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To describe the factors associated with epiretinal membrane (ERM) formation in eyes treated with pars plana vitrectomy (PPV) for rhegmatogenous retinal detachment (RRD).Nationwide, multicenter, clinical cohort study based on registry data.We reviewed 2239 cases treated with PPV for RRD repair registered in the Japan-Retinal Detachment Registry between February 2016 and March 2017. Associations of 13 baseline characteristics and 8 surgical procedures with ERM formation were evaluated using univariate analysis. We conducted a propensity score-matched analysis for the significantly associated clinical factor(s). The primary outcome measure was ERM formation after 6 months of vitrectomy.ERM had developed in 104 cases (4.6%) by 6 months. We found that drainage retinotomy was significantly associated with ERM after multiple testing correction (odds ratio [OR] 2.22 [95% confidence interval {CI} 1.50-3.31]; P.001). In the propensity score-matched analysis (n = 492 in each group), we confirmed a significant difference in the incidence of ERM after 6 months of vitrectomy (8.3% and 2.6% in cases with and without drainage retinotomy, respectively; OR 3.35 [95% CI 1.77-6.33]; P.001).Eyes treated with PPV combined with drainage retinotomy are more likely to develop ERM postoperatively.

Purpose To evaluate structural and angiographic neovascularization in patients with proliferative diabetic retinopathy (PDR) using volumetric three-dimensional optical coherence tomography angiography (OCTA). Methods This prospective, observational cross-sectional study included 29 eyes of 27 patients with PDR. The angiogenic structure, feeding vessel (epicenter), flow volume, and flow volume density (FVD) of the neovasculatures were evaluated using three-dimensional OCTA imaging. The flow area and the flow area density (FAD) were also measured using en face OCTA imaging. Results Sites of neovascularization were imaged successfully in 17 of the 29 eyes (58.6%). Three proposed types of neovascularization were identified on the basis of structural features seen on the three-dimensional OCTA images. Neovascularization of the adhesion type (9 of 17, 52.9%) adhered to the retinal vasculature. Those of the traction type (5 of 17, 29.4%) were partially separated from the retinal vascular plexus. Those of the mushroom type (3 of 17, 17.6%) were connected to the retinal vasculature via several epicenters. There was a significant difference between highly leaky (active) and faintly leaky (inactive) neovascularization for FVD, but not for flow area, flow volume, or FAD (P=0.01, 0.9, 0.6, and 0.1, respectively). Conclusions Volumetric three-dimensional OCTA revealed three types of neovascularization in PDR, and may be useful for assessing neovascular activity and planning vitrectomies.

Atsunobu Takeda,1,2 Eiichi Hasegawa,1 Shoji Notomi,1,2 Keijiro Ishikawa,1 Mitsuru Arima,1,2 Yusuke Murakami,1 Shintaro Nakao,1 Toshio Hisatomi,3 Koh-Hei Sonoda1,4 1Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 2Department of Ophthalmology, Clinical Research Institute, National Hospital Organization, Kyushu Medical Center, Fukuoka, Japan; 3Department of Ophthalmology, Chikushi Hospital, Fukuoka University, Fukuoka, Japan; 4Department of Ocular Pathology and Imaging Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, JapanCorrespondence: Atsunobu TakedaDepartment of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka, 812-8582, JapanTel +81-92-642-5648Fax +81-92-642-5663Email takeda.atsunobu.248@m.kyushu-u.ac.jpPurpose: To evaluate the pre- and post-operative outcomes of phacoemulsification in patients with uveitis-associated cataract in remission, such as conventional visual acuity (VA), photopic and mesopic contrast visual acuity (CVA), and flares in the anterior chamber objectively assessed as intraocular inflammation.Patients and Methods: This prospective study included 26 eyes of 19 patients with uveitis and 45 eyes of 26 controls who underwent cataract surgery at the Kyushu University Hospital and Kyushu Medical Center in Fukuoka, Japan, from October 2016 to December 2018. Conventional VA and flare values in the anterior chamber were evaluated preoperatively and 1 and 3 months postoperatively. Photopic and mesopic CVAs were assessed preoperatively and 3 months postoperatively.Results: The best-corrected VA (BCVA) was improved significantly from baseline to 1 and 3 months postoperatively in both groups (P < 0.01 in both groups). The mean preoperative 100% and 10% CVAs under the photopic condition were significantly lower in the uveitis group than in the control group (P < 0.05 for both CVA), whereas the mean preoperative 100% CVA under the mesopic condition was comparable between the two groups. Although the mean preoperative 100% and 10% CVAs improved significantly from baseline under both photopic and mesopic conditions in both groups (P < 0.01 in both groups), the postoperative contrast sensitivities under both photopic and mesopic conditions remained lower in the uveitis group than in the control group (P < 0.01 for both conditions). The postoperative complications included recurrence of active inflammation in five eyes and cystoid macular edema in one eye and were managed by topical steroid therapy alone.Conclusion: Cataract surgery for uveitis-associated cataracts during remission is well tolerated. However, the present results suggest that amelioration of hemeralopia and/or nyctalopia is not as good as expected after cataract surgery in patients with uveitis.Keywords: cataract, phacoemulsification, uveitis, contrast sensitivity, photopic and mesopic conditions

Drusen are known to be the important hallmark to predict the development of age-related macular degeneration (AMD). The prevalence of drusen is lower in Asians compared with Caucasians so that the role of signs constituting early AMD is not well established in Asian populations as in Western countries. In this study, we retrospectively investigated clinical characteristics and 5-year incidence of neovascular AMD (nAMD) in the fellow eye of unilateral nAMD patients. Of 296 consecutive unilateral nAMD patients who had been followed up more than 5 years, 170 typical AMD, 119 polypoidal choroidal vasculopathy, and 7 retinal angiomatous proliferation were included. To examine factors associated with nAMD occurrence in the fellow eye, drusen and pigmentary abnormality in the fellow eye were classified into 4 categories; Category 1: no or small drusen < 63 μm (37.2%), Category 2: 63–125 μm medium drusen or pigmentary abnormality (22.2%), Category 3: large drusen > 125 μm (25.0%), Category P: pachydrusen (15.5%). The mean sub-foveal choroidal thickness (SFCT) was Category 1: 276 μm, Category 2: 308 μm, Category 3: 246 μm, and Category P: 302 μm, respectively. Of note, SFCT in Category 2 and Category P was significantly larger than those of Category 3. Finally, the 5-year incidence of nAMD in the fellow eye was 32/296 (10.8%); Category 1: 0/110 (0%), Category 2: 12/66 (18.2%), Category 3: 20/74 (27.0%), and Category P: 0/46 (0%). Thus, signs of intermediate AMD (large drusen) as well as those of early AMD, especially the pigmentary abnormality, may contribute to development of bilateral nAMD in Japanese patients.

This study aims to investigate the changes in metamorphopsia after administering the treat-and-extend regimen of anti-vascular endothelial growth factor therapy for branch retinal vein occlusion-associated macular edema. We retrospectively examined 27 patients (27 eyes) with macula edema due to branch retinal vein occlusion who received intravitreal injections of anti-vascular endothelial growth factor agents using the treat-and-extend regimen for ≥18 months. We evaluated best-corrected visual acuity, central macular thickness, macular edema recurrence, and amount of metamorphopsia quantified by M-CHARTS. The best-corrected visual acuity (logarithm of minimum angle of resolution) and central macular thickness significantly improved at 18 months compared to baseline, the median value (interquartile range [IQR]), 0.30 (0.15-0.52) and 459 (373-542) μm at baseline, and 0 (-0.08-0.16) and 267 (232-306) μm at 18 months. The M-CHARTS score (the mean of vertical and horizontal scores) significantly decreased at 1, 6, and 12 months compared to baseline, but worsened at 18 month, the median value (IQR), 0.45 (0.250-0.925), 0.4 (0.15-0.70), 0.4 (0.150-0.625), 0.4 (0.225-0.550) and 0.45 (0.225-0.750) at baseline, 1 month, 6 months, 12 months and 18 months, respectively. The median cumulative number of macular edema recurrences was 2 (IQR, 0.5-3.0) at 18 months. Simple linear regression and multivariate analyses revealed that the change in the mean M-CHARTS score at 18 months was significantly correlated with the baseline score and the cumulative number of macular edema recurrences. Anti-vascular endothelial growth factor therapy using the treat-and-extend regimen improved metamorphopsia in branch retinal vein occlusion-related macular edema in the short to mid-term follow-up period, but not in the long term. Macular edema recurrence may be associated with persistent metamorphopsia.

Proliferative vitreoretinal diseases such as diabetic retinopathy, proliferative vitreoretinopathy (PVR), and age-related macular degeneration are a leading cause of decreased vision and blindness in developed countries. In these diseases, retinal fibro(vascular) membrane (FVM) formation above and beneath the retina plays an important role. Gene expression profiling of human FVMs revealed significant upregulation of periostin. Subsequent analyses demonstrated increased periostin expression in the vitreous of patients with both proliferative diabetic retinopathy and PVR. Immunohistochemical analysis showed co-localization of periostin with α-SMA and M2 macrophage markers in FVMs. In vitro, periostin blockade inhibited migration and adhesion induced by PVR vitreous and transforming growth factor-β2 (TGF-β2). In vivo, a novel single-stranded RNAi agent targeting periostin showed the inhibitory effect on experimental retinal and choroidal FVM formation without affecting the viability of retinal cells. These results indicated that periostin is a pivotal molecule for FVM formation and a promising therapeutic target for these proliferative vitreoretinal diseases.

Iori Wada, Shigeo Yoshida, Yoshiyuki Kobayashi, Yedi Zhou, Keijiro Ishikawa, Shintaro Nakao, Toshio Hisatomi, Yasuhiro Ikeda, Tatsuro Ishibashi, Koh-hei Sonoda Department of Ophthalmology, Kyushu University Graduate School ofMedical Sciences, Fukuoka, Japan Purpose: The purpose of this study was to determine the degree of metamorphopsia using M-CHARTS™ in patients with idiopathic macular hole before and after pars plana vitrectomy and internal limiting membrane (ILM) peeling. Patients and methods: The records of 22 eyes of 22 patients with a full-thickness macular hole who underwent pars plana vitrectomy and ILM peeling were reviewed. All patients underwent a complete ophthalmic examination including spectral-domain optical coherence tomography (OCT). Horizontal metamorphopsia (MH) and vertical metamorphopsia (MV) scores were determined using M-CHARTS at the same time. The time course of changes in metamorphopsia and the relationship between best-corrected visual acuity (BCVA) and OCT parameters were assessed. Results: Sealing of the macular hole was noted in all eyes after surgery. BCVA improved significantly from 1month after surgery (P

Retinal neovascularization (NV) due to retinal ischemia remains one of the principal causes of vision impairment in patients with ischemic retinal diseases. We recently reported that periostin (POSTN) may play a role in the development of preretinal fibrovascular membranes, but its role in retinal NV has not been determined. The purpose of this study was to examine the expression of POSTN in the ischemic retinas of a mouse model of oxygen-induced retinal NV. We also studied the function of POSTN on retinal NV using Postn KO mice and human retinal endothelial cells (HRECs) in culture. In addition, we used a novel RNAi agent, NK0144, which targets POSTN to determine its effect on the development of retinal NV. Our results showed that the expression of POSTN was increased in the vascular endothelial cells, pericytes, and M2 macrophages in ischemic retinas. POSTN promoted the ischemia-induced retinal NV by Akt phosphorylation through integrin αvβ3. NK0144 had a greater inhibitory effect than canonical double-stranded siRNA on preretinal pathological NV in vivo and in vitro. These findings suggest a causal relationship between POSTN and retinal NV, and indicate a potential therapeutic role of intravitreal injection of NK0144 for retinal neovascular diseases. Keywords: periostin, RNAi, neovascularization, diabetic retinopathy

Necrotizing retinopathies, including acute retinal necrosis (ARN) and progressive outer retinal necrosis (PORN), are a group of uncommon posterior segment complications associated with infections caused by the herpes virus. ARN was first described in 1971 by Urayama et al., Japanese ophthalmologists who termed the condition as Kirisawa uveitis and described it as acute unilateral panuveitis associated with optic neuritis and retinal panarteritis in healthy adults, with subsequent procession to diffuse necrotizing retinitis and/or subsequent rhegmatogenous retinal detachment (RRD) (Urayama et al. 1971). Standard diagnostic criteria for ARN were published by the American Uveitis Committee (Table 27.1) (Holland 1994).

We evaluated changes in the numbers of microaneurysms (MAs) on fluorescein angiography (FA) and indocyanine green angiography (IA) in eyes with diabetic macular edema (DME) following intravitreal injection of anti-vascular endothelial growth factor (VEGF) agents. Twenty-one eyes of 16 patients with DME were included in this retrospective study. All patients received an initial loading dose of three monthly injections of anti-VEGF agents; thereafter, they received a pro re nata regimen for at least 12 months of follow-up. FA and IA images were obtained before and at 6 months after the initial injection. The median numbers of MAs significantly decreased from six (interquartile range [IQR] 3–7) MAs in early-phase FA, three (IQR 3–5) leaky MAs in late-phase FA, and two (IQR 1–4) MAs in late-phase IA at baseline to two (IQR 1–3) MAs in early-phase FA, one (IQR 0–2) leaky MA in late-phase FA, and one (IQR 0–2) MA in late-phase IA at 6 months (P

Ischemic proliferative retinopathy (IPR), such as proliferative diabetic retinopathy (PDR), retinal vein occlusion and retinopathy of prematurity is a major cause of vision loss. Our previous studies demonstrated that periostin (PN) and tenascin-C (TNC) are involved in the pathogenesis of IPR. However, the interactive role of PN and TNC in angiogenesis associated with IPR remain unknown. We found significant correlation between concentrations of PN and TNC in PDR vitreous humor. mRNA and protein expression of PN and TNC were found in pre-retinal fibrovascular membranes excised from PDR patients. Interleukin-13 (IL-13) promoted mRNA and protein expression of PN and TNC, and co-immunoprecipitation assay revealed binding between PN and TNC in human microvascular endothelial cells (HRECs). IL-13 promoted angiogenic functions of HRECs. Single inhibition of PN or TNC and their dual inhibition by siRNA suppressed the up-regulated angiogenic functions. Pathological pre-retinal neovessels of oxygen-induced retinopathy (OIR) mice were attenuated in PN knock-out, TNC knock-out and dual knock-out mice compared to wild-type mice. Both in vitro and in vivo, PN inhibition had a stronger inhibitory effect on angiogenesis compared to TNC inhibition, and had a similar effect to dual inhibition of PN and TNC. Furthermore, PN knock-out mice showed scant TNC expression in pre-retinal neovessels of OIR retinas. Our findings suggest that interaction of PN and TNC facilitates pre-retinal angiogenesis, and PN is an effective therapeutic target for IPR such as PDR.

To investigate periostin (PN) and tenascin-C (TNC) expression in the aqueous humor and trabeculectomy specimens of patients with neovascular glaucoma (NVG) secondary to proliferative diabetic retinopathy (PDR). This study enrolled 37 eyes of 37 patients who were grouped into (1) NVG secondary to PDR (NVG; n = 8); (2) PDR without NVG (PDR; n = 9); (3) primary open-angle glaucoma (POAG; n = 11); and (4) cataract surgery patients as a control group (CG; n = 9). Aqueous humor samples were collected from the anterior chamber at the start of surgery or intravitreal injection of anti-VEGF drug. The concentrations of PN, TNC, VEGF, and TGF-β2 (transforming growth factor-beta 2) were measured by ELISA. Sclerostomy tissues containing trabecular meshwork were obtained from two NVG patients and a POAG patient who underwent trabeculectomy surgery. Immunohistochemical analyses were performed to determine the localization of PN and TNC expression in the sclerostomy tissues. PN and TNC-C levels were below detection threshold in the POAG and CG groups. The NVG group had significantly higher levels of PN and TNC compared with the PDR group (84.7 ng/ml vs 2.2 ng/ml and 18.5 ng/ml vs 4.6 ng/ml, respectively; p

Innate immune cells such as neutrophils, monocyte-macrophages and microglial cells are pivotal for the health and disease of the retina. For the maintenance of retinal homeostasis, these cells and immunosuppressive molecules in the eye actively regulate the induction and the expression of inflammation in order to prevent excessive activation and subsequent tissue damage. In the disease context, these regulatory mechanisms are modulated genetically and/or by environmental stimuli such as damage-associated molecular patterns (DAMPs), and a chronic innate immune response regulates or contributes to the formation of diverse retinal disorders such as uveitis, retinitis pigmentosa, retinal vascular diseases and retinal fibrosis. Here we summarize the recent knowledge regarding the innate immune response in both ocular immune regulation and inflammatory retinal diseases, and we describe the potential of the innate immune response as a biomarker and therapeutic target.

ObjectiveAccurate scleral marking of retinal breaks is essential for successful scleral buckling. This study aimed to investigate the use of wide-field fundus images obtained with an Optos for preoperative estimation of the distance from the limbus to the retinal breaks.Methods and analysisThis is a retrospective review of 29 eyes from 26 patients with rhegmatogenous retinal detachment who received scleral buckling with anatomically successful repair. They underwent wide-field fundus photography with Optos California. In the pre- and postoperative fundus images, we measured distances from the macula to the retinal tears (TM), to the center of the vortex veins (VM), to the optic disc (DM), and to the posterior edge of the scleral buckle (BM).Results(BM-VM) / DM was significantly correlated with the distance from the limbus to the posterior edge of the scleral buckle that had been determined intraoperatively. (r = 0.705; pConclusionPreoperative analysis of Optos images may be useful for estimating the distance from the limbus to retinal breaks, which might aid scleral marking during scleral buckling surgery.

PurposeTo determine whether the concentrations of vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein (MCP)-1 in the vitreous changed after vitrectomy in patients with proliferative diabetic retinopathy (PDR).ParticipantsTwenty-one eyes of 21 patients who needed a second surgery for PDR were included. The reasons for the second surgery were tractional retinal detachment (TRD), neovascular glaucoma, persistent vitreous hemorrhage, macular pucker, and secondary intraocular lens (IOL) implant.MethodsWe measured the VEGF and MCP-1 levels using sandwich enzyme-linked immunosorbent assays in vitreous samples collected from patients with PDR before pars plana vitrectomy (without IOL implantation), and from the same patients during the second surgery.ResultsThere was not significant change in mean VEGF concentrations when comparing first (0.81±0.88 ng/ml) and second surgeries (1.09±1.51 ng/ml). The MCP-1 level was significantly elevated at the time of second surgery (2.20±2.21 ng/ml) compared with the first vitrectomy (0.72±0.57 ng/ml). The MCP-1 levels of the second surgery cases with TRD (3.18±2.27 ng/ml) increased significantly compared with those with other complications (1.72±2.10 ng/ml).ConclusionsAt the second vitrectomy, VEGF did not change significantly in the vitreous of the patients examined. The MCP-1 concentration was markedly elevated at the second vitrectomy, implying an association between the prolonged inflammation after vitrectomy and complications, especially TRD.

Purpose Although vitrectomy has been reported to be effective for the treatment of macular retinoschisis associated with glaucoma in a few case series, the surgical techniques have yet to be established. This article aimed to describe the cases of two patients with macular retinoschisis who underwent vitrectomy with peripapillary internal limiting membrane peeling around the defective area of the retinal nerve fiber layer. Observations Both patients had been diagnosed with normal tension glaucoma and treated with eye drops to stabilize intraocular pressure. Progression of macular retinoschisis and accompanied vision loss were observed in both cases. Twelve months after the surgery, both patients had resolution of the retinoschisis and improvement in best corrected visual acuity. Conclusions and importance Our surgical technique may be effective for the resolution of macular retinoschisis in eyes with normal tension glaucoma.

SummaryAge is a major risk factor in age-related macular degeneration (AMD), but the underlying cause is unknown. We find increased Rho-associated kinase (ROCK) signaling and M2 characteristics in eyes of aged mice, revealing immune changes in aging. ROCK isoforms determine macrophage polarization into M1 and M2 subtypes. M2-like macrophages accumulated in AMD, but not in normal eyes, suggesting that these macrophages may be linked to macular degeneration. M2 macrophages injected into the mouse eye exacerbated choroidal neovascular lesions, while M1 macrophages ameliorated them, supporting a causal role for macrophage subtypes in AMD. Selective ROCK2 inhibition with a small molecule decreased M2-like macrophages and choroidal neovascularization. ROCK2 inhibition upregulated M1 markers without affecting macrophage recruitment, underlining the plasticity of these macrophages. These results reveal age-induced innate immune imbalance as underlying AMD pathogenesis. Targeting macrophage plasticity opens up new possibilities for more effective AMD treatment.

Age-related macular degeneration (AMD), a progressive chronic disease of the central retina, is associated with aging and is a leading cause of blindness worldwide. Here, we demonstrate that leukotriene B4 (LTB4) receptor 1 (BLT1) promotes laser-induced choroidal neovascularization (CNV) in a mouse model for wet-type AMD. CNV was significantly less in BLT1-deficient (BLT1-KO) mice compared with BLT1-WT controls. Expression of several proangiogenic and profibrotic factors was lower in BLT1-KO eyes than in BLT1-WT eyes. LTB4 production in the eyes was substantially increased in the early phase after laser injury. BLT1 was highly expressed in M2 macrophages in vitro and in vivo, and ocular BLT1+ M2 macrophages were increased in the aged eyes after laser injury. Furthermore, M2 macrophages were rapidly attracted by LTB4 and subsequently produced VEGF-A- through BLT1-mediated signaling. Consequently, intravitreal injection of M2 macrophages augmented CNV formation, which was attenuated by BLT1 deficiency. Thus, laser-induced injury to the retina triggered LTB4 production and attracted M2 macrophages via BLT1, leading to development of CNV. A selective BLT1 antagonist (CP105696) and 3 LTB4 inhibitors (zileuton, MK-886, and bestatin) reduced CNV in a dose-dependent manner. CP105696 also inhibited the accumulation of BLT1+ M2 macrophages in the laser-injured eyes of aged mice. Together, these results indicate that the LTB4-BLT1 axis is a potentially novel therapeutic target for CNV of wet-type AMD.

This work provides a comprehensive CpG methylation landscape of the different layers of the human eye that unveils the gene networks associated with their biological functions and how these are disrupted in common visual disorders. Herein, we firstly determined the role of CpG methylation in the regulation of ocular tissue-specification and described hypermethylation of retinal transcription factors (i.e., PAX6, RAX, SIX6) in a tissue-dependent manner. Second, we have characterized the DNA methylome of visual disorders linked to internal and external environmental factors. Main conclusions allow certifying that crucial pathways related to Wnt-MAPK signaling pathways or neuroinflammation are epigenetically controlled in the fibrotic disorders involved in retinal detachment, but results also reinforced the contribution of neurovascularization (ETS1, HES5, PRDM16) in diabetic retinopathy. Finally, we had studied the methylome in the most frequent intraocular tumors in adults and children (uveal melanoma and retinoblastoma, respectively). We observed that hypermethylation of tumor suppressor genes is a frequent event in ocular tumors, but also unmethylation is associated with tumorogenesis. Interestingly, unmethylation of the proto-oncogen RAB31 was a predictor of metastasis risk in uveal melanoma. Loss of methylation of the oncogenic mir-17-92 cluster was detected in primary tissues but also in blood from patients. The research leading to these results was supported by European Research Council Advanced Grant EPINORC, RecerCaixa Foundation, Federación Española de Enfermedades Raras (FEDER), Federación Española de Enfermedades Neuromusculares (ASEM), Fundación Isabel Gemio, COST CM1406, Instituto de Salud Carlos III (PI/00816) and Health and Sciences Departments of the Catalan Government (Generalitat de Catalunya). M.E. is an Institució Catalana de Recerca i Estudis Avançats (ICREA) Research Professor. We thank the staff of the Biobank Facility at the Bellvitge Biomedical Research Institute (IDIBELL), Spanish National Cancer Research Center (CNIO), Institute of Rare Diseases Research (BioNER-ISCIII), Vall d’Hebron Research Institute (VHIR) and Banc de Sang i Teixits (BST) of the Catalan Ministry of Health. We also thank Dr. Mercedes Hurtado (Department of Ophthalmology, University and Polytechnic Hospital La Fe) and Dr. Dolores Pinazo (Department of Ophthalmology, Dr. Peset University Hospital) for obtaining samples from glaucomatous patients. We thank the patients and their families. Sí

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).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).The concentrations of sCD163 and periostin in the vitreous were significantly higher in patients with PDR than in non-diabetic controls (p0.0001). 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.These findings indicate that the overexpression of CD163 by macrophages may be involved in the development of FVMs partly through periostin production.

Proliferative vitreoretinopathy (PVR) is a severe, vision-threatening disorder characterized by the fibrous membrane formation that leads to tractional 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 αV-mediated 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.

Purpose The purpose of this study was to evaluate the impact of intravitreal bevacizumab (IVB) on three cellular components (vascular endothelial cells, pericytes, and myofibroblasts) of the vascular microenvironment in fibrovascular membranes (FVMs) of patients with proliferative diabetic retinopathy. Methods Immunohistological studies with antibodies of CD34, αSMA, and transforming growth factor-β were performed on 20 surgical specimens obtained during a pars plana vitrectomy from 8 IVB-treated eyes, whereas 12 remained untreated. Four different indexes of vascular phenotype (vascular area, vascular major axis, CD34 endothelial area, and blood vessel density) and αSMA expression in vascular and stromal components were quantitatively analyzed. Results The intraluminal area of blood vessels, CD34 endothelial area, and the blood vessel density in IVB-treated FVMs were significantly less than in untreated FVMs. The number of CD34 blood vessels in IVB-treated FVMs was similar to that in untreated FVMs. Intravitreal bevacizumab could not affect vascular and stromal αSMA area significantly. However, the ratio of vascular αSMA area/CD34 area was significantly higher in IVB-treated FVMs than in untreated FVMs. Transforming growth factor-β expression could be observed in the IVB-treated FVM. Conclusion Intravitreal bevacizumab might primarily affect blood vessels, and the effects on pericytes and myofibroblasts might be secondary. Intravitreal bevacizumab treatment regulates vascular microenvironment by the contraction of blood vessels, the increasing pericyte ratio, and transforming growth factor-β expression in FVMs of patients with proliferative diabetic retinopathy.

Purpose To study the nature of retinal inflammatory response in rd10 mice, an animal model of retinitis pigmentosa (RP), and to investigate the effect of an antioxidant on retinal inflammation and photoreceptor apoptosis. Design Experimental study. Participants and Controls This study included 42 untreated rd10 mice, 30 N -acetylcysteine (NAC)–treated rd10 mice, and 20 C57BL/6 mice as controls. Methods Real-time polymerase chain reaction (PCR) was performed to evaluate the expression levels of inflammatory factors (proinflammatory cytokines and chemokines) in rd10 mouse retinas. Rd10 mice were treated with an antioxidant NAC, and its effect on retinal inflammation and photoreceptor apoptosis were examined by immunohistochemistry. Main Outcome Measures Real-time PCR and immunohistochemistry. Results We demonstrated sequential events involving increased expression of proinflammatory cytokines and chemokines, activation of microglia, and photoreceptor apoptosis during retinal degeneration of rd10 mice. Furthermore, antioxidant treatment with NAC prevented the photoreceptor cell death along with suppression of inflammatory factors and microglial activation. Conclusions Sustained chronic inflammatory reaction may contribute to the pathogenesis of retinal degeneration in rd10 mice, suggesting interventions for ocular inflammatory reaction using antioxidants as a potential treatment for patients with RP. Financial Disclosure(s) The authors have no proprietary or commercial interest in any of the materials discussed in this article.

To study the nature of inflammatory reaction in eyes of patients with retinitis pigmentosa (RP) and its possible role in the pathogenesis of RP.Retrospective, observational study.Three hundred seventy-one consecutive patients diagnosed with typical RP were included in this study. We included 165 patients without active inflammatory diseases, including 20 patients diagnosed with cataract, and 36 patients diagnosed with idiopathic epiretinal membrane as controls.Density of the inflammatory cells in the anterior vitreous cavity was measured and graded by slit-lamp biomicroscopy. A multiplex enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the concentration of cytokines and chemokines in aqueous humor and vitreous fluid of patients with RP and controls. In addition, we investigated the relationship between visual function and anterior vitreous cells in these patients.Slit-lamp biomicroscopic analysis, best-corrected visual acuity, visual field analysis, and multiplex ELISA.In 190 of 509 eyes with RP (37.3%), "1+" (5-9 cells per field) or more cells were observed in the anterior vitreous cavity. Strong inflammatory reaction with "2+" cells (10-30 cells per field) was associated with younger age. In the elderly patients with RP, significantly decreased visual function was seen in a group with "1+" or more cells (P0.05). Moreover, the levels of a variety of proinflammatory cytokines and chemokines, including monocyte chemotactic protein-1, were increased both in the aqueous humor and vitreous fluid of RP patients compared with the levels in control patients.Sustained chronic inflammatory reaction may underlie the pathogenesis of RP, suggesting interventions for ocular inflammatory reaction as a potential treatment for patients with RP.The authors have no proprietary or commercial interest in any of the materials discussed in this article.

Hypoxia-induced retinal neovascularization is a major pathological condition in many vision-threatening diseases. In the present study, we determined whether interleukin (IL)-12, a cytokine that regulates angiogenesis, plays a role in the neovascularization in a mouse model of oxygen-induced retinopathy (OIR). We found that the expressions of the mRNAs of both IL-12p35 and IL-12p40 were significantly reduced in the OIR retinas compared to that of the room air-raised control. The sizes of the avascular areas and neovascular tufts were larger in IL-12p40 knock-out (KO) mice than that in wild type (WT) mice. In addition, an intravitreal injection of recombinant IL-12 reduced both avascular areas and neovascular tufts. IL-12 injection enhanced the expressions of interferon-gamma (IFN-γ) and other downstream chemokines. In an in vitro system, IL-12 had no significant effect on tube formation of human retinal microvascular endothelial cells (HRECs). Moreover, a blockade of IFN-γ suppressed the inhibitory effect of IL-12 on pathological neovascularization. These results suggest that IL-12 plays important roles in inhibiting pathological retinal neovascularization.

Subretinal fibrosis is an end stage of neovascular age-related macular degeneration, characterized by fibrous membrane formation after choroidal neovascularization. An initial step of the pathogenesis is an epithelial-mesenchymal transition (EMT) of retinal pigment epithelium cells. αB-crystallin plays multiple roles in age-related macular degeneration, including cytoprotection and angiogenesis. However, the role of αB-crystallin in subretinal EMT and fibrosis is unknown. Herein, we showed attenuation of subretinal fibrosis after regression of laser-induced choroidal neovascularization and a decrease in mesenchymal retinal pigment epithelium cells in αB-crystallin knockout mice compared with wild-type mice. αB-crystallin was prominently expressed in subretinal fibrotic lesions in mice. In vitro , overexpression of αB-crystallin induced EMT, whereas suppression of αB-crystallin induced a mesenchymal-epithelial transition. Transforming growth factor-β2–induced EMT was further enhanced by overexpression of αB-crystallin but was inhibited by suppression of αB-crystallin. Silencing of αB-crystallin inhibited multiple fibrotic processes, including cell proliferation, migration, and fibronectin production. Bone morphogenetic protein 4 up-regulated αB-crystallin, and its EMT induction was inhibited by knockdown of αB-crystallin. Furthermore, inhibition of αB-crystallin enhanced monotetraubiquitination of SMAD4, which can impair its nuclear localization. Overexpression of αB-crystallin enhanced nuclear translocation and accumulation of SMAD4 and SMAD5. Thus, αB-crystallin is an important regulator of EMT, acting as a molecular chaperone for SMAD4 and as its potential therapeutic target for preventing subretinal fibrosis development in neovascular age-related macular degeneration.

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.

Proliferative vitreoretinopathy (PVR) is a serious complication of retinal detachment and ocular trauma and its recurrence may lead to irreversible vision loss. Epithelial to mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is a critical step in the pathogenesis of PVR, which is characterized by fibrotic membrane formation and traction retinal detachment. In this study, we investigated the potential impact of resveratrol (RESV) on EMT and the fibrotic process in cultured RPE cells and further examined the preventive effect of RESV on PVR development using a rabbit model of PVR. We found that RESV induces mesenchymal to epithelial transition (MET) and inhibits transforming growth factor-β2(TGF-β2)-induced EMT of RPE cells by deacetylating SMAD4. The effect of RESV on MET was dependent on sirtuin1 activation. RESV suppressed proliferation, migration and fibronectin synthesis induced by platelet-derived growth factor-BB or TGF-β2. In vivo, RESV inhibited the progression of experimental PVR in rabbit eyes. Histological findings showed that RESV reduced fibrotic membrane formation and decreased α-SMA expression in the epiretinal membranes. These results suggest the potential use of RESV as a therapeutic agent to prevent the development of PVR by targeting EMT of RPE.

Subretinal fibrosis is a result of a wound healing response that follows choroidal neovascularization in neovascular age-related macular degeneration (nAMD). Although anti-vascular endothelial growth factor therapy has become a standard treatment that improves visual acuity in many nAMD patients, unsuccessful treatment outcomes have often been attributed to the progression of subretinal fibrosis. In this review, we summarize the cellular and extracellular components of subretinal fibrous membranes and also discuss the possible molecular mechanisms including the functional involvement of growth factors and the inflammatory response in the process. Moreover, we present an murine animal model of subretinal fibrosis that might facilitate greater understanding of the pathophysiology and the development of novel therapeutic strategies for the inhibition of subretinal fibrosis in nAMD.

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.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.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 (p0.0001). 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 (p0.05). In contrast, the level of IL-8 was significantly lower after vitrectomy (p0.05). 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).The elevated levels of MCP-1 and IL-6 may indicate prolonged inflammation even after successful vitrectomy, which can cause postoperative DME.

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.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.The concentrations of M-CSF and IL-13 in the vitreous were significantly higher in patients with PDR than in non-diabetic controls (p0.0001). 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.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.

Case of novel PITX2 gene mutation associated with Peters' anomaly and persistent hyperplastic primary vitreous

Purpose Inflammation and immune cells regulate choroidal neovascularization (CNV) and could become therapeutic targets in age-related macular degeneration (AMD). Lymphangiogenesis is a key component of various inflammatory diseases. Whether lymphangiogenesis and lymph node-mediated immunity are involved in the pathogenesis of AMD is not understood. Methods To investigate lymphangiogenesis in CNV, we generated CNV in animals by laser and studied surgically removed CNV membranes from uveitis and AMD patients. Immunohistochemistry was performed with lymphatic vessel endothelial hyaluronate receptor 1 (LYVE-1) and podoplanin antibodies. VEGF-C and VEGFR-3 expressions were examined with immunohistochemistry and Western blotting. To examine the role of lymph node in CNV, we lasered lymphotoxin alpha-deficient mice (LTα-/-) and measured the CNV volume. Results Immunohistochemistry showed that LYVE-1(+) macrophages infiltrated in acutely induced CNV, although lymphatic tubes did not form. CNV membranes from patients did not show LYVE-1(+)podoplanin(+) vessels, suggesting the lack of lymphangiogenesis in AMD and uveitis. Western blots and immunostaining revealed VEGF-C and VEGFR-3 expression in CNV lesions, mainly in macrophages and angiogenic endothelial cells. Using fluorescent microsphere tracers, we show a path for cellular migration from the eye to the cervical lymph nodes (LNs) during CNV. However, CNV injury did not cause LN swelling. CNV volume did not differ between wild-type and LN-deficient mice, suggesting that LN is not a key component of early CNV formation. Conclusions Laser-induced CNV is not primarily dependent on acquired immunity, nor does the fundus injury affect peripheral LNs. Our results reveal a previously unknown cellular connection between the ocular fundus and the cervical LNs. This connection that in function resembles lymphatics is actively utilized in CNV.

PURPOSE. Anti–VEGF-A antibody (Ab) (e.g., bevacizumab, ranibizumab) is widely used as a treatment against retinal angiogenesis and edema. The purpose of this study was to evaluate whether intravitreal anti-VEGF Ab injection modulates inflammatory cells in retinal angiogenesis. METHODS. To investigate whether intravitreal bevacizumab injections affect the number of inflammatory cells in proliferative diabetic retinopathy (PDR) membranes in patients, immunohistochemical staining with CD45 Ab (pan-leukocyte marker) was performed using the surgically obtained membranes in pars plana vitrectomy with or without pretreatment with bevacizumab. To check whether anti–VEGF-A Ab affects leukocytes going in and out of blood vessels during retinal angiogenesis, the authors performed their novel leukocyte transmigration assay and CD45 immunostaining in a mouse model of oxygen-induced retinopathy (OIR). RESULTS. The authors’ new imaging approach revealed that intravitreal injection of anti–VEGF-A Ab blocks leukocyte infiltration as well as angiogenesis. The Ab injection inhibited leukocyte transmigration before affecting the angiogenenic area. CD45 staining showed no significant difference in the leukocyte number in the angiogenic retina or the human PDR membranes between the anti–VEGF-A Ab injected group and the control group. Furthermore, VEGF-A inhibition also affected leukocytes going out from the retina. CONCLUSIONS. Intravitreal injection of anti-VEGF-A Ab could inhibit leukocyte trafficking in the retina, suggesting that antiVEGF-A therapy could serve as a treatment in retinal inflammation. (Invest Ophthalmol Vis Sci. 2012;53:4323– 4328) DOI:10.1167/iovs.11-9119

Diabetic retinopathy (DR) is one of the leading causes of decreased vision and blindness in industrialized countries (Bhavsar, 2002). Much of the retinal damage that characterizes advanced proliferative diabetic retinopathy (PDR) results from retinal neovascularization (Simo, et al., 2006). When the newly formed vessels are associated with fibrous proliferations that form fibrovascular membranes (FVMs) on the surface of the neuroretina, traction retinal detachments can develop, resulting in potentially severe loss of vision (Hiscott, et al., 2000).

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 -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. (Invest Ophthalmol Vis Sci. 2011;52:5670‐5678) DOI:10.1167/iovs.10-6625

The purpose of this study was to determine whether vitrectomy alters the angiogenic profile in the vitreous of eyes with proliferative diabetic retinopathy (PDR).We measured the levels of angiopoietin-2, HGF, bFGF, PDGF, TIMP-1, and TIMP-2 by sandwich enzyme linked immunosorbent assay (ELISA) in vitreous samples from 27 eyes of 26 patients with PDR before pars plana vitrectomy (without IOL implantation) and in 12 fluid samples from 12 patients with PDR obtained during an IOL implantation 3.5 to 9 (mean 4.9) months after an earlier vitrectomy. The levels of these factors were also measured in 12 vitreous samples obtained from 12 eyes that had undergone epiretinal membrane (ERM) or macular hole (MH) surgeries.The mean vitreous levels of both angiopietin-2 (103 pg/ml) and HGF (1091 pg/ml) in the sample from eyes with PDR collected at the time of the IOL implantation were significantly lower than in those collected before the vitrectomy (P0.01). On the other hand, the changes in the levels of TIMP-1 and TIMP-2 were both not significant after vitrectomy.The significant decrease of angiopietin-2 and HGF in the vitreous fluid after vitrectomy suggests that vitrectomy shifts the eye towards an anti-angiogenic environment.

BackgroundProliferative 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 findingsA 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/significanceOur 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.

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.