TY - JOUR T1 - Investigation on the role of biallelic variants in VEGF‐C found in a patient affected by Milroy‐like lymphedema JF - Molecular Genetics & Genomic Medicine Y1 - 2020 VL - 00 UR - https://onlinelibrary.wiley.com/doi/abs/10.1002/mgg3.1389 JO - Mol Genet Genomic Med ER - TY - JOUR T1 - Lymphatics and the eye. [Finnish] JF - Duodecim Lääketieteellinen Aikakauskirja Y1 - 2020 A1 - Gucciardo, Erika A1 - Lehti, Timo A. A1 - Korhonen, Ani A1 - Salvén, Petri A1 - Lehti, Kaisa A1 - Jeltsch, Michael A1 - Loukovaara, Sirpa VL - 136 UR - https://www.duodecimlehti.fi/lehti/2020/16/duo15739 IS - 16 JO - Duodecim ER - TY - JOUR T1 - VEGF-C Protects the Integrity of Bone Marrow Perivascular Niche JF - Blood Y1 - 2020 AB - Key Points. Vegfc deletion in endothelial or LepR+ cells compromises the bone marrow perivascular niche and hematopoietic stem cell maintenance.Exogenous admin UR - https://ashpublications.org/blood/article/doi/10.1182/blood.2020005699/463465/VEGF-C-Protects-the-Integrity-of-Bone-Marrow JO - Blood ER - TY - JOUR T1 - KLK3/PSA and cathepsin D activate VEGF-C and VEGF-D JF - eLife Y1 - 2019 A1 - Jha, Sawan Kumar A1 - Rauniyar, Khushbu A1 - Chronowska, Ewa A1 - Mattonet, Kenny A1 - Maina, Eunice Wairimu A1 - Koistinen, Hannu A1 - Stenman, Ulf-Håkan A1 - Alitalo, Kari A1 - Jeltsch, Michael KW - cancer biology KW - Cathepsin D KW - kallikrein-related peptidases KW - KLK3/PSA KW - Lymphangiogenesis KW - mouse KW - VEGF-C KW - VEGF-D AB - Vascular endothelial growth factor-C (VEGF-C) acts primarily on endothelial cells, but also on non-vascular targets, e.g. in the CNS and immune system. Here we describe a novel, unique VEGF-C form in the human reproductive system produced via cleavage by kallikrein-related peptidase 3 (KLK3), aka prostate-specific antigen (PSA). KLK3 activated VEGF-C specifically and efficiently through cleavage at a novel N-terminal site. We detected VEGF-C in seminal plasma, and sperm liquefaction occurred concurrently with VEGF-C activation, which was enhanced by collagen and calcium binding EGF domains 1 (CCBE1). After plasmin and ADAMTS3, KLK3 is the third protease shown to activate VEGF-C. Since differently activated VEGF-Cs are characterized by successively shorter N-terminal helices, we created an even shorter hypothetical form, which showed preferential binding to VEGFR-3. Using mass spectrometric analysis of the isolated VEGF-C-cleaving activity from human saliva, we identified cathepsin D as a protease that can activate VEGF-C as well as VEGF-D. VL - 8 SN - 2050-084X UR - https://elifesciences.org/articles/44478 JO - eLife ER - TY - JOUR T1 - The Proteolytic Activation of Vascular Endothelial Growth Factor-C JF - Lymphologie in Forschung und Praxis Y1 - 2019 A1 - Lackner, Marcel A1 - Schmotz, Constanze A1 - Jeltsch, Michael KW - Lymphangiogenesis KW - proteinases KW - proteolysis KW - VEGF-C AB - 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. VL - 23 UR - https://doi.org/10.5281/zenodo.3629263 IS - 2 JO - LymphForsch ER - TY - JOUR T1 - Efficient activation of the lymphangiogenic growth factor VEGF-C requires the C-terminal domain of VEGF-C and the N-terminal domain of CCBE1 JF - Scientific Reports Y1 - 2017 VL - 7 UR - https://www.nature.com/articles/s41598-017-04982-1 IS - 1 ER - TY - CONF T1 - What you should know about VEGF-C when working with lymphatics [German] T2 - Lymphologie 2017 Y1 - 2017 A1 - Jeltsch, Michael JF - Lymphologie 2017 CY - Bad Soden (Frankfurt), Germany UR - https://jeltsch.org/Abstrakt-BadSoden2017 ER - TY - JOUR T1 - Factors regulating the substrate specificity of cytosolic phospholipase A2-alpha in vitro JF - Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids Y1 - 2016 VL - 1861 IS - 11 ER - TY - Generic T1 - From Molecular Genetics and Biology to Effective Treatments of Lymphatic Disorders T2 - 42nd Congress of the European Society of Lymphology Y1 - 2016 AB - 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. JF - 42nd Congress of the European Society of Lymphology CY - Mulhouse, France UR - http://www.eurolymphology.org/JOURNAL/VOL28-N74-2016/#p=14 ER - TY - JOUR T1 - Functional Importance of a Proteoglycan Co-Receptor in Pathologic Lymphangiogenesis JF - Circulation Research Y1 - 2016 AB - Rationale: Lymphatic vessel growth is mediated by major pro-lymphangiogenic factors such as VEGF-C and -D, among other endothelial effectors. Heparan sulfate is a linear polysaccharide expressed on proteoglycan core proteins on cell-membranes and matrix, playing roles in angiogenesis, although little is known regarding any function(s) in lymphatic remodeling in vivo. Objective: To explore the genetic basis and mechanisms whereby heparan sulfate proteoglycans mediate pathologic lymphatic remodeling. Methods and Results: Lymphatic endothelial deficiency in the major heparan sulfate biosynthetic enzyme N-deacetylase/N-sulfotransferase-1 (Ndst1; involved in glycan-chain sulfation) was associated with reduced lymphangiogenesis in pathologic models, including spontaneous neoplasia. Mouse mutants demonstrated tumor-associated lymphatic vessels with apoptotic nuclei. Mutant lymphatic endothelia demonstrated impaired mitogen (Erk) and survival (Akt) pathway signaling as well as reduced VEGF-C mediated protection from starvation-induced apoptosis. Lymphatic endothelial specific Ndst1 deficiency (in Ndst1f/fProx1+/CreERT2 mice) was sufficient to inhibit VEGF-C dependent lymphangiogenesis. Lymphatic heparan sulfate deficiency reduced phosphorylation of the major lymphatic growth receptor VEGFR-3 in response to multiple VEGF-C species. Syndecan-4 was the dominantly expressed heparan sulfate proteoglycan in mouse lymphatic endothelia, and pathologic lymphangiogenesis was impaired in Sdc4(-/-) mice. On the lymphatic cell surface, VEGF-C induced robust association between syndecan-4 and VEGFR-3 which was sensitive to glycan disruption. Moreover, VEGFR-3 mitogen and survival signaling was reduced in the setting of Ndst1 or Sdc4 deficiency. Conclusions: These findings demonstrate the genetic importance of heparan sulfate and the major lymphatic proteoglycan syndecan-4 in pathologic lymphatic remodeling. This may introduce novel future strategies to alter pathologic lymphatic-vascular remodeling. VL - 119 UR - http://circres.ahajournals.org/content/early/2016/05/25/CIRCRESAHA.116.308504 IS - 2 ER - TY - JOUR T1 - Lymphatic Vessels in Regenerative Medicine and Tissue Engineering JF - Tissue Engineering Part B Y1 - 2016 AB - 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. VL - 22 UR - http://online.liebertpub.com/doi/10.1089/ten.TEB.2016.0034 IS - 5 ER - TY - JOUR T1 - Functional Dissection of the CCBE1 Protein: A Crucial Requirement for the Collagen Repeat Domain. JF - Circ Res Y1 - 2015 AB -

RATIONALE: Collagen- and calcium-binding EGF domain-containing protein 1 (CCBE1) is essential for lymphangiogenesis in vertebrates and has been associated with Hennekam syndrome. Recently, CCBE1 has emerged as a crucial regulator of vascular endothelial growth factor-C (VEGFC) signaling.

OBJECTIVE: CCBE1 is a secreted protein characterized by 2 EGF domains and 2 collagen repeats. The functional role of the different CCBE1 protein domains is completely unknown. Here, we analyzed the functional role of the different CCBE1 domains in vivo and in vitro.

METHODS AND RESULTS: We analyzed the functionality of several CCBE1 deletion mutants by generating knock-in mice expressing these mutants, by analyzing their ability to enhance Vegfc signaling in vivo in zebrafish, and by testing their ability to induce VEGFC processing in vitro. We found that deleting the collagen domains of CCBE1 has a much stronger effect on CCBE1 activity than deleting the EGF domains. First, although CCBE1ΔCollagen mice fully phenocopy CCBE1 knock-out mice, CCBE1ΔEGF knock-in embryos still form rudimentary lymphatics. Second, Ccbe1ΔEGF, but not Ccbe1ΔCollagen, could partially substitute for Ccbe1 to enhance Vegfc signaling in zebrafish. Third, CCBE1ΔEGF, similarly to CCBE1, but not CCBE1ΔCollagen could activate VEGFC processing in vitro. Furthermore, a Hennekam syndrome mutation within the collagen domain has a stronger effect than a Hennekam syndrome mutation within the EGF domain.

CONCLUSIONS: We propose that the collagen domains of CCBE1 are crucial for the activation of VEGFC in vitro and in vivo. The EGF domains of CCBE1 are dispensable for regulation of VEGFC processing in vitro, however, they are necessary for full lymphangiogenic activity of CCBE1 in vivo.

VL - 116 UR - http://circres.ahajournals.org/content/116/10/1660.long IS - 10 ER - TY - CHAP T1 - The genetic causes of primary lymphedema. T2 - Erkrankungen des Lymphgefäßsystems Y1 - 2015 AB - 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. JF - Erkrankungen des Lymphgefäßsystems PB - Viavital Verlag CY - Cologne SN - 978-3-934371-53-8 ER - TY - JOUR T1 - Heterogeneity of the origin of the lymphatic system. [German]. JF - Lymphologie in Forschung und Praxis Y1 - 2015 AB - 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. VL - 19 UR - http://www.dglymph.de/fileadmin/global/pdfs/LymphForsch_2-15.pdf IS - 2 ER - TY - Generic T1 - Lymphangiogenesis in Health and Disease T2 - 41st European Societry of Lymphology (ESL) Congress Y1 - 2015 AB - 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. JF - 41st European Societry of Lymphology (ESL) Congress PB - European Group of Lymphology CY - Lausanne, Switzerland UR - http://www.eurolymphology.org/JOURNAL/VOL26-N72-2015/#p=10 ER - TY - JOUR T1 - Substrate efflux propensity is the key determinant of iPLA-β-mediated glycerophospholipid hydrolysised JF - Journal of Biological Chemistry Y1 - 2015 AB - 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. UR - http://www.jbc.org/content/early/2015/02/23/jbc.M115.642835.abstract ER - TY - CHAP T1 - The TIE Receptor Family T2 - Receptor Tyrosine Kinases: Family and Subfamilies Y1 - 2015 JF - Receptor Tyrosine Kinases: Family and Subfamilies PB - Springer International Publishing UR - https://link.springer.com/content/pdf/10.1007%2F978-3-319-11888-8_16.pdf ER - TY - JOUR T1 - CCBE1 enhances lymphangiogenesis via ADAMTS3-mediated VEGF-C activation JF - Circulation Y1 - 2014 AB - Background—Hennekam lymphangiectasia-lymphedema syndrome (OMIM 235510) is a rare autosomal recessive disease, which is associated with mutations in the collagen- and calcium-binding EGF domains 1 (CCBE1) gene. Because of the striking phenotypic similarity of embryos lacking either the Ccbe1 gene or the lymphangiogenic growth factor Vegfc gene, we searched for CCBE1 interactions with the VEGF-C growth factor signaling pathway, which is critical in embryonic and adult lymphangiogenesis. Methods and Results—By analyzing VEGF-C produced by CCBE1-transfected cells, we found that while CCBE1 itself does not process VEGF-C, it promotes proteolytic cleavage of the otherwise poorly active 29/31-kDa form of VEGF-C by the A disintegrin and metalloprotease with thrombospondin motifs-3 (ADAMTS3) protease, resulting in the mature 21/23-kDa form of VEGF-C, which induces increased VEGF-C receptor signaling. Adeno-associated viral vector (AAV) mediated transduction of CCBE1 into mouse skeletal muscle enhanced lymphangiogenesis and angiogenesis induced by AAV-VEGF-C. Conclusions—These results identify ADAMTS3 as a VEGF-C activating protease and reveal a novel type of regulation of a vascular growth factor by a protein that enhances its proteolytic cleavage and activation. The results suggest CCBE1 is a potential therapeutic tool for the modulation of lymphangiogenesis and angiogenesis in a variety of diseases that involve the lymphatic system, such as lymphedema or lymphatic metastasis. VL - 129 UR - http://circ.ahajournals.org/content/early/2014/02/19/CIRCULATIONAHA.113.002779.abstract IS - 19 ER - TY - JOUR T1 - The basis for the distinct biological activities of vascular endothelial growth factor receptor-1 ligands JF - Sci Signal Y1 - 2013 AB -

Vascular endothelial growth factors (VEGFs) regulate blood and lymphatic vessel development through VEGF receptors (VEGFRs). The VEGFR immunoglobulin homology domain 2 (D2) is critical for ligand binding, and D3 provides additional interaction sites. VEGF-B and placenta growth factor (PlGF) bind to VEGFR-1 with high affinity, but only PlGF is angiogenic in most tissues. We show that VEGF-B, unlike other VEGFs, did not require D3 interactions for high-affinity binding. VEGF-B with a PlGF-derived L1 loop (B-L1(P)) stimulated VEGFR-1 activity, whereas PlGF with a VEGF-B-derived L1 loop (P-L1(B)) did not. Unlike P-L1(B) and VEGF-B, B-L1(P) and PlGF were also angiogenic in mouse skeletal muscle. Furthermore, B-L1(P) also bound to VEGFR-2 and activated downstream signaling. These results establish a role for L1-mediated D3 interactions in VEGFR activation in endothelial cells and indicate that VEGF-B is a high-affinity VEGFR-1 ligand that, unlike PlGF, cannot efficiently induce signaling downstream of VEGFR-1.

VL - 6 IS - 282 ER - TY - JOUR T1 - Die lymphangiogenen Wachstumsfaktoren VEGF-C und VEGF-D JF - Lymphologie in Forschung und Praxis Y1 - 2013 A1 - Krebs, Rainer A1 - Jeltsch, Michael KW - Lymphangiogenese KW - VEGF-C KW - VEGF-D KW - Wachstumsfaktoren AB - 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. VL - 17 IS - 1 ER - TY - JOUR T1 - 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]. JF - Lymphologie in Forschung und Praxis Y1 - 2013 A1 - Krebs, Rainer A1 - Jeltsch, Michael KW - growth factors KW - Lymphangiogenesis KW - lymphedema KW - lymphogenic metastasis KW - VEGF-C KW - VEGF-D AB - 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. VL - 17 UR - http://jeltsch.org/sites/jeltsch.org/files/JeltschMichael_Lymphforsch2013_96.pdf IS - 2 JO - Lymphforsch ER - TY - JOUR T1 - The lymphangiogenic growth factors VEGF-C and VEGF-D. Part 1: Basic principles and embryonic development. [bilingual: English, German]. JF - Lymphologie in Forschung und Praxis Y1 - 2013 A1 - Krebs, Rainer A1 - Jeltsch, Michael KW - growth factors KW - Lymphangiogenesis KW - VEGF-C KW - VEGF-D AB - 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. VL - 17 UR - http://jeltsch.org/sites/jeltsch.org/files/JeltschMichael_Lymphforsch2013_30.pdf IS - 1 JO - Lymphforsch ER - TY - JOUR T1 - Receptor Tyrosine Kinase-Mediated Angiogenesis JF - Cold Spring Harbor Perspectives in Biology Y1 - 2013 AB - The endothelial cell is the essential cell type forming the inner layer of the vasculature. Two families of receptor tyrosine kinases (RTKs) are almost completely endothelial cell specific: the vascular endothelial growth factor (VEGF) receptors (VEGFR1-3) and the Tie receptors (Tie1 and Tie2). Both are key players governing the generation of blood and lymphatic vessels during embryonic development. Because the growth of new blood and lymphatic vessels (or the lack thereof) is a central element in many diseases, the VEGF and the Tie receptors provide attractive therapeutic targets in various diseases. Indeed, several drugs directed to these RTK signaling pathways are already on the market, whereas many are in clinical trials. Here we review the VEGFR and Tie families, their involvement in developmental and pathological angiogenesis, and the different possibilities for targeting them to either block or enhance angiogenesis and lymphangiogenesis. VL - 5 SN - , 1943-0264 UR - http://cshperspectives.cshlp.org/content/5/9/a009183 IS - 9 JO - Cold Spring Harb Perspect Biol ER - TY - JOUR T1 - Structural and mechanistic insights into VEGF receptor 3 ligand binding and activation JF - Proceedings of the National Academy of Sciences of the United States of America Y1 - 2013 AB - Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key drivers of blood and lymph vessel formation in development, but also in several pathological processes. VEGF-C signaling through VEGFR-3 promotes lymphangiogenesis, which is a clinically relevant target for treating lymphatic insufficiency and for blocking tumor angiogenesis and metastasis. The extracellular domain of VEGFRs consists of seven Ig homology domains; domains 1-3 (D1-3) are responsible for ligand binding, and the membrane-proximal domains 4-7 (D4-7) are involved in structural rearrangements essential for receptor dimerization and activation. Here we analyzed the crystal structures of VEGF-C in complex with VEGFR-3 domains D1-2 and of the VEGFR-3 D4-5 homodimer. The structures revealed a conserved ligand-binding interface in D2 and a unique mechanism for VEGFR dimerization and activation, with homotypic interactions in D5. Mutation of the conserved residues mediating the D5 interaction (Thr446 and Lys516) and the D7 interaction (Arg737) compromised VEGF-C induced VEGFR-3 activation. A thermodynamic analysis of VEGFR-3 deletion mutants showed that D3, D4-5, and D6-7 all contribute to ligand binding. A structural model of the VEGF-C/VEGFR-3 D1-7 complex derived from small-angle X-ray scattering data is consistent with the homotypic interactions in D5 and D7. Taken together, our data show that ligand-dependent homotypic interactions in D5 and D7 are essential for VEGFR activation, opening promising possibilities for the design of VEGFR-specific drugs. VL - 110 SN - 1091-6490 UR - http://www.pnas.org/content/110/32/12960.long IS - 32 JO - Proc. Natl. Acad. Sci. U.S.A. ER - TY - JOUR T1 - A truncation allele in vascular endothelial growth factor c reveals distinct modes of signaling during lymphatic and vascular development. JF - Development Y1 - 2013 AB -

Vascular endothelial growth factor C (Vegfc) is a secreted protein that guides lymphatic development in vertebrate embryos. However, its role during developmental angiogenesis is not well characterized. Here, we identify a mutation in zebrafish vegfc that severely affects lymphatic development and leads to angiogenesis defects on sensitized genetic backgrounds. The um18 mutation prematurely truncated Vegfc, blocking its secretion and paracrine activity but not its ability to activate its receptor Flt4. When expressed in endothelial cells, vegfc(um18) could not rescue lymphatic defects in mutant embryos, but induced ectopic blood vessel branching. Furthermore, vegfc-deficient endothelial cells did not efficiently contribute to tip cell positions in developing sprouts. Computational modeling together with assessment of endothelial cell dynamics by time-lapse analysis suggested that an autocrine Vegfc/Flt4 loop plays an important role in migratory persistence and filopodia stability during sprouting. Our results suggest that Vegfc acts in two distinct modes during development: as a paracrine factor secreted from arteries to guide closely associated lymphatic vasculature and as an autocrine factor to drive migratory persistence during angiogenesis.

VL - 140 IS - 7 ER - TY - JOUR T1 - Critical role of VEGF-C/VEGFR-3 signaling in innate and adaptive immune responses in experimental obliterative bronchiolitis. JF - Am J Pathol Y1 - 2012 AB -

Chronic 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.

VL - 181 IS - 5 ER - TY - JOUR T1 - Structural determinants of vascular endothelial growth factor-D receptor binding and specificity JF - Blood Y1 - 2011 A1 - Leppänen, Veli-Matti A1 - Jeltsch, Michael A1 - Anisimov, Andrey A1 - Tvorogov, Denis A1 - Aho, Kukka A1 - Kalkkinen, Nisse A1 - Toivanen, Pyry A1 - Ylä-Herttuala, Seppo A1 - Ballmer-Hofer, Kurt A1 - Alitalo, Kari AB - Vascular endothelial growth factors (VEGFs) and their tyrosine kinase receptors (VEGFR-1-3) are central mediators of angiogenesis and lymphangiogenesis. VEGFR-3 ligands VEGF-C and VEGF-D are produced as precursor proteins with long N- and C-terminal propeptides and show enhanced VEGFR-2 and VEGFR-3 binding on proteolytic removal of the propeptides. Two different proteolytic cleavage sites have been reported in the VEGF-D N-terminus. We report here the crystal structure of the human VEGF-D Cys117Ala mutant at 2.9 Å resolution. Comparison of the VEGF-D and VEGF-C structures shows similar extended N-terminal helices, conserved overall folds, and VEGFR-2 interacting residues. Consistent with this, the affinity and the thermodynamic parameters for VEGFR-2 binding are very similar. In comparison with VEGF-C structures, however, the VEGF-D N-terminal helix was extended by 2 more turns because of a better resolution. Both receptor binding and functional assays of N-terminally truncated VEGF-D polypeptides indicated that the residues between the reported proteolytic cleavage sites are important for VEGF-D binding and activation of VEGFR-3, but not of VEGFR-2. Thus, we define here a VEGFR-2-specific form of VEGF-D that is angiogenic but not lymphangiogenic. These results provide important new insights into VEGF-D structure and function. VL - 117 UR - http://view.ncbi.nlm.nih.gov/pubmed/21148085 IS - 5 JO - Blood ER - TY - JOUR T1 - Claudin-like protein 24 interacts with the VEGFR-2 and VEGFR-3 pathways and regulates lymphatic vessel development JF - Genes Dev Y1 - 2010 A1 - Saharinen, Pipsa A1 - Helotera, Hanna A1 - Miettinen, Juho A1 - Norrmen, Camilla A1 - D'Amico, Gabriela A1 - Jeltsch, Michael A1 - Langenberg, Tobias A1 - Vandevelde, Wouter A1 - Ny, Annelii A1 - Dewerchin, Mieke A1 - Carmeliet, Peter A1 - Alitalo, Kari AB - The Claudin-like protein of 24 kDa (CLP24) is a hypoxia-regulated transmembrane protein of unknown function. We show here that clp24 knockdown in Danio rerio and Xenopus laevis results in defective lymphatic development. Targeted disruption of Clp24 in mice led to enlarged lymphatic vessels having an abnormal smooth muscle cell coating. We also show that the Clp24(-/-) phenotype was further aggravated in the Vegfr2(+/LacZ) or Vegfr3(+/LacZ) backgrounds and that CLP24 interacts with vascular endothelial growth factor receptor-2 (VEGFR-2) and VEGFR-3 and attenuates the transcription factor CREB phosphorylation via these receptors. Our results indicate that CLP24 is a novel regulator of VEGFR-2 and VEGFR-3 signaling pathways and of normal lymphatic vessel structure. VL - 24 UR - http://view.ncbi.nlm.nih.gov/pubmed/20439428 IS - 9 JO - Genes & Development ER - TY - JOUR T1 - Effective suppression of vascular network formation by combination of antibodies blocking VEGFR ligand binding and receptor dimerization JF - Cancer Cell Y1 - 2010 A1 - Tvorogov, Denis A1 - Anisimov, Andrey A1 - Zheng, Wei A1 - Leppänen, Veli-Matti A1 - Tammela, Tuomas A1 - Laurinavicius, Simonas A1 - Holnthoner, Wolfgang A1 - Heloterä, Hanna A1 - Holopainen, Tanja A1 - Jeltsch, Michael A1 - Kalkkinen, Nisse A1 - Lankinen, Hilkka A1 - Ojala, Päivi M A1 - Alitalo, Kari AB - Antibodies that block vascular endothelial growth factor (VEGF) have become an integral part of antiangiogenic tumor therapy, and antibodies targeting other VEGFs and receptors (VEGFRs) are in clinical trials. Typically receptor-blocking antibodies are targeted to the VEGFR ligand-binding site. Here we describe a monoclonal antibody that inhibits VEGFR-3 homodimer and VEGFR-3/VEGFR-2 heterodimer formation, signal transduction, as well as ligand-induced migration and sprouting of microvascular endothelial cells. Importantly, we show that combined use of antibodies blocking ligand binding and receptor dimerization improves VEGFR inhibition and results in stronger inhibition of endothelial sprouting and vascular network formation in vivo. These results suggest that receptor dimerization inhibitors could be used to enhance antiangiogenic activity of antibodies blocking ligand binding in tumor therapy. VL - 18 UR - http://view.ncbi.nlm.nih.gov/pubmed/21130043 IS - 6 JO - Cancer Cell ER - TY - JOUR T1 - Structural determinants of growth factor binding and specificity by VEGF receptor 2 JF - Proceedings of the National Academy of Sciences of the United States of America Y1 - 2010 A1 - Leppänen, Veli-Matti A1 - Prota, Andrea E A1 - Jeltsch, Michael A1 - Anisimov, Andrey A1 - Kalkkinen, Nisse A1 - Strandin, Tomas A1 - Lankinen, Hilkka A1 - Goldman, Adrian A1 - Ballmer-Hofer, Kurt A1 - Alitalo, Kari AB - Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel formation through activation of three receptor tyrosine kinases, VEGFR-1, -2, and -3. The extracellular domain of VEGF receptors consists of seven immunoglobulin homology domains, which, upon ligand binding, promote receptor dimerization. Dimerization initiates transmembrane signaling, which activates the intracellular tyrosine kinase domain of the receptor. VEGF-C stimulates lymphangiogenesis and contributes to pathological angiogenesis via VEGFR-3. However, proteolytically processed VEGF-C also stimulates VEGFR-2, the predominant transducer of signals required for physiological and pathological angiogenesis. Here we present the crystal structure of VEGF-C bound to the VEGFR-2 high-affinity-binding site, which consists of immunoglobulin homology domains D2 and D3. This structure reveals a symmetrical 22 complex, in which left-handed twisted receptor domains wrap around the 2-fold axis of VEGF-C. In the VEGFs, receptor specificity is determined by an N-terminal alpha helix and three peptide loops. Our structure shows that two of these loops in VEGF-C bind to VEGFR-2 subdomains D2 and D3, while one interacts primarily with D3. Additionally, the N-terminal helix of VEGF-C interacts with D2, and the groove separating the two VEGF-C monomers binds to the D2/D3 linker. VEGF-C, unlike VEGF-A, does not bind VEGFR-1. We therefore created VEGFR-1/VEGFR-2 chimeric proteins to further study receptor specificity. This biochemical analysis, together with our structural data, defined VEGFR-2 residues critical for the binding of VEGF-A and VEGF-C. Our results provide significant insights into the structural features that determine the high affinity and specificity of VEGF/VEGFR interactions. VL - 107 UR - http://view.ncbi.nlm.nih.gov/pubmed/20145116 IS - 6 ER - TY - JOUR T1 - Suppressive effects of vascular endothelial growth factor-B on tumor growth in a mouse model of pancreatic neuroendocrine tumorigenesis JF - PLoS ONE Y1 - 2010 A1 - Albrecht, Imke A1 - Kopfstein, Lucie A1 - Strittmatter, Karin A1 - Schomber, Tibor A1 - Falkevall, Annelie A1 - Hagberg, Carolina E A1 - Lorentz, Pascal A1 - Jeltsch, Michael A1 - Alitalo, Kari A1 - Eriksson, Ulf A1 - Christofori, Gerhard A1 - Pietras, Kristian AB - BACKGROUND: The family of vascular endothelial growth factors (VEGF) contains key regulators of blood and lymph vessel development, including VEGF-A, -B, -C, -D, and placental growth factor. The role of VEGF-B during physiological or pathological angiogenesis has not yet been conclusively delineated. Herein, we investigate the function of VEGF-B by the generation of mouse models of cancer with transgenic expression of VEGF-B or homozygous deletion of Vegfb. METHODOLOGY/PRINCIPAL FINDINGS: Ectopic expression of VEGF-B in the insulin-producing β-cells of the pancreas did not alter the abundance or architecture of the islets of Langerhans. The vasculature from transgenic mice exhibited a dilated morphology, but was of similar density as that of wildtype mice. Unexpectedly, we found that transgenic expression of VEGF-B in the RIP1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis retarded tumor growth. Conversely, RIP1-Tag2 mice deficient for Vegfb presented with larger tumors. No differences in vascular density, perfusion or immune cell infiltration upon altered Vegfb gene dosage were noted. However, VEGF-B acted to increase blood vessel diameter both in normal pancreatic islets and in RIP1-Tag2 tumors. CONCLUSIONS/SIGNIFICANCE: Taken together, our results illustrate the differences in biological function between members of the VEGF family, and highlight the necessity of in-depth functional studies of VEGF-B to fully understand the effects of VEGFR-1 inhibitors currently used in the clinic. VL - 5 UR - http://view.ncbi.nlm.nih.gov/pubmed/21124841 IS - 11 JO - PLoS ONE ER - TY - JOUR T1 - Vascular endothelial growth factor-B acts as a coronary growth factor in transgenic rats without inducing angiogenesis, vascular leak, or inflammation JF - Circulation Y1 - 2010 A1 - Bry, Maija A1 - Kivelä, Riikka A1 - Holopainen, Tanja A1 - Anisimov, Andrey A1 - Tammela, Tuomas A1 - Soronen, Jarkko A1 - Silvola, Johanna A1 - Saraste, Antti A1 - Jeltsch, Michael A1 - Korpisalo, Petra A1 - Carmeliet, Peter A1 - Lemström, Karl B A1 - Shibuya, Masabumi A1 - Ylä-Herttuala, Seppo A1 - Alhonen, Leena A1 - Mervaala, Eero A1 - Andersson, Leif C A1 - Knuuti, Juhani A1 - Alitalo, Kari AB - BACKGROUND: Vascular endothelial growth factor-B (VEGF-B) binds to VEGF receptor-1 and neuropilin-1 and is abundantly expressed in the heart, skeletal muscle, and brown fat. The biological function of VEGF-B is incompletely understood. METHODS AND RESULTS: Unlike placenta growth factor, which binds to the same receptors, adeno-associated viral delivery of VEGF-B to mouse skeletal or heart muscle induced very little angiogenesis, vascular permeability, or inflammation. As previously reported for the VEGF-B(167) isoform, transgenic mice and rats expressing both isoforms of VEGF-B in the myocardium developed cardiac hypertrophy yet maintained systolic function. Deletion of the VEGF receptor-1 tyrosine kinase domain or the arterial endothelial Bmx tyrosine kinase inhibited hypertrophy, whereas loss of VEGF-B interaction with neuropilin-1 had no effect. Surprisingly, in rats, the heart-specific VEGF-B transgene induced impressive growth of the epicardial coronary vessels and their branches, with large arteries also seen deep inside the subendocardial myocardium. However, VEGF-B, unlike other VEGF family members, did not induce significant capillary angiogenesis, increased permeability, or inflammatory cell recruitment. CONCLUSIONS: VEGF-B appears to be a coronary growth factor in rats but not in mice. The signals for the VEGF-B-induced cardiac hypertrophy are mediated at least in part via the endothelium. Because cardiomyocyte damage in myocardial ischemia begins in the subendocardial myocardium, the VEGF-B-induced increased arterial supply to this area could have therapeutic potential in ischemic heart disease. VL - 122 UR - http://view.ncbi.nlm.nih.gov/pubmed/20937974 IS - 17 JO - Circulation ER - TY - JOUR T1 - Activated forms of VEGF-C and VEGF-D provide improved vascular function in skeletal muscle JF - Circ Res Y1 - 2009 A1 - Anisimov, Andrey A1 - Alitalo, Annamari A1 - Korpisalo, Petra A1 - Soronen, Jarkko A1 - Kaijalainen, Seppo A1 - Leppänen, Veli-Matti A1 - Jeltsch, Michael A1 - Ylä-Herttuala, Seppo A1 - Alitalo, Kari AB - The therapeutic potential of vascular endothelial growth factor (VEGF)-C and VEGF-D in skeletal muscle has been of considerable interest as these factors have both angiogenic and lymphangiogenic activities. Previous studies have mainly used adenoviral gene delivery for short-term expression of VEGF-C and VEGF-D in pig, rabbit, and mouse skeletal muscles. Here we have used the activated mature forms of VEGF-C and VEGF-D expressed via recombinant adeno-associated virus (rAAV), which provides stable, long-lasting transgene expression in various tissues including skeletal muscle. Mouse tibialis anterior muscle was transduced with rAAV encoding human or mouse VEGF-C or VEGF-D. Two weeks later, immunohistochemical analysis showed increased numbers of both blood and lymph vessels, and Doppler ultrasound analysis indicated increased blood vessel perfusion. The lymphatic vessels further increased at the 4-week time point were functional, as shown by FITC-lectin uptake and transport. Furthermore, receptor activation and arteriogenic activity were increased by an alanine substitution mutant of human VEGF-C (C137A) having an increased dimer stability and by a chimeric CAC growth factor that contained the VEGF receptor-binding domain flanked by VEGF-C propeptides, but only the latter promoted significantly more blood vessel perfusion when compared to the other growth factors studied. We conclude that long-term expression of VEGF-C and VEGF-D in skeletal muscle results in the generation of new functional blood and lymphatic vessels. The therapeutic value of intramuscular lymph vessels in draining tissue edema and lymphedema can now be evaluated using this model system. VL - 104 UR - http://view.ncbi.nlm.nih.gov/pubmed/19443835 IS - 11 JO - Circulation Research ER - TY - JOUR T1 - Overexpression of vascular endothelial growth factor-B in mouse heart alters cardiac lipid metabolism and induces myocardial hypertrophy JF - Circ Res Y1 - 2008 A1 - Karpanen, Terhi A1 - Bry, Maija A1 - Ollila, Hanna M A1 - Seppänen-Laakso, Tuulikki A1 - Liimatta, Erkki A1 - Leskinen, Hanna A1 - Kivelä, Riikka A1 - Helkamaa, Teemu A1 - Merentie, Mari A1 - Jeltsch, Michael A1 - Paavonen, Karri A1 - Andersson, Leif C A1 - Mervaala, Eero A1 - Hassinen, Ilmo E A1 - Ylä-Herttuala, Seppo A1 - Oresic, Matej A1 - Alitalo, Kari AB - Vascular endothelial growth factor (VEGF)-B is poorly angiogenic but prominently expressed in metabolically highly active tissues, including the heart. We produced mice expressing a cardiac-specific VEGF-B transgene via the alpha-myosin heavy chain promoter. Surprisingly, the hearts of the VEGF-B transgenic mice showed concentric cardiac hypertrophy without significant changes in heart function. The cardiac hypertrophy was attributable to an increased size of the cardiomyocytes. Blood capillary size was increased, whereas the number of blood vessels per cell nucleus remained unchanged. Despite the cardiac hypertrophy, the transgenic mice had lower heart rate and blood pressure than their littermates, and they responded similarly to angiotensin II-induced hypertension, confirming that the hypertrophy does not compromise heart function. Interestingly, the isolated transgenic hearts had less cardiomyocyte damage after ischemia. Significantly increased ceramide and decreased triglyceride levels were found in the transgenic hearts. This was associated with structural changes and eventual lysis of mitochondria, resulting in accumulation of intracellular vacuoles in cardiomyocytes and increased death of the transgenic mice, apparently because of mitochondrial lipotoxicity in the heart. These results suggest that VEGF-B regulates lipid metabolism, an unexpected function for an angiogenic growth factor. VL - 103 UR - http://view.ncbi.nlm.nih.gov/pubmed/18757827 IS - 9 JO - Circulation Research ER - TY - JOUR T1 - Reevaluation of the role of VEGF-B suggests a restricted role in the revascularization of the ischemic myocardium JF - Arterioscler Thromb Vasc Biol Y1 - 2008 A1 - Li, Xuri A1 - Tjwa, Marc A1 - Van Hove, Inge A1 - Enholm, Berndt A1 - Neven, Elke A1 - Paavonen, Karri A1 - Jeltsch, Michael A1 - Juan, Toni Diez A1 - Sievers, Richard E A1 - Chorianopoulos, Emmanuel A1 - Wada, Hiromichi A1 - Vanwildemeersch, Maarten A1 - Noel, Agnes A1 - Foidart, Jean-Michel A1 - Springer, Matthew L A1 - von Degenfeld, Georges A1 - Dewerchin, Mieke A1 - Blau, Helen M A1 - Alitalo, Kari A1 - Eriksson, Ulf A1 - Carmeliet, Peter A1 - Moons, Lieve AB - OBJECTIVE: The endogenous role of the VEGF family member vascular endothelial growth factor-B (VEGF-B) in pathological angiogenesis remains unclear. METHODS AND RESULTS: We studied the role of VEGF-B in various models of pathological angiogenesis using mice lacking VEGF-B (VEGF-B(-/-)) or overexpressing VEGF-B(167). After occlusion of the left coronary artery, VEGF-B deficiency impaired vessel growth in the ischemic myocardium whereas, in wild-type mice, VEGF-B(167) overexpression enhanced revascularization of the infarct and ischemic border zone. By contrast, VEGF-B deficiency did not affect vessel growth in the wounded skin, hypoxic lung, ischemic retina, or ischemic limb. Moreover, VEGF-B(167) overexpression failed to enhance vascular growth in the skin or ischemic limb. CONCLUSIONS: VEGF-B appears to have a relatively restricted angiogenic activity in the ischemic heart. These insights might offer novel therapeutic opportunities. VL - 28 UR - http://view.ncbi.nlm.nih.gov/pubmed/18511699 IS - 9 JO - Arteriosclerosis, Thrombosis, and Vascular Biology ER - TY - JOUR T1 - The tyrosine kinase inhibitor cediranib blocks ligand-induced vascular endothelial growth factor receptor-3 activity and lymphangiogenesis JF - Cancer Res Y1 - 2008 A1 - Heckman, Caroline A A1 - Holopainen, Tanja A1 - Wirzenius, Maria A1 - Keskitalo, Salla A1 - Jeltsch, Michael A1 - Ylä-Herttuala, Seppo A1 - Wedge, Stephen R A1 - Jürgensmeier, Juliane M A1 - Alitalo, Kari AB - Solid tumors express a range of factors required to sustain their growth and promote their dissemination. Among these are vascular endothelial growth factor-A (VEGF-A), the key angiogenic stimulant, and VEGF-C, a primary mediator of lymphangiogenesis. Small molecule tyrosine kinase inhibitors offer the potential to inhibit more than one kinase and impede tumor growth by multiple mechanisms. However, their potency toward individual targets can vary. Cediranib (RECENTIN; AZD2171) is an inhibitor of VEGF signaling that has been shown in experimental models to prevent VEGF-A-induced angiogenesis and primary tumor growth, yet the effects of cediranib on VEGF receptor (VEGFR)-3-mediated endothelial cell function and lymphangiogenesis are unknown. To better understand the activity of cediranib against VEGFR-3 and its associated signaling events compared with its activity against VEGFR-2, we used the receptor-specific ligands VEGF-E and VEGF-C156S. In human endothelial cells, cediranib inhibited VEGF-E-induced phosphorylation of VEGFR-2 and VEGF-C156S-induced phosphorylation of VEGFR-3 at concentrations of