[Role of energy metabolism in retinal pigment epithelium]. / Rolle des Energiestoffwechsels im retinalen Pigmentepithel.

The universal energy source adenosine triphosphate (ATP)is reduced by approximately 30 % in the retinal pigment epithelium (RPE) of elderly persons. Increased oxidative stress and decreased antioxidative capacity, such as glutathione in aging eyes cause impairment of energy-dependent RPE processes and lead to loss of visual function. We developed a cell culture model of aging RPE using atractyloside to inhibit mitochondrial ATP synthesis and tert-butyl hydroperoxide as oxidant. The ATP levels were reduced by 30 % and oxidative damaged proteins and DNA increased whereas antioxidative glutathione decreased. Autophagy as an internal cellular repair mechanism and phagocytosis of photoreceptors were impaired. Antioxidative and mitochondria-activating Ginkgo biloba extract EGb 761 increased the intracellular ATP level and antioxidative glutathione. This cell culture model seems to be suitable to investigate in vitro the effect of protective substances and their compounds on aging processes in RPE.

[Fundus screening by medical technicians]. / Fundusscreening durch Assistenzpersonal.

Ocular fundus photography allows detection of both ocular and systemic diseases. This study investigated the efficacy of a broad screening in a department of internal medicine using nonmydriatic digital fundus photography. For 8 weeks a medical technician was trained in using the camera as well as interpreting the photographs. The medical technician and an ophthalmologist evaluated the fundus photographs separately by using a self-developed questionnaire. The fundus camera was user-friendly and after several weeks of adjustment and practical application the medical technician was able to detect the majority of pathological fundus photographs. Out of 218 patients examined 148 (68%) were identified as pathological by the medical technician and 163 (75%) by the ophthalmologist (p = 0.0003). The medical technician missed 15 (7%) patients. Furthermore the diagnoses made by the medical technician were faulty. In summary an ophthalmological screening by a medical technician is feasible but the diagnosis still remains the responsibility of ophthalmologists. Such a compromise could facilitate the examination of a large number of patients and disclose previously unrecognized diseases.

[Intravitreal bevacizumab for choroidal neovascularization due to pathological myopia]. / Intravitreal Bevacizumab bei choroidaler Neovaskularisation infolge pathologischer Myopie.

PURPOSE: The aim of this study was to evaluate the safety and efficacy of intravitreal bevacizumab as a treatment for choroidal neovascularization (mCNV) due to pathological myopia. METHODS: A consecutive series of 23 patients with mCNV treated with 1.25 mg intravitreal bevacizumab after being thoroughly informed about the off-label character of the treatment. Of the patients 6 received additional photodynamic therapy at the time of first injection. Reinjections were given every 6 weeks if intraretinal or subretinal fluid persisted. RESULTS: The pre-injection mean visual acuity (VA) was 0.25 (0.58 logMAR+/-0.36). During a follow up of 16.6+/-13.7 weeks 1.4+/-0.8 injections were given. Complete resorption of subretinal or intraretinal fluid was achieved in all patients. VA improved by 2.3+/-3.5 lines on average, 9 patients (39.1%) had an increase of >or=3 lines, none lost more than 1 line. Patients with bevacizumab monotherapy (n=17) had an improvement of 2.59+/-3.9 lines, 7 patients (41.2%) had an increase of >or=3 lines. No intraocular or systemic side effects were observed. CONCLUSIONS: In this as yet largest series of patients with mCNV treated with intravitreal bevacizumab, the treatment seemed to be effective and safe.

[OCT-based re-injections for anti-VEGF-treatment for neovascular ARMD]. / OCT-Befund als Reinjektionskriterium bei der Anti-VEGF-Therapie für neovaskuläre AMD.

BACKGROUND: We present an optical coherence tomography (OCT)-based individual reinjection procedure for bevacizumab treatment in neovascular age-related macular degeneration (ARMD). METHODS: Thirty-two patients with active subfoveal occult choroidal neovascularisation in ARMD received a single intravitreal injection of 1.25 mg bevacizumab and were reinjected based on new or persisting subretinal or intraretinal fluid on OCT. Patient visits were every 6-8 weeks. RESULTS: After a single injection, 74% of patients demonstrated complete retinal fluid absorption, with 44% of patients showing no relapse during a follow-up of 30+/-13 weeks. Fifty-six percent of patients required a second injection after a mean of 19+/-8 weeks, with 82% of patients showing absorption of macular fluid thereafter with regain of their previous achieved best-corrected visual acuity. Thirty-two percent did not require any further injection (follow-up 32+/-12 weeks). Of those patients not showing retinal fluid absorption after the first injection (26%), 44% demonstrated retinal fluid absorption after the second injection. All patients achieved stabilisation of visual acuity during follow-up, with 30% of patients showing a significant gain of >or=3 lines. CONCLUSIONS: OCT-based reinjections of bevacizumab in neovascular ARMD reduce the number of injections and lead to anatomic and functional retinal stabilisation.

[Pathomechanisms for aging of retinal pigment epithelium (RPE) and prophylactic therapy options in regard to AMD]. / Pathomechanismen der Alterung des RPE und prophylaktische Therapieoptionen im Hinblick auf die AMD.

An intact retinal pigment epithelium (RPE) represents an essential condition for the visual process. This post-mitotic RPE monolayer combines different functions such as degradation of photoreceptor outer segments, vitamin A cycle, support of retinal metabolism and maintenance of the outer blood-retina barrier. As a consequence of excessive metabolism, high oxygen levels, exposition to light of short wave length and ensuing radical formation, the RPE is highly dependent on protective systems. In spite of differentiated defence mechanisms, aging processes cause cumulative RPE damage, representing a major component of age-related macular degeneration (AMD), the leading cause of irreversible severe vision loss in people over 50 years old. A better understanding of the underlying pathophysiology will help to develop new prophylactic options which is becoming more and more important with increasing life expectancy.

[OCT-guided reinjection of 2.5 mg bevacizumab for treating macular edema due to retinal vein occlusion]. / OCT-Befund als Reinjektionskriterium für die 2,5-mg-Bevacizumab-Therapie bei venösen retinalen Verschlüssen.

BACKGROUND: Macular edema (ME) due to retinal vein occlusion can be successfully treated with intravitreal bevacizumab therapy. There is no common recommendation concerning time intervals and criteria for reinjection. METHOD: Sixty-three patients (follow-up 30+/-18 weeks) received intravitreal injections of 2.5 mg bevacizumab. Reinjection was performed only if optical coherence tomography (OCT) showed persistent or recurrent ME. Check-ups were performed every 6-8 weeks. RESULTS: There was complete resolution of macular edema in 31 patients after the first injection (improvement in visual acuity 3.7+/-3.7 lines); 65.2% of these patients developed recurrence of ME within 13.3+/-4.4 weeks, which completely resolved again after a second injection. Visual acuity gained the same level as after the first injection. Another relapse of ME in this group occurred in 69% of patients after another 13.4+/-5.4 weeks. Patients with persistent ME after the first injection (n=32) received a second injection, initially leading to resolution of ME in 33.3%, but all of these patients had a relapse within 13.9+/-4.1 weeks. CONCLUSION: OCT-guided reinjection leads to anatomic and functional stabilization or improvement even if transient recurrence of ME occurs.

[Intravitreal anti-VEGF therapy with bevacizumab for neovascular AMD]. / Intravitreale Anti-VEGF-Therapie mit Bevacizumab bei neovaskulärer AMD.

PURPOSE: To report on the efficacy of intravitreal bevacizumab as off-label therapy in different angiographic subtypes in neovascular age-related macular degeneration (AMD). METHODS: Seventy-five patients with neovascular AMD and recent disease progression were classified into different angiographic subtypes and were treated with intravitreal bevacizumab (1.25 mg/0.05 ml) at 6-week intervals. Patients with subfoveal classic choroidal neovascularization (CNV) also received photodynamic therapy. ETDRS visual acuity, ophthalmic exams, and optic coherence tomography (OCT) were performed before treatment, 1 week after treatment, and then on a 6-week basis. Fluorescein angiographies and medical check-ups were also done. RESULTS: Bevacizumab led to stabilization of visual acuity (loss of less than 15 letters) in all angiographic subtypes during a follow-up of 37+/-13 weeks. Patients with occult extrafoveal CNV (n=6) profited the most and gained 2+/-2 lines. Treatment with intravitreal bevacizumab was very well tolerated in all patients, with neither systemic nor intraocular side effects, with the exception of one retinal pigment epithelium tear. CONCLUSION: Intravitreal bevacizumab treatment is efficacious in all angiographic CNV subtypes and leads to reduction of macular edema and stabilization or improvement in visual acuity.

[Photodynamic therapy of lid basal cell carcinomas in a 13-year-old patient with Gorlin Goltz syndrome]. / Photodynamische Therapie von Lidbasaliomen bei 13-jährigem Patienten mit Gorlin-Goltz-Syndrom.

BACKGROUND: Gorlin Goltz syndrome is a rare, autosomal dominant inherited disease that is characterised by multiple basal cell carcinomas (BCC) including the periorbital region and eye lids. We report a severe infantile manifestation with lid involvement treated by photodynamic therapy (PDT). PATIENT: A 13-year-old boy with Gorlin Goltz syndrome presented with multiple confluent BCC on both eye lids and the skin of neck and trunk. Multiple bilateral periorbital confluent and surgically not removable BCC were treated by topical PDT. RESULTS: Numerous superficial BCC were successfully treated by photodynamic therapy with remarkable cosmetic results. CONCLUSION: In cases of numerous confluent and surgically not removable BCC, PDT represents an effective therapy. Frequent monitoring is necessary to maintain the clinical outcome.

[N-acetylcysteine improves lysosomal function and enhances the degradation of photoreceptor outer segments in cultured RPE cells]. / N-Acetylcystein verbessert die lysosomale Funktion und beschleunigt den Abbau von Photorezeptoraussensegmenten in der RPE-Zellkultur.

BACKGROUND: In the retinal pigment epithelium (RPE) lipofuscin granules accumulate with age in the lysosomal compartment mainly as a byproduct of constant phagocytosis of oxidized membranous discs shed from photoreceptor outer segments. Antioxidative defiency and prooxidative conditions in the RPE play a key role in the pathogenesis of RPE dysfunction and macular degenerations such as ARMD. In human RPE cell cultures we investigated the antioxidative effect of N-acetylcysteine (ACC) on lysosomal functions. METHODS: Primary human RPE cell cultures were loaded with regular or oxidized human and porcine rod outer segments (ROS) and treated with ACC. Lysosomal volume and accumulation of autofluorescent material was measured using [14C] methylamine accumulation and FACS analysis. The regulation pattern of lysosomal proteins were investigated by proteome analysis. RESULTS: ACC reduced total lysosomal volume in control, ROS and oxidized ROS fed RPE cells. After ROS incubation increased accumulation of autofluorescent material was measured. ACC treatment decreased intracellular accumulation. Furthermore, incubation with ACC leads to a general down regulation of lysosomal proteins. CONCLUSION: In our cell culture model of ROS fed RPE cells simulating aged RPE ACC improves lysosomal volume and metabolism. Therefore ACC may represent a new prophylactic and causal treatment option for AMD.

[Photodynamic therapy for bilateral idiopathic detachment of the RPE]. / Fotodynamische Therapie bei beidseitiger idiopathischer RPE-Abhebung.

BACKGROUND: A bilateral monofocal detachment of the pigment epithelium (RPE) without any signs of ARMD or other retinal pathology represents a therapeutic challenge. PATIENT: An otherwise healthy 51-year-old woman presented first with a monocular, later a binocular decrease of visual acuity and metamorphopsia. Optical coherence tomography (OCT) showed a bilateral dome-shaped detachment of the RPE. Pooling beneath the detachment was documented using fluorescein angiography. A choroidal neovascularisation could be excluded by means of indocyanine green angiography (ICG). Due to the binocular decrease of visual acuity from 20/20 to 20/63 on the right eye and 20/100 on the left eye in spite of intensive systemic acetazolamide therapy both eyes were treated with photodynamic therapy (PDT) using verteporfin. During monthly controls, the visual acuity increased up to 20/20 on the right eye and 20/25 on the left eye. Metamorphopsia was also reduced. CONCLUSION: An idiopathic detachment of the pigment epithelium can effectively be treated using PDT whereas the underlying pathology remains unclear.

[Bevacizumab for the treatment of macular edema secondary to retinal vein occlusion]. / Bevacizumab zur Therapie des Makulaödems infolge venöser retinaler Gefässverschlüsse.

BACKGROUND: Retinal vein occlusion often leads to macular edema as a result of an elevated level of intravitreal VEGF. We report on the anatomic and functional results after intravitreal bevacizumab injections in patients with retinal vein occlusion. METHODS: In a prospective study, 18 patients with central, and 22 patients with branch retinal vein occlusion, all of whom had persistent macular edema (>300 microm) received 2.5 mg intravitreal bevacizumab. ETDRS visual acuity, ophthalmic examination and stratus OCT were performed at baseline, 1 week after injection and then monthly. Further injections were given every 6 weeks in patients with persistent or recurring macular edema. RESULTS: The findings did not deteriorate in any of the 40 patients. The injections (mean of 2.6+/-1.4 injections/patient) were very well tolerated in all cases during a mean follow-up of 23+/-13 weeks. On the last visit, 73.3% of patients with central retinal vein occlusion and 76.5% of those with branch retinal vein occlusion were found to have significantly improved visual acuity (by at least 3 lines). Mean central retinal thickness had decreased from 921+/-264 to 239+/-66.2 microm in patients with central retinal vein occlusion, and from 678+/-221 to 236+/-78 microm in patients with branch retinal vein occlusion. CONCLUSIONS: Neither intraocular nor systemic side-effects were observed in this study after repeated intravitreal injections of 2.5 mg bevacizumab. Current results suggest that intravitreal anti-VEGF therapy is a promising option in macular edema secondary to retinal vein occlusion.

Lipids and lipid peroxidation products in the pathogenesis of age-related macular degeneration.

In people over 50, age-related macular degeneration (ARMD) has become the most common cause for severe visual loss and legal blindness in all industrialized nations. Currently, there is no effective treatment for the majority of patients. To develop new and effective modes of therapy, understanding of the molecular basis of the disease in mandatory. However, the pathogenesis of ARMD is still poorly understood. Several lines of evidence suggest that aging changes of the retinal pigment epithelium (RPE), in particular the accumulation of autofluorescent lipofuscin granules in the lysosomal compartment of postmitotic RPE cells, play a key role in the pathogenesis of the disease. Recent studies indicate that lipidic compounds of lipofuscin, represented by the retinoid A2-E, and protein damage by lipid peroxidation products, in particular malondialdehyde and 4-hydroxynonenal, induce lysosomal dysfunction and lipofuscinogenesis in the RPE. The possible mechanisms underlying this lysosomal dysfunction and the resulting adverse effects on overall RPE function are discussed.

Inhibition of the ATP-driven proton pump in RPE lysosomes by the major lipofuscin fluorophore A2-E may contribute to the pathogenesis of age-related macular degeneration.

Lipofuscin accumulation in the retinal pigment epithelium (RPE) is associated with various blinding retinal diseases, including age-related macular degeneration (AMD). The major lipofuscin fluorophor A2-E is thought to play an important pathogenetic role. In previous studies A2-E was shown to severely impair lysosomal function of RPE cells. However, the underlying molecular mechanism remained obscure. Using purified lysosomes from RPE cells we now demonstrate that A2-E is a potent inhibitor of the ATP-driven proton pump located in the lysosomal membrane. Such inhibition of proton transport to the lysosomal lumen results in an increase of the lysosomal pH with subsequent inhibition of lysosomal hydrolases. An essential task of the lysosomal apparatus of postmitotic RPE for normal photoreceptor function is phagocytosis and degradation of membranous discs shed from photoreceptor outer segments (POS) and of biomolecules from autophagy. When the lysosomes of cultured RPE cells were experimentally loaded with A2-E, we observed intracellular accumulation of exogenously added POS with subsequent congestion of the phagocytic process. Moreover, the autophagic sequestration of cytoplasmic material was also markedly reduced after A2-E loading. These data support the hypothesis that A2-E-induced lysosomal dysfunction contributes to the pathogenesis of AMD and other retinal diseases associated with excessive lipofuscin accumulation.

Digital analysis of choroidal neovascularisation in consecutive fluorescein angiograms for use in longitudinal clinical trials.

BACKGROUND/AIMS: To document the natural history and to assess the efficacy of interventional therapies in neovascular age related macular degeneration (AMD), an accurate and reproducible method is required for analysis of consecutive fluorescence angiograms. The development and evaluation of an image analysis software for this purpose is described here. It allows for the quantitative analysis of changes in CNV and/or leakage area over time. METHODS: In digitised angiograms, a mouse driven arrow was used to delineate the CNV border. The ratio of the CNV area to the square of the distance between two vessels was automatically calculated by pixel count to compensate for variation in image sizes at different examination times. These results were directly transferred and stored in a database. To assess reproducibility, CNV areas in 20 patients with occult and 20 patients with classic CNV were determined independently by two readers. RESULTS: There was only marginal variability between observers with this method: the mean deviation was 0.01 pixels for classic CNV (95% CI -0.17 to +0.15, SD 0.35) and 0.55 pixels for occult CNV (95% CI -1.06 to -0.04, SD 1.14). CONCLUSIONS: This practical PC based method allows for quantification of angiographic features such as CNV size in early frames and area of leakage in late frames. Limitations include non-readily defined borders in angiograms of poor image quality or indistinct borders of the hyperfluorescent areas of interest. The software is applicable to future clinical trials where the analysis of neovascular complex changes is required, for example, following therapeutic intervention.

Intraocular microablation of choroidal tissue by a 308 nm AIDA excimer laser for RPE-transplantation in patients with age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of legal blindness in the western nations beyond 50 years of age. The most frequent cause for severe visual loss is the growth of neovascular membrances from the choroid into the subretinal space. This usually results in irreversible degeneration of the overlying retina. Surgical removal of the membrane is feasible, however, usually results in functional loss of apposing retinal photoreceptors since retinal pigment epithelial (RPE) cells are removed concurrently due to their tight adherence to the neovascular complex. Therefore, various attempts have been undertaken to fill the resulting RPE cell defect with either heterologous or autologous RPE cell transplants. So far cell survival, function and subsequent visual function has been disappointing. To minimize trauma and resulting dedifferentiation harvesting in the eye and transplantation in whole sheets and without temporary removal from the eyes would be desirable. This may be achieved by isolating grafts consisting of choroid, Bruch's membrance and RPE cells from the peripheral retina and transplantation of this graft under the neurosensory retina after removal of the choroidal neovascularization. However, the choroidal component of such a graft would be expected to interfere with diffusion of metabolites to and from the retina. Therefore, outcome would be expected to be better if the choroidal tissue would be removed before translocation. In preclinical experiments we used a 308 nm UV AIDA excimer laser to microablate choroidal tissue from such a graft in human donor eyes.

Changes in bronchoalveolar lavage fluid proteins in sarcoidosis: a proteomics approach.

In sarcoidosis, an inflammatory lung disease, the protein profile of bronchoalveolar lavage fluid (BALF) is altered. To study the BALF protein pattern changes in sarcoidosis, samples from six patients and four healthy individuals were analysed by two-dimensional polyacrylamide gel electrophoresis. A comparison of the protein-spot patterns showed a significantly higher number of protein spots in the pH range 5.5-6.7 in patients compared to controls (472 versus 384). Furthermore, the number of protein spots in the patients were significantly decreased in the acidic pH range 4.5-5.5 (399 versus 518). Measurement of the optical density in the gels showed varying expression levels for several protein spots. Seventeen of the altered protein spots were identified, of which seven have previously not been reported for BALF. Many of these are nonplasma proteins involved in the inflammatory and oxidant-antioxidant processes. In conclusion, the bronchoalveolar lavage fluid protein content is altered in sarcoidosis patients, especially for proteins that are not derived from plasma. The described proteomics approach will in the future be used to asses overall changes in the protein content associated with sarcoidosis and may offer the possibility of identifying disease-specific proteins.

[Detergent-like effects of the lipofuscin retinoid component A2-E in retinal pigment epithelial cells]. / Detergenzienwirkung des Lipofuszin-Retinoidbestandteils A2-E in retinalen Pigmentepithelzellen.

PURPOSE: Several lines of evidence suggest that excessive accumulation of lipofuscin in postmitotic retinal pigment epithelial (RPE) cells with age and in various hereditary retinal diseases, plays a pathogenetic role. The lipofuscin retinoid component A2-E (N-retinylidene-N-retinylethanolamine) inhibits lysosomal degradation. Here we sought to evaluate additional toxic mechanisms of A2-E, whereby possible detergent-like effects on various membranes in human RPE cells were investigated by latency measurements. METHODS: A postnuclear supernatant prepared from cultured human RPE cells was used to isolate intact lysosomes by fractionation of cellular organelles in two sequential gradients. Destabilization of the lysosomal membrane was tested by incubating the purified lysosomal fraction in the presence of A2-E and subsequent measurement of the latency of the lysosomal luminal marker beta-hexosaminidase. In order to compare the effect of A2-E on other cellular membranes, latencies of the specific markers succinate dehydrogenase and UDP-galactosyltransferase were assessed using partially purified mitochondria and microsomes. Intactness of the plasma membrane was tested by including A2-E in the culture medium before leakage of lactate dehydrogenase into the medium was determined. RESULTS: A more than 100-fold purification of the lysosomal fraction was achieved. Except for a minor activity of the mitochondrial marker, no contamination with other cell fractions was observed. Intactness of the purified lysosomes was well preserved during incubation in isotonic media and provided the basis for investigations on a possible detergent-like action of A2-E on lysosomal integrity. At concentrations above 2 microM A2-E, progressive leakage of the lysosomal marker was observed. In comparison leakage of the mitochondrial marker was induced at significantly lower concentrations (1 microM), whereas ER/Golgi membranes and the plasma membrane were relatively insensitive to a detergent effect of the retinoid. CONCLUSIONS: The described practical and fast methodology to obtain highly purified and intact lysosomes from RPE cells, provides a very suitable tool for investigations on compounds affecting the lysosomal structure. The results suggest that A2-E causes disintegration of the lysosomal membrane at relatively low concentrations which may implicate an involvement of such a mechanism in triggering lipofuscin-induced dysfunction of aged RPE in vivo. Secondary to disintegration of the lysosomal membrane, damage to mitochondria might be an additional pathogenic mechanism. Our data provide evidence for surfactant-like properties of A2-E on biomembranes which might be operative in retinal diseases associated with excessive lipofuscin accumulation including age-related macular degeneration.

Proteome analysis of lipofuscin in human retinal pigment epithelial cells.

Excessive accumulation of lipofuscin in postmitotic retinal pigment epithelial cells is a common pathogenetic pathway in various blinding retinal diseases including age-related macular degeneration, which is now the most common cause of registerable blindness in the industrialized nations. To better understand the role of lipofuscin accumulation and to manipulate the pathogenetic mechanisms on both experimental and therapeutic levels we analyzed the proteome of isolated human ocular lipofuscin granules from human RPE cells. After homogenization and fractionation by gradient ultracentrifugation of the RPE/choroid complex from 10 pairs of human donors, protein compounds were separated by 2D gel electrophoresis and analyzed using matrix-assisted laser desorption/ionization mass spectrometry and HPLC-coupled electrospray tandem mass spectrometry. Besides a better understanding of downstream pathways, this approach may provide new targets for therapeutic interventions in a currently untreatable disease.