PURPOSE To investigate the presence and the possible role of different matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in Tenon capsule fibroblasts. These enzymes are essential for the control of tissue remodeling in the context of wound repair. This aspect is important to further the understanding of and possibly to influence the scarring process of filtering blebs after glaucoma surgery. METHODS Untreated and latanoprost-treated human Tenon fibroblasts were examined for the presence of MMPs and TIMPs on the mRNA and protein levels. Assays performed included RT-PCR, real-time RT-PCR, immunocytochemistry, Western blot analysis, flow cytometry, and zymography. To investigate the changes in vivo, conjunctival specimens of rabbits treated with latanoprost eye drops were examined by immunohistochemistry. RESULTS In all assays, both MMP-3 and TIMP-2 were detected. With the real-time RT-PCR technique, MMP-1, -2, -3, -7, -9, and -14 and TIMP-1 and -2 were detected. An upregulation of MMP-3 and TIMP-2 after latanoprost treatment of the fibroblasts was shown and found to occur on the mRNA and the protein levels. The upregulation of MMP-3 and TIMP-2 was confirmed in vivo. CONCLUSIONS Tenon fibroblasts contain the ability on the mRNA level to synthesize all enzymes of the MMP and TIMP family that are related to remodeling of the extracellular matrix. The levels of MMP-3 and TIMP-2 increase after treatment with latanoprost. Tenon fibroblasts may be the target cells for attempts to influence the tissue levels of MMPs and TIMPs in the context of conjunctival wound healing after glaucoma surgery.

Despite the adjuvant use of mitomycin C during trabeculectomy, failures still occur. We investigated whether cultured human Tenon fibroblasts exposed to low-dose mitomycin C developed a multidrug resistance phenotype in vitro, and whether mitomycin C treatment during previous filtration surgery induces P-glycoprotein expression in vivo.

PURPOSE:In glaucoma surgery, scarring of the artificial fistula is the limiting factor for long-term control of intraocular pressure (IOP). Several devices and surgical techniques have been developed for artificial aqueous humor drainage in intractable glaucoma. The authors describe a novel surgical technique that uses a silicone tube as a shunt for aqueous flow from the anterior chamber to the suprachoroidal space.PATIENTS AND METHODS:Thirty-one eyes of 31 patients with uncontrollable refractory glaucoma were included in this prospective consecutive case-control study. Each eye had undergone an average of 3.5+/-1.9 previous interventions for glaucoma. The baseline IOP was 44.25+/-8.7 mm Hg despite maximum therapy. As in trabeculectomy, a limbus-based scleral flap was prepared. The suprachoroidal space was accessed via a deep posterior scleral flap. The silicone tube was inserted as an intrascleral connection from the anterior chamber to the suprachoroidal space. Cyclodialysis was avoided by this surgical approach. Success was defined as a lowering of IOP to below 21 mm Hg without the need for further medication or intervention.RESULTS:The mean functional shunt survival was 55.9+/-45.6 weeks. IOP was reduced to 12.9+/-5.2 mm Hg in 70% of all eyes after 30 weeks postoperatively. After 52 weeks, 60% of the eyes could be classified as representing success, and 76 weeks after surgery, 40% of the eyes still showed controlled IOP. In none of the eyes were severe postoperative hypotony or suprachoroidal bleeding observed. No localized or general inflammation or infection was seen in connection with the silicon tube. Two patients needed anterior chamber lavage because of bleeding. In 2 patients the tube had to be removed because of corneal endothelial contact. Shunt failure of the tube was caused in some cases by connective tissue formation at the posterior lumen of the tube.CONCLUSION:This novel surgical approach and the placement of the silicone tube described here have several advantages. Its intrascleral course minimizes the risk of conjunctival erosion and associated infections. No cyclodialysis is performed. Connection to the suprachoroidal space exploits the resorptive capability of the choroid. It guarantees drainage but also provides a natural counterpressure, avoiding severe postoperative hypotony. The suprachoroidal shunt presented here achieves good follow-up results in terms of IOP control. No serious complications have been observed. This new method promises to be an effective surgical technique and presents a new therapeutic option in intractable glaucoma. Fibroblast reaction obstructing the posterior lumen, seemed to be the only factor limiting drainage. Further studies and experiments will be needed to elucidate the exact physiologic mechanisms underlying the draining, the capacity and duration of the draining effect, and the histologic background of suprachoroidal scarring.

OBJECTIVETo report the functional and anatomical outcome of 20 patients who underwent surgical removal of choroidal neovascularization combined with transplantation of autologous iris pigment epithelial cells to the subretinal space 3 years after treatment.METHODSFreshly isolated autologous iris pigment epithelial cells were translocated to the subretinal space in 20 patients after membrane extraction. Patients were followed up by funduscopy, angiography, microperimetry, and visual acuity testing.RESULTSAfter a follow-up of 3 years, 1 patient showed improved visual acuity, 13 patients retained stable visual acuity, and 3 patients had reduced visual acuity. No macular edema or recurrent choroidal neovascularization was apparent at any time during the follow-up.CONCLUSIONSTransplanted autologous iris pigment epithelial cells were well tolerated for 3 years and stabilization of visual acuity was achieved in most patients. These results suggest that iris pigment epithelial cells may serve as a substitute for retinal pigment epithelial cells after choroidal neovascularization removal in patients with exudative macular degeneration; however, whether these cells will be of any value for the restoration of vision and possible protection against choroidal neovascularization recurrence awaits further clinical observation and additional research.

PURPOSE To characterize daunorubicin-induced cell death in cultured human retinal pigment epithelial (RPE) cells and its modulation by CD95 ligand (CD95L). METHODS In situ DNA end labeling and an ELISA for histone-associated DNA fragments were used to assess apoptosis. CD95 and CD95L expression were examined by immunohistochemistry, flow cytometry, immunoblot, and RT-PCR. Cell death was measured by crystal violet staining. YVAD- and DEVD-amc cleavage was used to measure caspase-1 and -3-like activity. Total RNA and protein synthesis was measured by incorporation level of [(3)H]-leucine and [(3)H]-uridine. RESULTS RPE cells expressed both CD95 and CD95L, but only CD95 was expressed at the cell surface. Daunorubicin-induced RPE cell apoptosis was associated with enhanced CD95 and CD95L expression. Enhanced CD95L expression was epiphenomenal to the death process, evidenced by the fact that neutralizing CD95L antibodies failed to modulate daunorubicin cytotoxicity. In contrast, the cytotoxic effects of daunorubicin were synergistically enhanced by exogenous CD95L. Synergy appeared to involve enhanced caspase-3-like activity as well as daunorubicin-mediated inhibition of RNA synthesis. CONCLUSIONS Apoptosis has been shown to be an important factor in the control of specific cell populations. The synergistic activity of an antiproliferative agent, daunorubicin, and a cytokine, CD95L, induces apoptosis in RPE cells. Such approaches provide a means to reduce the concentration of chemotherapeutic agents with a small therapeutic window owing to retinal toxicity, such as daunorubicin, in the adjuvant therapy of proliferative vitreoretinopathy.

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 cytometry. 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.

BACKGROUND:During surgical extraction of choroidal neovascular membranes (CNV) in age-related macular degeneration (AMD), the defective foveal retinal pigment epithelium (RPE) is removed. Subsequent translocation of the foveal neural retina to adjacent healthy RPE should result in stabilization and possibly improvement of visual acuity.METHODS:A prospective case series was carried out using controlled surgery and examination protocols with examinations made at fixed intervals. The surgical procedures combine counterrotation of the globe, phacoemulsification and implantation of a posterior chamber lens, complete vitrectomy, induction of a total retinal detachment, 360 degrees anterior retinotomy, removal of the subfoveal neovascular complex, foveal translocation outside the RPE defect, reattachment of the retina using F6H8, peripheral laser retinopexy and temporary silicone oil tamponade.PATIENTS:Macular translocation surgery was performed on 100 patients between December 1997 and December 1999. All patients had experienced recent visual loss due to exudative AMD and of these, 26 patients had major macular subretinal hemorrhage, 39 patients had occult and 25 patients classic subfoveal choroidal neovascularization. The preoperative findings in the remaining patients included tears in the pigment epithelium (n = 4), polypoidal choroidal vasculopathy (n = 1), recurrent subfoveal CNV following laser therapy (n = 2) and deep retinal vascular anomalous complexes (n = 3).RESULTS:A total of 97 patients completed the 12-month examination. Visual acuity increased by 15 or more ETDRS chart letters in 24 patients, remained stable in 42 patients and deteriorated by 15 or more EDTRS chart letters in 34 patients 12 months postoperatively. The silicone oil tamponade was removed in 97 patients, in 10 patients, silicone oil had to be reinjected because of severe complications. A secondary procedure was necessary in 25 patients, primary PVR was observed in 9 eyes, secondary PVR developed in 10 eyes, a macular pucker in 5 eyes and a macular hole in 1 patient. Other postoperative complications included persistent hypotonia, macular edema, IOL dislocation, keratopathy and recurrent CNV (n = 3).CONCLUSIONS:Macular translocation is a technically demanding operation, which requires a considerable learning curve. Although the procedure has a high rate of surgical and postoperative complications, the functional and anatomical results appear to be promising for selected patients with subfoveal CNV secondary to AMD.

OBJECTIVE:To assess long-term functional and morphological changes after macular translocation in patients with exudative age-related macular degeneration. METHODS:Evaluation of a noncomparative cohort study of 90 patients with a follow-up of 14 to 79 months (mean, 38.2 months). RESULTS:Visual acuity increased by 3 or more lines in 15 patients, remained stable in 35 patients, and deteriorated in 40 patients at final examination. Pigment epithelium atrophy extending to the new fovea was detected in 44 patients; in 25 patients this new atrophy was associated with loss of visual function. CONCLUSIONS:Long-term follow-up of macular translocation with 360 degrees retinotomy showed stabilization or improvement in half of the patients. Progressive atrophy of the pigment epithelium represented one major limiting factor of the beneficial effect of the treatment. Macular translocation may be an option for cases of exudative age-related macular degeneration that are not eligible for or do not respond to alternative treatments.

PURPOSE:To analyze the differential gene expression in cultured human retinal pigment epithelial (RPE) cells after treatment with vitreous.METHODS:Cultured human RPE cells were incubated for 48 hours with 25% human vitreous from donor eyes. Total RNA from treated and untreated cells was extracted. The gene expression was analyzed by differential expression analysis (DEmRNA-PCR). The differentially expressed genes were identified by gene bank searches. Differential expression was verified by a quantitative real-time RT-PCR fluorescent nucleic acid staining system. The in vivo mRNA expression of these genes in RPE cells was shown by gene-specific RT-PCR.RESULTS:Vitreous treatment of human RPE cells resulted in the reduced expression of NFIB2, KE03 (NY-REN-25ag), PIG-B, DKFZp564BC462, LKHA, G3BP, PAM, UEV-1, and MAP1B calibrated to the expression of GAPDH when compared with their expression in untreated cells. The reduced expression after vitreous treatment was quantified by gene-specific quantitative real-time RT-PCR and varied from 0.69 to 0.17 compared with untreated cells. The mRNA expression of UDP-GalNac mRNA remained constant. The mRNA expression of eight of these genes was demonstrated in this study for the first time in human RPE cells.CONCLUSIONS:Vitreous treatment of cultured RPE cells induces the differential expression of a variety of genes with functions in transcription, mediation of signal transduction and inflammation, glycosylation, ubiquitination and protein-protein interaction. Further examination of these genes may locate additional targets for treatment of diseases caused by contact of RPE cells with vitreous, typical in proliferative vitreoretinopathy.

Diabetic macular edema, resulting from increased microvascular permeability, is the most prevalent cause of vision loss in diabetes. The mechanisms underlying this complication remain poorly understood. In the current study, diabetic vascular permeability (blood-retinal barrier breakdown) is demonstrated to result from a leukocyte-mediated Fas-FasL-dependent apoptosis of the retinal vasculature. Following the onset of streptozotocin-induced diabetes, FasL expression was increased in rat neutrophils (P<0.005) and was accompanied by a simultaneous increase in Fas expression in the retinal vasculature. Static adhesion assays demonstrated that neutrophils from diabetic, but not control, rats induced endothelial cell apoptosis in vitro (P<0.005). The latter was inhibited via an antibody-based FasL blockade (P<0.005). In vivo, the inhibition of FasL potently reduced retinal vascular endothelial cell injury, apoptosis, and blood-retinal barrier breakdown (P<0.0001) but did not diminish leukocyte adhesion to the diabetic retinal vasculature. Taken together, these data are the first to identify leukocyte-mediated Fas-FasL-dependent retinal endothelial cell apoptosis as a major cause of blood-retinal barrier breakdown in early diabetes. These data imply that the targeting of the Fas-FasL pathway may prove beneficial in the treatment of diabetic retinopathy.

Background. 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.

Hintergrund. Mit dem Ziel einer Stabilisierung oder Verbesserung der visuellen Funktion von Patienten mit altersabhängiger Makuladegeneration (AMD) wurde die Translokation der Makula auf gesundes Pigmentepithel (RPE) nach Extraktion einer chorioidalen Neovaskularisation (CNV) entwickelt.

Background. During surgical extraction of choroidal neovascular membranes (CNV) in age-related macular degeneration (AMD), the defective foveal retinal pigment epithelium (RPE) is removed. Subsequent translocation of the foveal neural retina to adjacent healthy RPE should result in stabilization and possibly improvement of visual acuity. Methods. A prospective case series was carried out using controlled surgery and examination protocols with examinations made at fixed intervals.The surgical procedures combine counterrotation of the globe, phacoemulsification and implantation of a posterior chamber lens, complete vitrectomy, induction of a total retinal detachment, 360° anterior retinotomy, removal of the subfoveal neovascular complex, foveal translocation outside the RPE defect, reattachment of the retina using F6H8, peripheral laser retinopexy and temporary silicone oil tamponade. Patients. Macular translocation surgery was performed on 100 patients between December 1997 and December 1999. All patients had experienced recent visual loss due to exudative AMD and of these, 26 patients had major macular subretinal hemorrhage, 39 patients had occult and 25 patients classic subfoveal choroidal neovascularization.The preoperative findings in the remaining patients included tears in the pigment epithelium (n=4), polypoidal choroidal vasculopathy (n=1), recurrent subfoveal CNV following laser therapy (n=2) and deep retinal vascular anomalous complexes (n=3). Results. A total of 97 patients completed the 12-month examination.Visual acuity increased by 15 or more ETDRS chart letters in 24 patients, remained stable in 42 patients and deteriorated by 15 or more EDTRS chart letters in 34 patients 12 months postoperatively.The silicone oil tamponade was removed in 97 patients, in 10 patients, silicone oil had to be reinjected because of severe complications. A secondary procedure was necessary in 25 patients, primary PVR was observed in 9 eyes, secondary PVR developed in 10 eyes, a macular pucker in 5 eyes and a macular hole in 1 patient. Other postnen und Hamorrhagien, die zum Untergang von Photorezeptoren und in spateren Stadien zur Ausbildung disziformer Lasionen fuhren. Zum jetzigen Zeitpunkt sind die Laserphotokoagulation zur Behandlung extrafovealer CNV und die photodynamische Therapie (PDT), indiziert fur die kleine Gruppe subfovealer und uberwiegend klassischer CNV, die einzigen Therapieansatze, fur die ein positiver Effekt nachgewiesen wurde [27,28,29,40].Daruber hinaus sind bei beiden Verfahren auch bei erfolgreicher Therapie haufig eine oder mehrere Nachbehandlungen wegen eines CNV-Rezidives nach Laserkoagulation oder einer Reperfusion nach PDT erforderlich [30, 40]. Die zunehmende Pravalenz der Erkrankung und das Fehlen einer Therapie,die eine Funktionsverbesserung verspricht, fordert die Suche nach alternativen Behandlungsstrategien. Fur die Strahlentherapie in der Behandlung der subfovealen CNV konnte kein positiver Effekt beobachtet werden [16].Auf dem Gebiet der antiangiogenetischen medikamentosen Therapie ist bisher noch keine effektive Therapie zur Behandlung der exsudativen AMD nachgewiesen worden [33]. Die klassische chirurgische Entfernung subretinaler Membranen fuhrt aufgrund des geschadigten RPE nicht zu einer Funktionsverbesserung [4,9, 19, 35, 38]. Modifikationen der reinen Membranextraktion, die mit dem Ziel einer Verbesserung der Erfolgsrate entwickelt wurden, wie die Transplantation von Irispigmentepithelzellen (IPE) oder retinalen Pigmentepithelzellen (RPE), bedurfen weiterer experimenteller und klinischer Forschung [2, 20, 23, 39]. Die Idee, die Netzhaut zu drehen und so die Fovea auf gesunder erscheinendes RPE zu verlagern, wurde erstmalig von Lindsey et al. vorgeschlagen [24]. Machemer und Steinhorst prasentierten 1993 ihre ersten Ergebnisse nach Makulatranslokation mit 360°-Retinotomie bei Patienten mit AMD [25]. Inzwischen sind verschiedene Modifikationen dieses Operationsverfahrens beschrieben worden, einschlieslich der sog. limitierten Rotationen ohne 360°-Retinotomie [10, 14, 21, 26].Durch eine einoder zweizeitige Gegenrotation des Bulbus mit unterschiedlichen augenmuskelchirurgischen Masnahmen ist es moglich, die operativ induzierte Zyklotropie grostenteils zu kompensieren [10, 11, 13]. In einer prospektiven konsekutiven Fallserie mit kontrolliertem Operationsund Untersuchungsprotokoll untersuchten wir bei 100 Patienten mit subfovealer Neovaskularisation bei AMD funktionelle und angiographische Ergebnisse 12 Monate nach Makulatranslokation mit 360°-Retinotomie. Material und Methode