Toshihiro Kono

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

Long-term Follow-up of Visual Acuity in Eyes With Stage 5 Retinopathy of Prematurity After Closed Vitrectomy

PURPOSE This study was done to determine the efficacy of closed vitrectomy on long-term visual acuity of stage 5 retinopathy of prematurity. METHODS We studied the visual acuity of eyes that had undergone retinal reattachment by closed vitrectomy for stage 5 retinopathy of prematurity and were followed up for more than three years. RESULTS Forty-nine eyes of 31 patients among a total of 71 eyes of 48 consecutive patients were operated on and followed up for more than three years. Retinal reattachment occurred in 29 (59%) of the 49 eyes. Six patients (eight eyes) did not respond to visual acuity testing because of mental retardation or cerebral palsy. Measured visual acuity in the remaining 21 eyes varied from no light perception in one eye, light perception in four eyes, Recognizable hand movement in three eyes, 20/2,000 to 20/200 in seven eyes, 20/200 to 20/25 in five eyes, and 20/25 in one eye. No correlation was seen between visual outcome and factors such as gestational age, birth weight, and configuration of retinal detachment. Markedly better visual acuity was obtained in eyes that underwent initial surgery within four months of birth. CONCLUSIONS Useful vision can be obtained after reattachment of the retina by closed vitrectomy on some patients who have stage 5 retinopathy of prematurity. Better visual acuity may be obtained by earlier surgery.

Surgical Results and Visual Outcomes of Vitreous Surgery for Advanced Stages of Retinopathy of Prematurity

PURPOSE We evaluated the effectiveness of vitreous surgery for advanced stages of retinopathy of prematurity (ROP) with traction retinal detachment. METHODS Vitreous surgery was performed in 34 children (51 eyes) with total traction retinal detachment associated with stage 5 ROP between January 1989 and December 1991 at the Fukuoka University Hospital. The first phase of the procedure was composed of pars plicata lensectomy, followed by vitrectomy, membrane delamination, and hyaluronic acid injection. For unsuccessful cases, the second phase of the procedure, comprising scleral encircling, vitrectomy, membrane delamination, retinotomy fluid-air exchange, and SF6 or temporary silicone injection with endophotocoagulation, was performed. In cases with surgical success, visual acuity was measured using the Landolt ring test. RESULTS Of the 33 eyes with unsuccessful phase 1 surgery, 15 underwent the second phase procedure. Retinal reattachment was achieved in 7 of these 15 eyes (46.7%). Our composite rate of successful retinal reattachment for advanced stages of ROP was 47% (24 of 51 eyes). In 15 of the 24 eyes with surgical success, visual acuity was more than 20/600 in 3 eyes and no light perception in 1 eye. CONCLUSION In vitreous surgery for advanced stages of ROP, retinal breaks and vitreous hemorrhage are important factors leading to surgical failure. In this series, we could achieve visual acuity of more than light perception for several cases.

TEM7 (PLXDC1) in neovascular endothelial cells of fibrovascular membranes from patients with proliferative diabetic retinopathy.

PURPOSE Proliferative diabetic retinopathy (PDR) results from the formation of fibrovascular membranes (FVMs) in the posterior fundus that can lead to a severe decrease of vision. Tumor endothelial marker 7 (TEM7) is a protein that is highly expressed in the endothelial cells of tumors, but whether it plays a role in FVMs is unknown. The purpose of this study was to determine whether TEM7 is associated with the formation of FVMs. METHODS FVMs were obtained during vitrectomy from patients with PDR. RT-PCR was performed to determine the level of expression of the mRNA of TEM7. The splice variants of TEM7 were identified by direct sequencing. Immunohistochemical analyses and in situ hybridization was performed to determine the sites of TEM7 in the FVMs. RESULTS The level of the mRNA of TEM7 was high in 10 of 10 FVMs but was barely detectable in the five idiopathic epiretinal membranes. Direct sequencing of subcloned TEM7 PCR products revealed several splice variants (intracellular, secreted, and membrane-bound forms of TEM7) in the FVMs. Immunohistochemical analysis showed a colocalization of TEM7 and CD34, an endothelial cell marker, in most of the neovascular endothelial cells in the FVMs. Immunoelectron microscopy revealed that membrane-bound TEM7 was expressed on the luminal surfaces of the vascular endothelial cells of FVMs. CONCLUSIONS This study indicates that TEM7 may play a significant role in the proliferation and maintenance of neovascular endothelial cells in the FVMs. If correct, TEM7 may be a molecular target for new diagnostic and therapeutic strategies for PDR.

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.

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.

Gene expression profile of fibrovascular membranes from patients with proliferative diabetic retinopathy

Background/aims The purpose of this study was to generate a profile of genes expressed in preretinal fibrovascular membranes (FVMs) from patients with proliferative diabetic retinopathy. Methods A PCR-amplified complementary DNA (cDNA) library was constructed using the RNAs isolated from FVMs obtained during vitrectomy. The sequence from the 5′ end was obtained for randomly selected clones and used to generate expressed sequence tags (ESTs). Functional annotation was retrieved from Ensemble database and analysed by FatiGO. The web-based VisANT software was used to identify the molecular networks within the FVMs. Results A total of 2816 ESTs were assembled in 625 non-redundant clusters. Among these, 515 matched the human cDNA database. The 515 clusters were subdivided by functional subsets of genes related to ribosomal activity, oxidative phosphorylation, focal adhesion, cell adhesion and other functions. Querying against the VisANT database yielded 3175 possible physical relationships to other genes/proteins, which included an additional 2480 genes that were not detected in the FVM library. Conclusions The cDNA library constructed from human FVMs will be a valuable source of information. It should facilitate a wide range of studies that can establish the molecular mechanisms underlying the development of FVMs.