Deletion of Vascular Endothelial Growth Factor C (VEGF-C) and VEGF-D Is Not Equivalent to VEGF Receptor 3 Deletion in Mouse Embryos

ABSTRACT Lymphatic vessels play an important role in the regulation of tissue fluid balance, immune responses, and fat adsorption and are involved in diseases including lymphedema and tumor metastasis. Vascular endothelial growth factor (VEGF) receptor 3 (VEGFR-3) is necessary for development of the blood vasculature during early embryogenesis, but later, VEGFR-3 expression becomes restricted to the lymphatic vasculature. We analyzed mice deficient in both of the known VEGFR-3 ligands, VEGF-C and VEGF-D. Unlike the Vegfr3−/− embryos, the Vegfc−/−; Vegfd−/− embryos displayed normal blood vasculature after embryonic day 9.5. Deletion of Vegfr3 in the epiblast, using keratin 19 (K19) Cre, resulted in a phenotype identical to that of the Vegfr3−/− embryos, suggesting that this phenotype is due to defects in the embryo proper and not in placental development. Interestingly, the Vegfr3neo hypomorphic mutant mice carrying the neomycin cassette between exons 1 and 2 showed defective lymphatic development. Overexpression of human or mouse VEGF-D in the skin, under the K14 promoter, rescued the lymphatic hypoplasia of the Vegfc+/− mice in the K14-VEGF-D; Vegfc+/− compound mice, suggesting that VEGF-D is functionally redundant with VEGF-C in the stimulation of developmental lymphangiogenesis. Our results suggest VEGF-C- and VEGF-D-independent functions for VEGFR-3 in the early embryo.

[1]  Tatiana V. Petrova,et al.  Lymphangiogenesis in development and human disease , 2005, Nature.

[2]  H. Wolburg,et al.  Development of the Zebrafish Lymphatic System Requires Vegfc Signaling , 2006, Current Biology.

[3]  D. Hicklin,et al.  Cooperative and redundant roles of VEGFR‐2 and VEGFR‐3 signaling in adult lymphangiogenesis , 2007, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[4]  R. Kauppinen,et al.  A model for gene therapy of human hereditary lymphedema , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[5]  V. Luria,et al.  Maternally expressed PGK-Cre transgene as a tool for early and uniform activation of the Cre site-specific recombinase , 1998, Transgenic Research.

[6]  M. Tsai,et al.  Lineage tracing demonstrates the venous origin of the mammalian lymphatic vasculature. , 2007, Genes & development.

[7]  T. Veikkola,et al.  Signalling via vascular endothelial growth factor receptor‐3 is sufficient for lymphangiogenesis in transgenic mice , 2001, The EMBO journal.

[8]  R. Jain,et al.  Hyperplasia of lymphatic vessels in VEGF-C transgenic mice. , 1997, Science.

[9]  K. Alitalo,et al.  Adenoviral Expression of Vascular Endothelial Growth Factor-C Induces Lymphangiogenesis in the Skin , 2001, Circulation research.

[10]  B. Weinstein,et al.  Distinct genetic interactions between multiple Vegf receptors are required for development of different blood vessel types in zebrafish. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[11]  M. Taketo,et al.  Intestinal polyposis in mice with a dominant stable mutation of the β‐catenin gene , 1999, The EMBO journal.

[12]  R. Coffey,et al.  Keratin 19 gene drives Cre recombinase expression throughout the early postimplantation mouse embryo , 2005, Genesis.

[13]  S. Nishikawa,et al.  Involvement of vascular endothelial growth factor receptor-3 in maintenance of integrity of endothelial cell lining during tumor angiogenesis. , 2000, Blood.

[14]  J. Partanen,et al.  Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins , 2004, Nature Immunology.

[15]  J. Hitomi,et al.  Live imaging of lymphatic development in the zebrafish , 2006, Nature Medicine.

[16]  M. Shibuya,et al.  Distinct vascular endothelial growth factor signals for lymphatic vessel enlargement and sprouting , 2007, The Journal of experimental medicine.

[17]  Kris Vleminckx,et al.  A genetic Xenopus laevis tadpole model to study lymphangiogenesis , 2005, Nature Medicine.

[18]  Lena Claesson-Welsh,et al.  Ligand-induced Vascular Endothelial Growth Factor Receptor-3 (VEGFR-3) Heterodimerization with VEGFR-2 in Primary Lymphatic Endothelial Cells Regulates Tyrosine Phosphorylation Sites* , 2003, Journal of Biological Chemistry.

[19]  E. Fuchs,et al.  Tissue-specific and differentiation-specific expression of a human K14 keratin gene in transgenic mice. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Reynaldo Sequerra,et al.  High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP , 2000, Nature Genetics.

[21]  K. Alitalo,et al.  Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Philippe Soriano.  Generalized lacZ expression with the ROSA26 Cre reporter strain , 1999, Nature Genetics.

[23]  T. Veikkola,et al.  Lymphangiogenic growth factor responsiveness is modulated by postnatal lymphatic vessel maturation. , 2006, The American journal of pathology.

[24]  Yuquan Wei,et al.  A critical role of vascular endothelial growth factor D in zebrafish embryonic vasculogenesis and angiogenesis. , 2007, Biochemical and biophysical research communications.

[25]  P. Carmeliet,et al.  Heterodimerization with vascular endothelial growth factor receptor-2 (VEGFR-2) is necessary for VEGFR-3 activity. , 2004, Biochemical and biophysical research communications.

[26]  M. Halford,et al.  Vascular Endothelial Growth Factor D Is Dispensable for Development of the Lymphatic System , 2005, Molecular and Cellular Biology.

[27]  T. Veikkola,et al.  Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects , 2002, The Journal of experimental medicine.

[28]  S. V. D. van der Putte.  The early development of the lymphatic system in mouse embryos. , 1975, Acta morphologica Neerlando-Scandinavica.

[29]  M. Karkkainen,et al.  The Specificity of Receptor Binding by Vascular Endothelial Growth Factor-D Is Different in Mouse and Man* , 2001, The Journal of Biological Chemistry.

[30]  Antonio Duarte,et al.  Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation , 2008, Nature.

[31]  van der Putte Sc.  The early development of the lymphatic system in mouse embryos. , 1975 .

[32]  Robert E. Ferrell,et al.  Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema , 2000, Nature Genetics.

[33]  Seppo Ylä-Herttuala,et al.  Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3 , 2001, Nature Medicine.

[34]  Y Sakai,et al.  HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Involvement of vascular endothelial growth factor receptor-3 in maintenance of integrity of endothelial cell lining during tumor angiogenesis , 2022 .

[35]  K. Alitalo,et al.  A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases. , 1996, The EMBO journal.

[36]  G. Oliver,et al.  Prox1 Function Is Required for the Development of the Murine Lymphatic System , 1999, Cell.

[37]  K. Alitalo,et al.  Cardiovascular failure in mouse embryos deficient in VEGF receptor-3. , 1998, Science.

[38]  G. Mann,et al.  Targeting lymphangiogenesis to prevent tumour metastasis , 2006, British Journal of Cancer.

[39]  K. Alitalo,et al.  Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4). , 1998, Proceedings of the National Academy of Sciences of the United States of America.