Takahito Nakama

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

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.

M2 Macrophages Enhance Pathological Neovascularization in the Mouse Model of Oxygen-Induced Retinopathy

PURPOSE To investigate the roles played by M2 macrophages in a mouse model of oxygen-induced retinopathy (OIR). METHODS Oxygen-induced retinopathy was induced in C57BL/6J mice by exposing postnatal day seven (P7) pups to 75% oxygen and then returning them to room air at P12. Real-time RT-PCR and immunofluorescence staining were used to assess the levels and distributions of different macrophage markers. Bone marrow-derived M1 and M2 macrophages and mannosylated clodronate liposomes (MCLs) were injected into the vitreous on P12 to examine the effects at P17. M2 macrophages were cocultured with human retinal endothelial cells (HRECs) to examine their effects on proliferation and tube formation. RESULTS The results showed that the M2 macrophages, rather than M1 phenotype, were highly expressed in OIR mice. The number of M2 macrophages had increased significantly at P17, and the increase was closely associated with the presence of neovascular tufts in the OIR retinas. Selective depletion of M2 macrophages suppressed the pathological neovascularization and promoted physiological revascularization. In contrast, intravitreal injection of bone marrow-derived M2 macrophages or the culture supernatants promoted pathological neovascularization and inhibited physiological revascularization. In an in vitro coculture system, M2-polarized macrophages significantly promoted proliferation and tube formation of HRECs. CONCLUSIONS These results indicated that M2 macrophages, rather than M1, play an important role in promoting retinal pathological neovascularization probably by producing secreted factors. Thus, targeting M2 macrophages could be a potential therapeutic option for inhibiting retinal pathological neovascularization.

Comparison of Gene Expression Profile of Epiretinal Membranes Obtained from Eyes with Proliferative Vitreoretinopathy to That of Secondary Epiretinal Membranes

Background Proliferative vitreoretinopathy (PVR) is a destructive complication of retinal detachment and vitreoretinal surgery which can lead to severe vision reduction by tractional retinal detachments. The purpose of this study was to determine the gene expression profile of epiretinal membranes (ERMs) associated with a PVR (PVR-ERM) and to compare it to the expression profile of less-aggressive secondary ERMs. Methodology/Principal Findings A PCR-amplified complementary DNA (cDNA) library was constructed using the RNAs isolated from ERMs obtained during vitrectomy. The sequence from the 5′ end was obtained for randomly selected clones and used to generate expressed sequence tags (ESTs). We obtained 1116 nonredundant clusters representing individual genes expressed in PVR-ERMs, and 799 clusters representing the genes expressed in secondary ERMs. The transcriptome of the PVR-ERMs was subdivided by functional subsets of genes related to metabolism, cell adhesion, cytoskeleton, signaling, and other functions, by FatiGo analysis. The genes highly expressed in PVR-ERMs were compared to those expressed in the secondary ERMs, and these were subdivided by cell adhesion, proliferation, and other functions. Querying 10 cell adhesion-related genes against the STRING database yielded 70 possible physical relationships to other genes/proteins, which included an additional 60 genes that were not detected in the PVR-ERM library. Of these, soluble CD44 and soluble vascular cellular adhesion molecule-1 were significantly increased in the vitreous of patients with PVR. Conclusions/Significance Our results support an earlier hypothesis that a PVR-ERM, even from genomic points of view, is an aberrant form of wound healing response. Genes preferentially expressed in PVR-ERMs may play an important role in the progression of PVR and could be served as therapeutic targets.

Microarray analysis of gene expression in fibrovascular membranes excised from patients with proliferative diabetic retinopathy

PURPOSE We determined the profile of genes expressed in fibrovascular membranes (FVMs). METHODS Six FVMs were surgically removed from patients with proliferative diabetic retinopathy (PDR) during pars plana vitrectomy with membrane peeling. The FVMs were classified into three active FVMs or three inactive FVMs according to the presence or absence of neovascularization (NV) in the membranes. Total RNA was isolated from the six FVMs and also from three normal human retinas. The DNA microarray analysis was performed to compare the genes expressed in the FVMs to those in normal human retinas, and also between active and inactive FVMs. Ingenuity pathway analysis (IPA) was used to determine the key biological networks related to the genes that were significantly altered. Quantitative RT-PCR and immunohistochemistry were performed to validate the microarray analyses. RESULTS There were 87 genes expressed at significantly higher levels in FVMs than in normal human retinas. Functional classification of these genes showed that the most clustered genes were those related to extracellular matrix formation. The top biological network generated by the IPA was cellular assembly and organization involving nodes of genes related to extracellular matrix formation. These networks included the collagen family and matricellular proteins, THBS2, POSTN, and TNC. There were 91 genes significantly upregulated in active FVMs, and the most clustered functional category was angiogenesis. In contrast, 89 genes were significantly upregulated in inactive FVMs, and the most clustered functional category was metabolism. The IPA revealed that the top biological network related to the genes that were significantly altered in this comparison was cell-to-cell signaling, and interactions involving the PDGF and TGFβ families. The results of quantitative RT-PCR analyses and immunohistochemistry for several selected molecules were in good agreement with the microarray data. CONCLUSIONS Our data indicate that extracellular matrix-related molecules such as POSTN, TNC, TGFβ, and angiogenic factors have important roles in promoting the development of FVMs associated with PDR.

Increased expression of M-CSF and IL-13 in vitreous of patients with proliferative diabetic retinopathy: Implications for M2 macrophage-involving fibrovascular membrane formation

Purpose We recently demonstrated that M2 macrophages were involved in the development of fibrovascular membranes (FVM) associated with proliferative diabetic retinopathy (PDR) possibly through the induction of periostin. The purpose of this study was to determine whether macrophage colony-stimulating factor (M-CSF) and interleukin (IL)-13, inducers of the M2 polarisation of macrophages from monocytes, are elevated in the vitreous of patients with PDR, and whether M2-polarised macrophages induce periostin production. Methods We measured the levels of M-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, IL-13, soluble (s)CD163, periostin and vascular endothelial growth factor by sandwich ELISA in vitreous samples collected from 61 eyes of 47 patients with PDR, and 39 eyes of 36 patients with non-diabetic ocular diseases (control group). Human monocytes were polarised in vitro with GM-CSF, interferon-γ, and lipopolysaccharide for M1 macrophages, and M-CSF, IL-4, and IL-13 for M2 macrophages. Quantitative real-time PCR was used to determine the mRNA level of periostin. Results The concentrations of M-CSF and IL-13 in the vitreous were significantly higher in patients with PDR than in non-diabetic controls (p<0.0001). There was a strong positive correlation between the vitreous concentrations of M-CSF and sCD163 and periostin. The mean vitreous level of IL-13 was significantly higher in eyes with FVMs than in those without FVMs (epicentre only). In vitro studies showed that M2-polarlised macrophages significantly increased the expression of the mRNA of periostin. Conclusions These findings indicate that the M2 polarisation of macrophages is induced by M-CSF and IL-13 in diabetic retinas. The presence of M-CSF and IL-13 would then promote FVM formation by periostin production.

Overexpression of CD163 in vitreous and fibrovascular membranes of patients with proliferative diabetic retinopathy: possible involvement of periostin

Aim To determine whether CD163, a specific marker for M2 macrophages, is involved in the formation of preretinal fibrovascular membranes (FVMs) present in eyes with proliferative diabetic retinopathy (PDR). Methods We measured the levels of soluble (s)CD163, periostin and vascular endothelial growth factor by sandwich ELISA in vitreous samples from 74 eyes of 62 patients with PDR, 20 eyes of 18 patients with proliferative vitreoretinopathy, and 56 eyes of 54 patients with non-diabetic ocular diseases (control group). Immunohistochemical analyses were performed to determine the expressions of CD68, CD163 and periostin in the surgically resected FVMs and idiopathic epiretinal membranes (ERMs). Results The concentrations of sCD163 and periostin in the vitreous were significantly higher in patients with PDR than in non-diabetic controls (p<0.0001). There was a strong correlation between the vitreous concentrations of sCD163 and periostin. The mean vitreous level of sCD163 was significantly higher in eyes with FVMs than in those without FVMs (epicentre only). The number and percentage of CD163+ macrophages were significantly higher in the FVMs than in the idiopathic ERMs. Immunohistochemical analysis showed co-localisation of CD163 and periostin in FVM cells. Conclusions These findings indicate that the overexpression of CD163 by macrophages may be involved in the development of FVMs partly through periostin production.

Wide-Field Laser Ophthalmoscopy for Mice: A Novel Evaluation System for Retinal/Choroidal Angiogenesis in Mice

PURPOSE The purpose of this study was to investigate the application of wide-field laser ophthalmoscopy (Optos) for the evaluation of established models of angiogenesis and the healthy retina in mice. METHODS To investigate whether angiogenesis and leakage in the retina and choroid can be evaluated with Optos, we used two models of angiogenesis: oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV). Fundus imaging and fluorescein angiography (FAG) were performed with the Optos system without a contact lens. Furthermore, to evaluate in vivo leukocyte infiltration in these models, we injected acridine orange (AO) and performed imaging using Optos. RESULTS In vivo fundus imaging with Optos did not require any additional optical device. Additionally, Optos enabled us to repeatedly obtain high-resolution color images and FAG images in the OIR model as well as in the CNV model in mice. Through a combination of Optos imaging and AO fluorography, the number and location of the infiltrating leukocytes could be identified in these models. CONCLUSIONS Optos is a wide-viewing imaging tool for the noninvasive in vivo evaluation of common angiogenesis models, oxygen-induced retinopathy and laser-induced choroidal neovascularization, as well as the healthy retina in mice.