Mahdy Ranjbar

Fc Receptor Inhibition Reduces Susceptibility to Oxidative Stress in Human RPE Cells Treated with Bevacizumab, but not Aflibercept

Background/Aims: VEGF-A is induced by oxidative stress, and functions as a survival factor for various cell types, including retinal pigment epithelial (RPE) cells. Anti-vascular endothelial growth factor (VEGF) drugs like aflibercept and bevacizumab have shown to be most effective in treating neovascular age-related macular degeneration (AMD), however uptake of the drugs might lead to interference with cell physiology. Herein, we evaluated the significance of the Fc receptor (FcR) within this context and moreover explored the impact of VEGF inhibition under normal conditions as well as under oxidative stress, in terms of potential adverse effects. Methods: ARPE-19 (human RPE) cells were treated with aflibercept and bevacizumab in presence or absence of H2O2 as oxidative stress stimulus. After 24h cells were evaluated for drug uptake, VEGF-A expression and secretion, levels of intracellular reactive oxygen species (ROS) as well as cell proliferation. Experiments were repeated with cells being pre-incubated with an FcR inhibitor prior to drug application. Results: Both drugs inhibited extracellular levels of VEGF-A and were taken up into the RPE, resulting in significantly reduced intracellular levels of VEGF-A. When oxidative stress was applied, intracellular ROS levels in cells treated with both drugs rose, and cell proliferation was reduced. Prior incubation with the FcR inhibitor lessened the uptake of bevacizumab, but not aflibercept into RPE cells, and simultaneously enhanced cell survival under oxidative stress conditions. Conclusions: Our results indicate that uptake and accumulation of aflibercept and bevacizumab within RPE cells affect the intracellular VEGF-A metabolism negatively, leading to a biologically relevant reduced cell survival under oxidative stress. The FcR plays a substantial role in the uptake of bevacizumab, but not aflibercept, which allows an enhanced RPE cell survival through FcR blockage in an environment dominated by oxidative stress, as clinically significant for various inflammatory retinal disorders.

ApoA-I Mimetic Peptide 4F Reduces Age-Related Lipid Deposition in Murine Bruch’s Membrane and Causes Its Structural Remodeling

ABSTRACT Purpose: Accumulation of lipoprotein-derived lipids including esterified and unesterified cholesterol in Bruch’s membrane of human eyes is a major age-related change involved in initiating and sustaining soft drusen in age-related macular degeneration (AMD). The apolipoprotein (apo) A-I mimetic peptide 4F is a small anti-inflammatory and anti-atherogenic agent, and potent modifier of plasma membranes. We evaluated the effect of intravitreally-injected 4F on murine Bruch’s membrane. Methods: We tested single intravitreal injections of 4F doses (0.6 µg, 1.2 µg, 2.4 µg, and placebo scrambled peptide) in ApoEnull mice ≥10 months of age. After 30 days, mice were euthanized. Eyes were processed for either direct immunofluorescence detection of esterified cholesterol (EC) in Bruch’s membrane whole mounts via a perfringolysin O-based marker linked to green fluorescent protein or by transmission electron microscopic visualization of Bruch’s membrane integrity. Fluorescein isothiocyanate-conjugated 4F was traced after injection. Results: All injected eyes showed a dose-dependent reduction of Bruch’s membrane EC with a concomitant ultrastructural improvement compared to placebo treated eyes. At a 2.4 µg dose of 4F, EC was reduced on average by ~60% and Bruch’s membrane returned to a regular pentalaminar structure and thickness. Tracer studies confirmed that injected 4F reached intraocular targets. Conclusion: We demonstrated a highly effective pharmacological reduction of EC and restoration of Bruch’s membrane ultrastructure. The apoA-I mimetic peptide 4F is a novel way to treat a critical AMD disease process and thus represents a new candidate for treating the underlying cause of AMD.

Stereotactic radiotherapy in neovascular age-related macular degeneration: Real-life efficacy and morphological evaluation of the outer retina-choroid complex

AbstractStereotactic radiotherapy (SRT) is a new approach to treat neovascular age-related macular degeneration (nAMD). The INTREPID trial suggested that SRT could reduce the frequency of regular intravitreal injections (IVIs) with antivascular endothelial growth factor drugs, which are necessary to control disease activity. However, the efficacy of SRT in nAMD and resulting morphological changes have not been validated under real-life circumstances, an issue, which we would like to address in this retrospective analysis.Patients who met the INTREPID criteria for best responders were eligible for SRT. A total of 32 eyes of 32 patients were treated. Thereafter, patients were examined monthly for 12 months and received pro re nata IVI of aflibercept or ranibizumab. Outcome measures were: mean number of injections, best-corrected visual acuity, and morphological changes of the outer retina-choroid complex as well as patient safety.Mean number of IVI decreased by almost 50% during the 12 months after SRT compared to the year before, whereas visual acuity increased by one line (logMAR). Morphological evaluation showed that most changes affect outer retinal layers.Stereotactic radiotherapy significantly reduced IVI retreatment in nAMD patients under real-life circumstances. Therefore, SRT might be the first step to stop visual loss as a result of IVI undertreatment, which is a major risk.

Ranibizumab interacts with the VEGF-A/VEGFR-2 signaling pathway in human RPE cells at different levels.

Vascular endothelial growth factor (VEGF) secreted by the retinal pigment epithelium (RPE) plays an important role in ocular homeostasis, but also in diseases, most notably age-related macular degeneration (AMD). To date, anti-VEGF drugs like ranibizumab have been shown to be most effective in treating these pathologic conditions. However, clinical trials suggest that the RPE could degenerate and perish through anti-VEGF treatment. Herein, we evaluated possible pathways and outcomes of the interaction between ranibizumab and human RPE cells (ARPE-19). Results indicate that ranibizumab affects the VEGF-A metabolism in RPE cells from an extra- as well as intracellular site. The drug is taken up into the cells, with the VEGF receptor 2 (VEGFR-2) being involved, and decreases VEGF-A protein levels within the cells as well as extracellularly. Oxidative stress plays a key role in various inflammatory disorders of the eye. Our results suggest that oxidative stress inhibits RPE cell proliferation. This anti-proliferative effect on RPE cells is significantly enhanced through ranibizumab, which does not inhibit RPE cell proliferation substantially in absence of relevant oxidative stress. Therefore, we emphasize that anti-VEGF treatment should be selected carefully in AMD patients with preexistent extensive RPE atrophy.

The Concentration-Dependent Effects of Indocyanine Green on Retinal Function in the Electrophysiological ex vivo Model of Isolated Perfused Vertebrate Retina

Background: Dye solutions such as indocyanine green (ICG) are used for the staining of intraocular structures. The aim of the presented study was to investigate the effects of ICG on bovine retinal function using different concentrations of ICG. Methods: Bovine retina preparations were perfused with a standard solution and the electroretinogram was recorded. The nutrient solution was substituted by an ICG solution at varying concentrations for 45 min. Afterwards the preparations were reperfused with standard solution for at least 85 min. Results: Significant reductions in b-wave amplitude were found for concentrations of 0.0025% (p = 0.0099) and 0.025% (p = 0.0378). For the concentration of 0.025%, the b-wave amplitude remained significantly decreased (p = 0.0082) after the observation period, but a full recovery of the b-wave was observed for the concentration of 0.0025% (p = 0.1917). Conclusion: Intraocular application of sufficient ICG concentrations for internal limiting membrane staining seems not possible without interfering with retinal function.

Detection of esterified cholesterol in murine Bruch's membrane wholemounts with a perfringolysin O-based cholesterol marker.

PURPOSE To investigate the effects of Bruchs membrane (BrM) neutral lipid deposition in mouse models and its significance to aging and age-related macular degeneration, it is essential to reliably detect small quantities of neutral lipids including esterified cholesterol (EC). In chorioretinal sections and BrM wholemounts, we tested a novel fluorescent cholesterol marker based on the bacterial toxin perfringolysin O (PFO) and compared results with those obtained with the classic cholesterol dye filipin. METHODS An engineered plasmid containing the specific cholesterol binding domain (D4) of PFO fused to green fluorescent protein (GFP) was expressed in cultured E. coli, isolated, purified, and concentrated. A total of 150 BrM-choroid wholemounts and chorioretinal sections of 11- to 13-month-old ApoE(null) mice were prepared and stained with PFO/D4-GFP or filipin for EC. Samples were examined by epifluorescence microscopy. RESULTS The fluorescence intensity of PFO/D4-GFP was strong, stable, and, if small quantities of EC were present, superior to filipin. In all specimens, we could sharply locate the PFO/D4-GFP signal to BrM. A semiquantitative evaluation of BrM lipid deposition is possible by measuring PFO/D4-GFP fluorescence intensity. CONCLUSIONS The use of PFO/D4-GFP allowed a robust and direct detection of EC in aged murine BrM. In wholemount samples, its strong and stable fluorescence facilitated a semiquantitative evaluation of BrM-EC content over a large area. The patterns of EC deposition in murine BrM wholemounts are comparable with findings in human BrM wholemounts. Perfringolysin O/D4-GFP could be an important tool for investigating the effects of BrM lipid deposition in mouse models.

Impact of correct anatomical slab segmentation on foveal avascular zone measurements by optical coherence tomography angiography in healthy adults

Purpose To evaluate the impact of correct anatomical slab segmentation on foveal avascular zone (FAZ) dimensions in the superficial capillary plexus (SCP) and deep capillary plexus (DCP) using optical coherence tomography angiography (OCTA). Methods Participants with healthy retinas were recruited, and 5 × 5 mm OCTA images were acquired using the Canon HS-100 Angio eXpert module. FAZ size was measured in automatically (AS, manufacturer-based) and manually (MS, anatomical-based) segmented OCTA slabs by two experienced graders. FAZ dimensions, inter-rater agreement, and correlation to demographic and retinal parameters were evaluated. Results A total of 38 eyes from 20 healthy adult subjects were included in this cross-sectional study. While in AS slabs, the FAZ in the SCP was smaller than in the DCP, in MS images, it was the opposite. MS had a relevant impact on inter-rater agreement of FAZ measurements in the SCP. The FAZ area in both plexus correlated inversely with the central retinal thickness (CRT), irrespective of the segmentation applied. Furthermore, an enlargement of FAZ size in the DCP with increasing age was found. Finally, the FAZ in female participants was significantly larger than in their male counterparts, regardless of the evaluated plexus and chosen segmentation. Conclusions Correct anatomical slab segmentation has a significant impact on FAZ size measurements. Not adjusting the segmentation boundaries represents a significant source of error for measuring FAZ area and confounds comparisons across studies as well as OCTA devices.

The effect of disease-modifying antirheumatic drugs on retinal function in the electrophysiological ex vivo model of the isolated perfused vertebrate retina.

Background: Disease-modifying antirheumatic drugs (DMARDs) have been suggested in the treatment of inflammatory ophthalmological diseases. The aim of the present study was to investigate the effects of these DMARDs on bovine retinal function. Methods: Bovine retina preparations were perfused with a standard solution. After recording stable electroretinograms the nutrient solution was substituted by a DMARD medium with varying concentrations of different drugs (etanercept and infliximab) for 30 min. Afterwards b-wave recovery was observed. Results: Significant decreases in the b-wave amplitude (p < 0.05) were found for etanercept 0.5 mg/ml (p = 0.0022). Infliximab 2 mg/ml (p = 0.1276) did not result in any statistically significant b-wave reduction. Conclusion: The presented data suggest that infliximab might have the better safety profile than etanercept.

The effect of anakinra on retinal function in isolated perfused vertebrate retina

Purpose Blockage of the interleukin 1 (IL-1) signaling pathway has been proposed for treatment of inflammatory disorders like those affecting the retina and its adjacent tissue. Herein, we evaluated one of those inhibitory drugs, anakinra (Kineret®), based on its safety profile with emphasis on retinal function from an electrophysiological point of view. Methods Bovine retina preparations were perfused with two different concentrations of anakinra (1 mg/ml and 2 mg/ml). An electroretinogram (ERG) was recorded and b-wave recovery assessed. Results Exposure to anakinra at a concentration of 1 mg/ml did not decrease the b-wave amplitude, whereas 2 mg/ml resulted in a significant reduction. Conclusions Based on these preliminary results, anakinra at a dose as low as 1 mg/ml could be regarded as safe for retinal function. However, dosages of 2 mg/ml and more do have toxic electrophysiological effects, at least for the short-term.

Peripheral Hypertrophic Subepithelial Corneal Degeneration

A woman in her 40s with an unremarkable medical history presented with bilateral blurred vision. Visual acuity was 20/40 OU. Slitlamp examination revealed bilateral peripheral corneal fibrosis and nasal superficial vascularization resembling a pseudopterygium. Corneal topography identified irregular astigmatism (5.9 diopters [D] OD; 7.7 D OS), and optical coherence tomography showed focal hyperreflectivity as well as subepithelial nodules (Figure, A). Lesions were removed by superficial keratectomy plus intraoperative application of mitomycin C. Subsequent histopathologic evaluation demonstrated corneal changes without inflammation (Figure, B). At 3 months after surgery, the astigmatism had decreased (0.6 D OD; 1.8 D OS), while visual acuity had improved (20/20 OD; 20/25 OS). Peripheral hypertrophic subepithelial corneal degeneration is a typically bilateral, slowly progressive perilimbal disorder that, to our knowledge, has rarely been described in the literature and needs to be distinguished from Salzmann nodular degeneration.1,2 The lack of histologic inflammation is pathognomonic, yet the cause of peripheral hypertrophic subepithelial corneal degeneration is still unknown.3,4