Vegf-c signaling pathways through vegfr-2 and vegfr-3 in vasculo-angiogenesis and hematopoeesis

Embryonic development of blood vessels from endothelial cells consists of vasculogenesis and angiogenesis.Vasculo-angiogenesis is regulated by paracrine signals, many of which are protein ligands that bind to and modulate the activity of transmembrane receptor tyrosine kinases (RTKs). Among the vasculo-angiogenic factors, vascular endothelial growth factor (VEGF) family members and their receptors are known to be critical for vasculo-angiogenesis and hematopoiesis. To clarify these functional differences in VEGF-C signaling through VEGFR-2 and VEGFR-3, we analyzed using a coculture of para-aortic splanchnopleural mesoderm (PSp) expiants from mouse embryos with stromal cells (OP9). This culture system is used to assay both vasculo-angiogenesis and hematopoiesis (Immunity 9:677-86, 1998). Vasculogenesis and angiogenesis were evaluated by the extent of vascular bed and network formation, respectively. Addition of VEGF-C to the P-Sp culture enhanced vascular bed formation and suppressed definitive hematopoiesis. Both vascular bed and network formations were completely suppressed by addition of soluble VEGFR-1-Fc competitor protein. Formation of vascular beds but not networks could be rescued by VEGF-C in the presence of the competitor, while both were rescued by VEGF-A. VEGFR-3 deficient embryos show the abnormal vasculature and severe anemia. Consistent with these in vivo findings, vascular bed formation in the P-Sp from the VEGFR-3 deficient embryos was enhanced to that in wild-type or heterozygous embryos and hematopoiesis was severely suppressed. When VEGFR-3-Fc chimeric protein was added to trap endogenous VEGFC in the P-Sp culture of the VEGFR-3 deficient embryos, vascular bed formation was suppressed and hematopoiesis was partially rescued. These results demonstrate that since VEGF-C signaling through VEGFR-2 works synergistically with VEGF-A, the binding of VEGF-C to VEGFR-3 consequently regulates VEGFR-2 signaling. In VEGFR3 deficient embryos, an excess of VEGF-C signals through VEGFR-2 induced the disturbance of vasculogenesis and hematopoiesis during embryogenesis. This indicates that elaborated control through VEGFR-3 signaling is critical in vasculo-angiogenesis and hematopoiesis.