PURPOSE. In this study, we investigated the therapeutic potential of a Rho-associated coiledcoil-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.

These findings indicate that periostin may be involved in the development of FVMs.

Area-divided OCTA quantification shows the appearance of spatial biases of macular capillary dropout with the onset of DR, suggesting that DR-related macular capillary dropout occurs locally and randomly. Future studies are necessary to determine the clinical relevance of the spatial pattern of capillary dropout in DR.

Intravitreal injection of anti-VEGF-A Ab could inhibit leukocyte trafficking in the retina, suggesting that anti-VEGF-A therapy could serve as a treatment in retinal inflammation.

Approximately 40% of patients with diabetic macular edema (DME) are resistant to anti-vascular endothelial growth factor (VEGF) therapy (rDME). Here, we demonstrate that significant correlations between inflammatory cytokines and VEGF, as observed in naive DME, are lost in patients with rDME. VEGF overexpression in the mouse retina caused delayed inflammatory cytokine upregulation, monocyte/macrophage infiltration (CD11b 1 Ly6C 1 CCR2 1 cells), macrophage/microglia activation (CD11b 1 CD80 1 cells), and blood-retinal barrier disruption due to claudin-5 redistribution, which did not recover with VEGF blockade alone. Phosphorylated protein analysis of VEGFoverexpressed retinas revealed rho-associated coiled-coilcontaining protein kinase (ROCK) activation. Administration of ripasudil, a selective ROCK inhibitor, attenuated retinal inflammation and claudin-5 redistribution. Ripasudil also contributed to the stability of claudin-5 expression by both transcriptional enhancement and degradation suppression in inflammatory cytokine-stimulated endothelium. Notably, the anti-VEGF agent and the ROCK inhibitor were synergic in suppressing cytokine upregulation, monocyte/macrophage infiltration, macrophage/microglia activation, and claudin-5 redistribution. Furthermore, in vitro analysis confirmed that claudin-5 redistribution depends on ROCK2 but not on ROCK1. This synergistic effect was also confirmed in human rDME cases. Our results suggest that ROCKmediated claudin-5 redistribution by inflammation is a key mechanism in the anti-VEGF resistance of DME.