Search

Purpose: To provide new information on epiretinal cell proliferation and the cells' origin in idiopathic macular holes and to overcome the effects of embedding and sectioning preparation procedures on cell-distribution patterns., Methods: Interference and phase-contrast microscopy, immunocytochemistry, and scanning and transmission electron microscopy were performed on surgically excised whole-mounted internal limiting membrane (ILM) specimens removed from 60 eyes with idiopathic macular holes. Cell distribution and cell morphology were correlated with immunocytochemical staining characteristics. Twelve cell type-specific antibodies were used to detect glial cells, hyalocytes, retinal pigment epithelial cells, retinal ganglion cells, and immune cells. Cell viability was analyzed., Results: Epiretinal cell proliferation was found in all ILM specimens, irrespective of the stage of the macular hole. Cell density showed a broad variety. Immunocytochemistry frequently revealed simultaneous expression of GFAP/CD45, GFAP/CD64, GFAP/CD68, GFAP/CRALBP, and GFAP/CD90. Some cells presented with intracellular contractile filaments (anti-αSMA); others were not immunoreactive to any antibody examined. The percentage of viable cells showed a broad variety with a mean of 73% (SD 29%). Electron microscopy demonstrated glial cells, hyalocytes, and myofibroblast-like cells., Conclusions: The presence of epiretinal cells at the ILM in all macular hole stages strongly suggests a substantial involvement of cell migration and proliferation in the course of macular hole development. Glial cells and hyalocytes play the predominant role in epiretinal cell proliferation. Given the co-expression of glial cell and hyalocyte markers, transdifferentiation of epiretinal cells needs further elucidation, especially with respect to αSMA-positive cells leading to traction at the vitreoretinal interface.

Purpose: To determine the effect of alkylphosphocholines (APCs) on intraretinal proliferation induced by experimental retinal detachment in the rabbit., Methods: Retinal detachments were created in adult pigmented rabbits. APCs, either liposome bound (liposome, L-APC) or unbound (free, F-APC), were injected intravitreally on either day 1 or day 2 after detachment. BrdU was injected on day 3, 4 hours before death. After fixation, retinas were triple labeled with anti-BrdU, anti-vimentin, and the isolectin B4. The number of anti-BrdU-labeled cells was counted per millimeter of retina from sections imaged by laser scanning confocal microscopy. Toxicity was examined using toluidine blue-stained sections imaged by light microscopy and by electron microscopy for ultrastructural evaluation., Results: Retinal detachment initiated proliferation of all non-neuronal cells. After intravitreal injection on day 1 or 2 after experimental induction of retinal detachment, APCs significantly reduced the number of dividing cells at day 3. Liposome-bound drug given on day 2 was more effective on Müller cell proliferation than was unbound drug. Injection of F-APC on day 1 was more effective than when given on day 2. No apparent effect was seen on Müller cell hypertrophy as indicated by vimentin expression. In addition, no evidence of toxicity was observed in the retina at day 3 for any of the conditions., Conclusions: APCs significantly reduce the number of Müller cells that are stimulated to divide as a result of retinal detachment. The preliminary results indicate no evidence of significant toxicity; however, further studies are needed. APCs have the potential to be used as part of a therapeutic approach if they can be combined with other agents that can suppress the fibrosis that is also a critical event in the pathogenesis of proliferative vitreoretinal diseases such as proliferative vitreoretinopathy (PVR).

Purpose: To investigate the effect of alkylphosphocholines (APCs) on human retinal pigment epithelium (RPE) attachment, spreading, migration, and microfilament assembly in vitro., Methods: Cultured RPE cells of five human donors were treated with one of four APCs (C18:1-PC, C20:1-PC, C21:1-PC, or C22:1-PC) in the presence of fetal calf serum. Cell viability was tested by the trypan blue exclusion assay. Attachment was assessed after a 2-hour incubation of RPE cells on coated 96-well-plates and subsequent MTT testing. Cellular spreading is characterized by cytoplasmic halo formation and was quantified by counting four separate fields of RPE cells allowed to spread on coated 24-well plates for 4 hours. Migration was measured by a modification of the Boyden chamber method in microchemotaxis chambers with polycarbonated filters. Microfilament assembly was assessed by immunofluorescence analysis after incubation with rhodamine-phalloidin., Results: All four APCs inhibited RPE cell attachment by more than 70% of their IC50 (C18:1-PC: 30 microM; C20:1-PC: 10 microM; C21:1-PC: 10 microM; and C22:1-PC: 10 microM). Also, APCs inhibited RPE cell spreading by more than 80% and migration by more than 90% at similar concentrations. Trypan blue staining revealed a toxicity within control limits within the concentration interval tested. Microfilament organization was significantly disturbed after incubation of RPE cells with one of the four APCs close to its IC50., Conclusions: APCs inhibit RPE cell attachment and spreading in vitro at nontoxic concentrations. As a possible mechanism of action, APCs disturb microfilament assembly, since they are known to interfere with protein kinase C (PKC) function. This could represent a novel method of preventing even early stages of proliferative vitreoretinal diseases like proliferative vitreoretinopathy (PVR).

Purpose: To investigate the effect of alkylphosphocholines (APCs) on human Tenon fibroblast (HTF) proliferation, migration, and cell-mediated collagen gel contraction., Methods: HTFs were isolated from tissue samples of three patients obtained during surgery and cultured in DMEM and 10% fetal calf serum (FCS). HTFs (passage 3-6) were treated with one APC in different concentrations spanning the 50% inhibitory concentration (IC(50)), as determined previously. Inhibition of cell proliferation was assessed by the tetrazolium dye reduction assay. Migration was determined in chemoattractant chambers with fibronectin-coated polycarbonated membranes. For inhibition of contraction, three-dimensional collagen gels were seeded with HTFs, and the gel size was measured. Cell viability was determined by the trypan blue exclusion assay. For analysis of the mechanism of action, protein kinase C (PKC) activity was measured., Results: The IC(50) varied between 7.0 and 10.5 microM for all APCs tested. At this concentration, all four APCs inhibited HTF migration and cell-mediated collagen gel contraction in the presence of serum. The inhibitory effects on HTF proliferation, migration, and contraction were observed at nontoxic concentrations. PKC activity was reduced to 50% of control level at the IC(50) of all APCs applied., Conclusions: APCs are effective inhibitors of HTF proliferation, migration, and cell-mediated contraction of collagen gels at nontoxic concentrations. Their mechanism of action seems to involve the inhibition of the PKC pathway.

Purpose: To investigate the effect and mechanism of action of alkylphosphocholines (APCs) on proliferation of human retinal pigment epithelium (RPE) cells and RPE-mediated collagen matrix contraction in vitro., Methods: Cultured RPE cells of five human donors were treated with four APCs in the presence of fetal calf serum. Proliferation was assessed by the tetrazolium dye-reduction (MTT) assay and by counting the number of cells dividing in culture. The effect of APCs on RPE-mediated matrix contraction was determined in three-dimensional collagen gels. Cell viability was tested by the trypan blue exclusion assay. As a possible mechanism of APC action, protein kinase C (PKC) activity was quantified by scintillation counting of (32)P-labeled phosphate transferred to a PKC-specific substrate., Results: All APCs inhibited RPE proliferation and RPE-mediated collagen matrix contraction in a dose-dependent manner in vitro. The antiproliferative and anticontractile effect of APCs increased with elongation of the fatty acid chain beyond C20. IC(50)s of all APCs varied between 8.5 micro M (erucyl-phosphocholine, C22:1-PC), 9.0 micro M (Z)-12-heneicosenyl-phosphocholine, C21:1-PC), 11.0 micro M (Z)-10-eicosenyl-phosphocholine, C20:1-PC), and 26.5 micro M (oleyl-phosphocholine, C18:1-PC). Trypan blue staining revealed a toxicity below 5% for all APCs within the concentration interval tested. PKC activity was significantly reduced by all four APCs, with C22:1-PC being the most effective., Conclusions: APCs inhibit proliferation of RPE cells and RPE-mediated matrix contraction in vitro at nontoxic concentrations. This effect seems to be exerted through inhibition of PKC activity. Therefore, APCs are promising candidates for treatment of RPE-mediated proliferative processes such as proliferative vitreoretinopathy.