HGF/NK4 inhibited VEGF-induced angiogenesis in in vitro cultured endothelial cells and in vivo rabbit model.
OBJECTIVE
As vascular endothelial growth factor (VEGF) plays a pivotal role in the development of diabetic retinopathy, inhibition of angiogenesis induced by VEGF is crucial to treat diabetic retinopathy. HGF (hepatocyte growth factor)/NK4, containing the N-terminal hairpin domain and the four subsequent kringle domains of HGF, is considered as a specific antagonist for HGF. Our aim was to explore the inhibitory effects of HGF/NK4 on angiogenesis induced by VEGF.


METHODS
To analyze the in vivo angiogenesis, we used rabbit corneal micropocket assay. Proliferation and migration of human endothelial cells, expression of ets-1, an essential transcription factor for angiogenesis, and the phosphorylation of extracellular signal-regulated kinase (ERK) was examined with or without HGF/NK4.


RESULTS
Using corneal micropocket assay, in vivo administration of HGF/NK4 inhibited angiogenesis induced by VEGF. HGF/NK4 inhibited proliferation and migration of human endothelial cells induced by VEGF in a dose-dependent manner. Interestingly, VEGF-mediated phosphorylation of ERK was significantly attenuated by HGF/NK4. Of importance, HGF/NK4 attenuated the increase in ets-1 protein stimulated by VEGF. Nevertheless, HGF/NK4 did not affect phosphorylation of VEGF receptor-2 [kinase domain region (KDR)/foetal liver kinase (Flk)-1]. Although tyrosine phosphatase inhibitor (Na(3)VO(4)), or okadaic acid, serine-threonin kinase inhibitor, did not prevent the inhibition of ERK phosphorylation by HGF/NK4, co-incubation of HGF/NK4 with VEGF significantly diminished mitogen-activated protein (MAP) ERK kinase (MEK) phosphorylation (p<0.01).


CONCLUSIONS
Overall, HGF/NK4 inhibited angiogenesis induced by VEGF through inhibition of phosphorylation of ERK and ets-1 expression in in vitro cultured endothelial cells and in vivo rabbit model.
Nakabayashi
M
M
Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Suita, Japan.
Morishita
R
R
Nakagami
H
H
Kuba
K
K
Matsumoto
K
K
Nakamura
T
T
Tano
Y
Y
Kaneda
Y
Y
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D003315:Cornea / Q000098:blood supply*; D004305:Dose-Response Relationship, Drug; D016228:Endothelial Growth Factors / Q000008:administration & dosage / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000503:physiopathology*; D017228:Hepatocyte Growth Factor / Q000494:pharmacology*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000008:administration & dosage / Q000494:pharmacology*; D008222:Lymphokines / Q000008:administration & dosage / Q000494:pharmacology*; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000473:pathology*; D010766:Phosphorylation / Q000187:drug effects; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050783:Proto-Oncogene Proteins c-ets; D011817:Rabbits; D014157:Transcription Factors / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D042442:Vascular Endothelial Growth Factors
C494041:ETS1 protein, human; D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D017228:Hepatocyte Growth Factor; D040301:Vascular Endothelial Growth Factor Receptor-2; D020928:Mitogen-Activated Protein Kinases