The platelet activation receptor CLEC-2 plays crucial roles in thrombosis/hemostasis, tumor metastasis, and lymphangiogenesis, although its role in thrombosis/hemostasis remains controversial. An endogenous ligand for CLEC-2, podoplanin, is expressed in lymphatic endothelial cells (LECs). We and others have reported that CLEC-2-deficiency is lethal at mouse embryonic/neonatal stages associated with blood-filled lymphatics, indicating that CLEC-2 is essential for blood/lymphatic vessel separation. However, its mechanism, and whether CLEC-2 in platelets is necessary for this separation, remains unknown. We found that specific deletion of CLEC-2 from platelets leads to the misconnection of blood/lymphatic vessels. CLEC-2^+/+ platelets, but not by CLEC-2^−/− platelets, inhibited LEC migration, proliferation, and tube formation but had no effect on human umbilical vein endothelial cells. Additionally, supernatants from activated platelets significantly inhibited these three functions in LECs, suggesting that released granule contents regulate blood/lymphatic vessel separation. Bone morphologic protein-9 (BMP-9), which we found to be present in platelets and released upon activation, appears to play a key role in regulating LEC functions. Only BMP-9 inhibited tube formation, although other releasates including transforming growth factor-β and platelet factor 4 inhibited proliferation and/or migration. We propose that platelets regulate blood/lymphatic vessel separation by inhibiting the proliferation, migration, and tube formation of LECs, mainly because of the release of BMP-9 upon activation by CLEC-2/podoplanin interaction.

The lymphatic vasculature originates from the blood vasculature through a mechanism relying on Prox1 expression and VEGFC signalling, and is separated and kept separate from the blood vasculature in a Syk- and SLP76-dependent manner. However, the mechanism by which lymphatic vessels are separated from blood vessels is not known. To gain an understanding of the vascular partitioning, we searched for the affected gene in a spontaneous mouse mutant exhibiting blood-filled lymphatic vessels, and identified a null mutation of the Plcg2 gene, which encodes phospholipase Cγ2 (PLCγ2),by positional candidate cloning. The blood-lymph shunt observed in PLCγ2-null mice was due to aberrant separation of blood and lymphatic vessels. A similar phenotype was observed in lethally irradiated wild-type mice reconstituted with PLCγ2-null bone marrow cells. These findings indicate that PLCγ2 plays an essential role in initiating and maintaining the separation of the blood and lymphatic vasculature.

We have identified two novel human genes homologous to BAH (brain-specific angiogenesis inhibitor 1), an angiogenesis inhibitor that is a candidate for involvement in development of glioblastoma. Like BAH, these two genes, designated BAI2 and BAD, were specifically expressed in brain, and are likely to be expressed in the same type of cells. However, in spite of similar tissue specificity among the three BAI genes, only BAH is transcriptionally regulated by p53. BAD expression was absent in two of nine glioblastoma cell lines examined and was significantly reduced in three of the remaining seven. These data suggest that members of this novel gene family may play important roles in suppression of glioblastoma. BAH, BAI2 and BAI3 were mapped to 8q24, 1p35 and 6q12, respectively.

Mammalian Ras, which is encoded by three independent genes, has been thought to be a versatile component of intracellular signalling. However, when, where and how Ras signalling plays essential roles in development and whether the three Ras genes have overlapping functions in particular cells remain unclear. Here, we show that the three Ras proteins dose-dependently regulate lymphatic vessel growth in mice. We find that lymphatic vessel hypoplasia is a common phenotype in Ras compound knockout mice and that overexpressed normal Ras in an endothelial cell lineage selectively causes lymphatic vessel hyperplasia in vivo. Overexpression of normal Ras in lymphatic endothelial cells leads to sustained MAPK activation, cellular viability and enhanced endothelial network formation under serum-depleted culture conditions in vitro, and knockdown of endogenous Ras in lymphatic endothelial cells impairs cell proliferation, MAPK activation, cell migration and endothelial network formation. Ras overexpression and knockdown result in up- and downregulation of vascular endothelial growth factor receptor (VEGFR) 3 expression, respectively, in lymphatic endothelial cells in vitro. The close link between Ras and VEGFR3 in vitro is consistent with the result that Ras knockout and transgenic alleles are genetic modifiers in lymphatic vessel hypoplasia caused by Vegfr3 haploinsufficiency. Our findings demonstrate a cooperative function of the three Ras proteins in normal development, and also provide a novel aspect of VEGFR3 signalling modulated by Ras in lymphangiogenesis.

Lymphatic valves prevent the backflow of the lymph fluid and ensure proper lymphatic drainage throughout the body. Local accumulation of lymphatic fluid in tissues, a condition called lymphedema, is common in individuals with malformed lymphatic valves. The vascular endothelial growth factor receptor 3 (VEGFR3) is required for the development of lymphatic vascular system. The abundance of VEGFR3 in collecting lymphatic trunks is high before valve formation and, except at valve regions, decreases after valve formation. We found that in mesenteric lymphatics, the abundance of epsin 1 and 2, which are ubiquitin-binding adaptor proteins involved in endocytosis, was low at early stages of development. After lymphatic valve formation, the initiation of steady shear flow was associated with an increase in the abundance of epsin 1 and 2 in collecting lymphatic trunks, but not in valve regions. Epsin 1 and 2 bound to VEGFR3 and mediated the internalization and degradation of VEGFR3, resulting in termination of VEGFR3 signaling. Mice with lymphatic endothelial cell–specific deficiency of epsin 1 and 2 had dilated lymphatic capillaries, abnormally high VEGFR3 abundance in collecting lymphatics, immature lymphatic valves, and defective lymph drainage. Deletion of a single Vegfr3 allele or pharmacological suppression of VEGFR3 signaling restored normal lymphatic valve development and lymph drainage in epsin-deficient mice. Our findings establish a critical role for epsins in the temporal and spatial regulation of VEGFR3 abundance and signaling in collecting lymphatic trunks during lymphatic valve formation.