title: Vascular Normalization by ROCK Inhibitor: Therapeutic Potential of Ripasudil (K-115) Eye Drop in Retinal Angiogenesis and Hypoxia.
abstract: OBJECTIVE
  In this study, we investigated the therapeutic potential of a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor ripasudil (K-115) eye drop on retinal neovascularization and hypoxia.
  
  
  METHODS
  In vitro, human retinal microvascular endothelial cells (HRMECs) were pretreated with ripasudil and then stimulated with VEGF. ROCK activity was evaluated by phosphorylation of myosin phosphatase target protein (MYPT)-1. Endothelial migration and cell viability were assessed by cell migration and MTT assay, respectively. The concentration of ripasudil in the retina was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In vivo, normal saline, 0.4%, or 0.8% ripasudil were administered three times a day to mice with oxygen-induced retinopathy (OIR). The areas of neovascularization and avascular retina were also quantified with retinal flat-mounts at postnatal day (P) 15, P17, or P21. The retinal hypoxic area was evaluated using hypoxia-sensitive drug pimonidazole by immunohistochemistry at P17. The vascular normalization was also evaluated by immunohistochemistry at P17.
  
  
  RESULTS
  Ripasudil but not fasudil significantly reduced VEGF-induced MYPT-1 phosphorylation in HRMECs at 30 μmol/L. Ripasudil significantly inhibited VEGF-induced HRMECs migration and proliferation. The concentration of ripasudil in the retina was 3.8 to 10.4 μmol/L and 6.8 to 14.8 μmol/L after 0.4% and 0.8% ripasudil treatment, respectively. In the 0.4% and 0.8% ripasudil treated OIR mice, the areas of neovascularization as well as avascular area in the retina was significantly reduced compared with those of saline-treated mice at P17 and P21. Pimonidazole staining revealed that treatment with 0.4% and 0.8% ripasudil significantly inhibited the increase in the hypoxic area compared with saline. 0.8% ripasudil could cause intraretinal vascular sprouting and increase retinal vascular perfusion.
  
  
  CONCLUSIONS
  Novel ROCK inhibitor ripasudil eye drop has therapeutic potential in the treatment of retinal hypoxic neovascular diseases via antiangiogenic effects as well as vascular normalization.
authors.affiliation: Department of Ophthalmology Graduate School of Medical Sciences, Kyushu University, Higashi Ku, Fukuoka, Japan.
authors.forename: Muneo
authors.initials: M
authors.lastname: Yamaguchi
authors.affiliation: Department of Ophthalmology Graduate School of Medical Sciences, Kyushu University, Higashi Ku, Fukuoka, Japan.
authors.forename: Shintaro
authors.initials: S
authors.lastname: Nakao
authors.affiliation: Department of Ophthalmology Graduate School of Medical Sciences, Kyushu University, Higashi Ku, Fukuoka, Japan.
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Arita
authors.affiliation: Department of Ophthalmology Graduate School of Medical Sciences, Kyushu University, Higashi Ku, Fukuoka, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kaizu
authors.affiliation: Department of Ophthalmology Graduate School of Medical Sciences, Kyushu University, Higashi Ku, Fukuoka, Japan.
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Arima
authors.affiliation: Department of Ophthalmology Graduate School of Medical Sciences, Kyushu University, Higashi Ku, Fukuoka, Japan.
authors.forename: Yedi
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Department of Ophthalmology Graduate School of Medical Sciences, Kyushu University, Higashi Ku, Fukuoka, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kita
authors.affiliation: Department of Ophthalmology Graduate School of Medical Sciences, Kyushu University, Higashi Ku, Fukuoka, Japan.
authors.forename: Shigeo
authors.initials: S
authors.lastname: Yoshida
authors.affiliation: Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube City, Yamaguchi, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Kimura
authors.affiliation: Tokyo New Drug Research Laboratories, Kowa Company, Ltd., Higashimurayama, Tokyo, Japan.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Isobe
authors.affiliation: Tokyo New Drug Research Laboratories, Kowa Company, Ltd., Higashimurayama, Tokyo, Japan.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Kaneko
authors.affiliation: Department of Ophthalmology Graduate School of Medical Sciences, Kyushu University, Higashi Ku, Fukuoka, Japan.
authors.forename: Koh-Hei
authors.initials: KH
authors.lastname: Sonoda
authors.affiliation: Department of Ophthalmology Graduate School of Medical Sciences, Kyushu University, Higashi Ku, Fukuoka, Japan.
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Ishibashi
mesh_terms: D000818:Animals; D002465:Cell Movement / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002853:Chromatography, Liquid; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D000860:Hypoxia / Q000188:drug therapy; D066298:In Vitro Techniques; D007546:Isoquinolines / Q000008:administration & dosage / Q000032:analysis / Q000627:therapeutic use*; D051379:Mice; D008810:Mice, Inbred C57BL; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D009883:Ophthalmic Solutions; D010766:Phosphorylation / Q000187:drug effects; D012160:Retina / Q000737:chemistry; D015861:Retinal Neovascularization / Q000188:drug therapy*; D013449:Sulfonamides / Q000008:administration & dosage / Q000032:analysis / Q000627:therapeutic use*; D053719:Tandem Mass Spectrometry; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors*
chemical_list: D007546:Isoquinolines; C584679:K-115; D009883:Ophthalmic Solutions; D013449:Sulfonamides; D042461:Vascular Endothelial Growth Factor A; D054460:rho-Associated Kinases; D043263:Myosin-Light-Chain Phosphatase