bromochloroacetic-acid and Epiretinal-Membrane

We sought to characterize the angiofibrotic and apoptotic effects of vascular endothelial growth factor (VEGF)-inhibition on fibrovascular epiretinal membranes in eyes with traction retinal detachment because of proliferative diabetic retinopathy.. Membranes were excised from 20 eyes of 19 patients (10 randomized to intravitreal bevacizumab, 10 controls) at vitrectomy. Membranes were stained with antibodies targeting connective tissue growth factor (CTGF) or VEGF and colabeled with antibodies directed against endothelial cells (CD31), myofibroblasts, or retinal pigment epithelium markers. Quantitative and colocalization analyses of antibody labeling were obtained through immunofluorescence confocal microscopy. Masson trichrome staining, cell counting of hematoxylin and eosin sections, and terminal dUTP nick-end labeling staining were performed.. High levels of fibrosis were observed in both groups. Cell apoptosis was higher (P = 0.05) in bevacizumab-treated membranes compared with controls. The bevacizumab group had a nonsignificant reduction in colocalization in CD31-CTGF and cytokeratin-VEGF studies compared with controls. Vascular endothelial growth factor in extracted membranes was positively correlated with vitreous levels of VEGF; CTGF in extracted membranes was negatively correlated with vitreous levels of CTGF.. Bevacizumab suppresses vitreous VEGF levels, but does not significantly alter VEGF or CTGF in diabetic membranes that may be explained by high baseline levels of fibrosis. Bevacizumab may cause apoptosis within fibrovascular membranes.

Topics: Actins; Angiogenesis Inhibitors; Apoptosis; Bevacizumab; Cell Proliferation; Connective Tissue Growth Factor; Diabetic Retinopathy; Epiretinal Membrane; Fibrosis; Humans; Intravitreal Injections; Keratins; Platelet Endothelial Cell Adhesion Molecule-1; Prospective Studies; Receptors, Vascular Endothelial Growth Factor; Retina; Vascular Endothelial Growth Factor A; Vitrectomy

To develop a large animal model of proliferative vitreoretinopathy (PVR) in the swine to eventually study disease pathophysiology, as well as novel therapies.. PVR was induced in domestic swine by creation of a posterior vitreous detachment, creation of a retinal detachment by the injection of subretinal fluid, and intravitreal injection of green fluorescent protein-positive retinal pigment epithelial (GFP+ RPE) cells. Control eyes had the same surgical procedures without RPE cell injection. PVR was clinically graded on days 3, 7, and 14. Animals were euthanized on day 14, and enucleated eyes were analyzed by light microscopy and immunohistochemistry.. Injection of GFP+ RPE cells into the vitreous cavity produced localized, traction retinal detachments by day 14 in all eyes (14 of 14); in contrast, the retina spontaneously reattached by day 3 and remained attached in all control eyes (10 of 10). Contractile epiretinal membranes on the inner retinal surface that caused the traction retinal detachments consisted predominantly of GFP+ RPE cells. These cells stained positive for cytokeratin, confirming their epithelial origin, and also expressed α-SMA and fibronectin, markers for myofibroblasts and fibrosis, respectively.. We established a swine PVR model that recapitulates key clinical features found in humans and, thus, can be used to study the pathophysiology of PVR, as well as new novel therapies. GFP+ RPE cells injected into the vitreous cavity formed contractile membranes on the inner retinal surface and caused localized traction retinal detachments.

Topics: Actins; Animals; Disease Models, Animal; Epiretinal Membrane; Female; Fibronectins; Fibrosis; Green Fluorescent Proteins; Keratins; Luminescent Agents; Myofibroblasts; Retina; Retinal Detachment; Retinal Pigment Epithelium; Swine; Vitreoretinopathy, Proliferative

The Roundabout (Robo) family of proteins is related to the transmembrane receptors and plays a major role in neurogenesis. However, the role of the Robo proteins in proliferative retinopathy has not yet been defined. This study was conducted to determine whether Robo1 is expressed in the retina of patients with proliferative retinal disease and whether it has a pathobiological role in the disease.. Immunohistochemistry was used to determine the presence and distribution of Robo1 in the pathologic membranes in proliferative retinopathy. Small interfering (si)RNA technology was used to knockdown Robo1 expression and to study its effects on retinal pigment epithelial (RPE) cells in vitro. The impact on PVR development of blocking Robo1 expression was determined by applying specific siRNA in a PVR rabbit model. The prevalences of PVR and retinal detachment were determined by indirect ophthalmoscope on days 1, 3, 7, 14, 21, and 28 after the injection of RPE cells into the vitreous.. Immunohistochemistry showed that Robo1 expression was detected in GFAP-labeled glial cells and cytokeratin-labeled RPE cells in proliferative membranes. Robo1 expression was also detected in CD31-labeled vascular endothelial cells. Knockdown of Robo1 expression not only reduced human RPE cell proliferation in vitro but also effectively suppressed the development of PVR in a rabbit model.. Robo1 is present in the extracellular matrix of proliferative membranes and may be derived from dedifferentiated RPE cells. Silencing the expression of Robo1 in RPE cells inhibited cell proliferation and suppressed the development of PVR in an animal model, indicating a potential therapeutic usefulness in treating PVR.

Topics: Adolescent; Aged; Animals; Cell Line; Cell Movement; Cell Proliferation; Child; Diabetic Retinopathy; Disease Models, Animal; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Epiretinal Membrane; Female; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Gene Silencing; Glial Fibrillary Acidic Protein; Humans; Keratins; Male; Middle Aged; Nerve Tissue Proteins; Neuroglia; Platelet Endothelial Cell Adhesion Molecule-1; Rabbits; Receptors, Immunologic; Retinal Pigment Epithelium; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Roundabout Proteins; Vitreoretinopathy, Proliferative; Young Adult

The purpose of this study was to first determine whether hypoxia-inducible factor-1α (HIF-1 α) was detectable in diabetic preretinal membranes and to compare the presence of HIF-1α in fibrovascular proliferative diabetic retinopathy membranes with nondiabetic, idiopathic, epiretinal membranes.. Twelve patients with proliferative diabetic retinopathy membranes requiring pars plana vitrectomy and nine nondiabetic patients with idiopathic epiretinal membranes requiring pars plana vitrectomy underwent excision of these membranes. Immunohisto-chemical staining for the presence of HIF-1α was performed on the excised membranes. The degree of staining for HIF-1α (1+, 2+, and 3+ scale) and the cellular location of staining were determined for each specimen. Institutional Review Board approval and informed consent were obtained for all patients.. Eleven of 12 (92%) diabetic preretinal membranes were positive for HIF-1α, and most had intense (2+ to 3+) cytoplasmic staining with occasional focal nuclear positivity. Five of 9 (55%) nondiabetic epiretinal membranes were positive for HIF-1α with significantly weaker cytoplasmic staining (1+ to 2+) with occasional focal punctuate nuclear staining.. Hypoxia-inducible factor-1α is found more often and more intensely in diabetic preretinal membranes compared with nondiabetic idiopathic epiretinal membranes.

Topics: Actins; Adult; Aged; Animals; Diabetic Retinopathy; Epiretinal Membrane; Female; Fluorescent Antibody Technique, Indirect; Glial Fibrillary Acidic Protein; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Keratins; Male; Membranes; Microscopy, Fluorescence; Middle Aged; Platelet Endothelial Cell Adhesion Molecule-1; Rabbits; Retinal Neovascularization; Staining and Labeling; Vascular Endothelial Growth Factor A; Vitrectomy

To gain insight into the pathogenesis of neurofibromatosis type 2 (NF2) by investigating the ocular manifestations of this disease.. Using standard histologic techniques, immunohistochemistry, and electron microscopy, we described the ocular pathologic findings of a 34-year-old woman who died from complications of NF2.. We identified 3 types of NF2-associated lesions: juvenile posterior subcapsular cataracts, epiretinal membranes, and an intrascleral schwannoma.. Our analysis indicated that dysplastic lens cells accumulate just anterior to the posterior lens capsule in juvenile posterior subcapsular cataracts and that dysplastic Müller cells may be a major component of NF2-associated epiretinal membranes. Clinical Relevance Our findings suggest that a subset of glial cells with epithelial features (Schwann cells, ependymal cells, and Müller cells) may be particularly sensitive to loss of the NF2 gene. Understanding the molecular basis for this sensitivity may lead to novel strategies for treating NF2.

Topics: Adult; Cataract; Epiretinal Membrane; Eye Neoplasms; Fatal Outcome; Female; Glial Fibrillary Acidic Protein; Humans; Immunoenzyme Techniques; Keratins; Mucin-1; Neurilemmoma; Neurofibromatosis 2; S100 Proteins; Scleral Diseases

To elucidate the pathogenesis of macular hole formation, focusing in particular on the possible role of cellular migration on the cortical vitreous and internal limiting membrane (ILM) around the macular hole.. To gain a comprehensive overview of the ILM excised in macular hole surgery (n = 36), the ILMs were carefully unfolded and spread out onto glass slides as continuous flat sheets that each contained a macular hole. The specimens were observed by light microscopy and transmission electron microscopy (n = 9), and the cellular distribution was analyzed by scanning electron microscopy in a quantitative manner (n = 27). Immunohistochemistry for glial fibrillary acidic protein and cytokeratin 18 was carried out for cellular characterization. Cellular proliferation was assessed by immunohistochemistry for proliferating cell nuclear antigen and Ki-67.. Cellular migration was not apparent around the macular hole in the early stage of development of the macular hole (stage 2, 0 microm). As the macular hole passed through the later stages of development, cellular migration developed around the macular hole (stage 3, 84 microm) and the area of cellular migration gradually enlarged (stage 4, 420 microm). The immunophenotypic analysis showed that these cells were mainly glial fibrillary acidic protein-positive glial cells and cytokeratin 18-positive retinal pigment epithelial cells. The proliferating cell nuclear antigen and Ki-67 immunohistochemistry showed that some of these cells were proliferating on the ILM.. Cellular migration on the ILM is not necessary for the initial formation of a macular break. Cellular migration developed after the macular break occurred, and the migration and proliferation increased gradually from the macular hole.. This study provides a new method for understanding the ultrastructural analysis of the pathogenesis of the macular hole.

Topics: Basement Membrane; Cell Movement; Cell Proliferation; Epiretinal Membrane; Glial Fibrillary Acidic Protein; Humans; Keratins; Ki-67 Antigen; Microscopy, Electron, Scanning; Microscopy, Immunoelectron; Proliferating Cell Nuclear Antigen; Prospective Studies; Retinal Perforations

Endothelin one (ET-1) is a vasomodulator peptide that plays a role on ocular blood flow, glial proliferation, and collagen matrix contraction by retinal pigmented epithelial (RPE) cells. Both glial and RPE cells have been involved in the formation of epiretinal membranes (ERMs). This investigation was conducted to determine whether ET-1 may be associated with ERMs, either idiopathic (IERMs) or from proliferative vitreoretinopathy (PVR).. Plasma and vitreous samples were collected from patients classified by the presence of PVR membranes, retinal detachment (RD), and other ocular conditions, such as IERMs, that made the patients candidates for vitrectomy. Immunoreactive endothelin one (IR-ET-1) was tested in plasma and vitreous by radioimmunoassay. Immunoreactive-ET-1 was localized in IERMs and PVR membranes immunohistochemically. Expression of endothelin receptors A (ETA) and B (ETB) was confirmed by reverse transcription-polymerase chain reaction.. IR-ET-1 levels in plasma and vitreous were higher in patients with PVR and in patients with RD than in those of the control group. Eyes with IERMs also showed higher IR-ET-1 levels than the control group cases. IR-ET-1 levels in eyes with PVR were higher than those in eyes with IERMs. IR-ET-1 levels in eyes with RD were also higher than those of eyes with IERMs. Immunoreactive ET-1 was localized in the cellular and stromal components of both IERMs and PVR membranes. Furthermore, ETA and ETB receptors were expressed in both IERMs and PVR membranes.. IR-ET-1 in human vitreous is elevated in PVR, RD, and IERMs. ET-1 and its receptors ETA and ETB are present in epiretinal tissue of both idiopathic and PVR membranes. These data suggest an involvement of ET-1 in retinal disease.

Topics: Adult; Aged; Aged, 80 and over; Endothelin-1; Epiretinal Membrane; Female; Glial Fibrillary Acidic Protein; Humans; Keratins; Male; Middle Aged; Radioimmunoassay; Receptor, Endothelin A; Receptor, Endothelin B; Retinal Detachment; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Vitreoretinopathy, Proliferative; Vitreous Body

Atrial natriuretic peptide (ANP) has been recently described as an endogenous inhibitor of the synthesis and angiogenic action of vascular endothelial growth factor (VEGF). Given VEGF's key role in promoting neovascularization in proliferative diabetic retinopathy (PDR), this study was designed to evaluate the possibility that ANP could be involved in the neovascular and fibrotic complications of PDR.. We determined ANP by radioimmunoassay in plasma and vitreous humor samples collected from diabetic patients with and without PDR and from non-diabetic subjects. ANP was also immunohistochemically localized in the epiretinal membranes of patients with PDR.. Vitreous ANP concentrations were significantly higher in patients with active PDR compared to patients with quiescent PDR, diabetes without PDR or controls <0.05. Significant differences were also observed between vitreous ANP levels in diabetic patients without PDR and control subjects. There was no significant correlation between serum and vitreous ANP levels in any of the patient groups. ANP was detected in the fibrovascular epiretinal tissue of patients with PDR.. Diabetic patients with active neovascularization have significantly higher levels of ANP in the vitreous humor than those without active PDR. Diabetic patients without PDR were also found to have significantly higher vitreous ANP levels than non-diabetic patients. Since plasma and vitreous ANP concentrations were found to be unrelated, we suggest intraocular ANP synthesis and/or an increase in the release of ANP into the vitreous, as opposed to diffusion from the blood, as the main factors contributing to the high vitreous ANP levels observed in diabetic patients. In the fibrovascular epiretinal tissue of these patients, ANP was found to be localized in vascular, glial, fibroblast-like and retinal pigment epithelium cells. Our findings suggest a role for ANP in PDR.

Topics: Adult; Aged; Atrial Natriuretic Factor; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Epiretinal Membrane; Female; Glial Fibrillary Acidic Protein; Humans; Immunoenzyme Techniques; Keratins; Male; Middle Aged; Radioimmunoassay; Retinal Neovascularization; Vitrectomy; Vitreous Body

This study investigates the similarities and differences between epiretinal membranes in four clinically distinct types of vitreomaculopathy. We propose a hypothesis on the origin of the predominant cell type and its potential role in causing these conditions.. Epiretinal membranes (ERMs) surgically removed from a prospective, consecutive series of vitrectomies for macular pucker associated with an untreated peripheral horseshoe tear (MP), cellophane maculopathy (CM), stage 4 macular hole (MH) and vitreomacular traction syndrome (VMT) were examined by light microscopy and by immunocytochemistry (ICC) using antibodies marking type IV collagen, type II collagen, glial fibrillary acidic protein (GFAP), and low- and high-molecular-weight cytokeratin (MNF116). These specimens were compared with post-mortem control eyes with and without physiological posterior vitreous detachment (PVD). Light microscopy was carried out on 5-microm-thick sections cut from formalin-fixed, paraffin-embedded tissue blocks. Appropriate autoclave or enzyme pre-digestion steps were deployed to retrieve antigens for ICC. No patient had undergone previous vitreoretinal surgery or peripheral retinopexy.. From a series of 38 patients, (13 CM, 8 MP, 16 MH and 1 VMT) a total of 20 specimens contained sufficient tissue for histology and immunocytochemistry. All specimens contained portions of inner limiting membrane (ILM) coated by GFAP-positive cells. Specimens from patients with MP and CM exhibited hyperconvolution of the ILM, which was not found in the specimens from patients with MH or VMT or in the control eyes. Hyperconvolution was associated with increased glial cell density, GFAP staining intensity and duplication of ILM basement membrane. Three cases of ERMs from the MP group contained, in addition, cytokeratin-positive cells. In the control group; post-mortem eyes with PVDs showed patchy staining of the posterior hyaloid membrane for GFAP and type 4 collagen. Post-mortem eyes with attached gel showed weak positivity of the ILM for type 4 collagen, and a monolayer of GFAP-positive cells lined the vitreous aspect of the ILM.. These results indicate that glial cells are fundamentally important in the formation of ERMs found in this group of vitreomaculopathies. The hyperconvolution and duplication of the ILM in CM and MP were striking and distinctive features and suggest a mechanism by which these membranes exert tractional forces on the retina. Post-mortem control eyes contained a similar (but more dispersed) population of GFAP-positive cells in the region of the ILM, suggesting the primary aetiology for CM and MP may originate within the ILM. ERMs from MP cases may, in addition, contain cytokeratin-positive cells, of probable RPE origin.

Topics: Basement Membrane; Biomarkers; Collagen Type II; Collagen Type IV; Epiretinal Membrane; Eye Diseases; Female; Glial Fibrillary Acidic Protein; Humans; Immunohistochemistry; Keratins; Male; Prospective Studies; Retinal Diseases; Vitrectomy; Vitreous Body

Hepatocyte growth factor/scatter factor (HGF/SF) possesses mitogenic, motogenic, and morphogenic properties and has recently been implicated in various retinal diseases. The role of HGF/SF in proliferative vitreoretinal disease was investigated.. Sections of epiretinal membranes were stained immunohistochemically for cytokeratins, to identify HRPE cells, and for HGF/SF receptor (c-Met). Cultured HRPE cells were stained for c-Met and investigated for shape change in response to HGF/SF, by using image analysis. The dose-response relationship for HRPE cells to HGF/SF was investigated by a cell migration assay and the specificity of this response evaluated by a neutralization experiment. Subretinal fluid (SRF) and vitreous from patients with retinal detachment and proliferative vitreoretinopathy (PVR) plus vitreous from eyes obtained after death, eyes with macular hole, and eyes with proliferative diabetic retinopathy (PDR) were investigated for the presence of HGF/SF using an enzyme-linked immunosorbent assay (ELISA). HGF/SF activity was measured using an MDCK cell scatter assay.. HRPE cells in epiretinal membranes and in culture expressed c-Met. Cultured HRPE cells responded to HGF/SF by an epithelial-to-mesenchymal shape change and by cell migration, a response that increased with increasing concentrations of HGF/SF. This response was reduced in the presence of neutralizing antibody. There was evidence of HGF/SF in increasing concentrations in more severe PVR and in PDR when measured by ELISA, and, conversely, there was evidence of correspondingly decreasing HGF/SF activity when measured by MDCK cell scatter assay in these diseases.. HGF/SF is present in normal and pathologic vitreous. HRPE cells respond by shape change and cell migration to HGF/SF. Concentrations of HGF/SF increase in proliferative vitreoretinal disease and increase in turn with increased severity of the disease, but HGF/SF bioactivity decreases (consistent with activator depletion). These findings are consistent with the hypothesis that HGF/SF may play a role in the HRPE mesenchymal transformation that typifies PVR.

Topics: Adult; Aged; Aged, 80 and over; Animals; Cell Line; Cell Movement; Cell Size; Dogs; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Epiretinal Membrane; Hepatocyte Growth Factor; Humans; Immunoenzyme Techniques; Keratins; Kidney; Middle Aged; Pigment Epithelium of Eye; Proto-Oncogene Proteins c-met; Retinal Detachment; Vitreoretinopathy, Proliferative; Vitreous Body