Antiangiogenic properties of fasudil, a potent Rho-Kinase inhibitor.
OBJECTIVE
Vascular endothelial growth factor (VEGF) plays a pivotal role in pathological angiogenesis. In this study, we addressed the therapeutic potential of fasudil, a potent Rho-kinase inhibitor, for VEGF-elicited angiogenesis and also for the intracellular signalings induced by VEGF.


METHODS
In vitro, the inhibitory effects of fasudil on the VEGF-dependent VEGF receptor 2 (VEFGR2 or KDR), extracellular signal-related kinase (ERK) 1/2, Akt and myosin light chain (MLC) phosphorylation, as well as on the migration and proliferation of bovine retinal microvascular endothelial cells (BRECs) were analyzed with Western blotting, [3H]-thymidine uptake, and modified Boyden chamber assay. VEGF-elicited in vivo angiogenesis was analyzed with a mouse corneal micropocket assay coembedded with or without fasudil.


RESULTS
VEGF caused enhanced MLC phosphorylation of BRECs, which was almost completely attenuated by 10microM fasudil. VEGF-dependent phosphorylation of ERK1/2 and Akt were also partially but significantly attenuated by treatment with fasudil without affecting VEGFR2 (KDR) phosphorylation. Moreover, both VEGF-induced [3H]-thymidine uptake and the migration of BRECs were significantly inhibited in the presence of fasudil. Finally, VEGF-elicited angiogenesis in the corneal micropocket assay was potently attenuated by coembedding with fasudil (P < 0.01).


CONCLUSIONS
These findings indicate that fasudil might have a therapeutic potential for ocular angiogenic diseases. The antiangiogenic effect of fasudil appears to be mediated through the blockade not only of Rho-kinase signaling but also of ERK and Akt signaling.
Hata
Yasuaki
Y
Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. hatachan@med.kyushu-u.ac.jp
Miura
Muneki
M
Nakao
Shintaro
S
Kawahara
Shuhei
S
Kita
Takeshi
T
Ishibashi
Tatsuro
T
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D018929:Cell Culture Techniques; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D016510:Corneal Neovascularization / Q000517:prevention & control; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012171:Retinal Vessels / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors*
D020533:Angiogenesis Inhibitors; D018994:Myosin Light Chains; D047428:Protein Kinase Inhibitors; D042461:Vascular Endothelial Growth Factor A; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D040301:Vascular Endothelial Growth Factor Receptor-2; D051057:Proto-Oncogene Proteins c-akt; D054460:rho-Associated Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C049347:fasudil