Mitsuru Arima

Increased Expression of Periostin in Vitreous and Fibrovascular Membranes Obtained from Patients with Proliferative Diabetic Retinopathy

PURPOSE Preretinal fibrovascular membranes (FVMs) form as a sequela to proliferative diabetic retinopathy (PDR), and their presence can lead to a severe decrease of vision. The purpose of this study was to determine whether periostin, a matricellular protein that plays a role in cell adhesion and migration, is associated with the formation of FVMs. METHODS One hundred six vitreous samples and 15 FVMs were obtained during vitrectomy on patients with PDR. Semiquantitative RT-PCR was performed to determine the periostin level of the mRNA. Immunohistochemical analyses were performed to determine the sites of periostin expression in the FVMs. ELISA was used to measure the concentrations of periostin, bFGF, and VEGF in the vitreous. RESULTS The periostin level of the mRNA was high in 10 of 10 FVMs tested but was barely detectable in the control retinas. Sequencing of the periostin PCR products revealed three splice variants of the FVMs. Immunohistochemical analysis showed colocalization of periostin and α-SMA in FVM cells. The concentration of periostin in the vitreous was significantly higher in patients with PDR than in the 31 eyes of patients with a macular hole or an epiretinal membrane (P < 0.001). Among the PDR patients, the mean vitreous level of periostin in eyes with FVMs was significantly higher than in those without FVMs (epicenter only; P < 0.001). The correlation between the vitreous concentrations of periostin and of bFGF and VEGF was not significant. CONCLUSIONS These findings indicate that periostin may be involved in the development of FVMs.

Bone marrow-derived monocyte lineage cells recruited by MIP-1β promote physiological revascularization in mouse model of oxygen-induced retinopathy.

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-1β (MIP-1β) was the most upregulated protein. The purpose of this study was to investigate the role played by MIP-1β in recruiting bone marrow-derived monocyte lineage cells (BM-MLCs) in a mouse model of OIR. Our results showed that MIP-1β was upregulated, and its receptor, CCR5, was expressed in BM-MLCs in the hypoxic inner retina. Neutralizing Ab against MIP-1β 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-1β-regulated pathological NV strongly depended on VEGF-A, which was probably secreted by the hypoxic avascular retinas. These results indicate that MIP-1β 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-1β induction of BM-MLCs might possibly be used to promote intraretinal revascularization and thus prevent the abnormal NV in ischemic vision-threatening retinal diseases.

Antiangiogenic shift in vitreous after vitrectomy in patients with proliferative diabetic retinopathy

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.

Vascular Normalization by ROCK Inhibitor: Therapeutic Potential of Ripasudil (K-115) Eye Drop in Retinal Angiogenesis and Hypoxia.

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 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.

ADAM12 and ADAM17 are essential molecules for hypoxia-induced impairment of neural vascular barrier function.

Neural vascular barrier is essential for the life of multicellular organisms, and its impairment by tissue hypoxia is known to be a central of pathophysiology accelerating the progression of various intractable neural diseases. Therefore, the molecules involved in hypoxia-induced impairment of vascular barrier can be the targets to establish new therapies for intractable diseases. Here, we demonstrate that a disintegrin and metalloproteinases (ADAMs) 12 and 17 expressed in endothelial cells are the molecules responsible for the impairment of neural vascular barrier by hypoxia. Brain microvascular endothelial cells in vitro lost their barrier properties immediately after hypoxic stimulation through diminished localization of claudin-5, a tight junction molecule, on cell membranes. Hypoxic disappearance of claudin-5 from cell membranes and the consequent loss of barrier properties were completely suppressed by inhibition of the metalloproteinase activity which was found to be attributed to ADAM12 and ADAM17. Inhibition of either ADAM12 or ADAM17 was sufficient to rescue the in vivo neural vasculature under hypoxia from the loss of barrier function. This is the first report to specify the molecules which are responsible for hypoxia-induced impairment of neural vascular barrier and furthermore can be the targets of new therapeutic strategies for intractable neural diseases.

High-Resolution Imaging by Adaptive Optics Scanning Laser Ophthalmoscopy Reveals Two Morphologically Distinct Types of Retinal Hard Exudates

Histological studies from autopsy specimens have characterized hard exudates as a composition of lipid-laden macrophages or noncellular materials including lipid and proteinaceous substances (hyaline substances). However, the characteristics of hard exudates in living patients have not been examined due to insufficient resolution of existing equipment. In this study, we used adaptive optics scanning laser ophthalmoscopy (AO-SLO) to examine the characteristics of hard exudates in patients with retinal vascular diseases. High resolution imaging using AO-SLO enables morphological classification of retinal hard exudates into two types, which could not be distinguished either on fundus examination or by spectral domain optical coherence tomography (SD-OCT). One, termed a round type, consisted of an accumulation of spherical particles (average diameter of particles: 26.9 ± 4.4 μm). The other, termed an irregular type, comprised an irregularly shaped hyper-reflective deposition. The retinal thickness in regions with round hard exudates was significantly greater than the thickness in regions with irregular hard exudates (P = 0.01 →0.02). This differentiation of retinal hard exudates in patients by AO-SLO may help in understanding the pathogenesis and clinical prognosis of retinal vascular diseases.

Optical coherence tomography angiography reveals spatial bias of macular capillary dropout in diabetic retinopathy

Purpose Our purpose is to evaluate the spatial bias of macular capillary dropout accompanying diabetic retinopathy (DR) using optical coherence tomography angiography (OCTA). Methods This study included 47 patients with diabetes and 29 healthy individuals who underwent OCTA. Retinal capillary flow density (FD) of 2.6 × 2.6 or 5.2 × 5.2 mm foveal area as well as the four divided areas (superior, inferior, temporal, nasal) without a foveal avascular zone (FAZ) at the superficial capillary plexus and deep capillary plexus (DCP) were measured respectively using ImageJ and NI Vision. Spatial biases of FD (orientation bias ratio and hierarchical bias ratio) and the correlation between FAZ and FD were examined. Results OCTA showed focal capillary dropout in DR patients. The orientation bias of FD was significantly higher in NPDR compared to NDR in the DCP (P = 0.03). The hierarchical bias of FD was significantly shifted to a DCP dominance with progression of DR (P < 0.01). In addition, the FD and FAZ area were significantly inversely correlated in both plexus in DR patients but not in healthy subjects (P < 0.01). Conclusions 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.

Basigin can be a therapeutic target to restore the retinal vascular barrier function in the mouse model of diabetic retinopathy

Despite the advance in medical technology, diabetic retinopathy (DR) is still an intractable disease which leads to the damage of retinal cells and finally the visual loss. Impairment of retinal vascular barrier triggered by an admixture of multiple inflammatory cytokines is a core of pathophysiology of DR. Therefore, the molecules involved commonly in multiple cytokines-induced impairment of vascular barrier would be the targets of curative treatment of DR. Here, we demonstrate that basigin, a transmembrane molecule expressed in neural barrier-forming endothelial cells, is the molecule essential for vascular barrier impairment which is shared by various triggers including VEGF, TNFα and IL-1β. In vitro data with neural microvascular endothelial cells indicated that stimulation with cytokines decreases the levels of claudin-5 in cell membranes and consequently impairs the barrier function in a manner dependent on the interaction of claudin-5 with basigin and caveolin-1. In addition, the increased vascular permeability in retinas of streptozotocin-induced diabetic mice was shown to be clearly normalized by intravitreous injection of siRNAs specific for basigin. This study has highlighted basigin as a common essential molecule for various stimuli-induced impairment of retinal vascular barrier, which can be a target for strategies to establish a curative treatment of DR.

Tenascin-C secreted by transdifferentiated retinal pigment epithelial cells promotes choroidal neovascularization via integrin αV.

Tenascin-C is expressed in choroidal neovascular (CNV) membranes in eyes with age-related macular degeneration (AMD). However, its role in the pathogenesis of CNV remains to be elucidated. Here we investigated the role of tenascin-C in CNV formation. In immunofluorescence analyses, tenascin-C co-stained with α-SMA, pan-cytokeratin, CD31, CD34, and integrin αV in the CNV membranes of patients with AMD and a mouse model of laser-induced CNV. A marked increase in the expression of tenascin-C mRNA and protein was observed 3 days after laser photocoagulation in the mouse CNV model. Tenascin-C was also shown to promote proliferation and inhibit adhesion of human retinal pigment epithelial (hRPE) cells in vitro. Moreover, tenascin-C promoted proliferation, adhesion, migration, and tube formation in human microvascular endothelial cells (HMVECs); these functions were, however, blocked by cilengitide, an integrin αV inhibitor. Exposure to TGF-β2 increased tenascin-C expression in hRPE cells. Conditioned media harvested from TGF-β2-treated hRPE cell cultures enhanced HMVEC proliferation and tube formation, which were inhibited by pretreatment with tenascin-C siRNA. The CNV volume was significantly reduced in tenascin-C knockout mice and tenascin-C siRNA-injected mice. These findings suggest that tenascin-C is secreted by transdifferentiated RPE cells and promotes the development of CNV via integrin αV in a paracrine manner. Therefore, tenascin-C could be a potential therapeutic target for the inhibition of CNV development associated with AMD.

A case of metastatic choroidal tumor simulating a choroidal melanoma

The choroid is an area of the eye commonly affected both by metastatic tumors and by primary malignant melanomas [1]. Although the accuracy of the clinical diagnosis of uveal melanoma has improved [2], atypical ocular manifestations may lead to misdiagnosis in some cases. In the case presented here, we observed choroidal metastasis with melanocytic proliferation that mimicked malignant choroidal melanoma.