VASCULAR-PERMEABILITY FACTOR VASCULAR ENDOTHELIAL GROWTH FACTOR-MEDIATED SIGNALING IN MOUSE MESENTERY VASCULAR ENDOTHELIUM

Vascular permeability factor/vascular endothelial growth factor (VPF/V EGF) is a multifunctional cytokine and growth factor that has importan t roles in both pathological and physiological angiogenesis. VPF/VEGF induces vascular hyperpermeability, cell division, and other activitie s by interacting with two specific receptor tyrosine kinases, KDR/Flk- 1 and Flt-1, that are selectively expressed on vascular endothelium. T he signaling cascade that follows VPF/VEGF interaction with cultured e ndothelium is only partially understood but is known to result in incr eased intracellular calcium, activation of protein kinase C, and tyros ine phosphorylations of both receptors, phospholipase C-gamma (PLC-gam ma) and phosphatidylinositol 3'-kinase. For many reasons, signaling ev ents elicited in cultured endothelium mag not mimic mediator effects o n intact normal or tumor-induced microvessels in vivo. Therefore, we d eveloped a system that would allow measurement of VPF/VEGF-induced sig naling on intact microvessels, We used mouse mesentery, a tissue whose numerous microv essels are highly responsive to VPF/VEGF and that we found to express Flk-1 and Flt-1 selectively. At intervals after injec ting VPF/VEGF i.p., mesenteries were harvested, extracted, and immunop recipitated. Immunoblots confirmed that VPF/VEGF induced tyrosine phos phorylation of several proteins in mesenteric microvessels as in cultu red endothelium: Flk-1; PLC-gamma; and mitogen-activated protein kinas e, Similar phosphorylations were observed when mesentery was exposed t o VPF/VEGF in vitro, or when mesenteries were harvested from mice bear ing the mouse ovarian tumor ascites tumor, which itself secretes abund ant VPF/VEGF. Other experiments further elucidated the VPF/VEGF signal ing pathway, demonstrating phosphorylation of both PYK2 and focal adhe sion kinase, activation of c-jun-NH2-kinase with phosphorylation of c- Jun, and an association between Flk-1 and PLC-gamma. In addition, we d emonstrated translocation of mitogen-activated protein kinase to the c ell nucleus in cultured endothelium. Taken together, these experiments describe a new model system with the potential for investigating sign aling events in response to diverse mediators on intact microvessels i n vivo and have further elucidated the VPF/VEGF signaling cascade.