Neprilysin inhibits angiogenesis via proteolysis of fibroblast growth factor-2.
Neprilysin is a cell surface peptidase that catalytically inactivates neuropeptide substrates and functions as a tumor suppressor via its enzymatic function and multiple protein-protein interactions. We investigated whether neutral endopeptidase could inhibit angiogenesis in vivo utilizing a murine corneal pocket angiogenesis model and found that it reduced fibroblast growth factor-2-induced angiogenesis by 85% (p < 0.01) but had no effect on that of vascular endothelial growth factor. Treatment with recombinant neprilysin, but not enzymatically inactive neprilysin, resulted in a slight increase in basic fibroblast growth factor electrophoretic mobility from proteolytic cleavage between amino acids Leu-135 and Gly-136, which was inhibited by the neutral endopeptidase inhibitor CGS24592 and heparin. Cleavage kinetics were rapid, comparable with that of other known neprilysin substrates. Functional studies involving neprilysin-expressing vascular endothelial cells demonstrated that neutral endopeptidase inhibition significantly enhanced fibroblast growth factor-mediated endothelial cell growth, capillary array formation, and signaling, whereas exogenous recombinant neprilysin inhibited signaling. Recombinant constructs confirmed that cleavage products neither promoted capillary array formation nor induced signaling. Moreover, mutation of the cleavage site resulted in concomitant loss of cleavage and increased the potency of fibroblast growth factor-2 to induce capillary array formation. These data indicate that neprilysin proteolytically inactivates fibroblast growth factor-2, resulting in negative regulation of angiogenesis.
Goodman
Oscar B
OB
Urologic Oncology Research Laboratory, Department of Urology, Weil Medical College of Cornell University-New York Presbyterian Hospital, New York, NY 10021, USA.
Febbraio
Maria
M
Simantov
Ronit
R
Zheng
Rong
R
Shen
Ruoqian
R
Silverstein
Roy L
RL
Nanus
David M
DM
D002460:Cell Line; D042783:Endothelial Cells / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism; D006801:Humans; D007930:Leucine / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D018919:Neovascularization, Physiologic; D015260:Neprilysin / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015398:Signal Transduction
D016222:Fibroblast Growth Factor 2; D020928:Mitogen-Activated Protein Kinases; D015260:Neprilysin; D007930:Leucine; D005998:Glycine