Arno Hueber

Basic fibroblast growth factor mRNA, bFGF peptide and FGF receptor in epiretinal membranes of intraocular proliferative disorders (PVR and PDR).

Basic fibroblast growth factor (bFGF) has been shown to be involved in epiretinal membrane formation in proliferative vitreoretinal disorders. However, up to now, little knowledge exists, as to the actual cellular source of this potent mitogen.We examined 20 epiretinal membranes from patients with proliferative diabetic retinopathy (PDR) (n = 12) and proliferative vitreoretinopathy (PVR) (n = 8) for the presence of bFGF peptide, fibroblast growth factor receptor-1 (FGFR-1) and bFGF messenger ribonucleic acid (mRNA).Using a specific antibody, we detected bFGF peptide in most (8/10) examined PDR membranes and in all (8/8) PVR membranes. Moreover, we found positive staining for the corresponding receptor.Local production of bFGF in epiretinal membranes was confirmed by nonisotopic in situ hybridisation for bFGF mRNA in some (4/7) examined PDR membranes and some (3/4) examined PVR membranes. All membranes which contained bFGF mRNA were also positive for bFGF peptide.In conclusion, bFGF is produced and stored in epiretinal membranes. Together with the corresponding receptor, bFGF may play a role in the auto- and paracrine control of the proliferative processes at the vitroretinal interface.

Characteristics of Iris and Retinal Pigment Epithelial Cells Cultured on Collagen Type I Membranes

Purpose: Transplantation of pigment epithelial cells is a promising treatment modality to repair retinal damage in age-related macular degeneration. For this purpose, it is necessary to establish cell culture techniques that allow acquisition of proper functional and morphological characteristics by the cells to be transplanted. Methods: Primary retinal pigment epithelial (RPE) and iris pigment epithelial (IPE) cells grown to confluence on collagen membranes were examined for morphology, adhesion, proliferation, apoptosis, as well as viability after ex vivo transplantation. Results: Pigment epithelial cells adhere, proliferate, form monolayers, acquire differentiated properties, and remain viable during transplantation to the subretinal space. Conclusions: Pigment epithelial cells cultured on collagen membranes acquire differentiated characteristics and are amenable to be transplanted as cell monolayers.

The effect of previous surgery and topical eye drops for primary open-angle glaucoma on cytokine expression in aqueous humor

PurposeTo evaluate cytokine expression in the aqueous humor of patients with primary open-angle glaucoma (POAG) after previous glaucomatous and/or cataract surgery, and to determine the effect of intraocular pressure (IOP)-lowering eye drops on cytokine expression.MethodsThis prospective consecutive case study included 32 eyes diagnosed with POAG (19 with previous surgery and 13 without previous surgery, treated with topical antiglaucoma medication) and 12 eyes without signs of glaucoma. The Luminex 200 multiplex bead immunoassay was used to measure 27 cytokines in aqueous humor.ResultsEyes suffering from POAG, with previous surgery, had significantly elevated concentrations of IL-6, IL-8, CCL2, CXCL9, and HGF, and a significantly lower concentration of CCL5, compared to POAG eyes without previous surgery, treated only with topical antiglaucoma medication. When compared with cataract controls, eyes with POAG and previous surgery had significantly elevated levels of G-CSF, IL-8, IL-12, CXCL10, and HGF, and significantly decreased concentrations of IL-17, CCL5, and VEGF in aqueous humor. In a comparison between POAG eyes without previous surgery and cataract controls, the cataract control eyes had significantly higher levels of IL-6 and CCL2, as the only significant difference.ConclusionsPOAG is associated with an aqueous inflammatory response in the aqueous humor, which is significantly elevated in eyes with previous surgery. In contrast, preoperative IOP-lowering eye drops did not significantly alter the anterior chamber milieu. The results of the current study indicate that filtration surgery has a higher success rate in eyes that have not experienced previous surgery.

Canthaxanthin retinopathy: long-term observations.

Purpose: To describe the long-term outcome of canthaxanthin retinopathy. Methods: We identified 13 patients with small golden particles near the macular region among a group of 35 patients with known consumption of canthaxanthin somewhen between 1983 and 1988. One long-term follow-up examination was possible in 5 of 13 cases after 16–24 years. The examinations included determination of visual acuity, the Amsler grid, slit lamp examination, perimetry, electro-oculography, electroretinography, optical coherence tomography and fluorescein angiography. Results: Complete disappearance of the golden particles took approximately 20 years. The patients in our study were asymptomatic and no functional defect related to canthaxanthin could be detected. Conclusions: Ingestion of canthaxanthin causes no long-term adverse effects.

Multidrug resistance-associated proteins in glaucoma surgery

Abstract Background: Multidrug resistance (MDR) describes the phenomenon of cross-resistance between different cytostatic agents which are structurally and functionally dissimilar. Two recently discovered proteins, lung resistance protein (LRP) and the multidrug resistance-related protein (MRP) have been implicated in the development of MDR. Since resistance to chemotherapeutic agents is a common problem in filtration surgery, especially in cases of complicated glaucoma, we decided to investigate the presence of MRP and LRP in surgically removed Tenon specimens from glaucoma patients. Methods: The presence of MRP and LRP in surgically removed Tenon tissue (n=15) was analyzed by immunohistochemistry. The expression by cultured Tenon fibroblasts was assessed by reverse-transcriptase polymerase chain reaction (RT-PCR) and fluorocytometry. Results: LRP expression was detected in 8 of 10 Tenon specimens. Positive staining for MRP was obtained in 5 of 10 specimens. Negative controls with non-immune mouse IgG did not display any specific staining. RT-PCR and fluorocytometry revealed constitutive expression of MRP and LRP, at the RNA and protein level respectively, that was unaltered by pretreatment of the cells with mitomycin C or 5-fluorouracil. Conclusion: Our results demonstrate, that besides P-glycoprotein, other components of the MDR-system are present in conjunctival fibroblasts. Future developments in the use of chemotherapeutic agents in association with of filtration surgery need to take account of the presence of these counteracting mechanisms.

The neuroprotective potential of Rho-kinase inhibition in promoting cell survival and reducing reactive gliosis in response to hypoxia in isolated bovine retina.

Aims: To investigate the outcomes of Rho-kinase inhibition in the electrophysiological ex vivo model of the isolated perfused vertebrate retina under hypoxia. Methods: Bovine retinas were perfused with an oxygen saturated nutrient solution with or without the Rho-kinase inhibitor H-1152P. The retinas were stimulated repeatedly until stable amplitudes were reached and the electroretinogram was recorded at five minute intervals. Hypoxia was induced for 15, 30, and 45 minutes, after which the oxygen saturation was restored. The extent of the cell damage and glial reactivity was determined by Ethidium homodimer-1 staining, immunohistochemistry, and Western blot. Results: Hypoxia caused a time-dependent reduction of the b-wave amplitudes, which could not be prevented by the H-1152P. Although the Rho-kinase inhibitor maintained higher b-wave amplitudes, these effects did not reach statistical significance. Hypoxia also resulted in an increase in cell damage and the activation of the glial cells in the untreated retinas whereas the administration of H-1152P significantly reduced the extent of these events. Conclusion: H-1152P exerted a neuroprotective effect against necrosis on the isolated bovine retina under hypoxia together with a reduction in glial cell reactivity. However, the inhibitor could not prevent the hypoxia induced retinal dysfunction possibly due to the interference with synaptic modulation.

Apoptosis-mediating receptor–ligand systems in human retinal pigment epithelial cells

AbstractBackground. To establish new strategies for the treatment of proliferative vitreoretinopathy (PVR), we investigated new members of a recently discovered apoptosis-inducing receptor–ligand system in human retinal pigment epithelial (RPE) cells. TRAIL (Apo2-L) and Apo3-L are capable of inducing cell death via their receptors Trail-R1 to Trail-R4 and TRAMP. The goal of this study was to prove the existence of these new apoptosis-inducing receptors and ligands in RPE cells. Methods. Human RPE cells, cultured or prepared directly from the eye, were examined by RT-PCR. Immunohistochemistry of epiretinal membranes of traumatic PVR was performed for the detection of TRAIL and Trail-R1. Protein expression of Trail-R1 was examined in cultured human RPE cells by western blot. Cell death after TRAIL treatment of human RPE cells was measured by crystal violet staining. Results. For RPE cells derived directly from the eye, we detected mRNAs of Trail-R2, Trail-R3, TRAIL, and APO3-L, but not Trail-R1, Trail-R4, and TRAMP. All the examined transcripts were detected in human P0 RPE cells in vitro. Immunohistochemical studies on PVR membranes identified TRAIL and Trail-R1. Western blot confirmed the presence of Trail-R1 in cultured human RPE cells. TRAIL failed to kill RPE cells in vitro, but showed a strong synergistic killing effect when coincubated with protein (cycloheximide) or RNA (actinomycin D) synthesis inhibitor. Conclusions. We detected a novel apoptosis-inducing receptor–ligand system in RPE cells. An induction of apoptosis as a treatment of PVR seems to be possible. Further investigations are needed including an animal model of PVR.

Fulminant Endogenous Anterior Uveitis due to Listeria monocytogenes.

Purpose: To report an unusual case of fulminant anterior uveitis, confirmed as endogenous Listeria monocytogenes infection. Subject: A 67-year-old man with multiple comorbidities acutely developed a severe endogenous anterior uveitis. Results:L. monocytogenes, a ubiquitous Gram-positive bacillus, was directly indicated by culture and PCR. Early and aggressive treatment with intravenous antibiotics likely prevented the endophthalmitis which most cases on record experienced. Our patient regained satisfactory visual acuity. Conclusions: Prompt antimicrobial therapy is paramount when severe endogenous uveitis develops in a patient with comorbidities, especially with systemic immunosuppression. Treatment solely with corticosteroids should be avoided.

Lens epithelial cells express CD95 and CD95 ligand treatment induces cell death and DNA fragmentation in vitro

Purpose Despite advances in intraocular lens design and material, posterior capsule opacification remains one of the major problems in modern cataract surgery. Therefore, the use of antiproliferative agents has been advocated. CD95 ligand (CD95L, Fas, Apo-1) is a death ligand that triggers apoptosis in susceptible target cells. Apoptosis allows for the safe disposal of cells without damaging the surrounding tissue. The goal of this study was to characterize and evaluate CD95L-induced cell death in cultured lens epithelial cells (LEC). Methods Expression of CD95 in untreated porcine LEC was investigated by flow cyto metry. Cell death after CD95L or CD95 agonistic antibody treatment was assessed by crystal violet assay and DNA fragmentation was measured by comet assay. Results The presence of CD95 was observed in LEC. CD95L treatment resulted in a time-and concentration-dependent killing of LEC, which was synergistically enhanced by the addition of cyclohexamide. CD95L treatment induced DNA fragmentation. Conclusions The present study confirms the use of apoptosis-inducing CD95L in the inhibition of LEC proliferation. Further studies are needed before clinical application of CD95L to inhibit posterior capsule opacification will be feasible.

Synergistic effects of combined cytotoxic and apoptosis-inducing drugs on Tenon’s capsule fibroblasts in vitro and in vivo

BackgroundHighly toxic antimetabolites have gained access to routine clinical use to modulate and reduce the amount of postoperative scarring following glaucomatous filtering procedures. It could be speculated that by combining two different antiproliferative substances with different mechanisms of action total amounts of the substances could be decreased and side effects reduced.MethodsTwenty-two substances were tested that had antiproliferative effects by acting cytotoxically, inhibiting growth factors, or inducing apoptosis. With combinations of each two substances, cell culture experiments using 3T3 and human Tenon’s capsule fibroblasts were performed evaluating cell toxicity, proliferation and migration, the extent of free radicals, and the amount of apoptosis (TUNEL, electron microscopy). The five most potent combinations were used in an animal experiment with rabbits performing filtering procedures. The extent of episcleral scarring was evaluated by histopathology.ResultsThe results of the various assays revealed consistently strong effects in 5 of the 462 combinations. Of these five combinations, two were highly effective in the rabbit model. Substances with strong effects when applied in combination included staurosporine, mitomycin, and CD95L.ConclusionsWe found synergistic effects in assays that evaluated different aspects of cell function. The amount of scarring in an animal experiment was inhibited to a level comparable with a high single dose of mitomycin. Combination therapy of two antiproliferative acting substances may be a promising concept.