Vascular permeability factor/vascular endothelial growth factor-mediated signaling in mouse mesentery vascular endothelium.
Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a multifunctional cytokine and growth factor that has important roles in both pathological and physiological angiogenesis. VPF/VEGF induces vascular hyperpermeability, cell division, and other activities by interacting with two specific receptor tyrosine kinases, KDR/Flk-1 and Flt-1, that are selectively expressed on vascular endothelium. The signaling cascade that follows VPF/VEGF interaction with cultured endothelium is only partially understood but is known to result in increased intracellular calcium, activation of protein kinase C, and tyrosine phosphorylations of both receptors, phospholipase C-gamma (PLC-gamma) and phosphatidylinositol 3'-kinase. For many reasons, signaling events elicited in cultured endothelium may not mimic mediator effects on intact normal or tumor-induced microvessels in vivo. Therefore, we developed a system that would allow measurement of VPF/VEGF-induced signaling on intact microvessels. We used mouse mesentery, a tissue whose numerous microvessels are highly responsive to VPF/VEGF and that we found to express Flk-1 and Flt-1 selectively. At intervals after injecting VPF/VEGF i.p., mesenteries were harvested, extracted, and immunoprecipitated. Immunoblots confirmed that VPF/VEGF induced tyrosine phosphorylation of several proteins in mesenteric microvessels as in cultured endothelium: Flk-1; PLC-gamma; and mitogen-activated protein kinase. Similar phosphorylations were observed when mesentery was exposed to VPF/VEGF in vitro, or when mesenteries were harvested from mice bearing the mouse ovarian tumor ascites tumor, which itself secretes abundant VPF/VEGF. Other experiments further elucidated the VPF/VEGF signaling pathway, demonstrating phosphorylation of both PYK2 and focal adhesion kinase, activation of c-jun-NH2-kinase with phosphorylation of c-Jun, and an association between Flk-1 and PLC-gamma. In addition, we demonstrated translocation of mitogen-activated protein kinase to the cell nucleus in cultured endothelium. Taken together, these experiments describe a new model system with the potential for investigating signaling events in response to diverse mediators on intact microvessels in vivo and have further elucidated the VPF/VEGF signaling cascade.
Mukhopadhyay
D
D
Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.
Nagy
J A
JA
Manseau
E J
EJ
Dvorak
H F
HF
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002478:Cells, Cultured; D016228:Endothelial Growth Factors / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000502:physiology*; D051418:Focal Adhesion Kinase 2; D006801:Humans; D008222:Lymphokines / Q000494:pharmacology*; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D009374:Neoplasms, Experimental / Q000098:blood supply; D019000:Phosphotyrosine / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D017978:Receptors, Growth Factor / Q000378:metabolism; D040262:Receptors, Vascular Endothelial Growth Factor; D015398:Signal Transduction; D013152:Splanchnic Circulation; D010738:Type C Phospholipases / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; D016755:Proto-Oncogene Proteins c-jun; D017978:Receptors, Growth Factor; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor; D051418:Focal Adhesion Kinase 2; C496053:Ptk2b protein, mouse; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010738:Type C Phospholipases