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.

Vascular endothelial growth factor receptor‐3 (VEGFR‐3/Flt4) binds two known members of the VEGF ligand family, VEGF‐C and VEGF‐D, and has a critical function in the remodelling of the primary capillary vasculature of midgestation embryos. Later during development, VEGFR‐3 regulates the growth and maintenance of the lymphatic vessels. In the present study, we have isolated and cultured stable lineages of blood vascular and lymphatic endothelial cells from human primary microvascular endothelium by using antibodies against the extracellular domain of VEGFR‐3. We show that VEGFR‐3 stimulation alone protects the lymphatic endothelial cells from serum deprivation‐induced apoptosis and induces their growth and migration. At least some of these signals are transduced via a protein kinase C‐dependent activation of the p42/p44 MAPK signalling cascade and via a wortmannin‐sensitive induction of Akt phosphorylation. These results define the critical role of VEGF‐C/VEGFR‐3 signalling in the growth and survival of lymphatic endothelial cells. The culture of isolated lymphatic endothelial cells should now allow further studies of the molecular properties of these cells.

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 .