Search

Endostatin, a naturally cleaved fragment of type XVIII collagen with antiangiogenic activity, has been involved in the regulation of neovascularization during diabetic retinopathy. Here, the intracellular distribution of endostatin in healthy mouse and human neuroretinas has been analyzed. In addition, to study the effect of experimental hyperglycemia on retinal endostatin, the db/db mouse model has been used. Endostatin protein expression in mouse and human retinas was studied by immunofluorescence and Western blot, and compared with db/db mice. Eye fundus angiography, histology, and immunofluorescence were used to visualize mouse retinal and intravitreal vessels. For the first time, our results revealed the presence of endostatin in neurons of mouse and human retinas. Endostatin was mainly expressed in bipolar cells and photoreceptors, in contrast to the optic disc, where endostatin expression was undetectable. Diabetic mice showed a reduction of endostatin in their retinas associated with the appearance of intravitreal vessels at the optic disc in 50% of db/db mice. Intravitreal vessels showed GFAP positive neuroglia sheath, basement membrane thickening by collagen IV deposition, and presence of MMP-2 and MMP-9 in the vascular wall. All together, these results point that decreased retinal endostatin during experimental diabetes is associated with optic disc intravitreal vascularization. Based on their phenotype, these intravitreal vessels could be neovessels. However, it cannot be ruled out the possibility that they may also represent persistent hyaloid vessels., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Persistent fetal vasculature (PFV) is a spectrum of congenital anomalies caused by complete or partial failure of the ocular fetal vasculature to regress. We report the visual and anatomic outcomes in a large cohort of patients who underwent early surgery for PFV., Methods: We retrospectively reviewed the medical records of patients who underwent lensectomy and anterior or core vitrectomy for unilateral PFV without primary intraocular lens implantation through limbal or pars plana/plicata approach. Inclusion criteria were surgery prior to 7 months of age, with at least 12 months of follow-up. Eyes with severe posterior segment involvement and retinal detachment deemed beyond repair were excluded., Results: A total of 58 patients met inclusion criteria. Mean age at surgery was 2.1 ± 1.5 months. Mean follow-up was 6.7 ± 4.2 years. At final follow-up, 19 eyes (33%) had visual acuity better than 1.0 logMAR. Thirty-three eyes (57%) developed 1 or more postoperative adverse events: glaucoma in 21 (36%) and retinal detachment in 11 (19%), 8 of which occurred in eyes that had pars plana or pars plicata incisions (P = 0.002). In patients with limbal incisions, 17 of 40 (43%) achieved a visual acuity better than 1.0 logMAR, compared with 2 of 18 patients (11%) with a pars plana/pars plicata incision (P = 0.03)., Conclusions: In our study cohort, early surgery for PFV achieved functional visual acuity in about one-third of patients. Limbal approach to surgery may result in better visual acuity and anatomic results., (Copyright © 2019 American Association for Pediatric Ophthalmology and Strabismus. Published by Elsevier Inc. All rights reserved.)

Purpose: To determine the underlying reasons for the non-visualization of polyps on en face optical coherence tomography angiography (OCTA) in patients with polypoidal choroidal vasculopathy (PCV)., Methods: A cross-sectional study of consecutive treatment-naïve 30 eyes with active PCV was included. Results of fundus photography, fundus fluorescein angiography (FFA), indocyanine green angiography (ICGA), spectral domain optical coherence tomography (SD-OCT), and en face OCTA were analyzed., Results: A total of 64 active polyps were found on FFA and ICGA in 30 eyes. On OCTA, 42/64 (65.6%) polyps were visualized, while 22/64 (34.4%) polyps were non-visualized. There were no significant differences in the size (P = 0.723) and filling time of polyps (P = 0.558) between the two groups. However, polypoidal lesions were less common in the non-visualized group (P < 0.001). The height of the polyps on SD-OCT was 243.95 ± 114.24 μm in the non-visualized group, which was higher than those (188.00 ± 87.93 μm) in the visualized group (P = 0.048). Moreover, more pulsatile polyps (72.7%) were found in the non-visualized group than those (2.4%) in the visualized group (P < 0.001). Four of the 22 polyps in the non-visualized group (18.2%) were located under a thick subretinal hemorrhage, and two of 22 invisible polyps (9.6%) located under and parallel to the retinal vessel in the inner layer of retina., Conclusions: Our results revealed that the height of the polyps, and not the size and pulsation of the polyps, correlated with the visualization of the polyps on OCTA. Polyps that were pulsating in early ICGA were difficult to be visualized on OCTA, which is the most possible reason for the non-visualization. Coverage with thick subretinal hemorrhage or retina vessels was another reason for the non-visualization of the polyps in active PCV on OCTA.

Purpose: Persistent fetal vasculature (PFV) is a unique ocular disorder usually presenting early in life. The unregressed embryonal hyaloid vasculature poses a risk of severe ocular complications leading to decreased visual acuity. Surgery is the mainstay of therapy in complicated cases. We describe the clinical presentation and surgical treatment of PFV managed at our center from 2012 to 2015., Methods: The study is a case series comprised eight patients who were diagnosed with complicated severe PFV. All were managed with a tailored surgical approach. The clinical characteristics, medical and surgical treatment, and follow-up findings of each case are described., Results: There were six males and two females. Surgical intervention involved anterior or posterior vitrectomy, lens extraction, and intraocular lens implantation. Hyaloid stalk removal with release of ciliary traction was variably utilized in selected cases. Endodiathermy controlled intraocular bleeding, and intraocular scissors proved helpful in anterior PFV for disinserting the ciliary process from an abnormally thickened posterior lens capsule. Visual outcomes differed in each case, depending on multiple clinical factors., Conclusion: Severe complex PFV presents a therapeutic challenge. A tailored surgical approach with meticulous postoperative management is essential for visual rehabilitation.

The vitreous of perinatal mice temporarily develops a unique vascular system, called the vasa hyaloidea propria (VHP). Observations showed the vessels possessed an extracellular matrix including the basement membrane in their entire length. Immunostaining of whole mount preparations of VHP with integrin β 1 antibody displayed a bush-like network consisting of long and straight fibers which were associated with the VHP but extended apart from the blood vessels. Electron microscopically, each fiber was composed of a bundle of thin filaments different from collagen fibrils. Macrophages associated with the VHP appeared to be arrested by the integrin bushes. The integrin bushes fragmented and disappeared by postnatal day 10, just before the regression of the VHP. Macrophages were involved in the digestion and clearance of integrin bushes. The vitreous integrin bushes appear to provide a scaffold for architectural maintenance of the hyaloid vessels and macrophages.

The hyaloid vasculature constitutes a transitory system nourishing the internal structures of the developing eye, but the mechanism of vascular regression and its cell biological characteristics are not fully understood. The present study aimed to reveal the specificity of the hyaloid vessels by a systematic immunohistochemical approach for marker substances of myeloid cells and the extracellular matrix (ECM) in neonatal mice. Macrophages immunoreactive for F4/80, cathepsin D, and LYVE-1 gathered around the vasa hyaloidea propria (VHP), while small round cells in vascular lumen of VHP were selectively immunoreactive for galectin-3; their segmented nuclei and immunoreactivities for Ly-6G, CD11b, and myeloperoxidase indicated their neutrophilic origin. VHP possessed thick ECM and a dense pericyte envelope as demonstrated by immunostaining for laminin, type IV collagen, integrin β1, and NG2. The galectin-3 + cells loosely aggregated with numerous erythrocytes in the lumen of hyaloid vessels in a manner reminiscent of vascular congestion. Galectin-3 is known to polymerize and form a complex with ECM and NG2 as well as recruit leukocytes on the endothelium. Observation of galectin-3 KO mice implicated the involvement of galectin-3 in the regression of hyaloid vasculature. Since macrophages may play central roles including blocking of the blood flow and the induction of apoptosis in the regression, galectin-3 + neutrophils may play a supportive role in the macrophage-mediated involution of the hyaloid vascular system., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Purpose: Matrix metalloproteinase-14 (MMP-14) is a transmembrane MMP that plays a critical role in promoting angiogenesis. We investigated the expression levels of MMP-14 and correlated the levels with clinical disease activity and with the levels of the angiogenic factors vascular endothelial growth factor (VEGF) and MMP-9 in proliferative diabetic retinopathy (PDR). To reinforce the findings at the functional level, we examined the expression of MMP-14 in the retinas of diabetic rats., Methods: Vitreous samples from 34 patients with PDR and 18 nondiabetic patients and epiretinal membranes from 13 patients with PDR and the retinas of rats were studied with enzyme-linked immunosorbent assay, immunohistochemistry, western blotting, and real-time reverse transcription PCR (RT-PCR)., Results: The MMP-14, VEGF, and MMP-9 levels were statistically significantly higher in the vitreous samples from patients with PDR than in the samples from the nondiabetic controls (p<0.001 for all comparisons). The MMP-14 levels in patients with PDR with active neovascularization were statistically significantly higher than those in patients with inactive PDR (p<0.001). There were statistically significant positive correlations between levels of MMP-14 and levels of VEGF (r = 0.3; p = 0.032) and MMP-9 (r = 0.54; p<0.001). In the epiretinal membranes, MMP-14 was expressed in vascular endothelial cells, leukocytes, and myofibroblasts. Statistically significant positive correlations were detected between the numbers of blood vessels expressing CD31 and the numbers of blood vessels (r = 0.74; p = 0.004) and stromal cells (r = 0.72; p = 0.005) expressing MMP-14. Statistically significant increases of MMP-14 mRNA and protein were detected in rat retinas after induction of diabetes., Conclusions: These results suggest that MMP-14 is involved in PDR angiogenesis.

Purpose: To evaluate the efficacy and the rate of side effects of the pegylated aptamer pegaptanib in the treatment of patients with choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD) and a history of previous arterial thromboembolic events (ATEs)., Methods: Twenty-three eyes of 23 patients with subfoveal CNV due to AMD and cerebrovascular accidents (n = 12) and myocardial infarction (n = 11) in the previous 6 months received intravitreal pegaptanib 0.3 mg according to a pro re nata regimen and were followed for 12 months. The paired Student t test was used to evaluate mean changes in best-corrected visual acuity (BCVA; primary outcome measure) and central foveal thickness (CFT)., Results: The mean patient age was 71.5 ± 4.6 years; there were 14 women and 9 men. The CNV was type 1, 2, and 3 in 18, 3, and 2 eyes, respectively. The mean BCVA improved from 0.67 ± 0.23 logMAR at baseline to 0.52 ± 0.31 logMAR at the end of 12-month follow-up (p = 0.044). Thirty-five percent of patients achieved ≥3 Early Treatment Diabetic Retinopathy Study lines improvement at 12 months. Mean CFT at baseline (381 ± 111 µm) decreased to 304 ± 82 µm at 12 months (p = 0.008). Patients received a mean of 4.3 ± 1.3 (range 3-7) injections. No systemic or ocular side effects occurred; no patient experienced further ATEs., Conclusions: Intravitreal pegaptanib can be considered a viable treatment option for patients with AMD-related CNV who are at high risk of ATEs.

The development of the ocular vasculatures is perfectly synchronized to provide the nutritional and oxygen requirements of the forming human eye. The fetal vasculature of vitreous, which includes the hyaloid vasculature, vasa hyaloidea propria, and tunica vasculosa lentis, initially develops around 4-6 weeks gestation (WG) by hemo-vasculogenesis (development of blood and blood vessels from a common progenitor, the hemangioblast). This transient fetal vasculature expands around 12 WG by angiogenesis (budding from primordial vessels) and remains until a retinal vasculature begins to form. The fetal vasculature then regresses by apoptosis with the assistance of macrophages/hyalocytes. The human choroidal vasculature also forms by a similar process and will supply nutrients and oxygen to outer retina. This lobular vasculature develops in a dense collagenous tissue juxtaposed with a cell constitutively producing vascular endothelial growth factor (VEGF), the retinal pigment epithelium. This epithelial/endothelial relationship is critical in maintaining the function of this vasculature throughout life and maintaining it's fenestrated state. The lobular capillary system (choriocapillaris) develops first by hemo-vasculogenesis and then the intermediate choroidal blood vessels form by angiogenesis, budding from the choriocapillaris. The human retinal vasculature is the last to develop. It develops by vasculogenesis, assembly of CXCR4 + /CD39 + angioblasts or vascular progenitors perhaps using Muller cell Notch1 or axonal neuropilinin-1 for guidance of semaphorin 3A-expressing angioblasts. The fovea never develops a retinal vasculature, which is probably due to the foveal avascular zone area of retina expressing high levels of antiangiogenic factors. From these studies, it is apparent that development of the mouse ocular vasculatures is not representative of the development of the human fetal, choroidal and retinal vasculatures., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Myosin-X (Myo10) is an unconventional myosin best known for its striking localization to the tips of filopodia. Despite the broad expression of Myo10 in vertebrate tissues, its functions at the organismal level remain largely unknown. We report here the generation of KO-first (Myo10 tm1a/tm1a ), floxed (Myo10 tm1c/tm1c ), and KO mice (Myo10 tm1d/tm1d ). Complete knockout of Myo10 is semi-lethal, with over half of homozygous KO embryos exhibiting exencephaly, a severe defect in neural tube closure. All Myo10 KO mice that survive birth exhibit a white belly spot, all have persistent fetal vasculature in the eye, and ~50% have webbed digits. Myo10 KO mice that survive birth can breed and produce litters of KO embryos, demonstrating that Myo10 is not absolutely essential for mitosis, meiosis, adult survival, or fertility. KO-first mice and an independent spontaneous deletion (Myo10 m1J/m1J ) exhibit the same core phenotypes. During retinal angiogenesis, KO mice exhibit a ~50% decrease in endothelial filopodia, demonstrating that Myo10 is required to form normal numbers of filopodia in vivo. The Myo10 mice generated here demonstrate that Myo10 has important functions in mammalian development and provide key tools for defining the functions of Myo10 in vivo.

Nanoparticles adsorb biomolecules to form corona upon entering the biological environment. In this study, tissue-specific corona formation is provided as a way of controlling protein interaction with nanoparticles in vivo. In the vitreous, the composition of the corona was determined by the electrostatic and hydrophobic properties of the associated proteins, regardless of the material (gold and silica) or size (20- and 100-nm diameter) of the nanoparticles. To control protein adsorption, we pre-incubate 20-nm gold nanoparticles with 5 selectively enriched proteins from the corona, formed in the vitreous, to produce nanoparticle-protein complexes. Compared to bare nanoparticles, nanoparticle-protein complexes demonstrate improved binding to vascular endothelial growth factor (VEGF) in the vitreous. Furthermore, nanoparticle-protein complexes retain in vitro anti-angiogenic properties of bare nanoparticles. In particular, priming the nanoparticles (gold and silica) with tissue-specific corona proteins allows nanoparticle-protein complexes to exert better in vivo therapeutic effects by higher binding to VEGF than bare nanoparticles. These results suggest that controlled corona formation that mimics in vivo processes may be useful in the therapeutic use of nanomaterials in local environment., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Purpose: The purpose of this study was to classify combined persistent fetal vasculature (PFV) on the basis of the ultrasonographic and Doppler characteristics. The potential clinical significance for both surgery design and prognosis determination was discussed., Design: A cross-sectional case series., Participants: The eyes of 54 children diagnosed with unilateral combined PFV were evaluated using B-mode ultrasound and color Doppler imaging (CDI)., Methods: Each participant's age at first presentation, diagnosis for referral, gender, family history, and systemic or other ocular anomalies were recorded. Retinal detachment, optic nerve abnormalities, and macular dislocation were also recorded in detail according to the RetCam (Clarity Medical Systems, Pleasanton, CA), ultrasound, and Doppler findings. The PFV eyes were divided into 4 groups on the basis of the ultrasound and CDI findings. Intergroup analysis was performed., Main Outcome Measures: Overall and intergroup analyses of the demographic features of the children with PFV were performed. The axial length, depth of the anterior chamber, and lens thickness were compared between the affected eyes and the fellow healthy eyes among the 4 groups., Results: Some 22.2%, 18.5%, 33.3%, and 25.9% of the eyes were grouped into type I ("I" shape), II ("Y" shape), III (inverted "Y" shape), and IV ("X" shape) combined PFV, respectively. The age at first presentation for type I was older than that for the other groups (P = 0.014). The axial length was reduced (P = 0.012) and the anterior chamber more shallow (P = 0.011) than in fellow healthy eyes for type IV eyes, but not for the other 3 groups., Conclusions: Ultrasound and CDI are informative screening and diagnostic tools that show characteristic flow patterns in the 4 types of combined PFV. This novel classification system provides new and important information for the diagnosis of PFV and, if validated, may play a role in guiding treatment recommendations in the future., (Copyright © 2016 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Objective: To characterize the clinical, diagnostic, and histopathologic findings in dogs with canine ocular gliovascular syndrome (COGS)., Procedures: The archives at the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW) were used to identify eyes with COGS. Histopathological inclusion criteria included: a neovascular membrane extending from the optic nerve head or retina, clusters of spindle cells lacking vascularization within the vitreous, and histological signs of glaucoma. Special and immunohistochemical (IHC) staining techniques were performed. Clinical data, treatments, and outcomes were obtained from case records and information provided by submitting veterinarians., Results: Thirty-seven eyes of 36 dogs were identified with COGS. The average age at diagnosis was 8.8 years (±2.2). The relative risk for a Labrador retriever affected by COGS was significantly greater (9.3 times) (P < 0.0001) when compared to all other dog breeds within the COPLOW database. Most dogs presented with hyphema and secondary glaucoma; average intraocular pressure was 39 mmHg (±19). Average time to enucleation or evisceration was 27 days. Vitreal cells stained positive with IHC for glial fibrillary acidic protein in 14 of 17 globes, and vascular endothelial growth factor was expressed in the vitreal cells in five of five globes., Conclusions: We have defined a syndrome associated with vitreal glial cell aggregates and neovascular proliferation from the optic nerve or retina, which leads to neovascular glaucoma. The inflammation and secondary glaucoma resulting from this syndrome appear poorly responsive to conventional medical therapies. The exact etiology of COGS remains undetermined, but a systemic etiology is unlikely., (© 2014 American College of Veterinary Ophthalmologists.)

A 17-month-old girl referred for a suspected ciliary body medulloepithelioma was found to have persistent fetal vasculature. Fluorescein angiography showed perfused hyaloid artery posterior tunica vasculosa lentis with brittle star appearance and nonperfused anterior pupillary membrane. Ultrasound biomicroscopy confirmed absence of iris or ciliary body solid tumor., (Copyright 2014, SLACK Incorporated.)

Objective: Choroidal neovascularization (CNV) is a major cause of vision loss in retinal diseases such as age-related macular degeneration (AMD). Previously, we demonstrated that semaphorin3A (Sema3A), which is a chemorepellent guidance molecule, inhibited the formation of retina neovascularization. In the present study, we investigated the antiangiogenic effects of Sema3A on transforming growth factor beta (TGF-β) in vitro and in vivo., Methods: Enzyme-linked immunosorbent assays (ELISAs) were used to measure the TGF-β levels in the vitreous humor of patients with AMD and controls. Human umbilical vein endothelial cells (HUVECs) were used for the in vitro study, and a laser-induced CNV mouse model was prepared for the in vivo study. The HUVECs were incubated with TGF-β and Sema3A. The proliferation, migration, apoptosis, and tube formation of the cells were then measured using BrdU, Transwell, flow cytometry, and Matrigel assays, respectively, and the SMAD2/3 signaling pathways were analyzed using western blot analysis. The C57BL/6J mouse retina was exposed to a laser to induce choroidal neovascularization (CNV), and Sema3A was injected intravitreously. After 14 days, fundus fluorescein angiography was performed to evaluate the leakage area of the CNV. The vascular endothelial growth factor (VEGF) and TGF-β concentrations in the retina-choroid complex were measured with ELISA. Components of the p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase-1/2 (ERK1/2), c-Jun NH2-terminal kinase (JNK), and SMAD2/3 signaling pathways in the Sema3A-treated groups were analyzed using western blotting., Results: In this study, we first verified that the vitreous TGF-β level was higher in patients with neovascular AMD than in the controls. We also showed that Sema3A inhibited TGF-β-induced HUVEC proliferation, migration, and tube formation and inhibited the downstream SMAD2/3 signaling pathway. Sema3A also induced TGF-β-stimulated HUVEC apoptosis and inhibited the response of TGF-β in vitro. In vivo, the TGF-β level was increased in the CNV mouse model. Sema3A not only inhibited laser-induced CNV formation but also inhibited the uptake of VEGF and TGF-β. In the western blot analysis, Sema3A was shown to inhibit the phosphorylation of p38 MAPK, ERK1/2, and JNK and to inhibit the SMAD2/3 signaling pathway after Sema3A treatment in CNV mice., Conclusions: Sema3A can be applied as a useful, adjunctive therapeutic strategy for preventing CNV formation.

Purpose: NADPH oxidase-generated reactive oxygen species (ROS) are implicated in angiogenesis. Isoforms of NADPH oxidase NOX1, NOX2, and NOX4 are reported to be expressed in endothelial cells (ECs). Of these, NOX1 and NOX2 have been reported to contribute to intravitreal neovascularization (IVNV) in oxygen-induced retinopathy (OIR) models. In this study, we tested the hypothesis that the isoform NOX4 in ECs contributed to vascular endothelial growth factor (VEGF)-induced angiogenesis and IVNV., Methods: Isoforms of NADPH oxidase MRNA were measured in several types of cultured vascular ecs: human retinal microvascular ECs (hRMVECs), choroidal ECs (CECs), and human umbilical vascular ECs (HUVECs) using real-time PCR. Newborn rat pups and dams were placed into an OIR model that cycled oxygen concentration between 50% and 10% every 24 h for 14 days, and then were placed in room air (RA) for an additional 4 days (rat OIR model). NOX4 expression in retinal lysates from the RA-raised pups at postnatal day 0 (P0), P14, and P18 was determined with western blots. STAT3 activation was determined as the ratio of phosphorylated STAT3 to total STAT3 with western blot analysis of retinal lysates from pups raised in RA or from the rat OIR model at P18. Semiquantitative assessment of the density of NOX4 colabeling with lectin-stained retinal ECs was determined by immunolabeling of retinal cryosections from P18 pups in OIR or in RA. In hRMVECs transfected with NOX4 siRNA and treated with VEGF or control, 1) ROS generation was measured using the 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester fluorescence assay and 2) phosphorylated VEGF receptor 2 and STAT3, and total VEGFR2 and STAT3 were measured in western blot analyses. VEGF-stimulated hRMVEC proliferation was measured following transfection with NOX4 siRNA or STAT3 siRNA, or respective controls., Results: NOX4 was the most prevalent isoform of NADPH oxidase in vascular ECs. NOX4 expression in retinal lysates was significantly decreased during development in RA. Compared to RA, the expression of retinal NOX4 increased at P18. At p18 OIR, semiquantitative assessment of the density of lectin and NOX4 colabeling in retinal vascular ECs was greater in retinal cryosections and activated STAT3 was greater in retinal lysates when compared to the RA-raised pups. In cultured hRMVECs, knockdown of NOX4 by siRNA transfection inhibited VEGF-induced ROS generation. VEGF induced a physical interaction of phosphorylated-VEGFR2 and NOX4. Knockdown of NOX4: 1) reduced VEGFR2 activation but did not abolish it and 2) abolished STAT3 activation in response to VEGF. Knockdown of either NOX4 or STAT3 inhibited VEGF-induced EC proliferation., Conclusions: Our data suggest that in a model representative of human retinopathy of prematurity, NOX4 was increased at a time point when IVNV developed. VEGF-activated NOX4 led to an interaction between VEGF-activated VEGFR2 and NOX4 that mediated EC proliferation via activation of STAT3. Altogether, our results suggest that NOX4 may regulate VEGFR2-mediated IVNV through activated STAT3.

Purpose: To compare tissue distribution of dye-drug surrogates after intravitreal (IVT) and suprachoroidal (SCS) delivery to determine the influence of drug lipophilicity and choroidal circulation., Methods: Thirty-two pig eyes were collected immediately after euthanasia. Sixteen eyes were perfused for 30 min through one long posterior ciliary artery with nondye containing nutrient media. An IVT or SCS injection was performed with either a 100 μL balanced salt solution (BSS, n=8), 1% sodium fluorescein (NaF, n=12) or 0.12% lipophilic carbocyanine dye (DiI, n=12). Globes were maintained at 37°C for 15 min, and then snap-frozen and dissected. Aqueous extraction and measurement of NaF or DiI concentration was performed using spectrophotometry and spectrofluorometry, respectively., Results: After SCS delivery of NaF scleral, iris-ciliary body, choroidal and vitreous dye levels were higher in nonperfused eyes compared to perfused eyes. After DiI SCS or IVT delivery, no significant differences were found in dye tissue concentrations in perfused eyes compared to nonperfused eyes. Following perfusion, a better and even drug distribution was found in the retinal pigmented epithelium (RPE)-choroid following IVT and SCS delivery of the hydrophilic drug and after IVT injection of the lipophilic drug compared to nonperfused eyes., Conclusions: Choroidal circulation reduces the tissue drug concentration of the hydrophilic drug suggesting an early clearance mechanism after SCS delivery. SCS injections of lipid and hydrophilic drugs allowed direct drug delivery to the retina and RPE-choroid with limited exposition to the anterior segment.

We determined the levels of the endogenous angiogenesis inhibitors soluble vascular endothelial growth factor receptor-1 (sVEGFR-1), thrombospondin (TSP)-1 and TSP-2 in the vitreous fluid from patients with proliferative diabetic retinopathy (PDR) and correlated their levels with clinical disease activity and the levels of vascular endothelial growth factor (VEGF). Vitreous samples from 30 PDR and 25 nondiabetic patients were studied by enzyme-linked immunosorbent assay. TSP-1 was not detected. VEGF and TSP-2 levels were significantly higher in PDR with active neovascularization compared with inactive PDR and nondiabetic patients (P < 0.001 for both comparisons). VEGF, sVEGFR-1 and TSP-2 levels were significantly higher in PDR with hemorrhage compared with PDR without hemorrhage and nondiabetic patients (P = 0.0063; 0.0144; <0.001, respectively). VEGF and sVEGFR-1 levels were significantly higher in PDR without traction retinal detachment (TRD) compared with PDR with TRD and nondiabetic patients (P = 0.038; 0.022, respectively). TSP-2 levels were significantly higher in PDR with TRD compared with PDR without TRD and nondiabetic patients (P < 0.001). There was a significant correlation between levels of VEGF and sVEGFR-1 (r = 0.427, P = 0.038). Our findings suggest that upregulation of sVEGFR-1 and TSP-2 may be a protective mechanism against progression of angiogenesis associated with PDR. TSP-2 might be associated with TRD.

The vitreous humor represents a significant barrier to the penetration of nanoparticulate-based ocular drug delivery systems. The gel structure and biochemical components of the vitreous will impact the rate of nanoparticle movement through the tissue to reach the retinal tissue. Also the structure of the vitreous, flow systems operating within it, age-related structural changes, and the presence of inflammation will also have a potential effect on movement. To effectively target the posterior retina, the physical properties of the nanoparticle formulation are a key element in the design of a system that will penetrate through the vitreous barrier.

Purpose: Neovascularization (NV) is a sight-threatening complication of retinal ischemia in diabetes, retinal vein occlusion, and retinopathy of prematurity. Current treatment modalities, including laser photocoagulation and repeated intraocular injection of VEGF antagonists, are invasive and not always effective, and may carry side effects. We studied the use of hyperoxia as an alternative therapeutic strategy for regressing established vitreous NV in a mouse model of oxygen-induced ischemic retinopathy., Methods: Hyperoxia treatment (HT, 75% oxygen) was initiated on postnatal day (P)17 after the onset of vitreous NV. Immunohistochemistry and quantitative PCR were used to assess retinal vascular changes in relation to apoptosis, and expression of VEGFR2 and inflammatory molecules. Effects of intravitreal injections of VEGF-A, VEGF-E, PlGF-1, and VEGF trap were also studied., Results: HT selectively reduced NV by 70% within 24 hours. It robustly increased the level of cleaved caspase-3 in the vitreous NV between 6 and 18 hours and promoted infiltration of macrophage/microglial cells. The HT-induced apoptosis was preceded by a significant reduction in VEGFR2 expression within the NV and an increase in VEGFR2 within the surrounding neural tissue. Intravitreal VEGF-A and VEGF-E (VEGFR2 agonist) but not PlGF-1 (VEGFR1 agonist) prevented HT-induced apoptosis and regression of NV. In contrast, VEGF trap and VEGFR2 blockers mimicked the effect of HT. However, intravitreal VEGF trap induced increases in inflammatory molecules while HT did not have such unwanted effect., Conclusions: HT may be clinically useful to specifically treat proliferative NV in ischemic retinopathy.

Photoreceptor degeneration is the most critical cause of visual impairment in age-related macular degeneration (AMD). In neovascular form of AMD, severe photoreceptor loss develops with subretinal hemorrhage due to choroidal neovascularization (CNV), growth of abnormal blood vessels from choroidal circulation. However, the detailed mechanisms of this process remain elusive. Here we demonstrate that neovascular AMD with subretinal hemorrhage accompanies a significant increase in extracellular ATP, and that extracellular ATP initiates neurodegenerative processes through specific ligation of Purinergic receptor P2X, ligand-gated ion channel, 7 (P2RX7; P2X7 receptor). Increased extracellular ATP levels were found in the vitreous samples of AMD patients with subretinal hemorrhage compared to control vitreous samples. Extravascular blood induced a massive release of ATP and photoreceptor cell apoptosis in co-culture with primary retinal cells. Photoreceptor cell apoptosis accompanied mitochondrial apoptotic pathways, namely activation of caspase-9 and translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, as well as TUNEL-detectable DNA fragmentation. These hallmarks of photoreceptor cell apoptosis were prevented by brilliant blue G (BBG), a selective P2RX7 antagonist, which is an approved adjuvant in ocular surgery. Finally, in a mouse model of subretinal hemorrhage, photoreceptor cells degenerated through BBG-inhibitable apoptosis, suggesting that ligation of P2RX7 by extracellular ATP may accelerate photoreceptor cell apoptosis in AMD with subretinal hemorrhage. Our results indicate a novel mechanism that could involve neuronal cell death not only in AMD but also in hemorrhagic disorders in the CNS and encourage the potential application of BBG as a neuroprotective therapy.

Purpose: The mode of development of the human hyaloid vascular system (HVS) remains unclear. Early studies suggested that these blood vessels formed by vasculogenesis, while the current concept seems to favor angiogenesis as the mode of development. We examined embryonic and fetal human HVS using a variety of techniques to gain new insights into formation of this vasculature., Methods: Embryonic and fetal human eyes from 5.5 to 12 weeks gestation (WG) were prepared for immunohistochemical analysis or for light and electron microscopy. Immunolabeling of sections with a panel of antibodies directed at growth factors, transcription factors, and hematopoietic stem cell markers was employed., Results: Light microscopic examination revealed free blood islands (BI) in the embryonic vitreous cavity (5.5-7 WG). Giemsa stain revealed that BI were aggregates of mesenchymal cells and primitive nucleated erythroblasts. Free cells were also observed. Immunolabeling demonstrated that BI were composed of mesenchymal cells that expressed hemangioblast markers (CD31, CD34, C-kit, CXCR4, Runx1, and VEGFR2), erythroblasts that expressed embryonic hemoglobin (Hb-ε), and cells that expressed both. Few cells were proliferating as determined by lack of Ki67 antigen. As development progressed (12 WG), blood vessels became more mature structurally with pericyte investment and basement membrane formation. Concomitantly, Hb-ε and CXCR4 expression was down-regulated and von Willebrand factor expression was increased with the formation of Weibel-Palade bodies., Conclusions: Our results support the view that the human HVS, like the choriocapillaris, develops by hemo-vasculogenesis, the process by which vasculogenesis, erythropoiesis, and hematopoiesis occur simultaneously from common precursors, hemangioblasts.

Purpose: A timely regression of the hyaloid vascular system (HVS) is required for the normal ocular development. Although macrophages have a critical role in this process, the exact mechanism remains undetermined. Periostin is a matricellular protein involved in tissue and vascular remodeling. The purpose of our study was to determine whether periostin is involved in the HVS regression., Methods: We used wild type (WT) and periostin knockout (KO) mice. Indocyanine green angiography and immunohistochemistry with isolectin B4 were used to evaluate the HVS regression. TUNEL-labeling was used to quantify the number of apoptotic hyaloid vascular endothelial cells. F4/80 and Iba-1 staining was performed to determine the number and location of macrophages in the vitreous. The location of periostin also was investigated by immunohistochemistry. To determine the functional role of periostin, the degree of adhesion of human monocytes to fibronectin was measured by an adhesion assay., Results: The HVS regression and peak in the number of TUNEL-positive apoptotic endothelial cells were delayed in periostin KO mice. The number of F4/80 positive cells in the vitreous was higher in periostin KO mice. Only a small number of Iba-1-positive cells near the hyaloid vessels was co-stained with periostin, and peripheral blood monocytes were not stained with periostin. Adhesion assay showed that periostin increased the degree of attachment of monocytes to fibronectin., Conclusions: These results suggest that periostin, which is secreted by the intraocular macrophages, enhances the HVS regression by intensifying the adhesion of macrophages to hyaloid vessels.

The efficacy of an antisense oligonucleotide (ODN17) cationic nanoemulsion directed at VEGF-R2 to reduce neovascularization was evaluated using rat corneal neovascularization and retinopathy of prematurity (ROP) mouse models. Application of saline solution or scrambled ODN17 solution on eyes of rats led to the highest extent of corneal neovascularization. The groups treated with blank nanoemulsion or scrambled ODN17 nanoemulsion showed moderate inhibition in corneal neovascularization with no significant difference with the saline and scrambled ODN17 control solution groups, while the groups treated with ODN17 solution or Avastin® (positive ODN17 control) clearly elicited marked significant inhibition in corneal neovascularization confirming the results reported in the literature. The highest significant corneal neovascularization inhibition efficiency was noted in the groups treated with ODN17 nanoemulsion (topical and subconjunctivally). However, in the ROP mouse model, the ODN17 in PBS induced a 34% inhibition of retinal neovascularization when compared to the aqueous-vehicle-injected eyes. A significantly higher inhibition of vitreal neovascularization (64%) was observed in the group of eyes treated with ODN17 nanoemulsion. No difference in extent of neovascularization was observed between blank nanoemulsion, scrambled ODN17 nanoemulsion, vehicle or non-treated eyes. The overall results indicate that cationic nanoemulsion can be considered a promising potential ocular delivery system and an effective therapeutic tool of high clinical significance in the prevention and forthcoming treatment of ocular neovascular diseases., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Nitric oxide (NO) is a multifunctional gaseous molecule that regulates various physiological functions in both neuronal and non-neuronal cells. NO is synthesized by nitric oxide synthases (NOSs), of which three isoforms have been identified. Neuronal NOS (nNOS) and endothelial NOS (eNOS) constitutively produce low levels of NO as a cell-signaling molecule in response to an increase in intracellular calcium concentration. Recent data have revealed a predominant role of eNOS in both angiogenesis and vasculogenesis., Methods: The immunohistochemical localization of nNOS and eNOS was investigated during embryonic and fetal ocular vascular development from 7 to 21 weeks gestation (WG) on sections of cryopreserved tissue., Results: eNOS was confined to endothelial cells of developing vessels at all ages studied. nNOS was prominent in nuclei of vascular endothelial and smooth muscle cells in the fetal vasculature of vitreous and choriocapillaris. nNOS was also prominent in the nuclei of CXCR4(+) progenitors in the inner retina and inner neuroblastic layer., Conclusions: These findings demonstrate co-expression of n- and eNOS isoforms in different compartments of vasoformative cells during development. Nuclear nNOS was present in vascular and nonvascular progenitors as well as endothelial cells and pericytes. This suggests that nNOS may play a role in the transcription regulatory systems in endothelial cells and pericytes during ocular hemo-vasculogenesis, vasculogenesis, and angiogenesis.

Angiopoietin 1 (Ang1) is an activating ligand for the endothelial receptor tyrosine kinase Tie2, whereas Ang2 acts as a context-dependent agonist or antagonist that has a destabilizing effect on the vasculature. The molecular mechanisms responsible for the versatile functions of Ang2 are poorly understood. We show here that Ang2, but not Ang1, induces Tie2 translocation to the specific cell-matrix contact sites located at the distal end of focal adhesions. The Ang2-specific Tie2 translocation was associated with distinct Tie2 activation and downstream signals which differed from those of Ang1, and led to impaired cell motility and weak cell-matrix adhesion. We demonstrate that the different oligomeric or multimeric forms of the angiopoietins induce distinct patterns of Tie2 trafficking; the lower oligomerization state of native Ang2 was crucial for the Ang2-specific Tie2 redistribution, whereas multimeric structures of Ang1 and Ang2 induced similar responses. The Ang2-specific Tie2 trafficking to cell-matrix contacts was also dependent on the cell substratum, α2β1-integrin-containing cell-matrix adhesion sites and intact microtubules. Our data indicate that the different subcellular trafficking of Tie2-Ang2 and Tie2-Ang1 complexes generates ligand-specific responses in the angiopoietin-Tie signaling pathway, including modulation of cell-matrix interactions.

Reports of intraocular bleeding in infants from nonabusive causes are rare. The authors report a case of visually significant vitreous hemorrhage in an infant with a persistent hyaloid artery who also had raised intracranial pressure secondary to an intraventricular bleed., (Copyright 2012, SLACK Incorporated.)

VEGF(165) b is an anti-angiogenic form of VEGF(165) produced by alternative splicing. The localization of pro-angiogenic VEGF(165) and anti-angiogenic VEGF(165) b was investigated during development of the vasculatures in fetal human eyes from 7 to 21 weeks gestation (WG). The fetal vasculature of vitreous, which includes tunica vasculosa lentis (TVL), had moderate VEGF(165) immunoreactivity at 7WG and very little VEGF(165) b. Both forms were elevated at 12WG. VEGF(165) then decreased around 17WG when the TVL regresses but VEGF(165) b remained elevated. In choroid, VEGF(165) was present in forming choriocapillaris (CC) and retinal pigment epithelium (RPE) at 7WG while VEGF165b was present in CC and mesenchymal precursors within the choroidal stroma. By 21WG, both forms were elevated in RPE and choroidal blood vessels but VEGF(165) b was apical and VEGF(165) basal in RPE. Diffuse VEGF(165) immunoreactivity was prominent in 12WG innermost retina where blood vessels will form while VEGF(165) b was present in most CXCR4(+) progenitors in the inner neuroblastic layer and migrating angioblasts in the putative nerve fiber layer. By 21WG, VEGF(165) was present in nerve fibers and VEGF(165) b in the inner Muller cell process. The localization of VEGF(165) b was distinctly different from VEGF(165) both spatially and temporally and it was often associated with nucleus in progenitors., (Copyright © 2012 Wiley Periodicals, Inc.)

The pecten oculi is a highly vascular and pigmented organ placed in the vitreous body of the avian eye. As no data are currently available on the morphological organization of the pecten in the Psittaciformes, the pecten oculi of the budgerigar (Melopsittacus undulatus) was studied. The eyes from adult male budgerigars were examined by light, transmission, and scanning electron microscopy and a morphometric study on both light and transmission electron microscopy specimens was also performed in the different parts of the organ. In the budgerigar, the type of the pecten oculi was pleated. Its basal part had a cranio-caudal and postero-anterior course; its body consisted of 10-12-folds joined apically by a densely pigmented bridge. The pecten showed many capillaries, whose wall was thick and formed by pericytes and endothelial cells. These latter had a large number of microfolds, rectilinear on their luminal surface and tortuous on their abluminal surface. Interstitial pigment cells were placed among the capillaries, filled with melanin granules and showed many cytoplasmic processes. The morphometric analysis demonstrated significant differences among the three parts of the organ relative to the length of the endothelial processes and to the number and size of the pigment granules. The morphological and morphometric analysis showed that the bridge of the budgerigar, different from the other birds, had a large number of capillaries, so that this part of the organ could also play a trophic role for the retina in addition to the choriocapillaris., (Copyright © 2012 Wiley Periodicals, Inc.)

Background: Valuable insights into the complex process of retinal vascular development can be gained using models with abnormal retinal vasculature. Two such models are the recently described mouse lines with mutations in Lama1, an important component of the retinal internal limiting membrane (ILM). These mutants have a persistence of the fetal vasculature of vitreous (FVV) but lack a primary retinal vascular plexus. The present study provides a detailed analysis of astrocyte and vascular development in these Lama1 mutants., Results: Although astrocytes and blood vessels initially migrate into Lama1 mutant retinas, both traverse the peripapillary ILM into the vitreous by P3. Once in the vitreous, blood vessels anastomose with vessels of the vasa hyaloidea propria, part of the FVV, and eventually re-enter the retina where they dive to form the inner and outer retinal capillary networks. Astrocytes continue proliferating within the vitreous to form a dense mesh that resembles epiretinal membranes associated with persistent fetal vasculature and proliferative vitreoretinopathy., Conclusions: Lama1 and a fully intact ILM are required for normal retinal vascular development. Mutations in Lama1 allow developing retinal vessels to enter the vitreous where they anastomose with vessels of the hyaloid system which persist and expand. Together, these vessels branch into the retina to form fairly normal inner retinal vascular capillary plexi. The Lama1 mutants described in this report are potential models for studying the human conditions persistent fetal vasculature and proliferative vitreoretinopathy.

Complex ultrasound examination was performed in 108 children with unilateral congenital cataract (UCC) and persisting hyperplastic primary vitreous syndrome (PHPVS) to reveal indications and contraindications for surgery. The presence of functioning vessels with linear blood flow velocity (LBFV) measurement was performed in fibrovascular band and retrolenticular membrane (RLM) in different stages of PHPVS using color Doppler and energetic mapping. The echographic density of fibrovascular band and RLM was estimated using echodensitometry. The results of the study showed correlation of PHPVS severity and fibrovascular band diameter, LBFV in the vessels of the band and echographic density of the band, RLM and their thickness as well.

Purpose: To study vascular endothelial growth factor (VEGF) regulation in the development of intravitreous neovascularization and peripheral avascular retina in peripheral severe retinopathy of prematurity (ROP)., Methods: The rat 50/10 model of ROP mimics zone II, stage 3 severe ROP and recreates fluctuations in transcutaneous oxygen levels in preterm infants. On postnatal (p) day ages p0, p8, p11-p14, and p18, retinas from the model or room-air (RA) age-matched pups were analyzed for mRNA of VEGF splice variants and receptors using real-time polymerase chain reaction or VEGF protein using enzyme-linked immunosorbent assay., Results: On p14, when retinas were only 70% vascularized in the model but fully vascularized in RA, VEGF₁₆₄ expression was threefold greater in the model compared to RA. On p18, intravitreous neovascularization was associated with a 5-fold increase in VEGF₁₆₄ mRNA in the model compared to RA. By analysis of variance, VEGF₁₆₄ and VEGFR2 mRNAs were up-regulated in association with increasing developmental age (P<.0001 for both comparisons) or exposure to the model compared to RA (P<.0001 and P=.0247, respectively), whereas increasing developmental age was associated only with up-regulated VEGF₁₂₀ (P=.0006), VEGF₁₈₈ (P=.0256), and VEGFR1 (P<.0001) mRNAs. VEGF protein increased significantly in the model and on p14 and p18 compared to RA (P<.0001)., Conclusions: The model mimics contemporary severe ROP in the United States unlike other models of oxygen-induced retinopathy. Compared to RA retinas, VEGF significantly increased in association with avascular retina and intravitreous neovascularization. A hypothesis is proposed that VEGF up-regulation plays a role in the development of both important features.

Purpose: The hyaloid vascular system (HVS) is a transient network nourishing developing eyes and has been widely used as a natural model to study blood vessel regression. Failure of its regression in humans leads to several blinding diseases. Lymphatic vessel endothelial hyaluronic acid receptor (LYVE-1) is a recently defined lymphatic marker that is also expressed by a subpopulation of macrophages. To date, there is no report on its expression in the HVS. This study was conducted to investigate whether LYVE-1 is expressed in the HVS and how it is associated with the vascular structure and macrophage phenotype., Methods: Normal C57BL/6 mouse eyeballs were sampled from embryonic day (E) 10.5 to postnatal (P) and adult stages for immunofluorescent microscopic studies with antibodies against LYVE-1, CD31 (panendothelial cell marker), and F4/80 (macrophage marker). Additionally, Angiopoietin-2 (Ang-2) knockout mice with abnormally persistent HVS were examined., Results: The LYVE-1 expression was detected on normal HVS between E12.5 and P14. The LYVE-1(+) cells were F4/80(+) but CD31(-), indicating a macrophage lineage. Additionally, LYVE-1(+) cells bud on CD31(+) vessels and constitute an integral part of the network in both normal developing and Ang-2 knockout mice., Conclusions: This study provides the first evidence that the HVS contains a LYVE-1(+) cellular component in both physiological and pathologic conditions. This novel finding not only provides a new concept in defining the embryogenesis and pathogenesis of the HVS, it also leads to a completely natural model in which to study the functions of the LYVE-1 pathway, an important topic for lymphatic research as well.

Background and Objective: To present spectral domain optical coherence tomography (SD-OCT), scanning laser ophthalmoscopy, and intraoperative images showing possible pathways of blood entrance into the eye and ways in which it may spread inside the eye in Terson syndrome., Patients and Methods: Nine eyes of 5 patients with Terson syndrome underwent pars plana vitrectomy. Surgeries were recorded and analyzed afterward. SD-OCT and scanning laser ophthalmoscopy examinations were performed after surgery., Results: Visual acuity improved in all cases. SD-OCT three-dimensional mode improved visualization of the internal limiting membrane (ILM), possibly due to sub-ILM blood spreading detachment near the macula and optic disc. Scanning laser ophthalmoscopy revealed blood spreading along vessels and blood under the retina was observed intraoperatively., Conclusion: SD-OCT, scanning laser ophthalmoscopy, and intraoperative images show that blood may enter the vitreous cavity around the retinal vessels near the optic disc. Inside the eye, the blood may spread intraretinally, sub-ILM, or along the retinal vessels., (Copyright 2010, SLACK Incorporated.)

Unlabelled: A 23-year-old man presented with bilateral persistent fetal vasculature (PFV). Examination revealed bilateral cataracts, lens subluxation, elongated ciliary processes, and persistent hyaloid vessel remnants with mild posterior segment involvement. Extracapsular cataract extraction combined with posterior capsulectomy and anterior vitrectomy was performed in the right eye; lensectomy, posterior capsulectomy, anterior vitrectomy, superior peripheral iridectomy, and anterior chamber intraocular lens implantation were performed in the left eye. Postoperatively, the corrected distance visual acuity improved (20/100 in the right eye and 20/40 in the left eye) and the cosmetic appearance was satisfactory. This case shows that in rare cases of bilateral PFV, the natural course can be benign, with functional, although amblyopic, vision. In selected cases, late surgical intervention can improve vision and cosmetic appearance., Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned., (Copyright 2010 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.)

Objectives: To determine expression of vascular endothelial growth factor (VEGF), pigment epithelium-derived factor, and their respective receptors in retinas using a model of retinopathy of prematurity., Methods: Retinas isolated from a 50/10 oxygen (inspired oxygen cycled between 50% oxygen and 10% oxygen every 24 hours)-induced rat model of retinopathy of prematurity (50/10 OIR model), and from room air-raised rat pups (RA) at birth, age 14 days (persistent peripheral avascular retina in the 50/10 OIR model and complete retinal vascularization in RA) and age 18 days (intravitreous neovascularization in the 50/10 OIR model) were analyzed for messenger RNA of VEGF(164), neuropilin 1, neuropilin 2, VEGF receptor 1, VEGF receptor 2, pigment epithelium-derived factor, and pigment epithelium-derived factor receptor by real-time polymerase chain reaction., Results: In the 50/10 OIR model compared with RA, fold changes in expression of VEGF(164), neuropilin 1, and neuropilin 2 were significantly increased at ages 14 and 18 days. A trend for increased fold change was noted in expression of VEGF receptor 2 at age 14 days and a significant increase at age 18 days in the 50/10 OIR model compared with RA. Pigment epithelium-derived factor receptor was significantly increased at age 14 days in the 50/10 OIR model compared with RA., Conclusion: Increased expression of VEGF(164) and angiogenic receptors were found in association with both avascular retina at day 14 and intravitreous neovascularization at day 18 in a relevant model of retinopathy of prematurity., Clinical Relevance: Increased VEGF and angiogenic receptors may have a role in the development of peripheral avascular retina and stage 3 retinopathy of prematurity.

The objective of this study is to determine growth factor expression and activation of signaling pathways associated with intravitreous neovascularization and peripheral avascular retina using a model of retinopathy of prematurity (ROP) relevant to today with oxygen monitoring in neonatal units. Studies using 50/10 oxygen-induced retinopathy (OIR) and 50/10 OIR+SO models were reviewed. Repeated fluctuations in oxygen increased retinal vascular endothelial growth factor (VEGF) even while peripheral avascular retina persisted and prior to the development of intravitreous neovascularization. Repeated fluctuations in oxygen increased VEGF(164) expression but not VEGF(120). Neutralizing VEGF bioactivity significantly reduced intravitreous neovascularization and arteriolar tortuosity without interfering with ongoing retinal vascularization. Repeated oxygen fluctuations led to retinal hypoxia and increased reactive oxygen species (ROS). Inhibiting ROS with NADPH oxidase inhibitor, apocynin, reduced avascular retina by interfering with apoptosis. Supplemental oxygen reduced retinal VEGF concentration and exacerbated NADPH oxidase activation to contribute to intravitreous neovascularization through activation of the JAK/STAT pathway. Oxygen stresses relevant to those experienced by preterm infants today trigger signaling of different pathways to cause avascular retina and intravitreous neovascularization. Increased signaling of VEGF appears important to the development of both avascular retina and intravitreous neovascularization.

Although a blockade of acetylcholine esterase has been reported to suppress neuronal cell death induced by exogenous glutamate and beta-amyloid, information is still limited regarding the neuroprotective effects of the acetylcholine esterase inhibitor donepezil. We histologically examined the effects of donepezil on neuronal injury induced by ischemia-reperfusion. Intravenous and intravitreous treatment with donepezil 15 min prior to ischemia dramatically reduced the retinal damage. The protective effect of donepezil in the ganglion cell layer was not affected by mecamylamine, a nicotinic acetylcholine-receptor antagonist, nor scopolamine, a muscarinic acetylcholine-receptor antagonist. The protective effect of donepezil in the inner plexiform layer was reduced not by mecamylamine, but by scopolamine. Neostigmine, a choline-esterase inhibitor, and pilocarpine, a muscarinic acetylcholine-receptor agonist, have protective effects in the inner plexiform layer and the inner nuclear layer. These results suggest that not only the activation of acetylcholine receptors but also a mechanism unrelated to acetylcholine-esterase inhibition contribute to the protective effect of donepezil on the ganglion cells in the ischemic-reperfused rat retina. Donepezil may be useful as a therapeutic drug against retinal diseases that cause neuronal cell death such as glaucoma with high intraocular pressure.

To determine the effect of a vascular endothelial growth factor receptor 2 tyrosine kinase (VEGFR2) inhibitor on intravitreous neovascularization (IVNV), endothelial tip cell filopodia, and intraretinal vascularization in a rat model of retinopathy of prematurity (ROP). Within 4h of birth, newborn Sprague-Dawley rat pups and their mothers were cycled between 50% and 10% oxygen daily until postnatal day (p)12. Pups were given intravitreous injections of VEGFR2 inhibitor, SU5416, or control (dimethyl sulfoxide, DMSO) and returned to oxygen cycling until p14, then placed into room air. Intravitreous neovascularization (IVNV), avascular/total retinal areas, and endothelial tip cell filopodial number and length were determined in lectin-labeled neurosensory retinal flat mounts. Cryosections or fresh tissue were analyzed for phospho-VEGFR1, phospho-VEGFR2, activated caspase-3, or phospho-beta3 integrin. Human umbilical venous (HUVECs) and human choroidal endothelial cells (ECs) were treated with VEGFR2 inhibitor to determine effect on VEGFR2 phosphorylation and on directed EC migration toward a VEGF gradient. Filopodial length and number of migrated ECs were also measured. Compared to control, the VEGFR2 inhibitor reduced VEGFR2 phosphorylation in HUVECs in vitro and clock hours and areas of IVNV but not percent avascular retina in vivo. Filopodial length and number of filopodia/EC tip cell were reduced in retinal flat mounts at doses that inhibited IVNV, whereas at lower doses, only a reduction in filopodial length/EC tip cell was found. There was no difference in phosphorylated beta3 integrin and cleaved caspase-3 labeling in VEGFR2 inhibitor-treated compared to control in vivo. Doses of the VEGFR2 inhibitor that reduced filopodial length and number of filopodia/migrating EC corresponded to reduced EC migration in in vitro models. VEGFR2 inhibitor reduced IVNV and filopodial number and length/EC tip cell without interfering with intraretinal vascularization. Reducing the number and length of filopodia/endothelial tip cell may reduce guidance cues for endothelial cells to migrate into the vitreous without interfering with migration into the retina toward a VEGF gradient.

In children with persistent hyperplastic primary vitreous body (PHPVB) concurrent with unilateral congenial cataracts (UCC), combined ultrasound study used to examine blood supply to and hemodynamics of the hyaloid system--a fibrovascular band. To determine the prognosis of a surgical intervention in the mode of color and energy Doppler mapping in the persistent band, retrolenticular membrane, the authors studied whether they had functioning vessels and linear blood flow velocity in different degrees of PHPVB. Based on the results of combined ultrasound study, the authors defined indications for and contraindications to surgical treatment of UCC concurrent with PHPVB in order to prevent hemorrhagic complications (hyphema, hemophthalmos, hyaloid vascular bleeding), as well as the optimal time of surgical treatment.

Purpose: To investigate whether oxygen stresses experienced in retinopathy of prematurity (ROP) trigger signaling through reactive oxygen species (ROS) and whether the Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway lead to intravitreous neovascularization (IVNV) in an oxygen-induced retinopathy (OIR) rat model., Methods: Newborn rat pups exposed to repeated fluctuations in oxygen and rescued in supplemental oxygen (28% O(2), 50/10 OIR+SO) were treated with apocynin, an NADPH oxidase and ROS inhibitor (10 mg/kg/d), AG490, a JAK2 inhibitor (5 mg/kg/d), or phosphate-buffered saline. Intraperitoneal injections were given from postnatal day (P)12 to P17 (apocynin), or from P3 to P17 (AG490). Outcomes were intravitreous neovascularization and avascular/total retinal areas, vascular endothelial growth factor, phosphorylated JAK2, and phosphorylated STAT3., Results: Apocynin significantly reduced phosphorylated STAT3 in 50/10 OIR+SO (P = 0.04), in association with previously reported inhibition of the IVNV area. Inhibition of JAK with AG490 significantly reduced phosphorylated JAK2 (P < 0.001), phosphorylated STAT3 (P = 0.002), and IVNV area (P = 0.033) in the 50/10 OIR+SO model compared with control., Conclusions: Activation of NADPH oxidase from supplemental oxygen works through activated STAT3 to lead to IVNV. In addition, inhibition of the JAK/STAT pathway reduces IVNV. Further studies are needed to determine the effects and relationships of oxygen stresses on JAK/STAT and NAPDH oxidase signaling.

Background: The present study aimed to determine the efficiency and safety of baculovirus-mediated intravitreal gene transfer in rabbit eye and to compare its efficiency with adenovirus. We also studied how an intravitreal injection of vectors producing vascular endothelial growth factor D (VEGF-D) impacts the vasculature of rabbit eye., Methods: Baculoviral (BacVEGF-D) or adenoviral VEGF-D (AdVEGF-D) were administered intravitreally into the right eye at different doses (10(8), 10(9) and 10(10) IU/ml) to 24 animals. Left eyes were injected with control viruses. To determine how long transgene expression lasted, we injected BacVEGF-D or BacLacZ to the vitreous humour of 11 animals and followed them for 4 weeks. Vitreous samples were taken after sacrifice for enzyme-linked immunosorbent assays and eyes were removed and fixed for histological analyses., Results: Both baculoviruses and adenoviruses caused efficient expression of VEGF-D in the rabbit eyes. BacVEGF-D caused a dose-dependent vascular leakage and a moderate dilation of the capillaries. The highest effect was seen 6 days after gene transfer and was detectable for 2 weeks. Intravitreal injection of baculovirus caused expression of VEGF-D in the inner retina, photoreceptor cells and in retinal pigment epithelium cells, whereas adenovirus-mediated VEGF-D expression was detected in the nerve fiber layer and ganglion cell layer. Baculovirus caused a transient inflammation similar to adenoviruses., Conclusions: The study suggests that baculoviruses are efficient vectors for ocular gene transfer, especially if deeper retinal layers need to be transduced. In addition, intravitreal VEGF-D gene transfer caused blood-retina barrier breakdown but not neovessel formation in the rabbit eye., ((c) 2009 John Wiley & Sons, Ltd.)

The purpose of the present study was to characterize rat organic anion transporter (Oat) 3 (Oat3, Slc22a8) in the efflux transport at the inner blood-retinal barrier (BRB). Reverse transcription-polymerase chain reaction analysis showed that rat (r) Oat3 mRNA is expressed in retinal vascular endothelial cells (RVECs), but not rOat1 and rOat2 mRNA. The expression of Oat3 in the retina and human cultured retinal endothelial cells was further confirmed by Western blot analysis. Immunohistochemical staining in RVECs showed that rOat3 is colocalized with glucose transporter 1, but not P-glycoprotein, suggesting that rOat3 is possibly located at the abluminal membrane of the RVEC. The contribution of rOat3 to the efflux of [(3)H]p-aminohippuric acid ([(3)H]PAH), [(3)H]benzylpenicillin ([(3)H]PCG), and [(14)C]6-mercaptopurine ([(14)C]6-MP), substrates of rOat3, from the vitreous humor/retina to the circulating blood across the inner BRB was evaluated using the microdialysis method. [(3)H]PAH, [(3)H]PCG, [(14)C]6-MP, and [(14)C] or [(3)H]d-mannitol, a bulk flow marker, were biexponentially eliminated from the vitreous humor after vitreous bolus injection. The elimination rate constant of [(3)H]PAH, [(3)H]PCG, and [(14)C]6-MP during the terminal phase was approximately 2-fold greater than that of d-mannitol. This efflux transport was reduced in the retinal presence of probenecid, PAH, and PCG, whereas it was not inhibited by digoxin. In conclusion, rOat3 is expressed at the inner BRB and involved in the vitreous humor/retina-to-blood transport of PAH, PCG, and 6-MP. This transport system is one mechanism to limit the retinal distribution of PAH, PCG, and 6-MP.

While angiogenesis inhibitors are already widely used to treat retinal disease in adults, only limited reports are currently available for the use of anti-VEGF in pediatric vitreoretinal diseases such as retinopathy of prematurity, Coats' disease, familial exudative vitreoretinopathy and retinopathy of incontinentia pigmenti. The limited trials of anti-VEGF therapy for pediatric vitreoretinal diseases are promising, although more extensive controlled trials will be needed to confirm their safety and efficacy. This paper will examine the current evidence for use of anti-VEGF therapy in a number of pediatric vitreoretinal disorders and describe a case of anti-VEGF therapy in retinopathy of incontinentia pigmenti.

Diabetic retinopathy (DR) remains the major threat to sight in the working age population. Diabetic macular edema (DME) is a manifestation of DR that produces loss of central vision. Macular edema within 1 disk diameter of the fovea is present in 9% of the diabetic population. Proliferative diabetic retinopathy (PDR) is a major cause of visual loss in diabetic patients. In PDR, the growth of new vessels from the retina or optic nerve, is thought to occur as a result of vascular endothelial growth factor (VEGF) release into the vitreous cavity as a response to ischemia. Furthermore, VEGF increases vessel permeability leading to deposition of proteins in the interstitium that facilitate the process of angiogenesis and macular edema. This review demonstrates multiple benefits of intravitreal bevacizumab on DR including DME and PDR. The results indicate that intravitreal bevacizumab injections may have a beneficial effect on macular thickness and visual acuity (VA), independent of the type of macular edema that is present. Therefore, in the future this new treatment modality could replace or complement focal/grid laser photocoagulation in DME. In addition, in PDR, this new option could be an adjuvant agent to PRP so that more selective therapy may be applied.