@article {441,
	title = {CCBE1 enhances lymphangiogenesis via ADAMTS3-mediated VEGF-C activation},
	journal = {Circulation},
	volume = {129},
	year = {2014},
	month = {05/2014},
	chapter = {1962-1971},
	abstract = {Background{\textemdash}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{\textemdash}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{\textemdash}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.},
	keywords = {ADAMTS3, angiogenesis, CCBE1, endothelium, growth factors and cytokines, Hennekam Syndrome, metalloproteinase, vasculature, VEGF-C},
	doi = {http://dx.doi.org/10.1161/CIRCULATIONAHA.113.002779},
	url = {http://circ.ahajournals.org/content/early/2014/02/19/CIRCULATIONAHA.113.002779.abstract},
	author = {Jeltsch, Michael and Jha, Sawan Kumar and Tvorogov, Denis and Anisimov, Andrey and Lepp{\"a}nen, Veli-Matti and Holopainen, Tanja and Kivel{\"a}, Riikka and Ortega, Sagrario and K{\"a}rp{\"a}nen, Terhi and Alitalo, Kari}
}
@article {424,
	title = {Vascular endothelial growth factor-angiopoietin chimera with improved properties for therapeutic angiogenesis},
	journal = {Circulation},
	volume = {127},
	year = {2013},
	month = {01/2013},
	pages = {424-434},
	chapter = {424},
	abstract = {BACKGROUND:

There is an unmet need for proangiogenic therapeutic molecules for the treatment of tissue ischemia in cardiovascular diseases. However, major inducers of angiogenesis such as vascular endothelial growth factor (VEGF/VEGF-A) have side effects that limit their therapeutic utility in vivo, especially at high concentrations. Angiopoietin-1 has been considered to be a blood vessel stabilization factor that can inhibit the intrinsic property of VEGF to promote vessel leakiness. In this study, we have designed and tested the angiogenic properties of chimeric molecules consisting of receptor-binding parts of VEGF and angiopoietin-1. We aimed at combining the activities of both factors into 1 molecule for easy delivery and expression in target tissues.
METHODS AND RESULTS:

The VEGF-angiopoietin-1 (VA1) chimeric protein bound to both VEGF receptor-2 and Tie2 and induced the activation of both receptors. Detailed analysis of VA1 versus VEGF revealed differences in the kinetics of VEGF receptor-2 activation and endocytosis, downstream kinase activation, and VE-cadherin internalization. The delivery of a VA1 transgene into mouse skeletal muscle led to increased blood flow and enhanced angiogenesis. VA1 was also very efficient in rescuing ischemic limb perfusion. However, VA1 induced less plasma protein leakage and myeloid inflammatory cell recruitment than VEGF. Furthermore, angioma-like structures associated with VEGF expression were not observed with VA1.
CONCLUSIONS:

The VEGF-angiopoietin-1 chimera is a potent angiogenic factor that triggers a novel mode of VEGF receptor-2 activation, promoting less vessel leakiness, less tissue inflammation, and better perfusion in ischemic muscle than VEGF. These properties of VA1 make it an attractive therapeutic tool.},
	author = {Andrey Anisimov and Denis Tvorogov and Annamari Alitalo and Veli-Matti Lepp{\"a}nen and Y An and EC Han and F Orsenigo and EI Ga{\'a}l and Tanja Holopainen and YJ Koh and Tuomas Tammela and P Korpisalo and Salla Keskitalo and Michael Jeltsch and Seppo Yl{\"a}-Herttuala and Elisabetta Dejana and GY Koh and C Choi and Pipsa Saharinen and Kari Alitalo}
}
@article {42,
	title = {Effective suppression of vascular network formation by combination of antibodies blocking VEGFR ligand binding and receptor dimerization},
	journal = {Cancer Cell},
	volume = {18},
	year = {2010},
	month = {2010/Dec/},
	pages = {630 - 40},
	abstract = {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.},
	url = {http://view.ncbi.nlm.nih.gov/pubmed/21130043},
	author = {Tvorogov, Denis and Anisimov, Andrey and Zheng, Wei and Lepp{\"a}nen, Veli-Matti and Tammela, Tuomas and Laurinavicius, Simonas and Holnthoner, Wolfgang and Heloter{\"a}, Hanna and Holopainen, Tanja and Jeltsch, Michael and Kalkkinen, Nisse and Lankinen, Hilkka and Ojala, P{\"a}ivi M and Alitalo, Kari}
}
@article {37,
	title = {Overexpression of vascular endothelial growth factor-B in mouse heart alters cardiac lipid metabolism and induces myocardial hypertrophy},
	journal = {Circ Res},
	volume = {103},
	year = {2008},
	month = {2008/Oct/},
	pages = {1018 - 26},
	abstract = {Vascular endothelial growth factor (VEGF)-B is poorly angiogenic but prominently expressed in metabolically highly active tissues, including the heart. We produced mice expressing a cardiac-specific VEGF-B transgene via the alpha-myosin heavy chain promoter. Surprisingly, the hearts of the VEGF-B transgenic mice showed concentric cardiac hypertrophy without significant changes in heart function. The cardiac hypertrophy was attributable to an increased size of the cardiomyocytes. Blood capillary size was increased, whereas the number of blood vessels per cell nucleus remained unchanged. Despite the cardiac hypertrophy, the transgenic mice had lower heart rate and blood pressure than their littermates, and they responded similarly to angiotensin II-induced hypertension, confirming that the hypertrophy does not compromise heart function. Interestingly, the isolated transgenic hearts had less cardiomyocyte damage after ischemia. Significantly increased ceramide and decreased triglyceride levels were found in the transgenic hearts. This was associated with structural changes and eventual lysis of mitochondria, resulting in accumulation of intracellular vacuoles in cardiomyocytes and increased death of the transgenic mice, apparently because of mitochondrial lipotoxicity in the heart. These results suggest that VEGF-B regulates lipid metabolism, an unexpected function for an angiogenic growth factor.},
	url = {http://view.ncbi.nlm.nih.gov/pubmed/18757827},
	author = {Karpanen, Terhi and Bry, Maija and Ollila, Hanna M and Sepp{\"a}nen-Laakso, Tuulikki and Liimatta, Erkki and Leskinen, Hanna and Kivel{\"a}, Riikka and Helkamaa, Teemu and Merentie, Mari and Jeltsch, Michael and Paavonen, Karri and Andersson, Leif C and Mervaala, Eero and Hassinen, Ilmo E and Yl{\"a}-Herttuala, Seppo and Oresic, Matej and Alitalo, Kari}
}
@article {31,
	title = {Functional interaction of VEGF-C and VEGF-D with neuropilin receptors},
	journal = {FASEB J},
	volume = {20},
	year = {2006},
	month = {2006/Jul/},
	pages = {1462 - 72},
	abstract = {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.},
	url = {http://view.ncbi.nlm.nih.gov/pubmed/16816121},
	author = {K{\"a}rp{\"a}nen, Terhi and Heckman, Caroline A and Keskitalo, Salla and Jeltsch, Michael and Ollila, Hanna and Neufeld, Gera and Tamagnone, Luca and Alitalo, Kari}
}
@article {23,
	title = {Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis},
	journal = {EMBO J},
	volume = {20},
	year = {2001},
	month = {2001/Feb/},
	pages = {672 - 82},
	abstract = {Metastasis is a frequent and lethal complication of cancer. Vascular endothelial growth factor-C (VEGF-C) is a recently described lymphangiogenic factor. Increased expression of VEGF-C in primary tumours correlates with dissemination of tumour cells to regional lymph nodes. However, a direct role for VEGF-C in tumour lymphangiogenesis and subsequent metastasis has yet to be demonstrated. Here we report the establishment of transgenic mice in which VEGF-C expression, driven by the rat insulin promoter (Rip), is targeted to beta-cells of the endocrine pancreas. In contrast to wild-type mice, which lack peri-insular lymphatics, RipVEGF-C transgenics develop an extensive network of lymphatics around the islets of Langerhans. These mice were crossed with Rip1Tag2 mice, which develop pancreatic beta-cell tumours that are neither lymphangiogenic nor metastatic. Double-transgenic mice formed tumours surrounded by well developed lymphatics, which frequently contained tumour cell masses of beta-cell origin. These mice frequently developed pancreatic lymph node metastases. Our findings demonstrate that VEGF-C-induced lymphangiogenesis mediates tumour cell dissemination and the formation of lymph node metastases.},
	url = {http://view.ncbi.nlm.nih.gov/pubmed/11179212},
	author = {Mandriota, S J and Jussila, L and Jeltsch, M and Compagni, A and Baetens, D and Prevo, R and Banerji, S and Huarte, J and Montesano, R and Jackson, D G and Orci, L and Alitalo, K and Christofori, G and Pepper, M S}
}
@article {19,
	title = {Current biology of VEGF-B and VEGF-C},
	journal = {Curr Opin Biotechnol},
	volume = {10},
	year = {1999},
	month = {1999/Dec/},
	pages = {528 - 35},
	abstract = {Endothelial growth factors and their receptors may provide important therapeutic tools for the treatment of pathological conditions characterised by defective or aberrant angiogenesis. Vascular endothelial growth factor (VEGF) is pivotal for vasculogenesis and for angiogenesis in normal and pathological conditions. VEGF-B and VEGF-C provide this gene family with additional functions, for example, VEGF-C also regulates lymphangiogenesis.},
	url = {http://view.ncbi.nlm.nih.gov/pubmed/10600689},
	author = {Olofsson, B and Jeltsch, M and Eriksson, U and Alitalo, K}
}
@article {17,
	title = {Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells},
	journal = {Proc Natl Acad Sci U S A},
	volume = {95},
	year = {1998},
	month = {1998/Sep/},
	pages = {11709 - 14},
	abstract = {The vascular endothelial growth factor (VEGF) family has recently expanded by the identification and cloning of three additional members, namely VEGF-B, VEGF-C, and VEGF-D. In this study we demonstrate that VEGF-B binds selectively to VEGF receptor-1/Flt-1. This binding can be blocked by excess VEGF, indicating that the interaction sites on the receptor are at least partially overlapping. Mutating the putative VEGF receptor-1/Flt-1 binding determinants Asp63, Asp64, and Glu67 to alanine residues in VEGF-B reduced the affinity to VEGF receptor-1 but did not abolish binding. Mutational analysis of conserved cysteines contributing to VEGF-B dimer formation suggest a structural conservation with VEGF and platelet-derived growth factor. Proteolytic processing of the 60-kDa VEGF-B186 dimer results in a 34-kDa dimer containing the receptor-binding epitopes. The binding of VEGF-B to its receptor on endothelial cells leads to increased expression and activity of urokinase type plasminogen activator and plasminogen activator inhibitor 1, suggesting a role for VEGF-B in the regulation of extracellular matrix degradation, cell adhesion, and migration.},
	url = {http://view.ncbi.nlm.nih.gov/pubmed/9751730},
	author = {Olofsson, B and Korpelainen, E and Pepper, M S and Mandriota, S J and Aase, K and Kumar, V and Gunji, Y and Jeltsch, M M and Shibuya, M and Alitalo, K and Eriksson, U}
}
@article {12,
	title = {Vascular endothelial growth factors VEGF-B and VEGF-C},
	journal = {J Cell Physiol},
	volume = {173},
	year = {1997},
	month = {1997/Nov/},
	pages = {211 - 5},
	url = {http://view.ncbi.nlm.nih.gov/pubmed/9365524},
	author = {Joukov, V and Kaipainen, A and Jeltsch, M and Pajusola, K and Olofsson, B and Kumar, V and Eriksson, U and Alitalo, K}
}
@article {11,
	title = {VEGF and VEGF-C: specific induction of angiogenesis and lymphangiogenesis in the differentiated avian chorioallantoic membrane},
	journal = {Dev Biol},
	volume = {188},
	year = {1997},
	month = {1997/Aug/},
	pages = {96 - 109},
	abstract = {The lymphangiogenic potency of endothelial growth factors has not been studied to date. This is partially due to the lack of in vivo lymphangiogenesis assays. We have studied the lymphatics of differentiated avian chorioallantoic membrane (CAM) using microinjection of Mercox resin, semi- and ultrathin sectioning, immunohistochemical detection of fibronectin and alpha-smooth muscle actin, and in situ hybridization with VEGFR-2 and VEGFR-3 probes. CAM is drained by lymphatic vessels which are arranged in a regular pattern. Arterioles and arteries are accompanied by a pair of interconnected lymphatics and form a plexus around bigger arteries. Veins are also associated with lymphatics, particularly larger veins, which are surrounded by a lymphatic plexus. The lymphatics are characterized by an extremely thin endothelial lining, pores, and the absence of a basal lamina. Patches of the extracellular matrix can be stained with an antibody against fibronectin. Lymphatic endothelial cells of differentiated CAM show ultrastructural features of this cell type. CAM lymphatics do not possess mediae. In contrast, the lymphatic trunks of the umbilical stalk are invested by a single but discontinuous layer of smooth muscle cells. CAM lymphatics express VEGFR-2 and VEGFR-3. Both the regular pattern and the typical structure of these lymphatics suggest that CAM is a suitable site to study the in vivo effects of potential lymphangiogenic factors. We have studied the effects of VEGF homo- and heterodimers, VEGF/PlGF heterodimers, and PlGF and VEGF-C homodimers on Day 13 CAM. All the growth factors containing at least one VEGF chain are angiogenic but do not induce lymphangiogenesis. PlGF-1 and PlGF-2 are neither angiogenic nor lymphangiogenic. VEGF-C is the first lymphangiogenic factor and seems to be highly chemoattractive for lymphatic endothelial cells. It induces proliferation of lymphatic endothelial cells and development of new lymphatic sinuses which are directed immediately beneath the chorionic epithelium. Our studies show that VEGF and VEGF-C are specific angiogenic and lymphangiogenic growth factors, respectively.},
	url = {http://view.ncbi.nlm.nih.gov/pubmed/9245515},
	author = {Oh, S J and Jeltsch, M M and Birkenh{\"a}ger, R and McCarthy, J E and Weich, H A and Christ, B and Alitalo, K and Wilting, J}
}
