Yukio Sassa

The characterisation of hyalocytes: the origin, phenotype, and turnover

Aim: To determine the characterisation of hyalocytes: the origin, phenotype, and turnover in the rodent. Methods: To characterise the ultrastructure and distribution of hyalocytes, transmission and scanning electron microscopy was performed in rat eyes. Immunophenotypical analysis was performed by either anti-ED1 or ED2 antibodies. To examine the origin of the hyalocytes, the chimeric mice were created and were used to transplant the bone marrow (BM) cells from enhanced green fluorescent protein (EGFP) transgenic mice. The turnover of hyalocytes was examined at 0, 4, 6, 7, and 12 months after BM transplantation. Results: Hyalocytes were distributed especially in the vitreous cortex and had an irregular shape with a spherical granule. Immunophenotypical studies demonstrated that most of the hyalocytes in rat eyes expressed ED2 but not ED1. In the chimeric mice, the hyalocytes were GFP negative right after BM transplantation. Interestingly, more than 60% of hyalocytes were replaced within 4 months and approximately 90% within 7 months after BM transplantation. Conclusions: The rodent hyalocytes were shown to express tissue macrophage marker, were derived from BM, and totally replaced within 7 months. These data provide the characterisation of hyalocytes in physiological conditions, especially their origin, distribution, and turnover, and may contribute to the better understanding of the pathogenesis of vitreoretinal disease.

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.

Periostin promotes the generation of fibrous membranes in proliferative vitreoretinopathy

Proliferative vitreoretinopathy (PVR) is a severe, vision‐threatening disorder characterized by the fibrous membrane formation that leads to trac‐tional retinal detachment. There has been no effective therapeutic approach other than vitreoretinal surgery. In this study, DNA microarray analysis of the fibrous membranes revealed significant up‐regulation of periostin. We also found increased periostin expression in the vitreous and retinal pigment epithelial (RPE) cells from fibrous membranes of PVR patients. In vitro, periostin increased proliferation, adhesion, migration, and collagen production in RPE cells through integrin αVmediated FAK and AKT phosphorylation. Periostin blockade suppressed migration and adhesion induced by TGFβ2 and PVR vitreous. In vivo, periostin inhibition had the inhibitory effect on progression of experimental PVR in rabbit eyes without affecting the viability of retinal cells. These results identified periostin as a pivotal molecule for fibrous membrane formation as well as a promising therapeutic target for PVR.—Ishikawa, K., Yoshida, S., Nakao, S., Nakama, T., Kita, T., Asato, R., Sassa, Y., Arita, R., Miyazaki, M., Enaida, H., Oshima, Y., Murakami, N., Niiro, H., Ono, J., Matsuda, A., Goto, Y., Akashi, K., Izuhara, K., Kudo, A., Kono, T., Hafezi‐Moghadam, A., Ishibashi, T. Periostin promotes the generation of fibrous membranes in proliferative vitreoretinopathy. FASEB J. 28, 131–142 (2014). www.fasebj.org

Interleukin‐18 regulates pathological intraocular neovascularization

Recently, the proinflammatory cytokine IL‐18 has been shown to have a role in angiogenesis. This study aimed to elucidate its role in abnormal neovascularization (NV) in an oxygen‐induced retinopathy (OIR) mouse model of the retinopathy seen in human premature newborns. IL‐18 was constitutively expressed in the retina in C57BL/6 mice, but expression transiently dropped on Day 17 after birth in mice exposed to 75% oxygen for 5 days between Days 7 and 12. Coincident with the IL‐18 reduction in oxygen‐treated mice, vascular endothelial growth factor was expressed in the retina, and OIR developed. By Day 24, NV in the retina had regressed to normal levels. By contrast, IL‐18 knockout mice, exposed to elevated oxygen concentrations, developed more severe OIR on Day 17, and it is important that this persisted until Day 24. This suggested that IL‐18 negatively regulated retinal NV. To investigate this further, we administrated recombinant IL‐18 to C57BL/6 mice during the development of OIR but found no significant inhibition of retinopathy. However, when IL‐18‐binding protein was administered during the OIR recovery phase to neutralize endogenous IL‐18, OIR was still apparent on Day 24. We therefore concluded that IL‐18 regulates pathogenic retinal NV by promoting its regression rather than inhibiting its development. This suggests some useful, new approaches to treating retinopathy in humans.

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 drugs in the management of ocular diseases: Focus on antivascular endothelial growth factor

Age-related macular degeneration (AMD) complications are the leading cause of severe vision loss among the aging population in the many western countries. The introduction of molecular inhibitors of vascular endothelial growth factor (VEGF), such as pegaptanib, ranibizumab, and bevacizumab, as treatments for wet AMD has provided new hope for affected patients. Now we have these treatment options, which have the possibility to improve or maintain visual acuity for patients suffering from AMD. The treatment needs to be optimized and this is in progress. Based on emerging evidence, adopting a variable VEGF inhibitor-dosing strategy guided by visual acuity assessment and optical coherence tomography are now being tried to reduce the frequency of injections. VEGF inhibitors in combination with photodynamic therapy are another way to optimize treatment. Physicians are waiting for new guidelines for the management of AMD and the results of current and upcoming trials systematically addressing these issues will be expected to provide it.

Abnormal retinal vascular development in IL-18 knockout mice

Recent studies have indicated that interleukin 18 (IL-18) might act as either an angiogenic or an angiostatic factor, but the true function of this protein in vascular development is unclear. We therefore investigated the role of IL-18 in the formation of retinal vessels. Development of the retinal vasculature was compared in IL-18 knockout (KO) and wild-type (WT) mice at several different time points. The formation of vessels was evaluated using angiography of flat-mounted retinal samples after inoculation with fluorescein dextran. Retinal samples from both groups were also evaluated through histological examinations, and the expression of angiogenic factors was examined using the reverse-transcription-polymerase chain reaction. The capillary retinal vessels in both WT and IL-18 KO mice had reached the peripheral retina by postnatal day (P) 7. However, IL-18 KO mice showed angiectasis and vascular leakage at P7, especially in the mid-peripheral retina. These symptoms were not observed in WT mice at any stage. Histopathological analysis confirmed abnormal vascular formation in IL-18 KO mice at P14. Interestingly, these abnormalities regressed over time and had disappeared by P84. Several angiogenesis-associated factors, including vascular endothelial growth factor (VEGF), basic fibroblast-growth factor (bFGF), platelet-derived growth factor (PDGF) and pigment epithelium-derived factor (PEDF), were overexpressed in the retinas of IL-18 KO mice compared with those of WT mice at P14. Interferon-γ was detected only in WT mouse retinas at P14. These results provide new evidence for the role of IL-18 in retinal vascular development.

Increased vitreous concentrations of MCP-1 and IL-6 after vitrectomy in patients with proliferative diabetic retinopathy: possible association with postoperative macular oedema

Purpose To determine whether vitreal concentrations of MCP-1, IL-6 and IL-8 are altered after vitrectomy in patients with proliferative diabetic retinopathy (PDR) and to investigate whether the altered levels of these cytokines are associated with postoperative macular oedema. Methods Vitreous samples were collected from 36 eyes of 33 patients with PDR before pars plana vitrectomy without intraocular lens (IOL) implantation, and also from the same 36 eyes during IOL implantation surgery approximately 7 months after the initial vitrectomy. Levels of MCP-1, IL-6, IL-8 and vascular endothelial growth factor were measured by flow cytometry using cytometric bead array (CBA) technology. Results The mean vitreous levels of MCP-1, IL-6 and IL-8 in the samples collected before vitrectomy were significantly higher in patients with PDR than in control patients (p<0.0001). The levels of MCP-1 and IL-6 in the samples collected at the time of IOL implantation were significantly higher than those collected before vitrectomy (p<0.05). In contrast, the level of IL-8 was significantly lower after vitrectomy (p<0.05). The levels of IL-6 and IL-8, but not MCP-1, in the vitreous from eyes with PDR were inversely correlated with the interval between the initial vitrectomy and the time of implantation surgery. Among the vitrectomised patients, the mean vitreous level of MCP-1 in eyes with diabetic macular oedema (DME) was significantly higher than in those without DME (p=0.028). Conclusions The elevated levels of MCP-1 and IL-6 may indicate prolonged inflammation even after successful vitrectomy, which can cause postoperative DME.

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.

Inhibition of choroidal fibrovascular membrane formation by new class of RNA interference therapeutic agent targeting periostin

Age-related macular degeneration (AMD) is a vision-threatening disease characterized by choroidal fibrovascular membrane (FVM) formation, choroidal neovascularization (CNV) and choroidal fibrosis. No safe and effective therapeutic method has been developed for the choroidal fibrosis, although anti-vascular endothelial growth factor therapy can partially shrink the CNV. We recently reported that periostin (POSTN), which is produced by retinal pigment epithelial cells, has an important role in the formation of preretinal FVMs, but its role in choroidal FVMs has not been determined. In this study, we used Postn knockout mice to investigate the role played by POSTN in choroidal FVM formation. In addition, we used a new class of RNA interference (RNAi) agent (NK0144) that targets POSTN and determined its effect on choroidal FVM development. Genetic ablation of Postn had an inhibitory effect not only on CNV formation but also on choroidal fibrosis in a mouse CNV model. NK0144 also had a greater inhibitory effect on both the CNV and choroidal fibrosis than control RNAi with no apparent adverse effects. These findings suggest a causal relationship between POSTN and choroidal FVM formation, and also a potential therapeutic role of intravitreal NK0144 for AMD.