@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 {426,
	title = {The basis for the distinct biological activities of vascular endothelial growth factor receptor-1 ligands},
	journal = {Sci Signal},
	volume = {6},
	year = {2013},
	month = {2013},
	pages = {ra52},
	abstract = {<p>Vascular endothelial growth factors (VEGFs) regulate blood and lymphatic vessel development through VEGF receptors (VEGFRs). The VEGFR immunoglobulin homology domain 2 (D2) is critical for ligand binding, and D3 provides additional interaction sites. VEGF-B and placenta growth factor (PlGF) bind to VEGFR-1 with high affinity, but only PlGF is angiogenic in most tissues. We show that VEGF-B, unlike other VEGFs, did not require D3 interactions for high-affinity binding. VEGF-B with a PlGF-derived L1 loop (B-L1(P)) stimulated VEGFR-1 activity, whereas PlGF with a VEGF-B-derived L1 loop (P-L1(B)) did not. Unlike P-L1(B) and VEGF-B, B-L1(P) and PlGF were also angiogenic in mouse skeletal muscle. Furthermore, B-L1(P) also bound to VEGFR-2 and activated downstream signaling. These results establish a role for L1-mediated D3 interactions in VEGFR activation in endothelial cells and indicate that VEGF-B is a high-affinity VEGFR-1 ligand that, unlike PlGF, cannot efficiently induce signaling downstream of VEGFR-1.</p>},
	keywords = {PlGF, receptor tyrosine kinase, Signal Transduction, VEGF-B, VEGFR-1},
	issn = {1937-9145},
	doi = {10.1126/scisignal.2003905},
	author = {Anisimov, Andrey and Lepp{\"a}nen, Veli-Matti and Tvorogov, Denis and Zarkada, Georgia and Jeltsch, Michael and Holopainen, Tanja and Kaijalainen, Seppo and Alitalo, Kari}
}
@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 {43,
	title = {Vascular endothelial growth factor-B acts as a coronary growth factor in transgenic rats without inducing angiogenesis, vascular leak, or inflammation},
	journal = {Circulation},
	volume = {122},
	year = {2010},
	month = {2010/Oct/},
	pages = {1725 - 33},
	abstract = {BACKGROUND: Vascular endothelial growth factor-B (VEGF-B) binds to VEGF receptor-1 and neuropilin-1 and is abundantly expressed in the heart, skeletal muscle, and brown fat. The biological function of VEGF-B is incompletely understood. METHODS AND RESULTS: Unlike placenta growth factor, which binds to the same receptors, adeno-associated viral delivery of VEGF-B to mouse skeletal or heart muscle induced very little angiogenesis, vascular permeability, or inflammation. As previously reported for the VEGF-B(167) isoform, transgenic mice and rats expressing both isoforms of VEGF-B in the myocardium developed cardiac hypertrophy yet maintained systolic function. Deletion of the VEGF receptor-1 tyrosine kinase domain or the arterial endothelial Bmx tyrosine kinase inhibited hypertrophy, whereas loss of VEGF-B interaction with neuropilin-1 had no effect. Surprisingly, in rats, the heart-specific VEGF-B transgene induced impressive growth of the epicardial coronary vessels and their branches, with large arteries also seen deep inside the subendocardial myocardium. However, VEGF-B, unlike other VEGF family members, did not induce significant capillary angiogenesis, increased permeability, or inflammatory cell recruitment. CONCLUSIONS: VEGF-B appears to be a coronary growth factor in rats but not in mice. The signals for the VEGF-B-induced cardiac hypertrophy are mediated at least in part via the endothelium. Because cardiomyocyte damage in myocardial ischemia begins in the subendocardial myocardium, the VEGF-B-induced increased arterial supply to this area could have therapeutic potential in ischemic heart disease.},
	url = {http://view.ncbi.nlm.nih.gov/pubmed/20937974},
	author = {Bry, Maija and Kivel{\"a}, Riikka and Holopainen, Tanja and Anisimov, Andrey and Tammela, Tuomas and Soronen, Jarkko and Silvola, Johanna and Saraste, Antti and Jeltsch, Michael and Korpisalo, Petra and Carmeliet, Peter and Lemstr{\"o}m, Karl B and Shibuya, Masabumi and Yl{\"a}-Herttuala, Seppo and Alhonen, Leena and Mervaala, Eero and Andersson, Leif C and Knuuti, Juhani and Alitalo, Kari}
}
@article {35,
	title = {The tyrosine kinase inhibitor cediranib blocks ligand-induced vascular endothelial growth factor receptor-3 activity and lymphangiogenesis},
	journal = {Cancer Res},
	volume = {68},
	year = {2008},
	month = {2008/Jun/},
	pages = {4754 - 62},
	abstract = {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.},
	url = {http://view.ncbi.nlm.nih.gov/pubmed/18559522},
	author = {Heckman, Caroline A and Holopainen, Tanja and Wirzenius, Maria and Keskitalo, Salla and Jeltsch, Michael and Yl{\"a}-Herttuala, Seppo and Wedge, Stephen R and J{\"u}rgensmeier, Juliane M and Alitalo, Kari}
}
@conference {625,
	title = {Inhibiton of VEGF-C-induced VEGFR-3 activity and lymphatic endothelial cell function by the tyrosine kinase inhibitor AZD2171},
	booktitle = {98th Annual Meeting of the American-Association-for-Cancer-Research},
	year = {2007},
	month = {2007///},
	publisher = {American Association for Cancer Research},
	organization = {American Association for Cancer Research},
	address = {Los Angeles, CA},
	abstract = {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},
	url = {http://dx.doi.org/10.1158/0008-5472.CAN-07-5809},
	author = {Heckman, Caroline A. and Holopainen, Tanja and Wirzenius, Maria and Keskitalo, Salla and Jeltsch, Michael and Wedge, Stephen R. and Jurgensmeier, Juliane M.}
}
@article {30,
	title = {Vascular endothelial cell growth factor receptor 3-mediated activation of lymphatic endothelium is crucial for tumor cell entry and spread via lymphatic vessels},
	journal = {Cancer Res},
	volume = {65},
	year = {2005},
	month = {2005/Jun/},
	pages = {4739 - 46},
	abstract = {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.},
	url = {http://view.ncbi.nlm.nih.gov/pubmed/15930292},
	author = {He, Yulong and Rajantie, Iiro and Pajusola, Katri and Jeltsch, Michael and Holopainen, Tanja and Yla-Herttuala, Seppo and Harding, Thomas and Jooss, Karin and Takahashi, Takashi and Alitalo, Kari}
}
