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.

Recent clinical observations have indicated that vascular endothelial growth factor (VEGF) is a key factor that stimulates the development of preretinal pathological neovascularization (NV). However, it has not been established how intraretinal physiological revascularization of hypoxic avascular areas is regulated. Our earlier study on the gene expression profile of hypoxic retinas in a mouse model of oxygen-induced retinopathy (OIR) showed that macrophage inflammatory protein-1b (MIP-1b) was the most upregulated protein. The purpose of this study was to investigate the role played by MIP-1b in recruiting bone marrow-derived monocyte lineage cells (BM-MLCs) in a mouse model of OIR. Our results showed that MIP-1b was upregulated, and its receptor, CCR5, was expressed in BM-MLCs in the hypoxic inner retina. Neutralizing Ab against MIP-1b reduced the infiltration of BM-MLCs into the OIR retinas and increased the avascular area and preretinal neovascular tufts. A very strong significant correlation was found between the area of the preretinal neovascular tufts and the avascular area, regardless of the extent of BM-MLC infiltration into the OIR retinas. Additional treatment with VEGF-A-neutralizing Ab showed that the MIP-1b-regulated pathological NV strongly depended on VEGF-A, which was probably secreted by the hypoxic avascular retinas. These results indicate that MIP-1b is involved in the recruitment of BM-MLCs, which have a significant role in the physiological revascularization of hypoxic avascular retinas. Overall, these findings indicate that the MIP-1b induction of BM-MLCs might possibly be used to promote intraretinal revascularization and thus prevent the abnormal NV in ischemic vision-threatening retinal diseases. Tissue hypoxia is believed to be the common mechanism that initiates a series of events leading to compensatory angiogenesis. Hypoxia-induced retinal neovascularization (NV) is the main contributor to proliferative vascular diseases, such as diabetic retinopathy, retinopathy of prematurity, and retinal vein occlusion. These diseases can lead to irreversible damage to visual function.To determine the factors that are activated during retinal hypoxia, we performed a gene expression profile of hypoxic retinas obtained from a mouse model of oxygen-induced retinopathy (OIR) using gene microarray analyses. The results identified the functional set of genes that are related to the post-ischemic inflammation, neural and vascular development, and subsequent pathological NV. The most upregulated gene among the differentially expressed genes in hypoxic retinas was the macrophage inflammatory protein1b (MIP-1b) and not vascular endothelial growth factor A (VEGF-A) and other chemokines. 1 MIP-1b, also known as chemokine CC motif ligand 4, is a member of the CC chemokine family that are characterized by their ability to direct migration of leukocytes into inflamed tissues. 2 MIP-1b was first isolated from the culture medium of LPS-activated macrophages, 3 and it recruited macrophages/microglia to the sites of...

The purpose of this study was to evaluate neurodevelopmental outcomes in 18-month old (corrected age) preterm infants who received an intravitreal bevacizumab (IVB) injection for the treatment of type 1 retinopathy of prematurity (ROP). Methods In this ten-year retrospective study, we reviewed the medical records of patients who underwent ROP screening at Kyushu University Hospital. Among the patients who received IVB or laser photocoagulation (LPC) for the treatment of type 1 ROP, we included infants whose neurodevelopmental examination (the Kyoto Scale of Psychological Development [KSPD]) results at 18 months corrected age were available. Then, the effect of IVB on the developmental quotient (DQ) in each KSPD domain (Postural-Movement, Cognitive-Adaptive, or Language-Social domain) or the overall DQ was investigated by performing linear regression analysis. Results Out of the 513 patients reviewed, 53 were included in the study. IVB and LPC were performed for 14 and 39 patients, respectively. Administration of IVB was significantly associated with neurodevelopmental delay in the Language-Social domain (p = 0.01). The observed association remained even after adjusting for gestational age and birth weight (p = 0.03). Conclusions Administration of IVB may introduce a risk of developmental impairment of interpersonal relationships, socializations, and/or verbal abilities of preterm children. We recommended

PURPOSE. We determined whether the concentrations of VEGF, erythropoietin, and endostatin in the vitreous are altered after vitrectomy in patient with proliferative diabetic retinopathy (PDR). METHODS.We measured the levels of VEGF, erythropoietin, and endostatin by sandwich ELISA in vitreous samples collected from 38 eyes of 33 patients with PDR before pars plana vitrectomy (without IOL implantation) and the same 38 eyes during IOL implantation 3.1 to 25.7 (mean 6.7) months after the initial vitrectomy. RESULTS.The mean vitreous levels of VEGF (964.5 pg/mL) and erythropoietin (1359.5 pg/mL) in the samples collected before vitrectomy were significantly higher in patients with PDR than in the control patients (0.68 and 70.7 pg/mL, respectively; P < 0.01). The levels of VEGF (292.5 pg/mL) and erythropoietin (557.9 pg/mL) in the samples from eyes with PDR collected at the time of IOL implantation were significantly lower than those collected before vitrectomy (P < 0.01). In contrast, the changes in the level of endostatin were not significant after vitrectomy. The VEGF and erythropoietin levels in the vitreous fluid from patients with PDR were correlated inversely with the interval between the initial vitrectomy and the time of the IOL implantation.CONCLUSIONS. The significant decrease in the intravitreal concentration of VEGF and erythropoietin, and an absence of a significant change in the endostatin indicated a shift in the antiangiogenic balance in the vitreous of patients with PDR after successful vitrectomy. (Invest Ophthalmol Vis Sci. 2012; 53:6997-7003)