%0 Journal Article %J Molecular Genetics & Genomic Medicine %D 2020 %T Investigation on the role of biallelic variants in VEGF‐C found in a patient affected by Milroy‐like lymphedema %B Molecular Genetics & Genomic Medicine %V 00 %P e1389 %8 Feb-06-2022 %G eng %U https://onlinelibrary.wiley.com/doi/abs/10.1002/mgg3.1389 %! Mol Genet Genomic Med %R 10.1002/mgg3.1389 %0 Journal Article %J Duodecim Lääketieteellinen Aikakauskirja %D 2020 %T Lymphatics and the eye. [Finnish] %A Gucciardo, Erika %A Lehti, Timo A. %A Korhonen, Ani %A Salvén, Petri %A Lehti, Kaisa %A Jeltsch, Michael %A Loukovaara, Sirpa %B Duodecim Lääketieteellinen Aikakauskirja %V 136 %P 1777-1788 %8 2020/02/10/ %G eng %U https://www.duodecimlehti.fi/lehti/2020/16/duo15739 %N 16 %9 review %! Duodecim %& 1777 %R 10.5281/zenodo.4005517 %0 Journal Article %J Blood %D 2020 %T VEGF-C Protects the Integrity of Bone Marrow Perivascular Niche %X Key Points. Vegfc deletion in endothelial or LepR+ cells compromises the bone marrow perivascular niche and hematopoietic stem cell maintenance.Exogenous admin %B Blood %P accepted - for publication %8 2020/07/31/ %G eng %U https://ashpublications.org/blood/article/doi/10.1182/blood.2020005699/463465/VEGF-C-Protects-the-Integrity-of-Bone-Marrow %! Blood %R 10.1182/blood.2020005699 %0 Journal Article %J eLife %D 2019 %T KLK3/PSA and cathepsin D activate VEGF-C and VEGF-D %A Jha, Sawan Kumar %A Rauniyar, Khushbu %A Chronowska, Ewa %A Mattonet, Kenny %A Maina, Eunice Wairimu %A Koistinen, Hannu %A Stenman, Ulf-Håkan %A Alitalo, Kari %A Jeltsch, Michael %K cancer biology %K Cathepsin D %K kallikrein-related peptidases %K KLK3/PSA %K Lymphangiogenesis %K mouse %K VEGF-C %K VEGF-D %X Vascular endothelial growth factor-C (VEGF-C) acts primarily on endothelial cells, but also on non-vascular targets, e.g. in the CNS and immune system. Here we describe a novel, unique VEGF-C form in the human reproductive system produced via cleavage by kallikrein-related peptidase 3 (KLK3), aka prostate-specific antigen (PSA). KLK3 activated VEGF-C specifically and efficiently through cleavage at a novel N-terminal site. We detected VEGF-C in seminal plasma, and sperm liquefaction occurred concurrently with VEGF-C activation, which was enhanced by collagen and calcium binding EGF domains 1 (CCBE1). After plasmin and ADAMTS3, KLK3 is the third protease shown to activate VEGF-C. Since differently activated VEGF-Cs are characterized by successively shorter N-terminal helices, we created an even shorter hypothetical form, which showed preferential binding to VEGFR-3. Using mass spectrometric analysis of the isolated VEGF-C-cleaving activity from human saliva, we identified cathepsin D as a protease that can activate VEGF-C as well as VEGF-D. %B eLife %V 8 %P e44478 %8 2019/05/17/ %@ 2050-084X %G eng %U https://elifesciences.org/articles/44478 %! eLife %0 Journal Article %J Lymphologie in Forschung und Praxis %D 2019 %T The Proteolytic Activation of Vascular Endothelial Growth Factor-C %A Lackner, Marcel %A Schmotz, Constanze %A Jeltsch, Michael %K Lymphangiogenesis %K proteinases %K proteolysis %K VEGF-C %X The enzymatic cleavage of the protein backbone (proteolysis) is integral to many biological processes, e.g. for the breakdown of proteins in the digestive system. Specific proteolytic cleavages are also used to turn on or off the activity of proteins. For example, the lymphangiogenic vascular endothelial growth factor-C (VEGF-C) is synthesized as a precursor molecule that must be converted to a mature form by the enzymatic removal of C- and N-terminal propeptides before it can bind and activate its receptors. The constitutive C-terminal cleavage is mediated by proprotein convertases such as furin. The subsequent activating cleavage can be mediated by at least four different proteases: by plasmin, ADAMTS3, prostate-specific antigen (PSA) and cathepsin D. Processing by different proteases results in distinct forms of "mature" VEGF-C, that differ in their affinity and their receptor activation potential. This processing is tightly regulated by the CCBE1 protein. CCBE1 regulates the activating cleavage of VEGFC by ADAMTS3 and PSA, but not by plasmin. During embryonic development of the lymphatic system, VEGF-C is activated primarily by the ADAMTS3 protease. In contrast, it is believed that plasmin is responsible for wound healing lymphangiogenesis and PSA for tumor-associated pathological lymphangiogenesis. Cathepsin D has also been implicated in tumor lymphangiogenesis. In addition, cathepsin D in saliva might activate latent VEGFC upon wound licking, thereby accelerating wound healing. The molecular details of proteolytic activation of VEGF-C are only recently extensively explored, and we likely do not know yet all activating proteases. It appears that the activity of VEGF-C is regulated for different specific functions by different proteinases. Although VEGF-C clearly plays a pivotal role for tumor progression and metastasis in experimental animal studies, the relevance of most correlative studies on the role of VEGF-C in human cancers is quite limited until now, also due to the lack of methods to differentiate between inactive and active forms. %B Lymphologie in Forschung und Praxis %V 23 %P 88 - 98 %8 2019/12/18/ %G eng %U https://doi.org/10.5281/zenodo.3629263 %N 2 %9 Review %! LymphForsch %( Die proteolytische Aktivierung des Vaskulären Endothelzellwachstumsfaktors-C %R 10.5281/zenodo.3629263 %0 Journal Article %J Scientific Reports %D 2017 %T Efficient activation of the lymphangiogenic growth factor VEGF-C requires the C-terminal domain of VEGF-C and the N-terminal domain of CCBE1 %B Scientific Reports %V 7 %P 4916 %8 2017/07/07/ %G eng %U https://www.nature.com/articles/s41598-017-04982-1 %N 1 %R 10.1038/s41598-017-04982-1 %0 Conference Paper %B Lymphologie 2017 %D 2017 %T What you should know about VEGF-C when working with lymphatics [German] %A Jeltsch, Michael %B Lymphologie 2017 %C Bad Soden (Frankfurt), Germany %8 06/2017 %G eng %U https://jeltsch.org/Abstrakt-BadSoden2017 %0 Journal Article %J Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids %D 2016 %T Factors regulating the substrate specificity of cytosolic phospholipase A2-alpha in vitro %B Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids %V 1861 %P 1597 %8 2016/07/01/ %G eng %N 11 %0 Conference Proceedings %B 42nd Congress of the European Society of Lymphology %D 2016 %T From Molecular Genetics and Biology to Effective Treatments of Lymphatic Disorders %X In 1971, Judah Folkman proposed the concept of anti-angiogenic tumor therapy. 12 years later, Harold Dvorak isolated the responsible growth factor VEGF. Nine years later, Napoleone Ferrara reported the generation of neutralizing monoclonal antibodies against VEGF. Another five years later, Phase I trials started with the humanized version of one of the monoclonals: bevacizumab. Since 2004, when it received FDA approval, it has been marketed under the brand name Avastin. The translation of basic biomedical research into tangible benefits for patients appears sometimes agonizingly slow. The public has been promised much by hyped scientific breakthroughs [4]. Scientific journals and scientists have played along in over-hyping scientific breakthroughs in the hope of impact factors and citations in order to secure and justify funding and fame. Not surprisingly, practitioners ask when the discoveries from basic research will finally improve the standard of care for their patients. Lymphatic research is no exception. Practitioners are largely still limited to symptomatic treatment and there seems to be still an invisible, but perceptible divide between those who do the molecular biology research and those who treat patients. The Avastin story is a plea for basic research: it might be complicated and it might take time, but it eventually does pay off. How is the lymphatic research community doing concerning the translation of research results into treatment options? Examples of lymphatic research in or shortly before the clinical trial stage include: - Growth factor enhanced lymph node transplantation to treat secondary lymphedema - Utilizing the Schlemm channel's lymphatic character in glaucoma treatment - Anti-angiogenic tumor treatment with anti-lymphangiogenic agents Treatment of primary lymphedema with VEGF-C has been proposed. However, our understanding of the physiological process of lymph vessel development is far from complete, despite significant recent progress in our understanding of developmental lymphangiogenesis and first attempts at tissue-engineering lymphatic vessels. If the results from high throughput cancer profilings are predictive of lymphatic conditions, then many patients will feature very individual, multifactorial disease profiles. Even more challenging than the identification of such causes will be the development of treatment regimens that rapidly can be tailored to such individual needs. %B 42nd Congress of the European Society of Lymphology %C Mulhouse, France %8 05/2016 %G eng %U http://www.eurolymphology.org/JOURNAL/VOL28-N74-2016/#p=14 %0 Journal Article %J Circulation Research %D 2016 %T Functional Importance of a Proteoglycan Co-Receptor in Pathologic Lymphangiogenesis %X 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. %B Circulation Research %V 119 %P 210-221 %8 05/2016 %G eng %U http://circres.ahajournals.org/content/early/2016/05/25/CIRCRESAHA.116.308504 %N 2 %R 10.1161/CIRCRESAHA.116.308504 %0 Journal Article %J Tissue Engineering Part B %D 2016 %T Lymphatic Vessels in Regenerative Medicine and Tissue Engineering %X 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. %B Tissue Engineering Part B %V 22 %P 1-13 %8 2016 %G eng %U http://online.liebertpub.com/doi/10.1089/ten.TEB.2016.0034 %N 5 %9 Review %R 10.1089/ten.TEB.2016.0034 %0 Journal Article %J Circ Res %D 2015 %T Functional Dissection of the CCBE1 Protein: A Crucial Requirement for the Collagen Repeat Domain. %X
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.
%B Circ Res %V 116 %P 1660-1669 %8 2015 May 8 %G eng %U http://circres.ahajournals.org/content/116/10/1660.long %N 10 %R 10.1161/CIRCRESAHA.116.304949 %0 Book Section %B Erkrankungen des Lymphgefäßsystems %D 2015 %T The genetic causes of primary lymphedema. %X 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: Primäre Lymphödeme sind behandelbar, aber nicht heilbar. Zudem ist die Diagnostik aufgrund heterogener Phänotypen oft nicht eindeutig. Um diese Probleme anzugehen, müssen die das Ödem 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 primärer Lymphödeme identifiziert. Für einen weiteren großen Anteil dieser Erkrankungen werden multifaktorielle genetische Dispositionen vermutet. Dieses Kapitel gibt einen Überblick über den derzeitigen Kenntnisstand der genetischen Ursachen, der Kategorisierung sowie der molekularbiologischen und biochemischen Grundlagen primärer Lymphödeme. %B Erkrankungen des Lymphgefäßsystems %7 6 %I Viavital Verlag %C Cologne %P 210-229 %@ 978-3-934371-53-8 %G eng %( Die genetischen Ursachen des primären Lymphödems. %& 5 %0 Journal Article %J Lymphologie in Forschung und Praxis %D 2015 %T Heterogeneity of the origin of the lymphatic system. [German]. %X The question “How does the lymphatic system develop?” may be a simple one, but it is fundamental to our understanding of lymphatic malformations in children and the regeneration of lymphatics in adults. The question is by no means new and was already explored in the early 20 century. This resulted in a long-lasting controversy, which until recently had been far from being settled. The interest in the lymphatic system has greatly increased in recent years due to its implications in a variety of diseases. Several studies published this year address the heterogeneity of lymphatic endothelial cell development and unite previous controversially discussed data in a coherent model. These remarkable results, as well as the studies that paved their way, are discussed in this review. %B Lymphologie in Forschung und Praxis %V 19 %P 84-88 %8 12/2015 %G eng %U http://www.dglymph.de/fileadmin/global/pdfs/LymphForsch_2-15.pdf %N 2 %9 Review %0 Conference Proceedings %B 41st European Societry of Lymphology (ESL) Congress %D 2015 %T Lymphangiogenesis in Health and Disease %X Despite the intensive research on the lymphangiogenic VEGF-C/VEGFR-3 signaling pathway in the last two decades, new and unexpected findings do not cease to be made. Diseases that involve the lymphatic system have helped to uncover mechanisms of its normal functioning and development. A recent example of new basic knowledge that resulted from the investigation of a human disease is Hennekam lymphangiectasia lymphedema syndrome (OMIM 235510). It is an autosomal recessive condition, which can co-segregate with mutations in the collagen- and calcium-binding EGF domains 1 (CCBEJ) or the protocadherin Fat 4 (FAT4) gene. Both CCBEI and the lymphangiogenic vascular endothelial growth factor C (VEGF-C) are necessary for the early lymphatic development, namely for the budding and migration of endothelial cells from the cardinal vein (CV) and for the formation of the early lymphatic structures. These processes fail in embryos deficient of either Ccbel or Vegfc. In Vegfc-deficient embryos pro-spective lymphatic endothelial cells fail to sprout from the CV, whereas in Ccbel-deficient embryos, the sprouting is abnormal and does not result in the formation of discrete lymphatic structures. The similar phenotypes of Ccbe- and Vegfc-deficient embryos result from the interaction of CCBEI with the VEGF-C growth factor signaling pathway, which is critical in embryonic and adult lymphangiogenesis. VEGF-C is synthesized as an inactive proprotein and needs to be processed by at least two distinct proteases to become fully active. The presence of CCBEI promotes VEGF-C by two independent mechanisms. The C-terminal domain of CCBEI boosts VEGF-C function via increased ADAMTS3-mediated proteolytic activation of VEGF-C, while the N-terminal domain of CCBEI concentrates pro-VEGF-C on endothelial cell-surfaces, where it can be activated in situ by cell-surface associated proteases. Both mechanisms lead to increased VEGFR-3 signaling and increased lymphangiogenesis. These results show that CCBEI is integral to lymphangiogenesis by increasing the levels of active VEGF-C at the endothelial cell surface. Because some forms of lymphedema appear to be treatable by increasing the amount of VEGFR-3 signaling, the first clinical trials designed around a pro-lymphangiogenic concept use VEGF-C. The goal in these trials is to enhance the integration of lymph nodes into the lymphatic vasculature after autologous transfer to treat postmastectomy lymphedema. On the other hand, VEGF-C induced lymphangiogenesis enhances tumor metastasis and VEGF-C-induced tumor angiogenesis in several mouse models. Blocking VEGF-C might be for these reasons an attractive adjuvant treatment to supplement current cancer treatment regimens that include anti angiogenic drugs. The right balance between pro- and anti-Iymphangiogenic stimuli might therefore differ between individuals and CCBEI is an attractive drug target to adjust pro- and anti-Iymphangiogenic stimuli by tuning the rate of VEGF-C activation. %B 41st European Societry of Lymphology (ESL) Congress %I European Group of Lymphology %C Lausanne, Switzerland %8 06/2015 %G eng %U http://www.eurolymphology.org/JOURNAL/VOL26-N72-2015/#p=10 %0 Journal Article %J Journal of Biological Chemistry %D 2015 %T Substrate efflux propensity is the key determinant of iPLA-β-mediated glycerophospholipid hydrolysised %X 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. %B Journal of Biological Chemistry %8 2015/02/23 %G eng %U http://www.jbc.org/content/early/2015/02/23/jbc.M115.642835.abstract %R 10.1074/jbc.M115.642835 %0 Book Section %B Receptor Tyrosine Kinases: Family and Subfamilies %D 2015 %T The TIE Receptor Family %B Receptor Tyrosine Kinases: Family and Subfamilies %I Springer International Publishing %P 743-775 %G eng %U https://link.springer.com/content/pdf/10.1007%2F978-3-319-11888-8_16.pdf %& 16 %R 10.1007/978-3-319-11888-8_16 %0 Journal Article %J Circulation %D 2014 %T CCBE1 enhances lymphangiogenesis via ADAMTS3-mediated VEGF-C activation %X Background—Hennekam lymphangiectasia-lymphedema syndrome (OMIM 235510) is a rare autosomal recessive disease, which is associated with mutations in the collagen- and calcium-binding EGF domains 1 (CCBE1) gene. Because of the striking phenotypic similarity of embryos lacking either the Ccbe1 gene or the lymphangiogenic growth factor Vegfc gene, we searched for CCBE1 interactions with the VEGF-C growth factor signaling pathway, which is critical in embryonic and adult lymphangiogenesis. Methods and Results—By analyzing VEGF-C produced by CCBE1-transfected cells, we found that while CCBE1 itself does not process VEGF-C, it promotes proteolytic cleavage of the otherwise poorly active 29/31-kDa form of VEGF-C by the A disintegrin and metalloprotease with thrombospondin motifs-3 (ADAMTS3) protease, resulting in the mature 21/23-kDa form of VEGF-C, which induces increased VEGF-C receptor signaling. Adeno-associated viral vector (AAV) mediated transduction of CCBE1 into mouse skeletal muscle enhanced lymphangiogenesis and angiogenesis induced by AAV-VEGF-C. Conclusions—These results identify ADAMTS3 as a VEGF-C activating protease and reveal a novel type of regulation of a vascular growth factor by a protein that enhances its proteolytic cleavage and activation. The results suggest CCBE1 is a potential therapeutic tool for the modulation of lymphangiogenesis and angiogenesis in a variety of diseases that involve the lymphatic system, such as lymphedema or lymphatic metastasis. %B Circulation %V 129 %8 05/2014 %G eng %U http://circ.ahajournals.org/content/early/2014/02/19/CIRCULATIONAHA.113.002779.abstract %N 19 %& 1962-1971 %R http://dx.doi.org/10.1161/CIRCULATIONAHA.113.002779 %0 Journal Article %J Sci Signal %D 2013 %T The basis for the distinct biological activities of vascular endothelial growth factor receptor-1 ligands %XVascular 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.
%B Sci Signal %V 6 %P ra52 %8 2013 %G eng %N 282 %R 10.1126/scisignal.2003905 %0 Journal Article %J Lymphologie in Forschung und Praxis %D 2013 %T Die lymphangiogenen Wachstumsfaktoren VEGF-C und VEGF-D %A Krebs, Rainer %A Jeltsch, Michael %K Lymphangiogenese %K VEGF-C %K VEGF-D %K Wachstumsfaktoren %X VEGF-C und VEGF-D sind die zwei zentralen Signalmoleküle, die für die Entwicklung und das Wachstum des Lymphgefäßsystems verantwortlich sind. Beide gehören zur VEGF-Proteinfamilie, deren Mitglieder hauptsächlich im Wachstum von Blutgefässen (Angiogenese) und Lymphgefässen (Lymphangiogenese) ihre Funktionen haben. Die VEGF-Familie umfasst in Säugetieren fünf Mitglieder: VEGF, PlGF, VEGF-B, VEGF-C und VEGF-D. Benannt wurde diese Familie nach ihrem zuerst entdeckten Mitglied VEGF („Vascular Endothelial Growth Factor”). VEGF-C und VEGF-D bilden funktionell und strukturell eine Untergruppe innerhalb der VEGF-Familie. Sie unterscheiden sich von den anderen VEGFs durch ihre besondere Biosynthese: sie werden als inaktive Vorgängermoleküle produziert, für deren Aktivierung ihre langen N- und C-terminalen Propeptide enzymatisch abgespalten werden müssen. Im Gegensatz zu den anderen VEGFs sind VEGF-C und VEGF-D direkte Stimulatoren für das Wachstum lymphatischer Gefäße. Ihre lymphangiogene Wirkung enfalten VEGF-C und VEGF-D über den VEGF-Rezeptor-3 (VEGFR-3), der im erwachsenen Organismus fast nur auf den Endothelzellen der Lymphvaskulatur zu finden ist. In diesem Artikel geben wir einen Überblick über die VEGF-Proteinfamilie und deren Rezeptoren mit dem Schwerpunkt auf den lymphangiogenen Mitgliedern VEGF-C und VEGF-D, über ihre Biosynthese und ihre Rolle in der Embryonalentwicklung. VEGF-C and VEGF-D are the two central signaling molecules that stimulate the development and the growth of lymphatic system. Both belong to the VEGF protein family which plays important roles in the growth of blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis). In mammals the VEGF family comprises five members: VEGF, PlGF, VEGF-B, VEGF-C and VEGF-D. The family was named after its first discovered member VEGF (“Vascular Endothelial Growth Factor”). VEGF-C and VEGF-D form functionally and structurally a subgroup within this family. They differ from the other VEGFs by their peculiar biosynthesis: they are produced as inactive precursors and need to be activated by proteolytic removal of their long N- and C-terminal propeptides. Unlike the other VEGFs, VEGF-C and VEGF-D are direct stimulators of lymphatic growth. They exert their lymphangiogenic function via VEGF receptor 3, which is expressed in the adult organism almost exclusively on lymphatic endothelial cells. In this review we give an overview of the VEGF protein family and their receptors with the emphasis on the lymphangiogenic VEGF-C and VEGF-D, and we discuss their biosynthesis and their role in embryonic lymphangiogenesis. %B Lymphologie in Forschung und Praxis %V 17 %P 30-37 %8 06/2013 %G eng %N 1 %9 Review %& 30 %0 Journal Article %J Lymphologie in Forschung und Praxis %D 2013 %T Die lymphangiogenic growth factors VEGF-C and VEGF-D. Part 2: The role of VEGF-C and VEGF-D in diseases of the lymphatic system. [bilingual: English, German]. %A Krebs, Rainer %A Jeltsch, Michael %K growth factors %K Lymphangiogenesis %K lymphedema %K lymphogenic metastasis %K VEGF-C %K VEGF-D %X VEGF-C and VEGF-D are the two central signaling molecules that stimulate the develop- ment and growth of the lymphatic system. Both belong to the vascular endothelial growth factor (VEGF) protein family, which plays important roles in the growth of blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis). In mammals, the VEGF family comprises five members: VEGF-A, PlGF, VEGF-B, VEGF-C and VEGF-D. The family was named after VEGF-A, the first member to be discovered. VEGF-C and VEGF-D form a subgroup within this family in terms of function and structure. Their distinctive biosynthesis differentiates them from the other VEGFs: they are produced as inactive precursors and need to be activated by proteolytic removal of their long N- and C-terminal propeptides. Unlike the other VEGFs, VEGF-C and VEGF-D are direct stimulators of lymphatic vessel growth. They exert their lymphangiogenic function via VEGF receptor 3, which is expressed in the adult organism almost exclusively on lymphatic endothelial cells. In this review, we provide an overview of the VEGF protein family and their receptors. We focus on the lymphangiogenic VEGF-C and VEGF-D, discussing their biosynthesis and their role in embryonic lymphangiogenesis. %B Lymphologie in Forschung und Praxis %V 17 %P 96 - 104 %8 11/2013 %G eng %U http://jeltsch.org/sites/jeltsch.org/files/JeltschMichael_Lymphforsch2013_96.pdf %N 2 %! Lymphforsch %0 Journal Article %J Lymphologie in Forschung und Praxis %D 2013 %T The lymphangiogenic growth factors VEGF-C and VEGF-D. Part 1: Basic principles and embryonic development. [bilingual: English, German]. %A Krebs, Rainer %A Jeltsch, Michael %K growth factors %K Lymphangiogenesis %K VEGF-C %K VEGF-D %X VEGF-C and VEGF-D are the two central signaling molecules that stimulate the develop- ment and growth of the lymphatic system. Both belong to the vascular endothelial growth factor (VEGF) protein family, which plays important roles in the growth of blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis). In mammals, the VEGF family comprises five members: VEGF-A, PlGF, VEGF-B, VEGF-C and VEGF-D. The family was named after VEGF-A, the first member to be discovered. VEGF-C and VEGF-D form a subgroup within this family in terms of function and structure. Their distinctive biosynthesis differentiates them from the other VEGFs: they are produced as inactive precursors and need to be activated by proteolytic removal of their long N- and C-terminal propeptides. Unlike the other VEGFs, VEGF-C and VEGF-D are direct stimulators of lymphatic vessel growth. They exert their lymphangiogenic function via VEGF receptor 3, which is expressed in the adult organism almost exclusively on lymphatic endothelial cells. In this review, we provide an overview of the VEGF protein family and their receptors. We focus on the lymphangiogenic VEGF-C and VEGF-D, discussing their biosynthesis and their role in embryonic lymphangiogenesis. %B Lymphologie in Forschung und Praxis %V 17 %P 30 - 37 %8 05/2013 %G eng %U http://jeltsch.org/sites/jeltsch.org/files/JeltschMichael_Lymphforsch2013_30.pdf %N 1 %! Lymphforsch %0 Journal Article %J Cold Spring Harbor Perspectives in Biology %D 2013 %T Receptor Tyrosine Kinase-Mediated Angiogenesis %X 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. %B Cold Spring Harbor Perspectives in Biology %V 5 %8 2013 %@ , 1943-0264 %G eng %U http://cshperspectives.cshlp.org/content/5/9/a009183 %N 9 %! Cold Spring Harb Perspect Biol %R 10.1101/cshperspect.a009183 %0 Journal Article %J Proceedings of the National Academy of Sciences of the United States of America %D 2013 %T Structural and mechanistic insights into VEGF receptor 3 ligand binding and activation %X 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. %B Proceedings of the National Academy of Sciences of the United States of America %V 110 %P 12960 - 12965 %8 08/2013 %@ 1091-6490 %G eng %U http://www.pnas.org/content/110/32/12960.long %N 32 %! Proc. Natl. Acad. Sci. U.S.A. %R 10.1073/pnas.1301415110 %0 Journal Article %J Development %D 2013 %T A truncation allele in vascular endothelial growth factor c reveals distinct modes of signaling during lymphatic and vascular development. %XVascular 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.
%B Development %V 140 %P 1497-506 %8 2013 Apr %G eng %N 7 %R 10.1242/dev.084152 %0 Journal Article %J Am J Pathol %D 2012 %T Critical role of VEGF-C/VEGFR-3 signaling in innate and adaptive immune responses in experimental obliterative bronchiolitis. %XChronic inflammation, a hallmark of obliterative bronchiolitis, is known to induce lymphangiogenesis. We therefore studied the role of lymphangiogenic vascular endothelial growth factor C (VEGF-C), its receptor VEGFR-3, and lymphangiogenesis during development of experimental obliterative bronchiolitis [ie, obliterative airway disease (OAD)] in rat tracheal allografts. The functional importance of VEGF-C was investigated by adenovirus-mediated overexpression of VEGF-C (AdVEGF-C), and by inhibition of VEGF-C activity with VEGFR-3-Ig (AdVEGFR-3-Ig). Analyses included histology, immunohistochemistry, and real-time RT-PCR 10 and 30 days after transplantation. In the course of OAD development, lymphangiogenesis was induced in the airway wall during the alloimmune response, which was reversed by cyclosporine A in a dose-dependent fashion. VEGF-C overexpression in tracheal allografts induced epithelial activation, neutrophil chemotaxis, and a shift toward a Th17 adaptive immune response, followed by enhanced lymphangiogenesis and the development of OAD. In contrast, inhibition of VEGF-C activity with VEGFR-3-Ig inhibited lymphangiogenesis and angiogenesis and reduced infiltration of CD4(+) T cells and the development of OAD. Lymphangiogenesis was linked to T-cell responses during the development of OAD, and VEGF-C/VEGFR-3 signaling modulated innate and adaptive immune responses in the development of OAD in rat tracheal allografts. Our results thus suggest VEGFR-3-signaling as a novel strategy to regulate T-cell responses in the development of obliterative bronchiolitis after lung transplantation.
%B Am J Pathol %V 181 %P 1607-20 %8 2012 Nov %G eng %N 5 %R 10.1016/j.ajpath.2012.07.021 %0 Journal Article %J Blood %D 2011 %T Structural determinants of vascular endothelial growth factor-D receptor binding and specificity %A Leppänen, Veli-Matti %A Jeltsch, Michael %A Anisimov, Andrey %A Tvorogov, Denis %A Aho, Kukka %A Kalkkinen, Nisse %A Toivanen, Pyry %A Ylä-Herttuala, Seppo %A Ballmer-Hofer, Kurt %A Alitalo, Kari %X Vascular endothelial growth factors (VEGFs) and their tyrosine kinase receptors (VEGFR-1-3) are central mediators of angiogenesis and lymphangiogenesis. VEGFR-3 ligands VEGF-C and VEGF-D are produced as precursor proteins with long N- and C-terminal propeptides and show enhanced VEGFR-2 and VEGFR-3 binding on proteolytic removal of the propeptides. Two different proteolytic cleavage sites have been reported in the VEGF-D N-terminus. We report here the crystal structure of the human VEGF-D Cys117Ala mutant at 2.9 Å resolution. Comparison of the VEGF-D and VEGF-C structures shows similar extended N-terminal helices, conserved overall folds, and VEGFR-2 interacting residues. Consistent with this, the affinity and the thermodynamic parameters for VEGFR-2 binding are very similar. In comparison with VEGF-C structures, however, the VEGF-D N-terminal helix was extended by 2 more turns because of a better resolution. Both receptor binding and functional assays of N-terminally truncated VEGF-D polypeptides indicated that the residues between the reported proteolytic cleavage sites are important for VEGF-D binding and activation of VEGFR-3, but not of VEGFR-2. Thus, we define here a VEGFR-2-specific form of VEGF-D that is angiogenic but not lymphangiogenic. These results provide important new insights into VEGF-D structure and function. %B Blood %V 117 %P 1507 - 15 %8 2011/Feb/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/21148085 %N 5 %! Blood %0 Journal Article %J Genes Dev %D 2010 %T Claudin-like protein 24 interacts with the VEGFR-2 and VEGFR-3 pathways and regulates lymphatic vessel development %A Saharinen, Pipsa %A Helotera, Hanna %A Miettinen, Juho %A Norrmen, Camilla %A D'Amico, Gabriela %A Jeltsch, Michael %A Langenberg, Tobias %A Vandevelde, Wouter %A Ny, Annelii %A Dewerchin, Mieke %A Carmeliet, Peter %A Alitalo, Kari %X The Claudin-like protein of 24 kDa (CLP24) is a hypoxia-regulated transmembrane protein of unknown function. We show here that clp24 knockdown in Danio rerio and Xenopus laevis results in defective lymphatic development. Targeted disruption of Clp24 in mice led to enlarged lymphatic vessels having an abnormal smooth muscle cell coating. We also show that the Clp24(-/-) phenotype was further aggravated in the Vegfr2(+/LacZ) or Vegfr3(+/LacZ) backgrounds and that CLP24 interacts with vascular endothelial growth factor receptor-2 (VEGFR-2) and VEGFR-3 and attenuates the transcription factor CREB phosphorylation via these receptors. Our results indicate that CLP24 is a novel regulator of VEGFR-2 and VEGFR-3 signaling pathways and of normal lymphatic vessel structure. %B Genes Dev %V 24 %P 875 - 80 %8 2010/May/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/20439428 %N 9 %! Genes & Development %0 Journal Article %J Cancer Cell %D 2010 %T Effective suppression of vascular network formation by combination of antibodies blocking VEGFR ligand binding and receptor dimerization %A Tvorogov, Denis %A Anisimov, Andrey %A Zheng, Wei %A Leppänen, Veli-Matti %A Tammela, Tuomas %A Laurinavicius, Simonas %A Holnthoner, Wolfgang %A Heloterä, Hanna %A Holopainen, Tanja %A Jeltsch, Michael %A Kalkkinen, Nisse %A Lankinen, Hilkka %A Ojala, Päivi M %A Alitalo, Kari %X Antibodies that block vascular endothelial growth factor (VEGF) have become an integral part of antiangiogenic tumor therapy, and antibodies targeting other VEGFs and receptors (VEGFRs) are in clinical trials. Typically receptor-blocking antibodies are targeted to the VEGFR ligand-binding site. Here we describe a monoclonal antibody that inhibits VEGFR-3 homodimer and VEGFR-3/VEGFR-2 heterodimer formation, signal transduction, as well as ligand-induced migration and sprouting of microvascular endothelial cells. Importantly, we show that combined use of antibodies blocking ligand binding and receptor dimerization improves VEGFR inhibition and results in stronger inhibition of endothelial sprouting and vascular network formation in vivo. These results suggest that receptor dimerization inhibitors could be used to enhance antiangiogenic activity of antibodies blocking ligand binding in tumor therapy. %B Cancer Cell %V 18 %P 630 - 40 %8 2010/Dec/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/21130043 %N 6 %! Cancer Cell %0 Journal Article %J Proceedings of the National Academy of Sciences of the United States of America %D 2010 %T Structural determinants of growth factor binding and specificity by VEGF receptor 2 %A Leppänen, Veli-Matti %A Prota, Andrea E %A Jeltsch, Michael %A Anisimov, Andrey %A Kalkkinen, Nisse %A Strandin, Tomas %A Lankinen, Hilkka %A Goldman, Adrian %A Ballmer-Hofer, Kurt %A Alitalo, Kari %X Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel formation through activation of three receptor tyrosine kinases, VEGFR-1, -2, and -3. The extracellular domain of VEGF receptors consists of seven immunoglobulin homology domains, which, upon ligand binding, promote receptor dimerization. Dimerization initiates transmembrane signaling, which activates the intracellular tyrosine kinase domain of the receptor. VEGF-C stimulates lymphangiogenesis and contributes to pathological angiogenesis via VEGFR-3. However, proteolytically processed VEGF-C also stimulates VEGFR-2, the predominant transducer of signals required for physiological and pathological angiogenesis. Here we present the crystal structure of VEGF-C bound to the VEGFR-2 high-affinity-binding site, which consists of immunoglobulin homology domains D2 and D3. This structure reveals a symmetrical 22 complex, in which left-handed twisted receptor domains wrap around the 2-fold axis of VEGF-C. In the VEGFs, receptor specificity is determined by an N-terminal alpha helix and three peptide loops. Our structure shows that two of these loops in VEGF-C bind to VEGFR-2 subdomains D2 and D3, while one interacts primarily with D3. Additionally, the N-terminal helix of VEGF-C interacts with D2, and the groove separating the two VEGF-C monomers binds to the D2/D3 linker. VEGF-C, unlike VEGF-A, does not bind VEGFR-1. We therefore created VEGFR-1/VEGFR-2 chimeric proteins to further study receptor specificity. This biochemical analysis, together with our structural data, defined VEGFR-2 residues critical for the binding of VEGF-A and VEGF-C. Our results provide significant insights into the structural features that determine the high affinity and specificity of VEGF/VEGFR interactions. %B Proceedings of the National Academy of Sciences of the United States of America %V 107 %P 2425 - 30 %8 02/2010 %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/20145116 %N 6 %0 Journal Article %J PLoS ONE %D 2010 %T Suppressive effects of vascular endothelial growth factor-B on tumor growth in a mouse model of pancreatic neuroendocrine tumorigenesis %A Albrecht, Imke %A Kopfstein, Lucie %A Strittmatter, Karin %A Schomber, Tibor %A Falkevall, Annelie %A Hagberg, Carolina E %A Lorentz, Pascal %A Jeltsch, Michael %A Alitalo, Kari %A Eriksson, Ulf %A Christofori, Gerhard %A Pietras, Kristian %X BACKGROUND: The family of vascular endothelial growth factors (VEGF) contains key regulators of blood and lymph vessel development, including VEGF-A, -B, -C, -D, and placental growth factor. The role of VEGF-B during physiological or pathological angiogenesis has not yet been conclusively delineated. Herein, we investigate the function of VEGF-B by the generation of mouse models of cancer with transgenic expression of VEGF-B or homozygous deletion of Vegfb. METHODOLOGY/PRINCIPAL FINDINGS: Ectopic expression of VEGF-B in the insulin-producing β-cells of the pancreas did not alter the abundance or architecture of the islets of Langerhans. The vasculature from transgenic mice exhibited a dilated morphology, but was of similar density as that of wildtype mice. Unexpectedly, we found that transgenic expression of VEGF-B in the RIP1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis retarded tumor growth. Conversely, RIP1-Tag2 mice deficient for Vegfb presented with larger tumors. No differences in vascular density, perfusion or immune cell infiltration upon altered Vegfb gene dosage were noted. However, VEGF-B acted to increase blood vessel diameter both in normal pancreatic islets and in RIP1-Tag2 tumors. CONCLUSIONS/SIGNIFICANCE: Taken together, our results illustrate the differences in biological function between members of the VEGF family, and highlight the necessity of in-depth functional studies of VEGF-B to fully understand the effects of VEGFR-1 inhibitors currently used in the clinic. %B PLoS ONE %V 5 %P e14109 %8 2010// %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/21124841 %N 11 %! PLoS ONE %0 Journal Article %J Circulation %D 2010 %T Vascular endothelial growth factor-B acts as a coronary growth factor in transgenic rats without inducing angiogenesis, vascular leak, or inflammation %A Bry, Maija %A Kivelä, Riikka %A Holopainen, Tanja %A Anisimov, Andrey %A Tammela, Tuomas %A Soronen, Jarkko %A Silvola, Johanna %A Saraste, Antti %A Jeltsch, Michael %A Korpisalo, Petra %A Carmeliet, Peter %A Lemström, Karl B %A Shibuya, Masabumi %A Ylä-Herttuala, Seppo %A Alhonen, Leena %A Mervaala, Eero %A Andersson, Leif C %A Knuuti, Juhani %A Alitalo, Kari %X BACKGROUND: Vascular endothelial growth factor-B (VEGF-B) binds to VEGF receptor-1 and neuropilin-1 and is abundantly expressed in the heart, skeletal muscle, and brown fat. The biological function of VEGF-B is incompletely understood. METHODS AND RESULTS: Unlike placenta growth factor, which binds to the same receptors, adeno-associated viral delivery of VEGF-B to mouse skeletal or heart muscle induced very little angiogenesis, vascular permeability, or inflammation. As previously reported for the VEGF-B(167) isoform, transgenic mice and rats expressing both isoforms of VEGF-B in the myocardium developed cardiac hypertrophy yet maintained systolic function. Deletion of the VEGF receptor-1 tyrosine kinase domain or the arterial endothelial Bmx tyrosine kinase inhibited hypertrophy, whereas loss of VEGF-B interaction with neuropilin-1 had no effect. Surprisingly, in rats, the heart-specific VEGF-B transgene induced impressive growth of the epicardial coronary vessels and their branches, with large arteries also seen deep inside the subendocardial myocardium. However, VEGF-B, unlike other VEGF family members, did not induce significant capillary angiogenesis, increased permeability, or inflammatory cell recruitment. CONCLUSIONS: VEGF-B appears to be a coronary growth factor in rats but not in mice. The signals for the VEGF-B-induced cardiac hypertrophy are mediated at least in part via the endothelium. Because cardiomyocyte damage in myocardial ischemia begins in the subendocardial myocardium, the VEGF-B-induced increased arterial supply to this area could have therapeutic potential in ischemic heart disease. %B Circulation %V 122 %P 1725 - 33 %8 2010/Oct/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/20937974 %N 17 %! Circulation %0 Journal Article %J Circ Res %D 2009 %T Activated forms of VEGF-C and VEGF-D provide improved vascular function in skeletal muscle %A Anisimov, Andrey %A Alitalo, Annamari %A Korpisalo, Petra %A Soronen, Jarkko %A Kaijalainen, Seppo %A Leppänen, Veli-Matti %A Jeltsch, Michael %A Ylä-Herttuala, Seppo %A Alitalo, Kari %X The therapeutic potential of vascular endothelial growth factor (VEGF)-C and VEGF-D in skeletal muscle has been of considerable interest as these factors have both angiogenic and lymphangiogenic activities. Previous studies have mainly used adenoviral gene delivery for short-term expression of VEGF-C and VEGF-D in pig, rabbit, and mouse skeletal muscles. Here we have used the activated mature forms of VEGF-C and VEGF-D expressed via recombinant adeno-associated virus (rAAV), which provides stable, long-lasting transgene expression in various tissues including skeletal muscle. Mouse tibialis anterior muscle was transduced with rAAV encoding human or mouse VEGF-C or VEGF-D. Two weeks later, immunohistochemical analysis showed increased numbers of both blood and lymph vessels, and Doppler ultrasound analysis indicated increased blood vessel perfusion. The lymphatic vessels further increased at the 4-week time point were functional, as shown by FITC-lectin uptake and transport. Furthermore, receptor activation and arteriogenic activity were increased by an alanine substitution mutant of human VEGF-C (C137A) having an increased dimer stability and by a chimeric CAC growth factor that contained the VEGF receptor-binding domain flanked by VEGF-C propeptides, but only the latter promoted significantly more blood vessel perfusion when compared to the other growth factors studied. We conclude that long-term expression of VEGF-C and VEGF-D in skeletal muscle results in the generation of new functional blood and lymphatic vessels. The therapeutic value of intramuscular lymph vessels in draining tissue edema and lymphedema can now be evaluated using this model system. %B Circ Res %V 104 %P 1302 - 12 %8 2009/Jun/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/19443835 %N 11 %! Circulation Research %0 Journal Article %J Circ Res %D 2008 %T Overexpression of vascular endothelial growth factor-B in mouse heart alters cardiac lipid metabolism and induces myocardial hypertrophy %A Karpanen, Terhi %A Bry, Maija %A Ollila, Hanna M %A Seppänen-Laakso, Tuulikki %A Liimatta, Erkki %A Leskinen, Hanna %A Kivelä, Riikka %A Helkamaa, Teemu %A Merentie, Mari %A Jeltsch, Michael %A Paavonen, Karri %A Andersson, Leif C %A Mervaala, Eero %A Hassinen, Ilmo E %A Ylä-Herttuala, Seppo %A Oresic, Matej %A Alitalo, Kari %X Vascular endothelial growth factor (VEGF)-B is poorly angiogenic but prominently expressed in metabolically highly active tissues, including the heart. We produced mice expressing a cardiac-specific VEGF-B transgene via the alpha-myosin heavy chain promoter. Surprisingly, the hearts of the VEGF-B transgenic mice showed concentric cardiac hypertrophy without significant changes in heart function. The cardiac hypertrophy was attributable to an increased size of the cardiomyocytes. Blood capillary size was increased, whereas the number of blood vessels per cell nucleus remained unchanged. Despite the cardiac hypertrophy, the transgenic mice had lower heart rate and blood pressure than their littermates, and they responded similarly to angiotensin II-induced hypertension, confirming that the hypertrophy does not compromise heart function. Interestingly, the isolated transgenic hearts had less cardiomyocyte damage after ischemia. Significantly increased ceramide and decreased triglyceride levels were found in the transgenic hearts. This was associated with structural changes and eventual lysis of mitochondria, resulting in accumulation of intracellular vacuoles in cardiomyocytes and increased death of the transgenic mice, apparently because of mitochondrial lipotoxicity in the heart. These results suggest that VEGF-B regulates lipid metabolism, an unexpected function for an angiogenic growth factor. %B Circ Res %V 103 %P 1018 - 26 %8 2008/Oct/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/18757827 %N 9 %! Circulation Research %0 Journal Article %J Arterioscler Thromb Vasc Biol %D 2008 %T Reevaluation of the role of VEGF-B suggests a restricted role in the revascularization of the ischemic myocardium %A Li, Xuri %A Tjwa, Marc %A Van Hove, Inge %A Enholm, Berndt %A Neven, Elke %A Paavonen, Karri %A Jeltsch, Michael %A Juan, Toni Diez %A Sievers, Richard E %A Chorianopoulos, Emmanuel %A Wada, Hiromichi %A Vanwildemeersch, Maarten %A Noel, Agnes %A Foidart, Jean-Michel %A Springer, Matthew L %A von Degenfeld, Georges %A Dewerchin, Mieke %A Blau, Helen M %A Alitalo, Kari %A Eriksson, Ulf %A Carmeliet, Peter %A Moons, Lieve %X OBJECTIVE: The endogenous role of the VEGF family member vascular endothelial growth factor-B (VEGF-B) in pathological angiogenesis remains unclear. METHODS AND RESULTS: We studied the role of VEGF-B in various models of pathological angiogenesis using mice lacking VEGF-B (VEGF-B(-/-)) or overexpressing VEGF-B(167). After occlusion of the left coronary artery, VEGF-B deficiency impaired vessel growth in the ischemic myocardium whereas, in wild-type mice, VEGF-B(167) overexpression enhanced revascularization of the infarct and ischemic border zone. By contrast, VEGF-B deficiency did not affect vessel growth in the wounded skin, hypoxic lung, ischemic retina, or ischemic limb. Moreover, VEGF-B(167) overexpression failed to enhance vascular growth in the skin or ischemic limb. CONCLUSIONS: VEGF-B appears to have a relatively restricted angiogenic activity in the ischemic heart. These insights might offer novel therapeutic opportunities. %B Arterioscler Thromb Vasc Biol %V 28 %P 1614 - 20 %8 2008/Sep/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/18511699 %N 9 %! Arteriosclerosis, Thrombosis, and Vascular Biology %0 Journal Article %J Cancer Res %D 2008 %T The tyrosine kinase inhibitor cediranib blocks ligand-induced vascular endothelial growth factor receptor-3 activity and lymphangiogenesis %A Heckman, Caroline A %A Holopainen, Tanja %A Wirzenius, Maria %A Keskitalo, Salla %A Jeltsch, Michael %A Ylä-Herttuala, Seppo %A Wedge, Stephen R %A Jürgensmeier, Juliane M %A Alitalo, Kari %X Solid tumors express a range of factors required to sustain their growth and promote their dissemination. Among these are vascular endothelial growth factor-A (VEGF-A), the key angiogenic stimulant, and VEGF-C, a primary mediator of lymphangiogenesis. Small molecule tyrosine kinase inhibitors offer the potential to inhibit more than one kinase and impede tumor growth by multiple mechanisms. However, their potency toward individual targets can vary. Cediranib (RECENTIN; AZD2171) is an inhibitor of VEGF signaling that has been shown in experimental models to prevent VEGF-A-induced angiogenesis and primary tumor growth, yet the effects of cediranib on VEGF receptor (VEGFR)-3-mediated endothelial cell function and lymphangiogenesis are unknown. To better understand the activity of cediranib against VEGFR-3 and its associated signaling events compared with its activity against VEGFR-2, we used the receptor-specific ligands VEGF-E and VEGF-C156S. In human endothelial cells, cediranib inhibited VEGF-E-induced phosphorylation of VEGFR-2 and VEGF-C156S-induced phosphorylation of VEGFR-3 at concentrations of =1nmol/L and inhibited activation of downstream signaling molecules. Additionally, cediranib blocked VEGF-C156S-induced and VEGF-E-induced proliferation, survival, and migration of lymphatic and blood vascular endothelial cells. In vivo, cediranib (6 mg/kg/d) prevented angiogenesis and lymphangiogenesis induced by VEGF-E-expressing and VEGF-C156S-expressing adenoviruses, respectively. Cediranib (6 mg/kg/day) also blocked angiogenesis and lymphangiogenesis induced by adenoviruses expressing VEGF-A or VEGF-C and compromised the blood and lymphatic vasculatures of VEGF-C-expressing tumors. Cediranib may, therefore, be an effective means of preventing tumor progression, not only by inhibiting VEGFR-2 activity and angiogenesis, but also by concomitantly inhibiting VEGFR-3 activity and lymphangiogenesis. %B Cancer Res %V 68 %P 4754 - 62 %8 2008/Jun/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/18559522 %N 12 %! Cancer Research %0 Journal Article %J Circ Res %D 2007 %T Distinct architecture of lymphatic vessels induced by chimeric vascular endothelial growth factor-C/vascular endothelial growth factor heparin-binding domain fusion proteins %A Tammela, Tuomas %A He, Yulong %A Lyytikkä, Johannes %A Jeltsch, Michael %A Markkanen, Johanna %A Pajusola, Katri %A Ylä-Herttuala, Seppo %A Alitalo, Kari %X Vascular endothelial growth factor (VEGF)-C and VEGF-D are composed of the receptor-binding VEGF homology domain and a carboxy-terminal silk homology domain that requires proteolytic cleavage for growth factor activation. Here, we explored whether the C-terminal heparin-binding domain of the VEGF(165) or VEGF(189) isoform also containing neuropilin-binding sequences could substitute for the silk homology domain of VEGF-C. Such VEGF-C/VEGF-heparin-binding domain chimeras were produced and shown to activate VEGF-C receptors, and, when expressed in tissues via adenovirus or adeno-associated virus vectors, stimulated lymphangiogenesis in vivo. However, both chimeras induced a distinctly different pattern of lymphatic vessels when compared with VEGF-C. Whereas VEGF-C-induced vessels were initially a dense network of small diameter vessels, the lymphatic vessels induced by the chimeric growth factors tended to form directly along tissue borders, along basement membranes that are rich in heparan sulfate. For example, in skeletal muscle, the chimeras induced formation of lumenized lymphatic vessels more efficiently than wild-type VEGF-C. We conclude that the matrix-binding domain of VEGF can target VEGF-C activity to heparin-rich basement membrane structures. These properties may prove useful for tissue engineering and attempts to regenerate lymphatic vessels in lymphedema patients. %B Circ Res %V 100 %P 1468 - 75 %8 2007/May/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/17478733 %N 10 %! Circulation Research %0 Journal Article %J Circ Res %D 2007 %T Enhanced capillary formation stimulated by a chimeric vascular endothelial growth factor/vascular endothelial growth factor-C silk domain fusion protein %A Keskitalo, Salla %A Tammela, Tuomas %A Lyytikka, Johannes %A Karpanen, Terhi %A Jeltsch, Michael %A Markkanen, Johanna %A Yla-Herttuala, Seppo %A Alitalo, Kari %X Vascular endothelial growth factor (VEGF)-C and VEGF-D require proteolytic cleavage of the carboxy terminal silk-homology domain for activation. To study the functions of the VEGF-C propeptides, we engineered a chimeric growth factor protein, VEGF-CAC, composed of the amino- and carboxy-terminal propeptides of VEGF-C fused to the receptor-activating core domain of VEGF. Like VEGF-C, VEGF-CAC underwent proteolytic cleavage, and like VEGF, it bound to and activated VEGF receptor-1 and VEGF receptor-2, but not the VEGF-C receptor VEGF receptor-3. VEGF-CAC also bound to neuropilins in a heparin-dependent manner. Strikingly, when VEGF-CAC was expressed via an adenovirus vector in the ear skin of immunodeficient mice, it proved to be a more potent inducer of capillary angiogenesis than VEGF. The VEGF-CAC-induced vessels differed greatly from those induced by VEGF, as they formed a very dense and fine network of pericyte and basement membrane-covered capillaries that were functional, as shown by lectin perfusion experiments. VEGF-CAC could prove useful in proangiogenic therapies in patients experiencing tissue ischemia. %B Circ Res %V 100 %P 1460 - 7 %8 2007/May/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/17478734 %N 10 %! Circulation Research %0 Conference Paper %B 98th Annual Meeting of the American-Association-for-Cancer-Research %D 2007 %T Inhibiton of VEGF-C-induced VEGFR-3 activity and lymphatic endothelial cell function by the tyrosine kinase inhibitor AZD2171 %X Solid tumors express a range of growth factors required to sustain their growth and promote their dissemination. Among these factors is vascular endothelial growth factor-A (VEGF-A), the key angiogenic stimulant, and VEGF-C, a primary mediator of lymphangiogenesis. Small molecule tyrosine kinase inhibitors can prevent VEGF signaling activity by targeting the VEGF receptors and are an effective approach to impede tumor progression. The indole-ether quinazoline AZD2171 is a highly potent ATP-competitive inhibitor of VEGFR-2 (KDR) kinase, with additional activity against VEGFR-1 (Flt-1) and -3 (Flt-4), that has been shown in experimental models to prevent VEGF-A-induced angiogenesis and primary tumor growth (Wedge et al. Cancer Res 2005;65:4389-4400). For these studies we wished to further assess the ability of AZD2171 to inhibit VEGFR-3 and its associated functions. Upon binding its ligands VEGF-C or -D, VEGFR-3 becomes activated with the resulting signaling cascade eventually translated into increased proliferation, survival and migration of lymphatic and blood vascular endothelial cells. At concentrations of ≤1 nM AZD2171 inhibited VEGFR-3 phosphorylation in porcine aortic endothelial cells selectively expressing the human receptor, and in human dermal microvascular endothelial cells (HDMVECs). In HDMVECs, AZD2171 prevented phosphorylation of signaling molecules downstream of VEGFR-2 and -3, ERK1/2, Akt and CREB, induced by the VEGFR-2 and -3-specific ligands VEGF-E and -C156S, respectively. Additionally, AZD2171 blocked VEGF-E- and -C156S-induced proliferation of both lymphatic and blood vascular endothelial cells at similar concentrations, and prevented ligand-induced endothelial cell cord formation in a Matrigel assay. The effects of AZD2171 on VEGF-C-induced lymphangiogenesis are currently being assessed in vivo. These studies, together with previous results, not only demonstrate that AZD2171 may be an effective means of preventing tumor progression by inhibition of VEGFR-2 activity and angiogenesis, but may also prevent further tumor spread by inhibiting VEGFR-3 activity %B 98th Annual Meeting of the American-Association-for-Cancer-Research %I American Association for Cancer Research %C Los Angeles, CA %8 2007/// %G eng %U http://dx.doi.org/10.1158/0008-5472.CAN-07-5809 %0 Journal Article %J FASEB J %D 2006 %T Functional interaction of VEGF-C and VEGF-D with neuropilin receptors %A Kärpänen, Terhi %A Heckman, Caroline A %A Keskitalo, Salla %A Jeltsch, Michael %A Ollila, Hanna %A Neufeld, Gera %A Tamagnone, Luca %A Alitalo, Kari %X Lymphatic vascular development is regulated by vascular endothelial growth factor receptor-3 (VEGFR-3), which is activated by its ligands VEGF-C and VEGF-D. Neuropilin-2 (NP2), known to be involved in neuronal development, has also been implicated to play a role in lymphangiogenesis. We aimed to elucidate the mechanism by which NP2 is involved in lymphatic endothelial cell signaling. By in vitro binding studies we found that both VEGF-C and VEGF-D interact with NP2, VEGF-C in a heparin-independent and VEGF-D in a heparin-dependent manner. We also mapped the domains of VEGF-C and NP2 required for their binding. The functional importance of the interaction of NP2 with the lymphangiogenic growth factors was demonstrated by cointernalization of NP2 along with VEGFR-3 in endocytic vesicles of lymphatic endothelial cells upon stimulation with VEGF-C or VEGF-D. NP2 also interacted with VEGFR-3 in coprecipitation studies. Our results show that NP2 is directly involved in an active signaling complex with the key regulators of lymphangiogenesis and thus suggest a mechanism by which NP2 functions in the development of the lymphatic vasculature. %B FASEB J %V 20 %P 1462 - 72 %8 2006/Jul/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/16816121 %N 9 %! The FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology %0 Journal Article %J J Biol Chem %D 2006 %T Vascular endothelial growth factor (VEGF)/VEGF-C mosaic molecules reveal specificity determinants and feature novel receptor binding patterns %A Jeltsch, Michael %A Karpanen, Terhi %A Strandin, Tomas %A Aho, Kukka %A Lankinen, Hilkka %A Alitalo, Kari %X Vascular endothelial growth factors (VEGFs) and their receptors play key roles in angiogenesis and lymphangiogenesis. VEGF activates VEGF receptor-1 (VEGFR-1) and VEGFR-2, whereas VEGF-C activates VEGFR-2 and VEGFR-3. We have created a library of VEGF/VEGF-C mosaic molecules that contains factors with novel receptor binding profiles, notably proteins binding to all three VEGF receptors ("super-VEGFs"). The analyzed super-VEGFs show both angiogenic and lymphangiogenic effects in vivo, although weaker than the parental molecules. The composition of the VEGFR-3 binding molecules and scanning mutagenesis revealed determinants of receptor binding and specificity. VEGFR-2 and VEGFR-3 showed striking differences in their requirements for VEGF-C binding; extracellular domain 2 of VEGFR-2 was sufficient, whereas in VEGFR-3, both domains 1 and 2 were necessary. %B J Biol Chem %V 281 %P 12187 - 95 %8 2006/Apr/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/16505489 %N 17 %! The Journal of Biological Chemistry %0 Book Section %B Sigma-RBI Handbook of Receptor Classification and Signal Transduction %D 2006 %T VEGF Receptors %B Sigma-RBI Handbook of Receptor Classification and Signal Transduction %7 5. %I Sigma %G eng %U http://www.sigmaaldrich.com/technical-documents/articles/biology/rbi-handbook.html %0 Journal Article %J J Clin Invest %D 2005 %T Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation %A Baluk, Peter %A Tammela, Tuomas %A Ator, Erin %A Lyubynska, Natalya %A Achen, Marc G %A Hicklin, Daniel J %A Jeltsch, Michael %A Petrova, Tatiana V %A Pytowski, Bronislaw %A Stacker, Steven A %A Ylä-Herttuala, Seppo %A Jackson, David G %A Alitalo, Kari %A McDonald, Donald M %X Edema occurs in asthma and other inflammatory diseases when the rate of plasma leakage from blood vessels exceeds the drainage through lymphatic vessels and other routes. It is unclear to what extent lymphatic vessels grow to compensate for increased leakage during inflammation and what drives the lymphangiogenesis that does occur. We addressed these issues in mouse models of (a) chronic respiratory tract infection with Mycoplasma pulmonis and (b) adenoviral transduction of airway epithelium with VEGF family growth factors. Blood vessel remodeling and lymphangiogenesis were both robust in infected airways. Inhibition of VEGFR-3 signaling completely prevented the growth of lymphatic vessels but not blood vessels. Lack of lymphatic growth exaggerated mucosal edema and reduced the hypertrophy of draining lymph nodes. Airway dendritic cells, macrophages, neutrophils, and epithelial cells expressed the VEGFR-3 ligands VEGF-C or VEGF-D. Adenoviral delivery of either VEGF-C or VEGF-D evoked lymphangiogenesis without angiogenesis, whereas adenoviral VEGF had the opposite effect. After antibiotic treatment of the infection, inflammation and remodeling of blood vessels quickly subsided, but lymphatic vessels persisted. Together, these findings suggest that when lymphangiogenesis is impaired, airway inflammation may lead to bronchial lymphedema and exaggerated airflow obstruction. Correction of defective lymphangiogenesis may benefit the treatment of asthma and other inflammatory airway diseases. %B J Clin Invest %V 115 %P 247 - 57 %8 2005/Feb/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/15668734 %N 2 %! The Journal of Clinical Investigation %0 Journal Article %J Cancer Res %D 2005 %T Vascular endothelial cell growth factor receptor 3-mediated activation of lymphatic endothelium is crucial for tumor cell entry and spread via lymphatic vessels %A He, Yulong %A Rajantie, Iiro %A Pajusola, Katri %A Jeltsch, Michael %A Holopainen, Tanja %A Yla-Herttuala, Seppo %A Harding, Thomas %A Jooss, Karin %A Takahashi, Takashi %A Alitalo, Kari %X Lymphangiogenic growth factors vascular endothelial growth factor (VEGF)-C and VEGF-D have been shown to promote lymphatic metastasis by inducing tumor-associated lymphangiogenesis. In this study, we have investigated how tumor cells gain access into lymphatic vessels and at what stage tumor cells initiate metastasis. We show that VEGF-C produced by tumor cells induced extensive lymphatic sprouting towards the tumor cells as well as dilation of the draining lymphatic vessels, suggesting an active role of lymphatic endothelial cells in lymphatic metastasis. A significant increase in lymphatic vessel growth occurred between 2 and 3 weeks after tumor xenotransplantation, and lymph node metastasis occurred at the same stage. These processes were blocked dose-dependently by inhibition of VEGF receptor 3 (VEGFR-3) signaling by systemic delivery of a soluble VEGFR-3-immunoglobulin (Ig) fusion protein via adenoviral or adeno-associated viral vectors. However, VEGFR-3-Ig did not suppress lymph node metastasis when the treatment was started at a later stage after the tumor cells had already spread out, suggesting that tumor cell entry into lymphatic vessels is a key step during tumor dissemination via the lymphatics. Whereas lymphangiogenesis and lymph node metastasis were significantly inhibited by VEGFR-3-Ig, some tumor cells were still detected in the lymph nodes in some of the treated mice. This indicates that complete blockade of lymphatic metastasis may require the targeting of both tumor lymphangiogenesis and tumor cell invasion. %B Cancer Res %V 65 %P 4739 - 46 %8 2005/Jun/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/15930292 %N 11 %! Cancer Research %0 Journal Article %J Nat Immunol %D 2004 %T Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins %A Karkkainen, Marika J %A Haiko, Paula %A Sainio, Kirsi %A Partanen, Juha %A Taipale, Jussi %A Petrova, Tatiana V %A Jeltsch, Michael %A Jackson, David G %A Talikka, Marja %A Rauvala, Heikki %A Betsholtz, Christer %A Alitalo, Kari %X Lymphatic vessels are essential for immune surveillance, tissue fluid homeostasis and fat absorption. Defects in lymphatic vessel formation or function cause lymphedema. Here we show that the vascular endothelial growth factor C (VEGF-C) is required for the initial steps in lymphatic development. In Vegfc-/- mice, endothelial cells commit to the lymphatic lineage but do not sprout to form lymph vessels. Sprouting was rescued by VEGF-C and VEGF-D but not by VEGF, indicating VEGF receptor 3 specificity. The lack of lymphatic vessels resulted in prenatal death due to fluid accumulation in tissues, and Vegfc+/- mice developed cutaneous lymphatic hypoplasia and lymphedema. Our results indicate that VEGF-C is the paracrine factor essential for lymphangiogenesis, and show that both Vegfc alleles are required for normal lymphatic development. %B Nat Immunol %V 5 %P 74 - 80 %8 2004/Jan/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/14634646 %N 1 %! Nature Immunology %0 Journal Article %J Cell Tissue Res %D 2003 %T Genesis and pathogenesis of lymphatic vessels %A Jeltsch, Michael %A Tammela, Tuomas %A Alitalo, Kari %A Wilting, Jörg %X The lymphatic system is generally regarded as supplementary to the blood vascular system, in that it transports interstitial fluid, macromolecules, and immune cells back into the blood. However, in insects, the open hemolymphatic (or lymphohematic) system ensures the circulation of immune cells and interstitial fluid through the body. The Drosophila homolog of the mammalian vascular endothelial growth factor receptor (VEGFR) gene family is expressed in hemocytes, suggesting a close relationship to the endothelium that develops later in phylogeny. Lymph hearts are typical organs for the propulsion of lymph in lower vertebrates and are still transiently present in birds. The lymphatic endothelial marker VEGFR-3 is transiently expressed in embryonic blood vessels and is crucial for their development. We therefore regard the question of whether the blood vascular system or the lymphatic system is primary or secondary as open. Future molecular comparisons should be performed without any bias based on the current prevalence of the blood vascular system over the lymphatic system. Here, we give an overview of the structure, function, and development of the lymphatics, with special emphasis on the recently discovered lymphangiogenic growth factors. %B Cell Tissue Res %V 314 %P 69 - 84 %8 2003/Oct/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/12942362 %N 1 %! Cell and Tissue Research %0 Journal Article %J FASEB J %D 2003 %T Intrinsic versus microenvironmental regulation of lymphatic endothelial cell phenotype and function %A Veikkola, Tanja %A Lohela, Marja %A Ikenberg, Kristian %A Mäkinen, Taija %A Korff, Thomas %A Saaristo, Anne %A Petrova, Tatania %A Jeltsch, Michael %A Augustin, Hellmut G %A Alitalo, Kari %X Vascular endothelial cells are characterized by a high degree of functional and phenotypic plasticity, which is controlled both by their pericellular microenvironment and their intracellular gene expression programs. To gain further insight into the mechanisms regulating the endothelial cell phenotype, we have compared the responses of lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BECs) to vascular endothelial growth factors (VEGFs). VEGFR-3-specific signals are sufficient for LEC but not BEC proliferation, as shown by the ability of the specific ligand VEGF-C156S to stimulate cell cycle entry only in LECs. On the other hand, we found that VEGFR-3 stimulation did not induce LEC cell shape changes typical of VEGFR-2-stimulated LECs, indicating receptor-specific differences in the cytoskeletal responses. Genes induced via VEGFR-2 also differed between BECs and LECs: angiopoietin-2 (Ang-2) was induced via VEGFR-2 in BECs and LECs, but the smooth muscle cell (SMC) chemoattractant BMP-2 was induced only in BECs. Both BECs and LECs were able to promote SMC chemotaxis, but contact with SMCs led to down-regulation of VEGFR-3 expression in BECs in a 3-dimensional coculture system. This was consistent with the finding that VEGFR-3 is down-regulated in vivo at sites of endothelial cell-pericyte/smooth muscle cell contacts. Collectively, these data show intrinsic cell-specific differences of BEC and LEC responses to VEGFs and identify a pericellular regulatory mechanism for VEGFR-3 down-regulation in endothelial cells. %B FASEB J %V 17 %P 2006 - 13 %8 2003/Nov/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/14597670 %N 14 %! The FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology %0 Journal Article %J J Cell Biol %D 2003 %T VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia %A Gerhardt, Holger %A Golding, Matthew %A Fruttiger, Marcus %A Ruhrberg, Christiana %A Lundkvist, Andrea %A Abramsson, Alexandra %A Jeltsch, Michael %A Mitchell, Christopher %A Alitalo, Kari %A Shima, David %A Betsholtz, Christer %X Vascular endothelial growth factor (VEGF-A) is a major regulator of blood vessel formation and function. It controls several processes in endothelial cells, such as proliferation, survival, and migration, but it is not known how these are coordinately regulated to result in more complex morphogenetic events, such as tubular sprouting, fusion, and network formation. We show here that VEGF-A controls angiogenic sprouting in the early postnatal retina by guiding filopodial extension from specialized endothelial cells situated at the tips of the vascular sprouts. The tip cells respond to VEGF-A only by guided migration; the proliferative response to VEGF-A occurs in the sprout stalks. These two cellular responses are both mediated by agonistic activity of VEGF-A on VEGF receptor 2. Whereas tip cell migration depends on a gradient of VEGF-A, proliferation is regulated by its concentration. Thus, vessel patterning during retinal angiogenesis depends on the balance between two different qualities of the extracellular VEGF-A distribution, which regulate distinct cellular responses in defined populations of endothelial cells. %B J Cell Biol %V 161 %P 1163 - 77 %8 2003/Jun/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/12810700 %N 6 %! The Journal of Cell Biology %0 Journal Article %J FASEB J %D 2002 %T Adenoviral VEGF-C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes %A Saaristo, Anne %A Veikkola, Tanja %A Enholm, Berndt %A Hytönen, Maija %A Arola, Johanna %A Pajusola, Katri %A Turunen, Païvi %A Jeltsch, Michael %A Karkkainen, Marika J %A Kerjaschki, Dontscho %A Bueler, Hansruedi %A Ylä-Herttuala, Seppo %A Alitalo, Kari %X Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are important regulators of blood and lymphatic vessel growth and vascular permeability. The VEGF-C/VEGFR-3 signaling pathway is crucial for lymphangiogenesis, and heterozygous inactivating missense mutations of the VEGFR-3 gene are associated with hereditary lymphedema. However, VEGF-C can have potent effects on blood vessels because its receptor VEGFR-3 is expressed in certain blood vessels and because the fully processed form of VEGF-C also binds to the VEGFR-2 of blood vessels. To characterize the in vivo effects of VEGF-C on blood and lymphatic vessels, we have overexpressed VEGF-C via adenovirus- and adeno-associated virus-mediated transfection in the skin and respiratory tract of athymic nude mice. This resulted in dose-dependent enlargement and tortuosity of veins, which, along with the collecting lymphatic vessels were found to express VEGFR-2. Expression of angiopoietin 1 blocked the increased leakiness of the blood vessels induced by VEGF-C whereas vessel enlargement and lymphangiogenesis were not affected. However, angiogenic sprouting of new blood vessels was not observed in response to AdVEGF-C or AAV-VEGF-C. These results show that virally produced VEGF-C induces blood vessel changes, including vascular leak, but its angiogenic potency is much reduced compared with VEGF in normal skin. %B FASEB J %V 16 %P 1041 - 9 %8 2002/Jul/ %G eng %U http://view.ncbi.nlm.nih.gov/pubmed/12087065 %N 9 %! The FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology %0 Thesis %B University of Helsinki %D 2002 %T VEGFR-3 Ligands and Lymphangiogenesis %A Jeltsch, Michael %X Most of us have seen their own blood at one or the other time. The occasion might have been a small accident or in unfortunate cases a severe blood loss caused by a major injury. We also can feel our heart beating and the resulting pressure wave, the pulse. The existence of the car- diovascular system is obvious to us. Unlike the cardiovascular system, the lymphatic system has, until recently, escaped notable attention not only by the laymen, but also by the scientific community. It is unclear why the lymphatic system originally developed in higher vertebrates. Now, its main function seems to be to collect fluid that has leaked from the blood vessels and to return it into the cardiovascular system. Much of our knowledge about the development and structure of the lymphatic system is of considerable age, and it has been said that there has not been any progress in our understanding since the fine structure of the lymphatics was described with the introduction of the electron microscope. Vascular endothelial growth factor (VEGF) is the principal direct inducer of blood vessel growth, but it does not promote the growth of lymphatic vessels. This study demonstrates for the first time specific lymphangiogenesis as a response to the VEGF homologue VEGF-C. Overexpression of full length VEGF-C under the keratin-14 promoter in the skin of transgenic mice caused a proliferation of the lymphatic endothelium and lymphatic vessel enlargement. In the chorioallantoic membrane assay, the mature form of VEGF-C was also largely specific for lymphatic endothelial cells. A newly discovered close homologue of VEGF-C, VEGF-D was then shown to have the same receptor-binding pattern as VEGF-C. Contrary to the interaction of VEGF with its receptors, VEGF-C interaction with VEGFR-3 has not been analyzed at the molecular level. The structural determinants of VEGFR-3 binding were characterized in relation to VEGF using a non-random family shuffling approach with VEGF and VEGF-C as parent molecules. This approach led to the identification of VEGF/VEGF-C mosaic molecules that showed novel receptor binding profiles and a panel of these molecules was used to delineate the requirements of specific receptors in the induction of angiogenesis versus lymphangiogenesis. %B University of Helsinki %V PhD Thesis %G English %U http://ethesis.helsinki.fi/julkaisut/mat/bioti/vk/jeltsch %1 HTML version: http://ethesis.helsinki.fi/julkaisut/mat/bioti/vk/jeltsch/vegfr3li.html %0 Thesis %B University of Helsinki %D 1997 %T Functional Analysis of VEGF-B and VEGF-C %X Vascular endothelial growth factor (VEGF) is an important regulator of endothelial cell proliferation and migration dur- ing embryonic vasculogenesis and angiogenesis as well as in pathological angiogenesis. The recently cloned new factors structurally homologous to VEGF were designated as VEGF-B/VRF and VEGF-C/VRP. The receptor for VEGF-B is unknown. VEGF-C is the ligand for FLT4, a receptor tyrosine kinase whose expression becomes restricted largely to lymphatic endothelia during development and that is related to VEGF receptors FLT1 and KDR. In this study keratin 14-promoter-directed VEGF-C overexpression in the basal epidermis of transgenic mice was capa- ble of promoting an abundant growth of extensive lymphatic-like vessel structures in the dermis, including large vessel lacunae resembling in their histopathology the human condition known as lymphangioma. Thus, VEGF-C appears to induce selective angiogenesis of the lymphatic vessels in vivo. In contrast, preliminary data on mice, which overexpress VEGF-B under the same promoter, does not yet allow us draw any conclusions about its possible biological function. Recombinant biologically active human VEGF-C was produced using the baculovirus system. Unpurified and purified VEGF-C were used to confirm the interaction of VEGF-C with KDR, a fact recently missed by others. The recombinant protein is going to be used in a large number of future experiments. The production of VEGF-B seems to be intrinsically difficult in non-mammalian cells. Although quantitatively satisfying results could not be obtained yet, the purified growth factor will be used in experiments to identify its receptor. %B University of Helsinki %I Univeristy of Helsinki %C Helsinki %V Master's Thesis %G English %U http://urn.fi/URN:NBN:fi-fe977347 %9 M.Sc.