bromochloroacetic-acid and Vitreoretinopathy--Proliferative

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

Our aim was to establish a rat model of proliferative vitreoretinopathy (PVR) induced by macrophages and investigate whether macrophages can be a cell origin of fibroblast-like cells present in PVR.. One eye of each rat received an intravitreal injection of macrophages. Clinical examination was performed to evaluate the development of PVR. Histological study was carried out to observe the pathological progression. Immunohistochemical staining with vimentin (VIM), glial fibrillary acidic protein (GFAP), α-smooth-muscle actin (α-SM actin), cytokeratin (CK) and CD68 characterized the cell types within the PVR membranes. The distribution, morphological change of prelabeled macrophages, as well as their colocalization with CD68, VIM, GFAP, α-SM actin and CK, were observed on days 3, 14 and 28 after injection.. In response to intravitreal injection of macrophages, 90% of the experimental rats developed PVR from postoperative day 7. The histological progression of PVR was characterized by the sequential appearance of inflammatory cell invasion, fibroblast proliferation and scar formation. The dominating cells comprising the proliferative membranes at the advanced stage were fibroblasts. Injected macrophages retained round shape and positive staining with CD68 on day 3. On day 28, they acquired elongated/spindle shape combined with intense staining of VIM but absence of CD68, GFAP, α-SM actin and CK, and became the primary constituent of fibrocellular membranes.. Macrophages effectively and reproducibly induce the development of proliferative fibrocellular membranes in rats. In this PVR model, macrophages acquire fibroblast-like cell phenotype and contribute to fibrocellular membranes directly, suggesting that macrophages may be a cell origin of fibroblast-like cells involved in PVR.

Topics: Actins; Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Cell Transdifferentiation; Disease Models, Animal; Fibroblasts; Glial Fibrillary Acidic Protein; Intravitreal Injections; Keratins; Macrophages, Peritoneal; Phenotype; Rats; Rats, Sprague-Dawley; Vimentin; 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

To compare the relative levels of connective tissue growth factor (CTGF), platelet-derived growth factor alpha (PDGF-AA), and hepatocyte growth factor (HGF) in glial and retinal pigment epithelial (RPE) cells of epiretinal membranes from proliferative vitreoretinopathy (PVR).. A total of 37 PVR membranes, of various stages, underwent fluorescent immunohistochemisty and confocal laser scanning microscopy to localize CTGF, HGF, and PDGF-AA in RPE and glial cells.. Numerous RPE, and relatively fewer glial cells, were found in all stages of PVR. CTGF immunoreactivity increased from early to late stage PVR and was principally expressed by RPE cells in early stage, and by glial cells in late stage PVR. HGF, expressed by both RPE and glial cells, was principally expressed in mid-stage PVR. PDGF-AA, expressed by both cell types, demonstrated a uniform level of staining throughout all stages of PVR.. RPE and glial cells contribute to the expression of CTGF, HGF, and PDGF-AA during PVR, but with specific developmental patterns. PDGF-AA is expressed uniformly throughout all stages of PVR, while HGF expression peaks during mid stage, and CTGF expression is highest during late stage PVR. These results allow for the development of stage-specific therapeutics for PVR that may allow targeting of the early proliferative and/or the late tractional stages of PVR.

Topics: Biomarkers; Connective Tissue Growth Factor; Fluorescent Antibody Technique; Glial Fibrillary Acidic Protein; Hepatocyte Growth Factor; Humans; Immediate-Early Proteins; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Keratins; Membranes; Microscopy, Confocal; Neuroglia; Pigment Epithelium of Eye; Platelet-Derived Growth Factor; Retina; Vitreoretinopathy, Proliferative

To report the major intraretinal pathological changes in retinas with proliferative vitreoretinopathy (PVR) and retinal shortening, 13 human retinal samples from postoperative PVR after primary surgery for retinal detachment were immunostained for vimentin, glial fibrillary acidic protein (GFAP), cytokeratins, and CD68. One more sample was studied with electron microscopy. Retinal disorganization, neuronal loss, and gliosis were observed in 12 out of 13 samples, but all 13 were positive for GFAP. Muller cell processes showed different degrees of intermediate filament hyperplasia. CD68-positive cells were present in 11 of 13 retinal samples.. A gliotic response plays a major role in retinal shortening in PVR. In addition, the presence of macrophage-like cells in retinal tissues suggests a possible role of these cells in the pathogenesis of this variety of PVR.

Topics: Adult; Aged; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Biomarkers; Female; Glial Fibrillary Acidic Protein; Gliosis; Humans; Immunohistochemistry; Keratins; Male; Microscopy, Electron; Middle Aged; Prognosis; Retina; Vitreoretinopathy, Proliferative

The purpose of this work was to investigate the effect of c-fos antisense oligonucleotide (c-fos-AS-ON) on proliferative vitreoretinopathy (PVR). Cultures of human retinal pigment epithelial (hRPE) cells were established from adult human corneal donors. These cells were positively stained for cytokeratins. C-fos-AS-ON effect on serum-stimulated cell proliferation was estimated by evaluating the incorporation of 5-bromo-2'-deoxy-uridine (BrdU) into cellular DNA. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were respectively performed to quantify the serum-stimulated c-fos gene mRNA and protein expression in hRPE cells. Eight rabbits (16 eyes) were divided into c-fos-AS-ON treatment group and control group. 2.5 x 10(5) cultured hRPE cells were injected into the vitreous cavity of eyes to establish a PVR model. Prevalence of PVR and retinal detachment were determined by indirect ophthalmoscopy on days 1, 3, 7, 14, 21 and 28 post-injection and by pathological study on days 28 post-injection. The results showed that blocking the expression of c-fos by the addition of c-fos-AS-ON to the culture medium significantly inhibited the hRPE cells proliferation. This effect of c-fos-AS-ON was found to be sequence specific (the use of a sense or a mismatch sense oligonucleotide had no such an effect) and dose-dependent (0.375 microM was the lowest effective dose tested). Growth inhibition by c-fos-AS-ON remained for at least 72 h. By using RT-PCR and Western blotting, we found that the c-fos-AS-ON could specifically inhibit c-fos mRNA and protein synthesis in cultured hRPE cells. Though the eyes injected with c-fos-AS-ON also developed features of PVR, the severities of days 14, 21 and 28 post-injection were significantly lower than those in the control eyes (P<0.05). We conclude that c-fos-AS-ON can inhibit cultured hRPE cell proliferation, which mechanism may relate to blocking the expression of c-fos and can reduce the prevalence of experimental PVR. These findings establish a rationale for investigating the potential use of a c-fos-AS-ON as a novel therapeutical tool in the treatment of PVR.

Topics: Animals; Blotting, Western; Cell Proliferation; Cells, Cultured; Disease Progression; Gene Targeting; Genes, fos; Genetic Therapy; Humans; Keratins; Male; Oligonucleotides, Antisense; Pigment Epithelium of Eye; Proto-Oncogene Proteins c-fos; Rabbits; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transfection; Vitreoretinopathy, Proliferative

It has been reported that retinoic acid (RA) may inhibit the growth of RPE and be used in the treatment of proliferative vitreoretinopathy (PVR). However, previous reports in this field have been conflicting. The main reason for these contradictory findings is that different methods for evaluating the effects of RA on RPE from different species have been used. In human specimens, only RPE from the donor eye (stationary) but not RPE from the PVR membrane (already at active proliferation status) have been tested. This study tested the effects of RA on the growth of RPE using a novel in vitro model: RPE from the PVR membranes, which simulates the in vivo situation of PVR patients better than RPE from the donor eyes. This study also used various methods to solve the conflicting results reported previously. We found that both all transretinoic acid (all-RA) and 13-cis-retinoic acid (cis-RA) can promptly (though not completely) inhibit proliferation of RPE (inhibition rate of 89%-90%) over a very wide range of concentrations (10(-9)-10(-5) M) and various lengths of periods (2-12 days) in a dose-dependent and time-dependent manner and without evident cytotoxic effects. Previously reported disadvantages discovered from the study of RPE from donor eyes, e.g., the absence of inhibitory effects on the early passages of cultured cells and inhibition occurring only after long-term treatment, do not present in RPE cells from the PVR membrane. The proliferation of RPE recovered from the inhibition by RA rapidly after the discontinuation of treatment, indicating that a continuous supply of the drug over a long period, i.e., until the breakdown of the blood-retinal barrier has been repaired, is essential for the success of drug treatment of PVR.

Topics: Basement Membrane; Bromodeoxyuridine; Cell Count; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Glial Fibrillary Acidic Protein; Humans; Immunohistochemistry; Keratins; Pigment Epithelium of Eye; S100 Proteins; Time Factors; Tretinoin; Vitreoretinopathy, Proliferative

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

To understand molecular events that lead to retinal pigment epithelial (RPE) cell proliferation and migration during the early phases of proliferative vitreoretinopathy (PVR) in a rabbit model.. Retinal holes were created and interleukin-1beta(IL-1beta) was injected intravitreally. Eyes were examined by indirect ophthalmoscopy and eyecup pieces containing retinal holes were analyzed at different times after the surgery up to 4 weeks. RPE proliferation and migration were examined by immunohistochemistry. Tyrosine phosphorylation of extracellular signal regulated kinase (ERK) and hepatocyte growth factor receptor (HGFR or c-met) was determined by immunoprecipitation and western blot analysis. Tyrosine phosphorylation of c-met and morphological studies was performed on vitreous treated ARPE-19 cells. Expression of c-jun was determined by Northern blot analysis. Matrix metalloproteinase (MMP) content in vitreous was assessed by zymography.. Indirect ophthalmoscopy identified formation of epiretinal membrane and immunohistochemistry identified proliferative and migratory RPE and other cells in the posterior segment containing retinal holes at 4 weeks post-surgery. Tyrosine phosphorylation of ERK and c-met occurred in this segment within 30 min of surgery. ARPE-19 cells treated with vitreous from the 24 h post-surgical eyes, but not with control vitreous or IL-1beta, showed morphological changes and tyrosine phosphorylation of c-met. Northern blot analysis in this segment identified upregulation of c-jun within 30 min of surgery and the expression peaked at 72 h. Zymographic analysis of vitreous identified MMP-9 in 12-72 h post-surgery.. These data suggest that the presence of retinal holes and IL-1beta may lead to activation of HGF, mitogen activated protein kinases (MAPK), c-jun and extracellular matrix remodeling, resulting in proliferative and migratory cells in the wounded retina.

Topics: Animals; Blotting, Northern; Blotting, Western; Cell Division; Cell Movement; Disease Models, Animal; Hepatocyte Growth Factor; Interleukin-1; Keratins; Matrix Metalloproteinases; Mitogen-Activated Protein Kinases; Phosphorylation; Pigment Epithelium of Eye; Precipitin Tests; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-jun; Proto-Oncogene Proteins c-met; Rabbits; Retinal Perforations; Tyrosine; Vitreoretinopathy, Proliferative; 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

To clarify factor(s) involved in morphological dedifferentiation of retinal pigment epithelial (RPE) cells in vitro from mitotically quiescent hexagonal cells to flattened cells that lack epithelial characteristics and concurrent myoid differentiation.. RPE cells which retained their differentiated hexagonal morphology were isolated from bovine eyes by mechanical pipetting. Dedifferentiation and myoid differentiation of RPE cells were examined by microscopic observation and immunohistochemical analysis using antibodies against cytokeratin, an epithelial marker, and alpha smooth muscle actin, a marker of myoid differentiation. The contractile ability of RPE cells was evaluated by collagen gel contraction assay.. Platelet derived growth factor (PDGF) enhanced morphological changes in the RPE from hexagonal-shaped cells to flattened cells. Coincident with this morphological alteration, the expression of cytokeratin in RPE cells decreased and expression of alpha smooth muscle actin began and was increased in a time dependent manner. These alterations were completely blocked by collagen synthesis inhibitors. Interleukin 1beta, transforming growth factor beta1, insulin-like growth factor I, and basic fibroblast growth factor had little or no effect on the dedifferentiation. PDGF also potentiated the RPE induced collagen gel contraction.. These results demonstrate that PDGF enhanced the dedifferentiation of RPE cells, the initial step of proliferative vitreoretinopathy (PVR), as well as myoid differentiation and collagen gel contraction. PDGF may have a versatile role in the pathogenesis of PVR involving collagen synthesis.

Topics: Actins; Animals; Blotting, Western; Cattle; Cell Differentiation; Collagen; Epithelial Cells; Fluorescent Antibody Technique; Keratins; Muscle, Smooth; Pigment Epithelium of Eye; Platelet-Derived Growth Factor; Vitreoretinopathy, Proliferative

To determine whether syngeneic retinal cells injected in the vitreous cavity of the rat are able to initiate a proliferative process and whether the ocular inflammation induced in rats by lipopolysaccharide (LPS) promotes this proliferative vitreoretinopathy (PVR).. Primary cultured differentiated retinal Müller glial (RMG) and retinal pigmented epithelial (RPE) cells isolated from 8 to 12 postnatal Lewis rats were injected into the vitreous cavity of 8- to 10-week-old Lewis rats (10(5) cells/eye in 2 microlieter sterile saline), with or without the systemic injection of 150 microgram LPS to cause endotoxin-induced uveitis (EIU). Control groups received an intravitreal injection of 2 microliter saline. At 5, 15, and 28 days after cell injections, PVR was clinically quantified, and immunohistochemistry for OX42, ED1, vimentin (VIM), glial fibrillary acidic protein (GFAP), and cytokeratin was performed.. The injection of RMG cells, alone or in combination with RPE cells, induced the preretinal proliferation of a GFAP-positive tissue, that was enhanced by the systemic injection of LPS. Indeed, when EIU was induced at the time of RMG cell injection into the vitreous cavity, the proliferation led to retinal folds and localized tractional detachments. In contrast, PVR enhanced the infiltration of inflammatory cells in the anterior segment of the eye.. In the rat, syngeneic retinal cells of glial origin induce PVR that is enhanced by the coinduction of EIU. In return, vitreoretinal glial proliferation enhanced the intensity and duration of EIU.

Topics: Animals; Cell Transplantation; Cells, Cultured; Fluorescent Antibody Technique, Indirect; Glial Fibrillary Acidic Protein; Injections; Keratins; Lipopolysaccharides; Neuroglia; Pigment Epithelium of Eye; Rats; Rats, Inbred Lew; Receptors, Complement 3b; Retina; Retinal Detachment; Salmonella typhimurium; Transplantation, Isogeneic; Uveitis; Vimentin; Vitreoretinopathy, Proliferative; Vitreous Body

To identify the cellular components of vitreous samples obtained during vitrectomy for proliferative vitreoretinopathy (PVR).. With the use of three intermediate filament (IF) proteins, vimentin, glial fibrillary acidic protein (GFAP), and cytokeratin (CK), cytocentrifuge slides of 14 fresh vitreous aspirates were detected with immunohistochemical technique.. All the specimens contained epithelial-like proliferative cells with or without pigment and some membrane-like pieces. Immunocytochemical staining showed that 76.0-90.0% cells stained for CK, 17.4-29.6% cells expressed GFAP, and 80.1-91.0% cells were positive for vimentin.. Majority of cells in the vitreous samples originated from retinal pigment epithelial cells (RPE) and glial cells in PVR. Expression of IF proteins may be determined by tissue of origin and local microenvironment.

Topics: Adolescent; Adult; Female; Glial Fibrillary Acidic Protein; Humans; Immunohistochemistry; Intermediate Filaments; Keratins; Male; Middle Aged; Neuroglia; Pigment Epithelium of Eye; Vimentin; Vitreoretinopathy, Proliferative; Vitreous Body

Adjuvant intravitreal daunomycin is frequently used for the management of proliferative vitreoretinopathy (PVR). In this study the authors examined whether daunomycin could induce multidrug resistance (MDR), mediated by the mdr-1 gene product P-glycoprotein, in the cells responsible for reproliferation in vivo and in human retinal pigment epithelial (RPE) cells in vitro.. Expression of P-glycoprotein was examined by immunohistochemistry in surgically removed epiretinal membranes. The cellular source of P-glycoprotein was examined by colabeling for cytokeratin, glial fibrillary acidic protein, and the macrophage marker EBM-11. P-glycoprotein expression by cultured RPE cells was assessed by reverse transcription-polymerase chain reaction and immunoblot analysis. Daunomycin toxicity was quantified by crystal violet assay.. P-glycoprotein expression was detected in 10 of 10 patients pre-exposed to intravitreal daunomycin. In contrast, epiretinal membranes from only 2 of 13 patients never exposed to daunomycin showed faint P-glycoprotein expression. P-glycoprotein expression was strong within 8 months after daunomycin treatment and faded thereafter. Colocalization studies demonstrated predominant expression of P-glycoprotein by RPE cells. Pre-exposure of cultured human RPE cells to subtoxic concentrations of daunomycin induced resistance to daunomycin that was sensitive to the MDR inhibitor, verapamil. Induction of the MDR phenotype in RPE cells by daunomycin was associated with a minor increase in the mdr-1 mRNA level but a prominent increase in P-glycoprotein expression, thus suggesting a primarily translational mechanism of MDR development in human RPE cells.. Intravitreal daunomycin induced P-glycoprotein expression in PVR. Reproliferation in daunomycin-pretreated patients probably necessitates cotreatment with daunomycin and inhibitors of multidrug resistance such as verapamil or administration of antiproliferative drugs such as 5-fluorouracil, which act in a MDR-independent fashion.

Topics: Adolescent; Adult; Aged; Antibiotics, Antineoplastic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Culture Techniques; Chemotherapy, Adjuvant; Child, Preschool; Daunorubicin; Drug Resistance, Multiple; Female; Glial Fibrillary Acidic Protein; Humans; Immunoenzyme Techniques; Keratins; Male; Middle Aged; Pigment Epithelium of Eye; Polymerase Chain Reaction; RNA, Messenger; Transcription Factors; Vitreoretinopathy, Proliferative; Vitreous Body

The purpose of this work was to investigate if MYC-dependent intracellular mitogenic pathway is active in cultures of human retinal pigment epithelial (hRPE) cells and whether myc antisense phosphorotioate oligonucleotides (c-myc-AS-ODN) are useful tools for inhibiting the proliferation of hRPE cells. Cultures of hRPE cells were established from adult human corneal donors. These cells were positively stained for cytokeratins and vimentin. Myc mRNA expression was determined by Northern blot analysis and it was determined by means of immunofluorescence if MYC was expressed. C-myc-AS-ODN effect on cell proliferation was estimated by evaluating the incorporation of 5-bromo-2'-deoxy-uridine into cellular DNA. Cell number was estimated by using a tetrazolium bromide based colorimetric method. Human RPE cells in culture expressed MYC and myc mRNA as well as prothymosin alpha mRNA--a gene whose transcription is under MYC control--indicating that MYC-dependent intracellular mitogenic pathway is active in these cells. In accordance with this, we found that blocking the expression of myc by the addition of c-myc-AS-ODN to the culture medium inhibited hRPE cell proliferation. The effect of the c-myc-AS-ODN was found to be sequence specific (the use of a control oligonucleotide with the same sequence but in an opposite direction had no effect) and dose-dependent (4 microM was the lowest effective dose tested). By using RT-PCR we found that the c-myc-AS-ODN inhibition of cell proliferation was related to a diminution in c-myc mRNA expression, and by immunofluorescence we detected a diminution in c-MYC protein staining in RPE cells after 48 hr of treatment with c-myc-AS-ODN. Furthermore, growth inhibition remained for at least 5 days after addition of a single dose of the c-myc-AS-ODN to the culture. We conclude that hRPE cell proliferation is under MYC control. Blocking the expression of myc by c-myc-AS-ODN inhibited hRPE cell proliferation. These findings establish a rationale for investigating the potential use of a c-myc-AS-ODN as a novel therapeutical tool in the treatment of Proliferative Vitreoretinopathy.

Topics: Adult; Blotting, Northern; Cell Division; Cells, Cultured; Fluorescent Antibody Technique; Gene Expression; Humans; Keratins; Oligonucleotides, Antisense; Pigment Epithelium of Eye; Polymerase Chain Reaction; Proto-Oncogene Proteins c-myc; RNA, Messenger; Vimentin; Vitreoretinopathy, Proliferative

An experimental model of proliferative vitreoretinopathy was developed in the rabbit eye by injecting a solution of human platelet-rich plasma. In this model we evaluated the progression with time of intraocular inflammation and the rate and origin of cell proliferation. A sterile solution adjusted to 107 platelets was injected into the right eye of a total of 46 pigmented and 14 albino rabbits. Animals were sequentially sacrificed at days 7, 14, 21 and 1 month after injection. Clinical evaluation of vitreoretinal proliferation, using a classification in six grades, and of anterior segment inflammation assessed by a Laser Flare Meter, were done for 1 month after injection, before histopathological analysis. Eighty percent of eyes developed tractional retinal detachment in 1 month. Histopathology showed intense cell migration and proliferation in the area of the ciliary body, as early as the seventh day, then further increasing rapidly. Infiltrates were composed of cytokeratin- and vimentin-expressing cells. Abnormal expression of vimentin was also found in ciliary and retinal epithelia and in M¿ller cells. Inflammation measured by the Laser Flare Meter was maximal at day 11 and then reached a plateau at significantly higher levels than controls. Albino rabbits showed significantly lower grades of proliferation, as compared to pigmented rabbits. This study thus clarified some characteristics of experimental vitreoretinal proliferations that that proved similar to those in human diseases, such as the involvement of ciliary body and retinal pigment epithelium, the existence of inflammatory reactions preceding cell proliferation and strong changes in intermediate filaments. This may provide a simple and valuable model for antiproliferative assays and shed some light on the pathogenesis of intraocular proliferative disorders.

Topics: Albinism; Animals; Cell Division; Ciliary Body; Immunohistochemistry; Inflammation; Keratins; Lasers; Male; Rabbits; Retina; Vimentin; Vitreoretinopathy, Proliferative; Vitreous Body

To examine the gene expression for melanogenesis of retinal pigment epithelial cells during dedifferentiation and to compare the condition to that of eyes obtaining anatomical success after surgery for proliferative vitreoretinopathy.. Gene expression for melanogenesis was determined by reverse transcriptase-polymerase chain reaction of tyrosinase and tyrosinase-related protein-1 genes in normal and cultured retinal pigment epithelial cells and in proliferative membranes in patients with proliferative vitreoretinopathy.. Gene expression for melanogenesis was classified into three types during dedifferentiation of retinal pigment epithelial cells: (1) tyrosinase-related protein-1 gene expression, (2) tyrosinase and tyrosinase-related protein-1 gene expression and (3) no expression of these genes. The expression of these genes were maintained better in mediums with basic fibroblast growth factor than in medium without basic fibroblast growth factor. Of the anatomically unsuccessful patients with proliferative vitreoretinopathy treated by surgery, 76.9% showed both tyrosinase and tyrosinase-related protein-1 gene expression; only 20% of the anatomically successful patients showed the gene expression.. We reported three different conditions of retinal pigment epithelial cells based on gene expression for melanogenesis during dedifferentiation. The different condition of the retinal pigment epithelial cells may have some relationship to the anatomical results for proliferative vitreoretinopathy surgery.

Topics: Adolescent; Adult; Aged; Carrier Proteins; Cell Differentiation; Cells, Cultured; Child; DNA Primers; Female; Gene Expression; Humans; Immunoenzyme Techniques; Keratins; Male; Melanins; Membrane Glycoproteins; Membranes; Middle Aged; Monophenol Monooxygenase; Oxidoreductases; Pigment Epithelium of Eye; Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Vitreoretinopathy, Proliferative

During routine cell culture and under pathologic conditions, human retinal pigment epithelial (RPE) cells lose epithelial characteristics and change their morphology. In this study, changes in gene expression in RPE cells of different generations were evaluated by polymerase-chain-reaction-based differential display mRNA analysis (DD-RT-PCR).. Total RNA was prepared from freshly isolated and cultured human RPE cells of passages P0 and P3 and was subjected to DD-RT-PCR. One band with enhanced expression was excised, reamplified, and partially sequenced, using a modified dideoxy chain termination approach. Expression of the corresponding protein was ascertained by immunocytochemical analysis.. Differential display RT-PCR showed enhanced expression of a specific RNA in P3 cells compared with that in P0 cells. Sequence alignment revealed 98% identity with the 3' end of the coding sequence of human microtubule-associated protein 1B (MAP1B). Confirmation of induced expression of MAP1B mRNA was obtained by PCR with specific primers and by immunocytochemical analysis in cultured RPE cells and in surgically removed epiretinal membranes from patients with proliferative vitreoretinopathy. No expression of MAP1B mRNA or protein was detected in freshly isolated RPE cells.. Differential display RT-PCR in RPE cells with subsequent sequence analysis allows characterization of the maturation- and differentiation-dependent expression of previously undetected genes and gene products in cultured RPE cells.

Topics: Animals; Cell Differentiation; Cells, Cultured; DNA Primers; Fluorescent Antibody Technique, Indirect; Gene Expression; Humans; Keratins; Microtubule-Associated Proteins; Middle Aged; Pigment Epithelium of Eye; Polymerase Chain Reaction; RNA; RNA, Messenger; Swine; Up-Regulation; Vitreoretinopathy, Proliferative

To evaluate the characteristics of cellular proliferation in different retinal diseases.. Three specific antibodies [to cytokeratin (CK), glial fibrillary acidic protein (GFAP) and actin, respectively] were used. Vitrectomy specimens from 28 cases with different proliferative retinal diseases were studied immunohistochemically.. In the vitreous with proliferative vetreoretinopathy (PVR), most of the proliferative cells were derived from glial and retinal pigment epithelial (RPE) cells. In the vitreous of proliferative diabetic retinopathy (PDR), RPE cells might enhance the contraction of proliferative membranes. Increase in actin in firovascular membranes with PDR may play a positive role on the pericytes dropping out from microvessels.. The cell proliferation in the vitreous associated with the increase of levels of growth factors in pathological vitreous, possibly, is one of the mechanisms for the deterioration of proliferative retinal diseases.

Topics: Actins; Adult; Antibodies; Cell Division; Diabetic Retinopathy; Glial Fibrillary Acidic Protein; Humans; Immunohistochemistry; Keratins; Middle Aged; Pigment Epithelium of Eye; Retinal Vein Occlusion; Vitrectomy; Vitreoretinopathy, Proliferative; Vitreous Body

In the late stages of proliferative diabetic retinopathy (PDR) the vascularized posterior cortical gel (PCG) contracts leading to a partial posterior hyaloidal separation, hemorrhage, and traction retinal detachment (TRD). "Additional epiretinal membranes" have been described previously. These are thin, usually transparent epiretinal membranes which extend anteriorly from the point of attachment of the elevated posterior cortical gel to the edge of the TRD. The origin and frequency of the occurrence as well as the clinical significance of such additional epiretinal membranes are the subjects of controversy.. To quantitate the authors' clinical impression that additional epiretinal membranes are common in advanced PDR, to characterize them immunohistochemically, and to demonstrate the rationale for the authors' surgical approach.. Intraoperative observations for all patients undergoing diabetic vitrectomy and delamination over the last 2 years were reviewed retrospectively. The presence of additional epiretinal membrane was searched for in the initial stages of vitrectomy. When identified, their apparent continuity with the elevated portion of the PCG was confirmed. Surgical specimens were obtained from nine patients for immunohistochemical study.. Additional epiretinal membranes were observes in 145 (81%) of 179 consecutive eyes with PDR that underwent surgery for macular TRD. Immunohistochemical staining with type II collagen antibody was positive in all specimens, suggesting that these membranes were of vitreous origin.. It is likely that the additional epiretinal membranes represent the posterior leaf of a split PCG, the anterior leaf being the elevated portion of the PCG. The two leaves remain fused in the main, fibrovascular portion of the epiretinal membrane. These findings help explain the clinical experience that once the posterior leaf of the PCG is identified and elevated, it provides an accurate point of entry into the surgical plane facilitating delamination of the fused (vascularized) portion of the PCG from the detached retina.

Topics: Aged; Collagen; Diabetic Retinopathy; Eye Diseases; Female; Glial Fibrillary Acidic Protein; Humans; Immunoenzyme Techniques; Keratins; Male; Middle Aged; Retinal Detachment; Retrospective Studies; Vitrectomy; Vitreoretinopathy, Proliferative; Vitreous Body

To describe and evaluate retinal pigment epithelial (RPE) cell transdifferentiation in vitro and to determine its importance to the development of proliferative vitreoretinal disorders.. Porcine RPE cells from single animals were examined at different passages in culture. The authors examined cellular morphology, contraction of a collagenous matrix, and adhesion to fibronectin and type I collagen-coated substrata. These activities were correlated with loss of epithelial characteristics, redistribution of the actin cytoskeleton, and expression of alpha-smooth muscle actin (alpha-SMA), a marker of myoid differentiation.. During routine culture on tissue culture plastic, porcine RPE cells lose epithelial characteristics and acquire a mesenchymal cell-like phenotype. The ability of cultured porcine RPE cells to adhere to and exert tractional forces on an extracellular matrix increases with continued passage in vitro and transdifferentiation. This correlates with the loss of the differentiated epithelial morphology, decreased expression of the epithelial marker cytokeratin 18, redistribution of the actin cytoskeleton, and de novo expression of alpha-SMA.. Results indicate that RPE transdifferentiate in culture and that this transition is accompanied by a shift in biologic activities. Therefore, morphologic and behavioral transdifferentiation of these cells in culture are influencing factors in experimental pathology. The potential relevance of these extensive changes to the biology of proliferative vitreoretinal disorders is discussed.

Topics: Actins; Animals; Cell Adhesion; Cell Differentiation; Cells, Cultured; Collagen; Epithelial Cells; Epithelium; Fibronectins; Fluorescent Antibody Technique; Keratins; Mesoderm; Phenotype; Pigment Epithelium of Eye; Swine; Vitreoretinopathy, Proliferative