@techreport{bask_voiko_2021,
	address = {Helsinki},
	type = {Introduction to working life internship report},
	title = {Voiko 3d-tulostuksessa kytetyist muovimateriaaleista valmistettuja astioita käyttää solujen viljelyssä?},
	url = {https://bin.yhdistysavain.fi/1603755/ycRmgkivDwYtomTTG6ZN0VT-0G/1069%20Voiko%203d-tulostuksessa%20kytetyist%20muovimateriaaleista%20valmis.pdf},
	language = {Finnish},
	institution = {Käpylän Peruskoulu},
	author = {Bask, Pessi and Pouta, Rasmus and Siljander, Okko},
	year = {2021},
}

@article{kunnapuu_proteolytic_2021,
	title = {Proteolytic {Cleavages} in the {VEGF} {Family}: {Generating} {Diversity} among {Angiogenic} {VEGFs}, {Essential} for the {Activation} of {Lymphangiogenic} {VEGFs}},
	volume = {10},
	issn = {2079-7737},
	shorttitle = {Proteolytic {Cleavages} in the {VEGF} {Family}},
	url = {https://www.mdpi.com/2079-7737/10/2/167},
	doi = {10.3390/biology10020167},
	abstract = {Specific proteolytic cleavages turn on, modify, or turn off the activity of vascular endothelial growth factors (VEGFs). Proteolysis is most prominent among the lymph­angiogenic VEGF-C and VEGF-D, which are synthesized as precursors that need to undergo enzymatic removal of their C- and N-terminal propeptides before they can activate their receptors. At least five different proteases mediate the activating cleavage of VEGF-C: plasmin, ADAMTS3, prostate-specific antigen, cathepsin D, and thrombin. All of these proteases except for ADAMTS3 can also activate VEGF-D. Processing by different proteases results in distinct forms of the “mature” growth factors, which differ in affinity and receptor activation potential. The “default” VEGF-C-activating enzyme ADAMTS3 does not activate VEGF-D, and therefore, VEGF-C and VEGF-D do function in different contexts. VEGF-C itself is also regulated in different contexts by distinct proteases. During embryonic development, ADAMTS3 activates VEGF-C. The other activating proteases are likely important for non-developmental lymphangiogenesis during, e.g., tissue regeneration, inflammation, immune response, and pathological tumor-associated lymphangiogenesis. The better we understand these events at the molecular level, the greater our chances of developing successful therapies targeting VEGF-C and VEGF-D for diseases involving the lymphatics such as lymphedema or cancer.},
	language = {en},
	number = {2},
	urldate = {2021-03-10},
	journal = {Biology},
	author = {Künnapuu, Jaana and Bokharaie, Honey and Jeltsch, Michael},
	month = feb,
	year = {2021},
	pages = {167},
}

Specific proteolytic cleavages turn on, modify, or turn off the activity of vascular endothelial growth factors (VEGFs). Proteolysis is most prominent among the lymph­angiogenic VEGF-C and VEGF-D, which are synthesized as precursors that need to undergo enzymatic removal of their C- and N-terminal propeptides before they can activate their receptors. At least five different proteases mediate the activating cleavage of VEGF-C: plasmin, ADAMTS3, prostate-specific antigen, cathepsin D, and thrombin. All of these proteases except for ADAMTS3 can also activate VEGF-D. Processing by different proteases results in distinct forms of the “mature” growth factors, which differ in affinity and receptor activation potential. The “default” VEGF-C-activating enzyme ADAMTS3 does not activate VEGF-D, and therefore, VEGF-C and VEGF-D do function in different contexts. VEGF-C itself is also regulated in different contexts by distinct proteases. During embryonic development, ADAMTS3 activates VEGF-C. The other activating proteases are likely important for non-developmental lymphangiogenesis during, e.g., tissue regeneration, inflammation, immune response, and pathological tumor-associated lymphangiogenesis. The better we understand these events at the molecular level, the greater our chances of developing successful therapies targeting VEGF-C and VEGF-D for diseases involving the lymphatics such as lymphedema or cancer.

@techreport{rosich_just_generating_2020,
	address = {Barcelona},
	title = {Generating a fluorescence-tagged growth factor for in vivo detection and localization},
	institution = {University of Barcelona},
	author = {Rosich Just, Pau},
	month = sep,
	year = {2020},
}

@article{gucciardo_lymphatics_2020,
	title = {Lymphatics and the eye. [{Finnish}]},
	volume = {136},
	issn = {2242-3281},
	url = {https://www.duodecimlehti.fi/lehti/2020/16/duo15739},
	doi = {10.5281/zenodo.4005517},
	abstract = {The lymphatic system, fundamental for body fluid homeostasis and immune system functions, also participates in pathological processes including cancer, cardiovascular and neurodegenerative diseases. Many aspects of the lymphatic system are unclear, but great advancements have been made – e.g. the discovery of meningeal lymphatics and the lymphatic-like nature of Schlemm’s canal. Lymphatic-like structures were also recently discovered in proliferative diabetic retinopathy, a severe diabetic eye complication, where the treatment of pathological neovessel growth is not always effective. Novel findings in the field can help to develop new treatments for lymphatic disorders and for other diseases where lymphatic neovascular growth is involved.},
	language = {Finnish},
	number = {16},
	journal = {Duodecim Lääketieteellinen Aikakauskirja},
	author = {Gucciardo, Erika and Lehti, Timo A. and Korhonen, Ani and Salvén, Petri and Lehti, Kaisa and Jeltsch, Michael and Loukovaara, Sirpa},
	month = aug,
	year = {2020},
	pages = {1777--88},
}

The lymphatic system, fundamental for body fluid homeostasis and immune system functions, also participates in pathological processes including cancer, cardiovascular and neurodegenerative diseases. Many aspects of the lymphatic system are unclear, but great advancements have been made – e.g. the discovery of meningeal lymphatics and the lymphatic-like nature of Schlemm’s canal. Lymphatic-like structures were also recently discovered in proliferative diabetic retinopathy, a severe diabetic eye complication, where the treatment of pathological neovessel growth is not always effective. Novel findings in the field can help to develop new treatments for lymphatic disorders and for other diseases where lymphatic neovascular growth is involved.

@article{fang_vegf-c_2020,
	title = {{VEGF}-{C} protects the integrity of the bone marrow perivascular niche in mice},
	volume = {136},
	issn = {1528-0020},
	doi = {10.1182/blood.2020005699},
	abstract = {Hematopoietic stem cells (HSCs) reside in the bone marrow (BM) stem cell niche, which provides a vital source of HSC regulatory signals. Radiation and chemotherapy disrupt the HSC niche, including its sinusoidal vessels and perivascular cells, contributing to delayed hematopoietic recovery. Thus, identification of factors that can protect the HSC niche during an injury could offer a significant therapeutic opportunity to improve hematopoietic regeneration. In this study, we identified a critical function for vascular endothelial growth factor-C (VEGF-C), that of maintaining the integrity of the BM perivascular niche and improving BM niche recovery after irradiation-induced injury. Both global and conditional deletion of Vegfc in endothelial or leptin receptor-positive (LepR+) cells led to a disruption of the BM perivascular niche. Furthermore, deletion of Vegfc from the microenvironment delayed hematopoietic recovery after transplantation by decreasing endothelial proliferation and LepR+ cell regeneration. Exogenous administration of VEGF-C via an adenoassociated viral vector improved hematopoietic recovery after irradiation by accelerating endothelial and LepR+ cell regeneration and by increasing the expression of hematopoietic regenerative factors. Our results suggest that preservation of the integrity of the perivascular niche via VEGF-C signaling could be exploited therapeutically to enhance hematopoietic regeneration.},
	language = {eng},
	number = {16},
	journal = {Blood},
	author = {Fang, Shentong and Chen, Shuo and Nurmi, Harri and Leppänen, Veli-Matti and Jeltsch, Michael and Scadden, David and Silberstein, Lev and Mikkola, Hanna and Alitalo, Kari},
	month = oct,
	year = {2020},
	pmid = {32842144},
	pmcid = {PMC7568034},
	pages = {1871--1883},
}

Hematopoietic stem cells (HSCs) reside in the bone marrow (BM) stem cell niche, which provides a vital source of HSC regulatory signals. Radiation and chemotherapy disrupt the HSC niche, including its sinusoidal vessels and perivascular cells, contributing to delayed hematopoietic recovery. Thus, identification of factors that can protect the HSC niche during an injury could offer a significant therapeutic opportunity to improve hematopoietic regeneration. In this study, we identified a critical function for vascular endothelial growth factor-C (VEGF-C), that of maintaining the integrity of the BM perivascular niche and improving BM niche recovery after irradiation-induced injury. Both global and conditional deletion of Vegfc in endothelial or leptin receptor-positive (LepR+) cells led to a disruption of the BM perivascular niche. Furthermore, deletion of Vegfc from the microenvironment delayed hematopoietic recovery after transplantation by decreasing endothelial proliferation and LepR+ cell regeneration. Exogenous administration of VEGF-C via an adenoassociated viral vector improved hematopoietic recovery after irradiation by accelerating endothelial and LepR+ cell regeneration and by increasing the expression of hematopoietic regenerative factors. Our results suggest that preservation of the integrity of the perivascular niche via VEGF-C signaling could be exploited therapeutically to enhance hematopoietic regeneration.

@article{mukenge_investigation_2020,
	title = {Investigation on the {Role} of {Biallelic} {Variants} in {VEGF}-{C} {Found} in a {Patient} {Affected} by {Milroy}-like {Lymphedema}},
	volume = {8},
	url = {https://onlinelibrary.wiley.com/doi/full/10.1002/mgg3.1389},
	doi = {10.1002/mgg3.1389},
	number = {9},
	journal = {Molecular Genetics \& Genomic Medicine},
	author = {Mukenge, Sylvain and Jha, Sawan Kumar and Catena, Marco and Manara, Elena and Leppänen, Veli-Matti and Lenti, Elisa and Negrini, Daniela and Bertelli, Matteo and Brendolan, Andrea and Jeltsch, Michael and Aldrighetti, Luca},
	month = jun,
	year = {2020},
	pages = {e1389},
}

@phdthesis{jha_mechanism_2020,
	address = {Helsinki, Finland},
	type = {Doctoral {Thesis}},
	title = {Mechanism of {VEGF}-{C} {Activation} and {Effect} on {Lymphatic} {Vessel} {Growth} and {Regeneration}},
	copyright = {Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.},
	url = {https://helda.helsinki.fi/handle/10138/314714},
	abstract = {Lymphangiogenesis, the growth of the lymphatic vasculature, is a crucial process during embryonic development, and - if compromised by genetic damage - can lead to hereditary lymphedema. Although the molecular mechanisms that regulate the growth, development, and maintenance of the lymphatic vasculature have been researched with increasing intensity over the last 25 years, the therapeutic regeneration of lymphatic vessels is still a work in progress in the treatment of conditions such as lymphedema. Vascular endothelial growth factor-C (VEGF-C) is the primary growth factor responsible for the growth and development of the lymphatic vasculature. VEGF-C is activated by a complex process, which is indispensable for its ability to induce lymphangiogenesis via its primary receptor, VEGFR-3. The understanding of this process is a key factor for the development of VEGF-C as a drug target. The goal in my studies has been to increase our insights into VEGF-C activation at the molecular level, to identify its regulatory factors, and to establish its role in the lymphangiogenic process. 
 
Absence of the collagen- and calcium-binding EGF domains 1 (CCBE1) protein interrupts the lymphangiogenic process at about the same developmental stage when VEGF-C is first required. We utilized cell-based assays and adeno-associated viral-based gene transduction to investigate the role of CCBE1 on VEGF-C activation. In study I, we identified A disintegrin and metalloprotease with thrombospondin motifs-3 (ADAMTS3) as a protease that cleaves and activates VEGF-C, resulting in the major mature form of VEGF-C. We showed that CCBE1 acts as a cofactor in this process by enhancing the ability of ADAMTS3 to activate VEGF-C. Correspondingly, CCBE1 augmented the lymphangiogenic potential of VEGF-C in vivo.  
 
The presence of N- and C- terminal domains and their proteolytic cleavage characterize both CCBE1 and VEGF-C. In study II, we investigated the role of these domains for VEGF-C activation and the lymphangiogenic process. Our study demonstrated a requirement for the C-terminal domain of VEGF-C for the robust activation of VEGF-C both in vitro and in vivo. Moreover, we identified that the N- and C-terminal domains of CCBE1 have independent roles in the process of VEGF-C activation. The C-terminal domain accelerates the proteolytic cleavage, while the N-terminal domain aids in the assembly of the VEGF-C/ADAMTS3/CCBE1 cleavage complex by mobilizing VEGF-C to the endothelial cell surface. 
 
In study III, we searched for additional proteases that can cleave VEGF-C. We identified kallikrein-related peptidase 3 (KLK3) in seminal plasma and cathepsin D in saliva as proteases that cleave and activate VEGF-C. In human seminal plasma, we found substantial amounts of VEGF-C, which became activated concurrently with the semen liquefaction process. The newly identified VEGF-C cleavage sites are conserved in VEGF-D and we found that KLK3 and cathepsin D were able to activate VEGF-D as well. We also found that cleaved forms of VEGF-C and VEGF-D differ in their abilities to activate VEGFR-2 and VEGFR-3. When their N-termini were progressively shortened, the ability of VEGF-D to bind to and activate VEGFR-3 was decreased, while VEGF-C lost preferentially its ability to bind to and activate VEGFR-2. 
 
These findings contribute to the existing knowledge on the mechanisms of VEGF-C activation and the functional consequences thereof, and provide new opportunities to target VEGF-C for therapeutic purposes.},
	language = {en},
	urldate = {2020-06-01},
	school = {University of Helsinki},
	author = {Jha, Sawan Kumar},
	month = may,
	year = {2020},
	note = {Accepted: 2020-05-07T05:17:24Z
ISBN: 9789515160454
Publisher: Helsingin yliopisto},
}

Lymphangiogenesis, the growth of the lymphatic vasculature, is a crucial process during embryonic development, and - if compromised by genetic damage - can lead to hereditary lymphedema. Although the molecular mechanisms that regulate the growth, development, and maintenance of the lymphatic vasculature have been researched with increasing intensity over the last 25 years, the therapeutic regeneration of lymphatic vessels is still a work in progress in the treatment of conditions such as lymphedema. Vascular endothelial growth factor-C (VEGF-C) is the primary growth factor responsible for the growth and development of the lymphatic vasculature. VEGF-C is activated by a complex process, which is indispensable for its ability to induce lymphangiogenesis via its primary receptor, VEGFR-3. The understanding of this process is a key factor for the development of VEGF-C as a drug target. The goal in my studies has been to increase our insights into VEGF-C activation at the molecular level, to identify its regulatory factors, and to establish its role in the lymphangiogenic process. Absence of the collagen- and calcium-binding EGF domains 1 (CCBE1) protein interrupts the lymphangiogenic process at about the same developmental stage when VEGF-C is first required. We utilized cell-based assays and adeno-associated viral-based gene transduction to investigate the role of CCBE1 on VEGF-C activation. In study I, we identified A disintegrin and metalloprotease with thrombospondin motifs-3 (ADAMTS3) as a protease that cleaves and activates VEGF-C, resulting in the major mature form of VEGF-C. We showed that CCBE1 acts as a cofactor in this process by enhancing the ability of ADAMTS3 to activate VEGF-C. Correspondingly, CCBE1 augmented the lymphangiogenic potential of VEGF-C in vivo. The presence of N- and C- terminal domains and their proteolytic cleavage characterize both CCBE1 and VEGF-C. In study II, we investigated the role of these domains for VEGF-C activation and the lymphangiogenic process. Our study demonstrated a requirement for the C-terminal domain of VEGF-C for the robust activation of VEGF-C both in vitro and in vivo. Moreover, we identified that the N- and C-terminal domains of CCBE1 have independent roles in the process of VEGF-C activation. The C-terminal domain accelerates the proteolytic cleavage, while the N-terminal domain aids in the assembly of the VEGF-C/ADAMTS3/CCBE1 cleavage complex by mobilizing VEGF-C to the endothelial cell surface. In study III, we searched for additional proteases that can cleave VEGF-C. We identified kallikrein-related peptidase 3 (KLK3) in seminal plasma and cathepsin D in saliva as proteases that cleave and activate VEGF-C. In human seminal plasma, we found substantial amounts of VEGF-C, which became activated concurrently with the semen liquefaction process. The newly identified VEGF-C cleavage sites are conserved in VEGF-D and we found that KLK3 and cathepsin D were able to activate VEGF-D as well. We also found that cleaved forms of VEGF-C and VEGF-D differ in their abilities to activate VEGFR-2 and VEGFR-3. When their N-termini were progressively shortened, the ability of VEGF-D to bind to and activate VEGFR-3 was decreased, while VEGF-C lost preferentially its ability to bind to and activate VEGFR-2. These findings contribute to the existing knowledge on the mechanisms of VEGF-C activation and the functional consequences thereof, and provide new opportunities to target VEGF-C for therapeutic purposes.

@article{lackner_proteolytic_2019,
	title = {The {Proteolytic} {Activation} of {Vascular} {Endothelial} {Growth} {Factor}-{C}},
	volume = {23},
	url = {https://doi.org/10.5281/zenodo.3629263},
	doi = {10.5281/zenodo.3629263},
	number = {2},
	journal = {Lymphologie in Forschung und Praxis},
	author = {Lackner, Marcel and Schmotz, Constanze and Jeltsch, Michael},
	month = dec,
	year = {2019},
	note = {https://www.dglymph.de/fileadmin/user\_upload/Lymph\_02-19\_online.pdf},
	pages = {88--98},
}

@article{jha_klk3/psa_2019,
	title = {{KLK3}/{PSA} and cathepsin {D} activate {VEGF}-{C} and {VEGF}-{D}},
	volume = {8},
	issn = {2050-084X},
	url = {https://elifesciences.org/articles/44478},
	doi = {10.7554/eLife.44478},
	abstract = {Vascular endothelial growth factor-C (VEGF-C) acts primarily on endothelial cells, but also on non-vascular targets, e.g. in the CNS and immune system. Here we describe a novel, unique VEGF-C form in the human reproductive system produced via cleavage by kallikrein-related peptidase 3 (KLK3), aka prostate-specific antigen (PSA). KLK3 activated VEGF-C specifically and efficiently through cleavage at a novel N-terminal site. We detected VEGF-C in seminal plasma, and sperm liquefaction occurred concurrently with VEGF-C activation, which was enhanced by collagen and calcium binding EGF domains 1 (CCBE1). After plasmin and ADAMTS3, KLK3 is the third protease shown to activate VEGF-C. Since differently activated VEGF-Cs are characterized by successively shorter N-terminal helices, we created an even shorter hypothetical form, which showed preferential binding to VEGFR-3. Using mass spectrometric analysis of the isolated VEGF-C-cleaving activity from human saliva, we identified cathepsin D as a protease that can activate VEGF-C as well as VEGF-D.},
	language = {en},
	urldate = {2019-05-18},
	journal = {eLife},
	author = {Jha, Sawan Kumar and Rauniyar, Khushbu and Chronowska, Ewa and Mattonet, Kenny and Maina, Eunice Wairimu and Koistinen, Hannu and Stenman, Ulf-Håkan and Alitalo, Kari and Jeltsch, Michael},
	month = may,
	year = {2019},
	pages = {e44478},
}

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.

@article{jeltsch_was_2018,
	title = {Was man in der {Lymphologie} über {VEGF}-{C} wissen sollte [{What} you need to know as a lymphologist about {VEGF}-{C}]},
	volume = {30},
	issn = {0942-1181},
	url = {https://www.der-niedergelassene-arzt.de/suche/ergebnis/suche/was-man-in-der-lymphologie-ueber-vegf-c-wissen-sollte},
	language = {German},
	number = {4},
	journal = {Vasomed},
	author = {Jeltsch, Michael},
	month = jul,
	year = {2018},
	pages = {172--173},
}

@article{rauniyar_biology_2018,
	title = {Biology of {Vascular} {Endothelial} {Growth} {Factor} {C} in the {Morphogenesis} of {Lymphatic} {Vessels}},
	volume = {6},
	issn = {2296-4185},
	url = {https://www.frontiersin.org/articles/10.3389/fbioe.2018.00007/full?&utm_source=Email_to_authors_&utm_medium=Email&utm_content=T1_11.5e1_author&utm_campaign=Email_publication&field=&journalName=Frontiers_in_Bioengineering_and_Biotechnology&id=326326},
	doi = {10.3389/fbioe.2018.00007},
	abstract = {Because virtually all tissues contain blood vessels, the importance of hemevascularization has been long recognized in regenerative medicine and tissue engineering. However, the lymphatic vasculature has only recently become a subject of interest. Central to the task of growing a lymphatic network are lymphatic endothelial cells (LECs), which constitute the innermost layer of all lymphatic vessels. The central molecule that directs proliferation and migration of LECs during embryogenesis is Vascular Endothelial Growth Factor-C (VEGF-C). VEGF-C is, therefore, an important ingredient for LEC culture and attempts to (re)generate lymphatic vessels and networks. During its biosynthesis, VEGF-C undergoes a stepwise proteolytic processing, during which its properties and affinities for its interaction partners change. Many of these fundamental aspects of VEGF-C biosynthesis have only recently been uncovered. So far, most - if not all - applications of VEGF-C do not discriminate between different forms of VEGF-C. However, for lymphatic regeneration and engineering purposes, it appears mandatory to understand these differences, since they relate e.g. to such important aspects as biodistribution and receptor activation potential. In this review, we discuss the molecular biology of VEGF-C as it relates to the growth of LECs and lymphatic vessels. However, the properties of VEGF-C are similarly relevant for the cardiovascular system, since both old and recent data show that VEGF-C can have a profound effect on the blood vasculature.},
	language = {English},
	urldate = {2018-02-12},
	journal = {Frontiers in Bioengineering and Biotechnology},
	author = {Rauniyar, Khushbu and Jha, Sawan Kumar and Jeltsch, Michael},
	month = feb,
	year = {2018},
	keywords = {ADAMTS3, CCBE1, Lymphatic Vessels, Lymphedema, Tissue Engineering, VEGF receptors, VEGF-C, growth factor signaling, growth factors, proteolytic processing},
	pages = {7},
}

Because virtually all tissues contain blood vessels, the importance of hemevascularization has been long recognized in regenerative medicine and tissue engineering. However, the lymphatic vasculature has only recently become a subject of interest. Central to the task of growing a lymphatic network are lymphatic endothelial cells (LECs), which constitute the innermost layer of all lymphatic vessels. The central molecule that directs proliferation and migration of LECs during embryogenesis is Vascular Endothelial Growth Factor-C (VEGF-C). VEGF-C is, therefore, an important ingredient for LEC culture and attempts to (re)generate lymphatic vessels and networks. During its biosynthesis, VEGF-C undergoes a stepwise proteolytic processing, during which its properties and affinities for its interaction partners change. Many of these fundamental aspects of VEGF-C biosynthesis have only recently been uncovered. So far, most - if not all - applications of VEGF-C do not discriminate between different forms of VEGF-C. However, for lymphatic regeneration and engineering purposes, it appears mandatory to understand these differences, since they relate e.g. to such important aspects as biodistribution and receptor activation potential. In this review, we discuss the molecular biology of VEGF-C as it relates to the growth of LECs and lymphatic vessels. However, the properties of VEGF-C are similarly relevant for the cardiovascular system, since both old and recent data show that VEGF-C can have a profound effect on the blood vasculature.

@article{jha_key_2018,
	title = {Key molecules in lymphatic development, function, and identification},
	volume = {219},
	issn = {09409602},
	url = {http://linkinghub.elsevier.com/retrieve/pii/S0940960218300712},
	doi = {10.1016/j.aanat.2018.05.003},
	abstract = {While both blood and lymphatic vessels transport fluids and thus share many similarities, they also show functional and structural differences, which can be used to differentiate them. Specific visualization of lymphatic vessels has historically been and still is a pivot point in lymphatic research. Many of the proteins that are investigated by molecular biologists in lymphatic research have been defined as marker molecules, i.e. to visualize and distinguish lymphatic endothelial cells (LECs) from other cell types, most notably from blood vascular endothelial cells (BECs) and cells of the hematopoietic lineage.

Among the factors that drive the developmental differentiation of lymphatic structures from venous endothelium, Prospero homeobox protein 1 (PROX1) is the master transcriptional regulator. PROX1 maintains lymphatic identity also in the adult organism and thus is a universal LEC marker. Vascular endothelial growth factor receptor-3 (VEGFR-3) is the major tyrosine kinase receptor that drives LEC proliferation and migration. The major activator for VEGFR-3 is vascular endothelial growth factor-C (VEGF-C). However, before VEGF-C can signal, it needs to be proteolytically activated by an extracellular protein complex comprised of Collagen and calcium binding EGF domains 1 (CCBE1) protein and the protease A disintegrin and metallopeptidase with thrombospondin type 1 motif 3 (ADAMTS3).

This minireview attempts to give an overview of these and a few other central proteins that scientific inquiry has linked specifically to the lymphatic vasculature. It is limited in scope to a brief description of their main functions, properties and developmental roles.},
	language = {en},
	urldate = {2018-06-08},
	journal = {Annals of Anatomy - Anatomischer Anzeiger},
	author = {Jha, Sawan Kumar and Rauniyar, Khushbu and Jeltsch, Michael},
	month = sep,
	year = {2018},
	pages = {25--34},
}

While both blood and lymphatic vessels transport fluids and thus share many similarities, they also show functional and structural differences, which can be used to differentiate them. Specific visualization of lymphatic vessels has historically been and still is a pivot point in lymphatic research. Many of the proteins that are investigated by molecular biologists in lymphatic research have been defined as marker molecules, i.e. to visualize and distinguish lymphatic endothelial cells (LECs) from other cell types, most notably from blood vascular endothelial cells (BECs) and cells of the hematopoietic lineage. Among the factors that drive the developmental differentiation of lymphatic structures from venous endothelium, Prospero homeobox protein 1 (PROX1) is the master transcriptional regulator. PROX1 maintains lymphatic identity also in the adult organism and thus is a universal LEC marker. Vascular endothelial growth factor receptor-3 (VEGFR-3) is the major tyrosine kinase receptor that drives LEC proliferation and migration. The major activator for VEGFR-3 is vascular endothelial growth factor-C (VEGF-C). However, before VEGF-C can signal, it needs to be proteolytically activated by an extracellular protein complex comprised of Collagen and calcium binding EGF domains 1 (CCBE1) protein and the protease A disintegrin and metallopeptidase with thrombospondin type 1 motif 3 (ADAMTS3). This minireview attempts to give an overview of these and a few other central proteins that scientific inquiry has linked specifically to the lymphatic vasculature. It is limited in scope to a brief description of their main functions, properties and developmental roles.

@article{jha_efficient_2017,
	title = {Efficient activation of the lymphangiogenic growth factor {VEGF}-{C} requires the {C}-terminal domain of {VEGF}-{C} and the {N}-terminal domain of {CCBE1}},
	volume = {7},
	copyright = {2017 The Author(s)},
	issn = {2045-2322},
	url = {https://www.nature.com/articles/s41598-017-04982-1},
	doi = {10.1038/s41598-017-04982-1},
	abstract = {The collagen- and calcium-binding EGF domains 1 (CCBE1) protein is necessary for lymphangiogenesis. Its C-terminal collagen-like domain was shown to be required for the activation of the major lymphangiogenic growth factor VEGF-C (Vascular Endothelial Growth Factor-C) along with the ADAMTS3 (A Disintegrin And Metalloproteinase with Thrombospondin Motifs-3) protease. However, it remained unclear how the N-terminal domain of CCBE1 contributed to lymphangiogenic signaling. Here, we show that efficient activation of VEGF-C requires its C-terminal domain both in vitro and in a transgenic mouse model. The N-terminal EGF-like domain of CCBE1 increased VEGFR-3 signaling by colocalizing pro-VEGF-C with its activating protease to the lymphatic endothelial cell surface. When the ADAMTS3 amounts were limited, proteolytic activation of pro-VEGF-C was supported by the N-terminal domain of CCBE1, but not by its C-terminal domain. A single amino acid substitution in ADAMTS3, identified from a lymphedema patient, was associated with abnormal CCBE1 localization. These results show that CCBE1 promotes VEGFR-3 signaling and lymphangiogenesis by different mechanisms, which are mediated independently by the two domains of CCBE1: by enhancing the cleavage activity of ADAMTS3 and by facilitating the colocalization of VEGF-C and ADAMTS3. These new insights should be valuable in developing new strategies to therapeutically target VEGF-C/VEGFR-3-induced lymphangiogenesis.},
	language = {En},
	number = {1},
	urldate = {2017-07-07},
	journal = {Scientific Reports},
	author = {Jha, Sawan Kumar and Rauniyar, Khushbu and Karpanen, Terhi and Leppänen, Veli-Matti and Brouillard, Pascal and Vikkula, Miikka and Alitalo, Kari and Jeltsch, Michael},
	month = jul,
	year = {2017},
	pages = {4916},
}

The collagen- and calcium-binding EGF domains 1 (CCBE1) protein is necessary for lymphangiogenesis. Its C-terminal collagen-like domain was shown to be required for the activation of the major lymphangiogenic growth factor VEGF-C (Vascular Endothelial Growth Factor-C) along with the ADAMTS3 (A Disintegrin And Metalloproteinase with Thrombospondin Motifs-3) protease. However, it remained unclear how the N-terminal domain of CCBE1 contributed to lymphangiogenic signaling. Here, we show that efficient activation of VEGF-C requires its C-terminal domain both in vitro and in a transgenic mouse model. The N-terminal EGF-like domain of CCBE1 increased VEGFR-3 signaling by colocalizing pro-VEGF-C with its activating protease to the lymphatic endothelial cell surface. When the ADAMTS3 amounts were limited, proteolytic activation of pro-VEGF-C was supported by the N-terminal domain of CCBE1, but not by its C-terminal domain. A single amino acid substitution in ADAMTS3, identified from a lymphedema patient, was associated with abnormal CCBE1 localization. These results show that CCBE1 promotes VEGFR-3 signaling and lymphangiogenesis by different mechanisms, which are mediated independently by the two domains of CCBE1: by enhancing the cleavage activity of ADAMTS3 and by facilitating the colocalization of VEGF-C and ADAMTS3. These new insights should be valuable in developing new strategies to therapeutically target VEGF-C/VEGFR-3-induced lymphangiogenesis.

@article{mattonet_heterogeneity_2015,
	title = {Heterogeneity of the origin of the lymphatic system. [{German}].},
	volume = {19},
	issn = {1433-5255},
	url = {http://www.dglymph.de/fileadmin/global/pdfs/LymphForsch_2-15.pdf},
	doi = {10.5281/zenodo.4786280},
	abstract = {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.},
	number = {2},
	journal = {Lymphologie in Forschung und Praxis},
	author = {Mattonet, Kenny and Jeltsch, Michael},
	month = dec,
	year = {2015},
	pages = {84--88},
}

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.

@incollection{mattonet_genetischen_2015,
	address = {Cologne, Germany},
	edition = {6.},
	title = {Die genetischen {Ursachen} des primären {Lymphödems}},
	url = {https://www.der-niedergelassene-arzt.de/fileadmin/user_upload/Buecher/Leseproben/Leseprobe_Kap._5.10_Erkr._Lymph_6.pdf},
	abstract = {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 geneti- schen Ursachen gefunden, diagnostiziert und gezielt behandelt werden. Die hierzu notwendigen Techniken liefern die neuen Entwicklungen in der Molekularbiologie. Insbesondere durch die Technik der Exom-Sequenzie- rung wurden in den letzten Jahren die genetischen Ursachen vieler primä- rer 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 molekularbiologi- schen und biochemischen Grundlagen primärer Lymphödeme.},
	language = {German},
	booktitle = {Erkrankungen des {Lymphgefäßsystems}},
	publisher = {Viavital Verlag},
	author = {Mattonet, Kenny and Wilting, Jörg and Jeltsch, Michael},
	editor = {Weissleder, Horst and Schuchhardt, Christian},
	month = aug,
	year = {2015},
	pages = {210--229},
}

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 geneti- schen Ursachen gefunden, diagnostiziert und gezielt behandelt werden. Die hierzu notwendigen Techniken liefern die neuen Entwicklungen in der Molekularbiologie. Insbesondere durch die Technik der Exom-Sequenzie- rung wurden in den letzten Jahren die genetischen Ursachen vieler primä- rer 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 molekularbiologi- schen und biochemischen Grundlagen primärer Lymphödeme.

@article{batchu_substrate_2015,
	title = {Substrate {Efflux} {Propensity} {Is} the {Key} {Determinant} of {Ca2}+-independent {Phospholipase} {A}-β ({iPLAβ})-mediated {Glycerophospholipid} {Hydrolysis}},
	volume = {290},
	issn = {0021-9258, 1083-351X},
	url = {http://www.jbc.org/content/290/16/10093},
	doi = {10.1074/jbc.M115.642835},
	abstract = {The A-type phospholipases (PLAs) are key players in glycerophospholipid (GPL) homeostasis and in mammalian cells; Ca2+-independent PLA-β (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 whether 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 sn-1 and sn-2 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.},
	language = {en},
	number = {16},
	urldate = {2015-06-15},
	journal = {Journal of Biological Chemistry},
	author = {Batchu, Krishna Chaithanya and Hokynar, Kati and Jeltsch, Michael and Mattonet, Kenny and Somerharju, Pentti},
	month = apr,
	year = {2015},
	pmid = {25713085},
	keywords = {Homeostasis, Phospholipase A, mass spectrometry (MS), membrane, membrane bilayer},
	pages = {10093--10103},
}

The A-type phospholipases (PLAs) are key players in glycerophospholipid (GPL) homeostasis and in mammalian cells; Ca2+-independent PLA-β (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 whether 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 sn-1 and sn-2 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.

@article{roukens_functional_2015,
	title = {Functional {Dissection} of the {CCBE1} {Protein}. {A} {Crucial} {Requirement} for the {Collagen} {Repeat} {Domain}},
	volume = {116},
	issn = {0009-7330, 1524-4571},
	url = {http://circres.ahajournals.org/content/116/10/1660},
	doi = {10.1161/CIRCRESAHA.116.304949},
	abstract = {Rationale: Collagen- and calcium-binding EGF domain–containing protein 1 (CCBE1) is essential for lymphangiogenesis in vertebrates and has been associated with Hennekam syndrome. Recently, CCBE1 has emerged as a crucial regulator of vascular endothelial growth factor-C (VEGFC) signaling.
Objective: CCBE1 is a secreted protein characterized by 2 EGF domains and 2 collagen repeats. The functional role of the different CCBE1 protein domains is completely unknown. Here, we analyzed the functional role of the different CCBE1 domains in vivo and in vitro.
Methods and Results: We analyzed the functionality of several CCBE1 deletion mutants by generating knock-in mice expressing these mutants, by analyzing their ability to enhance Vegfc signaling in vivo in zebrafish, and by testing their ability to induce VEGFC processing in vitro. We found that deleting the collagen domains of CCBE1 has a much stronger effect on CCBE1 activity than deleting the EGF domains. First, although CCBE1ΔCollagen mice fully phenocopy CCBE1 knock-out mice, CCBE1ΔEGF knock-in embryos still form rudimentary lymphatics. Second, Ccbe1ΔEGF, but not Ccbe1ΔCollagen, could partially substitute for Ccbe1 to enhance Vegfc signaling in zebrafish. Third, CCBE1ΔEGF, similarly to CCBE1, but not CCBE1ΔCollagen could activate VEGFC processing in vitro. Furthermore, a Hennekam syndrome mutation within the collagen domain has a stronger effect than a Hennekam syndrome mutation within the EGF domain.
Conclusions: We propose that the collagen domains of CCBE1 are crucial for the activation of VEGFC in vitro and in vivo. The EGF domains of CCBE1 are dispensable for regulation of VEGFC processing in vitro, however, they are necessary for full lymphangiogenic activity of CCBE1 in vivo.},
	language = {en},
	number = {10},
	urldate = {2015-06-15},
	journal = {Circulation Research},
	author = {Roukens, M. Guy and Peterson-Maduro, Josi and Padberg, Yvonne and Jeltsch, Michael and Leppänen, Veli-Matti and Bos, Frank L. and Alitalo, Kari and Schulte-Merker, Stefan and Schulte, Dörte},
	month = may,
	year = {2015},
	pmid = {25814692},
	keywords = {CCBE1 protein, Endothelium, Vascular, Hennekam lymphangiectasia-lymphedema syndrome, lymphangiogenesis, vascular endothelial growth factor},
	pages = {1660--1669},
}

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.

@article{jeltsch_ccbe1_2014,
	title = {{CCBE1} {Enhances} {Lymphangiogenesis} via {A} {Disintegrin} and {Metalloprotease} {With} {Thrombospondin} {Motifs}-3–{Mediated} {Vascular} {Endothelial} {Growth} {Factor}-{C} {Activation}},
	volume = {129},
	copyright = {OK},
	issn = {0009-7322, 1524-4539},
	url = {https://www.ahajournals.org/doi/10.1161/CIRCULATIONAHA.113.002779},
	doi = {10.1161/CIRCULATIONAHA.113.002779},
	abstract = {Background—Hennekam lymphangiectasia–lymphedema syndrome (Online Mendelian Inheritance in Man 235510) is a rare autosomal recessive disease, which is associated with mutations in the 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 collagen- and calcium-binding epidermal growth factor domains 1 (CCBE1) interactions with the vascular endothelial growth factor-C (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, whereas 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 protease, resulting in the mature 21/23-kDa form of VEGF-C, which induces increased VEGF-C receptor signaling. Adeno-associated viral vector–mediated transduction of CCBE1 into mouse skeletal muscle enhanced lymphangiogenesis and angiogenesis induced by adeno-associated viral vector–VEGF-C.
Conclusions—These results identify A disintegrin and metalloprotease with thrombospondin motifs-3 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 that 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.},
	language = {en},
	number = {19},
	urldate = {2014-05-31},
	journal = {Circulation},
	author = {Jeltsch, Michael and Jha, Sawan Kumar and Tvorogov, Denis and Anisimov, Andrey and Leppänen, Veli-Matti and Holopainen, Tanja and Kivelä, Riikka and Ortega, Sagrario and Kärpanen, Terhi and Alitalo, Kari},
	month = may,
	year = {2014},
	pmid = {24552833},
	keywords = {angiogenesis effect, endothelium, growth substances, metalloproteinases},
	pages = {1962--1971},
}

Background—Hennekam lymphangiectasia–lymphedema syndrome (Online Mendelian Inheritance in Man 235510) is a rare autosomal recessive disease, which is associated with mutations in the 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 collagen- and calcium-binding epidermal growth factor domains 1 (CCBE1) interactions with the vascular endothelial growth factor-C (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, whereas 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 protease, resulting in the mature 21/23-kDa form of VEGF-C, which induces increased VEGF-C receptor signaling. Adeno-associated viral vector–mediated transduction of CCBE1 into mouse skeletal muscle enhanced lymphangiogenesis and angiogenesis induced by adeno-associated viral vector–VEGF-C. Conclusions—These results identify A disintegrin and metalloprotease with thrombospondin motifs-3 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 that 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.

@misc{jeltsch_disease_2014,
	title = {The disease they call fat - scientist/researcher episode 9: {Michael} {Jeltsch}},
	url = {https://diseasetheycallfat.lipedemaproject.org/product-tag/michael-jeltsch/},
	language = {en-US},
	urldate = {2019-05-03},
	author = {Jeltsch, Michael},
	month = mar,
	year = {2014},
	note = {https://www.imdb.com/title/tt5212746/},
}

@article{leppanen_structural_2013,
	title = {Structural and mechanistic insights into {VEGF} receptor 3 ligand binding and activation},
	volume = {110},
	issn = {0027-8424, 1091-6490},
	url = {http://www.pnas.org/content/110/32/12960},
	doi = {10.1073/pnas.1301415110},
	abstract = {Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key drivers of blood and lymph vessel formation in development, but also in several pathological processes. VEGF-C signaling through VEGFR-3 promotes lymphangiogenesis, which is a clinically relevant target for treating lymphatic insufficiency and for blocking tumor angiogenesis and metastasis. The extracellular domain of VEGFRs consists of seven Ig homology domains; domains 1–3 (D1-3) are responsible for ligand binding, and the membrane-proximal domains 4–7 (D4-7) are involved in structural rearrangements essential for receptor dimerization and activation. Here we analyzed the crystal structures of VEGF-C in complex with VEGFR-3 domains D1-2 and of the VEGFR-3 D4-5 homodimer. The structures revealed a conserved ligand-binding interface in D2 and a unique mechanism for VEGFR dimerization and activation, with homotypic interactions in D5. Mutation of the conserved residues mediating the D5 interaction (Thr446 and Lys516) and the D7 interaction (Arg737) compromised VEGF-C induced VEGFR-3 activation. A thermodynamic analysis of VEGFR-3 deletion mutants showed that D3, D4-5, and D6-7 all contribute to ligand binding. A structural model of the VEGF-C/VEGFR-3 D1-7 complex derived from small-angle X-ray scattering data is consistent with the homotypic interactions in D5 and D7. Taken together, our data show that ligand-dependent homotypic interactions in D5 and D7 are essential for VEGFR activation, opening promising possibilities for the design of VEGFR-specific drugs.},
	language = {en},
	number = {32},
	urldate = {2013-12-18},
	journal = {Proceedings of the National Academy of Sciences of the United States of America},
	author = {Leppänen, Veli-Matti and Tvorogov, Denis and Kisko, Kaisa and Prota, Andrea E. and Jeltsch, Michael and Anisimov, Andrey and Markovic-Mueller, Sandra and Stuttfeld, Edward and Goldie, Kenneth N. and Ballmer-Hofer, Kurt and Alitalo, Kari},
	month = aug,
	year = {2013},
	pmid = {23878260},
	keywords = {Receptor tyrosine kinase, signal transduction},
	pages = {12960--12965},
}

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.

@article{krebs_lymphangiogenen_2013,
	title = {Die lymphangiogenen {Wachstumsfaktoren} {VEGF}-{C} und {VEGF}-{D}. {Teil} 2. {Die} {Rolle} von {VEGF}-{C} und {VEGF}-{D} bei {Krankheiten} des {Lymphgefäßsystems}.},
	volume = {17},
	url = {http://jeltsch.org/sites/jeltsch.org/files/JeltschMichael_Lymphforsch2013_96.pdf},
	doi = {10.5281/zenodo.4438655},
	abstract = {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.},
	language = {German},
	number = {2},
	journal = {Lymphologie in Forschung und Praxis},
	author = {Krebs, Rainer and Jeltsch, Michael},
	month = dec,
	year = {2013},
	keywords = {VEGF-D, growth factors, lymphangiogenesis, lymphedema, lymphogenic metastasis, vegf-c},
	pages = {96--104},
}

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.

@article{krebs_lymphangiogenen_2013-1,
	title = {Die lymphangiogenen {Wachstumsfaktoren} {VEGF}-{C} und {VEGF}-{D}. {Teil} 1. {Grundlagen} und {Embryonalentwicklung}.},
	volume = {17},
	url = {http://jeltsch.org/sites/jeltsch.org/files/JeltschMichael_Lymphforsch2013_30.pdf},
	doi = {10.5281/zenodo.3611691},
	abstract = {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.},
	language = {German},
	number = {1},
	journal = {Lymphologie in Forschung und Praxis},
	author = {Krebs, Rainer and Jeltsch, Michael},
	month = jun,
	year = {2013},
	keywords = {VEGF-D, growth factors, lymphangiogenesis, vegf-c},
	pages = {30--37},
}

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.

@article{krebs_lymphangiogenic_2013,
	title = {The lymphangiogenic growth factors {VEGF}-{C} and {VEGF}-{D}. {Part} 1: {Fundamentals} and embryonic development},
	volume = {25},
	shorttitle = {Die lymphangiogenen wachstumsfaktoren {VEGF}-{C} und {VEGF}-{D} - {Teil} 1: {Grundlagen} und embryonalentwicklung},
	url = {http://www.scopus.com/inward/record.url?eid=2-s2.0-84891428885&partnerID=40&md5=72c1a2e9a50f7206f427bfb42e59bda3},
	number = {6},
	journal = {Vasomed},
	author = {Krebs, R. and Jeltsch, M.},
	month = jun,
	year = {2013},
	pages = {335--336},
}

@article{anisimov_basis_2013,
	title = {The {Basis} for the {Distinct} {Biological} {Activities} of {Vascular} {Endothelial} {Growth} {Factor} {Receptor}-1 {Ligands}},
	volume = {6},
	url = {http://stke.sciencemag.org/content/6/282/ra52},
	doi = {10.1126/scisignal.2003905},
	number = {282},
	urldate = {2014-01-08},
	journal = {Science Signaling},
	author = {Anisimov, Andrey and Leppanen, Veli-Matti and Tvorogov, Denis and Zarkada, Georgia and Jeltsch, Michael and Holopainen, Tanja and Kaijalainen, Seppo and Alitalo, Kari},
	month = jul,
	year = {2013},
	pages = {ra52},
}

@article{anisimov_vascular_2013,
	title = {Vascular {Endothelial} {Growth} {Factor}-{Angiopoietin} {Chimera} {With} {Improved} {Properties} for {Therapeutic} {AngiogenesisClinical} {Perspective}},
	volume = {127},
	issn = {0009-7322, 1524-4539},
	url = {http://circ.ahajournals.org/content/127/4/424},
	doi = {10.1161/CIRCULATIONAHA.112.127472},
	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.},
	language = {en},
	number = {4},
	urldate = {2013-04-04},
	journal = {Circulation},
	author = {Anisimov, Andrey and Tvorogov, Denis and Alitalo, Annamari and Leppänen, Veli-Matti and An, Yuri and Han, Eun Chun and Orsenigo, Fabrizio and Gaál, Emília Ilona and Holopainen, Tanja and Koh, Young Jun and Tammela, Tuomas and Korpisalo, Petra and Keskitalo, Salla and Jeltsch, Michael and Ylä-Herttuala, Seppo and Dejana, Elisabetta and Koh, Gou Young and Choi, Chulhee and Saharinen, Pipsa and Alitalo, Kari},
	month = jan,
	year = {2013},
	keywords = {Capillary Permeability, GENE therapy, Ischemia, angiogenesis inducers, vascular endothelium},
	pages = {424--434},
}

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.

@article{villefranc_truncation_2013,
	title = {A truncation allele in vascular endothelial growth factor c reveals distinct modes of signaling during lymphatic and vascular development},
	volume = {140},
	url = {http://dx.doi.org/10.1242/dev.084152},
	doi = {10.1242/dev.084152},
	number = {7},
	journal = {Development},
	author = {Villefranc, Jacques A. and Nicoli, Stefania and Bentley, Katie and Jeltsch, Michael and Zarkada, Georgia and Moore, John C. and Gerhardt, Holger and Alitalo, Kari and Lawson, Nathan D.},
	month = jan,
	year = {2013},
	note = {Self-archived version available from here: https://jeltsch.org/Villefranc2013
{\textless}a href="https://jeltsch.org/Villefranc2013"{\textgreater}Link{\textless}/a{\textgreater}},
	pages = {1497--1506},
}

@article{leppanen_structural_2011,
	title = {Structural determinants of vascular endothelial growth factor-{D} receptor binding and specificity},
	volume = {117},
	issn = {1528-0020},
	url = {http://dx.doi.org/10.1182/blood-2010-08-301549},
	doi = {10.1182/blood-2010-08-301549},
	abstract = {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.},
	number = {5},
	urldate = {2012-09-22},
	journal = {Blood},
	author = {Leppänen, Veli-Matti and Jeltsch, Michael and Anisimov, Andrey and Tvorogov, Denis and Aho, Kukka and Kalkkinen, Nisse and Toivanen, Pyry and Ylä-Herttuala, Seppo and Ballmer-Hofer, Kurt and Alitalo, Kari},
	month = feb,
	year = {2011},
	pmid = {21148085},
	keywords = {Amino Acid Sequence, Animals, Cell Proliferation, Cells, Cultured, Crystallography, X-Ray, Humans, Hydrogen Bonding, Immunoenzyme Techniques, Immunoprecipitation, Mice, Models, Molecular, Molecular Sequence Data, Muscle, Skeletal, Mutagenesis, Site-Directed, Mutation, Protein Binding, Protein Conformation, Recombinant Proteins, Sequence Homology, Amino Acid, Vascular Endothelial Growth Factor Receptor-2, Vascular Endothelial Growth Factor Receptor-3, vascular endothelial growth factor C, vascular endothelial growth factor D},
	pages = {1507--1515},
}

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.

@article{krebs_vegf-cvegfr-3_2011,
	title = {{VEGF}-{C}/{VEGFR}-3 {Signaling} {Regulates} {Inflammatory} {Response} in {Development} of {Obliterative} {Airway} {Disease}},
	volume = {30},
	url = {http://dx.doi.org/10.1016/j.healun.2011.01.348},
	abstract = {Purpose: The role of VEGF-C/VEGFR-3 signaling in the development of obliterative airway disease (OAD) as a manifestation of chronic rejection was investigated in the rat tracheal allograft model. Methods and Materials: Tracheal allografts were transplanted heterotopically from DA- to WF-rats (syngeneic controls: DA-{\textgreater}DA). Expression of VEGF-C and VEGFR-3 protein and lymphatic vessel marker LYVE-1 was analyzed from normal, syngeneic, and allogeneic tracheal transplants. Adenovirally mediated human VEGF-C (AdVEGF-C) and VEGFR-3 Ig (VEGF-C trap) gene transfer was used to overexpress VEGF-C and to suppress VEGF-C/VEGFR-3 signaling, respectively (controls: AdLacZ). Cyclosporine A (CsA) was used for background immunosuppression. Tracheal allografts were removed 10 and 30 days after transplantation for histology, immunohistochemistry, and real-time PCR analysis of cytokine expression. Results: Please, see figure for summary of results.
Conclusions: Our results show that VEGF-C/VEGFR-3-signaling induces Th17-mediated alloimmune activation leading to increased OAD. Inhibition of VEGF-C/VEGFR-3-signaling attenuates allograft inflammation and OAD development, suggesting a possible site of intervention in the development of obliterative bronchiolitis after lung transplantation.},
	number = {4},
	journal = {Journal of Heart and Lung Transplantation},
	author = {Krebs, R. and Tikkanen, J. M. and Ropponen, J. O. and Jeltsch, M. and Jokinen, J. J. and Yla-Herttuala, S. and Koskinen, P. K. and Nykanen, A. I. and Lemstrom, K. B.},
	month = mar,
	year = {2011},
	note = {undefined
APR 2011
VEGF-C/VEGFR-3 Signaling Regulates Inflammatory Response in Development of Obliterative Airway Disease
WOS:000288924300340},
	pages = {S118--S118},
}

Purpose: The role of VEGF-C/VEGFR-3 signaling in the development of obliterative airway disease (OAD) as a manifestation of chronic rejection was investigated in the rat tracheal allograft model. Methods and Materials: Tracheal allografts were transplanted heterotopically from DA- to WF-rats (syngeneic controls: DA-\textgreaterDA). Expression of VEGF-C and VEGFR-3 protein and lymphatic vessel marker LYVE-1 was analyzed from normal, syngeneic, and allogeneic tracheal transplants. Adenovirally mediated human VEGF-C (AdVEGF-C) and VEGFR-3 Ig (VEGF-C trap) gene transfer was used to overexpress VEGF-C and to suppress VEGF-C/VEGFR-3 signaling, respectively (controls: AdLacZ). Cyclosporine A (CsA) was used for background immunosuppression. Tracheal allografts were removed 10 and 30 days after transplantation for histology, immunohistochemistry, and real-time PCR analysis of cytokine expression. Results: Please, see figure for summary of results. Conclusions: Our results show that VEGF-C/VEGFR-3-signaling induces Th17-mediated alloimmune activation leading to increased OAD. Inhibition of VEGF-C/VEGFR-3-signaling attenuates allograft inflammation and OAD development, suggesting a possible site of intervention in the development of obliterative bronchiolitis after lung transplantation.

@article{leppanen_structural_2010,
	title = {Structural determinants of growth factor binding and specificity by {VEGF} receptor 2},
	volume = {107},
	issn = {0027-8424, 1091-6490},
	url = {http://www.pnas.org/content/107/6/2425},
	doi = {10.1073/pnas.0914318107},
	abstract = {Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel formation through activation of three receptor tyrosine kinases, VEGFR-1, -2, and -3. The extracellular domain of VEGF receptors consists of seven immunoglobulin homology domains, which, upon ligand binding, promote receptor dimerization. Dimerization initiates transmembrane signaling, which activates the intracellular tyrosine kinase domain of the receptor. VEGF-C stimulates lymphangiogenesis and contributes to pathological angiogenesis via VEGFR-3. However, proteolytically processed VEGF-C also stimulates VEGFR-2, the predominant transducer of signals required for physiological and pathological angiogenesis. Here we present the crystal structure of VEGF-C bound to the VEGFR-2 high-affinity-binding site, which consists of immunoglobulin homology domains D2 and D3. This structure reveals a symmetrical 2∶2 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.},
	language = {en},
	number = {6},
	urldate = {2012-09-22},
	journal = {Proceedings of the National Academy of Sciences of the United States of America},
	author = {Leppänen, Veli-Matti and Prota, Andrea E. and Jeltsch, Michael and Anisimov, Andrey and Kalkkinen, Nisse and Strandin, Tomas and Lankinen, Hilkka and Goldman, Adrian and Ballmer-Hofer, Kurt and Alitalo, Kari},
	month = feb,
	year = {2010},
	keywords = {Vascular Endothelial Growth Factor Receptor-2, angiogenesis, lymphangiogenesis, vascular endothelial growth factor C},
	pages = {2425--2430},
}

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 2∶2 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.

@article{bry_vascular_2010,
	title = {Vascular {Endothelial} {Growth} {Factor}-{B} {Acts} as a {Coronary} {Growth} {Factor} in {Transgenic} {Rats} {Without} {Inducing} {Angiogenesis}, {Vascular} {Leak}, or {Inflammation}},
	volume = {122},
	issn = {0009-7322, 1524-4539},
	url = {http://circ.ahajournals.org/content/122/17/1725},
	doi = {10.1161/CIRCULATIONAHA.110.957332},
	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-B167 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.},
	language = {en},
	number = {17},
	urldate = {2015-04-30},
	journal = {Circulation},
	author = {Bry, Maija and Kivelä, 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 Lemström, Karl B. and Shibuya, Masabumi and Ylä-Herttuala, Seppo and Alhonen, Leena and Mervaala, Eero and Andersson, Leif C. and Knuuti, Juhani and Alitalo, Kari},
	month = oct,
	year = {2010},
	pmid = {20937974},
	keywords = {Coronary Disease, angiogenesis, hypertrophy},
	pages = {1725--1733},
}

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-B167 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.

@article{tvorogov_effective_2010,
	title = {Effective suppression of vascular network formation by combination of antibodies blocking {VEGFR} ligand binding and receptor dimerization},
	volume = {18},
	issn = {1878-3686},
	url = {http://dx.doi.org/10.1016/j.ccr.2010.11.001},
	doi = {10.1016/j.ccr.2010.11.001},
	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.},
	number = {6},
	urldate = {2012-02-23},
	journal = {Cancer Cell},
	author = {Tvorogov, Denis and Anisimov, Andrey and Zheng, Wei and Leppänen, Veli-Matti and Tammela, Tuomas and Laurinavicius, Simonas and Holnthoner, Wolfgang and Heloterä, Hanna and Holopainen, Tanja and Jeltsch, Michael and Kalkkinen, Nisse and Lankinen, Hilkka and Ojala, Päivi M and Alitalo, Kari},
	month = dec,
	year = {2010},
	pmid = {21130043},
	keywords = {Angiogenesis Inhibitors, Antibodies, Monoclonal, Cells, Cultured, Humans, Morphogenesis, Protein Multimerization, Receptor, erbB-2, Vascular Endothelial Growth Factor Receptor-2, Vascular Endothelial Growth Factor Receptor-3, vascular endothelial growth factor C},
	pages = {630--640},
}

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.

@article{albrecht_suppressive_2010,
	title = {Suppressive {Effects} of {Vascular} {Endothelial} {Growth} {Factor}-{B} on {Tumor} {Growth} in a {Mouse} {Model} of {Pancreatic} {Neuroendocrine} {Tumorigenesis}},
	volume = {5},
	url = {http://dx.doi.org/10.1371/journal.pone.0014109},
	number = {11},
	journal = {PLoS ONE},
	author = {Albrecht, Imke and Kopfstein, Lucie and Strittmatter, Karin and Schomber, Tibor and Falkevall, Annelie and Hagberg, Carolina E. and Lorentz, Pascal and Jeltsch, Michael and Alitalo, Kari and Eriksson, Ulf and Christofori, Gerhard and Pietras, Kristian},
	month = nov,
	year = {2010},
	note = {undefined
NOV 24 2010
Suppressive Effects of Vascular Endothelial Growth Factor-B on Tumor Growth in a Mouse Model of Pancreatic Neuroendocrine Tumorigenesis
10.1371/journal.pone.0014109
WOS:000284572000011},
	pages = {e14109},
}

@article{saharinen_claudin-like_2010,
	title = {Claudin-like protein 24 interacts with the {VEGFR}-2 and {VEGFR}-3 pathways and regulates lymphatic vessel development},
	volume = {24},
	url = {http://dx.doi.org/10.1101/gad.565010},
	number = {9},
	journal = {Genes \& Development},
	author = {Saharinen, Pipsa and Helotera, Hanna and Miettinen, Juho and Norrmen, Camilla and D'Amico, Gabriela and Jeltsch, Michael and Langenberg, Tobias and Vandevelde, Wouter and Ny, Annelii and Dewerchin, Mieke and Carmeliet, Peter and Alitalo, Kari},
	month = mar,
	year = {2010},
	note = {undefined
MAY 1 2010
Claudin-like protein 24 interacts with the VEGFR-2 and VEGFR-3 pathways and regulates lymphatic vessel development
10.1101/gad.565010
WOS:000277244100004},
	pages = {875--880},
}

@article{anisimov_activated_2009,
	title = {Activated {Forms} of {VEGF}-{C} and {VEGF}-{D} {Provide} {Improved} {Vascular} {Function} in {Skeletal} {Muscle}},
	volume = {104},
	issn = {0009-7330, 1524-4571},
	url = {http://circres.ahajournals.org/content/104/11/1302},
	doi = {10.1161/CIRCRESAHA.109.197830},
	abstract = {The therapeutic potential of vascular endothelial growth factor (VEGF)-C and VEGF-D in skeletal muscle has been of considerable interest as these factors have both angiogenic and lymphangiogenic activities. Previous studies have mainly used adenoviral gene delivery for short-term expression of VEGF-C and VEGF-D in pig, rabbit, and mouse skeletal muscles. Here we have used the activated mature forms of VEGF-C and VEGF-D expressed via recombinant adeno-associated virus (rAAV), which provides stable, long-lasting transgene expression in various tissues including skeletal muscle. Mouse tibialis anterior muscle was transduced with rAAV encoding human or mouse VEGF-C or VEGF-D. Two weeks later, immunohistochemical analysis showed increased numbers of both blood and lymph vessels, and Doppler ultrasound analysis indicated increased blood vessel perfusion. The lymphatic vessels further increased at the 4-week time point were functional, as shown by FITC-lectin uptake and transport. Furthermore, receptor activation and arteriogenic activity were increased by an alanine substitution mutant of human VEGF-C (C137A) having an increased dimer stability and by a chimeric CAC growth factor that contained the VEGF receptor-binding domain flanked by VEGF-C propeptides, but only the latter promoted significantly more blood vessel perfusion when compared to the other growth factors studied. We conclude that long-term expression of VEGF-C and VEGF-D in skeletal muscle results in the generation of new functional blood and lymphatic vessels. The therapeutic value of intramuscular lymph vessels in draining tissue edema and lymphedema can now be evaluated using this model system.},
	language = {en},
	number = {11},
	urldate = {2012-09-15},
	journal = {Circulation Research},
	author = {Anisimov, Andrey and Alitalo, Annamari and Korpisalo, Petra and Soronen, Jarkko and Kaijalainen, Seppo and Leppänen, Veli-Matti and Jeltsch, Michael and Ylä-Herttuala, Seppo and Alitalo, Kari},
	month = jun,
	year = {2009},
	keywords = {VEGF-C, VEGF-D, adeno-associated virus, angiogenesis, lymphangiogenesis, skeletal muscle},
	pages = {1302--1312},
}

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.

@article{heckman_tyrosine_2008,
	title = {The tyrosine kinase inhibitor cediranib blocks ligand-induced vascular endothelial growth factor receptor-3 activity and lymphangiogenesis},
	volume = {68},
	url = {http://dx.doi.org/10.1158/0008-5472.CAN-07-5809},
	number = {12},
	journal = {Cancer Research},
	author = {Heckman, Caroline A. and Holopainen, Tanja and Wirzenius, Maria and Keskitalo, Salla and Jeltsch, Michael and Yla-Herttuala, Seppo and Wedge, Stephen R. and Jurgensmeier, Juliane M. and Alitalo, Kari},
	month = jun,
	year = {2008},
	note = {undefined
JUN 15 2008
The tyrosine kinase inhibitor cediranib blocks ligand-induced vascular endothelial growth factor receptor-3 activity and lymphangiogenesis
10.1158/0008-5472.CAN-07-5809
WOS:000256855700034},
	pages = {4754--4762},
}

@article{karpanen_overexpression_2008,
	title = {Overexpression of {Vascular} {Endothelial} {Growth} {Factor}-{B} in {Mouse} {Heart} {Alters} {Cardiac} {Lipid} {Metabolism} and {Induces} {Myocardial} {Hypertrophy}},
	volume = {103},
	url = {https://doi.org/10.1161%2FCIRCRESAHA.108.178459},
	number = {9},
	journal = {Circulation Research},
	author = {Karpanen, Terhi and Bry, Maija and Ollila, Hanna M. and Seppanen-Laakso, Tuulikki and Liimatta, Erkki and Leskinen, Hanna and Kivela, 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 Yla-Herttuala, Seppo and Oresic, Matej and Alitalo, Kari},
	month = oct,
	year = {2008},
	note = {undefined
OCT 24 2008
Overexpression of Vascular Endothelial Growth Factor-B in Mouse Heart Alters Cardiac Lipid Metabolism and Induces Myocardial Hypertrophy
10.1161/CIRCRESAHA.108.178459
WOS:000260308500018},
	pages = {1018--U247},
}

@article{li_reevaluation_2008,
	title = {Reevaluation of the role of {VEGF}-{B} suggests a restricted role in the revascularization of the ischemic myocardium},
	volume = {28},
	issn = {1524-4636},
	url = {http://www.ncbi.nlm.nih.gov/pubmed/18511699},
	doi = {10.1161/ATVBAHA.107.158725},
	abstract = {OBJECTIVE

The endogenous role of the VEGF family member vascular endothelial growth factor-B (VEGF-B) in pathological angiogenesis remains unclear.


METHODS AND RESULTS

We studied the role of VEGF-B in various models of pathological angiogenesis using mice lacking VEGF-B (VEGF-B(-/-)) or overexpressing VEGF-B(167). After occlusion of the left coronary artery, VEGF-B deficiency impaired vessel growth in the ischemic myocardium whereas, in wild-type mice, VEGF-B(167) overexpression enhanced revascularization of the infarct and ischemic border zone. By contrast, VEGF-B deficiency did not affect vessel growth in the wounded skin, hypoxic lung, ischemic retina, or ischemic limb. Moreover, VEGF-B(167) overexpression failed to enhance vascular growth in the skin or ischemic limb.


CONCLUSIONS

VEGF-B appears to have a relatively restricted angiogenic activity in the ischemic heart. These insights might offer novel therapeutic opportunities.},
	number = {9},
	urldate = {2012-02-23},
	journal = {Arteriosclerosis, Thrombosis, and Vascular Biology},
	author = {Li, Xuri and Tjwa, Marc and Van Hove, Inge and Enholm, Berndt and Neven, Elke and Paavonen, Karri and Jeltsch, Michael and Juan, Toni Diez and Sievers, Richard E and Chorianopoulos, Emmanuel and Wada, Hiromichi and Vanwildemeersch, Maarten and Noel, Agnes and Foidart, Jean-Michel and Springer, Matthew L and von Degenfeld, Georges and Dewerchin, Mieke and Blau, Helen M and Alitalo, Kari and Eriksson, Ulf and Carmeliet, Peter and Moons, Lieve},
	month = sep,
	year = {2008},
	pmid = {18511699},
	keywords = {Angiogenesis Inducing Agents, Animals, Coronary Vessels, Disease Models, Animal, GENE therapy, Gene Transfer Techniques, Hindlimb, Ischemia, Lung, Mice, Mice, Inbred C57BL, Mice, Knockout, Mice, Nude, Muscle, Skeletal, Myocardial Ischemia, Myocardium, Neovascularization, Physiologic, Recombinant Proteins, Retinal Vessels, Skin, Up-Regulation, Vascular Endothelial Growth Factor B, Vascular Endothelial Growth Factor Receptor-1},
	pages = {1614--1620},
}

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.

@inproceedings{heckman_inhibition_2007,
	address = {Los Angeles, CA},
	title = {Inhibition of {VEGF}-{C}-induced {VEGFR}-3 activity and lymphatic endothelial cell function by the tyrosine kinase inhibitor {AZD2171}},
	volume = {67},
	url = {http://cancerres.aacrjournals.org/content/67/9_Supplement/2999},
	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},
	booktitle = {Proc {AACR} {Ann} {Meet}},
	publisher = {American Association for Cancer Research},
	author = {Heckman, Caroline A. and Holopainen, Tanja and Wirzenius, Maria and Keskitalo, Salla and Jeltsch, Michael and Wedge, Stephen R. and Jurgensmeier, Juliane M.},
	month = may,
	year = {2007},
	note = {Poster 2999
undefined
JUN 15 2008
The tyrosine kinase inhibitor cediranib blocks ligand-induced vascular endothelial growth factor receptor-3 activity and lymphangiogenesis
10.1158/0008-5472.CAN-07-5809
WOS:000256855700034},
	pages = {2999},
}

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

@mastersthesis{aho_production_2006,
	address = {Helsinki. Finland},
	title = {Production and {Purification} of {Recombinant} {Human} {Vascular} {Endothelial} {Growth} {Factor} {D}},
	url = {https://helda.helsinki.fi/handle/10138/29574},
	abstract = {In multicellular organisms, complex signalling mechanisms have evolved to guide the behaviour of individual cells. Growth factors are secreted proteins that can stimulate the proliferation and/or differentiation of cells. Vascular endothelial growth factor D (VEGF-D) is a ligand for VEGF receptor 2 (VEGFR-2) and for VEGFR-3, which are predominantly expressed on blood vascular endothelial cells and on lymphatic endothelial cells, respectively. Thus VEGF-D can contribute to growth of both blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis). Although there have been many reports showing the angiogenic and lymphangiogenic effects of VEGF-D, its physiological role is still largely unknown. Most of these reports are severely hampered by incomplete characterization of the specific form of VEGF-D that was used. During or after secretion, VEGF-D undergoes complicated proteolytic processing. Alternative Nterminal cleavage results in two different fully processed forms, VEGF-D major and VEGF-D minor. Processing significantly increases the activity of VEGF-D towards its receptors. Surprisingly, it is still unknown whether the differential N-terminal cleavage of VEGF-D has any effect on receptor binding activity or on receptor activation. The goal of this study was to produce and purify high quality biologically active VEGF-D which is needed for studying the physiological role of this growth factor. Several different forms of recombinant human VEGF-D were produced using the Drosophila Schneider 2 insect cell system. A bioassay utilizing the Ba/F3 cells expressing chimeric VEGFR/EpoR receptors was used to determine the receptor binding activities of recombinant VEGF-Ds. Two constructs producing biologically active VEGF-Ds were chosen for chromatographic purification (untagged major and his-tagged major forms). During purification, the activity of both VEGF-D forms towards their receptors decreased significantly. In case of the untagged form, this was presumably due to some residual proteolytic activity during purifications. The results might indicate that only the major form is responsible for the activation of VEGFR-3. The fact that no activity of the minor forms was detected when screening the cell supernatants with Ba/F3-VEGFR-3-EpoR-bioassay, supports this explanation. If this explanation can be verified, the role of the alternative N-terminal cleavage becomes obvious: By proteolysis the activity of VEGF-D can be redirected from the lymphatics towards the blood vessels.},
	language = {English},
	school = {University of Helsinki},
	author = {Aho, Kukka},
	month = dec,
	year = {2006},
}

In multicellular organisms, complex signalling mechanisms have evolved to guide the behaviour of individual cells. Growth factors are secreted proteins that can stimulate the proliferation and/or differentiation of cells. Vascular endothelial growth factor D (VEGF-D) is a ligand for VEGF receptor 2 (VEGFR-2) and for VEGFR-3, which are predominantly expressed on blood vascular endothelial cells and on lymphatic endothelial cells, respectively. Thus VEGF-D can contribute to growth of both blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis). Although there have been many reports showing the angiogenic and lymphangiogenic effects of VEGF-D, its physiological role is still largely unknown. Most of these reports are severely hampered by incomplete characterization of the specific form of VEGF-D that was used. During or after secretion, VEGF-D undergoes complicated proteolytic processing. Alternative Nterminal cleavage results in two different fully processed forms, VEGF-D major and VEGF-D minor. Processing significantly increases the activity of VEGF-D towards its receptors. Surprisingly, it is still unknown whether the differential N-terminal cleavage of VEGF-D has any effect on receptor binding activity or on receptor activation. The goal of this study was to produce and purify high quality biologically active VEGF-D which is needed for studying the physiological role of this growth factor. Several different forms of recombinant human VEGF-D were produced using the Drosophila Schneider 2 insect cell system. A bioassay utilizing the Ba/F3 cells expressing chimeric VEGFR/EpoR receptors was used to determine the receptor binding activities of recombinant VEGF-Ds. Two constructs producing biologically active VEGF-Ds were chosen for chromatographic purification (untagged major and his-tagged major forms). During purification, the activity of both VEGF-D forms towards their receptors decreased significantly. In case of the untagged form, this was presumably due to some residual proteolytic activity during purifications. The results might indicate that only the major form is responsible for the activation of VEGFR-3. The fact that no activity of the minor forms was detected when screening the cell supernatants with Ba/F3-VEGFR-3-EpoR-bioassay, supports this explanation. If this explanation can be verified, the role of the alternative N-terminal cleavage becomes obvious: By proteolysis the activity of VEGF-D can be redirected from the lymphatics towards the blood vessels.

@article{karpanen_functional_2006,
	title = {Functional interaction of {VEGF}-{C} and {VEGF}-{D} with neuropilin receptors},
	volume = {20},
	issn = {0892-6638, 1530-6860},
	url = {http://www.fasebj.org/content/20/9/1462},
	doi = {10.1096/fj.05-5646com},
	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.—Kärpänen, T., Heckman, C. A., Keskitalo, S., Jeltsch, M., Ollila, H., Neufeld, G., Tamagnone, L., Alitalo, K. Functional interaction of VEGF-C and VEGF-D with neuropilin receptors.},
	language = {en},
	number = {9},
	urldate = {2012-12-20},
	journal = {FASEB Journal},
	author = {Karpanen, Terhi and Heckman, Caroline A. and Keskitalo, Salla and Jeltsch, Michael and Ollila, Hanna and Neufeld, Gera and Tamagnone, Luca and Alitalo, Kari},
	month = jul,
	year = {2006},
	keywords = {NP2, lymphatic endothelial cell},
	pages = {1462--1472},
}

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.—Kärpänen, T., Heckman, C. A., Keskitalo, S., Jeltsch, M., Ollila, H., Neufeld, G., Tamagnone, L., Alitalo, K. Functional interaction of VEGF-C and VEGF-D with neuropilin receptors.

@article{baluk_pathogenesis_2005,
	title = {Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation},
	volume = {115},
	issn = {0021-9738},
	url = {http://www.ncbi.nlm.nih.gov/pmc/articles/PMC544601/},
	doi = {10.1172/JCI200522037},
	abstract = {Edema occurs in asthma and other inflammatory diseases when the rate of plasma leakage from blood vessels exceeds the drainage through lymphatic vessels and other routes. It is unclear to what extent lymphatic vessels grow to compensate for increased leakage during inflammation and what drives the lymphangiogenesis that does occur. We addressed these issues in mouse models of (a) chronic respiratory tract infection with Mycoplasma pulmonis and (b) adenoviral transduction of airway epithelium with VEGF family growth factors. Blood vessel remodeling and lymphangiogenesis were both robust in infected airways. Inhibition of VEGFR-3 signaling completely prevented the growth of lymphatic vessels but not blood vessels. Lack of lymphatic growth exaggerated mucosal edema and reduced the hypertrophy of draining lymph nodes. Airway dendritic cells, macrophages, neutrophils, and epithelial cells expressed the VEGFR-3 ligands VEGF-C or VEGF-D. Adenoviral delivery of either VEGF-C or VEGF-D evoked lymphangiogenesis without angiogenesis, whereas adenoviral VEGF had the opposite effect. After antibiotic treatment of the infection, inflammation and remodeling of blood vessels quickly subsided, but lymphatic vessels persisted. Together, these findings suggest that when lymphangiogenesis is impaired, airway inflammation may lead to bronchial lymphedema and exaggerated airflow obstruction. Correction of defective lymphangiogenesis may benefit the treatment of asthma and other inflammatory airway diseases.},
	number = {2},
	urldate = {2012-09-15},
	journal = {Journal of Clinical Investigation},
	author = {Baluk, Peter and Tammela, Tuomas and Ator, Erin and Lyubynska, Natalya and Achen, Marc G. and Hicklin, Daniel J. and Jeltsch, Michael and Petrova, Tatiana V. and Pytowski, Bronislaw and Stacker, Steven A. and Ylä-Herttuala, Seppo and Jackson, David G. and Alitalo, Kari and McDonald, Donald M.},
	month = feb,
	year = {2005},
	pmid = {15668734},
	pmcid = {PMC544601},
	pages = {247--257},
}

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.

@article{he_vascular_2005,
	title = {Vascular endothelial cell growth factor receptor 3-mediated activation of lymphatic endothelium is crucial for tumor cell entry and spread via lymphatic vessels},
	volume = {65},
	url = {http://dx.doi.org/10.1158/0008-5472.CAN-04-4576},
	number = {11},
	journal = {Cancer Research},
	author = {He, Y. L. and Rajantie, I. and Pajusola, K. and Jeltsch, M. and Holopainen, T. and Yla-Herttuala, S. and Harding, T. and Jooss, K. and Takahashi, T. and Alitalo, K.},
	month = jun,
	year = {2005},
	note = {undefined
JUN 1 2005
Vascular endothelial cell growth factor receptor 3-mediated activation of lymphatic endothelium is crucial for tumor cell entry and spread via lymphatic vessels
10.1158/0008-5472.CAN-04-4576
WOS:000229407800033},
	pages = {4739--4746},
}

@article{krebs_dual_2005,
	title = {Dual role of vascular endothelial growth factor in experimental obliterative bronchiolitis},
	volume = {171},
	url = {http://dx.doi.org/ 10.1164/rccm.200408-1001OC},
	number = {12},
	journal = {American Journal of Respiratory and Critical Care Medicine},
	author = {Krebs, R. and Tikkanen, J. M. and Nykanen, A. I. and Wood, J. and Jeltsch, M. and Yla-Herttuala, S. and Koskinen, P. K. and Lemstrom, K. B.},
	month = mar,
	year = {2005},
	note = {undefined
JUN 15 2005
Dual role of vascular endothelial growth factor in experimental obliterative bronchiolitis
10.1164/rccm.200408-1001OC
WOS:000229711200016},
	pages = {1421--1429},
}

@article{karkkainen_vascular_2004,
	title = {Vascular endothelial growth factor {C} is required for sprouting of the first lymphatic vessels from embryonic veins},
	volume = {5},
	copyright = {© 2003 Nature Publishing Group},
	url = {http://dx.doi.org/10.1038/ni1013},
	doi = {10.1038/ni1013},
	language = {en},
	number = {1},
	urldate = {2012-09-15},
	journal = {Nature Immunology},
	author = {Karkkainen, Marika J. and Haiko, Paula and Sainio, Kirsi and Partanen, Juha and Taipale, Jussi and Petrova, Tatiana V. and Jeltsch, Michael and Jackson, David G. and Talikka, Marja and Rauvala, Heikki and Betsholtz, Christer and Alitalo, Kari},
	month = jan,
	year = {2004},
	pages = {74--80},
}

@article{veikkola_intrinsic_2003,
	title = {Intrinsic versus micro environmental regulation of lymphatic endothelial cell phenotype and function},
	volume = {17},
	url = {http://dx.doi.org/10.1096/fj.03-0179com},
	number = {14},
	journal = {FASEB Journal},
	author = {Veikkola, T. and Lohela, M. and Ikenberg, K. and Makinen, T. and Korff, T. and Saaristo, A. and Petrova, T. and Jeltsch, M. and Augustin, H. G. and Alitalo, K.},
	month = nov,
	year = {2003},
	note = {undefined
NOV 2003
Intrinsic versus micro environmental regulation of lymphatic endothelial cell phenotype and function
10.1096/fj.03-0179com
WOS:000186961200037},
	pages = {2006--2013},
}

@article{jeltsch_genesis_2003,
	title = {Genesis and pathogenesis of lymphatic vessels},
	volume = {314},
	url = {http://dx.doi.org/10.1007/s00441-003-0777-2},
	doi = {10.1007/s00441-003-0777-2},
	number = {1},
	journal = {Cell and Tissue Research},
	author = {Jeltsch, M. and Tammela, T. and Alitalo, K. and Wilting, J.},
	month = aug,
	year = {2003},
	note = {undefined
OCT 2003
Genesis and pathogenesis of lymphatic vessels
10.1007/s00441-003-0777-2
WOS:000186541800009},
	pages = {69--84},
}

@article{gerhardt_vegf_2003,
	title = {{VEGF} guides angiogenic sprouting utilizing endothelial tip cell filopodia},
	volume = {161},
	url = {http://dx.doi.org/},
	doi = {10.1083/jcb.200302047},
	number = {6},
	journal = {Journal of Cell Biology},
	author = {Gerhardt, H. and Golding, M. and Fruttiger, M. and Ruhrberg, C. and Lundkvist, A. and Abramsson, A. and Jeltsch, M. and Mitchell, C. and Alitalo, K. and Shima, D. and Betsholtz, C.},
	month = jun,
	year = {2003},
	note = {undefined
JUN 23 2003
VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia
10.1083/jcb.200302047
WOS:000183827400016},
	pages = {1163--1177},
}

@article{saaristo_adenoviral_2002,
	title = {Adenoviral {VEGF}-{C} overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes},
	volume = {16},
	url = {http://dx.doi.org/10.1096/fj.01-1042com},
	number = {9},
	journal = {FASEB Journal},
	author = {Saaristo, A. and Veikkola, T. and Enholm, B. and Hytonen, M. and Arola, J. and Pajusola, K. and Turunen, P. and Jeltsch, M. and Karkkainen, M. J. and Kerjaschki, D. and Bueler, H. and Yla-Herttuala, S. and Alitalo, K.},
	month = jul,
	year = {2002},
	note = {undefined
JUL 2002
Adenoviral VEGF-C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes
10.1096/fj.01-1042com
WOS:000177813100035},
	pages = {1041--1049},
}

@article{enholm_adenoviral_2001,
	title = {Adenoviral {Expression} of {Vascular} {Endothelial} {Growth} {Factor}-{C} {Induces} {Lymphangiogenesis} in the {Skin}},
	volume = {88},
	issn = {0009-7330, 1524-4571},
	url = {https://www.ahajournals.org/doi/10.1161/01.RES.88.6.623},
	doi = {10.1161/01.RES.88.6.623},
	language = {en},
	number = {6},
	urldate = {2017-05-03},
	journal = {Circulation Research},
	author = {Enholm, B. and Karpanen, T. and Jeltsch, M. and Kubo, H. and Stenback, F. and Prevo, R. and Jackson, D. G. and Yla-Herttuala, S. and Alitalo, K.},
	month = mar,
	year = {2001},
	pages = {623--629},
}

@article{veikkola_signalling_2001,
	title = {Signalling via vascular endothelial growth factor receptor‐3 is sufficient for lymphangiogenesis in transgenic mice},
	volume = {20},
	copyright = {Copyright © 2001 European Molecular Biology Organization},
	issn = {0261-4189, 1460-2075},
	url = {http://dx.doi.org/10.1093/emboj/20.6.1223},
	doi = {10.1093/emboj/20.6.1223},
	abstract = {Vascular endothelial growth factor receptor‐3 (VEGFR‐3) has an essential role in the development of embryonic blood vessels; however, after midgestation its expression becomes restricted mainly to the developing lymphatic vessels. The VEGFR‐3 ligand VEGF‐C stimulates lymphangiogenesis in transgenic mice and in chick chorioallantoic membrane. As VEGF‐C also binds VEGFR‐2, which is expressed in lymphatic endothelia, it is not clear which receptors are responsible for the lymphangiogenic effects of VEGF‐C. VEGF‐D, which binds to the same receptors, has been reported to induce angiogenesis, but its lymphangiogenic potential is not known. In order to define the lymphangiogenic signalling pathway we have created transgenic mice overexpressing a VEGFR‐3‐specific mutant of VEGF‐C (VEGF‐C156S) or VEGF‐D in epidermal keratinocytes under the keratin 14 promoter. Both transgenes induced the growth of lymphatic vessels in the skin, whereas the blood vessel architecture was not affected. Evidence was also obtained that these growth factors act in a paracrine manner in vivo. These results demonstrate that stimulation of the VEGFR‐3 signal transduction pathway is sufficient to induce specifically lymphangiogenesis in vivo.},
	language = {en},
	number = {6},
	urldate = {2015-06-15},
	journal = {The EMBO Journal},
	author = {Veikkola, Tanja and Jussila, Lotta and Makinen, Taija and Karpanen, Terhi and Jeltsch, Michael and Petrova, Tatiana V. and Kubo, Hajime and Thurston, Gavin and McDonald, Donald M. and Achen, Marc G. and Stacker, Steven A. and Alitalo, Kari},
	month = mar,
	year = {2001},
	pmid = {11250889},
	keywords = {VEGF receptors, angiogenesis, lymphangiogenesis, vascular endothelial growth factors (VEGFs)},
	pages = {1223--1231},
}

Vascular endothelial growth factor receptor‐3 (VEGFR‐3) has an essential role in the development of embryonic blood vessels; however, after midgestation its expression becomes restricted mainly to the developing lymphatic vessels. The VEGFR‐3 ligand VEGF‐C stimulates lymphangiogenesis in transgenic mice and in chick chorioallantoic membrane. As VEGF‐C also binds VEGFR‐2, which is expressed in lymphatic endothelia, it is not clear which receptors are responsible for the lymphangiogenic effects of VEGF‐C. VEGF‐D, which binds to the same receptors, has been reported to induce angiogenesis, but its lymphangiogenic potential is not known. In order to define the lymphangiogenic signalling pathway we have created transgenic mice overexpressing a VEGFR‐3‐specific mutant of VEGF‐C (VEGF‐C156S) or VEGF‐D in epidermal keratinocytes under the keratin 14 promoter. Both transgenes induced the growth of lymphatic vessels in the skin, whereas the blood vessel architecture was not affected. Evidence was also obtained that these growth factors act in a paracrine manner in vivo. These results demonstrate that stimulation of the VEGFR‐3 signal transduction pathway is sufficient to induce specifically lymphangiogenesis in vivo.

@inproceedings{hiltunen_vegf-c_2000,
	address = {Stockholm, Sweden},
	title = {{VEGF}-{C} adenovirus gene transfer reduces intima formation in rabbits},
	volume = {151},
	shorttitle = {Atherosclerosis},
	url = {http://linkinghub.elsevier.com/retrieve/pii/S0021915000803664},
	doi = {10.1016/S0021-9150(00)80366-4},
	language = {en},
	urldate = {2015-02-26},
	booktitle = {Atherosclerosis},
	author = {Hiltunen, Mikko O. and Laitinen, Marja and Turunen, Mikko P. and Jeltsch, Michael and Hartikainen, Juha and Rissanen, Tuomas T. and Laukkanen, Johanna and Niemi, Mari and Kossila, Maija and Häkkinen, Tomi P. and Kivelä, Antti and Enholm, Berndt and Mansukoski, Hannu and Turunen, Anna-Mari and Alitalo, Kari and ä-Herttuala, SeppoYl},
	month = jun,
	year = {2000},
	pages = {81},
}

@article{hiltunen_intravascular_2000,
	title = {Intravascular adenovirus-mediated {VEGF}-{C} gene transfer reduces neointima formation in balloon-denuded rabbit aorta},
	volume = {102},
	url = {http://dx.doi.org/10.1161/01.CIR.102.18.2262},
	number = {18},
	journal = {Circulation},
	author = {Hiltunen, M. O. and Laitinen, M. and Turunen, M. P. and Jeltsch, M. and Hartikainen, J. and Rissanen, T. T. and Laukkanen, J. and Niemi, M. and Kossila, M. and Hakkinen, T. P. and Kivela, A. and Enholm, B. and Mansukoski, H. and Turunen, A. M. and Alitalo, K. and Yla-Herttuala, S.},
	month = oct,
	year = {2000},
	note = {undefined
OCT 31 2000
Intravascular adenovirus-mediated VEGF-C gene transfer reduces neointima formation in balloon-denuded rabbit aorta
WOS:000090109400027},
	pages = {2262--2268},
}

@article{achen_vascular_1998,
	title = {Vascular endothelial growth factor {D} ({VEGF}-{D}) is a ligand for the tyrosine kinases {VEGF} receptor 2 ({Flk1}) and {VEGF} receptor 3 ({Flt4})},
	volume = {95},
	issn = {0027-8424, 1091-6490},
	url = {http://www.pnas.org/content/95/2/548},
	abstract = {We have identified a member of the VEGF family by computer-based homology searching and have designated it VEGF-D. VEGF-D is most closely related to VEGF-C by virtue of the presence of N- and C-terminal extensions that are not found in other VEGF family members. In adult human tissues, VEGF-D mRNA is most abundant in heart, lung, skeletal muscle, colon, and small intestine. Analyses of VEGF-D receptor specificity revealed that VEGF-D is a ligand for both VEGF receptors (VEGFRs) VEGFR-2 (Flk1) and VEGFR-3 (Flt4) and can activate these receptors. However, VEGF-D does not bind to VEGFR-1. Expression of a truncated derivative of VEGF-D demonstrated that the receptor-binding capacities reside in the portion of the molecule that is most closely related in primary structure to other VEGF family members and that corresponds to the mature form of VEGF-C. In addition, VEGF-D is a mitogen for endothelial cells. The structural and functional similarities between VEGF-D and VEGF-C define a subfamily of the VEGFs.},
	language = {en},
	number = {2},
	urldate = {2012-09-15},
	journal = {Proceedings of the National Academy of Sciences of the United States of America},
	author = {Achen, Marc G. and Jeltsch, Michael and Kukk, Eola and Mäkinen, Taija and Vitali, Angela and Wilks, Andrew F. and Alitalo, Kari and Stacker, Steven A.},
	month = jan,
	year = {1998},
	pages = {548--553},
}

We have identified a member of the VEGF family by computer-based homology searching and have designated it VEGF-D. VEGF-D is most closely related to VEGF-C by virtue of the presence of N- and C-terminal extensions that are not found in other VEGF family members. In adult human tissues, VEGF-D mRNA is most abundant in heart, lung, skeletal muscle, colon, and small intestine. Analyses of VEGF-D receptor specificity revealed that VEGF-D is a ligand for both VEGF receptors (VEGFRs) VEGFR-2 (Flk1) and VEGFR-3 (Flt4) and can activate these receptors. However, VEGF-D does not bind to VEGFR-1. Expression of a truncated derivative of VEGF-D demonstrated that the receptor-binding capacities reside in the portion of the molecule that is most closely related in primary structure to other VEGF family members and that corresponds to the mature form of VEGF-C. In addition, VEGF-D is a mitogen for endothelial cells. The structural and functional similarities between VEGF-D and VEGF-C define a subfamily of the VEGFs.

@article{olofsson_vascular_1998,
	title = {Vascular endothelial growth factor {B} ({VEGF}-{B}) binds to {VEGF} receptor-1 and regulates plasminogen activator activity in endothelial cells},
	volume = {95},
	issn = {0027-8424, 1091-6490},
	url = {http://www.pnas.org/content/95/20/11709},
	doi = {10.1073/pnas.95.20.11709},
	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.},
	language = {en},
	number = {20},
	urldate = {2015-04-30},
	journal = {Proceedings of the National Academy of Sciences of the United States of America},
	author = {Olofsson, Birgitta and Korpelainen, Eija and Pepper, Michael S. and Mandriota, Stefano J. and Aase, Karin and Kumar, Vijay and Gunji, Yuji and Jeltsch, Michael M. and Shibuya, Masabumi and Alitalo, Kari and Eriksson, Ulf},
	month = sep,
	year = {1998},
	pmid = {9751730},
	pages = {11709--11714},
}

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.

@article{pepper_vascular_1998,
	title = {Vascular endothelial growth factor ({VEGF})-{C} synergizes with basic fibroblast growth factor and {VEGF} in the induction of angiogenesis in vitro and alters endothelial cell extracellular proteolytic activity},
	volume = {177},
	copyright = {Copyright © 1998 Wiley-Liss, Inc.},
	issn = {1097-4652},
	url = {http://onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-4652(199812)177:3<439::AID-JCP7>3.0.CO;2-2/abstract},
	doi = {10.1002/(SICI)1097-4652(199812)177:3<439::AID-JCP7>3.0.CO;2-2},
	abstract = {Vascular endothelial growth factor-C (VEGF-C) is a recently characterized member of the VEGF family of angiogenic polypeptides. We demonstrate here that VEGF-C is angiogenic in vitro when added to bovine aortic or lymphatic endothelial (BAE and BLE) cells but has little or no effect on bovine microvascular endothelial (BME) cells. As reported previously for VEGF, VEGF-C and basic fibroblast growth factor (bFGF) induced a synergistic in vitro angiogenic response in all three cells lines. Unexpectedly, VEGF and VEGF-C also synergized in the in vitro angiogenic response when assessed on BAE cells. Characterization of VEGF receptor (VEGFR) expression revealed that BME, BAE, and BLE cell lines express VEGFR-1 and -2, whereas of the three cell lines assessed, only BAE cells express VEGFR-3. We also demonstrate that VEGF-C increases plasminogen activator (PA) activity in the three bovine endothelial cell lines and that this is accompanied by a concomitant increase in PA inhibitor-1. Addition of α2-antiplasmin to BME cells co-treated with bFGF and VEGF-C partially inhibited collagen gel invasion. These results demonstrate, first, that by acting in concert with bFGF or VEGF, VEGF-C has a potent synergistic effect on the induction of angiogenesis in vitro and, second, that like VEGF and bFGF, VEGF-C is capable of altering endothelial cell extracellular proteolytic activity. These observations also highlight the notion of context, i.e., that the activity of an angiogenesis-regulating cytokine depends on the presence and concentration of other cytokines in the pericellular environment of the responding endothelial cell. J. Cell. Physiol. 177:439–452, 1998. © 1998 Wiley-Liss, Inc.},
	language = {en},
	number = {3},
	urldate = {2015-04-30},
	journal = {Journal of Cellular Physiology},
	author = {Pepper, Michael S. and Mandriota, Stefano J. and Jeltsch, Michael and Kumar, Vijay and Alitalo, Kari},
	month = dec,
	year = {1998},
	pages = {439--452},
}

Vascular endothelial growth factor-C (VEGF-C) is a recently characterized member of the VEGF family of angiogenic polypeptides. We demonstrate here that VEGF-C is angiogenic in vitro when added to bovine aortic or lymphatic endothelial (BAE and BLE) cells but has little or no effect on bovine microvascular endothelial (BME) cells. As reported previously for VEGF, VEGF-C and basic fibroblast growth factor (bFGF) induced a synergistic in vitro angiogenic response in all three cells lines. Unexpectedly, VEGF and VEGF-C also synergized in the in vitro angiogenic response when assessed on BAE cells. Characterization of VEGF receptor (VEGFR) expression revealed that BME, BAE, and BLE cell lines express VEGFR-1 and -2, whereas of the three cell lines assessed, only BAE cells express VEGFR-3. We also demonstrate that VEGF-C increases plasminogen activator (PA) activity in the three bovine endothelial cell lines and that this is accompanied by a concomitant increase in PA inhibitor-1. Addition of α2-antiplasmin to BME cells co-treated with bFGF and VEGF-C partially inhibited collagen gel invasion. These results demonstrate, first, that by acting in concert with bFGF or VEGF, VEGF-C has a potent synergistic effect on the induction of angiogenesis in vitro and, second, that like VEGF and bFGF, VEGF-C is capable of altering endothelial cell extracellular proteolytic activity. These observations also highlight the notion of context, i.e., that the activity of an angiogenesis-regulating cytokine depends on the presence and concentration of other cytokines in the pericellular environment of the responding endothelial cell. J. Cell. Physiol. 177:439–452, 1998. © 1998 Wiley-Liss, Inc.

@article{chilov_genomic_1997,
	title = {Genomic organization of human and mouse genes for vascular endothelial growth factor {C}},
	volume = {272},
	url = {http://dx.doi.org/10.1074/jbc.272.40.25176},
	number = {40},
	journal = {Journal of Biological Chemistry},
	author = {Chilov, D. and Kukk, E. and Taira, S. and Jeltsch, M. and Kaukonen, J. and Palotie, A. and Joukov, V. and Alitalo, K.},
	month = oct,
	year = {1997},
	note = {undefined
OCT 3 1997
Genomic organization of human and mouse genes for vascular endothelial growth factor C
10.1074/jbc.272.40.25176
WOS:A1997XY97000069},
	pages = {25176--25183},
}

@article{jeltsch_hyperplasia_1997,
	title = {Hyperplasia of {Lymphatic} {Vessels} in {VEGF}-{C} {Transgenic} {Mice}},
	volume = {276},
	issn = {0036-8075, 1095-9203},
	url = {http://dx.doi.org/10.1126/science.276.5317.1423},
	doi = {10.1126/science.276.5317.1423},
	abstract = {No growth factors specific for the lymphatic vascular system have yet been described. Vascular endothelial growth factor (VEGF) regulates vascular permeability and angiogenesis, but does not promote lymphangiogenesis. Overexpression of VEGF-C, a ligand of the VEGF receptors VEGFR-3 and VEGFR-2, in the skin of transgenic mice resulted in lymphatic, but not vascular, endothelial proliferation and vessel enlargement. Thus, VEGF-C induces selective hyperplasia of the lymphatic vasculature, which is involved in the draining of interstitial fluid and in immune function, inflammation, and tumor metastasis. VEGF-C may play a role in disorders involving the lymphatic system and may be of potential use in therapeutic lymphangiogenesis.},
	language = {en},
	number = {5317},
	urldate = {2012-09-22},
	journal = {Science},
	author = {Jeltsch, Michael and Kaipainen, Arja and Joukov, Vladimir and Meng, Xiaojuan and Lakso, Merja and Rauvala, Heikki and Swartz, Melody and Fukumura, Dai and Jain, Rakesh K. and Alitalo, Kari},
	month = may,
	year = {1997},
	pages = {1423--1425},
}

No growth factors specific for the lymphatic vascular system have yet been described. Vascular endothelial growth factor (VEGF) regulates vascular permeability and angiogenesis, but does not promote lymphangiogenesis. Overexpression of VEGF-C, a ligand of the VEGF receptors VEGFR-3 and VEGFR-2, in the skin of transgenic mice resulted in lymphatic, but not vascular, endothelial proliferation and vessel enlargement. Thus, VEGF-C induces selective hyperplasia of the lymphatic vasculature, which is involved in the draining of interstitial fluid and in immune function, inflammation, and tumor metastasis. VEGF-C may play a role in disorders involving the lymphatic system and may be of potential use in therapeutic lymphangiogenesis.

@article{joukov_proteolytic_1997,
	title = {Proteolytic processing regulates receptor specificity and activity of {VEGF}-{C}},
	volume = {16},
	copyright = {© 1997 Nature Publishing Group},
	url = {http://dx.doi.org/10.1093/emboj/16.13.3898},
	doi = {10.1093/emboj/16.13.3898},
	abstract = {The recently identified vascular endothelial growth factor C (VEGF-C) belongs to the platelet-derived growth factor (PDGF)/VEGF family of growth factors and is a ligand for the endothelial-specific receptor tyrosine kinases VEGFR-3 and VEGFR-2. The VEGF homology domain spans only about one-third of the cysteine-rich VEGF-C precursor. Here we have analysed the role of post-translational processing in VEGF-C secretion and function, as well as the structure of the mature VEGF-C. The stepwise proteolytic processing of VEGF-C generated several VEGF-C forms with increased activity towards VEGFR-3, but only the fully processed VEGF-C could activate VEGFR-2. Recombinant 'mature' VEGF-C made in yeast bound VEGFR-3 (KD = 135 pM) and VEGFR-2 (KD = 410 pM) and activated these receptors. Like VEGF, mature VEGF-C increased vascular permeability, as well as the migration and proliferation of endothelial cells. Unlike other members of the PDGF/VEGF family, mature VEGF-C formed mostly non-covalent homodimers. These data implicate proteolytic processing as a regulator of VEGF-C activity, and reveal novel structure–function relationships in the PDGF/VEGF family.},
	language = {en},
	number = {13},
	urldate = {2012-08-22},
	journal = {EMBO Journal},
	author = {Joukov, Vladimir and Sorsa, Tarja and Kumar, Vijay and Jeltsch, Michael and Claesson-Welsh, Lena and Cao, Yihai and Saksela, Olli and Kalkkinen, Nisse and Alitalo, Kari},
	month = jul,
	year = {1997},
	keywords = {VEGF, VEGF-C, angiogenesis, growth factor, proteolytic processing},
	pages = {3898--3911},
}

The recently identified vascular endothelial growth factor C (VEGF-C) belongs to the platelet-derived growth factor (PDGF)/VEGF family of growth factors and is a ligand for the endothelial-specific receptor tyrosine kinases VEGFR-3 and VEGFR-2. The VEGF homology domain spans only about one-third of the cysteine-rich VEGF-C precursor. Here we have analysed the role of post-translational processing in VEGF-C secretion and function, as well as the structure of the mature VEGF-C. The stepwise proteolytic processing of VEGF-C generated several VEGF-C forms with increased activity towards VEGFR-3, but only the fully processed VEGF-C could activate VEGFR-2. Recombinant 'mature' VEGF-C made in yeast bound VEGFR-3 (KD = 135 pM) and VEGFR-2 (KD = 410 pM) and activated these receptors. Like VEGF, mature VEGF-C increased vascular permeability, as well as the migration and proliferation of endothelial cells. Unlike other members of the PDGF/VEGF family, mature VEGF-C formed mostly non-covalent homodimers. These data implicate proteolytic processing as a regulator of VEGF-C activity, and reveal novel structure–function relationships in the PDGF/VEGF family.

@article{oh_vegf_1997,
	title = {{VEGF} and {VEGF}-{C}: {Specific} {Induction} of {Angiogenesis} and {Lymphangiogenesis} in the {Differentiated} {Avian} {Chorioallantoic} {Membrane}},
	volume = {188},
	issn = {0012-1606},
	shorttitle = {{VEGF} and {VEGF}-{C}},
	url = {http://dx.doi.org/10.1006/dbio.1997.8639},
	doi = {10.1006/dbio.1997.8639},
	abstract = {The lymphangiogenic potency of endothelial growth factors has not been studied to date. This is partially due to the lack ofin vivolymphangiogenesis 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 α-smooth muscle actin, andin situhybridization 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 thein vivoeffects 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.},
	number = {1},
	urldate = {2012-09-22},
	journal = {Developmental Biology},
	author = {Oh, Su-Ja and Jeltsch, Markku M. and Birkenhäger, Ralf and McCarthy, John E.G. and Weich, Herbert A. and Christ, Bodo and Alitalo, Kari and Wilting, Jörg},
	month = aug,
	year = {1997},
	keywords = {Actins, Animals, Cell Differentiation, Cell Division, Chick Embryo, Chorion, Coturnix, DNA Probes, Endothelial Growth Factors, Fibronectins, Immunohistochemistry, In Situ Hybridization, Lymphatic System, Lymphokines, Microcirculation, Neovascularization, Physiologic, Receptor Protein-Tyrosine Kinases, Receptors, Cell Surface, Receptors, Growth Factor, Receptors, Vascular Endothelial Growth Factor, Recombinant Proteins, Vascular Endothelial Growth Factor A, Vascular Endothelial Growth Factor Receptor-3, Vascular Endothelial Growth Factors, vascular endothelial growth factor C},
	pages = {96--109},
}

The lymphangiogenic potency of endothelial growth factors has not been studied to date. This is partially due to the lack ofin vivolymphangiogenesis 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 α-smooth muscle actin, andin situhybridization 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 thein vivoeffects 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.

@mastersthesis{jeltsch_functional_1997,
	address = {Helsinki, Finland},
	title = {Functional {Analysis} of {VEGF}-{B} and {VEGF}-{C}},
	url = {http://urn.fi/URN:NBN:fi-fe977347},
	abstract = {Vascular endothelial growth factor (VEGF) is an important regulator of endothelial cell proliferation and migration during 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 capable 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 to 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.},
	school = {University of Helsinki},
	author = {Jeltsch, Michael},
	month = jan,
	year = {1997},
}

Vascular endothelial growth factor (VEGF) is an important regulator of endothelial cell proliferation and migration during 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 capable 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 to 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.