TY - JOUR
T1 - Vascular normalization by ROCK inhibitor
T2 - Therapeutic potential of ripasudil (K-115) eye drop in retinal angiogenesis and hypoxia
AU - Yamaguchi, Muneo
AU - Nakao, Shintaro
AU - Arita, Ryoichi
AU - Kaizu, Yoshihiro
AU - Arima, Mitsuru
AU - Zhou, Yedi
AU - Kita, Takeshi
AU - Yoshida, Shigeo
AU - Kimura, Kazuhiro
AU - Isobe, Tomoyuki
AU - Kaneko, Yoshio
AU - Sonoda, Koh hei
AU - Ishibashi, Tatsuro
N1 - Funding Information:
Supported by grants from JSPS KAKENHI (Tokyo, Japan), Grant-in-Aid for Young Scientists No. 25713057 (SN), No. 26861454 (RA) and in part by grants from Kowa Company, Ltd. (Tokyo, Japan).
Funding Information:
The authors thank Yukari Mizuno (Yamaguchi University Graduate School of Medicine) for technical assistance. Supported by grants from JSPS KAKENHI (Tokyo, Japan), Grant-in-Aid for Young Scientists No. 25713057 (SN), No. 26861454 (RA) and in part by grants from Kowa Company, Ltd. (Tokyo, Japan).
Publisher Copyright:
© 2016 Association for Research in Vision and Ophthalmology Inc.. All rights reserved.
PY - 2016
Y1 - 2016
N2 - PURPOSE. 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 lmol/L. Ripasudil significantly inhibited VEGF-induced HRMECs migration and proliferation. The concentration of ripasudil in the retina was 3.8 to 10.4 lmol/ L and 6.8 to 14.8 lmol/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.
AB - PURPOSE. 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 lmol/L. Ripasudil significantly inhibited VEGF-induced HRMECs migration and proliferation. The concentration of ripasudil in the retina was 3.8 to 10.4 lmol/ L and 6.8 to 14.8 lmol/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.
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U2 - 10.1167/iovs.15-17411
DO - 10.1167/iovs.15-17411
M3 - Article
C2 - 27124322
AN - SCOPUS:84978270693
SN - 0146-0404
VL - 57
SP - 2264
EP - 2276
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
IS - 4
ER -
