, title = {The TIE Receptor Family}, booktitle = {Receptor Tyrosine Kinases: Family and Subfamilies}, year = {2015}, pages = {743-775}, publisher = {Springer International Publishing}, organization = {Springer International Publishing}, chapter = {16}, keywords = {ANG, Angiopoietin, ANGPT, Endothelial cell, Lymphatic vessel, Neovascularization, TIE1, TIE2, Vascular dysfunction}, issn = {978-3-319-11888-8}, doi = {10.1007/978-3-319-11888-8_16}, url = {https://link.springer.com/content/pdf/10.1007\%2F978-3-319-11888-8_16.pdf}, author = {Saharinen, Pipsa and Jeltsch, Michael and Santoyo, MayteM. and Lepp{\"a}nen, Veli-Matti and Alitalo, Kari}, editor = {Wheeler, Deric L. and Yarden, Yosef} } @article {445, title = {Receptor Tyrosine Kinase-Mediated Angiogenesis}, journal = {Cold Spring Harbor Perspectives in Biology}, volume = {5}, year = {2013}, month = {2013}, abstract = {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.}, isbn = {, 1943-0264}, doi = {10.1101/cshperspect.a009183}, url = {http://cshperspectives.cshlp.org/content/5/9/a009183}, author = {Jeltsch, Michael and Lepp{\"a}nen, Veli-Matti and Saharinen, Pipsa and Alitalo, Kari} } @article {442, title = {Structural and mechanistic insights into VEGF receptor 3 ligand binding and activation}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {110}, year = {2013}, month = {08/2013}, pages = {12960 - 12965}, abstract = {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.}, keywords = {Amino Acid Sequence, Binding Sites, Binding, Competitive, Crystallography, X-Ray, Electrophoresis, Polyacrylamide Gel, Humans, Ligands, Microscopy, Electron, Models, Molecular, Molecular Sequence Data, Multiprotein Complexes, Mutation, Protein Binding, Protein Multimerization, Protein Structure, Tertiary, Scattering, Small Angle, Sequence Homology, Amino Acid, Thermodynamics, Vascular Endothelial Growth Factor C, Vascular Endothelial Growth Factor Receptor-3, X-Ray Diffraction}, isbn = {1091-6490}, doi = {10.1073/pnas.1301415110}, url = {http://www.pnas.org/content/110/32/12960.long}, author = {Lepp{\"a}nen, Veli-Matti and Tvorogov, Denis and Kisko, Kaisa and Prota, Andrea E and Jeltsch, Michael and Anisimov, Andrey and Markovic-Mueller, Sandra and Stuttfeld, Edward and Goldie, Kenneth N and Ballmer-Hofer, Kurt and Alitalo, Kari} } @article {424, title = {Vascular endothelial growth factor-angiopoietin chimera with improved properties for therapeutic angiogenesis}, journal = {Circulation}, volume = {127}, year = {2013}, month = {01/2013}, pages = {424-434}, chapter = {424}, abstract = {BACKGROUND: There is an unmet need for proangiogenic therapeutic molecules for the treatment of tissue ischemia in cardiovascular diseases. However, major inducers of angiogenesis such as vascular endothelial growth factor (VEGF/VEGF-A) have side effects that limit their therapeutic utility in vivo, especially at high concentrations. Angiopoietin-1 has been considered to be a blood vessel stabilization factor that can inhibit the intrinsic property of VEGF to promote vessel leakiness. In this study, we have designed and tested the angiogenic properties of chimeric molecules consisting of receptor-binding parts of VEGF and angiopoietin-1. We aimed at combining the activities of both factors into 1 molecule for easy delivery and expression in target tissues. METHODS AND RESULTS: The VEGF-angiopoietin-1 (VA1) chimeric protein bound to both VEGF receptor-2 and Tie2 and induced the activation of both receptors. Detailed analysis of VA1 versus VEGF revealed differences in the kinetics of VEGF receptor-2 activation and endocytosis, downstream kinase activation, and VE-cadherin internalization. The delivery of a VA1 transgene into mouse skeletal muscle led to increased blood flow and enhanced angiogenesis. VA1 was also very efficient in rescuing ischemic limb perfusion. However, VA1 induced less plasma protein leakage and myeloid inflammatory cell recruitment than VEGF. Furthermore, angioma-like structures associated with VEGF expression were not observed with VA1. CONCLUSIONS: The VEGF-angiopoietin-1 chimera is a potent angiogenic factor that triggers a novel mode of VEGF receptor-2 activation, promoting less vessel leakiness, less tissue inflammation, and better perfusion in ischemic muscle than VEGF. These properties of VA1 make it an attractive therapeutic tool.}, author = {Andrey Anisimov and Denis Tvorogov and Annamari Alitalo and Veli-Matti Lepp{\"a}nen and Y An and EC Han and F Orsenigo and EI Ga{\'a}l and Tanja Holopainen and YJ Koh and Tuomas Tammela and P Korpisalo and Salla Keskitalo and Michael Jeltsch and Seppo Yl{\"a}-Herttuala and Elisabetta Dejana and GY Koh and C Choi and Pipsa Saharinen and Kari Alitalo} } @article {39, title = {Claudin-like protein 24 interacts with the VEGFR-2 and VEGFR-3 pathways and regulates lymphatic vessel development}, journal = {Genes Dev}, volume = {24}, year = {2010}, month = {2010/May/}, pages = {875 - 80}, abstract = {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.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/20439428}, author = {Saharinen, Pipsa and Helotera, Hanna and Miettinen, Juho and Norrmen, Camilla and D{\textquoteright}Amico, Gabriela and Jeltsch, Michael and Langenberg, Tobias and Vandevelde, Wouter and Ny, Annelii and Dewerchin, Mieke and Carmeliet, Peter and Alitalo, Kari} } @article {40, title = {Structural determinants of growth factor binding and specificity by VEGF receptor 2}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {107}, year = {2010}, month = {02/2010}, pages = {2425 - 30}, abstract = {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.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/20145116}, author = {Lepp{\"a}nen, Veli-Matti and Prota, Andrea E and Jeltsch, Michael and Anisimov, Andrey and Kalkkinen, Nisse and Strandin, Tomas and Lankinen, Hilkka and Goldman, Adrian and Ballmer-Hofer, Kurt and Alitalo, Kari} } @article {41, title = {Suppressive effects of vascular endothelial growth factor-B on tumor growth in a mouse model of pancreatic neuroendocrine tumorigenesis}, journal = {PLoS ONE}, volume = {5}, year = {2010}, month = {2010//}, pages = {e14109}, abstract = {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.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/21124841}, author = {Albrecht, Imke and Kopfstein, Lucie and Strittmatter, Karin and Schomber, Tibor and Falkevall, Annelie and Hagberg, Carolina E and Lorentz, Pascal and Jeltsch, Michael and Alitalo, Kari and Eriksson, Ulf and Christofori, Gerhard and Pietras, Kristian} } @article {43, title = {Vascular endothelial growth factor-B acts as a coronary growth factor in transgenic rats without inducing angiogenesis, vascular leak, or inflammation}, journal = {Circulation}, volume = {122}, year = {2010}, month = {2010/Oct/}, pages = {1725 - 33}, abstract = {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.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/20937974}, author = {Bry, Maija and Kivel{\"a}, Riikka and Holopainen, Tanja and Anisimov, Andrey and Tammela, Tuomas and Soronen, Jarkko and Silvola, Johanna and Saraste, Antti and Jeltsch, Michael and Korpisalo, Petra and Carmeliet, Peter and Lemstr{\"o}m, Karl B and Shibuya, Masabumi and Yl{\"a}-Herttuala, Seppo and Alhonen, Leena and Mervaala, Eero and Andersson, Leif C and Knuuti, Juhani and Alitalo, Kari} } @article {38, title = {Activated forms of VEGF-C and VEGF-D provide improved vascular function in skeletal muscle}, journal = {Circ Res}, volume = {104}, year = {2009}, month = {2009/Jun/}, pages = {1302 - 12}, abstract = {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.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/19443835}, author = {Anisimov, Andrey and Alitalo, Annamari and Korpisalo, Petra and Soronen, Jarkko and Kaijalainen, Seppo and Lepp{\"a}nen, Veli-Matti and Jeltsch, Michael and Yl{\"a}-Herttuala, Seppo and Alitalo, Kari} } @article {37, title = {Overexpression of vascular endothelial growth factor-B in mouse heart alters cardiac lipid metabolism and induces myocardial hypertrophy}, journal = {Circ Res}, volume = {103}, year = {2008}, month = {2008/Oct/}, pages = {1018 - 26}, abstract = {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.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/18757827}, author = {Karpanen, Terhi and Bry, Maija and Ollila, Hanna M and Sepp{\"a}nen-Laakso, Tuulikki and Liimatta, Erkki and Leskinen, Hanna and Kivel{\"a}, Riikka and Helkamaa, Teemu and Merentie, Mari and Jeltsch, Michael and Paavonen, Karri and Andersson, Leif C and Mervaala, Eero and Hassinen, Ilmo E and Yl{\"a}-Herttuala, Seppo and Oresic, Matej and Alitalo, Kari} } @article {36, title = {Reevaluation of the role of VEGF-B suggests a restricted role in the revascularization of the ischemic myocardium}, journal = {Arterioscler Thromb Vasc Biol}, volume = {28}, year = {2008}, month = {2008/Sep/}, pages = {1614 - 20}, abstract = {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.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/18511699}, author = {Li, Xuri and Tjwa, Marc and Van Hove, Inge and Enholm, Berndt and Neven, Elke and Paavonen, Karri and Jeltsch, Michael and Juan, Toni Diez and Sievers, Richard E and Chorianopoulos, Emmanuel and Wada, Hiromichi and Vanwildemeersch, Maarten and Noel, Agnes and Foidart, Jean-Michel and Springer, Matthew L and von Degenfeld, Georges and Dewerchin, Mieke and Blau, Helen M and Alitalo, Kari and Eriksson, Ulf and Carmeliet, Peter and Moons, Lieve} } @article {32, title = {Vascular endothelial growth factor (VEGF)/VEGF-C mosaic molecules reveal specificity determinants and feature novel receptor binding patterns}, journal = {J Biol Chem}, volume = {281}, year = {2006}, month = {2006/Apr/}, pages = {12187 - 95}, abstract = {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.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/16505489}, author = {Jeltsch, Michael and Karpanen, Terhi and Strandin, Tomas and Aho, Kukka and Lankinen, Hilkka and Alitalo, Kari} } @article {29, title = {Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation}, journal = {J Clin Invest}, volume = {115}, year = {2005}, month = {2005/Feb/}, pages = {247 - 57}, abstract = {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.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/15668734}, author = {Baluk, Peter and Tammela, Tuomas and Ator, Erin and Lyubynska, Natalya and Achen, Marc G and Hicklin, Daniel J and Jeltsch, Michael and Petrova, Tatiana V and Pytowski, Bronislaw and Stacker, Steven A and Yl{\"a}-Herttuala, Seppo and Jackson, David G and Alitalo, Kari and McDonald, Donald M} } @article {28, title = {Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins}, journal = {Nat Immunol}, volume = {5}, year = {2004}, month = {2004/Jan/}, pages = {74 - 80}, abstract = {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.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/14634646}, author = {Karkkainen, Marika J and Haiko, Paula and Sainio, Kirsi and Partanen, Juha and Taipale, Jussi and Petrova, Tatiana V and Jeltsch, Michael and Jackson, David G and Talikka, Marja and Rauvala, Heikki and Betsholtz, Christer and Alitalo, Kari} } @article {27, title = {Intrinsic versus microenvironmental regulation of lymphatic endothelial cell phenotype and function}, journal = {FASEB J}, volume = {17}, year = {2003}, month = {2003/Nov/}, pages = {2006 - 13}, abstract = {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.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/14597670}, author = {Veikkola, Tanja and Lohela, Marja and Ikenberg, Kristian and M{\"a}kinen, Taija and Korff, Thomas and Saaristo, Anne and Petrova, Tatania and Jeltsch, Michael and Augustin, Hellmut G and Alitalo, Kari} } @article {26, title = {VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia}, journal = {J Cell Biol}, volume = {161}, year = {2003}, month = {2003/Jun/}, pages = {1163 - 77}, abstract = {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.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/12810700}, author = {Gerhardt, Holger and Golding, Matthew and Fruttiger, Marcus and Ruhrberg, Christiana and Lundkvist, Andrea and Abramsson, Alexandra and Jeltsch, Michael and Mitchell, Christopher and Alitalo, Kari and Shima, David and Betsholtz, Christer} } @article {24, title = {Adenoviral VEGF-C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes}, journal = {FASEB J}, volume = {16}, year = {2002}, month = {2002/Jul/}, pages = {1041 - 9}, abstract = {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.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/12087065}, author = {Saaristo, Anne and Veikkola, Tanja and Enholm, Berndt and Hyt{\"o}nen, Maija and Arola, Johanna and Pajusola, Katri and Turunen, Pa{\"\i}vi and Jeltsch, Michael and Karkkainen, Marika J and Kerjaschki, Dontscho and Bueler, Hansruedi and Yl{\"a}-Herttuala, Seppo and Alitalo, Kari} } @article {21, title = {Adenoviral expression of vascular endothelial growth factor-C induces lymphangiogenesis in the skin}, journal = {Circ Res}, volume = {88}, year = {2001}, month = {2001/Mar/}, pages = {623 - 9}, abstract = {The growth of blood and lymphatic vasculature is mediated in part by secreted polypeptides of the vascular endothelial growth factor (VEGF) family. The prototype VEGF binds VEGF receptor (VEGFR)-1 and VEGFR-2 and is angiogenic, whereas VEGF-C, which binds to VEGFR-2 and VEGFR-3, is either angiogenic or lymphangiogenic in different assays. We used an adenoviral gene transfer approach to compare the effects of these growth factors in adult mice. Recombinant adenoviruses encoding human VEGF-C or VEGF were injected subcutaneously into C57Bl6 mice or into the ears of nude mice. Immunohistochemical analysis showed that VEGF-C upregulated VEGFR-2 and VEGFR-3 expression and VEGF upregulated VEGFR-2 expression at 4 days after injection. After 2 weeks, histochemical and immunohistochemical analysis, including staining for the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), the vascular endothelial marker platelet-endothelial cell adhesion molecule-1 (PECAM-1), and the proliferating cell nuclear antigen (PCNA) revealed that VEGF-C induced mainly lymphangiogenesis in contrast to VEGF, which induced only angiogenesis. These results have significant implications in the planning of gene therapy using these growth factors.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/11282897}, author = {Enholm, B and Karpanen, T and Jeltsch, M and Kubo, H and Stenback, F and Prevo, R and Jackson, D G and Yla-Herttuala, S and Alitalo, K} } @article {22, title = {Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice}, journal = {EMBO J}, volume = {20}, year = {2001}, month = {2001/Mar/}, pages = {1223 - 31}, abstract = {Vascular endothelial growth factor receptor-3 (VEGFR-3) has an essential role in the development of embryonic blood vessels; however, after midgestation its expression becomes restricted mainly to the developing lymphatic vessels. The VEGFR-3 ligand VEGF-C stimulates lymphangiogenesis in transgenic mice and in chick chorioallantoic membrane. As VEGF-C also binds VEGFR-2, which is expressed in lymphatic endothelia, it is not clear which receptors are responsible for the lymphangiogenic effects of VEGF-C. VEGF-D, which binds to the same receptors, has been reported to induce angiogenesis, but its lymphangiogenic potential is not known. In order to define the lymphangiogenic signalling pathway we have created transgenic mice overexpressing a VEGFR-3-specific mutant of VEGF-C (VEGF-C156S) or VEGF-D in epidermal keratinocytes under the keratin 14 promoter. Both transgenes induced the growth of lymphatic vessels in the skin, whereas the blood vessel architecture was not affected. Evidence was also obtained that these growth factors act in a paracrine manner in vivo. These results demonstrate that stimulation of the VEGFR-3 signal transduction pathway is sufficient to induce specifically lymphangiogenesis in vivo.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/11250889}, author = {Veikkola, T and Jussila, L and Makinen, T and Karpanen, T and Jeltsch, M and Petrova, T V and Kubo, H and Thurston, G and McDonald, D M and Achen, M G and Stacker, S A and Alitalo, K} } @conference {653, title = {Signalling via VEGFR-3 is sufficient for lymphangiogenesis in transgenic mice.}, booktitle = {Molecular Targets and Cancer Therapeutics: Discovery, Biology, and Clinical Applications}, year = {2001}, month = {2001/10/29/}, address = {Miami Beach, Florida}, author = {Jussila, L. and Veikkola, T. and Jeltsch, M. and Thurston, G. and McDonald, D. and Achen, M. and Stacker, S. and Alitalo, K.} } @article {17, title = {Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells}, journal = {Proc Natl Acad Sci U S A}, volume = {95}, year = {1998}, month = {1998/Sep/}, pages = {11709 - 14}, abstract = {The vascular endothelial growth factor (VEGF) family has recently expanded by the identification and cloning of three additional members, namely VEGF-B, VEGF-C, and VEGF-D. In this study we demonstrate that VEGF-B binds selectively to VEGF receptor-1/Flt-1. This binding can be blocked by excess VEGF, indicating that the interaction sites on the receptor are at least partially overlapping. Mutating the putative VEGF receptor-1/Flt-1 binding determinants Asp63, Asp64, and Glu67 to alanine residues in VEGF-B reduced the affinity to VEGF receptor-1 but did not abolish binding. Mutational analysis of conserved cysteines contributing to VEGF-B dimer formation suggest a structural conservation with VEGF and platelet-derived growth factor. Proteolytic processing of the 60-kDa VEGF-B186 dimer results in a 34-kDa dimer containing the receptor-binding epitopes. The binding of VEGF-B to its receptor on endothelial cells leads to increased expression and activity of urokinase type plasminogen activator and plasminogen activator inhibitor 1, suggesting a role for VEGF-B in the regulation of extracellular matrix degradation, cell adhesion, and migration.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/9751730}, author = {Olofsson, B and Korpelainen, E and Pepper, M S and Mandriota, S J and Aase, K and Kumar, V and Gunji, Y and Jeltsch, M M and Shibuya, M and Alitalo, K and Eriksson, U} } @article {16, title = {Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4)}, journal = {Proc Natl Acad Sci U S A}, volume = {95}, year = {1998}, month = {1998/Jan/}, pages = {548 - 53}, abstract = {We have identified a member of the VEGF family by computer-based homology searching and have designated it VEGF-D. VEGF-D is most closely related to VEGF-C by virtue of the presence of N- and C-terminal extensions that are not found in other VEGF family members. In adult human tissues, VEGF-D mRNA is most abundant in heart, lung, skeletal muscle, colon, and small intestine. Analyses of VEGF-D receptor specificity revealed that VEGF-D is a ligand for both VEGF receptors (VEGFRs) VEGFR-2 (Flk1) and VEGFR-3 (Flt4) and can activate these receptors. However. VEGF-D does not bind to VEGFR-1. Expression of a truncated derivative of VEGF-D demonstrated that the receptor-binding capacities reside in the portion of the molecule that is most closely related in primary structure to other VEGF family members and that corresponds to the mature form of VEGF-C. In addition, VEGF-D is a mitogen for endothelial cells. The structural and functional similarities between VEGF-D and VEGF-C define a subfamily of the VEGFs.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/9435229}, author = {Achen, M G and Jeltsch, M and Kukk, E and M{\"a}kinen, T and Vitali, A and Wilks, A F and Alitalo, K and Stacker, S A} } @article {10, title = {Hyperplasia of lymphatic vessels in VEGF-C transgenic mice}, journal = {Science (80- )}, volume = {276}, year = {1997}, month = {1997/May/}, pages = {1423 - 5}, abstract = {No growth factors specific for the lymphatic vascular system have yet been described. Vascular endothelial growth factor (VEGF) regulates vascular permeability and angiogenesis, but does not promote lymphangiogenesis. Overexpression of VEGF-C, a ligand of the VEGF receptors VEGFR-3 and VEGFR-2, in the skin of transgenic mice resulted in lymphatic, but not vascular, endothelial proliferation and vessel enlargement. Thus, VEGF-C induces selective hyperplasia of the lymphatic vasculature, which is involved in the draining of interstitial fluid and in immune function, inflammation, and tumor metastasis. VEGF-C may play a role in disorders involving the lymphatic system and may be of potential use in therapeutic lymphangiogenesis.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/9162011}, author = {Jeltsch, M and Kaipainen, A and Joukov, V and Meng, X and Lakso, M and Rauvala, H and Swartz, M and Fukumura, D and Jain, R K and Alitalo, K} } @article {14, title = {Proteolytic processing regulates receptor specificity and activity of VEGF-C}, journal = {EMBO J}, volume = {16}, year = {1997}, month = {1997/Jul/}, pages = {3898 - 911}, abstract = {The recently identified vascular endothelial growth factor C (VEGF-C) belongs to the platelet-derived growth factor (PDGF)/VEGF family of growth factors and is a ligand for the endothelial-specific receptor tyrosine kinases VEGFR-3 and VEGFR-2. The VEGF homology domain spans only about one-third of the cysteine-rich VEGF-C precursor. Here we have analysed the role of post-translational processing in VEGF-C secretion and function, as well as the structure of the mature VEGF-C. The stepwise proteolytic processing of VEGF-C generated several VEGF-C forms with increased activity towards VEGFR-3, but only the fully processed VEGF-C could activate VEGFR-2. Recombinant {\textquoteright}mature{\textquoteright} VEGF-C made in yeast bound VEGFR-3 (K[D] = 135 pM) and VEGFR-2 (K[D] = 410 pM) and activated these receptors. Like VEGF, mature VEGF-C increased vascular permeability, as well as the migration and proliferation of endothelial cells. Unlike other members of the PDGF/VEGF family, mature VEGF-C formed mostly non-covalent homodimers. These data implicate proteolytic processing as a regulator of VEGF-C activity, and reveal novel structure-function relationships in the PDGF/VEGF family.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/9233800}, author = {Joukov, V and Sorsa, T and Kumar, V and Jeltsch, M and Claesson-Welsh, L and Cao, Y and Saksela, O and Kalkkinen, N and Alitalo, K} }